Archive for the ‘Hematopoiesis’ Category


Larry H. Bernstein, MD, FCAP, Curator



Hematopoietic Stem Cells Use a Simple Heirarchy




These papers challenge this model by arguing that the CMP does not exist. Let me say that again – the CMP, a cell that has been identified several times in mouse and human bone marrow isolates, does not exist. When CMPs were identified from mouse and human none marrow extracts, they were isolated by means of flow cytometry, which is a very powerful technique, but relies on the assumption that the cell type you want to isolate is represented by the cell surface protein you have chosen to use for its isolation. Once the presumptive CMP was isolated, it could recapitulate the myeloid lineage when implanted into the bone marrow of laboratory animals and it could also produce all the myeloid cells in cell culture. Sounds convincing doesn’t it?

In a paper in Science magazine, Faiyaz Notta and colleagues from the University of Toronto beg to differ. By using a battery of antibodies to particular cell surface molecules, Notta and others identified 11 different cell types from umbilical cord blood, bone marrow, and human fetal liver that isolates that would have traditionally been called the CMP. It turns out that the original CMP isolate was a highly heterogeneous mixture of different cell types that were all descended from the HSC, but had different developmental potencies.


Notta and others used single-cell culture assays to determine what kinds of cells these different cell types would make. Almost 3000 single-cell cultures later, it was clear that the majority of the cultured cells were unipotent (could differentiate into only one cell type) rather than multipotent. In fact, the cell that makes platelets, the megakarocyte, seems to derive directly from the MPP, which jives with the identification of megakarocyte progenitors within the HSC compartment of bone marrow that make platelets “speedy quick” in response to stress (see R. Yamamoto et al., Cell 154, 1112 (2013); S. Haas, Cell Stem Cell 17, 422 (2015)).

Another paper in the journal Cell by Paul and others from the Weizmann Institute of Science, Rehovot, Israel examined over 2700 mouse CMPs and subjected these cells to gene expression analyses (so-called single-cell transriptome analysis). If the CMP is truly multipotent, then you would expect it to express genes associated with lots of different lineages, but that is not what Paul and others found. Instead, their examination of 3461 genes revealed 19 different progenitor subpopulations, and each of these was primed toward one of the seven myeloid cell fates. Once again, the presumptive CMPs looked very unipotent at the level of gene expression.

One particular subpopulation of cells had all the trappings of becoming a red blood cell and there was no indication that these cells expressed any of the megakarocyte-specific genes you would expect to find if MEPS truly existed. Once again, it looks as though unipotency is the main rule once the MPP commits to a particular cell lineage.

Thus, it looks as though either the CMP is a very short-lived state or that it does not exist in mouse and human bone marrow. Paul and others did show that cells that could differentiate into more than one cell type can appear when regulation is perturbed, which suggests that under pathological conditions, this system has a degree of plasticity that allows the body to compensate for losses of particular cell lineages.

 A model of the changes in human My-Er-Mk differentiation that occur across developmental time points. Graphical depiction of My-Er-Mk cell differentiation that encompasses the predominant lineage potential of progenitor subsets; the standard model is shown for comparison. The redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent. The grayed planes represent theoretical tiers of differentiation.
A model of the changes in human My-Er-Mk differentiation that occur across developmental time points.
Graphical depiction of My-Er-Mk cell differentiation that encompasses the predominant lineage potential of progenitor subsets; the standard model is shown for comparison. The redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent. The grayed planes represent theoretical tiers of differentiation.

Fetal HSCs, however, are a bird of a different feather, since they divide quickly and reside in fetal liver.  Also, these HSCs seem to produce CMPs, which is more in line with the classical model.  Does the environmental difference or fetal liver and bone marrow make the difference?  In adult bone marrow, some HSCs nestle next to blood vessels where they encounter cells that hang around blood vessels known as “pericytes.”  These pericytes sport a host of cell surface molecules that affect the proliferative status of HSCs (e.g., nestin, NG2).  What about fetal liver?  That’s not so clear – until now.

In the same issue of Science magazine, Khan and others from the Albert Einstein College of Medicine in the Bronx, New York, report that fetal liver also has pericytes that express the same cell surface molecules as the ones in bone marrow, and the removal of these cells reduces the numbers of and proliferative status of fetal liver HSCs.

Now we have a conundrum, because the same cells in bone marrow do not drive HSC proliferation, but instead drive HSC quiescence.  What gives? Khan and others showed that the fetal liver pericytes are part of an expanding and constantly remodeling blood system in the liver and this growing, dynamic environment fosters a proliferative behavior in the fetal HSCs.

When umbilical inlet is closed at birth, the liver pericytes stop expressing Nestin and NG2, which drives the HSCs from the fetal liver to the other place were such molecules are found in abundance – the bone marrow.

These models give us a better view of the inner workings of HSC differentiation.  Since HSC transplantation is one of the mainstays of leukemia and lymphoma treatment, understanding HSC biology more perfectly will certainly yield clinical pay dirt in the future.


Distinct routes of lineage development reshape the human blood hierarchy across ontogeny

In a classical view of hematopoiesis, the various blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. We developed a cell-sorting scheme to resolve myeloid (My), erythroid (Er), and megakaryocytic (Mk) fates from single CD34+ cells and then mapped the progenitor hierarchy across human development. Fetal liver contained large numbers of distinct oligopotent progenitors with intermingled My, Er, and Mk fates. However, few oligopotent progenitor intermediates were present in the adult bone marrow. Instead, only two progenitor classes predominate, multipotent and unipotent, with Er-Mk lineages emerging from multipotent cells. The developmental shift to an adult “two-tier” hierarchy challenges current dogma and provides a revised framework to understand normal and disease states of human hematopoiesis.

Transcriptional Heterogeneity and Lineage Commitment in Myeloid Progenitors

Franziska Paul, et al.
Cell Dec 2015; Volume 163, Issue 7:1663–1677
Figure thumbnail fx1
  • Transcriptionally primed single-cell subpopulations in early myeloid progenitors
  • Transcription factors and epigenetic landscapes that regulate myeloid priming
  • Mixed lineage states are not observed but appear when regulation is perturbed
  • New reference model for studying hematopoiesis at single-cell resolution



Within the bone marrow, stem cells differentiate and give rise to diverse blood cell types and functions. Currently, hematopoietic progenitors are defined using surface markers combined with functional assays that are not directly linked with in vivo differentiation potential or gene regulatory mechanisms. Here, we comprehensively map myeloid progenitor subpopulations by transcriptional sorting of single cells from the bone marrow. We describe multiple progenitor subgroups, showing unexpected transcriptional priming toward seven differentiation fates but no progenitors with a mixed state. Transcriptional differentiation is correlated with combinations of known and previously undefined transcription factors, suggesting that the process is tightly regulated. Histone maps and knockout assays are consistent with early transcriptional priming, while traditional transplantation experiments suggest that in vivo priming may still allow for plasticity given strong perturbations. These data establish a reference model and general framework for studying hematopoiesis at single-cell resolution.



Fetal liver hematopoietic stem cell niches associate with portal vessels

Jalal A. Khan, et al.   Science  08 Jan 2016; 351(6269):176-180
How HSCs populate the fetal liver

Hematopoietic stem cells (HSCs) undergo dramatic expansion in the fetal liver before migrating to their definitive site in the bone marrow. Khan et al. identify portal vessel–associated Nestin+NG2+ pericytes as critical HSC niche components (see the Perspective by Cabezas-Wallscheid and Trumpp). The portal vessel niche and HSCs expand according to fractal geometries, suggesting that niche cells—rather than factors expressed by the niche—drive HSC proliferation. After birth, arterial portal vessels transform into portal veins, and lose Nestin+NG2+pericytes. When this happens, the niche is lost and HSCs migrate away from the neonatal liver.

Science, this issue p. 176; see also p. 126


Whereas the cellular basis of the hematopoietic stem cell (HSC) niche in the bone marrow has been characterized, the nature of the fetal liver niche is not yet elucidated. We show that Nestin+NG2+ pericytes associate with portal vessels, forming a niche promoting HSC expansion. Nestin+NG2+ cells and HSCs scale during development with the fractal branching patterns of portal vessels, tributaries of the umbilical vein. After closure of the umbilical inlet at birth, portal vessels undergo a transition from Neuropilin-1+Ephrin-B2+ artery to EphB4+ vein phenotype, associated with a loss of periportal Nestin+NG2+ cells and emigration of HSCs away from portal vessels. These data support a model in which HSCs are titrated against a periportal vascular niche with a fractal-like organization enabled by placental circulation.




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Neutrophil Serine Proteases in Disease and Therapeutic Considerations

Larry H. Bernstein, MD, FCAP, Curator



SERPINB1 Regulates the activity of the neutrophil proteases elastase, cathepsin G, proteinase-3, chymase,
chymotrypsin, and kallikrein-3. Belongs to the serpin family. Ov-serpin subfamily. Note: This description may
include information from UniProtKB.
Chromosomal Location of Human Ortholog: 6p25
Cellular Component: extracellular space; membrane; cytoplasm
Molecular Function: serine-type endopeptidase inhibitor activity
Reference #:  P30740 (UniProtKB)
Alt. Names/Synonyms: anti-elastase; EI; ELANH2; ILEU; LEI; Leukocyte elastase inhibitor; M/NEI; MNEI; Monocyte/neutrophil elastase inhibitor; Peptidase inhibitor 2; PI-2; PI2; protease inhibitor 2 (anti-elastase), monocyte/neutrophil derived; serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 1; Serpin B1; serpin peptidase inhibitor, clade B (ovalbumin), member 1; SERPINB1
Gene Symbols: SERPINB1
Molecular weight: 42,742 Da


Alternative titles; symbols
HGNC Approved Gene Symbol: SERPINB1
Cloning and Expression
Monocyte/neutrophil elastase inhibitor (EI) is a protein of approximately 42,000 Mr with serpin-like functional properties.
Remold-O’Donnell et al. (1992) cloned EI cDNA and identified 3 EI mRNA species of 1.5, 1.9, and 2.6 kb in monocyte-like cells
and no hybridizing mRNA in lymphoblastoid cells lacking detectable EI enzymatic activity. The cDNA open reading frame encoded
a 379-amino acid protein. Its sequence established EI as a member of the serpin superfamily. Sequence alignment indicated that
the reactive center P1 residue is cys-344, consistent with abrogation of elastase inhibitory activity by iodoacetamide and making
EI a naturally occurring cys-serpin.



In the course of studying 4 closely linked genes encoding members of the ovalbumin family of serine proteinase inhibitors
(Ov-serpins) located on 18q21.3, Schneider et al. (1995) investigated the mapping of elastase inhibitor. They prepared PCR
primer sets of the gene, and by using the NIGMS monochromosomal somatic cell hybrid panel, showed that the EI gene maps
to chromosome 6.

By amplifying DNA of a somatic cell hybrid panel, Evans et al. (1995) unambiguously localized ELANH2 to chromosome 6.
With the use of a panel of radiation and somatic cell hybrids specific for chromosome 6, they refined the localization to
the short arm telomeric of D6S89, F13A (134570), and D6S202 at 6pter-p24.



Evans, E., Cooley, J., Remold-O’Donnell, E. Characterization and chromosomal localization of ELANH2, the gene encoding human
monocyte/neutrophil elastase inhibitor. Genomics 28: 235-240, 1995. [PubMed: 8530031related citations] [Full Text]
Remold-O’Donnell, E., Chin, J., Alberts, M. Sequence and molecular characterization of human monocyte/neutrophil elastase inhibitor.
Proc. Nat. Acad. Sci. 89: 5635-5639, 1992. [PubMed: 1376927related citations][Full Text]
Schneider, S. S., Schick, C., Fish, K. E., Miller, E., Pena, J. C., Treter, S. D., Hui, S. M., Silverman, G. A. A serine proteinase inhibitor locus at
18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene. Proc. Nat. Acad. Sci. 92: 3147-3151, 1995.
[PubMed: 7724531,related citations] [Full Text]


Leukocyte elastase inhibitor (serpin B1) (IPR015557)

Short name: Serpin_B1

Family relationships

  • Serpin family (IPR000215)
    • Leukocyte elastase inhibitor (serpin B1) (IPR015557)


Leukocyte elastase inhibitor is also known as serpin B1. Serpins (SERine Proteinase INhibitors) belong to MEROPS inhibitor family I4 (clan ID)
[PMID: 14705960].

Serpin B1 regulates the activity of neutrophil serine proteases such as elastase, cathepsin G and proteinase-3 and may play a regulatory role to
limit inflammatory damage due to proteases of cellular origin [PMID: 11747453]. It also functions as a potent intracellular inhibitor of granzyme
H [PMID: 23269243]. In mouse, four different homologues of human serpin B1 have been described [PMID: 12189154].


The neutrophil serine protease inhibitor SerpinB1 protects against inflammatory lung injury and morbidity in influenza virus infection

Dapeng Gong1,2, Charaf Benarafa1,2, Kevan L Hartshorn3 and Eileen Remold-O’Donnell1,2
J Immunol April 2009; 182(Meeting Abstract Supplement) 43.10

SerpinB1 is an efficient inhibitor of neutrophil serine proteases. SerpinB1-/- mice fail to clear bacterial lung infection with increased inflammation and neutrophil death. Here, we investigated the role of serpinB1 in influenza virus infection, where infiltrating neutrophils and monocytes facilitate virus clearance but can also cause tissue injury. Influenza virus (H3N2 A/Phil/82) infection caused greater and more protracted body weight loss in serpinB1-/- vs. WT mice (20% vs. 15%; nadir on day 4 vs. day 3). Increased morbidity was not associated with defective virus clearance. Cytokines (IFN, TNF, IL-17, IFN, G-CSF) and chemokines (MIP-1, KC, MIP-2) were increased in serpinB1-/- mice vs. WT on days 2-7 post-infection but not on day 1. In WT mice, histology indicated large infiltration of neutrophils peaking on day 1 and maximal airway injury on day 2 that resolved on day 3 coincident with the influx of monocytes/macrophages. In serpinB1-/- mice, neutrophils also peaked on day 1; epithelial injury was severe and sustained with accumulation of dead cells on day 2 and 3. Immunophenotyping of lung digests on day 2 and 3 showed delayed recruitment of monocytes, macrophages and DC in serpinB1-/- mice, but increase of activated CD4 (day 2-3) and CD8 (day 3) T cells. Our findings demonstrate that serpinB1 protects against morbidity and inflammatory lung injury associated with influenza infection.


The neutrophil serine protease inhibitor serpinb1 preserves lung defense functions in Pseudomonas aeruginosainfection

Charaf Benarafa 1 , 2 Gregory P. Priebe 3 , 4 , and Eileen Remold-O’Donnell 1 , 2
JEM July 30, 2007; 204(8): 1901-1909

Neutrophil serine proteases (NSPs; elastase, cathepsin G, and proteinase-3) directly kill invading microbes. However, excess NSPs in the lungs play a central role in the pathology of inflammatory pulmonary disease. We show that serpinb1, an efficient inhibitor of the three NSPs, preserves cell and molecular components responsible for host defense against Pseudomonas aeruginosa. On infection, wild-type (WT) and serpinb1-deficient mice mount similar early responses, including robust production of cytokines and chemokines, recruitment of neutrophils, and initial containment of bacteria. However, serpinb1−/− mice have considerably increased mortality relative to WT mice in association with late-onset failed bacterial clearance. We found that serpinb1-deficient neutrophils recruited to the lungs have an intrinsic defect in survival accompanied by release of neutrophil protease activity, sustained inflammatory cytokine production, and proteolysis of the collectin surfactant protein–D (SP-D). Coadministration of recombinant SERPINB1 with the P. aeruginosa inoculum normalized bacterial clearance inserpinb1−/− mice. Thus, regulation of pulmonary innate immunity by serpinb1 is nonredundant and is required to protect two key components, the neutrophil and SP-D, from NSP damage during the host response to infection.


Neutrophils are the first and most abundant phagocytes mobilized to clear pathogenic bacteria during acute lung infection. Prominent among their antimicrobial weapons, neutrophils carry high concentrations of a unique set of serine proteases in their granules, including neu trophil elastase (NE), cathepsin G (CG), and proteinase-3. These neutrophil serine proteases (NSPs) are required to kill phagocytosed bacteria and fungi (12). Indeed, neutrophils lacking NE fail to kill phagocytosed pathogens, and mice deficient for NE and/or CG have increased mortality after infection with pulmonary pathogens (34). However, NSPs in the lung airspace can have a detrimental effect in severe inflammatory lung disease through degradation of host defense and matrix proteins (57). Thus, understanding of the mechanisms that regulate NSP actions during lung infections associated with neutrophilia will help identify strategies to balance host defense and prevent infection-induced tissue injury.


SERPINB1, also known as monocyte NE inhibitor (8), is an ancestral serpin super-family protein and one of the most efficient inhibitors of NE, CG, and proteinase-3 (910). SERPINB1 is broadly expressed and is at particularly high levels in the cytoplasm of neutrophils (1112). SERPINB1 has been found complexed to neutro phil proteases in lung fluids of cystic fibrosis patients and in a baboon model of bronchopulmonary dysplasia (1314). Although these studies suggest a role for SERPINB1 in regulating NSP activity, it is unclear whether these complexes reflect an important physiological role for SERPINB1 in the lung air space.


To define the physiological importance of SERPINB1 in shaping the outcome of bacterial lung infection, we generated mice deficient for serpinb1 (serpinb1−/−) by targeted mutagenesis in embryonic stem (ES) cells (Fig. 1, A–C). Crossings of heterozygous mice produced WT (+/+), heterozygous (+/−), and KO (−/−) mice for serpinb1 at expected Mendelian ratios (25% +/+, 51% +/−, and 24% −/−; n = 225; Fig. 1 D), indicating no embryonic lethality. Bone marrow neutrophils of serpinb1−/− mice lacked expression of the protein, whereas heterozygous serpinb1+/− mice had reduced levels compared with WT mice (Fig. 1 E). Importantly, levels of the cognate neutrophil proteases NE and CG, measured as antigenic units, were not altered by deletion of serpinb1 (Fig. 1 F). When maintained in a specific pathogen-free environment, serpinb1−/− mice did not differ from WT littermates in growth, litter size, or life span (followed up to 12 mo), and no gross or histopathological defects were observed at necropsy in 8-wk-old mice.

6–8-wk-old animals were intranasally inoculated with the nonmucoid Pseudomonas aeruginosa strain PAO1. Using two infection doses (3 × 106 and 7 × 106 CFU/mouse),serpinb1−/− mice had a significantly lower survival probability and a shorter median survival time compared with WT mice (Fig. 2 A). Further groups of infected mice were used to evaluate bacterial clearance. At 6 h after infection, the bacteria were similarly restricted in mice of the two genotypes, suggesting that the serpinb1−/− mice have a normal initial response to infection. At 24 h, the median bacterial count in the lungs of serpinb1−/− mice was five logs higher than that of the WT mice (P < 0.001), and the infection had spread systemically in serpinb1−/− mice but not in WT mice, as shown by high median CFU counts in the spleen (Fig. 2 B). Histological examination at 24 h after infection revealed abundant neutrophil infiltration in the lungs of both WT and serpinb1−/− mice, and consistent with the bacteriological findings, numerous foci of bacterial colonies and large areas of alveolar exudates were found in serpinb1−/− mice only (Fig. 2 C). When challenged with the mucoid P. aeruginosa clinical strain PA M57-15 isolated from a cystic fibrosis patient, WT mice cleared >99.9% of the inoculum within 24 h, whereas serpinb1-deficient mice failed to clear the infection (Fig. 2 D). Thus, the NSP inhibitor serpinb1 is essential for maximal protection against pneumonia induced by mucoid and nonmucoid strains of P. aeruginosa.

Figure 2.

Serpinb1−/− mice fail to clear P. aeruginosalung infection. (A) Kaplan-Meier survival curves of WT (+/+) and serpinb1-deficient (−/−) mice intranasally inoculated with nonmucoid P. aeruginosa strain PAO1. Increased mortality of serpinb1−/− mice was statistically significant (P = 0.03 at 3 × 106CFU/mouse; P < 0.0001 at 7 × 106CFU/mouse). (B) CFUs per milligram of lung (left) and splenic (right) tissue determined 6 and 24 h after inoculation with 3 × 106 CFUP. aeruginosa PAO1 in WT (+/+, filled circles) and serpinb1−/− (−/−, open circles) mice. Each symbol represents a value for an individual mouse. Differences between median values (horizontal lines) were analyzed by the Mann-Whitney U test. Data below the limit of detection (dotted line) are plotted as 0.5 CFU × dilution factor. (C) Lung sections stained with hematoxylin and eosin show bacterial colonies (arrowheads) and alveolar exudate in lungs of serpinb1−/− mice 24 h after infection with P. aeruginosa PAO1. Bars, 50 μm. (D) Total CFUs in the lung and spleen 24 h after inoculation with 2 × 108 CFU of the mucoid P. aeruginosa strain PA M57-15 in WT (+/+, filled circles) and serpinb1−/− (−/−, open circles) mice. Differences between median values (horizontal lines) were analyzed by the Mann-Whitney U test.

To verify specificity of the gene deletion, we tested whether delivering rSERPINB1 would correct the defective phenotype. Indeed, intranasal instillation of rSERPINB1 to serpinb1−/− mice at the time of inoculation significantly improved clearance of P. aeruginosa PAO1 from the lungs assessed at 24 h and reduced bacteremia compared with infectedserpinb1−/− mice that received PBS instead of the recombinant protein (Fig. S1 A, available at We have previously demonstrated that rSERPINB1 has no effect on the growth of P. aeruginosa in vitro (15) and does not induce bacterial aggrega tion (16). Also, rSERPINB1 mixed with PAO1 had no effect on adherence of the bacteria to human bronchial epithelial and corneal epithelial cell lines (unpublished data). Therefore, the improved bacterial clearance in treated serpinb1−/− mice is not related to a direct antibacterial role for rSERPINB1 but rather to reducing injury induced by excess neutrophil proteases. In addition, previous in vivo studies in WT rats showed that rSERPINB1 can protect against elastase-induced lung injury (17) and accelerate bacterial clearance two- to threefold in the Pseudomonas agar bead model (15).

Evidence of excess NSP action was examined in the lungs of infected serpinb1−/− mice by measuring surfactant protein–D (SP-D). SP-D, a multimeric collagenous C-type lectin produced by alveolar epithelial cells, is highly relevant as a host defense molecule, because it functions as an opsonin in microbial clearance (18) and acts on alveolar macrophages to regulate pro- and antiinflammatory cytokine production (19). SP-D is also relevant as an NSP target because it is degraded in vitro by trace levels of each of the NSPs (1620). SP-D levels in lung homogenates of WT and serpinb1−/− mice were similar 6 h after P. aeruginosa infection. At 24 h, SP-D levels were reduced in the lungs ofserpinb1−/− mice compared with WT mice, as indicated by immunoblots. A lower molecular mass band indicative of proteolytic degradation is also apparent (Fig. 3 A). Densitometry analysis of the 43-kD SP-D band relative to β-actin indicated that the reduction of SP-D level was statistically significant (+/+, 45 ± 6 [n = 8]; −/−, 10 ± 2 [n = 8]; P < 0.0001 according to the Student’s t test). Furthermore, rSERPINB1 treatment ofP. aeruginosa–infected serpinb1−/− mice partly prevented the degradation of SP-D in lung homogenates compared with nontreated mice (Fig. S1 B). As a further test of the impact of serpinb1 deletion on NSP activity, isolated neutrophils of serpinb1−/− mice were treated with LPS and FMLP and tested for their ability to cleave recombinant rat SP-D (rrSP-D) in vitro. The extent of rrSP-D cleavage by serpinb1−/− neutrophils was fourfold greater than by WT neutrophils, as determined by densitometry. The cleavage was specific for NSPs because it was abrogated by rSERPINB1 and diisopropyl fluorophosphate (Fig. 3 B). Collectively, these findings indicate a direct role for serpinb1 in regulating NSP activity released by neutrophils and in preserving SP-D, an important-host defense molecule.

Efficient clearance of P. aeruginosa infection requires an early cytokine and chemokine response coordinated by both resident alveolar macrophages and lung parenchymal cells (2122). The IL-8 homologue keratinocyte-derived chemokine (KC) and the cytokines TNF-α, IL-1β, and G-CSF were measured in cell-free bronchoalveolar (BAL) samples. Although the tested cytokines were undetectable in sham-infected mice of both genotypes (unpublished data), comparable induc tion of these cytokines was observed in BAL of WT and serpinb1−/− mice at 6 h after infection, demonstrating that there is no early defect in cytokine production in serpinb1−/− mice. At 24 h, levels of TNF-α, KC, and IL-1β were sustained or increased in serpinb1−/− mice and significantly higher than cytokine levels in WT mice. G-CSF levels at 24 h were elevated to a similar extent in BAL of WT and KO mice (Fig. 3 C). However, G-CSF levels were significantly higher in the serum of serpinb1−/− mice (WT, 336 ± 80 ng/ml; KO, 601 ± 13 ng/ml; n = 6 of each genotype; P < 0.01). In addition, serpinb1−/− mice that were treated at the time of infection with rSERPINB1 had cytokine levels in 24-h lung homogenates that were indistinguishable from those of infected WT mice (Fig. S1 C). The increased cytokine production in the lungs of infected serpinb1−/− mice may be caused by failed bacterial clearance but also by excess NSPs, which directly induce cytokine and neutrophil chemokine production in pulmonary parenchymal cells and alveolar macrophages (2324).

Neutrophil recruitment to the lungs was next examined as a pivotal event of the response to P. aeruginosa infection (25). Lung homogenates were assayed for the neutrophil-specific enzyme myeloperoxidase (MPO) to quantify marginating, interstitial, and alveolar neutrophils. Neutrophils in BAL fluid were directly counted as a measure of neutrophil accumulation in the alveolar and airway lumen. MPO in lung homo genates was undetectable in uninfected mice and was comparably increased in mice of both genotypes at 6 h, suggesting normal early serpinb1−/− neutrophil margination and migration into the interstitium. However, by 24 h after infection, MPO levels in lung homogenates remained high in WT mice but were significantly decreased in serpinb1−/− mice (Fig. 4 A). Importantly, the content of MPO per cell was the same for isolated neutrophils of WT andserpinb1−/− mice (+/+, 369 ± 33 mU/106 cells; −/−, 396 ± 27 mU/106 cells). The numbers of neutrophils in BAL were negligible in uninfected mice and were similarly increased in WT and serpinb1−/− mice at 6 h after infection. Neutrophil counts in BAL further increased at 24 h, but the mean BAL neutrophil numbers were significantly lower in serpinb1−/− mice compared with WT mice (Fig. 4 B). The evidence from the 6-h quantitation of MPO in homogenates and neutrophils in BAL strongly suggests that neutrophil recruitment is not defective in infected serpinb1−/− mice. Moreover, the high levels of cytokines and neutrophil chemoattractant KC in serpinb1−/− mice at 24 h (Fig. 3 C) also suggest that, potentially, more neutrophils should be recruited. Therefore, to examine neutrophil recruitment in serpinb1−/− mice, we used a noninfectious model in which neutrophils are mobilized to migrate to the lung after intranasal delivery of P. aeruginosa LPS. MPO levels in lung homogenate and neutrophil numbers in BAL were not statistically different in WT and serpinb1−/− mice 24 h after LPS instillation (Fig. 4, C and D). Furthermore, the number of circulating blood neutrophils and recruited peritoneal neutrophils after injection of sterile irritants glycogen and thioglycollate did not differ in WT and serpinb1−/− mice (unpublished data). Alveolar macrophage numbers were similar in uninfected mice of both genotypes (∼5 × 105 cells/mouse) and did not substantially change upon infection. Collectively, these findings show that neutrophil recruitment to the lungs in response to P. aeruginosa infection is not defective in serpinb1−/− mice, and therefore, the recovery of lower numbers of serpinb1−/− neutrophils at 24 h after infection suggests their decreased survival.

To examine the putative increased death of serpinb1−/− neutrophils in the lungs after P. aeruginosa infection, lung sections were analyzed by immunohistochemistry. Caspase-3–positive leukocytes were more relevant in the alveolar space of serpinb1−/− mice compared with WT mice at 24 h after infection, suggesting increased neutrophil apoptosis (Fig. 5 A). The positive cells were counted in 50 high power fields (hpf’s), and mean numbers of caspase-3–stained cells were increased in the lungs of serpinb1/− mice (1.8 ± 0.2 cells/hpf) compared with WT mice (0.4 ± 0.1 cells/hpf; P < 0.0001). To characterize neutrophils in the alveoli and airways, neutrophils in BAL were identified in flow cytometry by forward scatter (FSC) and side scatter and were stained with annexin V (AnV) and propidium iodide (PI). At 24 h after infection, the proportion of late apoptotic/necrotic neutrophils (AnV+PI+) was increased at the expense of viable neutrophils (AnVPI) in the BAL of serpinb1−/− mice compared with WT mice (Fig. 5 B). Neutrophil fragments in BAL were also identified in flow cytometry by low FSC (FSClow) within the neutrophil population defined by the neutrophil marker Gr-1. The number of neutrophil fragments (FSClow, Gr-1+) relative to intact neutrophils was increased two- to threefold at 24 h after infection for serpinb1−/− compared with WT mice (Fig. 5 C). Moreover, free MPO in BAL supernatants was increased in serpinb1−/− mice compared with WT mice at 24 h after infection, indicating increased PMN lysis or degranulation (Fig. 5 D).

Finally, we questioned whether the enhanced death of serpinb1−/− pulmonary neutrophils was a primary effect of gene deletion or a secondary effect caused by, for example, bacteria or components of inflammation. To address this, neutrophils were collected using the noninfectious LPS recruitment model and were cultured in vitro to allow for spontaneous cell death. After 24 h, the percentages of apoptotic and necrotic neutrophils evaluated by microscopy were increased in serpinb1−/− neutrophils compared with WT neutrophils (Fig. 6, A–C). A similar increase in apoptotic cells was observed using AnV/PI staining and measurements of hypodiploid DNA (unpublished data). Moreover, live cell numbers from serpinb1−/− mice remaining in culture after 24 h were significantly decreased compared with WT mice (Fig. 6 D). The in vitro findings indicate that enhanced death of pulmonary neutrophils of infected serpinb1−/− mice is at least in part a cell-autonomous defect likely mediated by unchecked NSP actions.


In this paper, we have demonstrated that serpinb1, an intracellular serpin family member, regulates the innate immune response and protects the host during lung bacterial infection. Serpinb1 is among the most potent inhibitors of NSPs and is carried at high levels within neutrophils. Serpinb1-deficient mice fail to clear P. aeruginosa PAO1 lung infection and succumb from systemic bacterial spreading. The defective immune function in serpinb1−/− mice stems at least in part from an increased rate of neutrophil necrosis, reducing the number of phagocytes and leading to increased NSP activity in the lungs with proteolysis of SP-D. In addition, serpinb1-deficient mice also have impaired clearance of the mucoid clinical strain PA M57-15. Interestingly, mucoid strains of P. aeruginosa are cleared with a very high efficiency from the lungs of WT and cystic fibrosis transmembrane conductance regulator–deficient mice (26). The phenotype of serpinb1−/− mice reproduces major pathologic features of human pulmonary diseases characterized by excessive inflammation, massive neutrophil recruitment to the air space, and destruction of cellular and molecular protective mechanisms. Importantly, serpinb1 deficiency may be helpful as an alternative or additional model of the inflammatory lung pathology of cystic fibrosis.

The present study documents a key protective role for serpinb1 in regulating NSP actions in the lung. This role has previously been attributed to the NSP inhibitors α1-antitrypsin and secretory leukocyte protease inhibitor, which are found in the airway and alveolar lining fluid (2728). However, patients with α1-antitrypsin deficiency do not present with pulmonary infection secondary to innate immune defects despite increased NSP activity that leads to reduced lung elasticity and emphysema. Moreover, there is so far no evidence that deficiency in secretory leukocyte protease inhibitor results in failure to clear pulmonary infection. Because synthesis and storage of NSPs in granules is an event that exclusively takes place in bone marrow promyelocytes (29), the regulation of NSPs in the lung relies entirely on NSP inhibitors. Thus, the extent of the innate immune defect inserpinb1−/− mice and the normalization of bacterial clearance with topical rSERPINB1 treatment indicate that serpinb1 is required to regulate NSP activity in the airway fluids and that, during acute lung infection associated with high neutrophilic recruitment, there is insufficient compensation by other NSP inhibitors. The devastating effects of NSPs when released in the lungs by degranulating and necrotic neutrophils are well documented in human pulmonary diseases (5630). Therefore, our findings clearly establish a physiological and nonredundant role for serpinb1 in regulating NSPs during pulmonary infection.

NSPs also cleave molecules involved in apoptotic cell clearance, including the surfactant protein SP-D and the phosphatidylserine receptor on macrophages (3132), thereby tipping the balance further toward a detrimental outcome. The increased numbers of leukocytes with active caspase-3 in the alveolar space of P. aeruginosa–infectedserpinb1−/− mice suggest that the removal of apoptotic cells may be inadequate during infection. SP-D has been shown to stimulate phagocytosis of P. aeruginosa by alveolar macrophages in vitro (33), and SP-D–deficient mice were found to have defective early (6-h) clearance of P. aeruginosa from the lung (34). Although the destruction of SP-D alone may not entirely account for the defective phenotype of serpinb1−/− mice, loss of SP-D likely diminishes bacterial clearance and removal of apop totic neutrophils.

Given that NSPs also mediate bacterial killing, why would NSP excess lead to a failed bacterial clearance? In the NE KO mice, the decreased killing activity of neutrophils is a direct consequence of the loss of the bactericidal activity of NE. The absence of an early bacterial clearance defect at 6 h after infection in serpinb1−/− mice suggests that there is initially normal bacterial killing. The current understanding is that the compartmentalization of the NSPs is crucial to the outcome of their actions: on the one hand, NSPs are protective when killing microbes within phagosomes, and on the other hand, extracellular NSPs destroy innate immune defense molecules such as lung collectins, immunoglobulins, and complement receptors. We have shown that the regulation of NSP activity is essential and that cytoplasmic serpinb1 provides this crucial shield. Neutrophils undergoing cell death gradually transition from apoptosis, characterized by a nonpermeable plasma membrane, to necrosis and lysis, where cellular and granule contents, including NSPs, are released. The increased pace of serpinb1−/− neutrophil cell death strongly suggests that unopposed NSPs may precipitate neutrophil demise and, therefore, reduce the neutrophil numbers leading to a late-onset innate immune defect. High levels of G-CSF, a prosurvival cytokine for neutrophils, also indicate that increased cell death is likely independent or downstream of G-CSF.

In conclusion, serpinb1 deficiency unleashes unbridled proteolytic activity during inflammation and thereby disables two critical components of the host response to bacterial infection, the neutrophil and the collectin SP-D. The phenotype of the infectedserpinb1-deficient mouse, characterized by a normal early antibacterial response that degenerates over time, highlights the delicate balance of protease–antiprotease systems that protect the host against its own defenses as well as invading microbes during infection-induced inflammation.



Proteinase 3 and neutrophil elastase enhance inflammation in mice by inactivating antiinflammatory progranulin

K Kessenbrock,1 LFröhlich,2 M Sixt,3 …., A Belaaouaj,5 J Ring,6,7 M Ollert,6 R Fässler,3 and DE. Jenne1
J Clin Invest. 2008 Jul 1; 118(7): 2438–2447.

Neutrophil granulocytes form the body’s first line of antibacterial defense, but they also contribute to tissue injury and noninfectious, chronic inflammation. Proteinase 3 (PR3) and neutrophil elastase (NE) are 2 abundant neutrophil serine proteases implicated in antimicrobial defense with overlapping and potentially redundant substrate specificity. Here, we unraveled a cooperative role for PR3 and NE in neutrophil activation and noninfectious inflammation in vivo, which we believe to be novel. Mice lacking both PR3 and NE demonstrated strongly diminished immune complex–mediated (IC-mediated) neutrophil infiltration in vivo as well as reduced activation of isolated neutrophils by ICs in vitro. In contrast, in mice lacking just NE, neutrophil recruitment to ICs was only marginally impaired. The defects in mice lacking both PR3 and NE were directly linked to the accumulation of antiinflammatory progranulin (PGRN). Both PR3 and NE cleaved PGRN in vitro and during neutrophil activation and inflammation in vivo. Local administration of recombinant PGRN potently inhibited neutrophilic inflammation in vivo, demonstrating that PGRN represents a crucial inflammation-suppressing mediator. We conclude that PR3 and NE enhance neutrophil-dependent inflammation by eliminating the local antiinflammatory activity of PGRN. Our results support the use of serine protease inhibitors as antiinflammatory agents.


Neutrophils belong to the body’s first line of cellular defense and respond quickly to tissue injury and invading microorganisms (1). In a variety of human diseases, like autoimmune disorders, infections, or hypersensitivity reactions, the underlying pathogenic mechanism is the formation of antigen-antibody complexes, so-called immune complexes (ICs), which trigger an inflammatory response by inducing the infiltration of neutrophils (2). The subsequent stimulation of neutrophils by C3b-opsonized ICs results in the generation of ROS and the release of intracellularly stored proteases leading to tissue damage and inflammation (3). It is therefore important to identify the mechanisms that control the activation of infiltrating neutrophils.

Neutrophils abundantly express a unique set of neutrophil serine proteases (NSPs), namely cathepsin G (CG), proteinase 3 (PR3; encoded by Prtn3), and neutrophil elastase (NE; encoded by Ela2), which are stored in the cytoplasmic, azurophilic granules. PR3 and NE are closely related enzymes, with overlapping and potentially redundant substrate specificities different from those of CG. All 3 NSPs are implicated in antimicrobial defense by degrading engulfed microorganisms inside the phagolysosomes of neutrophils (48). Among many other functions ascribed to these enzymes, PR3 and NE were also suggested to play a fundamental role in granulocyte development in the bone marrow (911).

While the vast majority of the enzymes is stored intracellularly, minor quantities of PR3 and NE are externalized early during neutrophil activation and remain bound to the cell surface, where they are protected against protease inhibitors (1213). These membrane presented proteases were suggested to act as path clearers for neutrophil migration by degrading components of the extracellular matrix (14). This notion has been addressed in a number of studies, which yielded conflicting results (1517). Thus, the role of PR3 and NE in leukocyte extravasation and interstitial migration still remains controversial.

Emerging data suggest that externalized NSPs can contribute to inflammatory processes in a more complex way than by simple proteolytic tissue degradation (18). For instance, recent observations using mice double-deficient for CG and NE indicate that pericellular CG enhances IC-mediated neutrophil activation and inflammation by modulating integrin clustering on the neutrophil cell surface (1920). Because to our knowledge no Prtn3–/– mice have previously been generated, the role of this NSP in inflammatory processes has not been deciphered. Moreover, NE-dependent functions that can be compensated by PR3 in Ela2–/–animals are still elusive.

One mechanism by which NSPs could upregulate the inflammatory response has recently been proposed. The ubiquitously expressed progranulin (PGRN) is a growth factor implicated in tissue regeneration, tumorigenesis, and inflammation (2123). PGRN was previously shown to directly inhibit adhesion-dependent neutrophil activation by suppressing the production of ROS and the release of neutrophil proteases in vitro (23). This antiinflammatory activity was degraded by NE-mediated proteolysis of PGRN to granulin (GRN) peptides (23). In contrast, GRN peptides may enhance inflammation (23) and have been detected in neutrophil-rich peritoneal exudates (24). In short, recent studies proposed PGRN as a regulator of the innate immune response, but the factors that control PGRN function are still poorly defined and its relevance to inflammation needs to be elucidated in vivo.

In the present study, we generated double-deficient Prtn3–/–Ela2–/– mice to investigate the role of these highly similar serine proteases in noninfectious neutrophilic inflammation. We established that PR3 and NE are required for acute inflammation in response to subcutaneous IC formation. The proteases were found to be directly involved in early neutrophil activation events, because isolated Prtn3–/–Ela2–/– neutrophils were poorly activated by ICs in vitro. These defects in Prtn3–/–Ela2–/– mice were accompanied by accumulation of PGRN. We demonstrated that PGRN represents a potent inflammation-suppressing factor that is cleaved by both PR3 and NE. Our data delineate what we believe to be a previously unknown proinflammatory role for PR3 and NE, which is accomplished via the local inactivation of antiinflammatory PGRN.


Generation of Prtn3–/–Ela2–/– mice.

To analyze the role of PR3 and NE in neutrophilic inflammation, we generated a Prtn3–/–Ela2–/– mouse line by targeted gene disruption in embryonic stem cells (see Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI34694DS1). Positive recombination of the Prtn3/Ela2locus was proven by Southern blotting of embryonic stem cell clones (Figure ​(Figure1A).1A). Prtn3–/–Ela2–/– mice showed no expression of mRNA for PR3 and NE in bone marrow cells, as assessed by RT-PCR (Figure ​(Figure1B).1B). The successful elimination of PR3 and NE was confirmed at the level of proteolytic activity in neutrophil lysates using a PR3/NE-specific chromogenic substrate (Supplemental Figure 3) as well as by casein zymography (Figure ​(Figure1C).1C). The substantially reduced casein degradation by heterozygous neutrophils indicates gene-dosage dependence of PR3/NE activities. Furthermore, PR3 and NE deficiency was proven by Western blotting using cell lysates from bone marrow–derived neutrophils, while other enzymes stored in azurophilic granula, such as CG and myeloperoxidase (MPO), were normally detected (Figure ​(Figure1D).1D). Crossing of heterozygous Prtn3+/–Ela2+/– mice resulted in regular offspring of WT, heterozygous, and homozygous genotype according to the Mendelian ratio. Despite the absence of 2 abundant serine proteases, and in contrast to expectations based on previous reports (911), we found unchanged neutrophil morphology (Figure ​(Figure1E)1E) and regular neutrophil populations in the peripheral blood of the mutant mice, the latter as assessed via flow cytometry to determine the differentiation markers CD11b and Gr-1 (Figure ​(Figure1F)1F) (2526). Moreover, Prtn3–/–Ela2–/– mice demonstrated normal percentages of the leukocyte subpopulations in the peripheral blood, as determined by the Diff-Quick staining protocol and by hemocytometric counting (Supplemental Figure 2, A and B). Hence, the proteases are not crucially involved in granulopoiesis, and ablating PR3 and NE in the germ line represents a valid approach to assess their biological significance in vivo.


Figure 1

Generation and characterization of Prtn3–/–Ela2–/– mice.

PR3 and NE are dispensable for neutrophil extravasation and interstitial migration.

To examine neutrophil infiltration into the perivascular tissue, we applied phorbol esters (croton oil) to the mouse ears. At 4 h after stimulation, we assessed the neutrophil distribution in relation to the extravascular basement membrane (EBM) by immunofluorescence microscopy of fixed whole-mount specimens (Figure ​(Figure2A).2A). We found that Prtn3–/–Ela2–/– neutrophils transmigrated into the interstitium without retention at the EBM (Figure ​(Figure2B),2B), resulting in quantitatively normal and widespread neutrophil influx compared with WT mice (Figure ​(Figure2C).2C). Moreover, we analyzed chemotactic migration of isolated neutrophils through a 3-dimensional collagen meshwork in vitro (Supplemental Video 1) and found unhampered chemotaxis toward a C5a gradient, based on the directionality (Figure ​(Figure2D)2D) and velocity (Figure ​(Figure2E)2E) of Prtn3–/–Ela2–/–neutrophils. These findings led us to conclude that PR3 and NE are not principally required for neutrophil extravasation or interstitial migration.


Figure 2

PR3 and NE are not principally required for neutrophil extravasation and interstitial migration.

Reduced inflammatory response to ICs in Prtn3–/–Ela2–/– mice.

The formation of ICs represents an important trigger of neutrophil-dependent inflammation in many human diseases (2). To determine the role of PR3 and NE in this context, we induced a classic model of subcutaneous IC-mediated inflammation, namely the reverse passive Arthus reaction (RPA) (27). At 4 h after RPA induction, we assessed the cellular inflammatory infiltrates by histology using H&E-stained skin sections (Figure ​(Figure3A).3A). Neutrophils, which were additionally identified by Gr-1 immunohistochemistry, made up the vast majority of all cellular infiltrates (Figure ​(Figure3A).3A). We found that neutrophil infiltration to the sites of IC formation was severely diminished in Prtn3–/–Ela2–/– mice. Indeed, histological quantification revealed significantly reduced neutrophil influx in Prtn3–/–Ela2–/– mice compared with WT mice, while Ela2–/– mice showed marginally reduced neutrophil counts (Figure ​(Figure3B).3B). These results indicate that PR3 and NE fulfill an important proinflammatory function during IC-mediated inflammation.

Figure 3

Impaired inflammatory response to locally formed ICs inPrtn3–/–Ela2–/– mice.

(A) Representative photomicrographs of inflamed skin sections 4 h after IC formation. Neutrophils were identified morphologically (polymorphic nucleus) in H&E stainings and by Gr-1 staining (red). The cellular infiltrates were located to the adipose tissue next to the panniculus carnosus muscle (asterisks) and were primarily composed of neutrophil granulocytes. Scale bars: 200 μm. (B) Neutrophil infiltrates in lesions from Prtn3–/–Ela2–/– mice were significantly diminished compared with Ela2–/– mice and WT mice. Neutrophil influx in Ela2–/–mice was slightly, but not significantly, diminished compared with WT mice. Results are mean ± SEM infiltrated neutrophils per HPF. *P < 0.05.

PR3 and NE enhance neutrophil activation by ICs in vitro.

PR3 and NE enhance neutrophil activation by ICs in vitro.

Because PR3 and NE were required for the inflammatory response to IC (Figure ​(Figure3),3), but not to phorbol esters (Figure ​(Figure2),2), we considered the enzymes as enhancers of the neutrophil response to IC. We therefore assessed the oxidative burst using dihydrorhodamine as a readout for cellular activation of isolated, TNF-α–primed neutrophils in the presence of ICs in vitro. While both WT and Prtn3–/–Ela2–/– neutrophils showed a similar, approximately 20-min lag phase before the oxidative burst commenced, the ROS production over time was markedly reduced, by 30%–40%, in the absence of PR3 and NE (Figure ​(Figure4A).4A). In contrast, oxidative burst triggered by 25 nM PMA was not hindered in Prtn3–/–Ela2–/– neutrophils (Figure ​(Figure4B),4B), which indicated no general defect in producing ROS. We also performed a titration series ranging from 0.1 to 50 nM PMA and found no reduction in oxidative burst activity in Prtn3–/–Ela2–/– neutrophils at any PMA concentration used (Supplemental Figure 4). These data are consistent with our in vivo experiments showing that neutrophil influx to ICs was impaired (Figure ​(Figure3),3), whereas the inflammatory response to phorbol esters was normal (Figure ​(Figure2,2, A–C), in Prtn3–/–Ela2–/– mice. To compare neutrophil priming in WT and Prtn3–/–Ela2–/–neutrophils, we analyzed cell surface expression of CD11b after 30 min of incubation at various concentrations of TNF-α and found no difference (Supplemental Figure 5). Moreover, we observed normal neutrophil adhesion to IC-coated surfaces (Supplemental Figure 6A) and unaltered phagocytosis of opsonized, fluorescently labeled E. coli bacteria (Supplemental Figure 6, B and C) in the absence of both proteases. We therefore hypothesized that PR3 and NE enhance early events of adhesion-dependent neutrophil activation after TNF-α priming and binding of ICs. It is important to note that Ela2–/– neutrophils were previously shown to react normally in the same setup (20). Regarding the highly similar cleavage specificities of both proteases, we suggested that PR3 and NE complemented each other during the process of neutrophil activation and inflammation.

Figure 4

Impaired oxidative burst and PGRN degradation by IC-activatedPrtn3–/–Ela2–/– neutrophils.

Oxidative burst as the readout for neutrophil activation by ICs was measured over time. (A) While no difference was observed during the initial 20-min lag phase of the oxidative burst, Prtn3–/–Ela2–/– neutrophils exhibited diminished ROS production over time compared with WT neutrophils. (B) Bypassing receptor-mediated activation using 25 nM PMA restored the diminished oxidative burst of Prtn3–/–Ela2–/–neutrophils. Results are presented as normalized fluorescence in AU (relative to maximum fluorescence produced by WT cells). Data (mean ± SD) are representative of 3 independent experiments each conducted in triplicate. (C) Isolated mouse neutrophils were activated by ICs in vitro and tested for PGRN degradation by IB. In the cellular fraction, the PGRN (~80 kDa) signal was markedly increased in Prtn3–/–Ela2–/–cells compared with WT and Ela2–/– neutrophils. Intact PGRN was present in the supernatant (SN) of IC-activated Prtn3–/–Ela2–/–neutrophils only, not of WT or Ela2–/– cells. (D and E) Exogenous administration of 100 nM PGRN significantly reduced ROS production of neutrophils activated by ICs (D), but not when activated by PMA (E). Data (mean ± SD) are representative of 3 independent experiments each conducted in triplicate.

Antiinflammatory PGRN is degraded by PR3 and NE during IC-mediated neutrophil activation.

PGRN inhibits neutrophil activation by ICs in vitro.

Both PR3 and NE process PGRN in vitro.

Figure 5

PR3 and NE are major PGRN processing enzymes of neutrophils.

PGRN inhibits IC-mediated inflammation in vivo.

Figure 6

PGRN is a potent inhibitor of IC-stimulated inflammation in vivo.

PR3 and NE cleave PGRN during inflammation in vivo.

Finally, we aimed to demonstrate defective PGRN degradation in Prtn3–/–Ela2–/– mice during neutrophilic inflammation in vivo. For practical reasons, we harvested infiltrated neutrophils from the inflamed peritoneum 4 h after casein injection and subjected the lysates of these cells to anti-PGRN Western blot. Intact, inhibitory PGRN was detected in Prtn3–/–Ela2–/– neutrophils, but not in WT cells (Figure ​(Figure6D).6D). These data prove that neutrophilic inflammation is accompanied by proteolytic removal of antiinflammatory PGRN and that the process of PGRN degradation is essentially impaired in vivo in the absence of PR3 and NE.


Chronic inflammatory and autoimmune diseases are often perpetuated by continuous neutrophil infiltration and activation. According to the current view, the role of NSPs in these diseases is mainly associated with proteolytic tissue degradation after their release from activated or dying neutrophils. However, recent observations suggest that NSPs such as CG may contribute to noninfectious diseases in a more complex manner, namely as specific regulators of inflammation (18). Here, we demonstrate that PR3 and NE cooperatively fulfilled an important proinflammatory role during neutrophilic inflammation. PR3 and NE directly enhanced neutrophil activation by degrading oxidative burst–suppressing PGRN. These findings support the use of specific serine protease inhibitors as antiinflammatory agents.

Much attention has been paid to the degradation of extracellular matrix components by NSPs. We therefore expected that ablation of both PR3 and NE would cause impaired neutrophil extravasation and interstitial migration. Surprisingly, we found that the proteases were principally dispensable for these processes:Prtn3–/–Ela2–/– neutrophils migrated normally through a dense, 3-dimensional collagen matrix in vitro and demonstrated regular extravasation in vivo when phorbol esters were applied (Figure ​(Figure2).2). This finding is in agreement with recent reports that neutrophils preferentially and readily cross the EBM through regions of low matrix density in the absence of NE (28).

Conversely, we observed that PR3 and NE were required for the inflammatory response to locally formed ICs (Figure ​(Figure3).3). Even isolated Prtn3–/–Ela2–/– neutrophils were challenged in performing oxidative burst after IC stimulation in vitro (Figure ​(Figure4A),4A), showing that the proteases directly enhanced the activation of neutrophils also in the absence of extracellular matrix. However, when receptor-mediated signal transduction was bypassed by means of PMA, neutrophils from Prtn3–/–Ela2–/– mice performed normal oxidative burst (Figure ​(Figure4B),4B), indicating that the function of the phagocyte oxidase (phox) complex was not altered in the absence of PR3 and NE. These findings substantiate what we believe to be a novel paradigm: that all 3 serine proteases of azurophilic granules (CG, PR3, and NE), after their release in response to IC encounter, potentiate a positive autocrine feedback on neutrophil activation.

In contrast to CG, the highly related proteases PR3 and NE cooperate in the effacement of antiinflammatory PGRN, leading to enhanced neutrophil activation. Previous studies already demonstrated that PGRN is a potent inhibitor of the adhesion-dependent oxidative burst of neutrophils in vitro, which can be degraded by NE (23). Here, we showed that PR3 and NE play an equally important role in the regulation of PGRN function. Ela2–/– neutrophils were sufficiently able to degrade PGRN. Only in the absence of both PR3 and NE was PGRN degradation substantially impaired, resulting in the accumulation of antiinflammatory PGRN during neutrophil activation in vitro (Figure ​(Figure4C)4C) and neutrophilic inflammation in vivo (Figure ​(Figure6D).6D). Moreover, we provided in vivo evidence for the crucial role of PGRN as an inflammation-suppressing mediator, because administration of recombinant PGRN potently inhibited the neutrophil influx to sites of IC formation (Figure ​(Figure6,6, A–C). Hence, the cooperative degradation of PGRN by PR3 and NE is a decisive step for the establishment of neutrophilic inflammation.

The molecular mechanism of PGRN function is not yet completely understood, but it seems to interfere with integrin (CD11b/CD18) outside-in signaling by blocking the function of pyk2 and thus dampens adhesion-related oxidative burst even when added after the initial lag phase of oxidase activation (23). PGRN is produced by neutrophils and stored in highly mobile secretory granules (29). It was recently shown that PGRN can bind to heparan-sulfated proteoglycans (30), which are abundant components of the EBM and various cell surfaces, including those of neutrophils. Also, PR3 and NE are known to interact with heparan sulfates on the outer membrane of neutrophils, where the enzymes appear to be protected against protease inhibitors (121331). These circumstantial observations support the notion that PGRN cleavage by PR3 and NE takes place at the pericellular microenvironment of the neutrophil cell surface.

Impaired outside-in signaling most likely reduced the oxidative burst in Prtn3–/–Ela2–/– neutrophils adhering to ICs. In support of this hypothesis, we excluded an altered response to TNF-α priming (Supplemental Figure 5) as well as reduced adhesion to immobilized ICs and defective endocytosis of serum-opsonized E. coli in Prtn3–/–Ela2–/– neutrophils (Supplemental Figure 6). MPO content and processing was also unchanged in Prtn3–/–Ela2–/– neutrophils (Figure ​(Figure1D);1D); hence, the previously discussed inhibitory effect of MPO on phox activity (3233) does not appear to be stronger in neutrophils lacking PR3 and NE. Because there was no difference in the lag phase of the oxidative burst, initial IC-triggered receptor activation was probably not affected by either PRGN or PR3/NE. Our concept is consistent with all these observations and takes into account that PGRN unfolds its suppressing effects in the second phase, when additional membrane receptors, endogenous PGRN, and some PR3/NE from highly mobile intracellular pools are translocated to the cell surface. The decline and cessation of ROS production suggested to us that outside-in signaling was not sustained and that active oxidase complexes were no longer replenished in the absence of PR3 and NE. Our present findings, however, do not allow us to exclude other potential mechanisms, such as accelerated disassembly of the active oxidase complex.

Proposed function of PR3 and NE in IC-mediated inflammation.

TNF-α–primed neutrophils extravasate from blood vessels, translocate PR3/NE to the cellular surface, and discharge PGRN to the pericellular environment (i). During transmigration of interstitial tissues (ii), neutrophil activation is initially suppressed by relatively high pericellular levels of antiinflammatory PGRN (green shading), which is also produced locally by keratinocytes and epithelial cells of the skin. Until IC depots are reached, neutrophil activation is inhibited by PGRN. Surface receptors (e.g., Mac-1) recognize ICs, which results in signal transduction (black dotted arrow) and activation of the phox. The molecular pathway of PGRN-mediated inhibition is not completely understood, but it may interfere with integrin signaling after IC encounter (green dotted line inside the cell). Adherence of neutrophils to ICs (iii) further increases pericellular PR3 and NE activity. PR3 and NE cooperatively degrade PGRN in the early stage of neutrophilic activation to facilitate optimal neutrophil activation (red shading), resulting in sustained integrin signaling (red arrow) and robust production of ROS by the phox system. Subsequently, neutrophils release ROS together with other proinflammatory mediators and chemotactic agents, thereby enhancing the recruitment of further neutrophils and establishing inflammation (iv). In the absence of PR3/NE, the switch from inflammation-suppressing (ii) to inflammation-enhancing (iii) conditions is substantially delayed, resulting in diminished inflammation in response to ICs (iv).


NSPs are strongly implicated as effector molecules in a large number of destructive diseases, such as emphysema or the autoimmune blistering skin disease bullous pemphigoid (143537). Normally, PR3/NE activity is tightly controlled by high plasma levels of α1-antitrypsin. This balance between proteases and protease inhibitors is disrupted in patients with genetic α1-antitrypsin deficiency, which represents a high risk factor for the development of emphysema and certain autoimmune disorders (38). The pathogenic effects of NSPs in these diseases have so far been associated with tissue destruction by the proteases after their release from dying neutrophils. Our findings showed that PR3 and NE were already involved in much earlier events of the inflammatory process, because the enzymes directly regulated cellular activation of infiltrating neutrophils by degrading inflammation-suppressing PGRN. This concept is further supported by previous studies showing increased inflammation in mice lacking serine protease inhibitors such as SERPINB1 or SLPI (3940). Blocking PR3/NE activity using specific inhibitors therefore represents a promising therapeutic strategy to treat chronic, noninfectious inflammation. Serine protease inhibitors as antiinflammatory agents can interfere with the disease process at 2 different stages, because they attenuate both early events of neutrophil activation and proteolytic tissue injury caused by released NSPs.





Editorial: Serine proteases, serpins, and neutropenia

David C. Dale

J Leuko Biol July 2011;  90(1): 3-4

Cyclic neutropenia and severe congenital neutropenia are autosomal-dominant diseases usually attributable to mutations in the gene for neutrophil elastase orELANE. Patients with these diseases are predisposed to recurrent and life-threatening infections [1]. Neutrophil elastase, the product of the ELANE gene, is a serine protease that is synthesized and packaged in the primary granules of neutrophils. These granules are formed at the promyelocytes stage of neutrophil development. Synthesis of mutant neutrophil elastase in promyelocytes triggers the unfolded protein response and a cascade of intracellular events, which culminates in death of neutrophil precursors through apoptosis [2]. This loss of cells causes the marrow abnormality often referred to as “maturation arrest” [34].

Neutrophil elastase is one of the serine proteases normally inhibited by serpinB1. In this issue of JLB, Benarafa and coauthors [5] present their intriguing studies of serpinB1 expression in human myeloid cells and their extensive investigations ofSERPINB1−/− mice. They observed that serpinB1 expression parallels protease expression. The peak of serpinB1 expression occurs in promyelocytes. Benarafa et al. [5] found that SERPINB1−/− mice have a deficiency of postmitotic neutrophils in the bone marrow. This change was accompanied by an increase in the plasma levels of G-CSF. The decreased supply of marrow neutrophils reduced the number of neutrophils that could be mobilized to an inflammatory site. Using colony-forming cell assays, they determined that the early myeloid progenitor pool was intact. Separate assays showed that maturing myeloid cells were being lost through accelerated apoptosis of maturing neutrophils in the marrow. The authors concluded that serpinB1 is required for maintenance of a healthy reserve of marrow neutrophils and a normal acute immune response [5].

This paper provides new and fascinating insights for understanding the mechanism for neutropenia. It also suggests opportunities to investigate potential therapies for patients with neutropenia and prompts several questions. As inhibition of the activity of intracellular serine proteases is the only known function of serpinB1, the findings reported by Benarafa et al. [5] suggest that uninhibited serine proteases perturbed neutrophil production severely. The SERPINB1−/− mice used in their work have accelerated apoptosis of myeloid cells, a finding suggesting that uninhibited serine proteases or mutant neutrophil elastase perturb myelopoiesis by similar mechanisms. It is now important to determine whether the defect in the SERPINB1−/− mice is, indeed, attributable to uninhibited activity of normal neutrophil elastase, other neutrophil proteases, or another mechanism. ″Double-knockout″ studies in mice deficient in neutrophil elastase and serpinB1 might provide an answer.

This report provides evidence regarding the intracellular mechanisms for the apoptosis of myeloid cells and indicates that other studies are ongoing. The key antiapoptotic proteins, Mcl-1, Bcl-XL, and A1/Bfl-I, are apparently not involved. A more precise understanding of the mechanisms of cell death is important for development of targeted therapies for neutropenia. It is also important to discover whether only cells of the neutrophil lineage are involved or whether monocytes are also affected. In cyclic and congenital neutropenia, patients failed to produce neutrophils, but they can produce monocytes; in fact, they overproduce monocytes and have significantly elevated blood monocyte counts. Neutropenia with monocytosis is probably attributable to differences in the expression of ELANE in the two lineages. Benarafa et al. [5] reported that human bone marrow monocytes contain substantially less serpinB1 than marrow neutrophils, suggesting that the expression of serpinB1 and the serine proteases are closely coordinated.

This report shows the importance of the marrow neutrophil reserves in the normal response to infections. Compared with humans, healthy mice are always neutropenic, but they have a bigger marrow neutrophil reserve, and their mature neutrophils in the marrow and blood look like human band neutrophils. These differences are well known, but they are critical for considering the clinical inferences that can be made from this report. For example, although theSERPINB1−/− mice were not neutropenic, human SERPINB1−/− might cause neutropenia because of physiological differences between the species. If some but not all mutations in SERPINB1 cause neutropenia, we might gain a better understanding about how serpinB1 normally inhibits the neutrophil’s serine proteases.

We do not know if some or all of the mutant neutrophil elastases can be inhibited by serpinB1. We do not know whether cyclic or congenital neutropenia are attributable to defects in this interaction. However, we do know that there are chemical inhibitors of neutrophil elastase that can abrogate apoptosis of myeloid cells in a cellular model for congenital neutropenia [6]. It would be interesting to see if these chemical inhibitors can replace the natural inhibitor and normalize neutrophil production in the SERPINB1−/− mice. This would provide evidence to support use of chemical protease inhibitors as a treatment for cyclic and congenital neutropenia.

Concerns with the use of G-CSF for the treatment of cyclic and congenital neutropenia are how and why some of these patients are at risk of developing leukemia. Are the SERPINB1−/− mice with a hyperproliferative marrow and high G-CSF levels also at risk of developing myeloid leukemia?

This is a very provocative paper, and much will be learned from further studies of the SERPINB1−/− mice.


SerpinB1 is critical for neutrophil survival through cell-autonomous inhibition of cathepsin G

Mathias Baumann1,2, Christine T. N. Pham3, and Charaf Benarafa1

Blood May 9, 2013; 121(19)

Key Points

  • Serine protease inhibitor serpinB1 protects neutrophils by inhibition of their own azurophil granule protease cathepsin G.
  • Granule permeabilization in neutrophils leads to cathepsin G–mediated death upstream and independent of apoptotic caspases.


Bone marrow (BM) holds a large reserve of polymorphonuclear neutrophils (PMNs) that are rapidly mobilized to the circulation and tissues in response to danger signals. SerpinB1 is a potent inhibitor of neutrophil serine proteases neutrophil elastase (NE) and cathepsin G (CG). SerpinB1 deficiency (sB1−/−) results in a severe reduction of the BM PMN reserve and failure to clear bacterial infection. Using BM chimera, we found that serpinB1 deficiency in BM cells was necessary and sufficient to reproduce the BM neutropenia ofsB1−/− mice. Moreover, we showed that genetic deletion of CG, but not NE, fully rescued the BM neutropenia in sB1−/− mice. In mixed BM chimera and in vitro survival studies, we showed that CG modulates sB1−/− PMN survival through a cell-intrinsic pathway. In addition, membrane permeabilization by lysosomotropic agent L-leucyl-L-leucine methyl ester that allows cytosolic release of granule contents was sufficient to induce rapid PMN death through a CG-dependent pathway. CG-mediated PMN cytotoxicity was only partly blocked by caspase inhibition, suggesting that CG cleaves a distinct set of targets during apoptosis. In conclusion, we have unveiled a new cytotoxic function for the serine protease CG and showed that serpinB1 is critical for maintaining PMN survival by antagonizing intracellular CG activity.


Polymorphonuclear neutrophil (PMN) granulocytes are essential components of the innate immune response to infection. PMNs are relatively short-lived leukocytes that originate from hematopoietic stem cells in the bone marrow (BM) in a process called granulopoiesis. Granulopoiesis proceeds through a proliferative phase followed by a maturation phase. After maturation, the BM retains a large reserve of mature PMNs, which includes over 90% of the mature PMNs in the body while only a small proportion (1%-5%) is in the blood.1,2 Even in noninflammatory conditions, granulopoiesis is remarkable as >1011 PMNs are produced daily in an adult human, only to be disposed of, largely unused, a few hours later.3 There is evidence that the majority of PMNs produced never reach circulation and die within the BM.4 Congenital or acquired forms of neutropenia are associated with the highest risks of bacterial and fungal infection,5 indicating a strong evolutionary pressure to maintain granulopoiesis at high levels and sustain a large mobilizable pool of PMNs in the BM.

In steady state, PMNs die by apoptosis, a form of programmed cell death that allows for the safe disposal of aging PMNs and their potentially toxic cargo. Like in other cells, caspases participate in the initiation, amplification, and execution steps of apoptosis in PMNs.6,7 Interestingly, noncaspase cysteine proteases calpain and cathepsin D were reported to induce PMN apoptosis through activation of caspases.811 In addition, PMNs carry a unique set of serine proteases (neutrophil serine proteases [NSPs]) including elastase (NE), cathepsin G (CG), and proteinase-3 (PR3) stored active in primary granules. There is strong evidence for a role of NSPs in killing pathogens and inducing tissue injury when released extracellularly.1214 In contrast, the function of NSPs in PMN homeostasis and cell death remains elusive. In particular, no defects in granulopoiesis or PMN homeostasis have been reported in mice deficient in cathepsin G (CG−/−),15 neutrophil elastase (NE−/−),16,17 or dipeptidylpeptidase I (DPPI−/−), which lack active NSPs.18 We have recently shown that mice lacking the serine protease inhibitor serpinB1 (sB1−/−) have reduced PMN survival in the lungs following Pseudomonas infection and that these mice have a profound reduction in mature PMN numbers in the BM.19,20SerpinB1, also known as monocyte NE inhibitor, is expressed at high levels in the cytoplasm of PMNs and is one of the most potent inhibitors of NE, CG, and PR3.21,22 In this study, we tested the hypothesis that serpinB1 promotes PMN survival by inhibiting 1 or several NSPs, and we discovered a novel regulatory pathway in PMN homeostasis in vivo.

Figure 1

Defective PMN reserve in BM chimera depends on serpinB1 deficiency in the hematopoietic compartment. Flow cytometry analysis of major BM leukocyte subsets of lethally irradiated mice was performed 8 to 10 weeks after BM transfer. (A) Irradiated WT (CD45.1) mice were transferred with WT (●) or sB1−/− (○) BM cells. (B) Irradiated WT (●) andsB1−/− (○) mice both CD45.2 were transferred with WT (CD45.1) BM cells. Each circle represents leukocyte numbers for 1 mouse and horizontal line indicates the median. Median subsets numbers were compared by the Mann-Whitney test (*P < .05; ***P < .001).

CG regulates neutrophil numbers in the BM

Because serpinB1 is an efficient inhibitor of NE, CG, and PR3, we then examined PMN numbers in mice deficient in 1 or several NSPs in combination with serpinB1 deletion. As expected, sB1−/− mice had significantly reduced numbers and percentage of mature PMNs in the BM compared with WT and heterozygous sB1+/− mice. In addition, PMN numbers were normal in mice deficient in either DPPI, NE, or CG (Figure 2A). DPPI is not inhibited by serpinB1 but is required for the activation of all NSPs, and no NSP activity is detectable in DPPI−/− mice.18,23 PMN counts in DPPI−/−.sB1−/− BM were significantly higher than in sB1−/− BM, suggesting that 1 or several NSPs contribute to the PMN survival defect. To examine the role of NSPs in this process, we crossed several NSP-deficient strains with sB1−/− mice. We found that NE.CG.sB1−/− mice had normal PMN numbers indicating that these NSPs play a key role in the defective phenotype of sB1−/− PMNs (Figure 2A). Furthermore, CG.sB1−/− mice showed normal PMN numbers whereasNE.sB1−/− mice retained the BM neutropenia phenotype indicating that CG, but not NE, plays a significant role in the death of sB1−/− PMNs (Figure 2A). In addition, the double-deficient NE.sB1−/− mice had significantly lower BM myelocyte numbers than sB1−/− mice while the myelocyte numbers in singly deficient NE−/− and sB1−/− BM were normal (Figure 2B). These results suggest that NE may promote myeloid cell proliferation, an activity that is revealed only when serpinB1 is absent. This complex interaction between sB1 and NE requires further investigation. On the other hand, B-cell and monocyte numbers and relative percentage in the BM were largely similar in all genotypes (supplemental Figure 2). Total numbers of blood leukocytes, erythrocytes, and platelets were normal in mice deficient in NSPs and/or serpinB1 (supplemental Figure 3). PMN numbers in blood were normal insB1−/− mice in steady state and combined deficiency of NSPs did not significantly alter these numbers (Figure 2C). Taken together, our results indicate that serpinB1 likely sustains the survival of postmitotic PMNs through its interaction with CG.

Figure 2

PMN and myelocyte numbers in BM and blood of mice deficient in NSPs and serpinB1.

CG-mediated PMN death proceeds independent of caspase activity

Figure 4

sB1−/− PMN death mediated by CG does not require caspase activity

Granule membrane permeabilization induces CG-mediated death in PMNs

To test whether granule disruption contributes to the serpinB1-regulated CG-dependent cell death, BM cells were treated with the lysosomotropic agent LLME. LLME accumulates in lysosomes where the acyl transferase activity of DPPI generates hydrophobic (Leu-Leu)n-OMe polymers that induce lysosomal membrane permeabilization (LMP) and cytotoxicity in granule-bearing cells such as cytotoxic T lymphocytes, NK cells, and myeloid cells.29,30

Figure 5

LMP induces CG-mediated death in PMNs

G-CSF therapy increases sB1−/− PMN numbers via enhanced granulopoiesis

G-CSF therapy is an effective long-term treatment in many cases of severe congenital neutropenia and it is also used to prevent chemotherapy-induced febrile neutropenia by enhancing PMN production. In addition, G-CSF delays neutrophil apoptosis by differentially regulating proapoptotic and antiapoptotic factors.10 To test whether G-CSF could rescue sB1−/− PMN survival defect, WT and sB1−/− mice were treated with therapeutic doses of G-CSF or saline for 5 days and BM and blood PMNs were analyzed 24 hours after the last injection. Total counts of myelocytes and PMNs were significantly increased in the BM of treated mice compared with their respective untreated genotype controls (Figure 6A-B). The increase in myelocyte numbers was identical in G-CSF–treated WT and sB1−/− mice, indicating that G-CSF–induced granulopoiesis proceeds normally in sB1−/−myeloid progenitors (Figure 6B).

Figure 6

In vivo G-CSF therapy increases PMN numbers in BM of sB1−/− mice.


SerpinB1 is a member of the clade B serpins, a subfamily composed of leaderless proteins with nucleocytoplasmic localization. Clade B serpins are often expressed in cells that also carry target proteases, which led to the hypothesis that intracellular serpins protect against misdirected granule proteases and/or protect bystander cells from released proteases.31 We previously reported that deficiency in serpinB1 is associated with reduced PMN survival in the BM and at inflammatory sites.19,20 The evidence presented here demonstrates that the cytoprotective function of serpinB1 in PMNs is based on the inhibition of granule protease CG. Deficiency in CG was sufficient to rescue the defect of sB1−/− mice as illustrated by normal PMN counts in the BM of double knockout CG.sB1−/− mice. We also showed that the protease-serpin interaction occurred within PMNs. Indeed, WT PMNs had a greater survival over sB1−/− PMNs in mixed BM chimera, whereas the survival of CG.sB1−/− PMNs was similar to WT PMNs after BM transfer. SerpinB1 is an ancestral clade B serpin with a conserved specificity determining reactive center loop in all vertebrates.32 Furthermore, human and mouse serpinB1 have the same specificity for chymotrypsin-like and elastase-like serine proteases.21,22 Likewise, human and mouse CG have identical substrate specificities and the phenotype of CG−/− murine PMN can be rescued by human CG.33 Therefore, it is highly likely that the antagonistic functions of CG and serpinB1 in cellular homeostasis observed in mice can be extended to other species.

Extracellular CG was previously reported to promote detachment-induced apoptosis (anoikis) in human and mouse cardiomyocytes.34 This activity is mediated through the shedding and transactivation of epidermal growth factor receptor and downregulation of focal adhesion signaling.35,36 In our study, exogenous human CG also induced PMN death in vitro but these effects were not enhanced in sB1−/− PMNs and the neutropenia associated with serpinB1 deficiency was principally cell intrinsic. How intracellular CG induces PMN death remains to be fully investigated. However, our studies provide some indications on the potential pathways. Like other NSPs, the expression of CG is transcriptionally restricted to the promyelocyte stage during PMN development and NSPs are then stored in active form in primary azurophil granules.37 Because serpinB1 is equally efficient at inhibiting NE, CG, and PR3, it was surprising that deletion of CG alone was sufficient to achieve a complete reversal of the PMN survival defect in CG.sB1−/− mice. A possible explanation would be that CG gains access to targets more readily than other granule proteases. There is evidence that binding to serglycin proteoglycans differs between NE and CG resulting in altered sorting of NE but not CG into granules of serglycin-deficient PMNs.38 Different interactions with granule matrix may thus contribute to differential release of CG from the granules compared with other NSPs. However, because sB1−/− PMNs have similar levels of CG and NE as WT PMNs20 and because LLME-induced granule permeabilization likely releases all granule contents equally, we favor an alternative interpretation where CG specifically targets essential cellular components that are not cleaved by the other serpinB1-inhibitable granule proteases. Upon granule permeabilization, we found that CG can induce cell death upstream of caspases as well as independent of caspases. CG was previously shown to activate caspase-7 in vitro and it functions at neutral pH, which is consistent with a physiological role in the nucleocytoplasmic environment.39 Cell death induced by lysosomal/granule membrane permeabilization has previously been linked to cysteine cathepsins in other cell types. However, these proteases appear to depend on caspase activation to trigger apoptosis and they function poorly at neutral pH, questioning their potential role as regulators of cell death.40 In contrast, CG-mediated cell death is not completely blocked by caspase inhibition, which is a property reminiscent of granzymes in cytotoxic T cells.41 In fact, CG is phylogenetically most closely related to serine proteases granzyme B and H.42 Granzymes have numerous nuclear, mitochondrial, and cytoplasmic target proteins leading to cell death41 and we anticipate that this may also be the case for CG.


G-CSF therapy is successfully used to treat most congenital and acquired neutropenia through increased granulopoiesis, mobilization from the BM, and increased survival of PMNs. Prosurvival effects of G-CSF include the upregulation of antiapoptotic Bcl-2 family members, which act upstream of the mitochondria and the activation of effector caspases. In sB1−/− mice, G-CSF levels in serum are fourfold higher than in WT mice in steady state and this is accompanied by an upregulation of the antiapoptotic Bcl-2 family member Mcl-1 in sB1−/− PMNs.19 Here, G-CSF therapy significantly increased granulopoiesis in both WT and sB1−/− mice. However, the PMN numbers in treated sB1−/− BM and blood were significantly lower than those of treated WT mice, indicating only a partial rescue of the survival defect. This is consistent with our findings that CG-mediated death can proceed independent of caspases and can thus bypass antiapoptotic effects mediated by G-CSF.

CG has largely been studied in association with antimicrobial and inflammatory functions due to its presence in PMNs.1214,49 In this context, we have previously shown that serpinB1 contributes to prevent increased mortality and morbidity associated with production of inflammatory cytokines upon infection with Pseudomonas aeruginosa and influenza A virus.20,50 In this study, we demonstrate that serpinB1 inhibition of the primary granule protease CG in PMNs is essential for PMN survival and this ultimately regulates PMN numbers in vivo. Our findings also extend the roles of CG from antimicrobial and immunoregulatory functions to a novel role in inducing cell death.


Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

Brice KorkmazMarshall S. HorwitzDieter E. Jenne and Francis Gauthier
Pharma Rev Dec 2010; 62(4):726-759

Polymorphonuclear neutrophils are the first cells recruited to inflammatory sites and form the earliest line of defense against invading microorganisms. Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes. These proteases are also externalized in an active form during neutrophil activation at inflammatory sites, thus contributing to the regulation of inflammatory and immune responses. As multifunctional proteases, they also play a regulatory role in noninfectious inflammatory diseases. Mutations in the ELA2/ELANE gene, encoding neutrophil elastase, are the cause of human congenital neutropenia. Neutrophil membrane-bound proteinase 3 serves as an autoantigen in Wegener granulomatosis, a systemic autoimmune vasculitis. All three proteases are affected by mutations of the gene (CTSC) encoding dipeptidyl peptidase I, a protease required for activation of their proform before storage in cytoplasmic granules. Mutations of CTSC cause Papillon-Lefèvre syndrome. Because of their roles in host defense and disease, elastase, proteinase 3, and cathepsin G are of interest as potential therapeutic targets. In this review, we describe the physicochemical functions of these proteases, toward a goal of better delineating their role in human diseases and identifying new therapeutic strategies based on the modulation of their bioavailability and activity. We also describe how nonhuman primate experimental models could assist with testing the efficacy of proposed therapeutic strategies.


Human polymorphonuclear neutrophils represent 35 to 75% of the population of circulating leukocytes and are the most abundant type of white blood cell in mammals (Borregaard et al., 2005). They are classified as granulocytes because of their intracytoplasmic granule content and are characterized by a multilobular nucleus. Neutrophils develop from pluripotent stem cells in the bone marrow and are released into the bloodstream where they reach a concentration of 1.5 to 5 × 109 cells/liter. Their half-life in the circulation is only on the order of a few hours. They play an essential role in innate immune defense against invading pathogens and are among the primary mediators of inflammatory response. During the acute phase of inflammation, neutrophils are the first inflammatory cells to leave the vasculature, where they migrate toward sites of inflammation, following a gradient of inflammatory stimuli. They are responsible for short-term phagocytosis during the initial stages of infection (Borregaard and Cowland, 1997Hampton et al., 1998Segal, 2005). Neutrophils use complementary oxidative and nonoxidative pathways to defend the host against invading pathogens (Kobayashi et al., 2005).

The three serine proteases neutrophil elastase (NE1), proteinase 3 (PR3), and cathepsin G (CG) are major components of neutrophil azurophilic granules and participate in the nonoxidative pathway of intracellular and extracellular pathogen destruction. These neutrophil serine proteases (NSPs) act intracellularly within phagolysosomes to digest phagocytized microorganisms in combination with microbicidal peptides and the membrane-associated NADPH oxidase system, which produces reactive oxygen metabolites (Segal, 2005). An additional extracellular antimicrobial mechanism, neutrophil extracellular traps (NET), has been described that is made of a web-like structure of DNA secreted by activated neutrophils (Papayannopoulos and Zychlinsky, 2009) (Fig. 1). NETs are composed of chromatin bound to positively charged molecules, such as histones and NSPs, and serve as physical barriers that kill pathogens extracellularly, thus preventing further spreading. NET-associated NSPs participate in pathogen killing by degrading bacterial virulence factors extracellularly (Brinkmann et al., 2004;Papayannopoulos and Zychlinsky, 2009).

Fig. 1.

Polymorphonuclear neutrophil. Quiescent (A) and chemically activated (B) neutrophils purified from peripheral blood. C, PMA-activated neutrophils embedded within NET and neutrophil spreading on insoluble elastin.

In addition to their involvement in pathogen destruction and the regulation of proinflammatory processes, NSPs are also involved in a variety of inflammatory human conditions, including chronic lung diseases (chronic obstructive pulmonary disease, cystic fibrosis, acute lung injury, and acute respiratory distress syndrome) (Lee and Downey, 2001Shapiro, 2002Moraes et al., 2003Owen, 2008b). In these disorders, accumulation and activation of neutrophils in the airways result in excessive secretion of active NSPs, thus causing lung matrix destruction and inflammation. NSPs are also involved in other human disorders as a consequence of gene mutations, altered cellular trafficking, or, for PR3, autoimmune disease. Mutations in the ELA2/ELANE gene encoding HNE are the cause of human cyclic neutropenia and severe congenital neutropenia (Horwitz et al., 19992007). Neutrophil membrane-bound proteinase 3 (mPR3) is the major target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA), which are associated with Wegener granulomatosis (Jenne et al., 1990). All three proteases are affected by mutation of the gene (CTSC) encoding dipeptidyl peptidase I (DPPI), which activates several granular hematopoietic serine proteases (Pham and Ley, 1999Adkison et al., 2002). Mutations of CTSC cause Papillon-Lefèvre syndrome and palmoplantar keratosis (Hart et al., 1999Toomes et al., 1999).


Fully processed mature HNE, PR3, and CG isolated from azurophilic granules contain, respectively, 218 (Bode et al., 1986Sinha et al., 1987), 222 (Campanelli et al., 1990b), and 235 (Salvesen et al., 1987Hof et al., 1996) residues. They are present in several isoforms depending on their carbohydrate content, with apparent mass of 29 to 33 kDa upon SDS-polyacrylamide gel electrophoresis (Twumasi and Liener, 1977Watorek et al., 1993). HNE and PR3 display two sites of N-glycosylation, whereas CG possesses only one. NSPs are stored mainly in neutrophil azurophilic granules, but HNE is also localized in the nuclear envelope, as revealed by immunostaining and electron microscopy (Clark et al., 1980;Benson et al., 2003), whereas PR3 is also found in secretory vesicles (Witko-Sarsat et al., 1999a). Upon neutrophil activation, granular HNE, PR3, and CG are secreted extracellularly, although some molecules nevertheless remain at the cell surface (Owen and Campbell, 1999Owen, 2008a). The mechanism through which NSPs are sorted from the trans-Golgi network to the granules has not been completely defined, even though an intracellular proteoglycan, serglycin, has been identified as playing a role in elastase sorting and packaging into azurophilic granules (Niemann et al., 2007). Unlike HNE and CG, PR3 is constitutively expressed on the membranes of freshly isolated neutrophils (Csernok et al., 1990Halbwachs-Mecarelli et al., 1995). Stimulation of neutrophils at inflammatory sites triggers intracytoplasmic granules to translocate to the phagosomes and plasma membrane, thereby liberating their contents. The first step of the translocation to the target membrane depends on cytoskeleton remodeling and microtubule assembly (Burgoyne and Morgan, 2003). This is followed by a second step of granule tethering and docking, which are dependent on the sequential intervention of SNARE proteins (Jog et al., 2007).


Exposure of neutrophils to cytokines (TNF-α), chemoattractants (platelet-activating factor, formyl-Met-Leu-Phe, or IL-8), or bacterial lipopolysaccharide leads to rapid granule translocation to the cell surface with secretion of HNE, PR3, and CG into the extracellular medium (Owen and Campbell, 1999). A fraction of secreted HNE, PR3, and CG is detected at the surface of activated neutrophils (Owen et al., 1995a1997Campbell et al., 2000). Resting purified neutrophils from peripheral blood express variable amounts of PR3 on their surface. A bimodal, apparently genetically determined, distribution has been observed with two populations of quiescent neutrophils that express or do not express the protease at their surface (Halbwachs-Mecarelli et al., 1995Schreiber et al., 2003). The percentage of mPR3-positive neutrophils ranges from 0 to 100% of the total neutrophil population within individuals. Furthermore, the percentage of mPR3-positive neutrophils remains stable over time and is not affected by neutrophil activation (Halbwachs-Mecarelli et al., 1995).

The mechanism through which HNE and CG are associated with the outer surface of the plasma membrane of neutrophils mainly involves electrostatic interactions with the sulfate groups of chondroitin sulfate- and heparan sulfate-containing proteoglycans (Campbell and Owen, 2007). These two proteases are released from neutrophil cell surfaces by high concentrations of salt (Owen et al., 1995b1997;Korkmaz et al., 2005a) and after treatment with chondroitinase ABC and heparinase (Campbell and Owen, 2007). Membrane PR3 is not solubilized by high salt concentrations, which means that its membrane association is not charge dependant (Witko-Sarsat et al., 1999aKorkmaz et al., 2009). Unlike HNE and CG, PR3 bears at its surface a hydrophobic patch formed by residues Phe166, Ile217, Trp218, Leu223, and Phe224 that is involved in membrane binding (Goldmann et al., 1999Hajjar et al., 2008) (Fig. 3B). Several membrane partners of PR3 have been identified, including CD16/FcγRIIIb (David et al., 2005Fridlich et al., 2006), phospholipid scramblase-1, a myristoylated membrane protein with translocase activity present in lipid rafts (Kantari et al., 2007), CD11b/CD18 (David et al., 2003), and human neutrophil antigen NB1/CD177 (von Vietinghoff et al., 2007Hu et al., 2009), a 58- to 64-kDa glycosyl-phosphatidylinositol anchored surface receptor belonging to the urokinase plasminogen activator receptor superfamily (Stroncek, 2007). NB1 shows a bimodal distribution that superimposes with that of PR3 on purified blood neutrophils (Bauer et al., 2007). Active, mature forms of PR3 but not pro-PR3 can bind to the surface of NB1-transfected human embryonic kidney 293 cells (von Vietinghoff et al., 2008) and Chinese hamster ovary cells (Korkmaz et al., 2008b). Interaction involves the hydrophobic patch of PR3 because specific amino acid substitutions disrupting this patch in the closely related gibbon PR3 prevent binding to NB1-transfected cells (Korkmaz et al., 2008b). Decreased interaction of pro-PR3 with NB1-transfected cells is explained by the topological changes affecting the activation domain containing the hydrophobic patch residues. Together, these results support the hydrophobic nature of PR3-membrane interaction.


Roles in Inflammatory Process Regulation

NSPs are abundantly secreted into the extracellular environment upon neutrophil activation at inflammatory sites. A fraction of the released proteases remain bound in an active form on the external surface of the plasma membrane so that both soluble and membrane-bound NSPs are able to proteolytically regulate the activities of a variety of chemokines, cytokines, growth factors, and cell surface receptors. Secreted proteases also activate lymphocytes and cleave apoptotic and adhesion molecules (Bank and Ansorge, 2001Pham, 2006Meyer-Hoffert, 2009). Thus, they retain pro- and anti-inflammatory activities, resulting in a modulation of the immune response at sites of inflammation.


Processing of Cytokines, Chemokines, and Growth Factors.

Processing and Activation of Cellular Receptors.

Induction of Apoptosis by Proteinase 3.

Physiological Inhibitors of Elastase, Proteinase 3, and Cathepsin G

During phagocytosis and neutrophil turnover, HNE, PR3, and CG are released into the extracellular space as active proteases. The proteolytic activity of HNE, PR3, and CG seems to be tightly regulated in the extracellular and pericellular space to avoid degradation of connective tissue proteins including elastin, collagen, and proteoglycans (Janoff, 1985). Protein inhibitors that belong to three main families, the serpins, the chelonianins, and the macroglobulins, ultimately control proteolytic activity of HNE, PR3, and CG activities. The individual contributions of these families depend on their tissue localization and that of their target proteases. The main characteristics of HNE, PR3, and CG physiological inhibitors are presented in Table 2.


Serine Protease Inhibitors

Serpins are the largest and most diverse family of protease inhibitors; more than 1000 members have been identified in human, plant, fungi, bacteria, archaea, and certain viruses (Silverman et al., 2001Mangan et al., 2008). They share a similar highly conserved tertiary structure and similar molecular weight of approximately 50 kDa. Human serpins belong to the first nine clades (A–I) of the 16 that have been described based on phylogenic relationships (Irving et al., 2000Silverman et al., 2001Mangan et al., 2008). For historical reasons, α1-protease inhibitor (α1-PI) was assigned to the first clade. Clade B, also known as the ov-serpin clan because of the similarity of its members to ovalbumin (a protein that belongs to the serpin family but lacks inhibitory activity), is the second largest clan in humans, with 15 members identified so far. Ov-serpin clan members are generally located in the cytoplasm and, to a lesser extent, on the cell surface and nucleus (Remold-O’Donnell, 1993).

Serpins play important regulatory functions in intracellular and extracellular proteolytic events, including blood coagulation, complement activation, fibrinolysis, cell migration, angiogenesis, and apoptosis (Potempa et al., 1994). Serpin dysfunction is known to contribute to diseases such as emphysema, thrombosis, angioedema, and cancer (Carrell and Lomas, 1997Lomas and Carrell, 2002). Most inhibitory serpins target trypsin-/chymotrypsin-like serine proteases, but some, termed “cross-class inhibitors,” have been shown to target cysteine proteases (Annand et al., 1999). The crystal structure of the prototype plasma inhibitor α1-PI revealed the archetype native serpin fold (Loebermann et al., 1984). All serpins typically have three β-sheets (termed A, B, and C) and eight or nine α-helices (hA–hI) arranged in a stressed configuration. The so-called reactive center loop (RCL) of inhibitory molecules determines specificity and forms the initial encounter complex with the target protease (Potempa et al., 1994Silverman et al., 2001). Serpins inhibit proteases by a suicide substrate inhibition mechanism. The protease initially recognizes the serpin as a potential substrate using residues of the reactive center loop and cleaves it between P1 and P1′ This cleavage allows insertion of the cleaved RCL into the β-sheet A of the serpin, dragging the protease with it and moving it over 71 Å to the distal end of the serpin to form a 1:1 stoichiometric covalent inhibitory complex (Huntington et al., 2000). Such cleavage generates a ∼4-kDa C-terminal fragment that remains noncovalently bound to the cleaved serpin. Displacement of the covalently attached active site serine residue from its catalytic partner histidine explains the loss of catalytic function in the covalent complex. The distortion of the catalytic site structure prevents the release of the protease from the complex, and the structural disorder induces its proteolytic inactivation (Huntington et al., 2000). Covalent complex formation between serpin and serine proteases triggers a number of conformational changes, particularly in the activation domain loops of the bound protease (Dementiev et al., 2006).


Pathophysiology of Elastase, Proteinase 3 and Cathepsin G in Human Diseases

In many instances, the initiation and propagation of lung damage is a consequence of an exaggerated inappropriate inflammatory response, which includes the release of proteases and leukocyte-derived cytotoxic products (Owen, 2008b;Roghanian and Sallenave, 2008). Inflammation is a physiological protective response to injury or infection consisting of endothelial activation, leukocyte recruitment and activation, vasodilation, and increased vascular permeability. Although designed to curtail tissue injury and facilitate repair, the inflammatory response sometimes results in further injury and organ dysfunction. Inflammatory chronic lung diseases, chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, and cystic fibrosis are syndromes of severe pulmonary dysfunction resulting from a massive inflammatory response and affecting millions of people worldwide. The histological hallmark of these chronic inflammatory lung diseases is the accumulation of neutrophils in the microvasculature of the lung. Neutrophils are crucial to the innate immune response, and their activation leads to the release of multiple cytotoxic products, including reactive oxygen species and proteases (serine, cysteine, and metalloproteases). The physiological balance between proteases and antiproteases is required for the maintenance of the lung’s connective tissue, and an imbalance in favor of proteases results in lung injury (Umeki et al., 1988Tetley, 1993). A number of studies in animal and cell culture models have demonstrated a contribution of HNE and related NSPs to the development of chronic inflammatory lung diseases. Available preclinical and clinical data suggest that inhibition of NSP in lung diseases suppresses or attenuates the contribution of NSP to pathogenesis (Chughtai and O’Riordan, 2004Voynow et al., 2008Quinn et al., 2010). HNE could also participate in fibrotic lung remodeling by playing a focused role in the conversion of latent transforming growth factor-β into its biologically active form (Chua and Laurent, 2006Lungarella et al., 2008).

Anti-Neutrophil Cytoplasmic Autoantibody-Associated Vasculitides

ANCA-associated vasculitides encompasses a variety of diseases characterized by inflammation of blood vessels and by the presence of autoantibodies directed against neutrophil constituents. These autoantibodies are known as ANCAs (Kallenberg et al., 2006). In Wegener granulomatosis (WG), antibodies are mostly directed against PR3. WG is a relatively uncommon chronic inflammatory disorder first described in 1931 by Heinz Karl Ernst Klinger as a variant of polyarteritis nodosa (Klinger, 1931). In 1936, the German pathologist Friedrich Wegener described the disease as a distinct pathological entity (Wegener, 19361939). WG is characterized by necrotizing granulomatous inflammation and vasculitis of small vessels and can affect any organ (Fauci and Wolff, 1973Sarraf and Sneller, 2005). The most common sites of involvement are the upper and lower respiratory tract and the kidneys. WG affects approximately 1 in 20,000 people; it can occur in persons of any age but most often affects those aged 40 to 60 years (Walton, 1958Cotch et al., 1996). Approximately 90% of patients have cold or sinusitis symptoms that fail to respond to the usual therapeutic measures and that last considerably longer than the usual upper respiratory tract infection. Lung involvement occurs in approximately 85% of the patients. Other symptoms include nasal membrane ulcerations and crusting, saddle-nose deformity, inflammation of the ear with hearing problems, inflammation of the eye with sight problems, and cough (with or without hemoptysis).

Hereditary Neutropenias

Neutropenia is a hematological disorder characterized by an abnormally low number of neutrophils (Horwitz et al., 2007). The normal neutrophil count fluctuates across human populations and within individual patients in response to infection but typically lies in the range of 1.5 to 5 × 109 cells/liter. Neutropenia is categorized as severe when the cell count falls below 0.5 × 109 cells/liter. Hence, patients with neutropenia are more susceptible to bacterial infections and, without prompt medical attention, the condition may become life-threatening. Common causes of neutropenia include cancer chemotherapy, drug reactions, autoimmune diseases, and hereditary disorders (Berliner et al., 2004Schwartzberg, 2006).

Papillon-Lefèvre Syndrome


New Strategies for Fighting Neutrophil Serine Protease-Related Human Diseases

Administration of therapeutic inhibitors to control unwanted proteolysis at inflammation sites has been tested as a therapy for a variety of inflammatory and infectious lung diseases (Chughtai and O’Riordan, 2004). Depending on the size and chemical nature of the inhibitors, they may be administered orally, intravenously, or by an aerosol route. Whatever the mode of administration, the access of therapeutic inhibitors to active proteases is often hampered by physicochemical constraints in the extravascular space and/or by the partitioning of proteases between soluble and solid phases.


Concluding Remarks

NSPs were first recognized as protein-degrading enzymes but have now proven to be multifunctional components participating in a variety of pathophysiological processes. Thus, they appear as potential therapeutic targets for drugs that inhibit their active site or impair activation from their precursor. Overall, the available preclinical and clinical data suggest that inhibition of NSPs using therapeutic inhibitors would suppress or attenuate deleterious effects of inflammatory diseases, including lung diseases. Depending on the size and chemical nature of inhibitors, those may be administered orally, intravenously, or by aerosolization. But the results obtained until now have not been fully convincing because of the poor knowledge of the biological function of each protease, their spatiotemporal regulation during the course of the disease, the physicochemical constraints associated with inhibitor administration, or the use of animal models in which NSP regulation and specificity differ from those in human. Two different and complementary approaches may help bypass these putative problems. One is to target active proteases by inhibitors at the inflammatory site in animal models in which lung anatomy and physiology are close to those in human to allow in vitro and in vivo assays of human-directed drugs/inhibitors. The other is to prevent neutrophil accumulation at inflammatory sites by impairing production of proteolytically active NSPs using an inhibitor of their maturation protease, DPPI. Preventing neutrophil accumulation at the inflammatory sites by therapeutic inhibition of DPPI represents an original and novel approach, the exploration of which has just started (Méthot et al., 2008). Thus pharmacological inactivation of DPPI in human neutrophils could well reduce membrane binding of PR3 and, as a consequence, neutrophil priming by pathogenic auto-antibodies in WG. In addition, it has been recognized that the intracellular level of NSPs depends on their correct intracellular trafficking. In the future, pharmacological targeting of molecules specifically involved in the correct intracellular trafficking of each NSP could possibly regulate their production and activity, a feature that could be exploited as a therapeutic strategy for inflammatory diseases.










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Understanding the Stem Cell Niche: A Webinar by The Scientist

Reporter: Stephen J. Williams, Ph.D.


The Scientist

nature stem cell

Schematic diagram showing some of the factors implicated in each process. Haematopoietic stem cells (HSCs) bound to the bone-marrow niche are mobilized in response to granulocyte colony-stimulating factor (G-CSF) or cyclophosphamide, or after peripheral myeloablation following treatment with 5-fluorouracil (5-FU). After extravasation from the bone-marrow cords into the microvasculature, HSCs enter the circulation and are distributed to peripheral tissues such as the spleen or liver. HSCs locate close to endothelial cells in the splenic red pulp. They home to the bone-marrow cords through the circulation, a process that is controlled by a number of adhesion molecules such as very late antigen 4 (VLA4), VLA5, lymphocyte function-associated antigen 1 (LFA1) or selectins. After entering the bone marrow, HSCs specifically lodge in the niche, a process requiring membrane-bound stem-cell factor (SCF), CXC-chemokine ligand 12 (CXCL12), osteopontin (OPN), hyaluronic acid, and their corresponding receptors. CXCR4, CXC-chemokine receptor 4; E-selectin, endothelial-cell selectin; P-selectin, platelet selectin; PSGL1, P-selectin glycoprotein ligand 1.


Understanding the Stem Cell Niche

  This presentation will begin on Tuesday, December 01, 2015 at 02:30 PM Eastern Standard Time.

Free Webinar
Tuesday December 1, 2015
2:30 – 4:00 PM EST

Stem cells provide an attractive model to study human physiology and disease. However, technical challenges persist in the biological characterization and manipulation of stem cells in their native microenvironment. The Scientist brings together a panel of experts to discuss interactions between stem cells and external cues, and the role of the stem cell niche in development and disease. Topics to be covered include the molecular mechanisms of hematopoietic stem cell niche interactions and techniques for engineering 3-D stem-cell microenvironments. Following the presentations, attendees will have an opportunity to ask questions concerning their specific applications and receive answers in real-time.


Dr. Jon Hoggatt, Assistant Professor of Medicine, Cancer Center and Center for Transplantation Sciences, Harvard Medical School/Massachusetts General Hospital.

Dr. Todd McDevitt, Senior Investigator, Gladstone Institute of Cardiovascular Disease, Professor, Department of Bioengineering & Therapeutic Sciences, UCSF.


Understanding the Stem Cell Niche
Click Here To Watch The Video

To find out about our upcoming events follow us on Twitter @LabMgrEvents


Notes from Webinar:

Hematopoetic stem cells good model since now we have liquid biopsies (as a result field has skyrocketed).

Two processes involved with stem cells finding their niche

  1. Homing; CXCR4-SDK1 dependent process into the bone marrow.
  2. Mobilization: stem cells moving from bone into blood (found that GMCSF main factor responsible for this process)

Dr. Raymond Schofield was one of the first to propose the existence of this stem cell niche (each progenitor will produce a unique factor {possibly a therapeutic target} for example leptin+ receptor target perivascular cells so one target is good for only a small subset of stem cells)

Therefore it may be possible or advantageous to target the whole stem cell milieu. One such possible target they are investigating is CD26 (dipeptyl peptidase). The diabetes drug Januvia is an inhibitor of CD26.

It was also noticed if inhibit the GMCSF receptor complex can inhibit the whole stem cell niche.

Prostoglandins and stem cell niche

  • Indomethacin blocks the mobilization step
  • Prostaglandin E increases homing
  • GMCSF and malaxocam (COX2 inhibitor) flattens osteoblast cells and may be a mechanism how inhibition of prostaglandin synthesis blocks mobilization
  • Found that the PGE4 receptor is ultimately responsible for the NSAID effect

The niche after G-CSF

Dr. Hoggat found that macrophages are supplying the factors that support the niche. He will be presenting the findings at 2015 Hematology conference. (See information about his conference presentation here).

From the 57th Annual American Society of Hematology Meeting (2015) please see Dr. Hoggat’s moderated section Hematopoiesis and Stem Cells: Microenvironment, Cell Adhesion and Stromal Stem Cells: Hematopoietic Stem Cell Niche


Relevant articles from Dr. Hoggat

Anti-CD47 Therapy Is More Than a Dinner Bell October 19, 2015

Dr. Hoggatt looks at the therapeutic effects of blocking CD47 aside from alerting macrophages to devour tumor cells.

Hematopoietic Stem Cells Should Hold Their Breath August 12, 2015

Dr. Hoggatt and Hannah Rasmussen discuss new approaches to the use of hematopoietic stem cells considering observer effects that emerge due to our experimental systems for HSCs.

Prostaglandin E2 enhances hematopoietic stem cell homing, survival, and proliferation. Hoggatt J, Singh P, Sampath J, Pelus LM. Blood. 2009 May 28;113(22):5444-55. doi: 10.1182/blood-2009-01-201335. Epub 2009 Mar 26.


Prostaglandin E2 enhances long-term repopulation but does not permanently alter inherent stem cell competitiveness. Hoggatt J, Mohammad KS, Singh P, Pelus LM. Blood. 2013 Oct 24;122(17):2997-3000. doi: 10.1182/blood-2013-07-515288. Epub 2013 Sep 18.


Pharmacologic increase in HIF1α enhances hematopoietic stem and progenitor homing and engraftment. Speth JM, Hoggatt J, Singh P, Pelus LM. Blood. 2014 Jan 9;123(2):203-7. doi: 10.1182/blood-2013-07-516336. Epub 2013 Oct 28.


Blockade of prostaglandin E2 signaling through EP1 and EP3 receptors attenuates Flt3L-dependent dendritic cell development from hematopoietic progenitor cells. Singh P, Hoggatt J, Hu P, Speth JM, Fukuda S, Breyer RM, Pelus LM. Blood. 2012 Feb 16;119(7):1671-82. doi: 10.1182/blood-2011-03-342428. Epub 2011 Nov 22.


Recovery from hematopoietic injury by modulating prostaglandin E(2) signaling post-irradiation. Hoggatt J, Singh P, Stilger KN, Plett PA, Sampson CH, Chua HL, Orschell CM, Pelus LM. Blood Cells Mol Dis. 2013 Mar;50(3):147-53. doi: 10.1016/j.bcmd.2012.11.006. Epub 2012 Nov 30.


Pulse exposure of haematopoietic grafts to prostaglandin E2 in vitro facilitates engraftment and recovery. Pelus LM, Hoggatt J, Singh P. Cell Prolif. 2011 Apr;44 Suppl 1:22-9. doi: 10.1111/j.1365-2184.2010.00726.x.


Pleiotropic effects of prostaglandin E2 in hematopoiesis; prostaglandin E2 and other eicosanoids regulate hematopoietic stem and progenitor cell function. Pelus LM, Hoggatt J. Prostaglandins Other Lipid Mediat. 2011 Nov;96(1-4):3-9. doi: 10.1016/j.prostaglandins.2011.06.004. Epub 2011 Jun 21. Review.


Differential stem- and progenitor-cell trafficking by prostaglandin E2. Hoggatt J, Mohammad KS, Singh P, Hoggatt AF, Chitteti BR, Speth JM, Hu P, Poteat BA, Stilger KN, Ferraro F, Silberstein L, Wong FK, Farag SS, Czader M, Milne GL, Breyer RM, Serezani CH, Scadden DT, Guise TA, Srour EF, Pelus LM. Nature. 2013 Mar 21;495(7441):365-9. doi: 10.1038/nature11929. Epub 2013 Mar 13.


Eicosanoid regulation of hematopoiesis and hematopoietic stem and progenitor trafficking.Hoggatt J, Pelus LM. Leukemia. 2010 Dec;24(12):1993-2002. doi: 10.1038/leu.2010.216. Epub 2010 Sep 30. Review.


Hematopoietic stem cell mobilization with agents other than G-CSF. Hoggatt J, Pelus LM. Methods Mol Biol. 2012;904:49-67. doi: 10.1007/978-1-61779-943-3_4.


Mobilization of hematopoietic stem cells from the bone marrow niche to the blood compartment. Hoggatt J, Pelus LM. Stem Cell Res Ther. 2011 Mar 14;2(2):13. doi: 10.1186/scrt54. Review.


Engineering 3D Pluripotent Stem Cell Microenvironments by Todd McDevitt, Ph.D.

In recent years, it has finally been shown how to produce centrally derived (self assembling) organoids (microtissues).


How to specifically deliver specific morphogens in 3D organoids


  1. Microparticle (MP)-mediated delivery (can do in mouse and human): reduces the amount needed to be delivered



What are other effects of introduced MP in ES (embryonic stem cell) aggregates?

  1. a) physiocomechanical changes –mechanical effects of materials
  2. b) how changes in local presentation of factors affect bioavailbility and binding properties







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Biochemistry and Dysmetabolism of Aging and Serious Illness

Curator: Larry H. Bernstein, MD, FCAP


White Matter Lipids as a Ketogenic Fuel Supply in Aging Female Brain: Implications for Alzheimer’s Disease

Lauren P. Klosinski, Jia Yao, Fei Yin, Alfred N. Fonteh, Michael G. Harrington, Trace A. Christensen, Eugenia Trushina, Roberta Diaz Brinton      DOI:
  • Mitochondrial dysfunction activates mechanisms for catabolism of myelin lipids to generate ketone bodies for ATP production.
  • Mechanisms leading to ketone body driven energy production in brain coincide with stages of reproductive aging in females.
  • Sequential activation of myelin catabolism pathway during aging provides multiple therapeutic targets and windows of efficacy.

The mechanisms underlying white matter degeneration, a hallmark of multiple neurodegenerative diseases including Alzheimer’s, remain unclear. Herein we provide a mechanistic pathway, spanning multiple transitions of aging, that links mitochondrial dysfunction early in aging with later age white matter degeneration. Catabolism of myelin lipids to generate ketone bodies can be viewed as an adaptive survival response to address brain fuel and energy demand. Women are at greatest risk of late-onset-AD, thus, our analyses in female brain address mechanisms of AD pathology and therapeutic targets to prevent, delay and treat AD in the sex most affected with potential relevance to men.


White matter degeneration is a pathological hallmark of neurodegenerative diseases including Alzheimer’s. Age remains the greatest risk factor for Alzheimer’s and the prevalence of age-related late onset Alzheimer’s is greatest in females. We investigated mechanisms underlying white matter degeneration in an animal model consistent with the sex at greatest Alzheimer’s risk. Results of these analyses demonstrated decline in mitochondrial respiration, increased mitochondrial hydrogen peroxide production and cytosolic-phospholipase-A2 sphingomyelinase pathway activation during female brain aging. Electron microscopic and lipidomic analyses confirmed myelin degeneration. An increase in fatty acids and mitochondrial fatty acid metabolism machinery was coincident with a rise in brain ketone bodies and decline in plasma ketone bodies. This mechanistic pathway and its chronologically phased activation, links mitochondrial dysfunction early in aging with later age development of white matter degeneration. The catabolism of myelin lipids to generate ketone bodies can be viewed as a systems level adaptive response to address brain fuel and energy demand. Elucidation of the initiating factors and the mechanistic pathway leading to white matter catabolism in the aging female brain provides potential therapeutic targets to prevent and treat demyelinating diseases such as Alzheimer’s and multiple sclerosis. Targeting stages of disease and associated mechanisms will be critical.

3. Results

  1. 3.1. Pathway of Mitochondrial Deficits, H2O2 Production and cPLA2 Activation in the Aging Female Brain
  2. 3.2. cPLA2-sphingomyelinase Pathway Activation in White Matter Astrocytes During Reproductive Senescence
  3. 3.3. Investigation of White Matter Gene Expression Profile During Reproductive Senescence
  4. 3.4. Ultra Structural Analysis of Myelin Sheath During Reproductive Senescence
  5. 3.5. Analysis of the Lipid Profile of Brain During the Transition to Reproductive Senescence
  6. 3.6. Fatty Acid Metabolism and Ketone Generation Following the Transition to Reproductive Senescence


4. Discussion

Age remains the greatest risk factor for developing AD (Hansson et al., 2006, Alzheimer’s, 2015). Thus, investigation of transitions in the aging brain is a reasoned strategy for elucidating mechanisms and pathways of vulnerability for developing AD. Aging, while typically perceived as a linear process, is likely composed of dynamic transition states, which can protect against or exacerbate vulnerability to AD (Brinton et al., 2015). An aging transition unique to the female is the perimenopausal to menopausal conversion (Brinton et al., 2015). The bioenergetic similarities between the menopausal transition in women and the early appearance of hypometabolism in persons at risk for AD make the aging female a rational model to investigate mechanisms underlying risk of late onset AD.

Findings from this study replicate our earlier findings that age of reproductive senescence is associated with decline in mitochondrial respiration, increased H2O2 production and shift to ketogenic metabolism in brain (Yao et al., 2010, Ding et al., 2013, Yin et al., 2015). These well established early age-related changes in mitochondrial function and shift to ketone body utilization in brain, are now linked to a mechanistic pathway that connects early decline in mitochondrial respiration and H2O2 production to activation of the cPLA2-sphingomyelinase pathway to catabolize myelin lipids resulting in WM degeneration (Fig. 12). These lipids are sequestered in lipid droplets for subsequent use as a local source of ketone body generation via astrocyte mediated beta-oxidation of fatty acids. Astrocyte derived ketone bodies can then be transported to neurons where they undergo ketolysis to generate acetyl-CoA for TCA derived ATP generation required for synaptic and cell function (Fig. 12).

Thumbnail image of Fig. 12. Opens large image

Fig. 12

Schematic model of mitochondrial H2O2 activation of cPLA2-sphingomyelinase pathway as an adaptive response to provide myelin derived fatty acids as a substrate for ketone body generation: The cPLA2-sphingomyelinase pathway is proposed as a mechanistic pathway that links an early event, mitochondrial dysfunction and H2O2, in the prodromal/preclinical phase of Alzheimer’s with later stage development of pathology, white matter degeneration. Our findings demonstrate that an age dependent deficit in mitochondrial respiration and a concomitant rise in oxidative stress activate an adaptive cPLA2-sphingomyelinase pathway to provide myelin derived fatty acids as a substrate for ketone body generation to fuel an energetically compromised brain.

Biochemical evidence obtained from isolated whole brain mitochondria confirms that during reproductive senescence and in response to estrogen deprivation brain mitochondria decline in respiratory capacity (Yao et al., 2009, Yao et al., 2010, Brinton, 2008a, Brinton, 2008b, Swerdlow and Khan, 2009). A well-documented consequence of mitochondrial dysfunction is increased production of reactive oxygen species (ROS), specifically H2O2 (Boveris and Chance, 1973, Beal, 2005, Yin et al., 2014, Yap et al., 2009). While most research focuses on the damage generated by free radicals, in this case H2O2 functions as a signaling molecule to activate cPLA2, the initiating enzyme in the cPLA2-sphingomyelinase pathway (Farooqui and Horrocks, 2006, Han et al., 2003, Sun et al., 2004). In AD brain, increased cPLA2 immunoreactivity is detected almost exclusively in astrocytes suggesting that activation of the cPLA2-sphingomyelinase pathway is localized to astrocytes in AD, as opposed to the neuronal or oligodendroglial localization that is observed during apoptosis (Sun et al., 2004, Malaplate-Armand et al., 2006, Di Paolo and Kim, 2011, Stephenson et al., 1996,Stephenson et al., 1999). In our analysis, cPLA2 (Sanchez-Mejia and Mucke, 2010) activation followed the age-dependent rise in H2O2 production and was sustained at an elevated level.

Direct and robust activation of astrocytic cPLA2 by physiologically relevant concentrations of H2O2 was confirmed in vitro. Astrocytic involvement in the cPLA2-sphingomyelinase pathway was also indicated by an increase in cPLA2 positive astrocyte reactivity in WM tracts of reproductively incompetent mice. These data are consistent with findings from brains of persons with AD that demonstrate the same striking localization of cPLA2immunoreactivity within astrocytes, specifically in the hippocampal formation (Farooqui and Horrocks, 2004). While neurons and astrocytes contain endogenous levels of cPLA2, neuronal cPLA2 is activated by an influx of intracellular calcium, whereas astrocytic cPLA2 is directly activated by excessive generation of H2O2 (Sun et al., 2004, Xu et al., 2003, Tournier et al., 1997). Evidence of this cell type specific activation was confirmed by the activation of cPLA2 in astrocytes by H2O2 and the lack of activation in neurons. These data support that astrocytic, not neuronal, cPLA2 is the cellular mediator of the H2O2 dependent cPLA2-sphingomyelinase pathway activation and provide associative evidence supporting a role of astrocytic mitochondrial H2O2 in age-related WM catabolism.

The pattern of gene expression during the shift to reproductive senescence in the female mouse hippocampus recapitulates key observations in human AD brain tissue, specifically elevation in cPLA2, sphingomyelinase and ceramidase (Schaeffer et al., 2010, He et al., 2010, Li et al., 2014). Further, up-regulation of myelin synthesis, lipid metabolism and inflammatory genes in reproductively incompetent female mice is consistent with the gene expression pattern previously reported from aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). In these analyses of gene expression in aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus down regulation of genes related to mitochondrial function, and up-regulation in multiple genes encoding for enzymes involved in ketone body metabolism occurred (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). The comparability across data derived from aging female mouse hippocampus reported herein and those derived from male rodent brain, female nonhuman brain and human AD brain strongly suggest that cPLA2-sphingomyelinase pathway activation, myelin sheath degeneration and fatty acid metabolism leading to ketone body generation is a metabolic adaptation that is generalizable across these naturally aging models and are evident in aged human AD brain. Collectively, these data support the translational relevance of findings reported herein.

Data obtained via immunohistochemistry, electron microscopy and MBP protein analyses demonstrated an age-related loss in myelin sheath integrity. Evidence for a loss of myelin structural integrity emerged in reproductively incompetent mice following activation of the cPLA2-sphingomyelinase pathway. The unraveling myelin phenotype observed following reproductive senescence and aging reported herein is consistent with the degenerative phenotype that emerges following exposure to the chemotherapy drug bortezomib which induces mitochondrial dysfunction and increased ROS generation (Carozzi et al., 2010, Cavaletti et al., 2007,Ling et al., 2003). In parallel to the decline in myelin integrity, lipid droplet density increased. In aged mice, accumulation of lipid droplets declined in parallel to the rise in ketone bodies consistent with the utilization of myelin-derived fatty acids to generate ketone bodies. Due to the sequential relationship between WM degeneration and lipid droplet formation, we posit that lipid droplets serve as a temporary storage site for myelin-derived fatty acids prior to undergoing β-oxidation in astrocytes to generate ketone bodies.

Microstructural alterations in myelin integrity were associated with alterations in the lipid profile of brain, indicative of WM degeneration resulting in release of myelin lipids. Sphingomyelin and galactocerebroside are two main lipids that compose the myelin sheath (Baumann and Pham-Dinh, 2001). Ceramide is common to both galactocerebroside and sphingomyelin and is composed of sphingosine coupled to a fatty acid. Ceramide levels increase in aging, in states of ketosis and in neurodegeneration (Filippov et al., 2012, Blazquez et al., 1999, Costantini et al., 2005). Specifically, ceramide levels are elevated at the earliest clinically recognizable stage of AD, indicating a degree of WM degeneration early in disease progression (Di Paolo and Kim, 2011,Han et al., 2002, Costantini et al., 2005). Sphingosine is statistically significantly elevated in the brains of AD patients compared to healthy controls; a rise that was significantly correlated with acid sphingomyelinase activity, Aβ levels and tau hyperphosphorylation (He et al., 2010). In our analyses, a rise in ceramides was first observed early in the aging process in reproductively incompetent mice. The rise in ceramides was coincident with the emergence of loss of myelin integrity consistent with the release of myelin ceramides from sphingomyelin via sphingomyelinase activation. Following the rise in ceramides, sphingosine and fatty acid levels increased. The temporal sequence of the lipid profile was consistent with gene expression indicating activation of ceramidase for catabolism of ceramide into sphingosine and fatty acid during reproductive senescence. Once released from ceramide, fatty acids can be transported into the mitochondrial matrix of astrocytes via CPT-1, where β-oxidation of fatty acids leads to the generation of acetyl-CoA (Glatz et al., 2010). It is well documented that acetyl-CoA cannot cross the inner mitochondrial membrane, thus posing a barrier to direct transport of acetyl-CoA generated by β-oxidation into neurons. In response, the newly generated acetyl-CoA undergoes ketogenesis to generate ketone bodies to fuel energy demands of neurons (Morris, 2005,Guzman and Blazquez, 2004, Stacpoole, 2012). Because astrocytes serve as the primary location of β-oxidation in brain they are critical to maintaining neuronal metabolic viability during periods of reduced glucose utilization (Panov et al., 2014, Ebert et al., 2003, Guzman and Blazquez, 2004).

Once fatty acids are released from myelin ceramides, they are transported into astrocytic mitochondria by CPT1 to undergo β-oxidation. The mitochondrial trifunctional protein HADHA catalyzes the last three steps of mitochondrial β-oxidation of long chain fatty acids, while mitochondrial ABAD (aka SCHAD—short chain fatty acid dehydrogenase) metabolizes short chain fatty acids. Concurrent with the release of myelin fatty acids in aged female mice, CPT1, HADHA and ABAD protein expression as well as ketone body generation increased significantly. These findings indicate that astrocytes play a pivotal role in the response to bioenergetic crisis in brain to activate an adaptive compensatory system that activates catabolism of myelin lipids and the metabolism of those lipids into fatty acids to generate ketone bodies necessary to fuel neuronal demand for acetyl-CoA and ATP.

Collectively, these findings provide a mechanistic pathway that links mitochondrial dysfunction and H2O2generation in brain early in the aging process to later stage white matter degeneration. Astrocytes play a pivotal role in providing a mechanistic strategy to address the bioenergetic demand of neurons in the aging female brain. While this pathway is coincident with reproductive aging in the female brain, it is likely to have mechanistic translatability to the aging male brain. Further, the mechanistic link between bioenergetic decline and WM degeneration has potential relevance to other neurological diseases involving white matter in which postmenopausal women are at greater risk, such as multiple sclerosis. The mechanistic pathway reported herein spans time and is characterized by a progression of early adaptive changes in the bioenergetic system of the brain leading to WM degeneration and ketone body production. Translationally, effective therapeutics to prevent, delay and treat WM degeneration during aging and Alzheimer’s disease will need to specifically target stages within the mechanistic pathway described herein. The fundamental initiating event is a bioenergetic switch from being a glucose dependent brain to a glucose and ketone body dependent brain. It remains to be determined whether it is possible to prevent conversion to or reversal of a ketone dependent brain. Effective therapeutic strategies to intervene in this process require biomarkers of bioenergetic phenotype of the brain and stage of mechanistic progression. The mechanistic pathway reported herein may have relevance to other age-related neurodegenerative diseases characterized by white matter degeneration such as multiple sclerosis.

Blood. 2015 Oct 15;126(16):1925-9. Epub 2015 Aug 14.
Targeting the leukemia cell metabolism by the CPT1a inhibition: functional preclinical effects in leukemias.
Cancer cells are characterized by perturbations of their metabolic processes. Recent observations demonstrated that the fatty acid oxidation (FAO) pathway may represent an alternative carbon source for anabolic processes in different tumors, therefore appearing particularly promising for therapeutic purposes. Because the carnitine palmitoyl transferase 1a (CPT1a) is a protein that catalyzes the rate-limiting step of FAO, here we investigated the in vitro antileukemic activity of the novel CPT1a inhibitor ST1326 on leukemia cell lines and primary cells obtained from patients with hematologic malignancies. By real-time metabolic analysis, we documented that ST1326 inhibited FAO in leukemia cell lines associated with a dose- and time-dependent cell growth arrest, mitochondrial damage, and apoptosis induction. Data obtained on primary hematopoietic malignant cells confirmed the FAO inhibition and cytotoxic activity of ST1326, particularly on acute myeloid leukemia cells. These data suggest that leukemia treatment may be carried out by targeting metabolic processes.
Oncogene. 2015 Oct 12. [Epub ahead of print]
Tumour-suppression function of KLF12 through regulation of anoikis.
Suppression of detachment-induced cell death, known as anoikis, is an essential step for cancer metastasis to occur. We report here that expression of KLF12, a member of the Kruppel-like family of transcription factors, is downregulated in lung cancer cell lines that have been selected to grow in the absence of cell adhesion. Knockdown of KLF12 in parental cells results in decreased apoptosis following cell detachment from matrix. KLF12 regulates anoikis by promoting the cell cycle transition through S phase and therefore cell proliferation. Reduced expression levels of KLF12 results in increased ability of lung cancer cells to form tumours in vivo and is associated with poorer survival in lung cancer patients. We therefore identify KLF12 as a novel metastasis-suppressor gene whose loss of function is associated with anoikis resistance through control of the cell cycle.
Mol Cell. 2015 Oct 14. pii: S1097-2765(15)00764-9. doi: 10.1016/j.molcel.2015.09.025. [Epub ahead of print]
PEPCK Coordinates the Regulation of Central Carbon Metabolism to Promote Cancer Cell Growth.
Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid, and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.
Mol Cancer Res. 2015 Oct;13(10):1408-20. Epub 2015 Jun 16.
Disruption of Proline Synthesis in Melanoma Inhibits Protein Production Mediated by the GCN2 Pathway.
Many processes are deregulated in melanoma cells and one of those is protein production. Although much is known about protein synthesis in cancer cells, effective ways of therapeutically targeting this process remain an understudied area of research. A process that is upregulated in melanoma compared with normal melanocytes is proline biosynthesis, which has been linked to both oncogene and tumor suppressor pathways, suggesting an important convergent point for therapeutic intervention. Therefore, an RNAi screen of a kinase library was undertaken, identifying aldehyde dehydrogenase 18 family, member A1 (ALDH18A1) as a critically important gene in regulating melanoma cell growth through proline biosynthesis. Inhibition of ALDH18A1, the gene encoding pyrroline-5-carboxylate synthase (P5CS), significantly decreased cultured melanoma cell viability and tumor growth. Knockdown of P5CS using siRNA had no effect on apoptosis, autophagy, or the cell cycle but cell-doubling time increased dramatically suggesting that there was a general slowdown in cellular metabolism. Mechanistically, targeting ALDH18A1 activated the serine/threonine protein kinase GCN2 (general control nonderepressible 2) to inhibit protein synthesis, which could be reversed with proline supplementation. Thus, targeting ALDH18A1 in melanoma can be used to disrupt proline biosynthesis to limit cell metabolism thereby increasing the cellular doubling time mediated through the GCN2 pathway.  This study demonstrates that melanoma cells are sensitive to disruption of proline synthesis and provides a proof-of-concept that the proline synthesis pathway can be therapeutically targeted in melanoma tumors for tumor inhibitory efficacy. Mol Cancer Res; 13(10); 1408-20. ©2015 AACR.
SDHB-Deficient Cancers: The Role of Mutations That Impair Iron Sulfur Cluster Delivery.
BACKGROUND:  Mutations in the Fe-S cluster-containing SDHB subunit of succinate dehydrogenase cause familial cancer syndromes. Recently the tripeptide motif L(I)YR was identified in the Fe-S recipient protein SDHB, to which the cochaperone HSC20 binds.
METHODS:   In order to characterize the metabolic basis of SDH-deficient cancers we performed stable isotope-resolved metabolomics in a novel SDHB-deficient renal cell carcinoma cell line and conducted bioinformatics and biochemical screening to analyze Fe-S cluster acquisition and assembly of SDH in the presence of other cancer-causing SDHB mutations.


We found that the SDHB(R46Q) mutation in UOK269 cells disrupted binding of HSC20, causing rapid degradation of SDHB. In the absence of SDHB, respiration was undetectable in UOK269 cells, succinate was elevated to 351.4±63.2 nmol/mg cellular protein, and glutamine became the main source of TCA cycle metabolites through reductive carboxylation. Furthermore, HIF1α, but not HIF2α, increased markedly and the cells showed a strong DNA CpG island methylator phenotype (CIMP). Biochemical and bioinformatic screening revealed that 37% of disease-causing missense mutations in SDHB were located in either the L(I)YR Fe-S transfer motifs or in the 11 Fe-S cluster-ligating cysteines.


These findings provide a conceptual framework for understanding how particular mutations disproportionately cause the loss of SDH activity, resulting in accumulation of succinate and metabolic remodeling in SDHB cancer syndromes.


SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMPK-mTOR Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

  1. L. Figarola, J. Singhal, J. D. Tompkins, G. W. Rogers, C. Warden, D. Horne, A. D. Riggs, S. Awasthi and S. S. Singhal.

J Biol Chem. 2015 Nov 3, [epub ahead of print]


CD47 Receptor Globally Regulates Metabolic Pathways That Control Resistance to Ionizing Radiation

  1. W. Miller, D. R. Soto-Pantoja, A. L. Schwartz, J. M. Sipes, W. G. DeGraff, L. A. Ridnour, D. A. Wink and D. D. Roberts.

J Biol Chem. 2015 Oct 9, 290 (41): 24858-74.


Knockdown of PKM2 Suppresses Tumor Growth and Invasion in Lung Adenocarcinoma

  1. Sun, A. Zhu, L. Zhang, J. Zhang, Z. Zhong and F. Wang.

Int J Mol Sci. 2015 Oct 15, 16 (10): 24574-87.


EglN2 associates with the NRF1-PGC1alpha complex and controls mitochondrial function in breast cancer

  1. Zhang, C. Wang, X. Chen, M. Takada, C. Fan, X. Zheng, H. Wen, Y. Liu, C. Wang, R. G. Pestell, K. M. Aird, W. G. Kaelin, Jr., X. S. Liu and Q. Zhang.

EMBO J. 2015 Oct 22, [epub ahead of print]


Mitochondrial Genetics Regulate Breast Cancer Tumorigenicity and Metastatic Potential.

Current paradigms of carcinogenic risk suggest that genetic, hormonal, and environmental factors influence an individual’s predilection for developing metastatic breast cancer. Investigations of tumor latency and metastasis in mice have illustrated differences between inbred strains, but the possibility that mitochondrial genetic inheritance may contribute to such differences in vivo has not been directly tested. In this study, we tested this hypothesis in mitochondrial-nuclear exchange mice we generated, where cohorts shared identical nuclear backgrounds but different mtDNA genomes on the background of the PyMT transgenic mouse model of spontaneous mammary carcinoma. In this setting, we found that primary tumor latency and metastasis segregated with mtDNA, suggesting that mtDNA influences disease progression to a far greater extent than previously appreciated. Our findings prompt further investigation into metabolic differences controlled by mitochondrial process as a basis for understanding tumor development and metastasis in individual subjects. Importantly, differences in mitochondrial DNA are sufficient to fundamentally alter disease course in the PyMT mouse mammary tumor model, suggesting that functional metabolic differences direct early tumor growth and metastatic efficiency. Cancer Res; 75(20); 4429-36. ©2015 AACR.


Cancer Lett. 2015 Oct 29. pii: S0304-3835(15)00656-4. [Epub ahead of print]
Carboxyamidotriazole inhibits oxidative phosphorylation in cancer cells and exerts synergistic anti-cancer effect with glycolysis inhibition.

Targeting cancer cell metabolism is a promising strategy against cancer. Here, we confirmed that the anti-cancer drug carboxyamidotriazole (CAI) inhibited mitochondrial respiration in cancer cells for the first time and found a way to enhance its anti-cancer activity by further disturbing the energy metabolism. CAI promoted glucose uptake and lactate production when incubated with cancer cells. The oxidative phosphorylation (OXPHOS) in cancer cells was inhibited by CAI, and the decrease in the activity of the respiratory chain complex I could be one explanation. The anti-cancer effect of CAI was greatly potentiated when being combined with 2-deoxyglucose (2-DG). The cancer cells treated with the combination of CAI and 2-DG were arrested in G2/M phase. The apoptosis and necrosis rates were also increased. In a mouse xenograft model, this combination was well tolerated and retarded the tumor growth. The impairment of cancer cell survival was associated with significant cellular ATP decrease, suggesting that the combination of CAI and 2-DG could be one of the strategies to cause dual inhibition of energy pathways, which might be an effective therapeutic approach for a broad spectrum of tumors.


Cancer Immunol Res. 2015 Nov;3(11):1236-47. Epub 2015 May 29.
Inhibition of Fatty Acid Oxidation Modulates Immunosuppressive Functions of Myeloid-Derived Suppressor Cells and Enhances Cancer Therapies.

Myeloid-derived suppressor cells (MDSC) promote tumor growth by inhibiting T-cell immunity and promoting malignant cell proliferation and migration. The therapeutic potential of blocking MDSC in tumors has been limited by their heterogeneity, plasticity, and resistance to various chemotherapy agents. Recent studies have highlighted the role of energy metabolic pathways in the differentiation and function of immune cells; however, the metabolic characteristics regulating MDSC remain unclear. We aimed to determine the energy metabolic pathway(s) used by MDSC, establish its impact on their immunosuppressive function, and test whether its inhibition blocks MDSC and enhances antitumor therapies. Using several murine tumor models, we found that tumor-infiltrating MDSC (T-MDSC) increased fatty acid uptake and activated fatty acid oxidation (FAO). This was accompanied by an increased mitochondrial mass, upregulation of key FAO enzymes, and increased oxygen consumption rate. Pharmacologic inhibition of FAO blocked immune inhibitory pathways and functions in T-MDSC and decreased their production of inhibitory cytokines. FAO inhibition alone significantly delayed tumor growth in a T-cell-dependent manner and enhanced the antitumor effect of adoptive T-cell therapy. Furthermore, FAO inhibition combined with low-dose chemotherapy completely inhibited T-MDSC immunosuppressive effects and induced a significant antitumor effect. Interestingly, a similar increase in fatty acid uptake and expression of FAO-related enzymes was found in human MDSC in peripheral blood and tumors. These results support the possibility of testing FAO inhibition as a novel approach to block MDSC and enhance various cancer therapies. Cancer Immunol Res; 3(11); 1236-47. ©2015 AACR.


Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase

  1. L. Judge, K. M. Owens, S. J. Pollock, C. F. Woeller, T. H. Thatcher, J. P. Williams, R. P. Phipps, P. J. Sime and R. M. Kottmann.

Am J Physiol Lung Cell Mol Physiol. 2015 Oct 15, 309 (8): L879-87.


Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH

  1. Yun, E. Mullarky, C. Lu, K. N. Bosch, A. Kavalier, K. Rivera, J. Roper, Chio, II, E. G. Giannopoulou, C. Rago, A. Muley, J. M. Asara, J. Paik, O. Elemento, Z. Chen, D. J. Pappin, L. E. Dow, N. Papadopoulos, S. S. Gross and L. C. Cantley.

Science. 2015 Nov 5, [epub ahead of print]


Down-regulation of FBP1 by ZEB1-mediated repression confers to growth and invasion in lung cancer cells

  1. Zhang, J. Wang, H. Xing, Q. Li, Q. Zhao and J. Li.

Mol Cell Biochem. 2015 Nov 6, [epub ahead of print]


J Mol Cell Cardiol. 2015 Oct 23. pii: S0022-2828(15)30073-0. [Epub ahead of print]
GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels.

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.


Br J Pharmacol. 2015 Oct 27. doi: 10.1111/bph.13377. [Epub ahead of print]
All-trans retinoic acid protects against doxorubicin-induced cardiotoxicity by activating the Erk2 signalling pathway.

Doxorubicin (Dox) is a powerful antineoplastic agent for treating a wide range of cancers. However, doxorubicin cardiotoxicity of the heart has largely limited its clinical use. It is known that all-trans retinoic acid (ATRA) plays important roles in many cardiac biological processes, however, the protective effects of ATRA on doxorubicin cardiotoxicity remain unknown. Here, we studied the effect of ATRA on doxorubicin cardiotoxicity and underlying mechanisms.


Cellular viability assays, western blotting and mitochondrial respiration analyses were employed to evaluate the cellular response to ATRA in H9c2 cells and primary cardiomyocytes. Quantitative PCR (Polymerase Chain Reaction) and gene knockdown were performed to investigate the underlying molecular mechanisms of ATRA’s effects on doxorubicin cardiotoxicity.


ATRA significantly inhibited doxorubicin-induced apoptosis in H9c2 cells and primary cardiomyocytes. ATRA was more effective against doxorubicin cardiotoxicity than resveratrol and dexrazoxane. ATRA also suppressed reactive oxygen species (ROS) generation, and restored the expression level of mRNA and proteins in phase II detoxifying enzyme system: Nrf2 (nuclear factor-E2-related factor 2), MnSOD (manganese superoxide dismutase), HO-1 (heme oxygenase1) as well as mitochondrial function (mitochondrial membrane integrity, mitochondrial DNA copy numbers, mitochondrial respiration capacity, biogenesis and dynamics). Both Erk1/2 (extracellular signal-regulated kinase1/2) inhibitor (U0126) and Erk2 siRNA, but not Erk1 siRNA, abolished the protective effect of ATRA against doxorubicin-induced toxicity in H9c2 cells. Remarkably, ATRA did not compromise the anticancer efficacy of doxorubicin in gastric carcinoma cells.


ATRA protected cardiomyocytes against doxorubicin-induced toxicity by activating the Erk2 pathway without compromising the anticancer efficacy of doxorubicin. Therefore, ATRA may be a promising candidate as a cardioprotective agent against doxorubicin cardiotoxicity.


Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation

  1. Colak, O. Pougovkina, L. Dai, M. Tan, H. Te Brinke, H. Huang, Z. Cheng, J. Park, X. Wan, X. Liu, W. W. Yue, R. J. Wanders, J. W. Locasale, D. B. Lombard, V. C. de Boer and Y. Zhao.

Mol Cell Proteomics. 2015 Nov 1, 14 (11): 3056-71.


Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

  1. Pancrazi, G. Di Benedetto, L. Colombaioni, G. Della Sala, G. Testa, F. Olimpico, A. Reyes, M. Zeviani, T. Pozzan and M. Costa.

Proc Natl Acad Sci U S A. 2015 Oct 27, 112(45): 13910-5.


Evidence of Mitochondrial Dysfunction within the Complex Genetic Etiology of Schizophrenia

  1. E. Hjelm, B. Rollins, F. Mamdani, J. C. Lauterborn, G. Kirov, G. Lynch, C. M. Gall, A. Sequeira and M. P. Vawter.

Mol Neuropsychiatry. 2015 Nov 1, 1 (4): 201-219.


Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation

  1. Bernard, N. J. Logsdon, S. Ravi, N. Xie, B. P. Persons, S. Rangarajan, J. W. Zmijewski, K. Mitra, G. Liu, V. M. Darley-Usmar and V. J. Thannickal.

J Biol Chem. 2015 Oct 16, 290 (42): 25427-38.


J Biol Chem. 2015 Oct 23;290(43):25834-46. Epub 2015 Sep 4.
Kinome Screen Identifies PFKFB3 and Glucose Metabolism as Important Regulators of the Insulin/Insulin-like Growth Factor (IGF)-1 Signaling Pathway.

The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterized by marked disruption of both metabolism and growth factor signaling.


FASEB J. 2015 Oct 19. fj.15-276360. [Epub ahead of print]
Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

Skeletal muscle mitochondrial content and oxidative capacity are important determinants of muscle function and whole-body health. Mitochondrial content and function are enhanced by endurance exercise and impaired in states or diseases where muscle function is compromised, such as myopathies, muscular dystrophies, neuromuscular diseases, and age-related muscle atrophy. Hence, elucidating the mechanisms that control muscle mitochondrial content and oxidative function can provide new insights into states and diseases that affect muscle health. In past studies, we identified Perm1 (PPARGC1- and ESRR-induced regulator, muscle 1) as a gene induced by endurance exercise in skeletal muscle, and regulating mitochondrial oxidative function in cultured myotubes. The capacity of Perm1 to regulate muscle mitochondrial content and function in vivo is not yet known. In this study, we use adeno-associated viral (AAV) vectors to increase Perm1 expression in skeletal muscles of 4-wk-old mice. Compared to control vector, AAV1-Perm1 leads to significant increases in mitochondrial content and oxidative capacity (by 40-80%). Moreover, AAV1-Perm1-transduced muscles show increased capillary density and resistance to fatigue (by 33 and 31%, respectively), without prominent changes in fiber-type composition. These findings suggest that Perm1 selectively regulates mitochondrial biogenesis and oxidative function, and implicate Perm1 in muscle adaptations that also occur in response to endurance exercise.-Cho, Y., Hazen, B. C., Gandra, P. G., Ward, S. R., Schenk, S., Russell, A. P., Kralli, A. Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.


A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells.
Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.


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Blocking Differentiation to Produce Stem Cells

Larry H. Bernstein, MD, FCAP, Curator



Blocking Differentiation is Enough to Turn Mature Cells into Stem Cells



ID3 inhibitor of DNA binding 3, dominant negative helix-loop-helix protein [ Homo sapiens (human) ]

Gene ID: 3399, updated on 15-Nov-2015


Official Symbol ID3 provided by HGNC 

Official Full Name inhibitor of DNA binding 3, dominant negative helix-loop-helix protein provided by HGNC

Primary source HGNC:HGNC:5362 See related Ensembl:ENSG00000117318; HPRD:02608; MIM:600277; Vega:OTTHUMG00000003229

Gene type protein coding

RefSeq status REVIEWED

OrganismHomo sapiens

LineageEukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo

Also known as HEIR-1; bHLHb25

Summary The protein encoded by this gene is a helix-loop-helix (HLH) protein that can form heterodimers with other HLH proteins. However, the encoded protein lacks a basic DNA-binding domain and therefore inhibits the DNA binding of any HLH protein with which it interacts. [provided by RefSeq, Aug 2011]

Orthologs mouse all


Exon count:
Annotation release Status Assembly Chr Location
107 current GRCh38.p2 (GCF_000001405.28) 1 NC_000001.11 (23557930..23559794, complement)
105 previous assembly GRCh37.p13 (GCF_000001405.25) 1 NC_000001.10 (23884421..23886285, complement)

Chromosome 1 – NC_000001.11Genomic Context describing neighboring genes

Related articles in PubMed


Induced Developmental Arrest of Early Hematopoietic Progenitors Leads to the Generation of Leukocyte Stem Cells

Tomokatsu Ikawa, Kyoko Masuda, Mirelle J.A.J. Huijskens, Rumi Satoh, Kiyokazu Kakugawa, Yasutoshi Agata, Tomohiro Miyai, Wilfred T.V. Germeraad, Yoshimoto Katsura, Hiroshi Kawamoto
Stem Cell Reports Nov 10, 2015; Volume 5, Issue 5, 716–727.   DOI:
  • Overexpression of ID3 endows hematopoietic progenitors with self-renewal activity
  • A simple block of cell differentiation is sufficient to induce stem cells
  • Induced leukocyte stem (iLS) cells exhibit robust multi-lineage reconstitution
  • Equivalent progenitors were produced from human cord blood hematopoietic stem cells

Self-renewal potential and multipotency are hallmarks of a stem cell. It is generally accepted that acquisition of such stemness requires rejuvenation of somatic cells through reprogramming of their genetic and epigenetic status. We show here that a simple block of cell differentiation is sufficient to induce and maintain stem cells. By overexpression of the transcriptional inhibitor ID3 in murine hematopoietic progenitor cells and cultivation under B cell induction conditions, the cells undergo developmental arrest and enter a self-renewal cycle. These cells can be maintained in vitro almost indefinitely, and the long-term cultured cells exhibit robust multi-lineage reconstitution when transferred into irradiated mice. These cells can be cloned and re-expanded with 50% plating efficiency, indicating that virtually all cells are self-renewing. Equivalent progenitors were produced from human cord blood stem cells, and these will ultimately be useful as a source of cells for immune cell therapy.

Figure thumbnail fx1


Somatic tissues with high turnover rates, such as skin, intestinal epithelium, and hematopoietic cells, are maintained by the activity of self-renewing stem cells, which are present in only limited numbers in each organ (Barker et al., 2012,Copley et al., 2012, Fuchs and Chen, 2013). For example, the frequency of hematopoietic stem cells (HSCs) in the mouse is about 1 in 105 of total bone marrow (BM) cells (Spangrude et al., 1988). Once HSCs begin the differentiation process, their progeny cells have hardly any self-renewal capacity, indicating that self-renewal is a special feature endowed only to stem cells.

Cells such as embryonic stem (ES) cells that retain self-renewal potential and multipotency only in vitro can also be included in the category of stem cells. Such stemness of ES cells is thought to be maintained by formation of a core transcriptional network and an epigenetic status unique to ES cells (Lund et al., 2012, Meissner, 2010, Ng and Surani, 2011). A stem cell equivalent to ES cells, called induced pluripotent stem (iPS) cells, can be produced from somatic cells by overexpression of only a few specific transcription factors (OCT3/4, SOX2, KLF4, and C-MYC), which are thought to be the essential components in forming the core network of transcriptional factors that define the status of ES cells (Takahashi et al., 2007, Takahashi and Yamanaka, 2006, Yamanaka, 2012). It is thus generally conceived that acquisition of such a network for a somatic cell depends on the reprogramming of the epigenetic status of that cell.

On the other hand, it could be envisioned that the self-renewing status of cells represents a state in which their further differentiation is inhibited. It is known, for example, that to maintain ES/iPS cells, factors such as leukemia inhibitory factor and basic fibroblast growth factor, for mouse and human cultures, respectively (Williams et al., 1988, Xu et al., 2005), are required, and these factors are thought to block further differentiation of the cells. In this context, it has previously been shown that systemic disruption of transcription factors essential for the B cell lineage, such as PAX5, E2A, and EBF1, leads to the emergence of self-renewing multipotent hematopoietic progenitors, which can be maintained under specific culture conditions (Ikawa et al., 2004a, Nutt et al., 1999, Pongubala et al., 2008). It has recently been shown that the suppression of lymphoid lineage priming promotes the expansion of both mouse and human hematopoietic progenitors (Mercer et al., 2011, van Galen et al., 2014). Therefore, it would seem theoretically possible to make a stem cell by inducing inactivation of these factors at particular developmental stages. Conditional depletion of PAX5 in B cell lineage committed progenitors, as well as mature B cells, resulted in the generation of T cells from the B lineage cells (Cobaleda et al., 2007, Nutt et al., 1999, Rolink et al., 1999). These studies, however, were mainly focused on the occurrence of cell-fate conversion by de-differentiation of target cells. Therefore, the minimal requirement for the acquisition of self-renewal potential remains undetermined.

Our ultimate goal is to obtain sufficient number of stem cells by expansion to overcome the limitation of cell numbers for immune therapies. We hypothesize that stem cells can be produced by simply blocking differentiation. As mentioned earlier, self-renewing multipotent progenitors (MPPs) can be produced by culturing E2A-deficient hematopoietic progenitors in B cell-inducing conditions (Ikawa et al., 2004a). Because it remains unclear at which developmental stage the acquisition of self-renewing potential has occurred in the case of such a systemic deletion, we thought to develop a method in which E2A function could be inactivated and reactivated in an inducible manner. We decided to use the ID3 protein for this purpose, because it is known that ID proteins serve as dominant-negative inhibitors of E proteins (Norton et al., 1998, Sayegh et al., 2003). Here we show that the overexpression of ID3 into HSCs or hematopoietic progenitor cells (HPCs) in both mouse and human induces the stemness of the progenitors and that the cells acquire the self-renewal activity. The ID3-expressing cells can be maintained in vitro as MPPs with T, B, and myeloid lineage potentials.



Jump to Section
  Generation of ID3-Transduced Hematopoietic Progenitors
  IdHP Cells Are Multipotent, Maintaining T, B, and Myeloid Lineage Potentials
  IdHP Cells Are Multipotent at a Clonal Level
  Generation of IdHP Cells from Mouse BM
  Generation of Inducible IdHP Cells Using ID3-ER Retrovirus
  Generation of IdHP Cells from Human Cord Blood Hematopoietic Progenitors
Experimental Procedures
  Growth Factors
  Isolation of Hematopoietic Progenitors
  Retroviral Constructs, Viral Supernatants, and Transduction
  In Vitro Differentiation Culture System
  Cloning of mIdHP Cells
  Colony-Forming Unit in Culture Assay
  Cell Cycle Assay
  Adoptive Transfer of mIdHP and hIdHP Cells
  PCR Analysis of Igh Gene Rearrangement
  RNA Extraction and qRT-PCR
  Microarray Analysis
Author Contributions
Supplemental Information

Generation of ID3-Transduced Hematopoietic Progenitors

IdHP Cells Are Multipotent, Maintaining T, B, and Myeloid Lineage Potentials

IdHP Cells Are Multipotent at a Clonal Level

Generation of IdHP Cells from Mouse BM

Generation of Inducible IdHP Cells Using ID3-ER Retrovirus

Generation of IdHP Cells from Human Cord Blood Hematopoietic Progenitors

Thumbnail image of Figure 1. Opens large image


Identification of cellular and molecular events regulating self-renewal or differentiation of the cells is a fundamental issue in the stem cell biology or developmental biology field. In the present study, we revealed that the simple inhibition of differentiation in HSCs or HPCs by overexpressing ID proteins and culturing them in suitable conditions induced the self-renewal of hematopoietic progenitors and allowed the extensive expansion of the multipotent cells. The reduction of ID proteins in MPPs resulted in the differentiation of the cells into lymphoid and myeloid lineage cells. Thus, it is possible to manipulate the cell fate by regulating E-protein or ID-protein activities. This inducible system will be a useful tool to figure out the genetic and epigenetic program controlling the self-renewal activity of multipotent stem cells.

Previous studies have shown that hematopoietic progenitors deficient for E2A, EBF1, and PAX5 maintain multilineage differentiation potential without losing their self-renewing capacity (Ikawa et al., 2004a, Nutt et al., 1999, Pongubala et al., 2008), indicating that the inhibition of the differentiation pathway at certain developmental stages leads to the expansion of multipotent stem cells. However, the MPPs were not able to differentiate into B cells because they lacked the activities of transcription factors essential for the initiation of the B lineage program. In addition, a restriction point regulating the lineage-specific patterns of gene expression during B cell specification remained to be determined because of the lack of an inducible system that regulates B cell differentiation. Here we have established the multipotent iLS cells using ID3-ER retrovirus, which can be maintained and differentiated into B cells in an inducible manner by simply adding or withdrawing 4-OHT. The data indicated the essential role of E2A for initiation of the B cell program that restricts other lineage potentials, because the depletion of 4-OHT from the culture immediately leads to the activation of E proteins, such as E2A, HEB, and E2-2, that promote B cell differentiation. This strategy is useful in understanding the cues regulating the self-renewal or differentiation of uncommitted progenitors to the B cell pathway. Analysis of genome-wide gene expression patterns and histone modifications will determine the exact mechanisms that underlie the B cell commitment process.

The iLS cells can also be generated from human CB hematopoietic progenitors. Human iLS cells exhibited differentiation potential and self-renewal activity similar to those of murine iLS cells, suggesting a similar developmental program during human B cell fate specification. Our data are consistent with a study demonstrating the critical role of the activity of ID and E proteins for controlling the status of human HSCs and progenitors (van Galen et al., 2014). This study reported that the overexpression of ID2 in human CB HSCs enhanced the myeloid and stem cell program at the expense of lymphoid commitment. Specifically, ID2 overexpression resulted in a 10-fold expansion of HSCs, suggesting that the inhibition of E-protein activities promotes the self-renewal of HSCs by antagonizing the differentiation. This raises a question about the functional differences between ID2 and ID3. Id3 seems to suppress the B cell program and promote the myeloid program more efficiently than does ID2, because the ID2-expressing HPCs appear to retain more B cell potential than ID3-expressing iLS cells (Mercer et al., 2011, van Galen et al., 2014). The self-renewal activity and differentiation potential of ID2-HPCs derived from murine HSCs in the BM seemed to be limited both in vivo and in vitro analysis (Mercer et al., 2011). In our study, the iLS cells retained more myeloid potential and self-renewal capacity during the culture. Strikingly, the multipotent iLS cells enormously proliferated (>103-fold in 1 month) as long as the cells were cultured in undifferentiated conditions. This could be due to the functional differences among Id family genes. Alternatively, combination with additional environmental signals, such as cytokines or chemokines, may affect the functional differences of ID proteins, although any ID proteins can repress the activation of the E2A targets, such as Ebf1 and Foxo1, that are essential for B cell differentiation. ID1 and ID3, but not ID2, are demonstrated to be negative regulators of the generation of hematopoietic progenitors from human pluripotent stem cells (Hong et al., 2011). Further analysis is required to determine the physiological role of ID proteins in regulating hematopoietic cell fate. It also remains to be determined whether the ID3-ER system can be applied to human progenitors. It would be informative to compare the regulatory networks that control B cell differentiation in mouse and human.

Immune cell therapy has become a major field of interest in the last two decades. However, the required high cell numbers restrain the application and success of immune reconstitution or anti-cancer treatment. For example, DCs are already being used in cell therapy against tumors. One of the major limitations of DC vaccine therapy is the difficulty in obtaining sufficient cell numbers, because DCs do not proliferate in the currently used systems. The method of making iLS cells could be applied to such cell therapies. Taken together, the simplicity of this method and the high expansion rate and retention of multilineage potential of the cells make this cell source appealing for regenerative medicine or immune cell therapy.

In summary, we showed that an artificially induced block of differentiation in uncommitted progenitors is sufficient to produce multipotent stem cells that retain self-renewal activity. Once the differentiation block is released, the cells start differentiating into mature cells both in vivo and in vitro. Thus, this method could be applicable for establishing somatic stem cells from other organs in a similar manner, which would be quite useful for regenerative medicine. The relative ease of making stem cells leads us to conceive that a block in differentiation is essential not only in other types of artificially engineered stem cells, such as ES cells and iPS cells, but also in any type of physiological somatic stem cell. In this context, it is tempting to speculate that it could have been easy for a multicellular organism to establish somatic stem cells by this mechanism during evolution.

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Cancer Companion Diagnostics

Curator: Larry H. Bernstein, MD, FCAP


Companion Diagnostics for Cancer: Will NGS Play a Role?

Patricia Fitzpatrick Dimond, Ph.D.

Companion diagnostics (CDx), in vitro diagnostic devices or imaging tools that provide information essential to the safe and effective use of a corresponding therapeutic product, have become indispensable tools for oncologists.  As a result, analysts expect the global CDx market to reach $8.73 billion by 2019, up from from $3.14 billion in 2014.

Use of CDx during a clinical trial to guide therapy can improve treatment responses and patient outcomes by identifying and predicting patient subpopulations most likely to respond to a given treatment.

These tests not only indicate the presence of a molecular target, but can also reveal the off-target effects of a therapeutic, predicting toxicities and adverse effects associated with a drug.

For pharma manufacturers, using CDx during drug development improves the success rate of drugs being tested in clinical trials. In a study estimating the risk of clinical trial failure during non-small cell lung cancer drug development in the period between 1998 and 2012 investigators analyzed trial data from 676 clinical trials with 199 unique drug compounds.

The data showed that Phase III trial failure proved the biggest obstacle to drug approval, with an overall success rate of only 28%. But in biomarker-guided trials, the success rate reached 62%. The investigators concluded from their data analysis that the use of a CDx assay during Phase III drug development substantially improves a drug’s chances of clinical success.

The Regulatory Perspective

According to Patricia Keegen, M.D., supervisory medical officer in the FDA’s Division of Oncology Products II, the agency requires a companion diagnostic test if a new drug works on a specific genetic or biological target that is present in some, but not all, patients with a certain cancer or disease. The test identifies individuals who would benefit from the treatment, and may identify patients who would not benefit but could also be harmed by use of a certain drug for treatment of their disease. The agency classifies companion diagnosis as Class III devices, a class of devices requiring the most stringent approval for medical devices by the FDA, a Premarket Approval Application (PMA).

On August 6, 2014, the FDA finalized its long-awaited “Guidance for Industry and FDA Staff: In Vitro Companion Diagnostic Devices,” originally issued in July 2011. The final guidance stipulates that FDA generally will not approve any therapeutic product that requires an IVD companion diagnostic device for its safe and effective use before the IVD companion diagnostic device is approved or cleared for that indication.

Close collaboration between drug developers and diagnostics companies has been a key driver in recent simultaneous pharmaceutical-CDx FDA approvals, and partnerships between in vitro diagnostics (IVD) companies have proliferated as a result.  Major test developers include Roche Diagnostics, Abbott Laboratories, Agilent Technologies, QIAGEN), Thermo Fisher Scientific, and Myriad Genetics.

But an NGS-based test has yet to make it to market as a CDx for cancer.  All approved tests include PCR–based tests, immunohistochemistry, and in situ hybridization technology.  And despite the very recent decision by the FDA to grant marketing authorization for Illumina’s MiSeqDx instrument platform for screening and diagnosis of cystic fibrosis, “There still seems to be a number of challenges that must be overcome before we see NGS for targeted cancer drugs,” commented Jan Trøst Jørgensen, a consultant to DAKO, commenting on presentations at the European Symposium of Biopathology in June 2013.

Illumina received premarket clearance from the FDA for its MiSeqDx system, two cystic fibrosis assays, and a library prep kit that enables laboratories to develop their own diagnostic test. The designation marked the first time a next-generation sequencing system received FDA premarket clearance. The FDA reviewed the Illumina MiSeqDx instrument platform through its de novo classification process, a regulatory pathway for some novel low-to-moderate risk medical devices that are not substantially equivalent to an already legally marketed device.

Dr. Jørgensen further noted that “We are slowly moving away from the ‘one biomarker: one drug’ scenario, which has characterized the first decades of targeted cancer drug development, toward a more integrated approach with multiple biomarkers and drugs. This ‘new paradigm’ will likely pave the way for the introduction of multiplexing strategies in the clinic using gene expression arrays and next-generation sequencing.”

The future of CDxs therefore may be heading in the same direction as cancer therapy, aimed at staying ahead of the tumor drug resistance curve, and acknowledging the reality of the shifting genomic landscape of individual tumors. In some cases, NGS will be applied to diseases for which a non-sequencing CDx has already been approved.

Illumina believes that NGS presents an ideal solution to transforming the tumor profiling paradigm from a series of single gene tests to a multi-analyte approach to delivering precision oncology. Mya Thomae, Illumina’s vice president, regulatory affairs, said in a statement that Illumina has formed partnerships with several drug companies to develop a universal next-generation sequencing-based oncology test system. The collaborations with AstraZeneca, Janssen, Sanofi, and Merck-Serono, announced in 2014 and 2015 respectively, seek to  “redefine companion diagnostics for oncology  focused on developing a system for use in targeted therapy clinical trials with a goal of developing and commercializing a multigene panel for therapeutic selection.”

On January 16, 2014 Illumina and Amgen announced that they would collaborate on the development of a next-generation sequencing-based companion diagnostic for colorectal cancer antibody Vectibix (panitumumab). Illumina will develop the companion test on its MiSeqDx instrument.

In 2012, the agency approved Qiagen’s Therascreen KRAS RGQ PCR Kit to identify best responders to Erbitux (cetuximab), another antibody drug in the same class as Vectibix. The label for Vectibix, an EGFR-inhibiting monoclonal antibody, restricts the use of the drug for those metastatic colorectal cancer patients who harbor KRAS mutations or whose KRAS status is unknown.

The U.S. FDA, Illumina said, hasn’t yet approved a companion diagnostic that gauges KRAS mutation status specifically in those considering treatment with Vectibix.  Illumina plans to gain regulatory approval in the U.S. and in Europe for an NGS-based companion test that can identify patients’ RAS mutation status. Illumina and Amgen will validate the test platform and Illumina will commercialize the test.

Treatment Options

Foundation Medicine says its approach to cancer genomic characterization will help physicians reveal the alterations driving the growth of a patient’s cancer and identify targeted treatment options that may not have been otherwise considered.

FoundationOne, the first clinical product from Foundation Medicine, interrogates the entire coding sequence of 315 cancer-related genes plus select introns from 28 genes often rearranged or altered in solid tumor cancers.  Based on current scientific and clinical literature, these genes are known to be somatically altered in solid cancers.

These genes, the company says, are sequenced at great depth to identify the relevant, actionable somatic alterations, including single base pair change, insertions, deletions, copy number alterations, and selected fusions. The resultant fully informative genomic profile complements traditional cancer treatment decision tools and often expands treatment options by matching each patient with targeted therapies and clinical trials relevant to the molecular changes in their tumors.

As Foundation Medicine’ s NGS analyses are increasingly applied, recent clinical reports describe instances in which comprehensive genomic profiling with the FoundationOne NGS-based assay result in diagnostic reclassification that can lead to targeted drug therapy with a resulting dramatic clinical response. In several reported instances, NGS found, among the spectrum of aberrations that occur in tumors, changes unlikely to have been discovered by other means, and clearly outside the range of a conventional CDx that matches one drug to a specific genetic change.

TRK Fusion Cancer

In July 2015, the University of Colorado Cancer Center and Loxo Oncology published a research brief in the online edition of Cancer Discovery describing the first patient with a tropomyosin receptor kinase (TRK) fusion cancer enrolled in a LOXO-101 Phase I trial. LOXO-101 is an orally administered inhibitor of the TRK kinase and is highly selective only for the TRK family of receptors.

While the authors say TRK fusions occur rarely, they occur in a diverse spectrum of tumor histologies. The research brief described a patient with advanced soft tissue sarcoma widely metastatic to the lungs. The patient’s physician submitted a tumor specimen to Foundation Medicine for comprehensive genomic profiling with FoundationOne Heme, where her cancer was demonstrated to harbor a TRK gene fusion.

Following multiple unsuccessful courses of treatment, the patient was enrolled in the Phase I trial of LOXO-101 in March 2015. After four months of treatment, CT scans demonstrated almost complete tumor disappearance of the largest tumors.

The FDA’s Elizabeth Mansfield, Ph.D., director, personalized medicine staff, Office of In Vitro Diagnostics and Radiological Health, said in a recent article,  “FDA Perspective on Companion Diagnostics: An Evolving Paradigm” that “even as it seems that many questions about co-development have been resolved, the rapid accumulation of new knowledge about tumor biology and the rapid evolution of diagnostic technology are challenging FDA to continually redefine its thinking on companion diagnostics.” It seems almost inevitable that a consolidation of diagnostic testing should take place, to enable a single test or a few tests to garner all the necessary information for therapeutic decision making.”

Whether this means CDx testing will begin to incorporate NGS sequencing remains to be seen.

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 Update on Chronic Myeloid Leukemia

Curator: Larry H Bernstein, MD, FCAP


Diagnosis and Treatment of Chronic Myeloid Leukemia in 2015
Philip A. Thompson, Hagop M. Kantarjian, Jorge E. Cortes,
Department of Leukemia, The University of Texas, MD Anderson Cancer Center, Houston

Target Audience: The target audience for Mayo Clinic Proceedings is primarily internal medicine physicians and other clinicians who wish to advance their current knowledge of clinical medicine and who wish to stay abreast of advances in medical research.
Statement of Need: General internists and primary care physicians must maintain an extensive knowledge base on a wide variety of topics covering all body systems as well as common and uncommon disorders. Mayo Clinic Proceedings aims to leverage the expertise of its authors to help physicians understand best practices in diagnosis and management of conditions encountered in the clinical setting.
Learning Objectives: On completion of this article, you should be able to (1) identify clinical and laboratory features consistent with a diagnosis of chronic myeloid leukemia (CML) and diagnose the disease based on laboratory testing; (2) identify factors associated with poor treatment outcomes and how these are identified and managed; and (3) describe standard first and second line therapeutic options for CML and their adverse effects.
In their editorial and administrative roles, William L. Lanier, Jr, MD, Terry L. Jopke, Kimberly D. Sankey, and Nicki M. Smith, MPA, have control of the content of this program but have no relevant financial relationship(s) with industry.
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Article Outline
I. Clinical Features
A. Presenting Hematologic Parameters
II. Differential Diagnosis
A. Chronic Myelomonocytic Leukemia
B. Atypical CML
C. Chronic Neutrophilic Leukemia
D. Essential Thrombocythemia
E. Diagnostic Workup
III. Determining Prognosis in CP-CML at Baseline
IV. Response Definitions
V. Routine Monitoring Schedule
VI. Initial Treatment of CP-CML
A. Which TKI and Dose?
VII. Treatment Objectives
A. Achievement of CCyR and MMR/MR3.0
B. Definitions of Treatment Failure
VIII. Treatment of Patients With Primary or Secondary Treatment Failure Who Remain in CP
A. Switching to a Second TKI
IX. Stopping Treatment in Patients With Prolonged CMR
X. Treatment of AP-CML
XI. Treatment of BP-CML
A. Treatment of LBP
B. Treatment of MBP
C. Which TKI Should Be Used in BP-CML?
D. Treatment of Refractory and Relapsed BP
XII. Conclusion
XIII. References

Few neoplastic diseases have undergone a transformation in a relatively short period like chronic myeloid leukemia (CML) has in the last few years. In 1960, CML was the first cancer in which a unique chromosomal abnormality was identified and a pathophysiologic correlation suggested. Landmark work followed, recognizing the underlying translocation between chromosomes 9 and 22 that gave rise to this abnormality and, shortly afterward, the specific genes involved and the pathophysiologic implications of this novel rearrangement. Fast forward a few years and this knowledge has given us the most remarkable example of a specific therapy that targets the dysregulated kinase activity represented by this molecular change. The broad use of tyrosine kinase inhibitors has resulted in an improvement in the overall survival to the point where the life expectancy of patients today is nearly equal to that of the general population. Still, there are challenges and unanswered questions that define the reasons why the progress still escapes many patients, and the details that separate patients from ultimate cure. In this article, we review our current understanding of CML in 2015, present recommendations for optimal management, and discuss the unanswered questions and what could be done to answer them in the near future.
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, characterized by the unrestrained expansion of pluripotent bone marrow stem cells.1 The hallmark of the disease is the presence of a reciprocal t(9;22)(q34;q11.2), resulting in a derivative 9q+ and a small 22q−. The latter, known as the Philadelphia (Ph) chromosome, results in a BCR-ABL fusion gene and production of a BCR-ABL fusion protein2; BCR-ABL has constitutive tyrosine kinase activity3 and is necessary and sufficient for production of the disease.4 In a few cases (5%-10%), the Ph chromosome is cytogenetically cryptic, often due to a complex translocation, and the diagnosis requires fluorescence in situ hybridization (FISH) to reveal the BCR-ABL fusion gene or polymerase chain reaction (PCR) to reveal the BCR-ABL messenger RNA transcript.5 A 210-kDa BCR-ABL transcript (p210) transcribed from the most common rearrangements between exons 13 or 14 of BCR and exon 2 of ABL (known as e13a2 [or b2a2] and e14a2 [or b3a2], respectively) is most common, but rare cases will have alternative BCR-ABL breakpoints, leading to a p190 transcript (from the e1a2 rearrangement, most typically seen in Ph-positive acute lymphoblastic leukemia [ALL]) or a p230 transcript.5 Indication of the typical hematopathologic features and the t(9;22)(q34;q11.2) by conventional cytogenetics or FISH and/or BCR-ABL by PCR is required for diagnosis.5

Clinical Features
Up to 50% of patients are asymptomatic and have their disease diagnosed incidentally after routine laboratory evaluation.6 Clinical features, when present, are generally nonspecific. Splenomegaly is present in 46% to 76%6, 7 and may cause left upper quadrant pain or early satiety, fatigue, night sweats, symptoms of anemia, and bleeding due to platelet dysfunction may occur, the last occurring most commonly in patients with marked thrombocytosis. Less than 5% of patients present with symptoms of hyperviscosity, including priapism, which are generally seen when the presenting white cell count exceeds 250,000/μL.7
The disease is classically staged into chronic phase (CP, most patients at presentation), accelerated phase (AP), and blast phase (BP).5 Many definitions have been used for these stages, but all the data generated from the tyrosine kinase inhibitor (TKI) studies have used the historically standard definition in which AP is defined by the presence of one or more of the following: 15% or more blasts in peripheral blood and bone marrow, 20% or more basophils in peripheral blood, and platelet counts less than 100,000/μL unrelated to treatment or the development of cytogenetic evolution. The blast phase is defined by the presence of 30% or more blasts in the peripheral blood or bone marrow, the presence of clusters of blasts in marrow, or the presence of extramedullary disease with immature cells (ie, a myeloid sarcoma).8 Progression to BP occurs at a median of 3 to 5 years from diagnosis in untreated patients, with or without an intervening identifiable AP.6

Presenting Hematologic Parameters
Characteristic complete blood cell count features are as follows: absolute leukocytosis (median leukocyte count of 100,000/μL) with a left shift and classic “myelocyte bulge” (more myelocytes than the more mature metamyelocytes seen on the blood smear); usual blast counts of less than 2%; nearly universal absolute basophilia, with absolute eosinophilia in 90% of cases5; monocytosis but generally not an increased monocyte percentage; absolute monocytosis in the unusual cases with a p190 BCR-ABL9; normal or elevated platelet count; and thrombocytopenia, which suggests an alternative diagnosis or the presence of AP, rather than CP, disease.

Differential Diagnosis
The differential diagnosis for chronic phase CML (CP-CML) includes the following Ph-negative conditions.

Chronic Myelomonocytic Leukemia
Chronic myelomonocytic leukemia is a myelodysplastic/myeloproliferative neoplasm that can be distinguished from CML by the presence of dysplastic features, more prominent cytopenias, more prominent monocytosis, and lack of basophilia. Chronic myelomonocytic leukemia is Ph negative and may have other cytogenetic abnormalities.5

Atypical CML
Atypical CML is a Ph-negative myelodysplastic/myeloproliferative neoplasm.

Chronic Neutrophilic Leukemia
Rare cases of CML with a p230 BCR-ABL transcript may be mistaken for chronic neutrophil leukemia (CNL) because of the predominant neutrophilia associated with this version of CML, but cytogenetics revealing the Ph chromosome will easily distinguish them. Importantly, this and other atypical rearrangements might not be detected by some standard PCR methods. The presence of these abnormalities should be suspected in instances where the Ph chromosome is detected by routine karyotype but with PCR “negative” for BCR-ABL, hence the importance of cytogenetic evaluation in all patients at baseline.

Essential Thrombocythemia
Rare cases of CML may present with isolated thrombocytosis, without leukocytosis. Basophilia is often present as a diagnostic clue. These cases will be distinguished by cytogenetics and molecular studies revealing Ph positivity and BCR-ABL positivity.10

Diagnostic Workup
The diagnosis will usually be suspected from the complete blood cell count and blood smear. FISH for t(9;22)(q34;q11.2) and quantitative reverse transcriptase PCR (qRT-PCR) for BCR-ABL can be performed on peripheral blood. However, bone marrow aspirate and unilateral biopsy with conventional cytogenetics and flow cytometry are essential at the time of diagnosis to exclude unrecognized advanced-stage disease and to detect rare cases with an alternative BCR-ABL transcript not detected by routine BCR-ABL PCR. Flow cytometry will identify cases with unrecognized progression to lymphoid blast crisis by their phenotypic features, whereas conventional karyotyping may identify additional cytogenetic abnormalities (cytogenetic clonal evolution).

Determining Prognosis in CP-CML at Baseline
The prognosis of CP-CML has markedly improved since the development of TKIs. The stage of disease is the most important prognostic feature. Most patients presenting with CP-CML achieve long-term control, and stem cell transplant is only needed in a few. Several prognostic scoring systems have been developed to assess the risk of poor outcome at presentation: the Sokal score11 and Hasford score were developed in the pre-imatinib era12 but retain prognostic significance in imatinib-treated patients. An online calculator is available to compute both these scores at Approximately 25% of high-risk patients fail to achieve complete cytogenetic response (CCyR) with imatinib-based treatment by 18 months; this and other important therapeutic milestones are discussed in detail subsequently. A simpler system based on basophil percentage in peripheral blood and spleen size, the European Treatment and Outcome Study (EUTOS) system, found that 34% of high-risk patients fail to achieve CCyR by 18 months.13Notwithstanding the appeal of the simplicity of the EUTOS score, its predictive value has not been universally confirmed.14, 15 The prognostic relevance of these classifications is ameliorated but not completely eliminated among patients treated with second-generation TKIs. Currently, we do not make treatment decisions based solely on these risk scores.
Other proposed pretreatment predictors include the level of CML cell membrane expression of the organic cation transporter-1 (OCT-1). OCT-1 is required for entry of imatinib into the cell; this protein (and its corresponding RNA) can be measured, and higher levels of expression and/or activity are associated with superior survival in imatinib-treated patients.16 Importantly, patients with lesser OCT-1 activity may benefit more from higher starting doses of imatinib.16 OCT-1 activity is not important for nilotinib-17 or dasatinib-treated patients because these drugs are not OCT-1 substrates.18

Response Definitions
Dynamic response assessment is essential to identify patients at high risk of disease progression, who may benefit from a change of therapy. Response definitions are given in Table 1.19 A complete hematologic response (CHR) is defined by clinical and peripheral blood criteria. Cytogenetic response is classified according to the percentage of Ph-positive metaphases by routine karyotype on bone marrow aspiration. A CCyR has also been defined in some instances by interphase FISH on peripheral blood as the absence of detectable BCR-ABL fusion in at least 200 examined nuclei.20 Molecular testing for BCR-ABL transcripts using qRT-PCR is more sensitive for low-level residual disease than cytogenetics or FISH (sensitivity of 10−4 to 10−5). Levels of response in this qRT-PCR assay are clinically relevant and reported as a percentage of the transcript levels of a normal housekeeper gene, such as ABL1 or BCR. Given that assays used by different laboratories have significantly different sensitivities, attempts have been made to harmonize reporting by developing the international scale. By parallel testing of samples with a reference laboratory, laboratory-specific conversion factors are produced to correct for differing sensitivities and allow a laboratory to report BCR-ABL transcript levels in a more uniform way.21 A World Health Organization standard material has also been developed for assay calibration.22 Major molecular response (MMR or MR3.0) corresponds to less than 0.1% BCR-ABL on the international scale, which represents a 3-log reduction from the standardized baseline rather than a 3-log reduction from the individual patient’s baseline BCR-ABL transcript level (which can vary significantly).23 MR4.0is less than 0.01% bcr-abl on the international scale, and MR4.5 is 0.0032% or less on the international squale (equivalent to a ≥4.5-log reduction), which is the limit of sensitivity of many assays. There is also a fair correlation between transcript levels and depth of cytogenetic response such that transcript levels of 1% on the international scale are grossly equivalent to a CCyR.
Table 1Definitions of Response
Response Definition
CHR Leukocyte count <10 × 109/L, basophils <5%, platelets <450 × 109/L, the absence of immature granulocytes, impalpable spleen
Minor CyR 36%-95% Ph+ metaphases in bone marrow
Major CyR 1%-35% Ph+ metaphases in bone marrow
CCyR 0% Ph+ metaphases in bone marrow
MMR BCR-ABL International Scale ≤0.1%
CMR Undetectable BCR-ABL with assay sensitivity ≥4.5 or 5.0 logs
CHR = complete hematologic response; CMR = complete molecular response; CyR = cytogenetic response; MMR = major molecular response; Ph = Philadelphia chromosome.

Routine Monitoring Schedule
Different monitoring schedules have been proposed, with the aim of early identification of patients who are not achieving therapeutic milestones and are therefore at higher risk of disease progression. Our own practice is to monitor the complete blood cell count every 1 to 2 weeks for the first 2 to 3 months to identify treatment-related cytopenias and the achievement of complete hematologic response. Most instances of grade 3 to 4 myelosuppression occur in the first few months. Thus, once the peripheral blood cell counts become stable, monitoring with complete blood cell count can be reduced to every 4 to 6 weeks and eventually every 3 to 6 months. In addition, BCR-ABL qRT-PCR is performed from peripheral blood every 3 months until the achievement of MMR then every 6 months thereafter. We perform a bone marrow aspiration with cytogenetics every 6 months until achievement of stable CCyR. This allows not only for the confirmation of CCyR but also for the discovery of chromosomal abnormalities in the emerging Ph-negative metaphases, a phenomenon that occurs in 10% to 15% of patients and may be associated with eventual development of myelodysplastic syndrome or acute myeloid leukemia.24, 25, 26 Subsequently, bone marrow examination only need be performed in the following circumstances: failure to achieve therapeutic milestones; evaluation of a significant, unexplained increase in BCR-ABL after initial response, not attributable to lack of adherence (see later sections for evaluation of suboptimal response or loss of response); to monitor known chromosomal abnormalities in Ph-negative metaphases; and to investigate unexplained cytopenia(s).

Initial Treatment of CP-CML
The TKIs have transformed outcomes in CML. The pivotal International Randomized Study of Interferon and STI571 (IRIS) study found far superior rates of CHR, CCyR, and MMR in imatinib- compared with interferon-treated patients and a superior progression-free survival (PFS).27, 28 Before IRIS, other than interferon-based therapy, allogeneic stem cell transplant (alloSCT) was the treatment of choice for eligible patients and achieved long-term disease-free survival (DFS) in approximately 50% to 85% of patients29, 30, 31, 32, 33 due to a graft-vs-leukemia effect.34 AlloSCT is associated with a unique toxicity profile, particularly opportunistic infections and graft-vs-host disease, resulting in treatment-related mortality of 5% to 20% and significant morbidity in many long-term survivors. Combined with the marked success of TKIs, alloSCT is now reserved for patients with advanced-stage disease or treatment failure; this is discussed in more detail in later sections.

Which TKI and Dose?
Three TKIs are now approved by the Food and Drug Administration for initial treatment of CP-CML: imatinib, nilotinib and dasatinib. Debate continues regarding the optimal initial TKI and dose, with compelling arguments supporting each. A number of studies have attempted to improve on results achieved with 400 mg/d of imatinib.
Shortly after imatinib was introduced as frontline therapy for CML, studies focused on use of higher doses to improve outcome.35 The single-arm TIDEL (Therapeutic Intensification in De Novo Leukemia) study, in which patients were treated with 600 mg/d of imatinib, found superior rates of MMR at 12 and 24 months in those patients able to maintain a daily mean of 600 mg of imatinib for the first 6 months36; in our experience, 400 mg of imatinib twice daily was associated with superior cumulative rates of CCyR and MMR relative to a historical control cohort and was generally well tolerated, with 82% of patients continuing to take at least 600 mg/d.37, 38In a confirmatory randomized study, the German CML study group reported that an initial imatinib dose of 800 mg was associated with higher rates of MMR at 12 months than 400 mg of imatinib or 400 mg of imatinib plus interferon alfa (59% vs 44% vs 46%). The mean daily dose tolerated in the group assigned to 800 mg of imatinib was 628 mg because of the higher adverse event profile of higher doses.39 The higher initial dose was also associated with more rapid achievement of MR4.5. There was, however, no event-free survival (EFS) or survival benefit, relative to 400 mg/d of imatinib.
Combinations of imatinib and interferon have been reported in several randomized trials, with mixed results. The French SPIRIT (STI571 Prospective International Randomized Trial) study and a Nordic group found higher rates of MMR at 12 months for patients receiving imatinib plus pegylated interferon alfa 2a or pegylated interferon alfa 2b, respectively, but no difference in CCyR.40, 41 In contrast, the German CML study group found no difference in MMR at 12 months between 400 mg of imatinib with or without nonpegylated interferon alfa,39and there was no difference in the rates of CCyR or MMR when pegylated interferon alfa 2b was combined with 800 mg/d of imatinib compared with imatinib alone.42 All studies have found poor tolerability of interferons with high rates of discontinuation, and none have found PFS or survival benefit.
Ten years ago, the first studies using second-generation TKI as initial therapy for CML were initiated, which found very high rates of CCyR and MMR using first-line dasatinib, 100 mg/d or 50 mg twice daily,43 and nilotinib, 400 mg twice daily.44, 45 Two major company-sponsored randomized studies later confirmed these results, comparing second-generation TKIs to imatinib, 400 mg/d. The Evaluating Nilotinib Efficacy and Safety in Clinical Trials-Newly Diagnosed Patients study (ENEST-nd) compared imatinib, 400 mg, to nilotinib, 300 mg, twice daily and nilotinib, 400 mg, twice daily. Nilotinib at both doses was associated with more, faster, and deeper responses and higher freedom from progression. The 400-mg twice-daily dose was associated with a small, but statistically significant, improvement in overall survival (OS) compared with 400 mg of imatinib; however, the results were also notable for a significantly higher incidence of major arteriothrombotic events, including ischemic heart disease, cerebrovascular accidents, and peripheral arterial disease (especially at 400 mg twice daily).46 The Dasatinib versus Imatinib Study in Treatment-Naive CML Patients (DASASION) compared 400 mg/d of imatinib with 100 mg/d of dasatinib. More, faster, and deeper responses were seen, with fewer transformations to AP and BP, but at 5 years of follow-up (the end of the study), there was still no PFS or OS benefit.47 A frequent (although usually grade 1 or 2) adverse effect of dasatinib is the development of pleural effusions, which may require dose adjustments and occasionally thoracentesis. Of some concern, also, was the development of pulmonary hypertension, which was diagnosed in 8 patients by echocardiographic criteria; however, only one patient had right-heart catheterization, which did not confirm pulmonary hypertension.
Imatinib, 400 mg, has also been compared with bosutinib, 500 mg. Faster and deeper responses, with a higher rate of MMR (but not CCyR, the primary end point) at 12 months, were seen in the bosutinib group, leading to fewer transformations. There was a higher rate of treatment discontinuation in the bosutinib arm, particularly due to diarrhea and liver function test abnormalities.48 A second randomized trial, using a lower starting dose of bosutinib (400 mg/d), has been initiated, seeking regulatory approval for this indication.
Ponatinib is a highly potent TKI and is the only TKI with activity in patients with the T315I mutation in ABL1. Because of the high level of preclinical and clinical activity of ponatinib in the salvage setting,49 it was also investigated as frontline therapy. Both a single-arm, phase 2 study50 and a randomized phase 3 study were conducted, the latter comparing 400 mg of imatinib with 45 mg of ponatinib.51 Ponatinib treatment resulted in faster and deeper responses, including very high rates of early MR4.5. Both studies reported a 3-month rate of BCR-ABL/ABL less than 10% of 94%, the highest of any study with TKI. Unfortunately, the high rate of major arterial thrombotic events (7% in the ponatinib arm vs 1% in the imatinib arm) and pancreatitis led to the 2 studies being terminated early at a median follow-up of 23 and 5.1 months, respectively.51
We believe that imatinib, dasatinib, and nilotinib all constitute adequate treatment options for patients with CML at the time of diagnosis. Outside clinical trials, the decision regarding which TKI to use should be tailored to an individual patient and depends on an assessment of factors such as the relative risk of the disease, risk factors for specific adverse events (eg, arteriothrombotic events, pleural effusion, pulmonary hypertension, poorly controlled diabetes, and pancreatitis), possible effect of the dose administration schedule, and cost. In patients with a poorer likelihood of responding to 400 mg of imatinib (eg, those with high Sokal scores or those with e1a2 CML), a second-generation TKI might be preferred. Patients with low OCT-1 activity may also benefit from high-dose imatinib or a second-generation TKI, but this test is not clinically available in the United States. In contrast, in patients with lower-risk disease or those with a higher risk for arteriothrombotic events, imatinib might be preferred. Higher doses of imatinib might offer similar efficacy benefits as dasatinib or nilotinib (eg, similar rates of early responses and transformation to AP and BP).50 Although higher-dose imatinib is associated with increased incidence of some adverse events, these usually consist of peripheral edema, muscle cramps, and gastrointestinal toxicity, but not arteriothrombotic events. Specific agents may be avoided because of their particular toxicity profiles; for example, it may be preferable not to use nilotinib in a patient with a history of coronary artery disease or with several coronary risk factors, and dasatinib may be avoided in patients who have tenuous respiratory function because of the risk of pleural effusions. Increasingly, pharmacoeconomic concerns may drive therapeutic decision making; generic imatinib will soon be available, and there will be a substantial cost differential between imatinib and second-generation TKIs, which no doubt will be a factor in the decision-making process.

Treatment Objectives
Response definitions according to hematologic, cytogenetic, and molecular criteria are given in Table 1.19, 52 It is important to remember that different laboratories have different BCR-ABL qRT-PCR sensitivity, and quantitative results may differ markedly.53 If the laboratory does not report results on the international scale, BCR-ABL should be monitored in the same laboratory for consistency. In addition, MMR cannot be adequately identified if a laboratory does not report on the international scale, increasing the importance of cytogenetic analysis for response assessment.

Achievement of CCyR and MMR/MR3.0
The European LeukemiaNet (ELN) 2013 guidelines (Table 2) place a strong emphasis on the importance of achieving MMR, ideally by the 12-month time point. This is achieved by 1 year in 18% to 58% of patients taking 400 mg of imatinib and 43% to 77% taking 600 to 800 mg.19 This is based on data from long-term follow-up of IRIS,54 which found that, of patients in MMR at 18 months, only 3% lost CCyR, compared with 26% of patients with BCR-ABL levels of greater than 0.1% to less than 1.0%. The key transcript levels at the 6-, 12-, and 18-month landmarks found to be associated with favorable EFS were 10% or less, 1% or less, and 0.1% or less, respectively.54, 55 Despite the importance of achieving MMR with imatinib, however, there are no data to indicate that switching therapy in a patient in the ELN warning (formerly suboptimal response) category improves outcome.56 In addition, our own data suggest that achievement of MMR offers no EFS or survival advantage over the achievement of CCyR by 12 or 18 months during frontline treatment with second-generation TKIs; achievement of CCyR by 3 months should be considered optimal response in this setting, with PCyR considered suboptimal.57 In a combined analysis of patients receiving either imatinib or second-generation TKIs, patients achieving CCyR by 6 months have a 97% 3-year EFS on landmark analysis, which was the major point of difference, and this did not differ according to subsequent achievement of MMR or not.58
Table 2European LeukemiaNet (ELN) Response Criteriaa
Optimal Warning Failure
Baseline NA High risk or CCA/Ph+, major route NA
3 mo BCR-ABL1 ≤10% and/or Ph+ ≤35% BCR-ABL1 >10% and/or Ph+ 36%-95% Non-CHR and/or Ph+ >95%
6 mo BCR-ABL1 <1% and/or Ph+ 0 BCR-ABL1 1%-0% and/or Ph+ 1%-35% BCR-ABL1 >10% and/or Ph+ >35%
12 mo BCR-ABL1 ≤0.1% (ie, MMR) BCR-ABL1 >0.1%-1% (ie, lack of MMR) BCR-ABL1 >1% and/or Ph+ >0% (ie, lack of CCyR)
Then and at any time BCR-ABL1 ≤0.1% CCA/Ph− (−7, or 7q−) Loss of CHR

Loss of CCyR

Confirmed loss of MMRb


aCCA/Ph+ = clonal cytogenetic abnormalities in Ph-positive cells; CCA/Ph− = clonal cytogenetic abnormalities in Ph-negative cells; CCyR = complete cytogenetic response; MMR = major molecular response; NA = not applicable; Ph = Philadelphia chromosome.
bIn 2 consecutive tests, at least one of which has BCR-ABL transcripts of 1% or greater.
Several studies38, 47, 55, 56, 59 also support achievement of BCR-ABL of 10% or less at 3 months as an important goal. Patients with this level of response at 3 months have an improved long-term outcome (EFS and OS) compared with those who have more than 10% transcripts. Although this has triggered recommendations for change in therapy for patients without this depth of response, no data suggest that the change in therapy alters the long-term outcome. Furthermore, even when those with slower responses have a worse outcome, the EFS at 5 years is approximately 80% in all series. Changing therapy for all represents an overreaction for most patients who will still have a favorable outcome. In fact, with additional assessment at 6 months, 30% to 50% will catch up in their response, and these patients have a similarly favorable outcome as if they had achieved the less than 10% BCR-ABL/ABL at 3 months.60 Adding more than one time point thus improves the prognostication abilities of early response. The rate of change of BCR-ABL transcripts in the first 3 months of therapy may also be important; patients with BCR-ABL greater than 10% at 3 months had superior outcomes if they had a halving time of less than 76 days.61 Patients who receive less than 80% of the target dose of imatinib, either because of dose reductions or because of missed doses, have a significantly lower probability of achieving the optimal response. Thus, at the moment, it is most prudent to minimize unnecessary treatment interruptions and dose reductions and to monitor patients carefully at early time points. No change in therapy is indicated until there is clear evidence of failure as defined by the ELN.

Definitions of Treatment Failure
Primary treatment failure can be defined as failure to achieve CHR and less than 95% Ph positive at 3 months, less than 10% BCR-ABL and Ph less than 35% at 6 months, or less than 1% BCR-ABL and CCyR at 12 months. This occurs in approximately 25% of imatinib-treated patients.19 Progression to AP and BP defines treatment failure at any point. Secondary treatment failure is loss of response after initially meeting treatment targets. Loss of response is defined as loss of CCyR, loss of CHR, or progression to AP and BP. Loss of response should not be defined on the basis of a single qRT-PCR result due to potential fluctuations inherent in testing method. Increasing molecular markers on 2 occasions should prompt further investigation.62, 63However, only 11% of patients in CCyR who have increasing molecular markers develop clinical events (loss of CCyR, loss of CHR, development of AP and BC), and switching TKIs has not been found to benefit patients with only molecular relapse but without loss of CCyR.64 Similarly, although ELN recommends the appearance of mutations to be considered treatment failure, it is not advised to investigate the presence of mutations unless there is clinical evidence of treatment failure. Furthermore, if a mutation were to be identified in a patient with adequate response, there is no evidence suggesting that change of therapy at that time improves outcome compared with change when clinical failure becomes evident, further supporting the recommendation to only test for mutations in instances of clinical failure.
Causes of treatment failure are diverse.65 Poor adherence is the most frequent cause of treatment failure and must be carefully evaluated. BCR-ABL mutations, which alter drug binding by directly altering an amino acid at the drug-binding site (eg, T315I, F317L, F359C/V) or indirectly by altering protein conformation (eg, G250E, Q252H, E255K/V), are crucial to identify because they determine sensitivity to salvage therapy and the subsequent choice of TKI. Other potential causes include pharmacokinetic interactions, such as accelerated TKI metabolism due to use of CYP3A4 hepatic enzyme inducers, or the use of proton pump inhibitors, which inhibit drug absorption. Diverse mechanisms may result in lower drug concentration within the cell despite adequate plasma levels, such as p-glycoprotein or ABCG2 drug efflux protein overexpression (affecting imatinib, nilotinib, and dasatinb), or low OCT1 activity, which is required for imatinib transportation into the cell (see earlier). Finally, overexpression of the Src kinase Lyn66 has been reported in some instances of resistance, but the incidence of this phenomenon is unknown.
Changes in BCR-ABL transcript levels may be associated with disease progression or development of resistance. However, identification of a sustained increase and an increasing trend are more important than a single increase, given the fluctuation that can occur in the assay results. In addition, the kinetics of change in BCR-ABL may vary according to the type of loss of response: patients with a rapid increase in BCR-ABL generally have disease progression to AP and BP or are nonadherent with therapy; in contrast, patients who have developed BCR-ABL1 mutations generally have a more gradual increase in BCR-ABL transcripts.63 An increase in BCR-ABL on a single occasion, particularly if the increase is greater than 5-fold or if MMR is lost, should prompt questioning regarding adherence to therapy and an early additional BCR-ABL qRT-PCR. If the rise is confirmed and adherence is not thought responsible, bone marrow aspiration should be performed to assess for the presence of disease progression, cytogenetic evolution, and BCR-ABL1 mutations. As mentioned previously, routine testing for mutations in patients with adequate response is not warranted. Even in the instance of suboptimal response or warning, mutations are identified in less than 5% of instances.

Treatment of Patients With Primary or Secondary Treatment Failure Who Remain in CP
Treatment of patients with refractory disease still in CP depends on several factors, particularly the type of initial therapy, the presence of BCR-ABL1 mutations, adherence, comorbidities, and eligibility for alloSCT.67 Patients who meet the definition of failure per the ELN have a shortened survival, with a median of approximately 5 years, and thus need a change in therapy.68 No randomized comparisons of switching to a second TKI compared with performing alloSCT exist, but our practice is to treat with at least a second TKI; patients are closely monitored. Although eligible patients for alloSCT should be considered for this approach if meeting failure criteria after a second TKI, in practice, most patients prefer to try a third TKI; still, a discussion about alloSCT should be held after initial failure.

Switching to a Second TKI
Six-year results of switching to dasatinib after imatinib failure or intolerance have been reported and reveal PFS and OS of 49% and 71% at 6 years, respectively. The CCyR rates were less than 50%, and the MMR rate was approximately 40% in long-term follow-up69; importantly, early responses (BCR-ABL <10% at 3 months) predicted longer-term outcomes. Comparable results have been reported with nilotinib, 400 mg twice daily, with the option to escalate to 600 mg twice daily, with a 4-year OS of 78%, PFS of 57%, and CCyR rate of 45%.70 Finally, bosutinib is active in imatinib-resistant patients, including all those with ABL1 mutations except T315I, at a dose of 500 mg/d; CCyR was achieved in 41% of patients with a 2-year PFS of 73% in imatinib-refractory patients and 95% in imatinib-intolerant patients. Bosutinib has an adverse effect profile that does not overlap substantially with the other TKIs, with the most frequent adverse events being diarrhea, rash, and biochemical liver function abnormalities.48 The drug is approved by the Food and Drug Administration for patients in whom at least one TKI has previously failed. Higher response rates to second-line TKI after imatinib failure are seen in patients with a low baseline Sokal risk score, greater depth of initial cytogenetic response with imatinib (particularly if CCyR was achieved), lack of recurrent neutropenia during imatinib therapy, and a good performance status.71, 72
Identification of specific BCR-ABL1 mutations is critical to subsequent TKI choice. Patients with a T315I mutation are resistant to all TKIs except ponatinib. Patients with the F317L mutation are resistant to dasatinib but responsive to nilotinib. Y253H, E255K/V, and F359V/C mutations are resistant to nilotinib but sensitive to dasatinib. There are no randomized studies to guide choice of subsequent TKI; however, changing to dasatinib is superior to increasing imatinib dose.73 Although the three second-generation TKIs have never been compared head to head, it appears that they have somewhat equivalent efficacy and can be selected based on known mutations, risk factors for toxicity, and schedule preferences. Still, despite the overall good results, less than 50% of patients achieve a CCyR with either of these drugs. Thus, better second-line treatment options are needed. In addition, for patients treated with second-generation TKI as frontline therapy, the results with any of these agents as second-line therapy are not known but are expected to be inferior to what is achieved when used after imatinib failure. Ponatinib is a logical candidate to fill this void, but unfortunately there is limited experience in this setting. Still, in instances of resistance to a second-generation TKI used as initial therapy, we usually select ponatinib as second line provided the patient does not have excessive risk factors for arteriothrombotic events. It is clear then that, despite the many good treatment options available in CML, new drugs or new approaches would still be welcome for the relatively small percentage of patients facing this clinical scenario.
Patients in whom 2 TKIs failed have more limited options, and treatment should be individualized. In the absence of BCR-ABL1 mutations predicted to produce resistance, nilotinib or dasatinib could be used, although there is limited, mostly retrospective, data with these agents. Bosutinib was prospectively investigated and is active in patients with failure of 2 previous TKIs, with a CCyR rate of 22% to 40% and 2-year PFS of 73%.74, 75 The PACE (Ponatinib Ph ALL and CML Evaluation) study found that 45 mg/d of ponatinib is highly active, achieving a 63% CCyR rate in a heavily pretreated population (>90% of patients had previously received at least 2 TKIs, and nearly 60% had previously received at least 3 TKIs). A subgroup analysis of the PACE study found equivalent efficacy for patients with the T315I mutation, who are resistant to all other TKIs.76Ponatinib has therefore been approved for patients with the T315I mutation or for whom no other TKI is indicated, under a risk evaluation and mitigation strategy, due to the risk of arterial thrombotic events.77Omacetaxine is a non-TKI protein synthesis inhibitor, given by subcutaneous injection for 14 days in a 28-day cycle, that is approved by the Food and Drug Administration for patients in whom 2 or more TKIs have failed.78In a phase 2 study in patients in whom 2 TKIs had previously failed, the rates of CHR, minor cytogenic response, and CCyR were 67%, 22%, and 4%, respectively79; in addition, the drug is active in patients with T315I mutation. In a separate phase 2 study, the rates of CHR and CCyR were 77% and 16%, respectively. However, PFS was only 7.7 months.80 The drug is associated with substantial myelosuppression.79, 80Although these results are more modest than those seen with TKIs, we use omacetaxine in instances where TKIs have failed or may not be indicated because of unacceptably high risk of specific adverse events.
AlloSCT should be considered for patients with CP-CML in whom 2 TKIs have failed. There are no data to guide the choice between third-line TKI or alloSCT, and this decision must therefore be individualized. However, the relatively low rates of CCyR and 2-year PFS with bosutinib and the risk of cardiovascular toxicity of ponatinib suggest that alloSCT should be considered in eligible patients; conversely, there are limited data on transplant outcomes in these heavily pretreated patients. A recent German CML study group study found that, provided they remain in CP, patients who undergo transplant after imatinib failure have excellent results post-alloSCT, with an 89% achievement of CMR after transplant, a treatment-related mortality of 6%, and a 3-year survival of 94%.81 Whether these impressive results can be replicated in patients who have experienced resistance to 2 or more TKIs remains to be seen.

Stopping Treatment in Patients With Prolonged CMR
Overall, 41% to 47% of patients who have been in continuous CMR for at least 24 months may remain with stable undetectable transcripts after ceasing imatinib.82, 83, 84 If recurrence up to the level of MMR is tolerated, the success rate increases to approximately 60%. Predictors of increased relapse likelihood in this setting include a high baseline Sokal risk score and a duration of imatinib therapy of less than 5 years.82 Continuous CMR for more than 64 months and treatment with a second-generation TKI may be associated with lower incidence of relapse after TKI cessation.84 Relapses occur most frequently within approximately 6 months; notably, most patients remain imatinib sensitive and regain CMR when use of the drug is recommenced.85However, the follow-up is still relatively short. Therefore, one needs to consider that late relapses after interferon therapy or alloSCT occurring more than 10 years after cessation of therapy may occur, and these are often in the lymphoid BP. Thus, continued monitoring is required, perhaps indefinitely, through peripheral blood PCR. Most patients who stop taking imatinib and maintain undetectable transcripts by standard qRT-PCR still have evidence of low-level disease when more sensitive, patient-specific DNA-based PCR assays are used.83 In addition, some patients have low-level fluctuation of BCR-ABL levels detected by standard RNA-based qRT-PCR, without experiencing true molecular relapse.85 The reasons for the lack of relapses in these patients are unclear, but it has been suggested that these patients may have an increased number of natural killer cells that may contribute to keeping the disease at bay.86
Although reported to be safe in relatively small numbers of patients in the clinical trial setting, this approach should only be undertaken in a clinical study or where a protocol for prospective, very close monitoring of patients is implemented to allow detection of early relapses and intervene promptly.

Treatment of AP-CML
Criteria for the diagnosis of AP-CML have been outlined earlier. ABL1 mutations increase in frequency in advanced-stage disease; mutational evaluation should therefore be performed and TKI choice based on this.62, 67 The optimal therapeutic approach in AP-CML differs according to whether the patient is TKI naive or has progressed from CP while taking a TKI. Eighty to ninety percent of treatment-naive patients will achieve CCyR with TKI87, 88 and have a similar EFS and OS to patients presenting in CP, particularly when treated with second-generation TKI. Those patients with cytogenetic clonal evolution as the only criterion for AP also have superior outcomes to those with hematologic and clinical features of AP.89 In contrast, much lower response rates and inferior EFS, with continued relapses, have been seen in studies of second-generation TKIs in patients with imatinib failure and AP disease.90, 91
Treatment options include a TKI or alloSCT (either de novo or after initial TKI therapy). There are no randomized data to guide the choice or dose of TKI. However, there is a suggestion from nonrandomized studies that second-generation TKIs have superior response rates to imatinib,87 and ponatinib provides perhaps the best outcome.
There are also no randomized data to guide the decision to perform alloSCT for patients with AP-CML. In the pre-imatinib era, patients transplanted in AP had 30% to 40% DFS at 4 years compared with 70% to 80% for CP.92, 93 Nonrandomized data suggest superior outcomes in patients treated with imatinib followed by alloSCT compared with imatinib alone, but there is the standard selection bias in this study.94
In summary, patients with de novo AP-CML may have good outcomes, particularly if treated with a second-generation TKI. We treat these patients following the same guidelines we use for CP patients, and alloSCT is only considered on failure of 2 TKIs. However, patients with AP developing after imatinib failure have significantly poorer outcomes and may be best treated more aggressively with a second-generation TKI followed by alloSCT when eligible. Patients with excellent, rapid responses to the second TKI may be followed up closely and alloSCT considered only if showing recurrence. Another important question for which there are no data to guide decisions is the role of maintenance TKI after transplant. Our practice is to continue prescribing TKI after transplant after count recovery for patients who previously progressed to AP or BP.

Treatment of BP-CML
Criteria for BP progression were outlined above. Approximately 50% to 60% of patients have myeloid blast phase (MBP) and 20% to 30% lymphoid blast phase (LBP). The remaining 10% to 30% are mixed.5 The aim of treatment is to achieve reversion to CP, then perform alloSCT with or without posttransplant TKI maintenance.

Treatment of LBP
Induction chemotherapy is given as per de novo ALL, with the addition of a TKI. Chemotherapy with hyperfractionated cyclophosphamide, vincristine, doxorubicin (adriamycin), and dexamethasone (hyper-CVAD) with a TKI can achieve CHR in approximately 90% of patients.95 Most patients will have previously received a TKI. However, in patients presenting with de novo transformation, it is important (although sometimes difficult) to distinguish CML in LBP from Ph-positive ALL. Morphologic criteria to suggest preexisting CML, such as monolobated megakaryocytes and basophilia, may be useful, as is the BCR-ABL transcript type; p210 BCR-ABL is present in most CML-LBP, whereas most Ph-positive ALL has the p190 transcript. Mutations in BCR-ABL1 in patients in whom imatinib therapy has failed are more frequent in BP (73%) relative to C and/AP96; the use of ABL1 mutational analysis to guide treatment is therefore essential. T315I is very frequent and, in contrast to CP, may be identified even before exposure to a TKI. These patients require treatment with ponatinib, usually combined with chemotherapy (hyper-CVAD, in our hands). Additional chromosomal abnormalities are frequent (particularly monosomy 7),95 and outcomes are generally poor. AlloSCT after initial response appears to improve outcomes, but selection bias in such studies is inevitable.96

Treatment of MBP
CML-MBP has a poor response to standard acute myeloid leukemia induction regimens.97 Patients with de novo MBP may respond to TKI monotherapy, but responses are shallow and transient.98, 99 There are few studies of AML induction chemotherapy or low-dose cytarabine combined with TKI.100, 101 Our general approach is to give standard acute myeloid leukemia induction chemotherapy with the addition of a TKI and perform alloSCT in responding patients.102 Although outcome for patients with prior BP is better when there is only minimal residual disease or no detectable disease even by PCR, we recommend alloSCT as soon as a patient is back to CP or has CHR because continued chemotherapy is no guarantee of improved response and may cause complications that can disqualify the patient for a later transplant.

Which TKI Should Be Used in BP-CML?
There are no head-to-head data in this area, and much existing data concern use of single-agent TKIs, which are rarely used in practice. Imatinib, 600 mg, results in shallow and transient single-agent responses.98, 99Imatinib does not cross the blood brain barrier and so is inadequate when central nervous system involvement exists.103, 104 Dasatinib, 140 mg/d, achieves a significantly higher rate of CCyR (26% and 46% in MBP and LBP, respectively), but responses are again transient, with a median survival of less than 12 months for MBP and less than 6 months for LBP.105 Although dasatinib crosses the blood brain barrier, we do not rely on this for prophylaxis or management of central nervous system disease and give standard treatment with intrathecal chemotherapy, high-dose systemic chemotherapy, and occasionally radiotherapy to approach this issue. Nilotinib, 400 mg twice daily, is associated with no better results compared with dasatinib and is not approved for this indication.70 Bosutinib is also approved for BP and may induce hematologic response in 28% and minor cytogenic response in 37%.106 Ponatinib has resulted in favorable response in heavily pretreated patients and patients with T315I mutations. Approximately 50% patients had a hematologic response after failure of dasatinib or nilotinib in MBP or LBP,107 and 18% achieved CCyR. The 1-year survival was an impressive 55%. Whenever possible, we use ponatinib because this might be the most effective agent and covers all mutations. Dasatinib and bostunib are suitable alternatives.

Treatment of Refractory and Relapsed BP
Novel agents for ALL, such as the CD22-immunoconjugate inotuzumab ozogamicin and the CD3/19 bispecific antibody blinatumomab, are yet to be evaluated because major studies have excluded Ph-positive patients. However, these could potentially be effective, as could CAR T cells directed against CD19, although there would likely be potential for antigen-negative escape or development of frank myeloid reversion, and any response would require consolidation with alloSCT.

Although CML remains one of the great success stories in modern oncology treatment, a number of challenges remain. Pre-treatment identification of patients likely to have poor outcomes is crude at best, and predictive tools to guide the optimal choice of TKI at baseline are not widely available, making treatment decisions largely empiric. Patients with failure of more than 1 TKI have relatively poor outcomes, and no data exist for second-line therapy for patients treated initially with a second-generation TKI. The mechanisms underlying the risk of arteriothrombotic events seen with several of the TKIs need to be better understood so that prevention and management can be approached more rationally. Finally, most patients require indefinite suppressive therapy, with an associated cumulative risk of potential toxic effects, particularly cardiovascular disease, as well as chronic, low-grade toxic effects that affect quality of life. Strategies to produce eradication of MRD, with minimal toxic effects, are essential to address these issues and to reduce the long-term pharmacoeconomic burden of indefinite TKI therapy.

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Leukemia’s Molecular Biography Uncovered in New Genomic Study

GEN 10/15/2015

  • Click Image To Enlarge +
    A new study offers a glimpse of the wealth of information that can be gleaned by combing the genome of a large collection of leukemia tissue samples. [hidesy/iStock]

    Researchers from the Dana-Farber Cancer Institute and the Broad Institute of MIT and Harvard have harnessed the power of next-generation sequencing to analyze a large collection of leukemia tissue samples. Using whole exome sequencing (WES), the investigators screened genetic material from more than 500 samples of chronic lymphocytic leukemia (CLL) and normal tissue—identifying dozens of genetic drivers for the disease, including two genes that had previously not been linked to human cancer.

    The investigators began to trace how some mutations affect the course of the disease and its susceptibility to treatment. Moreover, they started tracking the evolutionary path of CLL, as its dynamic genome spawns new groups and subgroups of tumor cells within a single patient.

    “Sequencing the DNA of CLL has taught us a great deal about the genetic basis of the disease,” explained senior author Catherine Wu, M.D., physician at Dana-Farber and associate professor of medicine at Harvard Medical School. “Previous studies, however, were limited by the relatively small number of tumor tissue samples analyzed, and by the fact that those samples were taken at different stages of the treatment process, from patients treated with different drug agents.

    Dr. Wu continued, stating “in our new study, we wanted to determine if analyzing tissue samples from a large, similarly-treated group of patients provides the statistical power necessary to study the disease in all its genetic diversity—to draw connections between certain mutations and the aggressiveness of the disease, and to chart the emergence of new mutations and their role in helping the disease advance.  Our results demonstrate the range of insights to be gained by this approach.”

    The findings from this study were published recently in Nature through an article entitled “Mutations driving CLL and their evolution in progression and relapse.”

    The researchers collected tumor and normal tissue samples from 538 patients with CLL, 278 of whom had participated in a German clinical trial that helped determine the standard treatment for the disease. After WES analysis, they uncovered dozens of genetic abnormalities that may play a role in CLL, including 44 mutated genes and 11 genes that were over- or under-copied in CLL cells. Interestingly, two of the mutated genes—RPS15 and IKZF3—have not previously been associated with human cancer.

    “This study also provides a vision of what the next phase of large-scale genomic sequencing efforts may look like,” noted lead author Dan Landau M.D., Ph.D., research fellow at Dana-Farber and the Broad Institute. “The growing sample size allows us to start engaging deeply with the complex interplay between different mutations found in any individual tumor, as well as reconstructs the evolutionary trajectories in which these mutations are acquired to allow the malignancy to thrive and overcome therapy.”

    Another fascinating discovery was that certain gene mutations were particularly common in tumor tissue from patients who had already undergone treatment, suggesting that these mutations help the disease rebound after initial therapy. In addition, the investigators found that therapy tends to produce shorter remissions in patients whose tumors carry mutations in the genes TP53 or SF3B1.

    “We found that genomic evolution after therapy is the rule rather than the exception,” Dr. Wu remarked. “Certain mutations were present in a greater number of leukemia cells within a sample after relapse, showing that these mutations, presumably, allow the tumor to persevere.”

    Dr. Wu and her colleagues hope that the findings from their studies will continue the push initiated by precision medicine to help personalize cancer treatments and develop new therapeutics.

    “The breadth of our findings shows what we will be able to achieve as we systematically sequence and analyze large cohorts of tumor tissue samples with defined clinical status,” stated co-senior author Gad Getz, Ph.D., director of the Cancer Genome Computational Analysis group at the Broad Institute. “Our work has enabled us to discover novel cancer genes, begin to chart the evolutionary path of CLL, and demonstrate specific mutations affect patients’ response to therapy. These discoveries will form the basis for precision medicine of CLL and other tumor types.”

Aurelian Udristioiu commented on Update on Chronic Myeloid Leukemia

 Update on Chronic Myeloid Leukemia  Larry H Bernstein, MD, FCAP, Curator LPBI Diagnosis and Treatment of Chronic …

In previous work the diagnosis of LAM-3 I made based on blood smears, the examination of bone marrow (BM) aspirates, the evaluation of promyeloblasts (greater than 30% in BM), and the presence of a specific immune phenotype. Immunocytochemical detection was performed to confirm the diagnosis of LAM-3 using FAR Leukemia kits (Italy), and there were positive results for the peroxidase reaction for promyelocytes, myelocytes, granulocytes, and peripheral blood cells (POX+) and negative results for the peroxidase reaction for the blast cells. We performed the leukocyte alkaline phosphatase reaction using a SIGMA kit ( to determine the neutrophil alkaline phosphatase (NAP) levels in granulocytes (negative or low values in LAM-3) and to evaluate the alpha-naphthyl-esterase reaction in monocytes cells (positive results indicate CMoL).
Lactate dehydrogenase (LDH) is an enzyme that is localized to the cytosol of human cells and catalyzes the reversible reduction of pyruvate to lactate via using hydrogenated nicotinamide deaminase (NADH) as co-enzyme. The causes of high LDH and high Mg levels in the serum include neoplastic states that promote the high production of intracellular LDH and the increased use of Mg²+ during molecular synthesis: Pyruvate acid>> LDH/NADH >>Lactate acid + NAD).
LDH is released from tissues in patients with physiological or pathological conditions and is present in the serum as a tetramer that is composed of the two monomers LDH-A and LDH-B, which can be combined into 5 isoenzymes: LDH-1 (B4), LDH-2 (B3-A1), LDH-3 (B2-A2), LDH-4 (B1-A3) and LDH-5 (A4). The LDH-A gene is located on chromosome 11, whereas the LDH-B gene is located on chromosome 12. The monomers differ based on their sensitivity to allosteric modulators. They facilitate adaptive metabolism in various tissues. The LDH-4 isoform predominates in the myocardium, is inhibited by pyruvate and is guided by the anaerobic conversion to lactate. Total LDH, which is derived from hemolytic processes, is used as a marker for monitoring the response to chemotherapy in patients with advanced neoplasm with or without metastasis.
The number of chromosome copies in malignant tumors can be correlated with the total serum LDH values. LDH levels in patients with malignant disease are increased as the result of high levels of the isoenzyme LDH-3 in patients with hematological malignant diseases and of the high level of the isoenzymes LDH-4 and LDH-5, which are increased in patients with other malignant diseases of tissues such as the liver, muscle, lungs, and conjunctive tissues. High concentrations of serum LDH damage the cell membrane.
In aerobic glucose metabolism, the oxidation of citric acid requires ADP and Mg²+, which will increase the speed of the reaction: Iso-citric acid + NADP (NAD) — isocitrate dehydrogenase (IDH) = alpha-ketoglutaric acid.
In the Krebs cycle (the citric cycle), IDH1 and IDH2 are NADP+-dependent enzymes that normally catalyze the inter-conversion of D-isocitrate and alpha-ketoglutarate (α-KG). The IDH1 and IDH2 genes are mutated in > 75% of different malignant diseases. Two distinct alterations are caused by tumor-derived mutations in IDH1 or IDH2: the loss of normal catalytic activity in the production of α-ketoglutarate (α-KG) and the gain of catalytic activity to produce 2-hydroxyglutarate (2-HG).
This product is a competitive inhibitor of multiple α-KG-dependent dioxygenases, including demethylases, prolyl-4-hydroxylase and the TET enzymes family (Ten-Eleven Translocation-2), resulting in genome-wide alternations in histones and DNA methylation.
IDH1 and IDH2 mutations have been observed in myeloid malignancies, including de novo and secondary AML (15%–30%), and in pre-leukemic clone malignancies, including myelodysplastic syndrome and myeloproliferative neoplasm (85% of the chronic phase and 20% of transformed cases in acute leukemia).
Today, the oncologists try a new drug, anti-IDH mutant, to treat AML..

Dr. Aurelian Udristioiu, MD,
Emergency County Hospital TARGU-JIU &USB University,
Primary Physician of Laboratory Medicine,
General Chemical Pathology, EuSpLM,
City Targu Jiu, Romania
National Academy of Biochemical Chemistry (NACB) Member,
Washington D.C, USA.

Aurelian Udristioiu commented on your update
” The IDH1 and IDH2 genes are mutated in > 75% of different malignant diseases. Two distinct alterations are caused by tumor-derived mutations in IDH1 or IDH2: the loss of normal catalytic activity. IDH1 and IDH2 mutations have been observed in myeloid malignancies, including de novo and secondary AML (15%–30%), and in pre-leukemic clone malignancies, including myelodysplastic syndrome and myeloproliferative neoplasm (85% of the chronic phase and 20% of transformed cases in acute leukemia). Today, the oncologists try a new drug, anti-IDH mutant, to treat AML.”

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