Posts Tagged ‘therapeutics’

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Recently, researchers at Mount Sinai were able to develop a therapeutic agent that shows high levels of effectiveness in Vitro disrupting a biological pathway that allow cancer to survive. This finding is according to a paper which was published in Cancer Discovery, which is a Journal of the American Association of cancer research in July 2021.

The therapy in which they focus on is a molecule named MS21, which causes the degradation of AKT which is an enzyme that is very active and present in cancers. In this study there was much evidence that pharmacological degradation of AKT is a feasible treatment for cancer’s which have a mutation in certain genes. 

AKT is a cancer gene that encodes an enzyme that is abnormally activated in cancer cells to stimulate tumor growth. The degradation of AKT reverses all these processes which ultimately inhibits further tumor growth.

“Our study lays a solid foundation for the clinical development of an AKT degrader for the treatment of human cancers with certain gene mutations,” said Ramon Parsons, MD, Ph.D., Director of The Tisch Cancer Institute and Ward-Coleman Chair in Cancer Research and Chair of Oncological Sciences at the Icahn School of Medicine at Mount Sinai. “Examination of 44,000 human cancers identified that 19 percent of tumors have at least one of these mutations, suggesting that a large population of cancer patients could benefit from therapy with an AKT degrader such as MS21.”


MS21 was tested and human cancer derived cell lines, is used in Laboratories as a model to study the efficacy of different cancer therapies.

At Mount Sinai they were looking to develop MS21 with an industry partner in order to open clinical trials for patients. 

“Translating these findings into effective cancer therapies for patients is a high priority because the mutations and the resulting cancer-driving pathways that we lay out in this study are arguably the most commonly activated pathways in human cancer, but this effort has proven to be particularly challenging,” said Jian Jin, Ph.D., Mount Sinai Professor in Therapeutics Discovery and Director of the Mount Sinai Center for Therapeutics Discovery at Icahn Mount Sinai. “We look forward to an opportunity to develop this molecule into a therapy that is ready to be studied in clinical trials.”


Image credit: National Cancer Institute

Original article: 

Researchers develop novel therapy that could be effective in many cancers

staff, S. X. (2021, July 23). R. Medical Xpress – by The Mount Sinai Hospital


UPDATE 12/12/2022

From Mt. Sinai

Advancing cancer precision medicine by creating a better toolbox for cancer therapy

Jian Jin1,2,3,4,5*, Arvin C. Dar1,2,3,4, Deborah Doroshow1


mong approximately 20,000 proteins in the human proteome, 627 have been identified by cancer-dependency studies as priority can­cer targets, which are functionally important for various cancers. Of these 600-plus priority targets, 232 are enzymes and 395 are nonenzyme proteins (1). Tremendous progress has been made over the past several decades in targeting enzymes, in particular kinas-es, which have suitable binding pockets that can be occupied by small-molecule inhibitors, leading to U.S. Food and Drug Administration (FDA) approvals of many small-molecule drugs as targeted anticancer thera-

1Tisch Cancer Institute; 2Department of Oncological Sciences; 3Department of Pharmacological Sciences; 4Mount Sinai Center for Therapeutics Discovery; 5Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY

*Corresponding author: jian.jin@mssm.edu


pies. However, most of the 395 nonenzyme protein targets, including tran­scription factors (TFs), do not have suitable binding pockets that can be effectively targeted by small molecules. These targets have consequently been considered undruggable; however, new cutting-edge approaches and technologies have recently been developed to target some of these “un-druggable” proteins in order to advance precision oncology.

TPD, a promising approach to precision cancer therapeutics

Targeted protein degradation (TPD) refers to the process of chemical­ly eliminating proteins of interest (POIs) by utilizing small molecules, which are broadly divided into two types of modalities: PROteolysis Tar­geting Chimeras (PROTACs) and molecular glues (2). PROTACs are het-erobifunctional small molecules that contain two moieties: one binding the POI, linked to another binding an ubiquitin E3 ligase. The induced proximity between the POI and ubiquitination machinery leads to selec­tive polyubiquitylation of the POI and its subsequent degradation by the ubiquitin–proteasome system (UPS). Molecular glues are monovalent small molecules, which, when built for TPD, directly induce interactions between the POI and an E3 ligase, also resulting in polyubiquitylation and subsequent degradation of the POI by the UPS. One of the biggest poten­tial advantages of these therapeutic modalities over traditional inhibitors is that PROTACs and molecular glues can target undruggable proteins. Explosive growth has been seen in the TPD field over recent years (2, 3). Here, we highlight several recent advancements.

TF-PROTAC, a novel platform for targeting undruggable

tumorigenic TFs

Many undruggable TFs are tumorigenic. To target them, TF-PROTAC was developed (4), which exploits the fact that TFs bind DNA in a sequence-specific manner. TF-PROTAC was created to selectively bind a TF and E3 ligase simultaneously, by conjugating a DNA oligonucleotide specific for the TF of interest to a selective E3 ligase ligand. As stated ear­lier, this simultaneous binding and induced proximity leads to selective polyubiquitination of the TF and its subsequent degradation by the UPS. TF-PROTAC is a cutting-edge technology that could potentially provide a universal strategy for targeting most undruggable tumorigenic TFs.

Development of novel PROTAC degraders

WDR5, an important scaffolding protein, not an enzyme, is essential for sustaining tumorigenesis in multiple cancers, including MLL-rearranged (MLL-r) leukemia. However, small-molecule inhibitors that block the pro-tein–protein interaction (PPI) between WDR5 and its binding partners ex­hibit very modest cancer cell–killing effects, likely due to the confounding fact that these PPI inhibitors target only some—but not all—of WDR5’s on-cogenic functions. To address this shortcoming, a novel WDR5 PROTAC, MS67, was recently created using a powerful approach that effectively eliminates the protein and thereby all WDR5 functions via ternary com­plex structure-based design (Figure 1) (5). MS67 is a highly effective WDR5 degrader that potently and selectively degrades WDR5 and effec­tively suppresses the proliferation of tumor cells both in vitro and in vivo. This study provides strong evidence that pharmacological degradation of WDR5 as a novel therapeutic strategy is superior to WDR5 PPI inhibition for treating WDR5-dependent cancers.

EZH2 is an oncogenic methyltransferase that catalyzes histone H3 ly­sine 27 trimethylation, mediating gene repression. In addition to this ca­nonical function, EZH2 has numerous noncanonical tumorigenic func­tions. EZH2 enzymatic inhibitors, however, are generally ineffective in

suppressing tumor growth in triple-negative breast cancer (TNBC) and MLL-r leukemia models and fail to phenocopy antitumor effects induced by EZH2 knockdown strategies. To target both canonical and noncanon-ical oncogenic functions of EZH2, several novel EZH2 degraders were recently developed, including MS1943, a hydrophobic tag–based EZH2 degrader (6), and MS177, an EZH2 PROTAC (7). MS1943 and MS177 effectively degrade EZH2 and suppress in vitro and in vivo growth in TNBC and MLL-r leukemia, respectively, suggesting that EZH2 degrad­ers could provide a novel and effective therapeutic strategy for EZH2-dependent tumors.

MS21, a novel AKT PROTAC degrader, was developed to target acti­vated AKT, the central node of the PI3K–AKT–mTOR signaling pathway (8). MS21 effectively suppresses the proliferation of PI3K–PTEN pathway-mutant cancers with wild-type KRAS and BRAF, which represent a large percentage of all human cancers. Another recent technology that expands the bifunctional toolbox for TPD is the demonstration that the E3 ligase KEAP1 can be leveraged for PROTAC development using a selective KEAP1 ligand (9). Overall, tremendous progress has been made in discov­ering novel degraders, some of which have advanced to clinical develop­ment as targeted therapies (2, 3).

Novel approaches to selective TPD in cancer cells

To minimize uncontrolled protein degradation in normal tissues, which may cause potential toxicity, a new technology was developed that incor­porates a light-inducible switch, termed “opto-PROTAC” (10). This switch serves as a caging group that renders opto-PROTAC inactive in all cells in the absence of ultraviolet (UV) light. Upon UV irradiation, however, the caging group is removed, resulting in the release of the active degrader and spatiotemporal control of TPD in cancer cells. Another strategy to achieve selective TPD in cancer over normal cells is to cage degraders with a folate group (11, 12). Folate-caged degraders are inert and selectively concen­trated within cancer cells, which overexpress folate receptors compared to normal cells. The caging group is subsequently removed inside tumor cells, releasing active degraders and achieving selective TPD in these cells. These novel approaches potentially enable degraders to be precision can­cer medicines.


Frontiers of Medical Research: Cancer

Trametiglue, a novel and atypical molecular glue

The RAS–RAF–MEK–ERK signaling pathway, one of the most frequent­ly mutated pathways in cancer, has been intensively targeted. Several drugs, such as the KRAS G12C inhibitor sotorasib and the MEK inhib­itor trametinib, have been approved by the FDA. A significant advance­ment in this area is the discovery that trametinib unexpectedly binds a pseudokinase scaffold termed “KSR” in addition to MEK through inter­facial contacts (13). Based on this structural and mechanistic insight, tra-metiglue, an analog of trametinib, was created as a novel molecular glue to limit adaptive resistance to MEK inhibition by enhancing interfacial binding between MEK, KSR, and the related homolog RAF. This study provides a strong foundation for developing next-generation drugs that target the RAS pathway.

TF-DUBTAC, a novel technology to stabilize undruggable tumor-suppressive TFs

Complementary to degrading tumorigenic TFs, stabilizing tumor-suppressive TFs could provide another effective approach for treating can­cer. While most tumor-suppressive TFs are undruggable, TF-DUBTAC was recently developed as a generalizable platform to stabilize tumor-sup­pressive TFs (14). Deubiquitinase-targeting chimeras (DUBTACs) are heterobifunctional small molecules with a deubiquitinase (DUB) ligand linked to a POI ligand, which stabilize POIs by harnessing the deubiq-uitination machinery (15). Similar to TF-PROTAC, TF-DUBTAC exploits the fact that most TFs bind specific DNA sequences. TF-DUBTAC links a DNA oligonucleotide specific to a tumor-suppressive TF with a selective DUB ligand, resulting in simultaneous binding of the TF and DUB. The induced proximity between the TF and DUB leads to selective deubiquiti-

Putting a bull’s-eye on cancer’s back

Scientists are aiming the immune systems’ “troops” directly at tumors to better treat cancer

Joshua D. Brody, Brian D. Brown


mmunotherapy has transformed the treatment of several types of can­cers. In particular, immune checkpoint blockade (ICB), which reinvig­orates killer T cells, has helped extend the lives of many patients with advanced-stage lung, bladder, kidney, or skin cancers. Unfortunately, ~80% of patients do not respond to current immunotherapies or even-tually relapse. Emerging data indicate that one of the most profound ways cancers resist immunotherapy is by keeping killer T cells out of the tumor and putting other immune cells in a suppressed state (1). This un­derstanding is giving rise to a new frontier in immunotherapy that is using synthetic biology and other approaches to reprogram the tumor from im­mune “cold” to immune “hot,” so T cells can be recruited to the tumor, and enter, target, and destroy the cancer cells (2) (Figure 1).

Cancers protect themselves by keeping out immune cells

Cancers grow in tissues like foreign invaders. Though they start from healthy cells, mutations turn cells malignant and allow them to grow un­checked. T cells can kill malignant cells that express mutated proteins, but cancers employ strategies to fend off the T cells. One way they do this is


nation of the TF and its stabilization. As an exciting new technology, TF-DUBTAC provides a potential general strategy to stabilize most undrugga-ble tumor-suppressive TFs for treating cancer.

Future outlook

The breathtaking pace we are seeing in the development of innovative approaches and technologies for advancing cancer therapies is only ex­pected to accelerate. The promising clinical results achieved by PROTACs with established targets are particularly encouraging and pave the way for development of PROTACs for newer and more innovative targets. These groundbreaking discoveries have now put opportunities to fully realize cancer precision medicine within our reach.


  1. F. M. Behan et al., Nature 568, 511–516 (2019).
  2. B. Dale et al., Nat. Rev. Cancer 21, 638–654 (2021).
  3. A. Mullard, Nat. Rev. Drug Discov. 20, 247–250 (2021).
  4. J. Liu et al., J. Am. Chem. Soc. 143, 8902–8910 (2021).
  5. X. Yu et al., Sci. Transl. Med. 13, eabj1578 (2021).
  6. A. Ma et al., Nat. Chem. Biol. 16, 214–222 (2020).
  7. J. Wang et al., Nat. Cell Biol. 24, 384–399 (2022).
  8. J. Xu et al., Cancer Discov. 11, 3064–3089 (2021).
  9. J. Wei et al., J. Am. Chem. Soc. 143, 15073–15083 (2021).
  10. J. Liu et al., Sci. Adv. 6, eaay5154 (2020).
  11. J. Liu et al., J. Am. Chem. Soc. 143, 7380–7387 (2021).
  12. H. Chen et al., J. Med. Chem. 64, 12273–12285 (2021).
  13. Z. M. Khan et al., Nature 588, 509–514 (2020).
  14. J. Liu et al., J. Am. Chem. Soc. 144, 12934–12941 (2022).

N. J. Henning et al., Nat. Chem. Biol. 18, 412–421 (2022

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Absolute Antibody Ltd., a leader of the market in recombinant antibody products announced a partnership with University of Zurich to offer synthetic nanobodies against the receptor binding domain (RBD) of SARS-CoV-2. Under the partnership, the original nanobodies and recently engineered formats are now accessible to the global research community for use as serological controls and in COVID-19 therapeutic development. The synthetic nanobodies hold a particular potential for the development of inhalable drugs, which could suggest a convenient treatment option for the COVID-19 pandemic.

The laboratory of Markus Seeger at University of Zurich designs a rapid in vitro selection platform to generate synthetic nanobodies, sybodies, against the receptor binding domain (RBD) of SARS-CoV-2. Within a two-week timeframe, the lab had recognized more than 60 unique anti-RBD sybodies from combinatorial display libraries. The sybodies are “designed to mimic the natural shape diversity of camelid nanobodies, consequently allowing for an optimal surface complementarity to the limited hydrophilic epitopes on membrane proteins. Due to their high thermal stabilities and low production costs, sybodies demonstrate a promise for diagnostic and therapeutic applications.

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Reporter: Irina Robu, PhD

Recent research from University of Alberta is looking at the role of fibrinogen, the substrate of thrombin in regulating a natural defense mechanism in the body. Fibrinogen is a well-known protein that is essential for wound healing and blood clotting in the body. Levels of fibrinogen increase in inflammatory states as part of the acute-phase response.

However, daily variation in plasma fibrinogen levels weakens its potential as a biomarker of cardiovascular risk. The discovery is expected to contribute to enhanced diagnosis and treatments for patients in a variety of diseases ranging from inflammation, to heart failure, to cancer.

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The Journey of Antibiotic Discovery

Reporter and Curator: Dr. Sudipta Saha, Ph.D.


The term ‘antibiotic’ was introduced by Selman Waksman as any small molecule, produced by a microbe, with antagonistic properties on the growth of other microbes. An antibiotic interferes with bacterial survival via a specific mode of action but more importantly, at therapeutic concentrations, it is sufficiently potent to be effective against infection and simultaneously presents minimal toxicity. Infectious diseases have been a challenge throughout the ages. From 1347 to 1350, approximately one-third of Europe’s population perished to Bubonic plague. Advances in sanitary and hygienic conditions sufficed to control further plague outbreaks. However, these persisted as a recurrent public health issue. Likewise, infectious diseases in general remained the leading cause of death up to the early 1900s. The mortality rate shrunk after the commercialization of antibiotics, which given their impact on the fate of mankind, were regarded as a ‘medical miracle’. Moreover, the non-therapeutic application of antibiotics has also greatly affected humanity, for instance those used as livestock growth promoters to increase food production after World War II.


Currently, more than 2 million North Americans acquire infections associated with antibiotic resistance every year, resulting in 23,000 deaths. In Europe, nearly 700 thousand cases of antibiotic-resistant infections directly develop into over 33,000 deaths yearly, with an estimated cost over €1.5 billion. Despite a 36% increase in human use of antibiotics from 2000 to 2010, approximately 20% of deaths worldwide are related to infectious diseases today. Future perspectives are no brighter, for instance, a government commissioned study in the United Kingdom estimated 10 million deaths per year from antibiotic resistant infections by 2050.


The increase in antibiotic-resistant bacteria, alongside the alarmingly low rate of newly approved antibiotics for clinical usage, we are on the verge of not having effective treatments for many common infectious diseases. Historically, antibiotic discovery has been crucial in outpacing resistance and success is closely related to systematic procedures – platforms – that have catalyzed the antibiotic golden age, namely the Waksman platform, followed by the platforms of semi-synthesis and fully synthetic antibiotics. Said platforms resulted in the major antibiotic classes: aminoglycosides, amphenicols, ansamycins, beta-lactams, lipopeptides, diaminopyrimidines, fosfomycins, imidazoles, macrolides, oxazolidinones, streptogramins, polymyxins, sulphonamides, glycopeptides, quinolones and tetracyclines.


The increase in drug-resistant pathogens is a consequence of multiple factors, including but not limited to high rates of antimicrobial prescriptions, antibiotic mismanagement in the form of self-medication or interruption of therapy, and large-scale antibiotic use as growth promotors in livestock farming. For example, 60% of the antibiotics sold to the USA food industry are also used as therapeutics in humans. To further complicate matters, it is estimated that $200 million is required for a molecule to reach commercialization, with the risk of antimicrobial resistance rapidly developing, crippling its clinical application, or on the opposing end, a new antibiotic might be so effective it is only used as a last resort therapeutic, thus not widely commercialized.


Besides a more efficient management of antibiotic use, there is a pressing need for new platforms capable of consistently and efficiently delivering new lead substances, which should attend their precursors impressively low rates of success, in today’s increasing drug resistance scenario. Antibiotic Discovery Platforms are aiming to screen large libraries, for instance the reservoir of untapped natural products, which is likely the next antibiotic ‘gold mine’. There is a void between phenotanypic screening (high-throughput) and omics-centered assays (high-information), where some mechanistic and molecular information complements antimicrobial activity, without the laborious and extensive application of various omics assays. The increasing need for antibiotics drives the relentless and continuous research on the foreground of antibiotic discovery. This is likely to expand our knowledge on the biological events underlying infectious diseases and, hopefully, result in better therapeutics that can swing the war on infectious diseases back in our favor.


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Gene-Silencing and Gene-Disabling in Pharmaceutical Development

Larry H. Bernstein, MD, FCAP, Curator


Gene-Silencing and Gene-Disabling in Pharmaceutical Development, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

Down and Out with RNAi and CRISPR


RNA interference (RNAi) silences, or knocks down, the translation of a gene by inducing degradation of a gene target’s transcript. To advance RNAi applications, Thermo Fisher Scientific has developed two types of small RNA molecules: short interfering RNAs and microRNAs. The company also offers products for RNAi analysis in vitro and in vivo, including libraries for high-throughput applications.

Genes can be knocked down with RNA interference (RNAi) or knocked out with CRISPR-Cas9. RNAi, the screening workhorse, knocks down the translation of genes by inducing rapid degradation of a gene target’s transcript.

CRISPR-Cas9, the new but already celebrated genome-editing technology, cleaves specific DNA sequences to render genes inoperative. Although mechanistically different, the two techniques complement one another, and when used together facilitate discovery and validation of scientific findings.

RNAi technologies along with other developments in functional genomics screening were discussed by industry leaders at the recent Discovery on Target conference. The conference, which emphasized the identification and validation of novel drug targets and the exploration of unknown cellular pathways, included a symposium on the development of CRISPR-based therapies.

RNAi screening can be performed in either pooled or arrayed formats. Pooled screening provides an affordable benchtop option, but requires back-end deconvolution and deep sequencing to identify the shRNA causing the specific phenotype. Targets are much easier to identify using the arrayed format since each shRNA clone is in an individual well.

“CRISPR complements RNAi screens,” commented Ryan Raver, Ph.D., global product manager of functional genomics at Sigma-Aldrich. “You can do a whole genome screen with either small interfering RNA (siRNA) or small hairpin RNA (shRNA), then validate with individual CRISPRs to ensure it is a true result.”

A powerful and useful validation method for knockdown or knockout studies is to use lentiviral open reading frames (ORFs) for gene re-expression, for rescue experiments, or to detect whether the wild-type phenotype is restored. However, the ORF randomly integrates into the genome. Also, with this validation technique, gene expression is not acting under the endogenous promoter. Accordingly, physiologically relevant levels of the gene may not be expressed unless controlled for via an inducible system.

In the future, CRISPR activators may provide more efficient ways to express not only wild-type but also mutant forms of genes under the endogenous promoter.

Choice of screening technique depends on the researcher and the research question. Whole gene knockout may be necessary to observe a phenotype, while partial knockdown might be required to investigate functions of essential or lethal genes. Use of both techniques is recommended to identify all potential targets.

For example, recently, a whole genome siRNA screen on a human glioblastoma cell line identified a gene, known as FAT1, as a negative regulator of apoptosis. A CRISPR-mediated knockout of the gene also conferred sensitivity to death receptor–induced apoptosis with an even stronger phenotype, thereby validating FAT1’s new role and link to extrinsic apoptosis, a new model system.

Dr. Raver indicated that next-generation RNAi libraries that are microRNA-adapted might have a more robust knockdown of gene expression, up to 90–95% in some cases. Ultracomplex shRNA libraries help to minimize both false-negative and false-positive rates by targeting each gene with ~25 independent shRNAs and by including thousands of negative-control shRNAs.

Recently, a relevant paper emerged from the laboratory of Jonathan Weissman, Ph.D., a professor of cellular and molecular pharmacology at the University of California, San Francisco. The paper described how a new ultracomplex pooled shRNA library was optimized by means of a microRNA-adapted system. This system, which was able to achieve high specificity in the detection of hit genes, produced robust results. In fact, they were comparable to results obtained with a CRISPR pooled screen. Members of the Weissman group systematically optimized the promoter and microRNA contexts for shRNA expression along with a selection of guide strands.

Using a sublibrary of proteostasis genes (targeting 2,933 genes), the investigators compared CRISPR and RNAi pooled screens. Data showed 48 hits unique to RNAi, 40 unique to CRISPR, and an overlap of 21 hits (with a 5% false discovery rate cut-off). Together, the technologies provided a more complete research story.

“RNA screens are well accepted and will continue to be used, but it is important biologically that researchers step away from the RNA mechanism to further study and validate their hits to eliminate potential bias,” explained Louise Baskin, senior product manager, Dharmacon, part of GE Healthcare. “The natural progression is to adopt CRISPR in the later stages.”

RNAi uses the cell’s endogenous mechanism. All of the components required for gene knockdown are already within the cell, and the delivery of the siRNA starts the process. With the CRISPR gene-editing system, which is derived from a bacterial immune defense system, delivery of both the guide RNA and the Cas9 nuclease, often the rate limiter in terms of knockout efficiency, are required.

Arrayed CRISPR Screens

Synthetic crRNA:tracrRNA reagents can be used in a similar manner to siRNA reagents for assessment of phenotypes in a cell population. Top row: A reporter cell line stably expressing Cas9 nuclease was transfected with GE Dharmacon’s Edit-R synthetic crRNA:tracrRNA system, which was used to target three positive control genes (PSMD7, PSMD14, and VCP) and a negative control gene (PPIB). Green cells indicate EGFP signaling occuring as a result of proteasome pathway disruption. Bottom row: A siGENOME siRNA pool targeting the same genes was used in the same reporter cell line.

In pooled approaches, the cell has to either drop out or overexpress so that it is sortable, limiting the types of addressable biological questions. A CRISPR-arrayed approach opens up the door for use of other analytical tools, such as high-content imaging, to identify hits of interest.

To facilitate use of CRISPR, GE recently introduced Dharmacon Edit-R synthetic CRISPR RNA (crRNA) libraries that can be used to carry out high-throughput arrayed analysis of multiple genes. Rather than a vector- or plasmid-based gRNA to guide the targeting of the Cas9 cleavage, a synthetic crRNA and tracrRNA are used. These algorithm-designed crRNA reagents can be delivered into the cells very much like siRNA, opening up the capability to screen multiple target regions for many different genes simultaneously.

GE showed a very strong overlap between CRISPR and RNAi using this arrayed approach to validate RNAi screen hits with synthetic crRNA. The data concluded that CRISPR can be used for medium- or high-throughput validation of knockdown studies.

“We will continue to see a lot of cooperation between RNAi and gene editing,” declared Baskin. “Using the CRISPR mechanism to knockin could introduce mutations to help understand gene function at a much deeper level, including a more thorough functional analysis of noncoding genes.

“These regulatory RNAs often act in the nucleus to control translation and transcription, so to knockdown these genes with RNAi would require export to the cytoplasm. Precision gene editing, which takes place in the nucleus, will help us understand the noncoding transcriptome and dive deeper into how those genes regulate disease progression, cellular development and other aspects of human health and biology.”

Tool Selection

The functional genomics tool should fit the specific biology; the biology should not be forced to fit the tool. Points to consider include the regulation of expression, the cell line or model system, as well as assay scale and design. For example, there may be a need for regulatable expression. There may be limitations around the cell line or model system. And assay scale and design may include delivery conditions and timing to optimally complete perturbation and reporting.

“Both RNAi- and CRISPR-based gene modulation strategies have pros and cons that should be considered based on the biology of the genes being studied,” commented Gwen Fewell, Ph.D., chief commercial officer, Transomic Technologies.

RNAi reagents, which can produce hypomorphic or transient gene-suppression states, are well known for their use in probing drug targets. In addition, these reagents are enriching studies of gene function. CRISPR-Cas9 reagents, which produce clean heterozygous and null mutations, are important for studying tumor suppressors and other genes where complete loss of function is desired.

Schematic of a pooled shRNA screening workflow developed by Transomic Technologies. Cells are transduced, and positive or negative selection screens are performed. PCR amplification and sequencing of the shRNA integrated into the target cell genome allows the determination of shRNA representation in the population.

Timing to readout the effects of gene perturbation must be considered to ensure that the biological assay is feasible. RNAi gene knockdown effects can be seen in as little as 24–72 hours, and inducible and reversible gene knockdown can be realized. CRISPR-based gene knockout effects may become complete and permanent only after 10 days.

Both RNAi and CRISPR reagents work well for pooled positive selection screens; however, for negative selection screens, RNAi is the more mature tool. Current versions of CRISPR pooled reagents can produce mixed populations containing a fraction of non-null mutations, which can reduce the overall accuracy of the readout.

To meet the needs of varied and complex biological questions, Transomic Technologies has developed both RNAi and CRISPR tools with options for optimal expression, selection, and assay scale. For example, the company’s shERWOOD-UltramiR shRNA reagents incorporate advances in design and small RNA processing to produce increased potency and specificity of knockdown, particularly important for pooled screens.

Sequence-verified pooled shRNA screening libraries provide flexibility in promoter choice, in vitro formats, in vivo formats, and a choice of viral vectors for optimal delivery and expression in biologically relevant cell lines, primary cells or in vivo.

The company’s line of lentiviral-based CRISPR-Cas9 reagents has variable selectable markers such that guide RNA- and Cas9-expressing vectors, including inducible Cas9, can be co-delivered and selected for in the same cell to increase editing efficiency. Promoter options are available to ensure expression across a range of cell types.

“Researchers are using RNAi and CRISPR reagents individually and in combination as cross-validation tools, or to engineer CRISPR-based models to perform RNAi-based assays,” informs Dr. Fewell. “Most exciting are parallel CRISPR and RNAi screens that have tremendous potential to uncover novel biology.”

Converging Technologies

The convergence of RNAi technology with genome-editing tools, such as CRISPR-Cas9 and transcription activator-like effector nucleases, combined with next-generation sequencing will allow researchers to dissect biological systems in a way not previously possible.

“From a purely technical standpoint, the challenges for traditional RNAi screens come down to efficient delivery of the RNAi reagents and having those reagents provide significant, consistent, and lasting knockdown of the target mRNAs,” states Ross Whittaker, Ph.D., a product manager for genome editing products at Thermo Fisher Scientific. “We have approached these challenges with a series of reagents and siRNA libraries designed to increase the success of RNAi screens.”

Thermo Fisher’ provides lipid-transfection RNAiMax reagents, which effectively deliver siRNA. In addition, the company’s Silencer and Silencer Select siRNA libraries provide consistent and significant knockdown of the target mRNAs. These siRNA libraries utilize highly stringent bioinformatic designs that ensure accurate and potent targeting for gene-silencing studies. The Silencer Select technology adds a higher level of efficacy and specificity due to chemical modifications with locked nucleic acid (LNA) chemistry.

The libraries alleviate concerns for false-positive or false-negative data. The high potency allows less reagent use; thus, more screens or validations can be conducted per library.

Dr. Whittaker believes that researchers will migrate regularly between RNAi and CRISPR-Cas9 technology in the future. CRISPR-Cas9 will be used to create engineered cell lines not only to validate RNAi hits but also to follow up on the underlying mechanisms. Cell lines engineered with CRISPR-Cas9 will be utilized in RNAi screens. In the long term, CRISPR-Cas9 screening will likely replace RNAi screening in many cases, especially with the introduction of arrayed CRISPR libraries.

Validating Antibodies with RNAi

Unreliable antibody specificity is a widespread problem for researchers, but RNAi is assuaging scientists’ concerns as a validation method.

The procedure introduces short hairpin RNAs (shRNAs) to reduce expression levels of a targeted protein. The associated antibody follows. With its protein knocked down, a truly specific antibody shows dramatically reduced or no signal on a Western blot. Short of knockout animal models, RNAi is arguably the most effective method of validating research antibodies.

The method is not common among antibody suppliers—time and cost being the chief barriers to its adoption, although some companies are beginning to embrace RNAi validation.

“In the interest of fostering better science, Proteintech felt it was necessary to implement this practice,” said Jason Li, Ph.D., founder and CEO of Proteintech Group, which made RNAi standard protocol in February 2015. “When researchers can depend on reproducibility, they execute more thorough experiments and advance the treatment of human diseases and conditions.”

Down and Out with RNAi and CRISPR

Genes can be knocked down with RNA interference (RNAi) or knocked out with CRISPR-Cas9. RNAi, the screening workhorse, knocks down the translation of genes by inducing rapid degradation of a gene target’s transcript.

RNA-Based Therapeutics and Vaccines

RNA-based biopharmaceuticals, which includes therapeutics and vaccines, is a relatively new class of treatment and prophylactic for a number of chronic and rare diseases, including cancer, diabetes, tuberculosis, and certain cardiovascular conditions. The field holds great promise in the prevention and treatment of these diseases as demonstrated by early-phase clinical trials as well as significant investment by the drug development community.

Ready, Aim, CRISPR (or RNAi)

Recent progress in probing gene function via the RNAi and CRISPR methods were a strong theme of the Discovery On Target conference. Both methods enable researchers to impair the function of a targeted gene.

Masked RNAi Drug Slips through Membrane, Sheds Guise within Cell

For small interfering RNA, approaching a cell is like walking up to the door of an old speakeasy. Such doors were heavily reinforced and had a narrow, built-in sliding panel at eye level, and if the eyes peering out though the open panel didn’t like the look of you, well, you were not getting inside. Failing to gain entry is something that happens all too frequently to small interfering RNAs, which admittedly are anything but “life of the party” types.

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Accessing the Blood Brain Barrier for Chemotherapy

Larry H. Bernstein, MD, FCAP, Curator


Blood-Brain Barrier Opened Noninvasively with Focused Ultrasound for the First Time

Mon, 11/09/2015 – 9:26amby Focused Ultrasound Foundation

The blood-brain barrier has been non-invasively opened in a patient for the first time. A team at Sunnybrook Health Sciences Centre in Toronto used focused ultrasound to enable temporary and targeted opening of the blood-brain barrier (BBB), allowing the more effective delivery of chemotherapy into a patient’s malignant brain tumor.

The team, led by neurosurgeon Todd Mainprize, MD, and physicist Kullervo Hynynen, PhD, infused the chemotherapy agent doxorubicin, along with tiny gas-filled bubbles, into the bloodstream of a patient with a brain tumor. They then applied focused ultrasound to areas in the tumor and surrounding brain, causing the bubbles to vibrate, loosening the tight junctions of the cells comprising the blood-brain barrier and allowing high concentrations of the chemotherapy to enter targeted tissues.

“The blood-brain barrier has been a persistent impediment to delivering valuable therapies to treat tumors,” said Dr. Mainprize. “We are encouraged that we were able to open this barrier to deliver chemotherapy directly into the brain, and we look forward to more opportunities to apply this revolutionary approach.”

This patient treatment is part of a pilot study of up to 10 patients to establish the feasibility, safety and preliminary efficacy of focused ultrasound to temporarily open the blood-brain barrier to deliver chemotherapy to brain tumors. The Focused Ultrasound Foundation is currently funding this trial through their Cornelia Flagg Keller Memorial Fund for Brain Research.

“Breaching this barrier opens up a new frontier in treating brain disorders,” said Neal Kassell, MD, Chairman of the Focused Ultrasound Foundation. “We are encouraged by the momentum building for the use of focused ultrasound to non-invasively deliver therapies for a number of brain disorders.”

Opening the blood-brain barrier in a localized region to deliver chemotherapy to a tumor is a predicate for utilizing focused ultrasound for the delivery of other drugs, DNA-loaded nanoparticles, viral vectors, and antibodies to the brain to treat a range of neurological conditions, including various types of brain tumors, Parkinson’s, Alzheimer’s and some psychiatric diseases.

The procedure was conducted using Insightec’s ExAblate Neuro system. “This first patient treatment is a technological breakthrough that may lead to many clinical applications,” said Eyal Zadicario, Vice President for R&D and Director of Neuro Programs, Insightec.

While the current trial is a first-in-human achievement, Dr. Kullervo Hynynen, senior scientist at the Sunnybrook Research Institute, has been performing similar pre-clinical studies for about a decade. His research has shown that the combination of focused ultrasound and microbubbles may not only enable drug delivery, but might also stimulate the brain’s natural responses to fight disease. For example, the temporary opening of the blood-brain barrier appears to facilitate the brain’s clearance of a key pathologic protein related to Alzheimer’s and improves cognitive function.

recent study by Gerhard Leinenga and Jürgen Götz from the Queensland Brain Institute in Australia further corroborated Hynynen’s research, demonstrating opening the blood-brain barrier with focused ultrasound reduced brain plaques and improved memory in a mouse model of Alzheimer’s disease.

Based on these two pre-clinical studies, a pilot clinical trial using focused ultrasound to treat Alzheimer’s is being organized.

Blood-brain Barrier Opened Non-invasively for the First Time


Scientists, for the first time, have non-invasively opened the blood-brain barrier (BBB) in a patient.

A team at Sunnybrook Health Sciences Centre in Toronto, led by neurosurgeon Todd Mainprize, M.D., used focused ultrasound technology to more effectively introduce chemotherapy drugs into a patient’s malignant brain tumor.  The results were verified with a post procedure MRI scan, Mainprize said at a press conference Tuesday.

The blood-brain barrier is a protective layer that keeps harmful substances such as toxins from entering from the blood vessels into the brain.  Unfortunately, it also prevents many drugs from reaching the brain in adequate doses.

At the press conference, Mainprize stressed that this is a phase one safety and concept study to show that they could pass through the BBB. He noted the operation went smoothly and the patient, a 56-year-old women, who is the first of 10 to undergo the procedure for the study, is doing well.

To breach the BBB, doctors infused a chemotherapy drug, along with tiny gas-filled bubbles, into the blood stream. Then focused ultrasound was applied to the tumor and surrounding brain, causing the bubbles to vibrate, and open the BBB so high concentrations of the chemotherapy could enter targeted tissues.

The team is actively analyzing brain tissue samples to see how much of the drug was able to enter.  The findings have not been published yet, but were presented at the Focused Ultrasound Surgery Foundation meeting, according to Mainprize.

Mainprize described the device: It has 1,024 transducers that are arranged in a helmet shape that goes around the head and the forehead, and corrects for aberrations in the skull.

While the BBB has been non-invasively opened in animals, this was the first instance in humans.

“There have been hundreds and hundreds of animal models opening the blood-brain barrier, in mice, dogs, pigs, and primates, all of which have shown a very good safety profile with no changes in function behavior or hemorrhage,” Mainprize said at the press conference.

He noted that this is a reversible procedure, and the barrier is restored back to its normal function in 24 hours.

Nathan McDannold, Ph.D., associate professor of radiology at Brigham and Women’s Hospital said, “If you compare this to alternative methods, whatever risks there are, there much less than if you were invasively injecting drugs.”

The scientists believe the technology has applications beyond brain tumors, such as in Alzheimer’s and Parkinson’s diseases.

McDannold said that groups are in the process of planning a protocol that would deliver antibodies to clear amyloid proteins, associated with Alzheimer’s, and for Parkinson’s they are looking at neuroprotectives and potential gene therapies.

The trial is being funded by the Focused Ultrasound Foundation.

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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
DOI: http://dx.doi.org/10.1016/j.mayocp.2015.08.010

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 http://www.leukemia-net.org/content/leukemias/cml/cml_score/index_eng.html. 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.

1. Melo JV, Hughes TP, Apperley JF. Chronic Myeloid Leukemia. In: ASH Education Program Book2003:132–152.
2. Shtivelman, E., Lifshitz, B., Gale, R.P., and Canaani, E. Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature. 1985; 315: 550–554
o View in Article
o | CrossRef

o | PubMed

o | Scopus (807)
3. Lugo, T.G., Pendergast, A.M., Muller, A.J., and Witte, O.N. Tyrosine kinase activity and transformation potency of bcr-abl oncogene products. Science. 1990; 247: 1079–1082
o View in Article
o | CrossRef

o | PubMed
4. Daley, G.Q., Van Etten, R.A., and Baltimore, D. Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science. 1990; 247: 824–830
o View in Article
o | CrossRef

o | PubMed
5. in: S.H. Swerdlow, E. Campo, N.L. Harris, (Eds.) World Health Organization Classification of Tumors of Haematopoietics and Lymphoid Tissues. IARC Press, Lyon, France; 2008
o View in Article
6. Faderl, S., Talpaz, M., Estrov, Z., O’Brien, S., Kurzrock, R., and Kantarjian, H.M. The biology of chronic myeloid leukemia. N Engl J Med. 1999; 341: 164–172
o View in Article
o | CrossRef

o | PubMed

o | Scopus (757)
7. Savage, D.G., Szydlo, R.M., and Goldman, J.M. Clinical features at diagnosis in 430 patients with chronic myeloid leukaemia seen at a referral centre over a 16-year period. Br J Haematol. 1997; 96:111–116
o View in Article
o | CrossRef

o | PubMed

o | Scopus (113)
8. Kantarjian, H.M., Keating, M.J., Smith, T.L., Talpaz, M., and McCredie, K.B. Proposal for a simple synthesis prognostic staging system in chronic myelogenous leukemia. Am J Med. 1990; 88: 1–8
o View in Article
o | Abstract

o | Full Text PDF

o | PubMed

o | Scopus (100)
9. Melo, J.V., Myint, H., Galton, D.A., and Goldman, J.M. P190BCR-ABL chronic myeloid leukaemia: the missing link with chronic myelomonocytic leukaemia?. Leukemia. 1994; 8: 208–211
o View in Article
o | PubMed
10. Stoll, D.B., Peterson, P., Exten, R. et al. Clinical presentation and natural history of patients with essential thrombocythemia and the Philadelphia chromosome. Am J Hematol. 1988; 27: 77–83

Leukemia’s Molecular Biography Uncovered in New Genomic Study

GEN 10/15/2015

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    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 (www.sigmaaldrich.com) 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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Functional Correlates of Signaling Pathways

Author and Curator: Larry H. Bernstein, MD, FCAP


We here move on to a number of specific, key published work on signaling, and look at the possible therapeutic applications to disease states.

Scripps Research Professor Wolfram Ruf and colleagues have identified a key connection between

  • the signaling pathways and the immune system spiraling out of control involving
  • the coagulation system and vascular endothelium that,
  • if disrupted may be a target for sepsis. (Science Daily, Feb 29, 2008).

It may be caused by a bacterial infection that enters the bloodstream, but

  • we now recognize the same cascade not triggered by bacterial invasion.

The acute respiratory distress syndrome (ARDS) has been defined as

  • a severe form of acute lung injury featuring
  • pulmonary inflammation and increased capillary leak.

ARDS is associated with a high mortality rate and accounts for 100,000 deaths annually in the United States. ARDS may arise in a number of clinical situations, especially in patients with sepsis. A well-described pathophysiological model of ARDS is one form of

  • the acute lung inflammation mediated by
  1. neutrophils,
  2. cytokines, and
  3. oxidant stress.

Neutrophils are major effect cells at the frontier of

  • innate immune responses, and they play
  • a critical role in host defense against invading microorganisms.

The tissue injury appears to be related to

  • proteases and toxic reactive oxygen radicals
  • released from activated neutrophils.

In addition, neutrophils can produce cytokines and chemokines that

enhance the acute inflammatory response.

Neutrophil accumulation in the lung plays a pivotal role in the pathogenesis of acute lung injury during sepsis. Directed movement of neutrophils is

  • mediated by a group of chemoattractants,
  • especially CXC chemokines.

Local lung production of CXC chemokines is intensified during experimental sepsis induced by cecal ligation and puncture (CLP).

Sepsis, Multi-organ Dysfunction Syndrome, and Septic Shock: A Conundrum of Signaling Pathways Cascading Out of Control


Integrins and extracellular matrix in mechanotransduction

ligand binding of integrins

ligand binding of integrins

Integrins are a family of cell surface receptors which

mediate cell–matrix and cell–cell adhesions.

Among other functions they provide an important

mechanical link between the cells external and intracellular environments while

the adhesions that they form also have critical roles in cellular signal-transduction.

Cell–matrix contacts occur at zones in the cell surface where

adhesion receptors cluster and when activated

the receptors bind to ligands in the extracellular matrix.

The extracellular matrix surrounds the cells of tissues and forms the

structural support of tissue which is particularly important in connective tissues.

Cells attach to the extracellular matrix through

specific cell-surface receptors and molecules

including integrins and transmembrane proteoglycans.

The integrin family of αβ heterodimeric receptors act as

cell adhesion molecules

connecting the ECM to the actin cytoskeleton.

The actin cytoskeleton is involved in the regulation of

1.cell motility,

2.cell polarity,

3.cell growth, and

4.cell survival.

The combination of αβ subunits determines

binding specificity and

signaling properties.

Both α and β integrin subunits contain two separate tails, which

penetrate the plasma membrane and possess small cytoplasmic domains which facilitate

the signaling functions of the receptor.

There is some evidence that the β subunit is the principal site for

binding of cytoskeletal and signaling molecules,

whereas the α subunit has a regulatory role. The integrin tails

link the ECM to the actin cytoskeleton within the cell and with cytoplasmic proteins,

such as talin, tensin, and filamin. The extracellular domains of integrin receptors bind the ECM ligands.

binding of integrins depends on ECM divalent cations ch19

binding of integrins depends on ECM divalent cations ch19

integrin coupled to F-actin via linker

integrin coupled to F-actin via linker


Schematic of the ‘focal adhesion clutch’ on stiff (a) versus soft (b) extracellular matrix (ECM). In all cases, integrins are coupled to F-actin via linker proteins (for example, talin and vinculin). The linker proteins move backwards (as indicated by the small arrows) as F-actin also moves backwards, under pushing forces from actin polymerization and/or pulling forces from myosin II activity. This mechanism transfers force from actin to integrins, which pull on the ECM. A stiff ECM (a) resists this force so that the bound integrins remain immobile. A compliant matrix (b) deforms under this force (as indicated by the compressed ECM labelled as deformed matrix) so that the bound integrins can also move backwards. Their movement reduces the net loading rate on all the force-bearing elements, which results in altered cellular responses

The ECM is a complex mixture of matrix molecules, including –

  • glycoproteins, collagens, laminins, glycosaminoglycans, proteoglycans,
  • and nonmatrix proteins, – including growth factors

The integrin receptor formed from the binding of α and β subunits is

  • shaped like a globular head supported by two rod-like legs (Figure 1).

Most of the contact between the two subunits occurs in the head region, with

  • the intracellular tails of the subunits forming the legs of the receptor.

Integrin recognition of ligands is not constitutive but

  • is regulated by alteration of integrin affinity for ligand binding.

For integrin binding to ligands to occur

  • the integrin must be primed and activated, both of which involve
  • conformational changes to the receptor.

Linking integrin conformation to function

Figure  Integrin binding to extracellular matrix (ECM). Conformational changes to integrin structure and clustering of subunits which allow enhanced function of the receptor.

Integrins work alongside other proteins such as


immunoglobulin superfamily

cell adhesion molecules,

selectins, and


to mediate

cell–cell and

cell–matrix interactions and communication.

Activation of adhesion receptors triggers the formation of matrix contacts in which

bound matrix components,

adhesion receptors,

and associated intracellular cytoskeletal and signaling molecules

form large functional, localized multiprotein complexes.

Cell–matrix contacts are important in a variety of different cell and

tissue properties including

1.embryonic development,

2.inflammatory responses,

3.wound healing,

4.and adult tissue homeostasis.

Integrin extracellular binding activity is regulated from inside the cell and binding to the ECM induces signals that are transmitted into the cell. This bidirectional signaling requires


spatially, and

temporally regulated formation and

disassembly of multiprotein complexes that

form around the short cytoplasmic tails of integrins.

Ligand binding to integrin family members leads to clustering of integrin molecules in the plasma membrane and recruitment of actin filaments and intracellular signaling molecules to the cytoplasmic domain of the integrins. This forms focal adhesion complexes which are able to maintain

not only adhesion to the ECM

but are involved in complex signaling pathways

which include establishing

1.cell polarity,

2.directed cell migration, and

3.maintaining cell growth and survival.

Initial activation through integrin adhesion to matrix recruits up to around 50 diverse signaling molecules

to assemble the focal adhesion complex

which is capable of responding to environmental stimuli efficiently.

Mapping of the integrin

adhesome binding and signaling interactions

a network of 156 components linked together which can be modified by 690 interactions.

Genetic programming occurs with the binding of integrins to the ECM

Signal transduction pathway activation arising from integrin-ECM binding results in

  • changes in gene expression of cells and
  • leads to alterations in cell and tissue function.

Various different effects can arise depending on the

1.cell type,

2.matrix composition, and

3.integrins activated

It has been suggested that integrin-type I collagen interaction is necessary for

  • the phosphorylation and activation of osteoblast-specific transcription factors
  • present in committed osteoprogenitor cells.

During mechanical loading/stimulation of chondrocytes there is an

  1. influx of ions across the cell membrane resulting from
  2. activation of mechanosensitive ion channels
  3. which can be inhibited by subunit-specific anti-integrin blocking antibodies or RGD peptides.

Using these strategies it was identified that

  • α5β1 integrin is a major mechanoreceptor in articular chondrocyte
  • responses to mechanical loading/stimulation.

Osteoarthritic chondrocytes show a depolarization response to 0.33 Hz stimulation

  • in contrast to the hyperpolarization response of normal chondrocytes.

The mechanotransduction pathway in chondrocytes derived from normal and osteoarthritic cartilage

  • both involve recognition of the mechanical stimulus
  • by integrin receptors resulting in
  • the activation of integrin signaling pathways
  • leading to the generation of a cytokine loop.

Normal and osteoarthritic chondrocytes show differences

  • at multiple stages of the mechanotransduction cascade.

Signaling pathways activated in chondrocytes

Signaling pathways activated in chondrocytes


Chondrocyte integrins are important mediators of cell–matrix interactions in cartilage

  • by regulating the response of the cells to signals from the ECM that
  1. control cell proliferation,
  2. survival,
  3. differentiation,
  4. matrix remodeling.

Integrins participate in development and maintenance of the tissue but also

  • in pathological processes related to matrix destruction, where
  • they likely play a role in the progression of OA.

Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels

Cells exhibited four types of mechanical responses:

(1) an immediate viscoelastic response;

(2) early adaptive behavior characterized by pulse-to-pulse attenuation in response to oscillatory forces;

(3) later adaptive cell stiffening with sustained (>15 second) static stresses; and

(4) a large-scale repositioning response with prolonged (>1 minute) stress.

Importantly, these adaptation responses differed biochemically.

The immediate and early responses were affected by

chemically dissipating cytoskeletal prestress (isometric tension), whereas

the later adaptive response was not.

The repositioning response was prevented by

inhibiting tension through interference with Rho signaling,

similar to the case of the immediate and early responses, but it was also prevented by

blocking mechanosensitive ion channels or

by inhibiting Src tyrosine kinases.

All adaptive responses were suppressed by cooling cells to 4°C to slow biochemical remodeling. Thus, cells use multiple mechanisms to sense and respond to static and dynamic changes in the level of mechanical stress applied to integrins.

Microtubule-Stimulated ADP Release, ATP Binding, and Force Generation In Transport Kinesins

All three classes of molecular motor proteins are now known to be

  • large protein families with diverse cellular functions.

Both the kinesin family and the myosin family have been defined and their proteins grouped into subfamilies. Finally, the elusive cytoplasmic version of dynein was identified and a multigene family of flagellar and cytoplasmic dyneins defined. Members of a given motor protein family share

  • significant homology in their motor domains with the defining member,
  • kinesin, dynein or myosin; but they also contain
  • unique protein domains that are specialized for interaction with different cargoes.

This large number of motor proteins may reflect

  • the number of cellular functions that require force generation or movement,
  • ranging from mitosis to morphogenesis to transport of vesicles.

Kinesins are a large family of microtubule (MT)-based motors that play important roles in many cellular activities including


motility, and

intracellular transport

Their involvement in a range of pathological processes

  • also highlights their significance as therapeutic targets and
  • the importance of understanding the molecular basis of their function

They are defined by their motor domains that contain both

  • the microtubule (MT) and
  • ATP binding sites.

Three ATP binding motifs—

  1. the P-loop,
  2. switch I,
  3. switch II–

are highly conserved among

  1. kinesins,
  2. myosin motors, and
  • small GTPases.

They share a conserved mode of MT binding such that

  • MT binding,
  • ATP binding, and
  • hydrolysis

are functionally coupled for efficient MT-based work.

The interior of a cell is a hive of activity, filled with

  • proteins and other items moving from one location to another.

A network of filaments called microtubules forms tracks

  • along which so-called motor proteins carry these items.

Kinesins are one group of motor proteins, and a typical kinesin protein has

  • one end (called the ‘motor domain’) that can attach itself to the microtubules.

The other end links to the cargo being carried, and a ‘neck’ connects the two. When two of these proteins work together,

  • flexible regions of the neck allow the two motor domains to move past one another,
  • which enable the kinesin to essentially walk along a microtubule in a stepwise manner.

Although the two kinesins have been thought to move along the microtubule tracks in different ways, Atherton et al. find that the core mechanism used by their motor domains is the same.

When a motor domain binds to the microtubule, its shape changes,

  • first stimulating release of the breakdown products of ATP from the previous cycle.

This release makes room for a new ATP molecule to bind. The structural changes caused by ATP binding

  • produce larger changes in the flexible neck region that
  • enable individual motor domains within a kinesin pair to
  • co-ordinate their movement and move in a consistent direction.

The major and largely invariant point of contact between kinesin motor domains and the MT is helix-α4,

  • which lies at the tubulin intradimer interface.

The conformational changes in functionally important regions of each motor domain are described,

  • starting with the nucleotide-binding site,
  • from which all other conformational changes emanate.

The nucleotide-binding site (Figure 2) has three major elements:

(1) the P-loop (brown) is visible in all our reconstructions;

(2) loop9 (yellow, contains switch I) undergoes major conformational changes through the ATPase cycle; and

(3) loop11 (red, contains switch II) that connects strand-β7 to helix-α4, the conformation and flexibility of which is

  • determined by MT binding and motor nucleotide state.

Movement and extension of helix-α6 controls neck linker docking

the N-terminus of helix-α6 is closely associated with elements of the nucleotide binding site suggesting that

  • its conformation alters in response to different nucleotide states.


  • because the orientation of helix-α6 with respect to helix-α4 controls neck linker docking and
  • because helix-α4 is held against the MT during the ATPase cycle,
    • conformational changes in helix-α6 control movement of the neck linker.

Mechanical amplification and force generation involves conformational changes across the motor domain

A key conformational change in the motor domain following Mg-ATP binding is

  • peeling of the central β-sheet from the C-terminus of helix-α4 increasing their separation;
  • this is required to accommodate rotation of helix-α6 and consequent neck linker docking

ATP binding draws loop11 and loop9 closer together; causing

(1) tilting of most of the motor domain not contacting the MT towards the nucleotide-binding site,

(2) rotation, translation, and extension of helix-α6 which we propose contributes to force generation, and

(3) allows neck linker docking and biases movement of the 2nd head towards the MT plus end.

In both motors, microtubule binding promotes

ordered conformations of conserved loops that

stimulate ADP release,

enhance microtubule affinity and

prime the catalytic site for ATP binding.

ATP binding causes only small shifts of these nucleotide-coordinating loops but induces

large conformational changes elsewhere that

allow force generation and

neck linker docking towards the microtubule plus end.

The study presents evidence provide evidence for a conserved ATP-driven

mechanism for kinesins and

reveals the critical mechanistic contribution of the microtubule interface.

Phosphorylation at endothelial cell–cell junctions: Implications for VE-cadherin function

This review summarizes the role of VE-cadherin phosphorylation in the regulation of endothelial cell–cell junctions and highlights how this affects vascular permeability and leukocyte extravasation.

The vascular endothelium is the inner lining of blood vessels and

forms a physical barrier between the vessel lumen and surrounding tissue;

controlling the extravasation of fluids,

plasma proteins and leukocytes.

Changes in the permeability of the endothelium are tightly regulated. Under basal physiological conditions, there is a continuous transfer of substances across the capillary beds. In addition the endothelium can mediate inducible,

transient hyperpermeability

in response to stimulation with inflammatory mediators,

which takes place primarily in post-capillary venules

However, when severe, inflammation may result in dysfunction of the endothelial barrier

  • in various parts of the vascular tree, including large veins, arterioles and capillaries.

Dysregulated permeability is observed in various pathological conditions, such as

  • tumor-induced angiogenesis,
  • cerebrovascular accident and
  • atherosclerosis.

Two fundamentally different pathways regulate endothelial permeability,

  1. the transcellular and
  2. paracellular pathways.

Solutes and cells can pass through the body of endothelial cells via the transcellular pathway, which includes

  • vesicular transport systems,
  • fenestrae, and
  • biochemical transporters.

The paracellular route is controlled by

  • the coordinated opening and closing of endothelial junctions and
  • thereby regulates traffic across the intercellular spaces between endothelial cells.

Endothelial cells are connected by

tight, gap and

adherens junctions,

of which the latter, and particularly the adherens junction component,

vascular endothelial (VE)-cadherin,

are of central importance for the initiation and stabilization of cell–cell contacts.

Although multiple adhesion molecules are localized at endothelial junctions,

  • blocking the adhesive function of VE-cadherin using antibodies
  • is sufficient to disrupt endothelial junctions and
  • to increase endothelial monolayer permeability both in vitro and in vivo.

Like other cadherins, VE-cadherin mediates adhesion via

  • homophilic, calcium-dependent interactions.

This cell–cell adhesion

is strengthened by binding of cytoplasmic proteins, the catenins,

to the C-terminus of VE-cadherin.

VE-cadherin can directly bind

  • β-catenin and plakoglobin, which
  • both associate with the actin binding protein α-catenin.

Initially, α-catenin was thought to directly anchor cadherins to the actin cytoskeleton, but recently it became clear that

  • α-catenin cannot bind to both β-catenin and actin simultaneously.

Numerous lines of evidence indicate that p120-catenin

  • promotes VE-cadherin surface expression and stability at the plasma membrane.

Different models are proposed that describe how

  • p120-catenin regulates cadherin membrane dynamics, including the hypothesis
  • that p120-catenin functions as a ‘cap’ that prevents the interaction of VE-cadherin
  • with the endocytic membrane trafficking machinery.

In addition, p120-catenin might regulate VE-cadherin internalization

  • through interactions with small GTPases.

Cytoplasmic p120-catenin, which is not bound to VE-cadherin, has been shown to

decrease RhoA activity,

elevate active Rac1 and Cdc42, and thereby is thought

to regulate actin cytoskeleton organization and membrane trafficking.

The intact cadherin-catenin complex is required for proper functioning of the adherens junction.

Several mechanisms may be involved in the

  • regulation of the organization and function of the cadherin–catenin complex, including
  1. endocytosis of the complex,
  2. VE-cadherin cleavage and
  3. actin cytoskeleton reorganization.

The remainder of this review primarily focuses on the

role of tyrosine phosphorylation in the control of VE-cadherin-mediated cell–cell adhesion.

Regulation of the adhesive function of VE-cadherin by tyrosine phosphorylation

It is a widely accepted concept that tyrosine phosphorylation of

  • components of the VE–cadherin-catenin complex
  • Correlates with the weakening of cell–cell adhesion.

A general idea has emerged that

tyrosine phosphorylation of the VE-cadherin complex

leads to the uncoupling of VE-cadherin from the actin cytoskeleton

through dissociation of catenins from the cadherin.

However, tyrosine phosphorylation of VE-cadherin

  • is required for efficient transmigration of leukocytes.

This suggests that VE-cadherin-mediated cell–cell contacts

1.are not just pushed open by the migrating leukocytes, but play

2.a more active role in the transmigration process.

A schematic overview of leukocyte adhesion-induced signals leading to VE-cadherin phosphorylation

Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin.

Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin

Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin

N-glycosylation status of E-cadherin controls cytoskeletal dynamics through the organization of distinct β-catenin- and γ-catenin-containing AJs

N-glycosylation of E-cadherin has been shown to inhibit cell–cell adhesion.

Specifically, our recent studies have provided evidence that

  • the reduction of E-cadherin N-glycosylation
  • promoted the recruitment of stabilizing components,
  • vinculin and serine/ threonine protein phosphatase 2A (PP2A), to adherens junctions (AJs)
  • and enhanced the association of AJs with the actin cytoskeleton.

Here, we examined the details of how

N-glycosylation of E-cadherin affected the molecular organization of AJs and their cytoskeletal interactions.

Using the hypoglycosylated E-cadherin variant, V13, we show that

V13/β-catenin complexes preferentially interacted with PP2A and with the microtubule motor protein dynein.

This correlated with dephosphorylation of the microtubule-associated protein tau, suggesting that

increased association of PP2A with V13-containing AJs promoted their tethering to microtubules.

These studies provide the first mechanistic insights into how N-glycosylation of E-cadherin drives changes in AJ composition through

  • the assembly of distinct β-catenin- and γ-catenin-containing scaffolds that impact the interaction with different cytoskeletal components

Cytoskeletal Basis of Ion Channel Function in Cardiac Muscle

MacKinnon. Fig 1  Ion channels exhibit three basic properties

MacKinnon. Fig 1 Ion channels exhibit three basic properties

In order to contract and accommodate the repetitive morphological changes induced by the cardiac cycle, cardiomyocytes

depend on their highly evolved and specialized cytoskeletal apparatus.

Defects in components of the cytoskeleton, in the long term,

affect the ability of the cell to compensate at both functional and structural levels.

In addition to the structural remodeling,

the myocardium becomes increasingly susceptible to altered electrical activity leading to arrhythmogenesis.

The development of arrhythmias secondary to structural remodeling defects has been noted, although the detailed molecular mechanisms are still elusive.

subjects with severe left ventricular chamber dilation such as in DCM can have left bundle branch block (LBBB), while right bundle branch block (RBBB) is more characteristic of right ventricular failure.  LBBB and RBBB have both been repeatedly associated with AV block in heart failure.

The impact of volume overload on structural and electro-cardiographic alterations has been noted in cardiomyopathy patients treated with left ventricular assist device (LVAD) therapy, which puts the heart at mechanical rest.

In LVAD-treated subjects,

QRS- and both QT- and QTc duration decreased,

suggesting that QRS- and QT-duration are significantly influenced by mechanical load and

that the shortening of the action potential duration contributes to the improved contractile performance after LVAD support.

An early postoperative period study after cardiac unloading therapy in 17 HF patients showed that in the first two weeks after LVAD implantation,

HF was associated with a relatively high incidence of ventricular arrhythmias associated with QTc interval prolongation.

In addition, a recent retrospective study of 100 adult patients with advanced HF, treated with an axial-flow HeartMate LVAD suggested that

  • the rate of new-onset monomorphic ventricular tachycardia (MVT) was increased in LVAD treated patients compared to patients given only medical treatment,

The myocardium is exposed to severe and continuous biomechanical stress during each contraction-relaxation cycle. When fiber tension remains uncompensated or simply unbalanced,

it may represent a trigger for arrhythmogenesis caused by cytoskeletal stretching,

which ultimately leads to altered ion channel localization, and subsequent action potential and conduction alterations.

Cytoskeletal proteins not only provide the backbone of the cellular structure, but they also

maintain the shape and flexibility of the different sub-cellular compartments, including the

1.plasma membrane,

2.the double lipid layer, which defines the boundaries of the cell and where

ion channels are mainly localized.

The interaction between the sarcomere, which is the basic for the passive force during diastole and for the restoring force during systole.

Sarcomeric Proteins and Ion Channels

besides fiber stretch associated with mechanical and hemodynamic impairment, cytoskeletal alterations due to primary genetic defects or indirectly to alterations in response to cellular injury can potentially

1.affect ion channel anchoring, and trafficking, as well as

2.functional regulation by second messenger pathways,

3.causing an imbalance in cardiac ionic homeostasis that will trigger arrhythmogenesis.

Intense investigation of

the sarcomeric actin network,

the Z-line structure, and

chaperone molecules docking in the plasma membrane,

has shed new light on the molecular basis of

  • cytoskeletal interactions in regulating ion channels

Actin disruption using cytochalasin D, an agent that interferes with actin polymerization, increased Na+ channel activity in 90% of excised patches tested within 2 min, which indicated that

the integrity of the filamentous actin (F-actin) network was essential for the maintenance of normal Na+ channel function

These data were the first to support a role for the cytoskeleton in cardiac arrhythmias.

Molecular interactions between the cytoskeleton and ion channels

The figure illustrates the interactions between the ion channels on the sarcolemma, and the sarcomere in cardiac myocytes. Note that the Z-line is connected to the cardiac T-tubules. The diagram illustrates the complex protein-protein interactions that occur between structural components of the cytoskeleton and ion channels. The cytoskeleton is involved in regulating the metabolism of ion channels, modifying their expression, localization, and electrical properties.

sarcomere structure

sarcomere structure

It is important to be aware of the enormous variety of clinical presentations that derive from distinct variants in the same pool of genetic factors. Knowledge of these variants could facilitate tailoring the therapy of choice for each patient. In particular,

the recent findings of structural and functional links between

the cytoskeleton and ion channels

could expand the therapeutic interventions in

arrhythmia management in structurally abnormal myocardium, where aberrant binding

between cytoskeletal proteins can directly or indirectly alter ion channel function.

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