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New Mutant KRAS Inhibitors Are Showing Promise in Cancer Clinical Trials: Hope For the Once ‘Undruggable’ Target, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 1: Next Generation Sequencing (NGS)

New Mutant KRAS Inhibitors Are Showing Promise in Cancer Clinical Trials: Hope For the Once ‘Undruggable’ Target

Curator: Stephen J. Williams, Ph.D.

UPDATED 10/2/2021

A Newly Identified Mutant RAS G12C  GTPase Activating Protein (GAP) may lead to discovery of new class of RAS inhibitors 

From the journal Science in Filling in the GAPs in understanding RAS: A newly identified regulator increases the efficacy of a new class of targeted anti-RAS drugs.

Source:

SCIENCE•8 Oct 2021•Vol 374Issue 6564•pp. 152-153•DOI: 10.1126/science.abl3639

    According to canonical thinking, mutationally activated RAS (constitutive activation) is insensitive to GTPase-activating proteins (GAPs).  GAPs accelerated the conversion of GTP bound to RAS to GDP, a critical step in the inactivation of RAS signaling cycle.  However a small molecule has just been FDA approved, sotorasib, in cancer cells by binding only the GDP bound KRAS G12C mutant. A few non small cell lung cancers (NSCLC) are resistant to another such inhibitor, adagrasib.  In the same Science issue, another paper by Li et. al. explains the potential that a GAP, RGS3, may play in this conundrum and demonstrates that certain other inhibitors of the RAS cycle, mainly the GEF (guanine exchange factor) SOS1, in combination with MEK inhibitors may circumvent this resistance.

Inhibitors of mutant KRAS
The KRASG12C (Gly12→Cys) mutant is refractory to canonical GAPs, p120 RASGAP and neurofibromin, but not RGS3, which promotes GTP hydrolysis. The resulting GDP-bound KRASG12C is an anticancer drug target. Combination with inhibitors of SOS1 or with inhibitors of downstream signaling may further improve efficacy.
GRAPHIC: C. BICKEL/SCIENCE
 
First a discussion of the RAS signalling cycle is shown below in a good review of RAS activation and signal termination.
 
PMCID: PMC3124093
NIHMSID: NIHMS294865
PMID: 21102635
Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy?

Abstract

There is now considerable and increasing evidence for a causal role of aberrant activity of the Ras superfamily of small GTPases in human cancers. These GTPases act as GDP-GTP-regulated binary switches that control many fundamental cellular processes. A common mechanism of GTPase deregulation in cancer is the deregulated expression and/or activity of their regulatory proteins, guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound state and GTPase activating proteins (GAPs) that return the GTPase to its GDP-bound inactive state. We assess the association of GEFs and GAPs with cancer and their druggability for cancer therapeutics.

Box 1

Ras superfamily of small GTPases

The human Ras superfamily comprised of over 150 members which is divided into five major branches on the basis of sequence and functional similarities. In addition to the three Ras isoforms, other members of the Ras family with important roles in cancer include Rheb and Ral proteins. The ~20 kDa core G domain (corresponding to Ras residues 4–166) is conserved among all Ras superfamily proteins and is involved in GTP binding and hydrolysis. This domain is comprised of five conserved guanine nucleotide consensus sequence elements (Ras residue numbering) involved in binding phosphate/Mg2+ (PM) or the guanine base (G). The switch I (Ras residues 30–38) and II (59–76) regions change in conformation during GDP-GTP cycling and contribute to preferential effector binding to the GTP-bound state and the core effector domain (E; Ras residues 32–40). Ras and Rho family proteins have additional C-terminal hypervariable (HV) sequences that commonly terminate with a CAAX motif that signals for farnesyl or geranylgeranyl isoprenoid addition to the cysteine residue, proteolytic removal of the AAX residues and carboxylmethylation of the prenylated cysteine. Some are modified additionally by a palmitate fatty acid to cysteine residues in the HV sequence that contributes to membrane association. Rab proteins also contain a C-terminal HV region that terminates with cysteine-containing motifs that are modified by addition of geranylgeranyl lipids, with some undergoing carboxylmethylation. Arf family proteins are characterized by an N-terminal extension involved in membrane interaction, with some cotranslationally modified by addition of a myristate fatty acid. Ran is not lipid modified but contains a C-terminal extension that is essential for function. Rho proteins are characterized by an up to 13 amino acid “Rho insert” sequence positioned between Ras residues 122 and 123 involved in effector regulation.

 

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The GDP-GTP cycle

Ras superfamily proteins possess intrinsic guanine nucleotide exchange and GTP hydrolysis activities. However, these activities are too low to allow efficient and rapid cycling between their active GTP-bound and inactive GDP-bound states. GEFs and GAPs accelerate and regulate these intrinsic activities. Members of the different branches of the superfamily are regulated by GEFs and GAPs with structurally distinct catalytic domains. Here we have utilized the Rho family as an example to illustrate the complexity of this process, where multiple GEFs and GAPs may regulate one specific GTPase. For the 20 human Rho GTPases there are 83 GEFs and 67 GAPs and a subset of Rho GTPases are not likely regulated by GEFs and GAPs (e.g., Rnd3/RhoE). Rho GTPases are activated by distinct RhoGEF families. Dbl family RhoGEFs (68) possesses a tandem Dbl homology (DH) catalytic and pleckstrin homology (PH) regulatory domain topology. DOCK family RhoGEFs (11) are characterized by two regions of high sequence conservation that are designated Dock-homology region regulatory DHR-1 and catalytic DHR-2 domains. Two other RhoGEFs have been described (SWAP70 and SLAT) contain a PH but no DH domain (2) and smgGDS (1) is an unusual GEF in that it functions as a GEF for some Rho as well as non-Rho family GTPases. At least 24 Dbl RhoGEFs have been reported to activate RhoA. Rho (and Rab) GTPases are also controlled by a third class of regulatory proteins, Rho dissociation inhibitors (RhoGDI) (of which there are 3) whose main function involves regulation of Rho GTPase membrane association by masking the isoprenoid group.

 

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So GEFs like SOS 1,2,3 are responsible for activation of the G Protien RAS upon Receptor Tyrosine Kinase (RTK) activation by a multitude of growth factors.  The GAPs are responsible for termination of the activated RAS cycle and now RAS, in its normal unmuted version, can now be activated again.  The G12C mutant keeps RAS in its activated state as GAPs cannot fascilitate the hydroylsis of GTP.  

Li et al. (in this issue) theorized that there must be cellular factors that stimulate formation of GDP-bound KRAS G12C, enabling its vulnerability to KRAS G12C inhibitors, and discovered a regulator of G protein signaling 3 (RGS3), which had GAP activity previously for heterotrimeric G proteins GαI and Gαq with unexpected GAP activity for the KRAS G12C mutant.   They also found the fully activated GTP bound KRAS G12C is dependent on RTK-mediated activation of SOS1.  This suggested that SOS1 inhibitors with MEK inhibitors could be effective against mutant KRAS.

The paper by Li is summarized below:

The G protein signaling regulator RGS3 enhances the GTPase activity of KRAS

SCIENCE•8 Oct 2021•Vol 374Issue 6564•pp. 197-201•DOI: 10.1126/science.abf1730

Abstract

Recently reported to be effective in patients with lung cancer, KRASG12C inhibitors bind to the inactive, or guanosine diphosphate (GDP)–bound, state of the oncoprotein and require guanosine triphosphate (GTP) hydrolysis for inhibition. However, KRAS mutations prevent the catalytic arginine of GTPase-activating proteins (GAPs) from enhancing an otherwise slow hydrolysis rate. If KRAS mutants are indeed insensitive to GAPs, it is unclear how KRASG12C hydrolyzes sufficient GTP to allow inactive state–selective inhibition. Here, we show that RGS3, a GAP previously known for regulating G protein–coupled receptors, can also enhance the GTPase activity of mutant and wild-type KRAS proteins. Our study reveals an unexpected mechanism that inactivates KRAS and explains the vulnerability to emerging clinically effective therapeutics.

UPDATED 09/26/2021

The KRAS G12C Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients

Source: Hallin J, Engstrom LD, Hargis L, Calinisan A, Aranda R, Briere DM, Sudhakar N, Bowcut V, Baer BR, Ballard JA, Burkard MR, Fell JB, Fischer JP, Vigers GP, Xue Y, Gatto S, Fernandez-Banet J, Pavlicek A, Velastagui K, Chao RC, Barton J, Pierobon M, Baldelli E, Patricoin EF 3rd, Cassidy DP, Marx MA, Rybkin II, Johnson ML, Ou SI, Lito P, Papadopoulos KP, Jänne PA, Olson P, Christensen JG. The KRASG12C Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients. Cancer Discov. 2020 Jan;10(1):54-71. doi: 10.1158/2159-8290.CD-19-1167. Epub 2019 Oct 28. PMID: 31658955; PMCID: PMC6954325.

Abstract

Despite decades of research, efforts to directly target KRAS have been challenging. MRTX849 was identified as a potent, selective, and covalent KRASG12C inhibitor that exhibits favorable drug-like properties, selectively modifies mutant cysteine 12 in GDP-bound KRASG12C, and inhibits KRAS-dependent signaling. MRTX849 demonstrated pronounced tumor regression in 17 of 26 (65%) KRASG12C-positive cell line- and patient-derived xenograft models from multiple tumor types, and objective responses have been observed in patients with KRASG12C-positive lung and colon adenocarcinomas. Comprehensive pharmacodynamic and pharmacogenomic profiling in sensitive and partially resistant nonclinical models identified mechanisms implicated in limiting antitumor activity including KRAS nucleotide cycling and pathways that induce feedback reactivation and/or bypass KRAS dependence. These factors included activation of receptor tyrosine kinases (RTK), bypass of KRAS dependence, and genetic dysregulation of cell cycle. Combinations of MRTX849 with agents that target RTKs, mTOR, or cell cycle demonstrated enhanced response and marked tumor regression in several tumor models, including MRTX849-refractory models. SIGNIFICANCE: The discovery of MRTX849 provides a long-awaited opportunity to selectively target KRASG12C in patients. The in-depth characterization of MRTX849 activity, elucidation of response and resistance mechanisms, and identification of effective combinations provide new insight toward KRAS dependence and the rational development of this class of agents.

Introduction

KRAS is one of the most frequently mutated oncogenes in cancer; however, efforts to directly target KRAS have been largely unsuccessful due to its high affinity for GTP/GDP and the lack of a clear binding pocket (1–4). More recently, compounds were identified that covalently bind to KRASG12C at the cysteine 12 residue, lock the protein in its inactive GDP-bound conformation, inhibit KRAS-dependent signaling, and elicit antitumor responses in tumor models (5–7). Advances on early findings demonstrated that the binding pocket under the switch II region was exploitable for drug discovery, culminating in the identification of more potent KRASG12C inhibitors with improved physiochemical properties that are now entering clinical trials. The identification of KRASG12C inhibitors provides a renewed opportunity to develop a more comprehensive understanding of the role of KRAS as a driver oncogene and to explore the clinical utility of direct KRAS inhibition.

KRASG12C mutations are present in lung and colon adenocarcinomas as well as smaller fractions of other cancers. The genetic context of KRASG12C alteration can vary significantly among tumors and is predicted to affect response to KRAS inhibition. KRAS mutations are often enriched in tumors due to amplification of mutant or loss of wild-type allele (8, 9). In addition, KRAS mutations often co-occur with other key genetic alterations including TP53 and CDKN2A in multiple cancers, KEAP1 and/or STK11 in lung adenocarcinoma, or APC and PIK3CA in colon cancer (3, 8–12). Whether differences in KRAS-mutant allele fraction or co-occurrence with other mutations influence response to KRAS blockade is not yet well understood. In addition, due to the critical importance of the RAS pathway in normal cellular function, there is extensive pathway isoform redundancy and a comprehensive regulatory network in normal cells to ensure tight control of temporal pathway signaling. RAS pathway negative feedback signaling is mediated by ERK1/2 and receptor tyrosine kinases (RTK) as well as by ERK pathway target genes including dual-specificity phosphatases (DUSP) and Sprouty (SPRY) proteins (13–17). One important clinically relevant example is provided by the reactivation of ERK signaling observed following treatment of BRAFV600E-mutant cancers with selective BRAF inhibitors (18–20). The observed intertumoral heterogeneity and extensive feedback signaling network in KRAS-mutant cancers may necessitate strategies to more comprehensively block oncogenic signal transduction and deepen the antitumor response in concert with KRAS blockade (15, 21, 22).

Potential strategies to augment the response to KRASG12C inhibitor treatment are evident at multiple nodes of the signaling pathway regulatory machinery. RAS proteins are small GTPases that normally cycle between an active, GTP-bound state and an inactive, GDP-bound state. RAS proteins are loaded with GTP through guanine nucleotide exchange factors (e.g., SOS1) which are activated by upstream RTKs, triggering subsequent interaction with effector proteins that activate RAS-dependent signaling. RAS proteins hydrolyze GTP to GDP through their intrinsic GTPase activity, which is dramatically enhanced by GTPase-activating proteins (GAP). Mutations at codons 12 and 13 in RAS proteins impair GAP-stimulated GTP hydrolysis, leaving RAS predominantly in the GTP-bound, active state.

Potent covalent KRASG12C inhibitors described to date bind only GDP-bound KRAS (5–7). Although codon 12 and 13 mutations decrease the fraction of GDP-bound KRAS, recent biochemical analyses revealed that KRASG12C exhibits the highest intrinsic GTP hydrolysis rate and highest nucleotide exchange rate among KRAS mutants (23). Furthermore, the nucleotide-bound state of KRASG12C can be shifted toward the GDP-bound state by pharmacologically modulating upstream signaling with RTK inhibitors that increase the activity of KRASG12C inhibitors (7, 22, 24). Likewise, SHP2 is a phosphatase that positively transduces RTK signaling to KRAS. Accordingly, SHP2 inhibitors are active in cancers driven by KRAS mutations that are dependent on nucleotide cycling, including KRASG12C (25–27).

MRTX849 is among the first KRASG12C inhibitors to advance to clinical trials. The comprehensive and durable inhibition of KRASG12C by MRTX849 provides a unique opportunity to understand the extent to which KRAS functions as an oncogenic driver. In addition, the observation that the response to blockade of KRAS is markedly different in vitro and in vivo indicates that evaluation of the consequences of KRAS blockade in in vivo model systems is critical to understand the role of KRAS-driven tumor progression. The demonstration of partial responses in patients with lung and colon adenocarcinomas treated with MRTX849 in clinical trials indicates that results observed in tumor models extend to KRASG12C-positive human cancers. Our comprehensive molecular characterization of multiple tumor models at baseline and during response to KRAS inhibition has provided further insight toward the contextual role of KRAS mutation in the setting of genetic and tumoral heterogeneity. Finally, further interrogation of these genetic alterations and signaling pathways utilizing functional genomics strategies including CRISPR and combination approaches uncovered regulatory nodes that sensitize tumors to KRAS inhibition when cotargeted.

Introduction

KRAS is one of the most frequently mutated oncogenes in cancer; however, efforts to directly target KRAS have been largely unsuccessful due to its high affinity for GTP/GDP and the lack of a clear binding pocket (1–4). More recently, compounds were identified that covalently bind to KRASG12C at the cysteine 12 residue, lock the protein in its inactive GDP-bound conformation, inhibit KRAS-dependent signaling, and elicit antitumor responses in tumor models (5–7). Advances on early findings demonstrated that the binding pocket under the switch II region was exploitable for drug discovery, culminating in the identification of more potent KRASG12C inhibitors with improved physiochemical properties that are now entering clinical trials. The identification of KRASG12C inhibitors provides a renewed opportunity to develop a more comprehensive understanding of the role of KRAS as a driver oncogene and to explore the clinical utility of direct KRAS inhibition.

KRASG12C mutations are present in lung and colon adenocarcinomas as well as smaller fractions of other cancers. The genetic context of KRASG12C alteration can vary significantly among tumors and is predicted to affect response to KRAS inhibition. KRAS mutations are often enriched in tumors due to amplification of mutant or loss of wild-type allele (8, 9). In addition, KRAS mutations often co-occur with other key genetic alterations including TP53 and CDKN2A in multiple cancers, KEAP1 and/or STK11 in lung adenocarcinoma, or APC and PIK3CA in colon cancer (3, 8–12). Whether differences in KRAS-mutant allele fraction or co-occurrence with other mutations influence response to KRAS blockade is not yet well understood. In addition, due to the critical importance of the RAS pathway in normal cellular function, there is extensive pathway isoform redundancy and a comprehensive regulatory network in normal cells to ensure tight control of temporal pathway signaling. RAS pathway negative feedback signaling is mediated by ERK1/2 and receptor tyrosine kinases (RTK) as well as by ERK pathway target genes including dual-specificity phosphatases (DUSP) and Sprouty (SPRY) proteins (13–17). One important clinically relevant example is provided by the reactivation of ERK signaling observed following treatment of BRAFV600E-mutant cancers with selective BRAF inhibitors (18–20). The observed intertumoral heterogeneity and extensive feedback signaling network in KRAS-mutant cancers may necessitate strategies to more comprehensively block oncogenic signal transduction and deepen the antitumor response in concert with KRAS blockade (15, 21, 22).

Potential strategies to augment the response to KRASG12C inhibitor treatment are evident at multiple nodes of the signaling pathway regulatory machinery. RAS proteins are small GTPases that normally cycle between an active, GTP-bound state and an inactive, GDP-bound state. RAS proteins are loaded with GTP through guanine nucleotide exchange factors (e.g., SOS1) which are activated by upstream RTKs, triggering subsequent interaction with effector proteins that activate RAS-dependent signaling. RAS proteins hydrolyze GTP to GDP through their intrinsic GTPase activity, which is dramatically enhanced by GTPase-activating proteins (GAP). Mutations at codons 12 and 13 in RAS proteins impair GAP-stimulated GTP hydrolysis, leaving RAS predominantly in the GTP-bound, active state.

Potent covalent KRASG12C inhibitors described to date bind only GDP-bound KRAS (5–7). Although codon 12 and 13 mutations decrease the fraction of GDP-bound KRAS, recent biochemical analyses revealed that KRASG12C exhibits the highest intrinsic GTP hydrolysis rate and highest nucleotide exchange rate among KRAS mutants (23). Furthermore, the nucleotide-bound state of KRASG12C can be shifted toward the GDP-bound state by pharmacologically modulating upstream signaling with RTK inhibitors that increase the activity of KRASG12C inhibitors (7, 22, 24). Likewise, SHP2 is a phosphatase that positively transduces RTK signaling to KRAS. Accordingly, SHP2 inhibitors are active in cancers driven by KRAS mutations that are dependent on nucleotide cycling, including KRASG12C (25–27).

MRTX849 is among the first KRASG12C inhibitors to advance to clinical trials. The comprehensive and durable inhibition of KRASG12C by MRTX849 provides a unique opportunity to understand the extent to which KRAS functions as an oncogenic driver. In addition, the observation that the response to blockade of KRAS is markedly different in vitro and in vivo indicates that evaluation of the consequences of KRAS blockade in in vivo model systems is critical to understand the role of KRAS-driven tumor progression. The demonstration of partial responses in patients with lung and colon adenocarcinomas treated with MRTX849 in clinical trials indicates that results observed in tumor models extend to KRASG12C-positive human cancers. Our comprehensive molecular characterization of multiple tumor models at baseline and during response to KRAS inhibition has provided further insight toward the contextual role of KRAS mutation in the setting of genetic and tumoral heterogeneity. Finally, further interrogation of these genetic alterations and signaling pathways utilizing functional genomics strategies including CRISPR and combination approaches uncovered regulatory nodes that sensitize tumors to KRAS inhibition when cotargeted.

Results

MRTX849 Is a Potent and Selective Inhibitor of KRASG12C, KRAS-Dependent Signal Transduction, and Cell Viability In Vitro

A structure-based drug design approach, including optimization for favorable drug-like properties, led to the discovery of MRTX849 as a potent, covalent KRASG12C inhibitor (Fig. 1A; Supplementary Table S1). An LC/MS-based KRASG12C protein modification assay revealed that MRTX849 demonstrated much greater modification of KRASG12C when preloaded with GDP compared with GTP (Supplementary Table S2), supporting that MRTX849 binds to and stabilizes the inactive GDP-bound form of KRASG12C. Indeed, introducing a comutation that impairs the GTPase activity of KRASG12C (24) attenuated the inhibitory activity of MRTX1257, a close analogue of MRTX849 (Supplementary Fig. S1A). Secondary mutations that modulate the nucleotide exchange function of KRASG12C also affected inhibition by MRTX1257, supporting that the MRTX compound series is dependent on KRASG12C nucleotide cycling.

Figure 1.

 

MRTX849 is a potent, covalent KRASG12C inhibitor in vitroA, Structure of MRTX849. B, Immunoblot protein Western blot analyses of KRAS pathway targets in MIA PaCa-2 cells treated from 1 hours to 72 hours with MRTX849 at 100 nmol/L. C, Immunoblot protein Western blot analyses of KRAS pathway targets in MIA PaCa-2 cells treated for 24 hours with MRTX849 over a 13-point dose response. D, Left y-axis shows active RAS ELISA assay to determine the reduction in RAS-GTP abundance following MRTX849 treatment in MIA PaCa-2 cells for 24 hours. The vehicle value was normalized to 1 by dividing all average values by the vehicle value. Right y-axis shows quantitation of KRAS band shift by MRTX849 treatment in MIA PaCa-2 cells for 24 hours as assessed by Western blot and densitometry. E, In-cell Western blot assay to evaluate modulation of pERK in MIA PaCa-2 cells grown in standard tissue-culture conditions treated with MRTX849 over a time course. F, CellTiter-Glo assay to evaluate cell viability performed on seven KRASG12C-mutant cell lines and three non–KRASG12C-mutant cell lines grown in 2-D tissue-culture conditions in a 3-day assay (left plot) or 3-D conditions using 96-well, ULA plates in a 12-day assay (right plot).

 

We next determined the cellular activity of MRTX849 utilizing the KRASG12C-mutant H358 lung and MIA PaCa-2 pancreatic cancer cell lines. In both models, MRTX849 demonstrated an upward electrophoretic mobility shift of KRASG12C protein band migration by immunoblot, indicative of covalent modification of KRASG12C. A maximal mobility shift was observed by 1 hour, was maintained through 72 hours (Fig 1B; Supplementary Fig. S1B), and was evident at concentrations as low as 2 nmol/L with near-maximal modification observed at 15.6 nmol/L (Fig. 1C; Supplementary Fig. S1C). Comparable inhibition of active RAS was observed as determined by a RAF RAS-binding domain capture ELISA assay (Fig. 1D; 1D). MRTX849 also inhibited KRAS-dependent signaling targets including ERK1/2 phosphorylation (pERK; Thr202/Tyr204 ERK1), S6 phosphorylation (pS6; RSK-dependent Ser235/236), and expression of the ERK-regulated DUSP6, each with IC50 values in the single-digit nanomolar range in both cell lines (Fig. 1B and C; Supplementary Fig. S1B and S1C). The evaluation of pERK over a time course of 48 hours indicated maximal inhibition was observed at 24 hours (Fig. 1E; Supplementary Fig. S1E). Treatment with the des-acrylamide version of MRTX849, which is unable to covalently bind to KRASG12C, did not demonstrate significant inhibition of ERK phosphorylation (Supplementary Fig. S1F). The H358 cell line was selected for determination of MRTX849 cysteine selectivity utilizing an LC/MS-based proteomics approach able to detect approximately 6,000 cysteine-containing peptides. After treatment for 3 hours, decreased KRASG12C Cys12-free peptide was detected with treated-to-control ratios of 0.029 and 0.008 determined at 1 and 10 μmol/L, respectively, indicating near-complete engagement of the intended target (Supplementary Table S3). In contrast, the only other peptides identified were from lysine-tRNA ligase (KARS) at Cys209 near the detection limit, indicating a high degree of selectivity toward KRASCYS12.

To evaluate the breadth of MRTX849 activity, its effect on cell viability was determined across a panel of 17 KRASG12C-mutant and 3 non–KRASG12C-mutant cancer cell lines using 2-D (3-day, adherent cells) and 3-D (12-day, spheroids) cell-growth conditions. MRTX849 potently inhibited cell growth in the vast majority of KRASG12C-mutant cell lines with IC50 values ranging between 10 and 973 nmol/L in the 2-D format and between 0.2 and 1,042 nmol/L in the 3-D format (Supplementary Table S4; Fig. 1F). In agreement with prior KRASG12C inhibitor studies (5), MRTX849 demonstrated improved potency in the 3-D assay format, as all but one KRASG12C-mutant cell line exhibited an IC50 value below 100 nmol/L. Although MRTX849 was broadly effective in inhibiting viability of KRASG12C-mutant cell lines, IC50 values varied across the cell panel by 100-fold, suggesting a differential degree of sensitivity to treatment. All three non–KRASG12C-mutant cell lines tested demonstrated IC50 values greater than 1 μmol/L in 2-D conditions and greater than 3 μmol/L in 3-D conditions, suggesting the effect of MRTX849 on cell viability was dependent on the presence of KRASG12C.

To determine whether the difference in sensitivity across the cell panel correlated with the ability of MRTX849 to bind to KRAS or inhibit KRAS-dependent signal transduction, seven KRASG12C-mutant cancer cell lines were selected from the panel for further evaluation. In each cell line, MRTX849 demonstrated a very similar concentration-dependent electrophoretic mobility shift (IC50) for KRASG12C protein migration, suggesting that the ability to bind to and modify KRASG12C does not readily account for differences in response in viability studies (Fig. 1B and C; Supplementary Figs. S1B and S1C and S2A and S2B). The effect of MRTX849 on selected phosphoproteins implicated in mediating KRAS-dependent signaling was also evaluated across the cell panel by immunoblot and/or reverse-phase protein array (RPPA) following treatment for 6 or 24 hours. Notably, the concentration–response relationship and maximal effect of MRTX849 on inhibition of ERK and S6S235/236 phosphorylation varied across the cell panel (Supplementary Fig. S2A and S2C; Supplementary Table S7). MRTX849 demonstrated only partial inhibition of pERK in KYSE-410 and SW1573 cells and a minimal effect on pS6S235/236 in SW1573, H2030, and KYSE-410 cells (Supplementary Fig. S2A and S2C). Each of these cell lines were among those that exhibited a submaximal response to MRTX849 in both 2-D and 3-D viability assays (Fig. 1F). Although KRAS is implicated in mediating signal transduction through the PI3K and mTOR pathways, there was minimal evidence of a significant and/or durable effect of MRTX849 on AKT (S473, T308) or 4E-BP1 (T37/T46, S65, T70) phosphorylation at any time point in any cell lines evaluated (Supplementary Fig. S2D). However, MRTX849 demonstrated concentration-dependent partial inhibition of the mTOR-dependent signaling targets p70 S6 kinase (T412) and/or pS6 (S240/44) in the H358, MIA PaCa-2,H2122, and H1373 cell lines, each of which exhibited a maximal response to treatment. Together, these data suggest that maximizing inhibition of KRAS-dependent ERK and S6 signaling may be required to elicit a robust response in tumor-cell viability assays.

MRTX849 Treatment In Vivo Leads to Dose-Dependent KRASG12C Modification, KRAS Pathway Inhibition, and Antitumor Efficacy

Studies were conducted to evaluate MRTX849 antitumor activity along with its pharmacokinetic and pharmacodynamic properties in vivo, both to understand the clinical utility of this agent and to provide insight toward response to treatment. MRTX849 demonstrated moderate plasma clearance and prolonged half-life following oral administration (Supplementary Table S1; Supplementary Fig. S3). To evaluate the pharmacodynamic response to MRTX849 and to correlate drug exposure with target inhibition, MRTX849 was administered via oral gavage over a range of dose levels to H358 xenograft–bearing mice, and plasma and tumors were collected at defined time points. The fraction of covalently modified KRASG12C protein was proportional to the plasma concentration of MRTX849 (Fig. 2A). When evaluated over time after a single oral dose at 30 mg/kg, the modified fraction of KRASG12C was 74% at 6 hours after dose and gradually decreased to 47% by 72 hours (Fig. 2B). This extended pharmacodynamic effect, despite declining levels of MRTX849 in plasma, was consistent with the irreversible inhibition of KRASG12C by MRTX849 and the relatively long half-life for the KRASG12C protein (∼24–48 hours; Supplementary Table S5). The modification of KRASG12C was maximized after repeated daily dosing for 3 days at 30 mg/kg (Fig. 2B), and higher dose levels did not demonstrate additional KRASG12C modification in multiple tumor models (data not shown). The maximum level of modification of approximately 80%, despite increasing dose and plasma levels of MRTX849, suggests that accurate measurement of complete inhibition of KRASG12C utilizing LC/MS may not be attainable, potentially due to a pool of active, noncycling, or unfolded KRASG12C protein in tumors. Together, these studies demonstrated a dose-dependent increase in covalent modification of KRASG12C by MRTX849 and that the majority of targetable KRAS was covalently modified by MRTX849 over a repeated administration schedule at dose levels at or exceeding 30 mg/kg.

Figure 2.

 

MRTX849 modifies KRASG12C and inhibits KRAS signaling and tumor growth in vivoA, MRTX849 was administered orally as a single dose to mice bearing established H358 xenografts (average tumor volume ∼350 mm3) at 10, 30, and 100 mg/kg. KRAS modification and MRTX849 plasma concentration data from n = 3 mice are shown as mean ± SD. KRASG12C modification was statistically significant versus vehicle control using the two-tailed Student t test. **, P < 0.01. B, MRTX849 was administered orally as a single dose or daily (QD) for 3 days to mice bearing established H358 xenografts (average tumor volume ∼350 mm3) at 30 mg/kg. Plasma was collected at 0.5, 2, 6, 24, 48, and 72 hours after administration of the last dose, and tumors were collected at 6, 24, 48, and 72 hours after dose. KRASG12C modification and MRTX849 plasma concentration data are shown from n = 3 mice as mean ± SD. Induction of modified KRASG12C protein at all time points was determined to be statistically significant versus vehicle control using two-way ANOVA. In addition, induction of modified KRASG12C protein at 72 hours in day 1 samples and 48 and 72 hours in day 3 samples was statistically significant versus the 6-hour time point. Brackets indicate P < 0.05 as compared with left-most sample. C, MRTX849 was administered as in A. Tumors were collected 6 hours after dose, and total and phosphorylated ERK1/2 and total and phosphorylated S6 were analyzed by immunoblot and quantified by densitometric analysis. Relative fluorescence intensity of pERK1/2 and pS6 was normalized by dividing pERK1/2 and pS6 by total ERK1/2 and total S6, respectively. Vehicle-treated tumors were normalized to 1 by dividing all average values by the vehicle value. Average pERK1/2 and pS6 values were divided by the average value in vehicle-treated tumors. Data shown represent the average of 2 to 3 tumors per treatment group plus SD. Reduction of pS6 relative fluorescence intensity was determined to be statistically significant versus vehicle control using the two-tailed Student t test. Brackets indicate P < 0.05 compared with left-most sample. D, MRTX849 was administered as in B. Tumors were collected at 6, 24, 48, or 72 hours after administration of the last dose, and total and phosphorylated ERK1/2 and total and phosphorylated S6 were analyzed as in C. Data shown represent the average of 3 to 4 tumors per treatment group plus SD. Reduction of pS6 relative fluorescence intensity on day 3 was determined to be statistically significant versus vehicle control using two-way ANOVA. Brackets indicate P < 0.05 compared with left-most sample. E, MRTX849 was administered via daily oral gavage at the doses indicated to mice bearing established MIA PaCa-2 xenografts. Dosing was initiated when tumors were approximately 350 to 400 mm3.MRTX849 was administered to mice daily until day 16. Data are shown as mean tumor volume ± SEM. Tumor volumes at day 16 were determined to be statistically significant versus vehicle control via two-tailed Student t test. **, P < 0.01; *, P < 0.05.

 

To evaluate the effect of MRTX849 on KRAS-dependent signal transduction in vivo, a single dose of MRTX849 at 10, 30, or 100 mg/kg was administered to H358 tumor–bearing mice. Dose-dependent inhibition of ERK1/2 and pS6S235/36 phosphorylation was observed at 6 hours after dose based on immunoblot and densitometric analysis (Fig. 2C). MRTX849 also demonstrated marked inhibition of ERK1/2 and S6S235/36 phosphorylation after one or three daily doses at 6 or 24 hours, and levels gradually recovered by 72 hours after the final dose (Fig. 2D). pERK1/2 and pS6S235/36 were further evaluated in formalin-fixed, paraffin-embedded sections from vehicle-treated and MRTX849-treated xenografts in four tumor models utilizing IHC methods coupled with image analysis algorithms. These studies demonstrated increased pERK1/2 and pS6 in nontumor/stromal cells following MRTX849 administration, indicating that immunoblotting studies with bulk tumor lysate likely underrepresent the degree of pathway inhibition in tumor cells, whereas IHC-based evaluation may more accurately reflect both the degree and spatial impact of pathway inhibition. Maximal inhibition was observed for both ERK and S6S235/36 phosphorylation after a single dose at the 6-hour time point, with a rebound in signaling evident 24 hours after single dose in each model (Supplementary Fig. S4). Marked inhibition of ERK phosphorylation was observed at 6 hours after administration, with 89%, 94%, and 94% inhibition observed compared with vehicle controls in MIA PaCa-2, H1373, and H2122 tumors, respectively (H358 pERK not quantifiable). This indicates that dose levels at or exceeding 30 mg/kg dose maximized inhibition of ERK phosphorylation in multiple models (Supplementary Fig. S4A and S4B). Inhibition of S6 phosphorylation at 6 hours was more variable, with percent inhibition values of 76%, 50%, 86%, and 56% observed in MIA PaCa-2, H1373, H358, and H2122 tumors, respectively (Supplementary Fig. S4B). Together, these data indicate that consistent acute (6 hours) inhibition of KRAS-dependent ERK phosphorylation was maximized in all evaluated models, whereas inhibition of S6S235/36 was more variable, presumably due to varying degrees of KRAS-independent activation of this pathway in different tumor models.

MIA PaCa-2 and H358 were selected as MRTX849-responsive tumor models, thereby enabling a high-resolution understanding of dose–response relationships. Significant, dose-dependent, antitumor activity was observed at the 3, 10, 30, and 100 mg/kg dose levels in the MIA PaCa-2 model (Fig. 2E). Evidence of rapid tumor regression was observed at the earliest post-treatment tumor measurement, and animals in the 30 and 100 mg/kg cohorts exhibited evidence of a complete response at study day 15. Dosing was stopped at study day 16, and all 4 mice in the 100 mg/kg cohort and 2 of 7 mice in the 30 mg/kg cohort remained tumor-free through study day 70 (Supplementary Fig. S5A). In a second MIA PaCa-2 study, dose-dependent antitumor efficacy was observed at the 5, 10, and 20 mg/kg dose levels, and 2 of 5 mice at the 20 mg/kg dose level exhibited complete tumor regression (Supplementary Fig. S5B). Significant dose-dependent antitumor efficacy was also observed in the H358 model, including 61% and 79% tumor regression at the 30 and 100 mg/kg dose levels, respectively, at day 22 (Supplementary Fig. S5C). MRTX849 was well tolerated, and no effect on body weight was observed at all dose levels evaluated (Supplementary Fig. S5D). These studies indicated that MRTX849 demonstrated dose-dependent antitumor efficacy over a well-tolerated dose range and that the maximally efficacious dose of MRTX849 is between 30 and 100 mg/kg/day.

MRTX849 Demonstrates Broad-Spectrum Tumor Regression in KRASG12C Cell Line and Patient-Derived Xenograft Models

To evaluate the breadth of antitumor activity across genetically and histologically heterogeneous KRASG12C-mutant cancer models, MRTX849 was evaluated at a fixed dose of 100 mg/kg/day (a dose projected to demonstrate near-maximal target inhibition in most models) in a panel of human KRASG12C-mutant cell line–derived xenograft (CDX) and patient-derived xenograft (PDX) models. MRTX849 demonstrated tumor regression exceeding 30% volume reduction from baseline in 17 of 26 models (65%) at approximately 3 weeks of treatment (Fig. 3A; Supplementary Table S6). By comparison, MRTX849 did not exhibit significant antitumor activity at 100 mg/kg in three non–KRASG12C-mutant models (Fig. 3A; Supplementary Table S6). Together, these results indicate that KRASG12C-mutant tumors are broadly dependent upon mutant KRAS for tumor-cell growth and survival and that MRTX849 elicits antitumor activity through a KRASG12C-dependent mechanism.

Figure 3.

 

Antitumor activity of MRTX849 in KRASG12C-mutant and non–KRASG12C-mutant human tumor xenograft models. A, MRTX849 was administered via oral gavage at 100 mg/kg every day to mice bearing the CDX or PDX model indicated. Dosing was initiated when tumors were, on average, approximately 250 to 400 mm3. MRTX849 was formulated as a free base and resuspended as a solution in 10% Captisol and 50 mmol/L citrate buffer, pH 5.0. The percent change from baseline control was calculated at days 19 to 22 for most models. Statistical significance was determined for each model and is shown in Supplementary Table S6. Status of mutations and alterations in key genes is shown below each model. MAF (%), percent KRASG12C-mutant allele fraction by RNA-seq; CNV, copy-number variation; * denotes very high CDK4 expression by RNA-seq and possible amplification. HER family status was determined by averaging EGFRERBB2, and ERBB3 RNA-seq expression for CDX (CCLE) or PDX (Crown huBase) models. Positive HER family calls denote greater than the median expression of the models tested. CDX and PDX model HER family calls were determined independently. B, Tumor-growth inhibition plots from representative xenograft models that were categorized as sensitive, partially sensitive, or treatment refractory.

 

Although MRTX849 exhibited marked antitumor responses in the majority of models tested, a response pattern ranging from delayed tumor growth to complete regression was observed across the xenograft panel. The response to treatment was categorized as sensitive, partially sensitive, or treatment refractory (Fig. 3B). Rank order and Pearson statistical analyses were performed to evaluate the correlation between in vitro potency (IC50 in 2-D or 3-D viability assays) and antitumor response in vivo (% regression or progression on day 22), and a significant correlation between response in cell lines compared with tumor models was not observed (Supplementary Fig. S6A and S6B). Thus, we focused on a comprehensive analysis of correlates with MRTX849 tumor response in vivo, including tumor histology, co-occurring genetic alterations, as well as baseline or drug-induced changes in expression of KRAS-related genes [RNA sequencing (RNA-seq)] and/or protein signaling networks (RPPA in 18 models, ref. 28; Supplementary Fig. S7). No individual genetic alteration, including but not limited to KRAS-mutant allele frequency, TP53, STK11, or CDKN2A, predicted the antitumor activity of MRTX849. Interestingly, baseline gene and/or protein expression of selected members of the HER family of RTKs and of regulators of early cell-cycle transition did exhibit a trend with the degree of antitumor response, suggesting these pathways may influence the response to KRAS inhibitors (Supplementary Fig. S7A). Together, these data indicate that there are no individual binary biomarkers that clearly predict therapeutic response and that the molecular complexity and heterogeneity present in distinct KRAS-mutated tumors may contribute to the response to target blockade.

MRTX849 Antitumor Activity Translates to RECIST Responses in Patients with Cancer

A 45-year-old female former smoker diagnosed with stage IV lung adenocarcinoma and refractory to multiple lines of therapy including carboplatin/pemetrexed/pembrolizumab, docetaxel, and investigational treatment with binimetinib and palbociclib was enrolled onto the MRTX849-001 phase Ib clinical trial with two bilateral lung lesions and mediastinal lymph node as target lesions. Targeted next-generation sequencing (NGS) demonstrated a KRASG12C mutation (c.34G>T). In addition, loss-of-function KEAP1 (K97M) and STK11 (E223*) mutations were detected and are predicted to be deleterious to their respective proteins. The patient was administered MRTX849 (600 mg twice a day) and had marked clinical improvement within 2 weeks, including complete resolution of baseline cough and oxygen dependency. A RECIST-defined partial response of 33% reduction of target lesions was observed at cycle 3 day 1 (45 days), and the patient continues on study (Fig. 4A).

Figure 4.

 

Activity of MRTX849 in patients with lung and colon cancers. A, Pretreatment and 6-week scans of a heavily pretreated patient with a KRASG12C mutation–positive lung adenocarcinoma indicating 33% reduction of target lesions. Patient continues on study. The top plots show a coronal view, and bottom plots an axial view of CT chest images prior to MRTX849 treatment (left) and after two cycles of MRTX849 treatment (right). B, Baseline, 6-week (Cycle 2), and 12-week (Cycle 4) scans of a patient with a KRASG12C mutation–positive colon adenocarcinoma. Partial response (PR) was confirmed at Cycle 4, and patient continues on study. Four lesions (TL1–4) are shown with axial views of CT images prior to MRTX849 treatment (top), after two cycles of MRTX849 treatment (center), and after four cycles of MRTX849 treatment (bottom).

 

A 47-year-old female never-smoker with metastatic adenocarcinoma of the left colon who exhibited progressive disease after receiving multiple lines of systemic therapy, including FOLFOX plus bevacizumab, single-agent capecitabine, FOLFIRI plus bevacizumab, and an investigational antibody–drug conjugate, was enrolled into the MRTX849-001 phase Ib clinical trial. This patient had extensive metastases involving the liver, peritoneum, ovaries, and lymph nodes. Targeted NGS identified a KRASG12C mutation. The patient was administered MRTX849 (600 mg twice a day) and demonstrated marked clinical improvement within 3 weeks and a visible decrease in size of her umbilical Sister Mary Joseph nodule. Her carcinoembryonic antigen levels decreased from 77 ng/mLat baseline to 11 ng/mL at cycle 2 day 1 and 3 ng/mL by cycle 3 day 1 (normal range, 0–5 ng/mL). A RECIST-defined partial response with a 37% reduction of target lesions and complete response of a nontarget lesion was observed at cycle 3 day 1(day 42). Confirmatory CT scans were conducted at cycle 5, day 1 (day 84) and indicated a confirmed RECIST partial response with further reduction of target lesions at −47% from baseline (Fig. 4B). The patient remains on treatment through Cycle 6.

Temporal Effects of MRTX849 on KRAS-Dependent Signaling and Feedback Pathways and Relationship to Antitumor Activity Following Repeat Dosing in Xenograft Models

A comprehensive analysis was conducted to evaluate MRTX849-induced temporal molecular changes to further interrogate mechanisms of drug response across sensitive and partially sensitive models. To evaluate temporal changes in global gene expression, xenograft-bearing mice were administered vehicle or 100 mg/kg MRTX849, and RNA-seq was performed on tumors at 6 and 24 hours after treatment. Gene expression was evaluated at day 1 and day 5 for the sensitive models MIA PaCa-2 and H1373 to ensure sufficient tissue availability from regressing tumors, or at day 7 in the partially sensitive models H358, H2122, and H2030 to coordinate with tumor stasis plateau. The top differentially expressed gene set enrichment analysis (GSEA) hallmark gene sets, regardless of tumor response, in all five models were several KRAS-annotated gene sets confirming MRTX849 selectively inhibits multiple genes directly related to KRAS signaling. MYC, mTOR, cell cycle, and apoptosis/BCL2 pathway gene sets were also strongly differentially expressed, confirming MRTX849 broadly affected multiple well-established, KRAS-regulated pathways, several of which have proved difficult to directly inhibit with previous targeted therapies (Fig. 5A and B; Supplementary Fig. S8A—S8D). The marked impact of MRTX849 on a large number of genes that regulate cell cycle and apoptosis provides further insight into molecular mechanisms which mediate its antitumor activity.

Figure 5.

 

MRTX849 treatment in vivo regulates KRAS-dependent oncogenic signaling and feedback-inhibitory pathways. A, Volcano plots displaying differentially expressed genes in xenograft tumors 24 hours after oral administration of vehicle or 100 mg/kg MRTX849 in a representative MRTX849-sensitive (H1373) and MRTX849-partially sensitive (H358) model. Significance denoted in the legend (Padj < 0.01). B, GSEA heat maps depicting hallmark signature pathways differentially regulated in at least one model 24 hours following oral administration of a single 100 mg/kg MRTX849 dose compared with vehicle. Normalized enrichment score shown in all models 6 or 24 hours after a single dose (QD × 1) or 5 (QD × 5) or 7 (QD × 7) days dosing. C, Genes that feedback-inhibit MAP kinase signaling are downregulated following MRTX849 treatment in all five cell line xenografts assessed by RNA-seq. TPM, Transcripts Per Kilobase Million.

 

Targeted RNA-seq analysis was performed on genes implicated in the temporal regulation of external signaling inputs and feedback pathways which collectively temper signaling flux through the RAS–RAF–MEK–ERK MAP kinase (MAPK) pathway including DUSPSPRY, and PHLDA family genes (13, 18). These MAPK pathway–negative regulators were each ranked among the most strongly decreased genes following MRTX849 treatment, providing evidence that ERK-dependent transcriptional output is blocked and that pathways involved in reactivation of RTK- and ERK-dependent signaling were activated (Fig. 5C; Supplementary Fig. S4A).

On the basis of the observation of dynamic changes in transcriptional programs linked to KRAS pathway reactivation, IHC plus quantitative imaging of tumor cell–specific pERK and pS6 was evaluated over a range of time points. In the sensitive MIA PaCa-2 and H1373 tumor models, treatment with MRTX849 (100 mg/kg) demonstrated ≥90% inhibition of ERK phosphorylation at 6 and 24 hours on both days 1 and 5 (Supplementary Fig. S4). In contrast, in the partially sensitive H358 and H2122 models, robust inhibition of ERK phosphorylation was observed at 6 hours after a single dose; however, marked recovery of ERK phosphorylation was observed at 24 hours after single dose and at both 6 and 24 hours following 7 days of repeat-dose administration. Because DUSPSPRY, and ETV family transcripts remain downregulated through 5 to 7 days in all models, it is evident that other independent factors contribute to temporal reactivation of ERK (Fig. 5C). Similar to what was observed with single-dose administration, the effect of MRTX849 on pS6 was variable over time and did not track with the antitumor activity of MRTX849. Together, these results suggest that the extent and duration of inhibition of pERK may track with the magnitude of antitumor efficacy of KRASG12C inhibitors and that further evaluation of the role of S6 is required to understand if it plays a role in drug sensitivity.

The effect of MRTX849 on cell proliferation and apoptosis was characterized by IHC analysis of Ki-67 or cleaved caspase-3 after a single dose or repeat administration. The fraction of Ki-67–positive cells was significantly reduced in tumors after repeat administration in all four models tested, further supporting a broadly operative antiproliferative mechanism, independent of the magnitude of MRTX849 antitumor response (Supplementary Fig. S4). Induction of apoptosis as determined by cleaved caspase-3 immunostaining was also evident on day 1 of treatment (6 and/or 24 hours after treatment) in the sensitive H358, MIA PaCa-2, and H1373 models (79%–100% maximal regression) but not in the partially sensitive H2122 model (Supplementary Fig. S4). An expanded RPPA-based pathway analysis of several models also indicated a correlation between antitumor activity of MRTX849 and decreased survivin (statistically significant at days 5/7 in 7 models evaluated; Supplementary Fig. S7B) and a trend toward increased cleaved caspase-3 induction (day 1, P = 0.08, 16 models), supporting the induction of apoptosis as a key mediator of a cytoreductive antitumor response (Supplementary Fig. S7C). Interestingly, the magnitude of reduction of MYC and cyclin B1 protein levels at days 5/7 also closely correlated with MRTX849 antitumor activity, consistent with their roles as critical regulators of KRAS-mediated cell growth and survival pathways (Supplementary Fig. S7B). Collectively, these data support that durable inhibition of ERK activity and maximal inhibition of ERK-regulated outputs including MYC and E2F-mediated transcription are associated with induction of apoptosis and maximal response to MRTX849 treatment.

CRISPR/Cas9 Screen Identifies Vulnerabilities and Modifiers of Response to MRTX849 in KRASG12C-Mutant Cancer Cell Lines In Vitro and In Vivo

The correlative analysis of genomic or proteomic markers with response to MRTX849 in the defined panel of models provided only limited insight toward mechanism of therapeutic response or resistance. Therefore, we directly interrogated the role of selected genes in mediating therapeutic response utilizing a focused CRISPR/Cas9 knockout screen targeting approximately 400 genes including many genes involved in KRAS signaling. This was conducted in H358 and H2122 cells in vitro and in H2122 xenografts in vivo in the presence and absence of MRTX849 treatment (Supplementary Fig. S9A–S9F). In MRTX849-anchored screens in vitro, single guide RNAs (sgRNA) that target RAS signaling pathway genes including MYC, SHP2 (H2122), mTOR pathway (MTOR and RPS6), and cell-cycle genes (CDK1, CDK2, CDK4/6, and RB1) were identified to affect cell fitness. sgRNAs that target KEAP1 and CBL were enriched in the H2122 model, demonstrating cell-specific genetic routes toward improved fitness through loss of classic tumor-suppressor genes, including in the context of MRTX849 treatment. KRAS sgRNA dropout was less pronounced in the MRTX849-treated cells compared with DMSO control–treated cells, as would be expected with redundant depletion of the drug target (Supplementary Fig. S9C and S9D). To evaluate whether a distinct KRAS dependence or modulation of MRTX849 therapeutic response was observed in vitro versus in vivo, xenograft-bearing mice bearing H2122 cells (∼250 mm3) transduced with the sgRNA library were orally administered vehicle or MRTX849 for 2 weeks (Supplementary Fig. S9A, S9E, and S9F). In MRTX849-treated xenografts, sgRNAs targeting cell cycle, SHP2, MYC, and mTOR pathway genes remained among the top depleted sgRNAs, demonstrating that inhibition of these targets in vivo, in the context of KRAS inhibition, leads to further tumor-growth inhibition over and above the effects of KRAS inhibition alone (Supplementary Fig. S9E and S9F). sgRNAs targeting the tumor suppressor KEAP1 were enriched in MRTX849-treated xenografts, suggesting loss of KEAP1 may represent a mechanism of intrinsic or acquired resistance. Interestingly, NRAS was one of the top enriched genes in the vehicle-treated xenografts, suggesting NRAS functions as a tumor suppressor in this context; however, enrichment was not as pronounced in the MRTX849-treated xenografts, suggesting NRAS may compensate for KRAS in the context of KRAS inhibition (Supplementary Fig. S9F). Collectively, these data demonstrate the importance of selected proteins that regulate RTK- and RAS-dependent signaling and cell-cycle transition in mediating the oncogenic effects of mutant KRAS, and also provide a catalog of potentially druggable vulnerabilities that complement KRAS blockade.

Cancer Therapeutic Combination Screen to Identify Rational and Clinically Tractable Strategies to Address Feedback and Resistance Pathways

To further interrogate pathways that mediate the antitumor response to MRTX849 and to identify combinations capable of enhancing response to MRTX849, a combination screen was conducted in vitro using a focused library of small-molecule inhibitors across a panel of cell lines (Supplementary Fig. S10A and S10B; Supplementary Table S8). Approximately 70 compounds targeting relevant pathways (RTKs, MAPK/ERK, PI3K, mTOR, cell cycle) were tested in a 3- or 7-day viability assay, and synergistic combinations were identified and ranked. Multiple hits from this screen were then identified for additional evaluation in combination studies with MRTX849, including the HER family inhibitor afatinib, the CDK4/6 inhibitor palbociclib, the SHP2 inhibitor RMC-4550, and mTOR pathway inhibitors.

Combination Strategies That Target Upstream Signaling Pathways Implicated in Extrinsic Regulation of KRAS Nucleotide Cycling and Feedback/Bypass Pathways

MRTX849 in combination with HER family inhibitors synergistically inhibited tumor-cell viability in the majority of cell lines evaluated and were the top hit in the combination screen in vitro (Supplementary Fig. S10). Cell lines with the highest (top 50th percentile) average composite baseline RNA expression values of selected HER family members exhibited the highest synergy scores to these combinations (Supplementary Fig. S11A). Afatinib was selected as a prototype HER family inhibitor based on its broad in vitro combination activity. Combination studies were conducted with MRTX849 and afatinib in five tumor models that were partially sensitive or treatment refractory to single-agent MRTX849. The MRTX849 and afatinib combination demonstrated significantly greater antitumor efficacy compared with either single agent in all five models evaluated, including multiple models exhibiting complete or near-complete responses to the combination (Fig. 6A; Supplementary Fig. S11B).

Figure 6.

 

HER family and SHP2 inhibitor combinations further inhibit KRAS signaling and exhibit increased antitumor responses. A, MRTX849 at 100 mg/kg, afatinib at 12.5 mg/kg, or the combination was administered daily via oral gavage to mice bearing the H2122 or KYSE-410 cell line xenografts (n = 5). Combination treatment led to a statistically significant decrease in tumor growth compared with either single-agent treatment. *, Padj < 0.01. B, Quantification of KRAS mobility shift and pERK in H2122 cells treated for 24 hours with MRTX849 (0.1–73 nmol/L), afatinib (200 nmol/L), or the combination assessed by Western blot densitometry. C, MRTX849 at 100 mg/kg, afatinib at 12.5 mg/kg, or the combination was administered once or daily for 7 days via oral gavage to mice bearing H2122 cell line xenografts (n = 3/group). Tumors were harvested at 6 and 24 hours following the final dose. Tumor sections were stained for pERK and pS6 via IHC methods. Quantitation of images shown by H-score in tumor tissue. Reduction of pERK or pS6 staining intensity was determined to be statistically significant relative to vehicle or either single agent using one-way ANOVA. Brackets indicate P < 0.05 compared with left-most sample. D, Quantitation of KRAS band shift and pERK after 24-hour treatment with MRTX849 (0.1–73 nmol/L), RMC-4550 (1 μmol/L), or the combination in H358 cells assessed by Western blot densitometry. E, MRTX849 at 100 mg/kg, RMC-4550 at 30 mg/kg, or the combination was administered daily via oral gavage to mice bearing the KYSE-410 or H358 cell line xenografts (n = 5/group). Combination treatment led to a statistically significant reduction in tumor growth compared with either single agent on the last day of dosing. *, Padj < 0.05. F, MRTX849 at 100 mg/kg, RMC-4550 at 30 mg/kg, or the combination was administered via oral gavage to mice bearing KYSE-410 cell line xenografts (n = 3/group), and tumors were harvested at 6 and 24 hours post-dose. Tumor sections were stained with pERK or pS6 via IHC methods. Quantitation of images shown by H-score in tumor tissue. Reduction of pERK staining intensity was determined to be statistically significant relative to RMC-4550 alone using one-way ANOVA. Brackets indicate P < 0.05 compared with left-most sample.

 

To evaluate whether afatinib affected covalent modification of KRASG12C by MRTX849, partially sensitive H2122 cells were treated with increasing concentrations of MRTX849 alone or in the presence of afatinib (200 nmol/L, IC90), and the mobility shift in KRAS protein was densitometrically determined from immunoblots. A clear shift in the concentration response to MRTX849 was apparent in the presence of afatinib, indicating that the combination increased the fraction of modified KRASG12C consistent with the putative role of HER family receptors in extrinsic regulation of KRASG12C GTP loading (Fig. 6B). The concentration–response relationship for inhibition of ERK phosphorylation was also clearly shifted in the presence of afatinib. To further evaluate the effect of the combination on KRAS-dependent signaling, four cell lines (H2030, H2122, H358, and KYSE-410) were treated with a range of MRTX849 concentrations in the presence or absence of afatinib for 6 or 24 hours, and key signaling molecules were evaluated by RPPA. Afatinib demonstrated clear inhibition of EGFR (pY1068) and HER2 (pY1248) activity and partial inhibition of ERK, AKT (S473), and p70S6K phosphorylation at both time points (Supplementary Fig. S11C). The effect of afatinib on S6 (S235/236, S240/244) and p90 RSK (S380) phosphorylation was variable and exhibited only minimal inhibition in most of the cell lines evaluated. The combination of afatinib and MRTX849 demonstrated markedly enhanced concentration-dependent inhibition and/or a greater magnitude of effect on ERK, RSK, p70 S6K, and S6 (S235/236) phosphorylation compared with MRTX849 alone at both 6 and 24 hours. Of note, neither afatinib nor MRTX849 alone inhibited S6 phosphorylation at the S240/244 site regulated by mTOR/S6K, whereas the combination demonstrated marked inhibition at 24 hours.

In vivo, the combination also exhibited a trend toward increased pERK and pS6 (S235/236) inhibition in the partially sensitive H2122 model in combination groups as determined by quantitation of immunostaining after 1- or 7-day administration (Fig. 6C). Similar results were observed in the MRTX849-refractory KYSE-410 model, and the combination also increased the number of apoptotic cells in this model (Supplementary Fig. S12A–S12C). Collectively, these data indicate that upstream baseline HER family activation may limit the ability of MRTX849 to achieve robust inhibition of the ERK and mTOR–S6 signaling pathways. Accordingly, the combination of afatinib and MRTX849 can limit feedback reactivation of ERK and demonstrate complementary inhibition of AKT–mTOR–S6 signaling, resulting in significantly improved antitumor activity.

SHP2 inhibition has been shown to inhibit the growth of cells that harbor KRASG12C mutations, and this effect is likely mediated, in part, by decreasing KRAS GTP loading (25–27). To evaluate whether SHP2 inhibition enhanced covalent modification of KRASG12C by MRTX849, H358 and H2122 cells were incubated with increasing MRTX849 concentrations with or without the SHP2 inhibitor RMC-4550. In both cell lines, cotreatment with RMC-4550 (1 μmol/L, IC90) demonstrated a MRTX849 concentration-dependent increase in KRASG12C protein modification and a concomitant decrease in ERK phosphorylation compared with MRTX849 alone (Fig. 6D; Supplementary Fig. S13A). RPPA analysis of KRAS-dependent signaling was conducted at 6 or 24 hours after treatment in three cell lines (H358, H2030, H2122) over a range of MRTX849 concentrations in the presence or absence of RMC-4550. RMC-4550 demonstrated robust inhibition of ERK phosphorylation and partial inhibition of p90 RSK (S380) and p70 S6K (T412) at both time points (Supplementary Fig. S13B). The combination of RMC-4550 and MRTX849 demonstrated incrementally increased concentration-dependent inhibition of ERK and RSK phosphorylation in all cell lines at both 6 and 24 hours and markedly improved inhibition of S6 (S235/236) phosphorylation compared with MRTX849 alone in H2122 and H358 cells at 24 hours. In addition, the combination demonstrated near-complete inactivation of KRAS in MRTX849-refractory KYSE-410 xenografts as determined using an active RAS ELISA assay, and this was significant compared with single agents (Supplementary Fig. S13C). On the basis of these findings, combination studies were conducted with MRTX849 and RMC-4550 in six KRASG12C-mutated tumor models in vivo, and the combination demonstrated significantly greater antitumor efficacy compared with either single agent in 4 of 6 models evaluated (Fig. 6E; Supplementary Fig. S13D). Consistent with the in vitro data, the combination also demonstrated a significant decrease in ERK phosphorylation compared with either single agent in the KYSE-410 model as determined by quantitation of tumor-cell immunostaining on day 1 at 6 and 24 hours and day 7 at 6 hours after dose (Fig. 6F). Together, these data indicate that EGFR family and SHP2 blockade can augment the antitumor activity of KRASG12C inhibitors through enhancing covalent target modification and establishing a more comprehensive blockade of KRAS-dependent signaling.

Combinations That Inhibit Bypass Pathways Downstream of KRAS and Exhibit Increased Antitumor Activity in Xenograft Models

KRAS is implicated in regulation of the oncogenic S6 protein translation pathway through both ERK-dependent activation of RSK, which phosphorylates S6 at Ser235/236, and cross-talk with the PI3K and mTOR pathway that additionally phosphorylates S6 at Ser240/244 (29). However, the S6 pathway can also be activated independently of mutated KRAS in tumor cells through hyperactivated RTK signaling, PI3K activation, or STK11 mutations, each of which converge on mTOR-mediated activation of S6. In the in vitro combination screen, mTOR inhibitors demonstrated synergy in a subset of evaluated cell lines (Supplementary Fig. S14A). To further evaluate the effect of the combination on KRAS and mTOR pathway–dependent signaling, four cell lines were treated with MRTX849 in the presence or absence of the selective ATP-competitive mTOR inhibitor vistusertib (1 μmol/L), for 6 or 24 hours, and several signaling molecules were evaluated by RPPA. Vistusertib demonstrated clear and robust inhibition of several components of the PI3K–mTOR signaling pathway including AKT (S473), p70 S6K (T412), S6 (pS235/236, S240/244), and 4E-BP1 (S65, T70) phosphorylation in each cell line at both time points consistent with its mechanism of action (Supplementary Fig. S14B). MRTX849 alone did not affect 4E-BP1 or S6 (S240/244) activity, and it exhibited a variable and cell line–dependent effect on p70 S6K and S6 (pS235/236) phosphorylation in these cell lines. Vistusertib also demonstrated marked induction of ERK phosphorylation, often several-fold over vehicle control, at both time points in all four cell lines, consistent with prior reports (30). The combination of vistusertib and MRTX849 demonstrated a comparable level of inhibition of ERK phosphorylation compared with single-agent MRTX849, indicating that the activation of ERK signaling by vistusertib was impeded by the combination of the two agents. In addition, MRTX849 combined with vistusertib further inhibited p70 S6K and AKT S473 phosphorylation compared with either single agent. Near-complete inhibition of S6 (S235/236, S240/244) phosphorylation at limit of detection was observed for the combination in each cell line at evaluated time points.

Consequently, a cohort of tumor models was identified, and the combination of MRTX849 with the selective mTOR inhibitor vistusertib demonstrated marked tumor regression and significantly improved antitumor activity compared with either single agent in all six models evaluated (Fig. 7A; Supplementary Fig. S14C). MRTX849 in combination with a second, differentiated mTOR inhibitor, everolimus, which inhibits TORC1 but not TORC2, in the H2030 xenograft model also demonstrated a striking combination effect (Supplementary Fig. S14D). In the KRASG12C, STK11-mutant H2030 model, MRTX849 demonstrated marked inhibition of ERK phosphorylation through 24 hours, but exhibited only partial inhibition of pS6235/36 at 6 hours after dose, on days 1 and 7 (Fig. 7B and C). Vistusertib demonstrated marked inhibition of pS6235/36 at 6 hours after treatment with evidence of recovery by 24 hours. The combination of vistusertib and MRTX849 did not have a further effect on ERK phosphorylation but demonstrated a significant reduction in pS6235/36 on day 1 at 24 hours compared with vistusertib alone and a trend toward reduced pS6235/36 on both day 1 and day 7 at 6 hours compared with either single agent (Fig. 7B; Supplementary Fig. S14E). Together, these data indicate that MRTX849 and mTOR inhibitor combination demonstrates complementary inhibition of the ERK and mTOR–S6 signaling pathways, resulting in broad antitumor activity in KRASG12C-mutant tumor models.

Figure 7.

 

CDK4/6 and mTOR combinations suppress independently hyperactivated downstream pathways and exhibit increased antitumor responses. A, MRTX849 at 100 mg/kg, vistusertib at 15 mg/kg, or the combination was administered daily via oral gavage to mice bearing the H2122 or H2030 cell line xenografts (n = 5/group). Combination treatment led to a statistically significant decrease in tumor growth compared with either single-agent treatment. *, Padj < 0.05. B, MRTX849 at 100 mg/kg, vistusertib at 15 mg/kg, or the combination was administered once or daily for 7 days via oral gavage to mice bearing H2030 cell line xenografts (n = 3/group). Tumors were harvested at 6 and 24 hours following the final dose. Tumor sections were stained with pERK and pS6 via IHC methods. Quantitation of images shown by H-score in tumor tissue. Reduction of pERK or pS6 staining intensity was determined to be statistically significant relative to vehicle or either single agent using one-way ANOVA. Brackets indicate P < 0.05 compared with left-most sample. C, Protein Western blot analysis of KRAS pathway targets in H2030 xenografts treated with MRTX849 (100 mg/kg), vistusertib (15 mg/kg), or the combination, 6 or 24 hours after a single dose. D, Protein Western blot analysis of KRAS pathway and cell-cycle targets in H2122 cells treated for 24 hours with MRTX849, palbociclib, or the combination. E, Normalized RNA-seq gene-expression data on E2F targets in H2122 xenografts treated with MRTX849, palbociclib, or the combination, 6 and 24 hours after a single daily dose or seven daily doses. F, MRTX849 at 100 mg/kg, palbociclib at 130 mg/kg, or the combination was administered daily via oral gavage to mice bearing the H2122 or SW1573 cell line xenografts (n = 5). Combination treatment led to a statistically significant decrease in tumor growth compared with either single-agent treatment. *, Padj < 0.05.

 

Signaling through KRAS is known to mediate cell proliferation, at least in part, through the regulation of the cyclin D family and triggering RB/E2F-dependent entry of cells into cell cycle. Loss-of-function mutations and homozygous deletions in the cell-cycle tumor suppressor CDKN2A (p16) are coincident in a subset of KRAS-mutant non–small cell lung cancer (NSCLC) and hyperactivate CDK4/6-dependent RB phosphorylation and cell-cycle transition. In the CDKN2A-null H2122 and SW1573 cell lines in vitro, MRTX849 demonstrated concentration-dependent partial inhibition of RB phosphorylation (pRB pS807/811) and concurrent increase in p27 in H2122 cells, but not SW1573 cells, at 24 hours (Fig. 7D; Supplementary Fig. S15A). MRTX849 in combination with the CDK4/6 inhibitor palbociclib (1 μmol/L) demonstrated near-complete inhibition of pRB in both H2122 and SW1573 cells and further induced p27 in H2122 cells. Interestingly, pS6 (S235/236) was also much more effectively suppressed by the combination in both H2122 and SW1573 cells, which is consistent with a recent report (31). RNA expression of target genes and RPPA analysis of target protein signaling events were also used as a readout of cell-cycle inhibition in the H2122 tumor model in vivo, and the combination of MRTX849 and palbociclib significantly inhibited E2F1 and selected E2F family target genes, induced p27 protein expression to a greater degree compared with either single agent, and further reduced the number of Ki-67–positive cells after 7 days of administration (Fig. 7E; Supplementary Fig. S15B and S15C). In addition, the combination demonstrated a significant decrease in pRB (S780) compared with either single agent after 7 days of administration in SW1573 tumors in vivo (Supplementary Fig. S15D). This combination also induced tumor regression in five tumor xenograft models that was significant compared with either single-agent control (Fig. 7F; Supplementary Fig. S15E). Although not significant, a trend was noted in which models with CDKN2A homozygous deletion exhibited an increased antitumor response to the combination of MRTX849 and CDK4/6 inhibition compared with models lacking evidence of genetic dysregulation of key cell-cycle genes (Supplementary Fig. S15F and S15G).

Discussion

The identification of MRTX849 as a highly selective KRASG12C inhibitor capable of near-complete inhibition of KRAS in vivo provides a renewed opportunity to better understand the role of this mutation as an oncogenic driver in various cancers and to guide rational clinical trial design. The lack of a significant correlation between sensitivity to MRTX849 antitumor activity in in vitro versus in vivo model systems made it necessary to further study KRAS oncogene dependence in tumor models in vivo, a more clinically relevant setting. The demonstration that MRTX849 exhibited significant antitumor efficacy in all evaluated KRASG12C-mutated cancer models and demonstrated marked regression in the majority (65%) confirms that this mutation is a broadly operative oncogenic driver and that MRTX849 represents a compelling therapeutic opportunity. This evidence of activity extended to patients, as demonstrated by RECIST partial responses in 2 patients enrolled in a phase I clinical trial of MRTX849. Collectively however, these data also illustrate that the degree of dependence of cancer cells on the presence of a KRASG12C mutation for growth and survival can vary across tumors and that co-occurring genetic alterations observed in KRAS-mutated cancers may influence response to direct targeted therapy. The further observation that KRAS mutations occur across different cancers and that no single co-occurring genetic alteration predicted response to treatment illustrates the genetic heterogeneity of KRAS-driven cancers. Findings in the present studies are consistent with other functional genomics or therapeutic strategies to block KRAS function across panels of cell lines or models which demonstrated a highly significant response of KRAS-mutant cells to target knockdown, a heterogeneous magnitude of response, and no clear co-occurring aberrations that predict resistance to target blockade (5, 32, 33). Interestingly, despite the implication that certain mutations that co-occur with KRAS including TP53, STK11, and KEAP1 may limit therapeutic response in KRASG12C-positive lung cancers, none of these mutations correlated with response or resistance in the cell-line panel. In addition, the partial response we reported in the patient with lung adenocarcinoma was observed in a patient harboring deleterious comutations in both STK11 and KEAP1. Together, these data further illustrate the heterogeneity and complexity of KRAS-mutated cancers and suggest that no binary co-occurring genetic event may be predictive of therapeutic response.

Temporal and dose–response analysis indicated maximal modification of KRASG12C and durable inhibition of KRAS-dependent signaling was important in maximizing therapeutic response. The recovery of ERK signaling and the inability to inhibit mTOR–S6 signaling despite continued treatment were each associated with transient or submaximal response to MRTX849. ERK1/2 is implicated in direct phosphorylation and negative feedback regulation of EGFR (T669), FGFR1 (S777), and SOS1, and each of these targets may facilitate KRASG12C-independent resetting of ERK signaling flux (34–36). The rapid and remarkable suppression of ERK pathway–regulated transcripts such as DUSP and SPRY/SPRED family members by MRTX849 in all models evaluated is consistent with that observed for RAF inhibitors and is implicated in reactivation of ERK and RTK signaling (18, 19). The dual-specificity phosphatases DUSP4 and 6 were strongly suppressed by MRTX849 and are implicated in dephosphorylating and inactivating ERK1/2 (14, 18, 37), whereas SPRY family members are implicated in the negative regulation of RTKs and adaptor proteins (e.g., GRB2), and may participate in modifying RAS family nucleotide exchange and effector binding (e.g., RAF1; ref. 38). Although suppression of DUSP and SPRY/SPRED was broadly observed in all models, the magnitude of signaling reactivation and response to MRTX849 varied across models. This suggests some tumor models harbor additional factors that bypass KRAS dependence or affect RAS pathway signaling flux, such as expression or activation of selected RTKs (e.g., ERBB2 amplification in the KYSE-410 model) or STK11 loss-of-function mutations, and may be primed for feedback reactivation of RAS-dependent signaling and/or limit the degree of signaling inhibition by MRTX849. This phenomenon was observed for BRAFV600E-mutant colon cancer (but not melanoma) which exhibits high baseline EGFR expression, is primed for rapid feedback activation of this RTK, and is resistant to single-agent inhibition but highly responsive to cotargeting BRAF (and/or MEK) and EGFR (20). In addition, blockade of BRAF or MEK1/2 resulted in feedback-mediated activation of the PI3K–mTOR signaling pathway in concert with the coactivation of upstream RTKs (e.g., EGFR), resulting in bypass of ERK pathway dependence and therapeutic resistance (17, 20, 39). The observations that baseline expression of HER family RTKs trended with MRTX849 antitumor activity and that CRISPR-based drug-anchored screens implicated EGFR, SHP2, and mTOR–S6 pathways as cotargetable vulnerabilities both support the hypothesis that these targets act as conditional response modifiers.

Activation of RTK signaling in the context of KRASG12C-mutant cancer was predicted to limit MRTX849 therapeutic response both by enhancing extrinsic regulation of GTPase activity and initiating KRAS-independent ERK and mTOR–S6 pathway activation. Therefore, HER family and SHP2 inhibition were employed as strategies to either block the critical RTK family in KRAS-mutant cells or block collective RTK signaling downstream, respectively. As MRTX849 binds only GDP-KRASG12C, both HER family and SHP2 inhibition each enhanced KRASG12C modification by MRTX849 and significantly improved antitumor activity. This observation is consistent with the putative role of activated RTKs in the engagement of SHP2 to mediate SOS1-dependent RAS GTP loading and to diminish RAS GAP activity, each of which converge on enhanced RAS activation state (40). The afatinib combination demonstrated a clear and marked inhibition of both the ERK–RSK and AKT–mTOR–S6 signaling pathways, whereas the SHP2 inhibitor combination demonstrated a clear impact on ERK–RSK signaling and a relatively less prominent impact on mTOR–S6 signaling. Although afatinib may more effectively address mTOR–S6 bypass signaling, SHP2 inhibition should be an effective combinatorial strategy to combat other RTKs outside of the HER family, such as FGFRs or MET, that could affect KRAS dependence. To further address bypass signaling mediated by RTK activation or STK11 mutations, each of which activate the mTOR–S6 signaling pathway independently of KRAS, mTOR inhibition in combination with MRTX849 was also evaluated. MRTX849 in combination with vistusertib, in fact, demonstrated significantly improved antitumor activity in vivo compared with either single agent in all six tumor models evaluated, regardless of STK11 mutational status. Consistent with the mechanism of action of vistusertib, comprehensive inhibition of AKT–mTOR–S6 signaling was observed for vistusertib alone and near-complete inhibition of pS6S235–36 and pS6240–44 was observed in combination. In addition, the marked feedback reactivation of ERK by vistusertib was relieved by the combination. The induction of ERK activity has been observed in tumor cells following mTORC1 inhibition by rapalogs or ATP-competitive inhibitors and has been implicated in limiting antitumor activity of this class of agents (30, 41, 42), supporting the suppression of ERK signaling by MRTX849 as a key mechanism of response to the combination. Notably, all three combination strategies converge on more comprehensive inhibition of KRAS-dependent signaling, converging on ERK and S6 activity. In addition, although the inhibition of the AKT–mTOR–S6 pathway did not correlate with model response to MRTX849 (potentially due to tumor heterogeneity), the observations that both MTOR and RPS6 drop out in drug-anchored CRISPR screens and that effective combination strategies more comprehensively block this pathway illustrate its likely importance in maximizing therapeutic response in KRAS-mutated cancers.

Cell-cycle dysregulation due to genetic alterations in cell-cycle regulators identified additional factors that could modify the therapeutic response to MRTX849. In addition, CDKN2A, RB1, CDK4, and CDK6 were all identified as gene targets that affected cell fitness in CRISPR screens. Genetic alterations including homozygous deletion of CDKN2A or amplification of CDK4 or CCND1 comprise up to 20% of KRAS-mutated NSCLC (43). Combination studies with MRTX849 and palbociclib in vivo demonstrated more comprehensive inhibition of RB and E2F family target genes and increased antitumor activity compared with either single agent in NSCLC models. In addition, these studies indicated that the combination resulted in more effective inhibition of S6 (S235/236) phosphorylation, establishing a previously unappreciated connection between cell-cycle blockade and protein translation pathways. Notably, this combination was especially effective in CDKN2A-deleted models, suggesting that this combination strategy may be primarily beneficial in a molecularly defined subset of patients characterized by decoupling of cell-cycle regulation from KRAS.

Collectively, models exhibiting a cytoreductive response to single-agent MRTX849 demonstrated a more comprehensive and durable inhibition of KRAS-dependent signaling and induction of an apoptotic response. These data suggest that maintaining durable inhibition of KRAS-dependent signaling below a defined threshold is required to elicit tumor regression. The elucidation of mechanisms that limit the therapeutic response to single-agent KRAS inhibition has provided insight toward strategies to enhance therapeutic activity in KRAS-mutant tumors. Of the 35% of models (9/26) that did not exhibit durable regression with single-agent MRTX849 treatment, five models (KYSE410, SW1573, H2122, H2030, and LU6405) were selected for rational combination studies, and at least one combination demonstrated significant improvement in antitumor efficacy and elicited a >50% tumor regression in all five models evaluated. These results suggest that essentially all KRASG12C-mutated cancers can derive clinical benefit from direct KRAS inhibitor–directed therapy either alone or in combination. Furthermore, rational pathway-centric combination regimens directed at hallmark signaling nodes may be directed to genetically defined patient subsets. For example, KRAS-mutated NSCLC exhibits mutually exclusive, co-occurring genetic alterations in STK11 and CDKN2A (43). The present data suggest that KRASG12C/STK11-mutated NSCLC could be readily addressed by combining a KRASG12C inhibitor with an RTK or mTOR inhibitor, whereas KRASG12C/CDKN2A-mutated NSCLC could be more effectively addressed by combination with a CDK4/6 inhibitor. Collectively, the present studies support the broad utility of covalent KRASG12C inhibitors in treating KRASG12C-mutated cancers and provide defining strategies to identify patients likely to benefit from single-agent therapy or rationally directed combinations.

  

UPDATED 02/07/2021

The November 1st issue of Science highlights a series of findings which give cancer researchers some hope in finally winning a thirty year war with the discovery of drugs that target KRAS, one of the most commonly mutated oncogenes  (25% of cancers), and thought to be a major driver of tumorigenesis. Once considered an undruggable target, mainly because of the smooth surface with no obvious pockets to fit a drug in, as well as the plethora of failed attempts to develop such an inhibitor, new findings with recently developed candidates, highlighted in this article and other curated within, are finally giving hope to researchers and oncologists who have been hoping for a clinically successful inhibitor of this once considered elusive target.

For a great review on development of G12C KRas inhibitors please see Dr. Hobb’s and Channing Der’s review in Cell Selective Targeting of the KRAS G12C Mutant: Kicking KRAS When It’s Down

Figure 1Mechanism of Action of ARS853 showing that the inhibitors may not need bind to the active conformation of KRAS for efficacy

Abstract: Two recent studies evaluated a small molecule that specifically binds to and inactivates the KRAS G12C mutant. The new findings argue that the perception that mutant KRAS is persistently frozen in its active GTP-bound form may not be accurate.

Although the development of the KRASG12C-specific inhibitor, compound 12 (Ostrem et al., 2013), was groundbreaking, subsequent studies found that the potency of compound 12 in cellular assays was limited (Lito et al., 2016, Patricelli et al., 2016). A search for more-effective analogs led to the development of ARS853 (Patricelli et al., 2016), which exhibited a 600-fold increase of its reaction rate in vitro over compound 12 and cellular activities in the low micromolar range.

A Summary and more in-depth curation of the Science article is given below:

After decades, progress against an ‘undruggable’ cancer target

Summary

Cancer researchers are making progress toward a goal that has eluded them for more than 30 years: shrinking tumors by shutting off a protein called KRAS that drives growth in many cancer types. A new type of drug aimed at KRAS made tumors disappear in mice and shrank tumors in lung cancer patients, two companies report in papers published this week. It’s not yet clear whether the drugs will extend patients’ lives, but the results are generating a wave of excitement. And one company, Amgen, reports an unexpected bonus: Its drug also appears to stimulate the immune system to attack tumors, suggesting it could be even more powerful if paired with widely available immunotherapy treatments.

Jocelyn Kaiser. After decades, progress against an ‘undruggable’ cancer target. Science  01 Nov 2019: Vol. 366, Issue 6465, pp. 561 DOI: 10.1126/science.366.6465.561

The article highlights the development of three inhibitors: by Wellspring Biosciences, Amgen, and Mirati Therapeutics.

Wellspring BioSciences

In 2013, Dr. Kevan Shokat’s lab at UCSF discovered a small molecule that could fit in the groove of the KRAS mutant G12C.  The G12C as well as the G12D is a common mutation found in KRAS in cancers. KRAS p.G12C mutations predominate in NSCLC comprising 11%–16% of lung adenocarcinomas (45%–50% of mutant KRAS is p.G12C) (Campbell et al., 2016; Jordan et al., 2017), as well as 1%–4% of pancreatic and colorectal adenocarcinomas, respectively (Bailey et al., 2016; Giannakis et al., 2016).  This inhibitor was effective in shrinking, in mouse studies conducted by Wellspring Biosciences,  implanted tumors containing this mutant KRAS.

See Wellspring’s news releases below:

March, 2016 – Publication – Selective Inhibition of Oncogenic KRAS Output with Small Molecules Targeting the Inactive State

 

February, 2016 – Publication – Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism

 

Amgen

Amgen press release on AMG510 Clinical Trial at ASCO 2019

THOUSAND OAKS, Calif., June 3, 2019 /PRNewswire/ — Amgen (NASDAQ: AMGN) today announced the first clinical results from a Phase 1 study evaluating investigational AMG 510, the first KRASG12C inhibitor to reach the clinical stage. In the trial, there were no dose-limiting toxicities at tested dose levels. AMG 510 showed anti-tumor activity when administered as a monotherapy in patients with locally-advanced or metastatic KRASG12C mutant solid tumors. These data are being presented during an oral session at the 55th Annual Meeting of the American Society of Clinical Oncology (ASCO) in Chicago.

“KRAS has been a target of active exploration in cancer research since it was identified as one of the first oncogenes more than 30 years ago, but it remained undruggable due to a lack of traditional small molecule binding pockets on the protein. AMG 510 seeks to crack the KRAS code by exploiting a previously hidden groove on the protein surface,” said David M. Reese, M.D., executive vice president of Research and Development at Amgen. “By irreversibly binding to cysteine 12 on the mutated KRAS protein, AMG 510 is designed to lock it into an inactive state. With high selectivity for KRASG12C, we believe investigational AMG 510 has high potential as both a monotherapy and in combination with other targeted and immune therapies.”

The Phase 1, first-in-human, open-label multicenter study enrolled 35 patients with various tumor types (14 non-small cell lung cancer [NSCLC], 19 colorectal cancer [CRC] and two other). Eligible patients were heavily pretreated with at least two or more prior lines of treatment, consistent with their tumor type and stage of disease. 

Canon, J., Rex, K., Saiki, A.Y. et al. The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature 575, 217–223 (2019) doi:10.1038/s41586-019-1694-1

Besides blocking tumor growth, AMG510 appears to stimulate T cells to attack the tumor, thus potentially supplying a two pronged attack to the tumor, inhibiting oncogenic RAS and stimulating anti-tumor immunity.

Mirati Therapeutics

Mirati’s G12C KRAS inhibitor (MRTX849) is being investigated in a variety of solid malignancies containing the KRAS mutation.

For recent publication on results in lung cancer see Patricelli M.P., et al. Cancer Discov. 2016; (Published online January 6, 2016)

For more information on Mirati’s KRAS G12C inhibitor see https://www.mirati.com/pipeline/kras-g12c/

KRAS G12C Inhibitor (MRTX849)

Study 849-001 – Phase 1b/2 of single agent MRTX849 for solid tumors with KRAS G12C mutation

Phase 1b/2 clinical trial of single agent MRTX849 in patients with advanced solid tumors that have a KRAS G12C mutation.

See details for this study at clinicaltrials.gov

UPDATED 02/07/2021

Amgen scientists’ rapid work to challenge the undruggable KRAS G12C mutation in cancer

Inside a 40-year quest to challenge the KRAS G12C mutation in cancer
By Amgen Oncology
Amgen’s sotorasib, an investigational lung cancer treatment, has been submitted to the FDA for review

 

 

Nearly four decades have passed since researchers first identified the RAS gene family, which includes HRASNRAS and KRASRAS is the most frequently mutated family of oncogenes – or potentially cancerous genes – in human cancers.1,2 While research  efforts have been able to identify and develop treatments for other driver gene mutations that contribute to cancer growth, success with treating KRAS, the most frequently mutated variant of the RAS family, has remained elusive.2 But now there is hope.

Amgen, a leading biotechnology company, has taken on one of the toughest challenges of the last 40 years in cancer research.3 Chemical biologist Kevan Shokat’s lab at the University of California, San Francisco, identified a small molecule that could slip into a groove on a KRAS mutation called G12C in 2013.4 Building on their own research strategies and this new insight, scientists at Amgen used structural biology and medicinal chemistry to identify an adjacent groove, and by November 2017, made the initial decision to advance the molecule that would become investigational sotorasib.5

KRAS G12C is the most common KRAS mutation in NSCLC.6,7 In the U.S., about 13% of patients with NSCLC harbor the KRAS G12C mutation.8 There is a high unmet need and poor outcomes in the second-line treatment of KRAS G12C-driven non-small cell lung cancer (NSCLC) and, currently, there are no KRASG12C targeted therapies approved.

According to Amgen’s head of research and development David Reese, “the company’s scientists had an idea some time ago that the future of oncology would be led by the marriage of immuno-oncology and precision therapy. We wanted to go after high value targets, and RAS proteins are one of them.”

Because of this effort to rapidly accelerate the speed of innovation, investigational sotorasib entered clinical trials in humans less than 12 months.

 

 

At the same time that scientists discovered investigational sotorasib, the team was undertaking a project to map out every step it takes to progress a potential new treatment from an idea in a lab to being made available for patients. The goal was to shrink timelines and eliminate gaps to develop drugs more rapidly in order to reach patients with serious illnesses like NSCLC as quickly as possible.

Because of this effort to rapidly accelerate the speed of innovation, sotorasib entered clinical trials in humans less than 12 months.5 Sotorasib was the first investigational KRASG12C inhibitor to enter the clinic, and is now being studied in the broadest clinical program exploring 10 combinations with global investigational sites spanning five continents.9 In a little more than two years, the sotorasib clinical program has established a clinical data set of more than 700 patients studied across 13 tumor types.9

The investigational treatment was recently submitted to the FDA for review and was granted Breakthrough Therapy designation, a distinction designed to expedite the development and review of drugs.5 It was also accepted into the FDA’s Real-Time Oncology Review pilot program, which aims to explore a more efficient review process.5

To learn more about Amgen and how the speed of innovation is bringing new oncology treatments to patients with high unmet needs, visit Amgen.com/KnowKRAS.  

______________________

1 Ryan MB, et al. Nat Rev Clin Oncol. 2018;15:709-720.

2 Cox AD, et al. Nat Rev Drug Discov. 2014;13:828-851.

3 Kim D, et al. Cell. 2020. doi:10.1016/j.cell.2020.09.044.

4 Ostrem JM, et al. Nature. 2013 ; 503 :548-551.

5 AMGEN, 2020. Retrieved January 8, 2021, from https://www.amgen.com/stories/2020/12/rapidly-advancing-development-of-amgens-investigational-kras-g12c-inhibitor

6 Pakkala S, et al. JCI Insight. 2018;3:e120858.

7 Arbour KC, et al. Clin Cancer Res. 2018;24:334-340.

8 Amgen, Data on File. 2020.

9 ClinicalTrials.gov. NCT04185883, NCT04380753, NCT03600883, NCT04303780. https://clinicaltrials.gov/ct2/. Accessed January 20, 2020.

 
 
Members of the editorial and news staff of the USA TODAY Network were not involved in the creation of this content.
 
 
 

Additional References:

Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism.

Lito P et al. Science. (2016)

Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor.

Janes MR et al. Cell. (2018)

Potent and Selective Covalent Quinazoline Inhibitors of KRAS G12C.

Zeng M et al. Cell Chem Biol. (2017)

Campbell, J.D., Alexandrov, A., Kim, J., Wala, J., Berger, A.H., Pedamallu, C.S., Shukla, S.A., Guo, G., Brooks, A.N., Murray, B.A., et al.; Cancer Genome Atlas Research Network (2016). Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas. Nat. Genet.48, 607–616

Jordan, E.J., Kim, H.R., Arcila, M.E., Barron, D., Chakravarty, D., Gao, J., Chang, M.T., Ni, A., Kundra, R., Jonsson, P., et al. (2017). Prospective comprehensive molecular characterization of lung adenocarcinomas for efficient patient matching to approved and emerging therapies. Cancer Discov. 7, 596–609.

Bailey, P., Chang, D.K., Nones, K., Johns, A.L., Patch, A.M., Gingras, M.C., Miller, D.K., Christ, A.N., Bruxner, T.J., Quinn, M.C., et al.; Australian Pancreatic Cancer Genome Initiative (2016). Genomic analyses identify molecular subtypes of pancreatic cancer. Nature 531, 47–52.

Giannakis, M., Mu, X.J., Shukla, S.A., Qian, Z.R., Cohen, O., Nishihara, R., Bahl, S., Cao, Y., Amin-Mansour, A., Yamauchi, M., et al. (2016). Genomic correlates of immune-cell infiltrates in colorectal carcinoma. Cell Rep. 15, 857–865.

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Pfizer buys out Array BioPharma for $11.4 Billion to beef up its oncology offerings

Reporter: Stephen J. Williams, PhD

As reported in FiercePharma.com:

by Angus Liu |

Three years after purchasing Medivation for $14.3 billion, Pfizer is back with another hefty M&A deal. And once again, it’s betting on oncology.

In the first big M&A deal under new CEO Albert Bourla, Pfizer has agreed to buy oncology specialist Array BioPharma for a total value of about $11.4 billion, the two companies unveiled Monday. The $48-per-share offer represents a premium of about 62% to Array stock’s closing price on Friday.

With the acquisition, Pfizer will beef up its oncology offerings with two marketed drugs, MEK inhibitor Mektovi and BRAF inhibitor Braftovi, which are approved as a combo treatment for melanoma and recently turned up positive results in colon cancer.

The buy will enhance the Pfizer innovative drug business’ “long-term growth trajectory,” Bourla said in a Monday statement, dubbing Mektovi-Braftovi “a potentially industry-leading franchise for colorectal cancer.”

RELATED: Array’s ‘extremely compelling’ new colon cancer data spark blockbuster talk

In a recent interim analysis of a trial in BRAF-mutant metastatic colorectal cancer, the pair, used in tandem with Eli Lilly and Merck KGaA’s Erbitux, produced a benefit in 26% of patients, versus the 2% that chemotherapy helped. The combo also showed it could reduce the risk of death by 48%. SVB Leerink analysts at that time called the data “extremely compelling.”

Right now, one in every three new patients with mutated metastatic melanoma is getting the combo, despite its third-to-market behind combos from Roche and Novartis, Andy Schmeltz, Pfizer’s oncology global president, said during an investor briefing on Monday.

It is being studied in more than 30 clinical studies across several solid tumor indications. Moving forward, Pfizer believes the combo could potentially be used in the adjuvant setting to prevent tumor recurrence after surgery, Pfizer’s chief scientific officer, Mikael Dolsten, said on the call. The company is also keen to know how it could be paired up with Pfizer’s own investigational PD-1, he said, as the combo is already in studies with other PD-1/L1s.

But as Pfizer execs have previously said, the company’s current business development strategy no longer centers on adding revenues “now or soon,” but rather on strengthening Pfizer’s pipeline with earlier-stage assets. And Array can help there, too.

“We are very excited by Array’s impressive track record of successfully discovering and developing innovative small-molecules and targeted cancer therapies,” Dolsten said in a statement.

On top of Mektovi and Braftovi, Array has a long list of out-licensed drugs that could generate big royalties over time. For example, Vitrakvi, the first drug to get an initial FDA approval in tumors with a particular molecular feature regardless of their location, was initially licensed to Loxo Oncology—which was itself snapped up by Eli Lilly for $8 billion—but was taken over by pipeline-hungry Bayer. There are other drugs licensed to the likes of AstraZeneca, Roche, Celgene, Ono Pharmaceutical and Seattle Genetics, among others.

Those drugs are also a manifestation of Array’s strong research capabilities. To keep those Array scientists doing what they do best, Pfizer is keeping a 100-person team in Colorado as a standalone research unit alongside Pfizer’s existing hubs, Schmeltz said.

Pfizer is counting on Array to augment its leadership in breast cancer, an area championed by Ibrance, and prostate cancer, the pharma giant markets Astellas-partnered Xtandi. For 2018, revenues from the Pfizer oncology portfolio jumped to $7.20 billion—up from $6.06 billion in 2017—mainly thanks to those two drugs.

Source: https://www.fiercepharma.com/pharma/pfizer-never-say-never-m-a-buys-oncology-innovator-array-for-11-4b

 

About Array BioPharma

Array markets BRAFTOVI® (encorafenib) capsules in combination with MEKTOVI® (binimetinib)  tablets for the treatment of patients with unresectable or metastatic melanoma with a BRAFV600E or BRAFV600K  mutation in the United States and with partners in other major worldwide markets.* Array’s lead clinical programs, encorafenib and binimetinib, are being investigated in over 30 clinical trials across a number of solid tumor indications, including a Phase 3 trial in BRAF-mutant metastatic colorectal cancer. Array’s pipeline includes several additional programs being advanced by Array or current license-holders, including the following programs currently in registration trials: selumetinib (partnered with AstraZeneca), LOXO-292 (partnered with Eli Lilly), ipatasertib (partnered with Genentech), tucatinib (partnered with Seattle Genetics) and ARRY-797. Vitrakvi® (larotrectinib, partnered with Bayer AG) is approved in the United States and Ganovo® (danoprevir, partnered with Roche) is approved in China.

 

Other Articles of Note of Pfizer Merger and Acquisition deals on this Open Access Journal Include:

From Thalidomide to Revlimid: Celgene to Bristol Myers to possibly Pfizer; A Curation of Deals, Discovery and the State of Pharma

Pfizer Near Allergan Buyout Deal But Will Fed Allow It?

Pfizer offers legal guarantees over AstraZeneca bid

Re-Creation of the Big Pharma Model via Transformational Deals for Accelerating Innovations: Licensing vs In-house inventions

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Inactivation of an enzyme needed to metabolize glucose by Vitamic C deprives tumor cells of energy

Reporter: Aviva Lev-Ari, PhD, RN

 

 

Vitamin C did kill cultured colon cancer cells with BRAF or KRAS mutations by raising free radical levels, which in turn inactivate an enzyme needed to metabolize glucose, depriving the cells of energy.

 

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

Glucose Deprivation Contributes to the Development of KRAS Pathway Mutations in Tumor Cells

A few years ago, Jihye Yun, then a graduate student at Johns Hopkins University in Baltimore, Maryland, found that colon cancer cells whose growth is driven by mutations in the gene KRAS or a less commonly mutated gene,BRAF, make unusually large amounts of a protein that transports glucose across the cell membrane. The transporter, GLUT1, supplies the cells with the high levels of glucose they need to survive. GLUT1 also transports the oxidized form of vitamin C, dehydroascorbic acid (DHA), into the cell, bad news for cancer cells, because Yun found that DHA can deplete a cell’s supply of a chemical that sops up free radicals. Because free radicals can harm a cell in various ways, the finding suggested “a vulnerability” if the cells were flooded with DHA, says Lewis Cantley at Weill Cornell Medicine in New York City, where Yun is now a postdoc.

Cantley’s lab and collaborators found that large doses of vitamin C did indeed kill cultured colon cancer cells with BRAF or KRAS mutations by raising free radical levels, which in turn inactivate an enzyme needed to metabolize glucose, depriving the cells of energy. Then they gave daily high dose injections—equivalent to a person eating 300 oranges—to mice engineered to develop KRAS-driven colon tumors. The mice developed fewer and smaller colon tumors compared with control mice.

SOURCE
http://www.sciencemag.org/news/2015/11/vitamin-c-kills-tumor-cells-hard-treat-mutation

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FDA approves blood-based colorectal screen

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

FDA Clears First Blood-Based Colorectal Cancer Screening Test.
Lab Soft News by Bruce Friedman
https://www.linkedin.com/pulse/fda-clears-first-blood-based-colorectal-cancer-screening-joseph-colao

In the upcoming months, there surely will be an increasing number of blood-based genomic cancer tests approved by the FDA. This specific test market is just too attractive. In a recent note, I discussed some of these testing initiatives from the perspective of companion diagnostics (see: An Expanding Definition for Companion Diagnostics). This form of testing in used in collaboration with cancer therapy to select the right drug or monitor the effectiveness of drug therapy. Obviously and of equal importance are biomarkers intended for cancer screening. A recent article reported that the FDA has cleared the first blood-based screening test for colorectal cancer (see: FDA Clears First Blood-Based Colorectal Cancer Screening Test), Below is an excerpt from it:

The first blood-based colorectal cancer (CRC) screening test, Epi proColon...has been approved by the US Food and Drug Administration (FDA)….The Epi proColon test is a qualitative in vitro diagnostic test for detecting methylated Septin9 DNA, which has been associated with the occurrence of CRC, in plasma obtained from whole-blood specimens. It is indicated for use in average-risk patients who have chosen not to undergo other screening methods, such as colonoscopy or stool-based tests.The test was recommended for FDA approval in 2014 by the Molecular and Clinical Genetics Panel of the FDA’s Medical Devices Advisory Committee, but some of the experts were not convinced….The agency approved the Epi proColon test for CRC screening in average-risk patients (as defined by current screening guidelines) who choose not to be screened by colonoscopy or a stool-based FIT [fecal immunochemical test for occult blood in the stool].The Epi proColon blood test for CRC screening can be performed during routine office visits. It requires no dietary restrictions or alterations in medication use. The blood sample is analyzed by a local or regional diagnostic laboratory….The company will initiate a postapproval study to show the long-term benefit of blood-based CRC screening using Epi proColon, as required by the FDA.

Here’s more information about Septin9 DNA (see: Plasma methylated septin 9: a colorectal cancer screening marker):

The biomarker septin 9 has been found to be hypermethylated in nearly 100% of tissue neoplasia specimens and detected in circulating DNA fractions of CRC patients. A commercially available assay for septin 9 has been developed with moderate sensitivity (∼70%) and specificity (∼90%) and a second generation assay, Epi proColon 2.0 (Epigenomics AG), shows increased sensitivity (∼92%).The performance of the assay proved to be independent of tumor site and reaches a high sensitivity of 77%, even in early cancer stages (I and II). Furthermore, septin 9 was recently used in follow-up studies for detection of early recurrence of CRC. 

There is clearly a need for a blood-based biomarker for colorectal cancer screening. Patients tend to dislike the home stool collection that is required for fecal immunochemical tests for occult blood in the stool (FIT). Moreover, testing for blood in the stool offers a somewhat crude substitute for the identification of reliable cancer biomarkers in the blood. It must be noted, however, that some of the FDA experts in 2014 were not convinced that the septic 9 biomarker offered advantages over FIT.

I am not sure that septin 9 will be the final and most efficient biomarker for CRC but I am sure of two things. The first is that there will eventually be a high-specificity, high-sensitivity blood test for CRC. The second is that probably tens of billions of dollars would be saved by the elimination of screening colonoscopies for CRC by such a test. I found an article dating way back to 2002 about the number of screening endoscopies performed in the U.S. but the numbers are sill impressive  (see: How many endoscopies are performed for colorectal cancer screening?) Here is a quote from it: “Approximately 2.8 million flexible sigmoidoscopes and 14.2 million colonoscopies were estimated to have been performed in 2002.” Needless to say, many gastroenterologists and radiologists may be hoping that such a lab test does not reach the market soon.

Septin-9 is a protein that in humans is encoded by the SEPT9 gene.[1][2][3

SEPT9 has been shown to interact with SEPT2[4] and SEPT7.[4]

Along with AHNAK, eIF4E and S100A11, SEPT9 has been shown to be essential for pseudopod protrusion, tumor cell migration and invasion.[5]

The v2 region of the SEPT9 promoter has been shown to be methylated in colorectal cancer tissue compared with normal colonic mucosa.[6] Using highly sensitive real time PCR assays, methylated SEPT9 was detected in the blood of colorectal cancer patients. This alternate methylation pattern in cancer samples is suggestive of an aberrant activation or repression of the gene compared to normal tissue samples.[7][8]

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Colorectal cancer stemness and ERK

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

KCTD12 Regulates Colorectal Cancer Cell Stemness through the ERK Pathway

Liping Li, Tingmei Duan, Xin Wang, Ru-Hua Zhang, Meifang Zhang, Suihai Wang,Fen Wang, Yuanzhong Wu, Haojie Huang & Tiebang Kang

Scientific Reports 2016; 6(20460)    http://dx.doi.org:/10.1038/srep20460

Targeting cancer stem cells (CSCs) in colorectal cancer (CRC) remains a difficult problem, as the regulation of CSCs in CRC is poorly understood. Here we demonstrated that KCTD12, potassium channel tetramerization domain containing 12, is down-regulated in the CSC-like cells of CRC. The silencing of endogenous KCTD12 and the overexpression of ectopic KCTD12 dramatically enhances and represses CRC cell stemness, respectively, as assessed in vitro and in vivo using a colony formation assay, a spheroid formation assay and a xenograft tumor model. Mechanistically, KCTD12 suppresses CRC cell stemness markers, such as CD44, CD133 and CD29, by inhibiting the ERK pathway, as the ERK1/2 inhibitor U0126 abolishes the increase in expression of CRC cell stemness markers induced by the down-regulation of KCTD12. Indeed, a decreased level of KCTD12 is detected in CRC tissues compared with their adjacent normal tissues and is an independent prognostic factor for poor overall and disease free survival in patients with CRC (p = 0.007). Taken together, this report reveals that KCTD12 is a novel regulator of CRC cell stemness and may serve as a novel prognostic marker and therapeutic target for patients with CRC.

 

Colorectal carcinoma (CRC) remains one of the most aggressive cancers in the world. Every year, more than 1.2 million patients with CRC are diagnosed, and almost 50% die from the disease. Surgery, radiotherapy and chemotherapy are still the predominant therapeutic strategies1. Although surgery combined with chemoradiotherapy represents a viable treatment option for early stage tumors, the majority of patients are not diagnosed until the late stage, for which the 5 year survival rate post-surgery decreased from 69.2% to 11.7%2.

Cancer stem cells (CSCs) are considered to be responsible for recurrence and metastasis during CRC tumorigenesis3. CSCs, possessing self-renewal characteristics, initiate tumor growth and promote chemotherapy and radiation resistance, which are considered to be responsible for CRC progression and recurrence4,5. Targeting the determinants of CRC cell stemness has been proposed as a therapeutic strategy6.

KCTD12 (potassium channel tetramerization domain containing 12, pfetin), which contains a voltage-gated potassium (K+) channel tetramerization T1 domain and a BTB/POZ (Bric-a-brac, Tram-track, Broad complex poxvirus and zinc finger) domain, belongs to the KCTD family and was initially identified in cochlea. In addition to being a K+ channel protein that responds to membrane potential7, KCTD12 also acts as an auxiliary subunit of GABAB (γ-aminobutyric acid type B) receptors, which regulate emotionality and neuronal excitability8,9. Interestingly, high KCTD12 expression indicates a favorable prognosis and could act as an independent prognostic factor for GIST (gastrointestinal stromal tumors)10, most likely due to the control of tumor and tumor stem cell proliferation by GABA signaling11. In addition, other KCTD family members, such as KCTD21, 11, and 6, have been shown to regulate the growth of MB (medulloblastoma) stem cells through the histone deacetylase HDAC1 and Hh/Gli12,13,14. However, there is no information about whether KCTD family members play crucial roles in CRC cell stemness. As described here, using HT29 cells and their spheroids, we observed that KCTD12 was the most altered member of the KCTD family in the spheroids of HT29 cells, leading to our speculation that KCTD12 plays a crucial role in CRC cell stemness. In verification of this hypothesis, our data support that KCTD12 is a potential regulator of CRC cell stemness at a cellular level, in an animal model and in clinical samples.

 

KCTD12 is down-regulated in the spheroids of HT29 cells

To investigate whether KCTD family members are involved in the stemness of CRC cells, we first enriched for stemness characteristics of HT29 cells by culturing them as spheroids for 8 days15. As shown in Fig. 1A,B, the percentages of cells expressing CD133 and CD44, two well-known stemness markers, were dramatically increased as shown by flow cytometry assay and western blotting. Subsequently, the mRNA levels of KCTD family members were compared between the spheroids and the parental HT29 cells. As the results in Fig. 1C show, the mRNA levels of KCTD1, 5 and 12 were decreased, while the levels of KCTD21 were increased in spheroids of HT29 cells. Notably, KCTD12 was the most significantly decreased family member in spheroids of HT29 cells (Fig. 1C), which was further confirmed by the observation that the KCTD12 protein was also significantly decreased in the spheroids of HT29 cells (Fig. 1D). However, KCTD8 and KCTD19 could not be detected in HT29 cells. These findings indicate that KCTD12 may play a crucial role in the stemness of CRC cells.

Figure 1: KCTD12 is down-regulated in CSC-like HT29 cells.

Figure 1

http://www.nature.com/article-assets/npg/srep/2016/160205/srep20460/images_hires/w926/srep20460-f1.jpg

(A) Representative flow cytometry plots and quantitative analysis showing the percentages of CD44+ and CD133+ cells in normal adherent and spheroid cultures of HT29 cells. (B) CD44 and CD133 expressions were analyzed by Western blotting in adherent and spheroid cultures of HT29 cells. (C) Quantitative real time PCR analysis of the relative mRNA levels of the KCTD family members in adherent and spheroid cultures of HT29 cells. (D) KCTD12 expression was analyzed by Western blotting in adherent and spheroid cultures of HT29 cells. The results are presented as the means ± SD, and all data are representative of three independent experiments. *P < 0.05, **P < 0.01.

KCTD12 regulates CRC cell stemness in cell lines

Next, we asked whether KCTD12 influences stemness using CRC cell lines with varying levels of KCTD12 (Fig. 2A). HT29 cells and DLD1 cells were chosen to knockdown and overexpress KCTD12, respectively (Fig. 2B). As shown in Fig. 2C, the silencing and the overexpression of KCTD12 were capable of increasing and decreasing, respectively, the well-known CRC cell stemness markers CD44, CD133 and CD29 at the protein and mRNA levels. Consistently, the percentages of cells positive for CD44 or CD133 were dramatically increased when KCTD12 was knocked down in HT29 cells (Fig. 2D). In addition, the silencing and the overexpression of KCTD12 in HT29 or DLD1 cells increased and decreased the sizes of spheres, respectively, while having no effect on the numbers of spheres (Fig. 2E,F). Taken together, these results indicate that KCTD12 is critical to the stemness of CRC cells.

 

Figure 2: KCTD12 suppresses the stemness of CRC cells.

Figure 2

http://www.nature.com/article-assets/npg/srep/2016/160205/srep20460/images_hires/w926/srep20460-f2.jpg

(A) KCTD12 protein was analyzed by Western blotting in the indicated CRC cell lines. (B) The indicated stable cell lines with silencing or overexpression of KCTD12 were analyzed by Western blotting. α-tubulin or HSP70 was used as the loading control. (CE) CD44, CD133 and CD29 levels were analyzed by Western blotting, qRT-PCR and flow cytometry, in the indicated stable cell lines. Red lines indicating the mean intensity of fluorescence of CD44+ or CD133+ were quantified by Flow-J software in the flow cytometry analysis. The mean intensity of fluorescence of CD44+ or CD133+ was calculated in triplicates. (F,G) Images and quantification of the number and size of spheres formed from the indicated stable cell lines in the absence of serum for 7 days. Original magnification in F, 40×(upper), 400×(lower). Original magnification in G, 40×. Scale bars, 100 μm. The results are presented as the means ± SD, and all data are representative of three independent experiments. *P < 0.05, **P < 0.01.

 

KCTD12 is involved in the self-renewal ability of CRC cells in vitro and in the tumorigenesis of CRC cells in vivo

We further explored the functions of KCTD12 in the self-renewal and tumorigenesis of CRC cells. First, as shown in Fig. 3A by the colony formation assay, the knockdown of KCTD12 in HT29 cells significantly enhanced the cells’ colony formation capacity, whereas the overexpression of KCTD12 in both DLD1 and HCT116 cells reduced this capacity (Fig. 3B). However, the alteration of KCTD12 in these cells did not affect their proliferation (Fig. 3C). These results indicate that KCTD12 is involved in the self-renewal ability of CRC cells in vitro. Second, as shown inFig. 4, the knockdown of KCTD12 in HT29 cells promoted, whereas the overexpression of KCTD12 in DLD1 cells inhibited, tumor growth in nude mice, as measured by tumor volumes and weights. These results suggest that KCTD12 plays a crucial role in CRC tumorigenesis in vivo.

Figure 3: KCTD12 inhibits the colony formation of CRC cells in vitro.

Figure 3

http://www.nature.com/article-assets/npg/srep/2016/160205/srep20460/images_hires/w926/srep20460-f3.jpg

(A,B) The colony formation assays were performed in the indicated stable cell lines. (C) The cell proliferation was measured by MTT in the indicated stable cell lines. The results are presented as the means ± S.E. of three independent experiments. *P < 0.05, **P < 0.01.

 

Figure 4: KCTD12 represses the tumorigenicity of CRC cells in vivo.

Figure 4

http://www.nature.com/article-assets/npg/srep/2016/160205/srep20460/images_hires/w926/srep20460-f4.jpg

(A,B) A xenograft model consisting of nude mice with HT29 cells harboring KCTD12 silencing injected into the armpits of 4 week old mice (n = 7/group). The images of mice harboring tumors (left) and tumors from the mice (right). Tumor volumes were measured every two days (left). Mean tumor weights were calculated. (D,E) A xenograft model consisting of nude mice with DLD1 cells overexpressing KCTD12 were injected into the armpits of 4 week old mice (n = 5/group). The images of mice harboring tumors (left) and tumors from the mice (right). Tumor volumes were measured every two days (left). Mean tumor weights were calculated. The results are presented as the means ± SD. *P < 0.05, **P < 0.01. (C,F) H&E staining of tumors and IHC staining for KCTD12 protein in these cells. Original magnification, 200×. Scale bars, 100 μm.

 

Silencing of KCTD12 enhances the drug resistance of CRC cells

Figure 5: Silencing of KCTD12 enhances the drug resistance to both imatinib and 5-FU in HT29 cells.

http://www.nature.com/article-assets/npg/srep/2016/160205/srep20460/images_hires/m685/srep20460-f5.jpg

 

KCTD12 regulates CRC cell stemness via the ERK pathway

Given that KCTD12 acts as a component of the GABABcomplex, downstream of which is the ERK pathway, we sought to determine whether the ERK pathway is involved in the KCTD12-mediated regulation of CRC cell stemness. As shown in Fig. 6A,B, phosph-ERK1/2 levels were dramatically increased in HT29 cells with silenced KCTD12 and decreased in DLD1 cells with overexpressed KCTD12. Moreover, U0126, an ERK 1/2 inhibitor, abrogated the increases in CD44, CD133 and CD29 levels in HT29 cells induced by the knockdown of KCTD12 (Fig. 6C). Likewise, the inhibition of the ERK pathway by U0126 reduced the sizes of spheres in KCTD12 knockdown HT29 cells (Fig. 6D). These results indicate that KCTD12 regulates CRC cell stemness via the ERK pathway.

 

Figure 6: KCTD12 regulates stemness of CRC cells via the ERK signaling pathway

http://www.nature.com/article-assets/npg/srep/2016/160205/srep20460/images_hires/m685/srep20460-f6.jpg

(A,B) Phosphorylation of ERK1/2 (p-ERK1/2) and total ERK1/2 (t-ERK1/2) were detected using western blotting in the indicated stable cell lines. (C) HT29 cells with silenced KCTD12 were treated with U0126 (30 μM) for 24 h. Western blotting was performed to detect t-ERK1/2, p-ERK1/2, CD44, CD133 and CD29. Hsp70 was used as a loading control. (D) The sphere formation assays were performed in HT29 cells with silenced KCTD12 and treated with U0126 or DMSO for 7 days. Images and quantification of the numbers and sizes of spheres formed were calculated. The experiments were repeated three times. *P < 0.05, **P < 0.01. Scale bars, 200 μm (left) and 100 μm (right).

 

Low KCTD12 expression indicates a poor prognosis of patients with CRC

Finally, we analyzed the clinical relevance of KCTD12 in CRC samples. As shown in Fig. 7A, the protein level of KCTD12 was significantly higher in normal tissues than in CRC tumor tissues. …

Figure 7: Low expression of KCTD12 was detected in human colorectal cancer tissues.

http://www.nature.com/article-assets/npg/srep/2016/160205/srep20460/images_hires/m685/srep20460-f7.jpg

 

In this report, the down-regulation of KCTD12 is detected in colorectal CSC-like cells, and a low level of KCTD12 is associated with a poor prognosis of patients with CRC. Functionally, KCTD12 regulates CRC cell stemness characteristics, such as self-renewal, tumorigenesis and drug resistance, through the ERK pathway. This is the first report to reveal that KCTD12 regulates CRC cell stemness through the ERK pathway.

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Signaling of Immune Response in Colon Cancer, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 1: Next Generation Sequencing (NGS)

Signaling of Immune Response in Colon Cancer

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Revised 1/13/2016

STING Protein May Serve as Biomarker for Colorectal and Other Cancers

http://www.genengnews.com/gen-news-highlights/sting-protein-may-serve-as-biomarker-for-colorectal-and-other-cancers/81252165/

 

Scientists at University of Miami Miller School of Medicine’s Sylvester Comprehensive Cancer Center say they have discovered how the stimulator of interferon genes (STING) signaling pathway may play an important role in alerting the immune system to cellular transformation. They believe their finding will shed further light on the immune system’s response to cancer development.

In 2008, Glen N. Barber, Ph.D., leader of the viral oncology program at Sylvester, and professor and chairman of cell biology at the Miller School of Medicine, and colleagues published in Nature (“STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling”) the discovery of STING as a new cellular molecule that recognizes virus and bacteria infection to initiate host defense and immune responses. In the new study, published in Cell Reports (“Deregulation of STING Signaling in Colorectal Carcinoma Constrains DNA Damage Responses and Correlates With Tumorigenesis”), they describe STING’s role in the potential suppression of colorectal cancer.

“Since 2008 we’ve known that STING is crucial for antiviral and antibacterial responses,” said Dr. Barber. “But until now, little had been known about its function in human tumors. In this study we show, for the first time, that STING signaling is repressed in colorectal carcinoma and other cancers, an event which may enable transformed cells to evade the immune system.”

Colorectal cancer currently affects around 1.2 million people in the U.S. and 150,000 new cases are diagnosed every year, making it the third most common cancer in both men and women. Since most colon cancers develop from benign polyps, they can be treated successfully when detected early. However, if the tumor has already spread, survival rates are generally low.

Using disease models of colorectal cancer, the team of Sylvester scientists showed that loss of STING signaling negatively affected the body’s ability to recognize DNA-damaged cells. In particular, certain cytokines that facilitate tissue repair and antitumor priming of the immune system were not sufficiently produced to initiate a significant immune response to eradicate the colorectal cancer.

“We were able to show that impaired STING responses may enable damaged cells to elude the immune system,” continued Dr. Barber. “And if the body doesn’t recognize and attack cancer cells, they will multiply and, ultimately, spread to other parts of the body.”

He and his colleagues suggest evaluating STING signaling as a prognostic marker for the treatment of colorectal as well as other cancers. For example, Dr. Barber’s study showed that cancer cells with defective STING signaling were particularly prone to attack by oncolytic viruses presently being used as cancer therapies.

“Impaired STING responses may enable damaged cells to evade host immunosurveillance processes, although they provide a critical prognostic measurement that could help predict the outcome of effective oncoviral therapy,” wrote the investigators.

STING Protein Could be Used for Cancer Diagnosis

http://www.technologynetworks.com/Proteomics/news.aspx?ID=186674

 

This is the first detailed examination of how the stimulator of interferon genes (STING) signaling pathway, discovered by Glen N. Barber, Ph.D., Leader of the Viral Oncology Program at Sylvester Comprehensive Cancer Center, may play an important role in alerting the immune system to cellular transformation.

In 2008, Barber, who is also Professor and Chairman of Cell Biology at the University of Miami Miller School of Medicine, and colleagues published in Nature the discovery of STINGas a new cellular molecule that recognizes virus and bacteria infection to initiate host defense and immune responses. In the new study they describe STING’s role in the potential suppression of colorectal cancer.

“Since 2008 we’ve known that STING is crucial for antiviral and antibacterial responses,” said Barber. “But until now, little had been known about its function in human tumors. In this study we show, for the first time, that STING signaling is repressed in colorectal carcinoma and other cancers, an event which may enable transformed cells to evade the immune system.”

Colorectal cancer currently affects around 1.2 million people in the United States and 150.000 new cases are diagnosed every year, making it the third most common cancer in both men and women. Since most colon cancers develop from benign polyps, they can be treated successfully when detected early. However, if the tumor has already spread, survival rates are generally low.

Using disease models of colorectal cancer, the team of Sylvester scientists showed that loss of STING signaling negatively affected the body’s ability to recognize DNA-damaged cells. In particular, certain cytokines – small proteins important for cell signaling – that facilitate tissue repair and anti-tumor priming of the immune system were not sufficiently produced to initiate a significant immune response to eradicate the colorectal cancer.

“We were able to show that impaired STING responses may enable damaged cells to elude the immune system,” added Barber. “And if the body doesn’t recognize and attack cancer cells, they will multiply and, ultimately, spread to other parts of the body.”

Barber and his colleagues suggest evaluating STING signaling as a prognostic marker for the treatment of colorectal as well as other cancers. For example, Barber’s study showed that cancer cells with defective STING signaling were particularly prone to attack by oncolytic viruses presently being used as cancer therapies. Alternate studies with colleagues have also shown that activators of STING signaling are potent stimulators of anti-tumor immune responses. Collectively, the control of STING signaling may have important implications for cancer development as well as cancer treatment.

 

Every step you take: STING pathway key to tumor immunity

http://sciencelife.uchospitals.edu/2014/11/20/every-step-you-take-sting-pathway-key-to-tumor-immunity/

A recently discovered protein complex known as STING plays a crucial role in detecting the presence of tumor cells and promoting an aggressive anti-tumor response by the body’s innate immune system, according to two separate studies published in the Nov. 20 issue of the journal Immunity.

The studies, both from University of Chicago-based research teams, have major implications for the growing field of cancer immunotherapy. The findings show that when activated, the STING pathway triggers a natural immune response against the tumor. This includes production of chemical signals that help the immune system identify tumor cells and generate specific killer T cells. The research also found that targeted high-dose radiation therapy dials up the activation of this pathway, which promotes immune-mediated tumor control.

These findings could “enlarge the fraction of patients who respond to immunotherapy with prolonged control of the tumor,” according to a commentary on the papers by the University of Verona’s Vincenzo Bronte, MD. “Enhancing the immunogenicity of their cancers might expand the lymphocyte repertoire that is then unleashed by interference with checkpoint blockade pathways,” such as anti-PD-1.

STING, short for STimulator of INterferon Genes complex, is a crucial part of the process the immune system relies on to detect threats — such as infections or cancer cells — that are marked by the presence of DNA that is damaged or in the wrong place, inside the cell but outside the nucleus.

Detection of such “cytosolic” DNA initiates a series of interactions that lead to the STING pathway. Activating the pathway triggers the production of interferon-beta, which in turn alerts the immune system to the threat, helps the system detect cancerous or infected cells, and ultimately sends activated T cells into the battle.

“We have learned

“Innate immune sensing via the host STING pathway is critical for tumor control by checkpoint blockade,” Gajewski’s team noted in their paper. They found promising drugs known as checkpoint inhibitors — such as anti PD-1 or anti PD-L1, which can take the brakes off of an immune response — were not effective in STING-deficient mice. New agents that stimulate the STING pathway are being developed as potential cancer therapeutics.

A second University of Chicago team, led by cancer biologistYang-Xin Fu, MD, PhD, professor of pathology, and Ralph Weichselbaum, MD, chairman of radiation and cellular oncology and co-director of the Ludwig Center for Metastasis Research, found that high-dose radiation therapy not only kills targeted cancer cells but the resulting DNA damage drives a systemic immune response.

a great deal recently about what we call checkpoints, the stumbling blocks that prevent the immune system from ultimately destroying cancers,” said Thomas Gajewski, MD, PhD, professor of medicine and pathology at the University of Chicago and senior author of one of the studies. “Blockade of immune checkpoints, such as with anti-PD-1, is therapeutic in a subset of patients, but many individuals still don’t respond. Understanding the role of the STING pathway provides insights into how we can ‘wake up’ the immune response against tumors. This can be further boosted by checkpoint therapies.”

The two published studies, he said, help move this approach forward.

In a series of experiments in mice, both research teams found tumor cell-derived DNA could initiate an immune response against cancers. But when tested in mice that lacked a functional gene for STING, the immune system did not effectively respond.

“This result unifies traditional studies of DNA damage with newly identified DNA sensing of immune responses,” Fu said.

“This is a previously unknown mechanism,” Weichselbaum added.

In mice that lacked STING, however, there was no therapeutic immune response. The induction of interferons by radiation and consequent cancer cell killing, they conclude, depends on STING-pathway signaling.

They did find that combining cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), an earlier step in the STING pathway, with radiation, could greatly enhance the antitumor efficacy of radiation.

“This opens a new avenue to develop STING-related agonists for patients with radiation-resistant cancers,” Fu said.

 

 

STING-Dependent Cytosolic DNA Sensing Mediates Innate Immune Recognition of Immunogenic Tumors

Seng-Ryong Woo1Mercedes B. Fuertes1Leticia Corrales1, …., Maria-Luisa Alegre2Thomas F. Gajewski1, 2   

Immunity 20 Nov 2014; 41(5): 830–842    http://dx.doi.org:/10.1016/j.immuni.2014.10.017

 

Highlights

• Spontaneous T cell responses against tumors require the host STING pathway in vivo
• Tumor-derived DNA can induce type I interferon production via STING
• Tumor DNA can be identified in host APCs in the tumor microenvironment in vivo

Summary

Spontaneous T cell responses against tumors occur frequently and have prognostic value in patients. The mechanism of innate immune sensing of immunogenic tumors leading to adaptive T cell responses remains undefined, although type I interferons (IFNs) are implicated in this process. We found that spontaneous CD8+ T cell priming against tumors was defective in mice lacking stimulator of interferon genes complex (STING), but not other innate signaling pathways, suggesting involvement of a cytosolic DNA sensing pathway. In vitro, IFN-β production and dendritic cell activation were triggered by tumor-cell-derived DNA, via cyclic-GMP-AMP synthase (cGAS), STING, and interferon regulatory factor 3 (IRF3). In the tumor microenvironment in vivo, tumor cell DNA was detected within host antigen-presenting cells, which correlated with STING pathway activation and IFN-β production. Our results demonstrate that a major mechanism for innate immune sensing of cancer occurs via the host STING pathway, with major implications for cancer immunotherapy.

 

Image for unlabelled figure

http://ars.els-cdn.com/content/image/1-s2.0-S1074761314003938-fx1.jpg

 

Immunity Erratum STING-Dependent Cytosolic DNA Sensing Mediates Innate Immune Recognition of Immunogenic Tumors

Seng-Ryong Woo, Mercedes B. Fuertes, Leticia Corrales, Stefani Spranger, Michael J. Furdyna, Michael Y.K. Leung, Ryan Duggan, Ying Wang, Glen N. Barber, Katherine A. Fitzgerald, Maria-Luisa Alegre, and Thomas F. Gajewski* *Correspondence: tgajewsk@medicine.bsd.uchicago.edu http://dx.doi.org/10.1016/j.immuni.2014.12.015 (Immunity 41, 830–842; November 20, 2014)

The original Figure 3C accidentally contained a duplicated panel in the bright-field column, third row down, and this has now been replaced with the correct data. The change does not alter the conclusions of the paper. This mistake has now been corrected online, and the authors regret the error.

 

Cytosolic DNA Sensors (CDSs): a STING in the tail – Review

November 2012   http://www.invivogen.com/review-cds-ligands

The innate immune system provides the first line of defense against infectious pathogens and serves to limit their early proliferation. It is also vital in priming and activating the adaptive immune system.

Innate immune detection of intracellular DNA derived from viruses and invasive bacteria is important to initiate an effective protective response. This crucial step depends on cytosolic DNA sensors (CDSs), which upon activation trigger the production of type I interferons (IFNs) and the induction of IFN-responsive genes and proinflammatory chemokines.
Although the identity of these CDSs is not fully uncovered, much progress has been made in understanding the signaling pathways triggered by these sensors.

Cytosolic DNA-mediated production of type I IFNs (mainly IFN-β) requires the transcription factor IFN regulatory factor 3 (IRF3), which is activated upon phosphorylation by TANK-binding-kinase-1 (TBK1) [1].

STING in DNA sensing

Recently, a new molecule, STING (stimulator of IFN genes), has been shown to be essential for the TBK1-IRF3- dependent induction of IFN-β by transfected DNA ligands and intracellular DNA produced by pathogens after infection [2, 3].
STING (also known as MITA, MPYS and ERIS) is a transmembrane protein that resides in the endoplasmic reticulum (ER) [2-6]. In response to cytosolic DNA, STING forms dimers and translocates from the ER to the Golgi then to punctate cytosolic structures where it colocalizes with TBK-1, leading to the phosphorylation of IRF3.
How STING stimulates TBK1-dependent IRF3 activation was recently elucidated by Tanaka and Chen. They found that, upon cytosolic DNA sensing, the C-terminal tail of STING acts as a scaffold protein to promote the phosphorylation of IRF3 by TBK1 [7].

STING in the host response to intracellular pathogens. Linking type I IFN response and autophagy for better defense

STING in the host response to intracellular pathogens

http://www.invivogen.com/images/STING-autophagy.png

 

STING activates the IFN response

Until very recently, STING in addition to its role as an adaptor protein was also thought to function as a sensor of cyclic dinucleotides.
Burdette et al. first demonstrated that STING binds directly to the bacterial molecule cyclic diguanylate monophosphate (c-di-GMP) [8]. This finding was confirmed by several teams who examined the structure of STING bound to c-di-GMP [9-11], including Cheng and colleagues, however their data suggest that STING is not the primary sensor of c-di-GMP [12]. Rather, they indicate that DDX41, an identified CDS, functions as a direct receptor for cyclic dinucleotides upstream of STING. The authors hypothesized that DDX41 binds to c-di-GMP then forms a complex with STING to activate the IFN response.

STING induces autophagy

Exciting new developments reveal that STING participates in another aspect of innate immunity, autophagy.
Autophagy plays a critical role in host defense responses to pathogens by promoting the elimination of microbes that enter into the cytosol by their sequestration into autophagosomes and their delivery to the lysosome.

 

CDS pathway

http://www.invivogen.com/images/STING-CDS_pathway_small.jpg

Recent studies have reported that DNA viruses and intracellular bacteria induce autophagy and that this process is dependent on cytosolic genomic DNA and STING [13-15]. Robust induction of autophagy was also observed after transfection of various double stranded (ds) DNA species, such as poly(dA:dT), poly(dG:dC) or plasmid DNA, but not single stranded (ss) DNA, dsRNA or ssRNA [16].

Interestingly, activated STING was shown to relocate to unidentified membrame-bound compartments where it colocalizes with LC3, a hallmark of autophagy, and ATg9a. The latter protein was reported to regulate the interaction between STING and TBK1 after dsDNA stimulation [16]. The E3 ubiquitin ligases TRIM56 and TRIM32
were also found to regulate STING by mediating its dimerization through K63-linked ubiquitination [17, 18].

Several cytosolic DNA sensors upstream of STING have been proposed.
DNA-dependent activator of IRFs (DAI) was the first CDS discovered based on the ability of transfected poly(dA:dT) to induce IFN-β [19]. However, the role of DAI has been shown to be very cell-type specific and cells derived from DAI-deficient mice responded normally to dsDNA ligands [20].

While analyzing immune responses to dsDNA regions derived from vaccinia virus (VACV-70) or Herpes simplex virus 1 (HSV-60) genomes, Unterholzner et al. identified IFI16 as a DNA binding protein mediating IFN-β induction [21]. Interestingly, IFI16 belongs to a new family of pattern recognition receptors that contain the pyrin and HIN domain (PYHIN), termed AIM2-like receptors (ALRs).

AIM2 is a STING-independent cytosolic DNA sensor that forms an inflammasome with ASC to trigger caspase-1 activation and the secretion of the proinflammatory cytokines IL-1β and IL-18 [20].

Members of the DExD/H-box helicase superfamily have also been reported to function as cytosolic DNA sensors. While DHX36 and DHX9 were identified as STING-independent but MyD88-dependent sensors of CpG-containing DNA in plasmacytoid dendritic cells, DDX41 was found to bind various dsDNA ligands and localize with STING to promote IFN-β expression [22]. Other CDSs have been reported to function independently of STING: RNA Pol III, LRRFIP1 and Ku70 [20].

Unlike cytosolic RNA sensors (RIG-I, MDA-5), which detect structural moieties specific to pathogen RNA, such as 5’-triphosphates, it is not clear whether cytosolic DNA sensors can recognize any particular structural motif of DNA that would discriminate between self and non-self. This suggests that CDSs may have a role not only in anti-microbial innate immune responses but also in autoimmunity. A multitude of CDSs have been described but whether they are all true receptors remains an open question.

1. Stetson DB & Medzhitov R. 2006. Recognition of cytosolic DNA activates an IRF3-dependent innate immune response. Immunity. 24(1):93-103.
2. Ishikawa H. & Barber GN., 2008. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature. 455(7213):674-8.
3. Ishikawa H. et al., 2009. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature. 461(7265):788-92.
4. Zhong B. et al., 2008. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation. Immunity. 29(4):538-50.
5. Jin L. et al., 2008. MPYS, a novel membrane tetraspanner, is associated with major histocompatibility complex class II and mediates transduction of apoptotic signals. Mol Cell Biol. 28(16):5014-26.

 

UV Light Potentiates STING (Stimulator of Interferon Genes)-dependent Innate Immune Signaling through Deregulation of ULK1 (Unc51-like Kinase 1).

 J Biol Chem. 2015 May 8;290(19):12184-94.  http://dx.doi.org:/10.1074/jbc.M115.649301. Epub 2015 Mar 19.

The mechanism by which ultraviolet (UV) wavelengths of sunlight trigger or exacerbate the symptoms of the autoimmune disorder lupus erythematosus is not known but may involve a role for the innate immune system. Here we show that UV radiation potentiates STING (stimulator of interferon genes)-dependent activation of the immune signaling transcription factor interferon regulatory factor 3 (IRF3) in response to cytosolic DNA and cyclic dinucleotides in keratinocytes and other human cells. Furthermore, we find that modulation of this innate immune response also occurs with UV-mimetic chemical carcinogens and in a manner that is independent of DNA repair and several DNA damage and cell stress response signaling pathways. Rather, we find that the stimulation of STING-dependent IRF3 activation by UV is due to apoptotic signaling-dependent disruption of ULK1 (Unc51-like kinase 1), a pro-autophagic protein that negatively regulates STING. Thus, deregulation of ULK1 signaling by UV-induced DNA damage may contribute to the negative effects of sunlight UV exposure in patients with autoimmune disorders.

 

 

STING and the innate immune response to nucleic acids in the cytosol

Dara L Burdette & Russell E Vance

https://mcb.berkeley.edu/labs/vance/Resources/Burdette%20(2013)%20review.pdf

Cytosolic detection of pathogen-derived nucleic acids is critical for the initiation of innate immune defense against diverse bacterial, viral and eukaryotic pathogens. Conversely, inappropriate responses to cytosolic nucleic acids can produce severe autoimmune pathology. The host protein STING has been identified as a central signaling molecule in the innate immune response to cytosolic nucleic acids. STING seems to be especially critical for responses to cytosolic DNA and the unique bacterial nucleic acids called ‘cyclic dinucleotides’. Here we discuss advances in the understanding of STING and highlight the many unresolved issues in the field.

The detection of pathogen-derived nucleic acids is a central strategy by which the innate immune system senses microbes to then initiate protective responses1. Conversely, inappropriate recognition of self nucleic acids can result in debilitating autoimmune diseases such as systemic lupus erythematosus2. It is therefore important to understand the molecular basis of the detection of nucleic acids by the innate immune system. Studies have established that nucleic acids derived from extracellular sources are sensed mainly by endosomal Toll-like receptors (TLRs), such as TLR3, TLR7 and TLR9, whereas cytosolic nucleic acids are detected independently of TLRs by a variety of less-well-characterized mechanisms1.

Studies have identified STING (‘stimulator of interferon genes’; also known as TMEM173, MPYS, MITA and ERIS) as a critical signaling molecule in the innate response to cytosolic nucleic-acid ligands. STING was first described as a protein that interacts with major histocompatibility complex class II molecules3, but the relevance of this interaction remains unclear. Subsequent studies have instead focused on the role of STING in the transcriptional induction of type I interferons and coregulated genes in response to nucleic acids in the cytosol. Several groups have independently isolated STING by screening for proteins able to induce interferon-B (IFN-B) when overexpressed4–6. Studies of STING-deficient mice have subsequently confirmed the essential role of STING in innate responses to cytosolic nucleic-acid ligands, particularly double-stranded DNA (dsDNA) and unique bacterial nucleic acids called ‘cyclic dinucleotides’7–9. Several studies have also linked STING to the interferon response to cytosolic RNA5–7, but this has not been found consistently7,8,10,11; thus, we focus here on the role of STING in response to DNA and cyclic dinucleotides.

 

Protein Stimulator of interferon genes protein
Gene TMEM173
Organism Homo sapiens (Human)
Facilitator of innate immune signaling that acts as a sensor of cytosolic DNA from bacteria and viruses and promotes the production of type I interferon (IFN-alpha and IFN-beta). Innate immune response is triggered in response to non-CpG double-stranded DNA from viruses and bacteria delivered to the cytoplasm. Acts by recognizing and binding cyclic di-GMP (c-di-GMP), a second messenger produced by bacteria, and cyclic GMP-AMP (cGAMP), a messenger produced in response to DNA virus in the cytosol: upon binding of c-di-GMP or cGAMP, autoinhibition is alleviated and TMEM173/STING is able to activate both NF-kappa-B and IRF3 transcription pathways to induce expression of type I interferon and exert a potent anti-viral state. May be involved in translocon function, the translocon possibly being able to influence the induction of type I interferons. May be involved in transduction of apoptotic signals via its association with the major histocompatibility complex class II (MHC-II). Mediates death signaling via activation of the extracellular signal-regulated kinase (ERK) pathway. Essential for the induction of IFN-beta in response to human herpes simplex virus 1 (HHV-1) infection. Exhibits 2′,3′ phosphodiester linkage-specific ligand recognition. Can bind both 2′-3′ linked cGAMP and 3′-3′ linked cGAMP but is preferentially activated by 2′-3′ linked cGAMP (PubMed:26300263)
Stimulator of interferon genes protein (IPR029158)
Transmembrane protein 173, also known as stimulator of interferon genes protein (STING) or endoplasmic reticulum interferon stimulator (ERIS), is a transmembrane adaptor protein which is involved in innate immune signalling processes. It induces expression of type I interferons (IFN-alpha and IFN-beta) via the NF-kappa-B and IRF3, pathways in response to non-self cytosolic RNA and dsDNA [PMID: 18724357, PMID: 19776740,PMID: 18818105, PMID: 19433799].

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Glypican-1 identifies cancer exosomes

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Glypican-1 identifies cancer exosomes and detects early pancreatic cancer

Sonia A. MeloLinda B. LueckeChristoph KahlertAgustin F. FernandezSeth T. GammonJudith Kaye, et al.

Nature (09 July 2015); 523: 177–182     http://dx.doi.org:/10.1038/nature14581

Most cells shed so-called extracellular vesicles or exosomes consisting of proteins and nucleic acids enclosed in phospholipid bilayers. Exosomes derived from cancer cells can be isolated.

Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer-cell-derived exosomes in the circulation is currently lacking. Using mass spectrometry analyses, we identify a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer-cell-derived exosomes. GPC1+ circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of patients and mice with cancer. GPC1+ crExos were detected in the serum of patients with pancreatic cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreatic disease from patients with early- and late-stage pancreatic cancer. Levels of GPC1+ crExos correlate with tumour burden and the survival of pre- and post-surgical patients. GPC1+ crExos from patients and from mice with spontaneous pancreatic tumours carry specific KRAS mutations, and reliably detect pancreatic intraepithelial lesions in mice despite negative signals by magnetic resonance imaging. GPC1+ crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer to facilitate possible curative surgical therapy.

Figure 1: GPC1 is present on cancer exosomes.

GPC1 is present on cancer exosomes.

a, Venn diagram of proteins from NIH/3T3 (blue), MCF10A (red), HDF (green), E10 (yellow) and MDA-MB-231 (purple) exosomes. In total, 48 proteins were exclusively detected in MDA-MB-231 exosomes (n = 3 protein samples,…

Figure 2: GPC1+ crExos are a non-invasive biomarker for pancreatic cancer.

GPC1+ crExos are a non-invasive biomarker for pancreatic cancer.

a, Percentage of GPC1+crExo beads in healthy donors, patients with breast cancer and patients with PDAC (analysis of variance (ANOVA), post-hoc Tamhane T2, ****P < 0.0001). b, Frequency ofKRAS mutation in 47 tumours…

Figure 3: Levels of circulating GPC1+exosomes inform pancreatic cancer resection outcome.

Levels of circulating GPC1+ exosomes inform pancreatic cancer resection outcome.

a, Longitudinal blood collection pre- and post-operatively (day 7). b, Percentage of GPC1+crExo beads from patients with BPD (n = 4), PCPL (n = 4) or PDAC (n = 29) (paired two-tailed Student’s t-test, **P < 0.01, ****P < 0.0001). Data a…

Cancer: Diagnosis by extracellular vesicles

Nature (09 July 2015); 523: 161–162.   http://dx.doi.org:/10.1038/nature14626

The detection of a single molecule anchored to circulating extracellular vesicles allows late-stage pancreatic cancer to be identified from just one drop of a patient’s blood. See Article p.177

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Oxidative stress inhibits distant metastasis by human melanoma cells

Elena PiskounovaMichalis AgathocleousMalea M. MurphyZeping HuSara E. HuddlestunZhiyu Zhao, et al.

Nature 14 Oct 2015      http://dx.doi.org:/10.1038/nature15726

Solid cancer cells commonly enter the blood and disseminate systemically, but are highly inefficient at forming distant metastases for poorly understood reasons. Here we studied human melanomas that differed in their metastasis histories in patients and in their capacity to metastasize in NOD-SCID-Il2rg−/− (NSG) mice. We show that melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficiently metastasizing melanomas. Melanoma cells in the blood and visceral organs experienced oxidative stress not observed in established subcutaneous tumours. Successfully metastasizing melanomas underwent reversible metabolic changes during metastasis that increased their capacity to withstand oxidative stress, including increased dependence on NADPH-generating enzymes in the folate pathway. Antioxidants promoted distant metastasis in NSG mice. Folate pathway inhibition using low-dose methotrexate, ALDH1L2 knockdown, or MTHFD1 knockdown inhibited distant metastasis without significantly affecting the growth of subcutaneous tumours in the same mice. Oxidative stress thus limits distant metastasis by melanoma cells in vivo.

Lymph node-independent liver metastasis in a model of metastatic colorectal cancer

Ida B. EnquistZinaida GoodAdrian M. JubbGermaine FuhXi WangMelissa R. JunttilaErica L. Jackson & Kevin G. Leong

Nature Communications  26 Mar 2014; 3530(5)    http://dx.doi.org:/10.1038/ncomms4530

Deciphering metastatic routes is critically important as metastasis is a primary cause of cancer mortality. In colorectal cancer (CRC), it is unknown whether liver metastases derive from cancer cells that first colonize intestinal lymph nodes, or whether such metastases can form without prior lymph node involvement. A lack of relevant metastatic CRC models has precluded investigations into metastatic routes. Here we describe a metastatic CRC mouse model and show that liver metastases can manifest without a lymph node metastatic intermediary. Colorectal tumours transplanted onto the colonic mucosa invade and metastasize to specific target organs including the intestinal lymph nodes, liver and lungs. Importantly, this metastatic pattern differs from that observed following caecum implantation, which invariably involves peritoneal carcinomatosis. Anti-angiogenesis inhibits liver metastasis, yet anti-lymphangiogenesis does not impact liver metastasis despite abrogating lymph node metastasis. Our data demonstrate direct hematogenous spread as a dissemination route that contributes to CRC liver malignancy.

Comprehensive models of human primary and metastatic colorectal tumors in immunodeficient and immunocompetent mice by chemokine targeting

Huanhuan Joyce ChenJian SunZhiliang HuangHarry Hou JrMyra ArcillaNikolai RakhilinDaniel J JoeJiahn ChoiPoornima GadamsettyJeff MilsomGovind NandakumarRandy LongmanXi Kathy Zhou, et al.

Nature Biotechnology (2015);  33:656–660    http://dx.doi.org:/10.1038/nbt.3239

Current orthotopic xenograft models of human colorectal cancer (CRC) require surgery and do not robustly form metastases in the liver, the most common site clinically. CCR9 traffics lymphocytes to intestine and colorectum. We engineered use of the chemokine receptor CCR9 in CRC cell lines and patient-derived cells to create primary gastrointestinal (GI) tumors in immunodeficient mice by tail-vein injection rather than surgery. The tumors metastasize inducibly and robustly to the liver. Metastases have higher DKK4 and NOTCH signaling levels and are more chemoresistant than paired subcutaneous xenografts. Using this approach, we generated 17 chemokine-targeted mouse models (CTMMs) that recapitulate the majority of common human somatic CRC mutations. We also show that primary tumors can be modeled in immunocompetent mice by microinjecting CCR9-expressing cancer cell lines into early-stage mouse blastocysts, which induces central immune tolerance. We expect that CTMMs will facilitate investigation of the biology of CRC metastasis and drug screening.

Induction of the intestinal stem cell signature gene SMOC-2 is required for L1-mediated colon cancer progression

A Shvab, G Haase, A Ben-Shmuel, N Gavert, T Brabletz, S Dedhar and A Ben-Ze’ev

Oncogene , (27 April 2015) |       http://dx.doi.org:/10.1038/onc.2015.127

Overactivation of Wnt-β-catenin signaling, including β-catenin-TCF target gene expression, is a hallmark of colorectal cancer (CRC) development. We identified the immunoglobulin family of cell-adhesion receptors member L1 as a β-catenin-TCF target gene preferentially expressed at the invasive edge of human CRC tissue. L1 can confer enhanced motility and liver metastasis when expressed in CRC cells. This ability of L1-mediated metastasis is exerted by a mechanism involving ezrin and the activation of NF-κB target genes. In this study, we identified the secreted modular calcium-binding matricellular protein-2 (SMOC-2) as a gene activated by L1-ezrin-NF-κB signaling. SMOC-2 is also known as an intestinal stem cell signature gene in mice expressing Lgr5 in cells at the bottom of intestinal crypts. The induction of SMOC-2 expression in L1-expressing CRC cells was necessary for the increase in cell motility, proliferation under stress and liver metastasis conferred by L1. SMOC-2 expression induced a more mesenchymal like phenotype in CRC cells, a decrease in E-cadherin and an increase in Snail by signaling that involves integrin-linked kinase (ILK). SMOC-2 was localized at the bottom of normal human colonic crypts and at increased levels in CRC tissue with preferential expression in invasive areas of the tumor. We found an increase in Lgr5 levels in CRC cells overexpressing L1, p65 or SMOC-2, suggesting that L1-mediated CRC progression involves the acquisition of a stem cell-like phenotype, and that SMOC-2 elevation is necessary for L1-mediated induction of more aggressive/invasive CRC properties.

Global analysis of L1-transcriptomes identified IGFBP-2 as a target of ezrin and NF-κB signaling that promotes colon cancer progression

A Ben-Shmuel, A Shvab, N Gavert, T Brabletz and A Ben-Ze’ev

Oncogene 06 Aug 2012; Oncogene  (04 July 2013); 32: 3220-3230 |  http://dx.doi.org:/10.1038/onc.2012.340

L1, a neuronal cell adhesion receptor of the immunoglobulin-like protein family is expressed in invading colorectal cancer (CRC) cells as a target gene of Wnt/β-catenin signaling. Overexpression of L1 in CRC cells enhances cell motility and proliferation, and confers liver metastasis. We recently identified ezrin and the IκB-NF-κB pathway as essential for the biological properties conferred by L1 in CRC cells. Here, we studied the underlying molecular mechanisms and found that L1 enhances ezrin phosphorylation, via Rho-associated protein kinase (ROCK), and is required for L1–ezrin co-localization at the juxtamembrane domain and for enhancing cell motility. Global transcriptomes from L1-expressing CRC cells were compared with transcriptomes from the same cells expressing small hairpin RNA (shRNA) to ezrin. Among the genes whose expression was elevated by L1 and ezrin we identified insulin-like growth factor-binding protein 2 (IGFBP-2) and showed that its increased expression is mediated by an NF-κB-mediated transactivation of the IGFBP-2 gene promoter. Expression of a constitutively activated mutant ezrin (Ezrin567D) could also increase IGFBP-2 levels in CRC cells. Overexpression of IGFBP-2 in CRC cells lacking L1-enhanced cell proliferation (in the absence of serum), cell motility, tumorigenesis and induced liver metastasis, similar to L1 overexpression. Suppression of endogenous IGFBP-2 in L1-transfected cells inhibited these properties conferred by L1. We detected IGFBP-2 in a unique organization at the bottom of human colonic crypts in normal mucosa and at increased levels throughout human CRC tissue samples co-localizing with the phosphorylated p65 subunit of NF-κB. Finally, we found that IGFBP-2 and L1 can form a molecular complex suggesting that L1-mediated signaling by the L1–ezrin–NF-κB pathway, that induces IGFBP-2 expression, has an important role in CRC progression.

 

Exosome Scouts Help Tumors Populate Distant Organs

  • Click Image To Enlarge +
    This image shows exosomes (green) that have infiltrated the whole lung. [Ayuko Hoshino, David Lyden, Weill Cornell Medicine

    When certain types of cancer spread, they seem to prefer particular organs in the body, a choosiness that led Stephen Paget to propose the “seed and soil” hypothesis. This hypothesis, now more than 100 years old, suggests that different organs are somehow more receptive to certain types of cancer, just as different soils seem to allow some seeds, but not others, to find purchase.

    While this hypothesis is as expressive as ever, it still lacks detail. It doesn’t suggest what mechanisms might drive organ-specific metastasis, or organotropic metastasis. The hypothesis, however, is being taken farther by researchers based at Weill Cornell Medicine. These researchers suggest that the old seed-and-soil idea, which sounds as haphazard as the dispersal of seeds by uncultivated plants, could be updated to describe a process that is more directed.

    Essentially, a tumor metastasis may proceed the way settlers cultivate new land. First, scouts and pioneers are dispatched to identify fertile spots and develop basic infrastructure. Then, once the ground is prepared, settlers establish new communities.

    In this scenario, the scouts are tumor exosomes. These exosomes are released by tumors in the millions, and they carry samples of the tumors’ proteins and genetic content. They fuse preferentially with cells at specific locations, and they ensure that recipient organs are prepared to host the tumor cells they represent.

    Most important, this updated view of organotropic metastasis includes a mechanism to explain how exosomes are directed to specific organs. The exosomes, it turns out, are outfitted with particular sets of integrins, proteins that serve as a kind of destination label.

    Supportive findings appeared October 28 in the journal Nature, in an article entitled, “Tumour exosome integrins determine organotropic metastasis.” This article described how the Weill Cornell researchers, in collaboration with scientists from the Memorial Sloan Kettering Cancer center and the Spanish National Cancer Research Centre (CNIO), examined exosomes from mouse and human lung-, liver-, and brain-tropic tumor cells. These exosomes were seen to fuse preferentially with resident cells at their predicted destinations, namely, lung fibroblasts and epithelial cells, liver Kupffer cells, and brain endothelial cells.

    “Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis,” wrote the authors. “Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively.”

    In other words, the study demonstrated the importance of integrins in metastatic nesting by blocking specific integrins in tumors that metastasize to specific organs. For example, when integrins were blocked in breast cancer, metastasis to lungs was reduced. Similarly, when integrins were blocked in pancreatic cancer, metastasis to liver was reduced.

    In addition, the study showed that a tumor could be “tricked” by changing the integrin destination code of its exosomes. For example, a tumor that would normally go to the bones could be directed to the lungs instead.

    “The integrin-specific signature that we identified may have significant value clinically, serving as a prognostic indicator for metastasis to specific organ sites,” said senior author David Lyden, M.D., Ph.D., the Stavros S. Niarchos Professor in Pediatric Cardiology and a professor of pediatrics and of cell and developmental biology at Weill Cornell Medicine. “Instead of waiting for late-stage metastasis, we can now initiate preventative strategies at an earlier point of disease progression with the hope of preventing its spread. This really changes the treatment paradigm.”

     

  • Using CRISPR as a High-Throughput Cancer Screening and Modeling Tool
  • Click Image To Enlarge +
    Using CRISPR/Cas9, scientists created a new high-throughput screening tool for studying the development and progression of liver cancer in mice. [Ernesto del Aguila III, NHGRI]

    A contingent of researchers from the UK, Germany, and Spain have recently developed a novel CRISPR/Cas9 system that they believe can be utilized as a multiplexed screening approach to study and model cancer development in mice. In the current study, the investigators directly mutated genes within adult mouse livers to elucidate their role in cancer development and progression—simultaneously uncovering the gene combinations that coordinate to cause liver cancer.

    “We reasoned that, by targeting mutations directly to adult liver cells using CRISPR/Cas9, we could better study and understand the biology of this important cancer,” explained co-author Mathias Friedrich, Ph.D., research scientist at the Wellcome Trust Sanger Institute. “Other approaches to engineer mutations in mice, such as stem cell manipulation, are limited by the laborious process, the long time frames and large numbers of animals needed. And, our method better mimics important aspects of human cancer biology than many “classic” mouse models: as in most human cancers, the mutations occur in the adult and only affect a few cells”.

    The findings from this study were published online recently in PNAS through an article entitled “CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice.”

    This new approach is rapid, scalable, and extremely efficient, allowing the researchers to examine an array of genes or large regions of the genome concurrently. Moreover, this methodology affords scientists the ability to distinguish between cancer driver mutations and passenger mutations—those that occur as side-effects of cancer development.

    The research team developed a list of up to eighteen genes with known or unknown evidence for their importance in two forms of liver cancer. They then introduced the CRISPR/Cas9 molecules, targeting various combinations of these genes into mice, which subsequently developed liver or bile duct cancer within a few months.

    “Our approach enables us to simultaneously target multiple putative genes in individual cells,” noted co-author Roland Rad, Ph.D., project leader at the Technical University of Munich and the German Cancer Research Center Heidelberg. “We can now rapidly and efficiently screen which genes are cancer-causing and which ones are not. And, we can study how genes work together to cause cancers—a crucial piece of the puzzle we must solve to understand and tackle the disease.”

    The investigators were able to confirm that a set of DNA-binding proteins called ARID (AT-rich interactive domain), influence the organization of chromosomes and are important for liver cancer development. Furthermore, mutations in a second protein, TET2, were found to be causative in bile duct cancer: although TET2 has not been found to be mutated in human biliary cancers, the proteins that it interacts with have been, showing that the CRISPR/Cas9 method can identify human cancer genes that are not mutated, but whose function is disturbed by other events.

    “The new tools of targeting genes in combination and inducing insertions or deletions in chromosomes change our ability to identify new cancer-causing genes and to understand their role in cancer,” stated senior group leader and co-author Allan Bradley, Ph.D., director emeritus from the Sanger Institute. “Our results show that this approach is feasible and productive in liver cancer; we will now continue to study our new findings and try to extend the approach to other cancer types.”

    This CRISPR/Cas9 approach may also be favorable for an in-depth examination of genomic deserts —regions within the human genome that appear to be devoid of genes. Yet, recent data from the ENCODE Project suggests that deserts can be populated, if not by genes, then by DNA regulatory regions that influence the activity of genes.

    “Liver cancer has many DNA alterations in regions lacking genes: we don’t know which of these might be important for the disease,” said Dr. Rad. “However, we could show that it is now possible to delete such regions to systematically determine their role in liver cancer development.”

     

CRISPR Used to Create Mouse Models of Cancer

  • When scientists study the genetics of cancer, they often breed mice strains that carry selected cancer-associated mutations. But cultivating such strains, usually via transgenesis or gene targeting in embryonic stem cells, is often time-consuming and expensive. Could there be a better way—a faster, cheaper way—to create mice strains that carry particular genetic flaws?

    An alternative has been proposed by researchers from MIT. They have shown that the CRISPR gene editing system can introduce cancer-causing mutations into the livers of adult mice. The researchers anticipate that their method will allow for more rapid testing of any single genes or gene combinations that are suspected of being capable of initiating tumor formation in the liver.

    “The sequencing of human tumors has revealed hundreds of oncogenes and tumor suppressor genes in different combinations. The flexibility of this technology, as delivery gets better in the future, will give you a way to pretty rapidly test those combinations,” said Phillip Sharp, Ph.D., a professor at MIT’s Koch Institute for Integrative Cancer Research.

    Dr. Sharp was part of the MIT research team, which was led by Koch Institute director Tyler Jacks, Ph.D. Dr. Jacks noted that the CRISPR technique, which not only provides the ability to delete genes, but also to replace them with altered versions, “really opens up all sorts of new possibilities when you think about the kinds of genes that you would want to mutate in the future.” Both loss of function and gain of function, he explained, are possible.

    The MIT researchers presented their results August 6 in Nature, in an article entitled, “CRISPR-mediated direct mutation of cancer genes in the mouse liver.” It described how cancer models were generated using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice.

    “We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumor suppressor genes Pten and p53 (also known as TP53 and Trp53), alone and in combination,” wrote the authors. “CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre–LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumors that mimicked those caused by Cre–loxP-mediated deletion of Pten and p53.”

    Studies of such genetically engineered mice have yielded many important discoveries, but the process, which requires introducing mutations into embryonic stem cells, can take more than a year and costs hundreds of thousands of dollars. Using Cas enzymes targeted to cut snippets of p53 and Pten, the researchers were able to disrupt those two genes in about 3% of liver cells, enough to produce liver tumors within three months.

    With traditional techniques, genetically engineering such models is “a very long process,” commented Dr. Jacks. “And the more genes you’re working with, the longer and more complicated it becomes.

    The researchers also used CRISPR to create a mouse model with an oncogene called beta catenin, which makes cells more likely to become cancerous if additional mutations occur later on. To create this model, the researchers had to cut out the normal version of the gene and replace it with an overactive form, which was successful in about 0.5% of hepatocytes.

    In the Nature article, the authors emphasized that simplified methods of testing the oncogenic properties of candidates in vivo are critical. In particular, they cited the need to somehow evaluate the thousands of candidate cancer genes that are being discovered through next-generation sequencing efforts.

    Already looking forward to refining their method of generating cancer models, the authors suggested that it could attain greater sensitivity if CRISPR/Cas9-mediated mutagenesis could be performed on a “sensitized” background carrying constitutive or conditional mutations in a tumor suppressor gene such as p53. “More efficient delivery techniques, such as adenovirus or adeno-associated virus, more potent sgRNAs, and longer homologous recombination templates,” they wrote, “might also improve the overall efficiency of this method and expand the range of tissue that could be targeted.”

     

 

Bioinformatics beyond Genome Crunching  

Flow Cytometry, Workflow Development, and Other Information Stores Can Become Treasure Troves If You Use the Right IT Tools and Services

  • Click Image To Enlarge +
    Shown here is the FlowJo platform’s visualization of surface activation marker expression (CD38) on live lymphocyte CD8+ T cells. Colors represent all combinations of subsets positive and negative for interferon gamma (IFN?), perforin (Perf), and phosphorylated ERK (pERK).

    Advances in bioinformatics are no longer limited to just crunching through genomic and exosomic data. Bioinformatics, a discipline at the interface between biotechnology and information technology, also has lessons for flow cytometry and experimental design, as well as database searches, for both internal and external content.

    One company offering variations on traditional genome crunching is DNAnexus. With the advent of the $1,000 genome, researchers find themselves drowning in data. To analyze the terabytes of information, they must contract with an organization to provide the computing power, or they must perform the necessary server installation and maintenance work in house.

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Reporter: Aviva Lev-Ari, PhD, RN

Ca Prevention: Calcium May Protect Colon

Reviewed by Robert Jasmer, MD; Associate Clinical Professor of Medicine, University of California, San Francisco

WASHINGTON — Increasing calcium intake may lower the risk of colorectal adenomas in people who are at increased risk of the precancerous lesions due to variations in two genes, researchers reported here.

In a two-phase, case-control study of nearly 6,000 subjects, high calcium intake was associated with a significantly reduced risk of adenoma among those who carried variants in the KCNJ and SLC12A1 genes.

High calcium intake was not associated with a reduced risk of colorectal adenoma among those with no variants in KCNJ and SLC12A1, both of which are essential to calcium reabsorption in the kidney, reported Xiangzhu Zhu, MD, of Vanderbilt-Ingram Cancer Center at the American Association for Cancer Research meeting here.

The two-phase study was undertaken to explore whether 14 genes involved in calcium homeostasis are associated with the risk for colorectal adenoma. The researchers also wanted to determine whether intake of calcium and magnesium modified any such risks.

To do so, they utilized data from 1,818 cases and 3,992 controls enrolled in the Tennessee Colorectal Polyp Study. Of the 14 genes,KCNJ and SLC12A1 were found to modify the risk between calcium intake and adenomas.

Among the findings:

  • 52% of participants had a variant allele in one of the two genes, and 13% carried variant alleles in both genes.
  • In people with both gene variants, those the top tertile of calcium intake – consuming 1,300 mg a day or more – had a 69% lower risk of adenoma than people in the lowest tertile, who consumed less than 1,000 mg a day (for trend=0.039).
  • In patients who had one gene variant, there was a 39% reduction in adenomas for those in the highest tertile compared with those in the lowest tertile (for trend=0.046).

The risk for advanced or multiple adenomas were reduced by 89% among those with variants in both genes (for trend=.01).

If confirmed, the findings suggest that patients who carry one or both variants should increase their calcium intake to at least 1,300 mg per day, either through diet or supplementation, Zhu said.

The findings may also “provide one possible explanation for the inconsistency in previous studies on calcium intake and colorectal abnormalities,” she said.

Further study will be needed to confirm the findings, commented Susan T. Mayne, PhD, of Yale University School of Public Health.

Mayne said the study emphasizes that “one size does not always fit all” when it comes to optimal nutrient intakes.

James R. Marshall, PhD, senior vice president of cancer prevention and population sciences at Roswell Park Cancer Institute in Buffalo, N.Y., agreed, pointing out that studies like this are needed to find biomarkers that can pinpoint those patients most likely to benefit from prevention strategies.

“Case-control studies raise possibilities that help to define which patients to include in future trials,” Marshall said.

Kathleen Struck, MedPage Today Senior Editor, contributed to this article.

The possibility that a supplement such as calcium may prove to be a useful chemoprevention agent is intriguing, but a single study is just a single study — worthy of more investigation. Share your thoughts and read what your colleagues are saying about calcium and colon cancer by clicking the Add Your Knowledge link at the bottom of this article. — Sanjay Gupta, MD

The authors reported no relevant financial disclosures.

Mayne and Marshall reported no relevant financial disclosures.

Primary source: American Association for Cancer Research

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Author and curator: Ritu Saxena, Ph.D.

This post attempts to integrate three posts and to embed all comments made to all three papers, allowing the reader a critically thought compilation of evidence-based medicine and scientific discourse.

Dr. Dror Nir authored a post on October 16th titled “Knowing the tumor’s size and location, could we target treatment to THE ROI by applying imaging-guided intervention?” The article attracted over 20 comments from readers including researchers and oncologists debating the following issues:

  • imaging technologies in cancer
  • tumor size, and
  • tumor response to treatment.

The debate lead to several new posts authored by:

This post is a compilation of the views of authors representing different specialties including research and medicine. In medicine: Pathology, Oncology Surgery and Medical Imaging, are represented.

Dr. Nir’s post talked about an advanced technique developed by the researchers at Sunnybrook Health Sciences Centre, University of Toronto, Canada for cancer lesions’ detection and image-guided cancer treatment in the specific Region of Interest (ROI). The group was successfully able to show the feasibility and safety of magnetic resonance imaging (MRI) – controlled transurethral ultrasound therapy for prostate cancer in eight patients.

The dilemma of defining the Region of Interest for imaging-based therapy

Dr. Bernstein, one of the authors at Pharmaceuticalintelligence.com, a Fellow of the American College of Pathology, reiterated the objective of the study stating that “Their study’s objective was to prove that using real-time MRI guidance of HIFU treatment is possible and it guarantees that the location of ablated tissue indeed corresponds to the locations planned for treatment.” He expressed his opinion about the study by bringing into focus a very important issue i.e., given the fact that the part surrounding the cancer tissue is in the transition state, challenge in defining a ROI that could be approached by imaging-based therapy. Regarding the study discussed, he states – “This is a method demonstration, but not a proof of concept by any means.  It adds to the cacophany of approaches, and in a much larger study would prove to be beneficial in treatment, but not a cure for serious prostate cancer because it is unlikely that it can get beyond the margin, and also because there is overtreatment at the cutoff of PSA at 4.0. I think that the pathologist has to see the tissue, and the standard in pathology now is for any result that is cancer, two pathologists or a group sitting together should see it. It’s not an easy diagnosis.”

“The crux of the matter in terms of capability is that the cancer tissue, adjacent tissue, and the fibrous matrix are all in transition to the cancerous state. It is taught to resect leaving “free margin”, which is better aesthetically, and has had success in breast surgery. The dilemma is that the patient may return, but how soon?” concludes Dr. Larry.

Dr. Nir responded, “The philosophy behind lumpectomy is preserving quality of life. It was Prof. Veronesi (IEO) who introduced this method 30 years ago noticing that in the majority of cases; the patient will die from something else before presenting recurrence of breast cancer. It is well established that when the resection margins are declared by a pathologist (as good as he/she could be) as “free of cancer”, the probability of recurrence is much lower than otherwise. He explains further, “The worst enemy of finding solutions is doing nothing while using the excuse of looking for the “ultimate solution.” Personally, I believe in combining methods and improving clinical assessment based on information fusion. Being able to predict, and then timely track the response to treatment is a major issue that affects survival and costs!

In this discussion my view is expressed, below.

  • The paper that discusses imaging technique had the objective of finding out whether real-time MRI guidance of treatment was even possible and if yes, whether the treatment could be performed in accurate location of the ROI? The data reveals they were pretty successful in accomplishing their objective and of course that gives hope to the imaging-based targeted therapies.
  • Whether the ROI is defined properly and if it accounts for the real tumor cure, is a different question. Role of pathologists and the histological analysis and what they bring to the table cannot be ruled out, and the absence of a defined line between the tumor and the stromal region in the vicinity is well documented. However, that cannot rule out the value and scope of imaging-based detection and targeted therapy. After all, it is seminal in guiding minimally invasive surgery.
  • As another arm of personalized medicine-based cure for cancer, molecular biologists at MD Anderson have suggested molecular and genetic profiling of the tumor to determine genetic aberrations on the basis of which matched-therapy could be recommended to patients.
  • When phase I trial was conducted, the results were encouraging and the survival rate was better in matched-therapy patients compared to unmatched patients. Therefore, every time there is more to consider when treating a cancer patient and who knows a combination of views of oncologists, pathologists, molecular biologists, geneticists, surgeons would device improvised protocols for diagnosis and treatment. It is always going to be complicated and generalizations would never give an answer. Smart interpretations of therapies – imaging-based or others would always be required!

To read additional comments, including those from Dr. Williams, Dr. Lev-Ari, refers to:

Knowing the tumor’s size and location, could we target treatment to THE ROI by applying imaging-guided intervention? Author and Reporter: Dror Nir, Ph.D.

Dr. Lev-Ari in her paper linked three fields that bear weight in the determination of Tumor Response to Therapy:

  • Personalized Medicine
  • Cancer Cell Biology, and
  • Minimally Invasive Surgery (MIS)

Her objectives were to address research methodology, the heterogeneity innate to Cancer Cell Biology and Treatment choice in the Operating Room — all are related to the topic at hand: How to deliver optimal care with least invasive intervention course.

Any attempt aimed at approaching this desirable result, called Personalized Medicine,  involves engagement in three strategies:

  • prediction of Patient’s reaction to Drug induction
  • design of Clinical Trials to validate drug efficacy on small subset of patients predicted to react favorable to drug regimen, increasing validity and reliability
  • Genetical identification of patients at no need to have a drug administered if non sensitivity to the drug has been predicted

These method are to be applied to a list of 56 leading Cancer types.

While the executive task of the clinician remains to assess the differentiation in Tumor Response to Treatment, pursuit of  individualized histopathology, as well as tumor molecular, genetic and functional characteristics has to take into consideration the “total” individual patients’ characteristics: age, co-morbidities, secondary risks and allergies to drugs.

In Dr. Lev-Ari’s paper Minimally Invasive Treatment (MIT) is compared with Minimally Invasive Surgery (MIS) applied for tumor resection.  In many cases MIS is not the right surgical decision, yet, it is applied for a corollary of patient-centered care considerations. At present, facing the unknown of the future behavior of the tumor as its response to therapeutics bearing uncertainty related to therapy outcomes.

Forget me not – says the ‘Stroma’

Dr. Brücher, the author of review on tumor response criteria, expressed his views on the topic. He remembers that 10 years ago, every cancer researcher stated – “look at the tumor cells only – forget the stroma”. However, the times have changed, “now, everyone knows that it is a system we are looking at, and viewing and analyzing only tumor cells is really not enough.”

He went on to state “if we would be honest, we would have to declare that all data, which had been produced 8-13 years ago, dealing with laser capture microdissection, would need a rescrutinization, because the influence of the stroma was ‘forgotten’.”

He added, “the surgeon looks at the ‘free margin’ in a kind of reductionable model, the pathologist is more the control instance. I personally see the pathologist as ‘the control instance’ of surgical quality. Therefore, not the wish of the surgeon is important, the objective way of looking into problems or challenges. Can a pathologist always state if a R0-resection had been performed?”

What is the real RO-resection?

There have been many surrogate marker analysis, says Dr. Brücher, and that a substantially well thought through structured analysis has never been done: mm by mm and afterwards analyzing that by a ROC analysis. For information on genetic markers on cancer, refer to the following post by Dr. Lev-Ari’s: Personalized Medicine: Cancer Cell Biology and Minimally Invasive Surgery (MIS)

He also stated that there is no gold standard to compare the statistical ROC analysis to. Often it is just declared and stated but it is still not clear what the real RO-resection is?

He added, “in some organs it is very difficult and we all (surgeons, pathologists, clinicians) that we always get to the limit, if we try interpreting the R-classification within the 3rd dimension.”

Dr. Brücher explains regarding resectability classification, “If lymph nodes are negative it does not mean, lymph nodes are really negative. For example, up to 38% upper GI cancers have histological negative lymph nodes, but immunohistochemical positive lymph nodes. And, Stojadinovic et al have also shown similar observations at el in colorectal cancer. So the 4th dimension of cancer – the lymph nodes / the lymphatic vessel invasion are much more important than just a TNM classification, which unfortunately does often not reflect real tumor biology.”

The discussion regarding the transition state of the tumor surrounding tissue and the ‘free margin’ led to a bigger issue, the heterogeneity of tumors.

Dr. Bernstein quoted a few lines from the review article titled “Tumor response criteria: are they appropriate?, authored by Dr Björn LDM Brücher et al published in Future Oncology in 2012.

  • Tumor heterogeneity is a ubiquitous phemomenon. In particular, there are important differences among the various types of gastrointestinal (GI) cancers in terms of tumor biology, treatment response and prognosis.
  • This forms the principal basis for targeted therapy directed by tumor-specific testing at either the gene or protein level. Despite rapid advances in our understanding of targeted therapy for GI cancers, the impact on cancer survival has been marginal.
  • Can tumor response to therapy be predicted, thereby improving the selection of patients for cancer treatment?
  • In 2000, the NCI with the European Association for Research and Treatment of Cancer, proposed a replacement of 2D measurement with a decrease in the largest tumor diameter by 30% in one dimension. Tumor response as defined would translate into a 50% decrease for a spherical lesion
  • We must rethink how we may better determine treatment response in a reliable, reproducible way that is aimed at individualizing the therapy of cancer patients.
  • We must change the tools we use to assess tumor response. The new modality should be based on empirical evidence that translates into relevant and meaningful clinical outcome data.
  • This becomes a conundrum of sorts in an era of ‘minimally invasive treatment’.
  • Integrated multidisciplinary panel of international experts – not sure that that will do it.

Dr. Bernstein followed up by authoring a separate post on tumor response. His views on tumor response criteria have been quoted in the following paragraphs:

Can tumor response to therapy be predicted?

The goal is not just complete response. Histopathological response seems to be related post-treatment histopathological assessment but it is not free from the challenge of accurately determining treatment response, as this method cannot delineate whether or not there are residual cancer cells. Functional imaging to assess metabolic response by 18-fluorodeoxyglucose PET also has its limits, as the results are impacted significantly by several variables:

• tumor type
• sizing
• doubling time
• anaplasia?
• extent of tumor necrosis
• type of antitumor therapy and the time when response was determined.

The new modality should be based on individualized histopathology as well as tumor molecular, genetic and functional characteristics, and individual patients’ characteristics, a greater challenge in an era of ‘minimally invasive treatment’.

This listing suggests that for every cancer the following data has to be collected (except doubling time). If there were five variables, the classification based on these alone would calculate to be very sizable based on Eugene Rypka’s feature extraction and classification.

But looking forward, time to remission and disease free survival are additionally important. Treatment for cure is not the endpoint, but the best that can be done is to extend the time of survival to a realistic long term goal and retain a quality of life.

For detailed discussion on the topic of tumor response and comments refer to the following posts:

What can we expect of tumor therapeutic response?

Author: Larry H. Bernstein, MD, FCAP

Judging ‘Tumor response’-there is more food for thought

Reporter: Ritu Saxena, Ph.D.

Additional Sources:

Research articles:

Brücher BLDM  et al. Tumor response criteria: are they appropriate? Future Oncol. August Vol. 8, No. 8, Pages 903-906 (2012).

Brücher BLDM, Piso P, Verwaal V et al. Peritoneal carcinomatosis: overview and basics. Cancer Invest.30(3),209–224 (2012).


Brücher BLDM, Swisher S, Königsrainer A et al. Response to preoperative therapy in upper gastrointestinal cancers. Ann. Surg. Oncol.16(4),878–886 (2009).


Miller AB, Hoogstraten B, Staquet M, Winkler A. Reporting results of cancer treatment. Cancer47(1),207–214 (1981).


Therasse P, Arbuck SG, Eisenhauer EA et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J. Natl Cancer Inst.92(3),205–216 (2000).


Brücher BLDM, Becker K, Lordick F et al. The clinical impact of histopathological response assessment by residual tumor cell quantification in esophageal squamous cell carcinomas. Cancer106(10),2119–2127 (2006).

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Personalized Medicine: Cancer Cell Biology and Minimally Invasive Surgery (MIS)

Curator: Aviva Lev-Ari, PhD, RN

In the field of Cancer Research, Translational Medicine  will become Personalized Medicine when each of the cancer type, below will have a Genetic Marker allowing the Clinical Team to use the marker for:

  • prediction of Patient’s reaction to Drug induction
  • design of Clinical Trials to validate drug efficacy on small subset of patients predicted to react favorable to drug regimen, increasing validity and reliability
  • Genetical identification of patients at no need to have a drug administered if non sensitivity to the drug has been predicted

Current urgent need exists for Identification of Genetic Markers to predict Patient’s reaction to Drugs Induction for the following types of Cancer:

 

The executive task of the clinician remains to assess the differentiation in Tumor Response to Treatment.

Review of limitations for the current existing Tools used by clinicians in to be found in:

Brücher BLDM, Bilchik A, Nissan A, Avital I & Stojadinovic A. Can tumor response to therapy be predicted, thereby improving the selection of patients for cancer treatment?  Future Oncology 2012; 8(8): 903-906 , DOI 10.2217/fon.12.78 (doi:10.2217/fon.12.78)   The heterogeneity is a problem that will take at least another decade to unravel because of the number of signaling pathways and the crosstalk that is specifically at issue.

Future Oncology August 2012, Vol. 8, No. 8, Pages 903-906 ,

It is suggested that the new modality should be based on individualized histopathology as well as tumor molecular, genetic and functional characteristics, and individual patients’ characteristics. The new modality should be based on empirical evidence that translates into relevant and meaningful clinical outcome data.

Cancer is in particular a difficult to treat tissue type pathology. In “Tumor response criteria: are they appropriate?” that concern is addressed as follows:

“This becomes a conundrum of sorts in an era of ‘minimally invasive treatment’. One frequently encountered example is that of a patient with chronic gastric reflux and an ultrasound-staged T3N1 distal esophageal adenocarcinoma, who had complete sonographic tumor response to neoadjuvant chemoradiation. The physician may declare that, the tumor having disappeared, the patient requires no further treatment. The surgical oncologist recommends resection, recognizing the fact that up to 20% or more of these complete responders will have identifiable nests of tumor beyond the mucosal scar within the specimen – in other words: residual tumor. In other cases, patients with clinical, sonographic, functional (PET) and histopathological ‘complete’ tumor response to induction therapy experience recurrence within the first 2 years of resection, reminding us of the intricacy and enigma of tumor biology. We have yet to develop the tools needed to consistently delineate the response of a tumor to multimodality therapy.”

This described reality in the Oncology Operating Room is coupled with new trends in invasive treatment of tumor resection.

Minimally Invasive Surgery (MIS) vs. conventional surgery dissection applied to cancer tissue with the known pathophysiology of recurrence and remission cycles has its short term advantages. However, in many cases MIS is not the right surgical decision, yet, it is applied for a corollary of patient-centered care considerations. At present, facing the unknown of the future behavior of the tumor as its response to therapeutics bearing uncertainty related to therapy outcomes.

An increase in the desirable outcomes of MIS as a modality of treatment, will be strongly assisted in the future, with anticipated progress to be made in the field of Cancer Research, Translational Medicine and Personalized Medicine, when each of the cancer types, above,  will already have a Genetic Marker allowing the Clinical Team to use the marker(s) for:

  • prediction of Patient’s reaction to Drug induction
  • design of Clinical Trials to validate drug efficacy on small subset of patients predicted to react favorable to drug regimen, increasing validity and reliability
  • Genetical identification of patients at no need to have a drug administered if non sensitivity to the drug has been predicted by the genetic marker.

REFERENCES

Tumor response criteria: are they appropriate?

Björn LDM Brücher*1,2, Anton Bilchik2,3, Aviram Nissan2,4, Itzhak Avital2,5 & Alexander Stojadinovic2,6

 
Treatment for cure is not the endpoint, but the best that can be done is to extend the time of survival to a realistic long term goal and retain a quality of life.
 
Brücher BLDM, Piso P, Verwaal V et al. Peritoneal carcinomatosis: overview and basics. Cancer Invest.30(3),209–224 (2012).
 
Brücher BLDM, Swisher S, Königsrainer A et al. Response to preoperative therapy in upper gastrointestinal cancers. Ann. Surg. Oncol.16(4),878–886 (2009).
 
Miller AB, Hoogstraten B, Staquet M, Winkler A. Reporting results of cancer treatment. Cancer47(1),207–214 (1981).
 
 
 

Other research papers on Cancer and Cancer Therapeutics were published on this Scientific Web site as follows:

What can we expect of tumor therapeutic response?

PIK3CA mutation in Colorectal Cancer may serve as a Predictive Molecular Biomarker for adjuvant Aspirin therapy

Nanotechnology Tackles Brain Cancer

Response to Multiple Cancer Drugs through Regulation of TGF-β Receptor Signaling: a MED12 Control

Personalized medicine-based cure for cancer might not be far away

GSK for Personalized Medicine using Cancer Drugs needs Alacris systems biology model to determine the in silico effect of the inhibitor in its “virtual clinical trial”

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See comment written for:

Knowing the tumor’s size and location, could we target treatment to THE ROI by applying…..

http://pharmaceuticalintelligence.com/2012/10/16/knowing-the-tumors-size-and-location-could-we-target-treatment-to-the-roi-by-applying-imaging-guided-intervention/

24 Responses

  1. GREAT work.

    I’ll read and comment later on

  2. Highlights of The 2012 Johns Hopkins Prostate Disorders White Paper include:

    A promising new treatment for men with frequent nighttime urination.
    Answers to 8 common questions about sacral nerve stimulation for lower urinary tract symptoms.
    Surprising research on the link between smoking and prostate cancer recurrence.
    How men who drink 6 cups of coffee a day or more may reduce their risk of aggressive prostate cancer.
    Should you have a PSA screening test? Answers to important questions on the controversial USPSTF recommendation.
    Watchful waiting or radical prostatectomy for men with early-stage prostate cancer? What the research suggests.
    A look at state-of-the-art surveillance strategies for men on active surveillance for prostate cancer.
    Locally advanced prostate cancer: Will you benefit from radiation and hormones?
    New drug offers hope for men with metastatic castrate-resistant prostate cancer.
    Behavioral therapy for incontinence: Why it might be worth a try.

    You’ll also get the latest news on benign prostatic enlargement (BPE), also known as benign prostatic hyperplasia (BPH) and prostatitis:
    What’s your Prostate Symptom Score? Here’s a quick quiz you can take right now to determine if you should seek treatment for your enlarged prostate.
    Your surgical choices: a close look at simple prostatectomy, transurethral prostatectomy and open prostatectomy.
    New warnings about 5-alpha-reductase inhibitors and aggressive prostate cancer.

  3. Promising technique.

    INCORE pointed out in detail about the general problem judging response and the stil missing quality in standardization:

    http://www.futuremedicine.com/doi/abs/10.2217/fon.12.78?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dwww.ncbi.nlm.nih.gov

    I did research in response evaluation and prediction for about 15y now and being honest: neither the clinical, nor the molecular biological data proved significant benefit in changing a strategy in patient diagnosis and / or treatment. I would state: this brings us back on the ground and not upon the sky. Additionally it means: we have to ´work harder on that and the WHO has to take responsibility: clinicians use a reponse classification without knowing, that this is just related to “ONE” experiment from the 70′s and that this experiment never had been rescrutinized (please read the Editorial I provided – we use a clinical response classification since more than 30 years worldwide (Miller et al. Cancer 1981) but it is useless !

  4. Dr. BB

    Thank you for your comment.
    Dr. Nir will reply to your comment.
    Regarding the Response Classification in use, it seems that the College of Oncology should champion a task force to revisit the Best Practice in use in this domain and issue a revised version or a new effort for a a new classification system for Clinical Response to treatment in Cancer.

  5. I’m sorry that I was looking for this paper again earlier and didn’t find it. I answered my view on your article earlier.

    This is a method demonstration, but not a proof of concept by any means. It adds to the cacophany of approaches, and in a much larger study would prove to be beneficial in treatment, but not a cure for serious prostate cancer because it is unlikely that it can get beyond the margin, and also because there is overtreatment at the cutoff of PSA at 4.0. There is now a proved prediction model that went to press some 4 months ago. I think that the pathologist has to see the tissue, and the standard in pathology now is for any result that is cancer, two pathologist or a group sitting together should see it. It’s not an easy diagnosis.

    Björn LDM Brücher, Anton Bilchik, Aviram Nissan, Itzhak Avital, & Alexander Stojadinovic. Tumor response criteria: are they appropriate? Future Oncol. (2012) 8(8), 903–906. 10.2217/FON.12.78. ISSN 1479-6694.

    ..Tumor heterogeneity is a ubiquitous phemomenon. In particular, there are important differences among the various types of gastrointestinal (GI) cancers in terms of tumor biology, treatment response and prognosis.

    ..This forms the principal basis for targeted therapy directed by tumor-specific testing at either the gene or protein level. Despite rapid advances in our understanding of targeted therapy for GI cancers, the impact on cancer survival has been marginal.

    ..Can tumor response to therapy be predicted, thereby improving the selection of patients for cancer treatment?

    ..In 2000 theNCI with the European Association for Research and Treatment of Cancer, proposed a replacement of 2D measurement with a decrease in the largest tumor diameter by 30% in one dimension. Tumor response as defined would translate into a 50% decrease for a spherical lesion

    ..We must rethink how we may better determine treatment response in a reliable, reproducible way that is aimed at individualizing the therapy of cancer patients.

    ..we must change the tools we use to assess tumor response. The new modality should be based on empirical evidence that translates into relevant and meaningful clinical outcome data.

    ..This becomes a conundrum of sorts in an era of ‘minimally invasive treatment’.

    ..integrated multidisciplinary panel of international experts – not sure that that will do it

    Several years ago i heard Stamey present the totality of his work at Stanford, with great disappointment over hsPSA that they pioneered in. The outcomes were disappointing.

    I had published a review of all of our cases reviewed for 1 year with Marguerite Pinto.
    There’s a reason that the physicians line up outside of her office for her opinion.
    The review showed that a PSA over 24 ng/ml is predictive of bone metastasis. Any result over 10 was as likely to be prostatitis, BPH or cancer.

    I did an ordinal regression in the next study with Gustave Davis using a bivariate ordinal regression to predict lymph node metastasis using the PSA and the Gleason score. It was better than any univariate model, but there was no followup.

    I reviewed a paper for Clin Biochemistry (Elsevier) on a new method for PSA, very different than what we are familiar with. It was the most elegant paper I have seen in the treatment of the data. The model could predict post procedural time to recurrence to 8 years.

    • I hope we are in agreement on the fact that imaging guided interventions are needed for better treatment outcome. The point I’m trying to make in this post is that people are investing in developing imaging guided intervention and it is making progress.

      Over diagnosis and over treatment is another issue altogether. I think that many of my other posts are dealing with that.

  6. Tumor response criteria: are they appropriate?
    Future Oncology 2012; 8(8): 903-906 , DOI 10.2217/fon.12.78 (doi:10.2217/fon.12.78)
    Björn LDM Brücher, Anton Bilchik, Aviram Nissan, Itzhak Avital & Alexander Stojadinovic
    Tumor heterogeneity is a problematic because of differences among the metabolic variety among types of gastrointestinal (GI) cancers, confounding treatment response and prognosis.
    This is in response to … a group of investigators from Sunnybrook Health Sciences Centre, University of Toronto, Ontario, Canada who evaluate the feasibility and safety of magnetic resonance (MR) imaging–controlled transurethral ultrasound therapy for prostate cancer in humans. Their study’s objective was to prove that using real-time MRI guidance of HIFU treatment is possible and it guarantees that the location of ablated tissue indeed corresponds to the locations planned for treatment.
    1. There is a difference between expected response to esophageal or gastric neoplasms both biologically and in expected response, even given variability within a class. The expected time to recurrence is usually longer in the latter case, but the confounders are – age at time of discovery, biological time of detection, presence of lymph node and/or distant metastasis, microscopic vascular invasion.
    2. There is a long latent period in abdominal cancers before discovery, unless a lesion is found incidentally in surgery for another reason.
    3. The undeniable reality is that it is not difficult to identify the main lesion, but it is difficult to identify adjacent epithelium that is at risk (transitional or pretransitional). Pathologists have a very good idea about precancerous cervical neoplasia.

    The heterogeneity rests within each tumor and between the primary and metastatic sites, which is expected to be improved by targeted therapy directed by tumor-specific testing. Despite rapid advances in our understanding of targeted therapy for GI cancers, the impact on cancer survival has been marginal.

    The heterogeneity is a problem that will take at least another decade to unravel because of the number of signaling pathways and the crosstalk that is specifically at issue.

    I must refer back to the work of Frank Dixon, Herschel Sidransky, and others, who did much to develop a concept of neoplasia occurring in several stages – minimal deviation and fast growing. These have differences in growth rates, anaplasia, and biochemical. This resembles the multiple “hit” theory that is described in “systemic inflammatory” disease leading to a final stage, as in sepsis and septic shock.
    In 1920, Otto Warburg received the Nobel Prize for his work on respiration. He postulated that cancer cells become anaerobic compared with their normal counterpart that uses aerobic respiration to meet most energy needs. He attributed this to “mitochondrial dysfunction. In fact, we now think that in response to oxidative stress, the mitochondrion relies on the Lynen Cycle to make more cells and the major source of energy becomes glycolytic, which is at the expense of the lean body mass (muscle), which produces gluconeogenic precursors from muscle proteolysis (cancer cachexia). There is a loss of about 26 ATP ~Ps in the transition.
    The mitochondrial gene expression system includes the mitochondrial genome, mitochondrial ribosomes, and the transcription and translation machinery needed to regulate and conduct gene expression as well as mtDNA replication and repair. Machinery involved in energetics includes the enzymes of the Kreb’s citric acid or TCA (tricarboxylic acid) cycle, some of the enzymes involved in fatty acid catabolism (β-oxidation), and the proteins needed to help regulate these systems. The inner membrane is central to mitochondrial physiology and, as such, contains multiple protein systems of interest. These include the protein complexes involved in the electron transport component of oxidative phosphorylation and proteins involved in substrate and ion transport.
    Mitochondrial roles in, and effects on, cellular homeostasis extend far beyond the production of ATP, but the transformation of energy is central to most mitochondrial functions. Reducing equivalents are also used for anabolic reactions. The energy produced by mitochondria is most commonly thought of to come from the pyruvate that results from glycolysis, but it is important to keep in mind that the chemical energy contained in both fats and amino acids can also be converted into NADH and FADH2 through mitochondrial pathways. The major mechanism for harvesting energy from fats is β-oxidation; the major mechanism for harvesting energy from amino acids and pyruvate is the TCA cycle. Once the chemical energy has been transformed into NADH and FADH2 (also discovered by Warburg and the basis for a second Nobel nomination in 1934), these compounds are fed into the mitochondrial respiratory chain.
    The hydroxyl free radical is extremely reactive. It will react with most, if not all, compounds found in the living cell (including DNA, proteins, lipids and a host of small molecules). The hydroxyl free radical is so aggressive that it will react within 5 (or so) molecular diameters from its site of production. The damage caused by it, therefore, is very site specific. The reactions of the hydroxyl free radical can be classified as hydrogen abstraction, electron transfer, and addition.
    The formation of the hydroxyl free radical can be disastrous for living organisms. Unlike superoxide and hydrogen peroxide, which are mainly controlled enzymatically, the hydroxyl free radical is far too reactive to be restricted in such a way – it will even attack antioxidant enzymes. Instead, biological defenses have evolved that reduce the chance that the hydroxyl free radical will be produced and, as nothing is perfect, to repair damage.
    Currently, some endogenous markers are being proposed as useful measures of total “oxidative stress” e.g., 8-hydroxy-2’deoxyguanosine in urine. The ideal scavenger must be non-toxic, have limited or no biological activity, readily reach the site of hydroxyl free radical production (i.e., pass through barriers such as the blood-brain barrier), react rapidly with the free radical, be specific for this radical, and neither the scavenger nor its product(s) should undergo further metabolism.
    Nitric oxide has a single unpaired electron in its π*2p antibonding orbital and is therefore paramagnetic. This unpaired electron also weakens the overall bonding seen in diatomic nitrogen molecules so that the nitrogen and oxygen atoms are joined by only 2.5 bonds. The structure of nitric oxide is a resonance hybrid of two forms.
    In living organisms nitric oxide is produced enzymatically. Microbes can generate nitric oxide by the reduction of nitrite or oxidation of ammonia. In mammals nitric oxide is produced by stepwise oxidation of L-arginine catalyzed by nitric oxide synthase (NOS). Nitric oxide is formed from the guanidino nitrogen of the L-arginine in a reaction that consumes five electrons and requires flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) tetrahydrobiopterin (BH4), and iron protoporphyrin IX as cofactors. The primary product of NOS activity may be the nitroxyl anion that is then converted to nitric oxide by electron acceptors.
    The thiol-disulfide redox couple is very important to oxidative metabolism. GSH is a reducing cofactor for glutathione peroxidase, an antioxidant enzyme responsible for the destruction of hydrogen peroxide. Thiols and disulfides can readily undergo exchange reactions, forming mixed disulfides. Thiol-disulfide exchange is biologically very important. For example, GSH can react with protein cystine groups and influence the correct folding of proteins, and it GSH may play a direct role in cellular signaling through thiol-disulfide exchange reactions with membrane bound receptor proteins (e.g., the insulin receptor complex), transcription factors (e.g., nuclear factor κB), and regulatory proteins in cells. Conditions that alter the redox status of the cell can have important consequences on cellular function.
    So the complexity of life is not yet unraveled.

    Can tumor response to therapy be predicted, thereby improving the selection of patients for cancer treatment?
    The goal is not just complete response. Histopathological response seems to be related post-treatment histopathological assessment but it is not free from the challenge of accurately determining treatment response, as this method cannot delineate whether or not there are residual cancer cells. Functional imaging to assess metabolic response by 18-fluorodeoxyglucose PET also has its limits, as the results are impacted significantly by several variables:

    • tumor type
    • sizing
    • doubling time
    • anaplasia?
    • extent of tumor necrosis
    • type of antitumor therapy and the time when response was determined.
    The new modality should be based on individualized histopathology as well as tumor molecular, genetic and functional characteristics, and individual patients’ characteristics, a greater challenge in an era of ‘minimally invasive treatment’.
    This listing suggests that for every cancer the following data has to be collected (except doubling time). If there are five variables, the classification based on these alone would calculate to be very sizable based on Eugene Rypka’s feature extraction and classification. But looking forward, time to remission and disease free survival are additionally important. Treatment for cure is not the endpoint, but the best that can be done is to extend the time of survival to a realistic long term goal and retain a quality of life.

    Brücher BLDM, Piso P, Verwaal V et al. Peritoneal carcinomatosis: overview and basics. Cancer Invest.30(3),209–224 (2012).
    Brücher BLDM, Swisher S, Königsrainer A et al. Response to preoperative therapy in upper gastrointestinal cancers. Ann. Surg. Oncol.16(4),878–886 (2009).
    Miller AB, Hoogstraten B, Staquet M, Winkler A. Reporting results of cancer treatment. Cancer47(1),207–214 (1981).
    Therasse P, Arbuck SG, Eisenhauer EA et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J. Natl Cancer Inst.92(3),205–216 (2000).
    Brücher BLDM, Becker K, Lordick F et al. The clinical impact of histopathological response assessment by residual tumor cell quantification in esophageal squamous cell carcinomas. Cancer106(10),2119–2127 (2006).

    • Dr. Larry,

      Thank you for this comment.

      Please carry it as a stand alone post, Dr. Ritu will refer to it and reference it in her FORTHCOMING pst on Tumor Response which will integrate multiple sources.

      Please execute my instruction

      Thank you

    • Thank you Larry for this educating comment. It explains very well why the Canadian investigators did not try to measure therapy response!

      What they have demonstrated is the technological feasibility of coupling a treatment device to an imaging device and use that in order to guide the treatment to the right place.

      the issue of “choice of treatment” to which you are referring is not in the scope of this publication.
      The point is: if one treatment modality can be guided, other can as well! This should encourage others, to try and develop imaging-based treatment guidance systems.

  7. The crux of the matter in terms of capability is that the cancer tissue, adjacent tissue, and the fibrous matrix are all in transition to the cancerous state. It is taught to resect leaving “free margin”, which is better aesthetically, and has had success in breast surgery. The dilemma is that the patient may return, but how soon?

    • Correct. The philosophy behind lumpectomy is preserving quality of life. It was Prof. Veronesi (IEO) who introduced this method 30 years ago noticing that in the majority of cases, the patient will die from something else before presenting recurrence of breast cancer..

      It is well established that when the resection margins are declared by a pathologist (as good as he/she could be) as “free of cancer”, the probability of recurrence is much lower than otherwise.

  8. Dr. Larry,

    To assist Dr. Ritu, PLEASE carry ALL your comments above into a stand alone post and ADD to it your comment on my post on MIS

    Thank you

  9. Great post! Dr. Nir, can the ultrasound be used in conjunction with PET scanning as well to determine a spatial and functional map of the tumor. With a disease like serous ovarian cancer we typically see an intraperitoneal carcimatosis and it appears that clinicians are wanting to use fluorogenic probes and fiberoptics to visualize the numerous nodules located within the cavity Also is the technique being used mainy for surgery or image guided radiotherapy or can you use this for detecting response to various chemotherapeutics including immunotherapy.

    • Ultrasound can and is actually used in conjunction with PET scanning in many cases. The choice of using ultrasound is always left to the practitioner! Being a non-invasive, low cost procedure makes the use of ultrasound a non-issue. The down-side is that because it is so easy to access and operate, nobody bothers to develop rigorous guidelines about using it and the benefits remains the property of individuals.

      In regards to the possibility of screening for ovarian cancer and characterising pelvic masses using ultrasound I can refer you to scientific work in which I was involved:

      1. VAES (E.), MANCHANDA (R), AUTIER, NIR (R), NIR (D.), BLEIBERG (H.), ROBERT (A.), MENON (U.). Differential diagnosis of adnexal masses: Sequential use of the Risk of Malignancy Index and a novel computer aided diagnostic tool. Published in Ultrasound in Obstetrics & Gynecology. Issue 1 (January). Vol. 39. Page(s): 91-98.

      2. VAES (E.), MANCHANDA (R), NIR (R), NIR (D.), BLEIBERG (H.), AUTIER (P.), MENON (U.), ROBERT (A.). Mathematical models to discriminate between benign and malignant adnexal masses: potential diagnostic improvement using Ovarian HistoScanning. Published in International Journal of Gynecologic Cancer (IJGC). Issue 1. Vol. 21. Page(s): 35-43.

      3. LUCIDARME (0.), AKAKPO (J.-P.), GRANBERG (S.), SIDERI (M.), LEVAVI (H.), SCHNEIDER (A.), AUTIER (P.), NIR (D.), BLEIBERG (H.). A new computer aided diagnostic tool for non-invasive characterisation of malignant ovarian masses: Results of a multicentre validation study. Published in European Radiology. Issue 8. Vol. 20. Page(s): 1822-1830.

      Dror Nir, PhD
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  10. totally true and i am very thankfull for these briliant comments.

    Remember: 10years ago: every cancer researcher stated: “look at the tumor cells only – forget the stroma”. The era of laser-captured tumor-cell dissection started. Now , everyone knows: it is a system we are looking at and viewing and analyzing tumor cells only is really not enough.

    So if we would be honest, we would have to declare, that all data, which had been produced 13-8years ago, dealing with laser capture microdissection, that al these data would need a re-scrutinization, cause the influence of the stroma was “forgotten”. I ‘d better not try thinking about the waisted millions of dollars.

    If we keep on being honest: the surgeon looks at the “free margin” in a kind of reductionable model, the pathologist is more the control instance. I personally see the pathologist as “the control instance” of surgical quality. Therefore, not the wish of the surgeon is important, the objective way of looking into problems or challenges. Can a pathologist always state, if a R0-resection had been performed ?

    The use of the Resectability Classification:
    There had been many many surrogate marker analysis – nothing new. BUT never a real substantial well tought through structured analysis had been done: mm by mm by mm by mm and afterwards analyzing that by a ROC analysis. BUt against which goldstandard ? If you perform statistically a ROC analysis – you need a golstandard to compare to. Therefore what is the real R0-resectiòn? It had been not proven. It just had been stated in this or that tumor entity that this or that margin with this margin free mm distance or that mm distance is enough and it had been declared as “the real R0-classification”. In some organs it is very very difficult and we all (surgeons, pathologists, clinicians) that we always get to the limit, if we try interpretating the R-classification within the 3rd dimension. Often it is just declared and stated.

    Otherwise: if lymph nodes are negative it does not mean, lymph nodes are really negative, cause up to 38% for example in upper GI cancers have histological negative lymph nodes, but immunohistochemical positive lymph nodes. And this had been also shown by Stojadinovic at el analyzing the ultrastaging in colorectal cancer. So the 4th dimension of cancer – the lymph nodes / the lymphatic vessel invasion are much more important than just a TNM classification, which unfortunately does often not reflect real tumor biology.

    AS we see: cancer has multifactorial reasons and it is necessary taking the challenge performing high sophisticated research by a multifactorial and multidisciplinary manner.

    Again my deep and heartly thanks for that productive and excellent discussion !

    • Dr. BB,

      Thank you for your comment.

      Multidisciplinary perspectives have illuminated the discussion on the pages of this Journal.

      Eager to review Dr. Ritu’s forthcoming paper – the topic has a life of its own and is embodied in your statement:

      “the 4th dimension of cancer – the lymph nodes / the lymphatic vessel invasion are much more important than just a TNM classification, which unfortunately does often not reflect real tumor biology.”

    • Thank you BB for your comment. You have touched the core limitation of healthcare professionals: how do we know that we know!

      Do we have a reference to each of the test we perform?

      Do we have objective and standardise quality measures?

      Do we see what is out-there or are we imagining?

      The good news: Everyday we can “think” that we learned something new. We should be happy with that, even if it is means that we learned that yesterday’s truth is not true any-more and even if we are likely to be wrong again…:)

      But still, in the last decades, lots of progress was made….

  11. Dr. Nir,
    I thoroughly enjoyed reading your post as well as the comments that your post has attracted. There were different points of view and each one has been supported with relevant examples in the literature. Here are my two cents on the discussion:
    The paper that you have discussed had the objective of finding out whether real-time MRI guidance of treatment was even possible and if yes, and also if the treatment could be performed in accurate location of the ROI? The data reveals they were pretty successful in accomplishing their objective and of course that gives hope to the imaging-based targeted therapies.
    Whether the ROI is defined properly and if it accounts for the real tumor cure, is a different question. Role of pathologists and the histological analysis they bring about to the table cannot be ruled out, and the absence of a defined line between the tumor and the stromal region in the vicinity is well documented. However, that cannot rule out the value and scope of imaging-based detection and targeted therapy. After all, it is seminal in guiding minimally invasive surgery. As another arm of personalized medicine-based cure for cancer, molecular biologists at MD Anderson have suggested molecular and genetic profiling of the tumor to determine genetic aberrations on the basis of which matched-therapy could be recommended to patients. When phase I trial was conducted, the results were obtained were encouraging and the survival rate was better in matched-therapy patients compared to unmatched patients. Therefore, everytime there is more to consider when treating a cancer patient and who knows a combination of views of oncologists, pathologists, molecular biologists, geneticists, surgeons would device improvised protocols for diagnosis and treatment. It is always going to be complicated and generalizations would never give an answer. Smart interpretations of therapies – imaging-based or others would always be required!

    Ritu

    • Dr. Nir,
      One of your earlier comments, mentioned the non invasiveness of ultrasound, thus, it’s prevalence in use for diagnosis.

      This may be true for other or all areas with the exception of Mammography screening. In this field, an ultrasound is performed only if a suspected area of calcification or a lump has been detected in the routine or patient-initiated request for ad hoc mammography secondery to patient complain of pain or patient report of suspected lump.

      Ultrasound in this field repserents ascalation and two radiologists review.

      It in routine use for Breast biopsy.

    • Thanks Ritu for this supporting comment. The worst enemy of finding solutions is doing nothing while using the excuse of looking for the “ultimate solution” . Personally, I believe in combining methods and improving clinical assessment based on information fusion. Being able to predict, and then timely track the response to treatment is a major issue that affects survival and costs!

Judging the ‘Tumor response’-there is more food for thought

http://pharmaceuticalintelligence.com/2012/12/04/judging-the-tumor-response-there-is-more-food-for-thought/

13 Responses

  1. Dr. Sanexa
    you have brought up an interesting and very clinically relevant point: what is the best measurement of response and 2) how perspectives among oncologists and other professionals differ on this issues given their expertise in their respective subspecialties (immunologist versus oncologist. The advent of functional measurements of tumors (PET etc.) seems extremely important in the therapeutic use AND in the development of these types of compounds since usually a response presents (in cases of solid tumors) as either a lack of growth of the tumor or tumor shrinkage. Did the authors include an in-depth discussion of the rapidity of onset of resistance with these types of compounds?
    Thanks for the posting.

  2. Dr. Williams,
    Thanks for your comment on the post. The editorial brings to attention a view that although PET and other imaging methods provide vital information on tumor growth, shrinkage in response to a therapy, however, there are more aspects to consider including genetic and molecular characteristics of tumor.
    It was an editorial review and the authors did not include any in-depth discussion on the rapidity of onset of resistance with these types of compounds as the focus was primarily on interpreting tumor response.
    I am glad you found the contents of the write-up informative.
    Thanks again!
    Ritu

  3. Thank you for your wonderful comment and interpretation. Dr.Sanexa made a brilliant comment.

    May I allow myself putting my finger deeper into this wound ? Cancer patients deserve it.

    It had been already pointed out by international experts from Munich, Tokyo, Hong-Kong and Houston, dealing with upper GI cancer, that the actual response criteria are not appropriate and moreover: the clinical response criteria in use seem rather to function as an alibi, than helping to differentiate and / or discriminate tumor biology (Ann Surg Oncol 2009):

    http://www.ncbi.nlm.nih.gov/pubmed/19194759

    The response data in a phase-II-trial (one tumor entity, one histology, one treatment, one group) revealed: clinical response evaluation according to the WHO-criteria is not appropriate to determine response:

    http://www.ncbi.nlm.nih.gov/pubmed/15498642

    Of course, there was a time, when it seemed to be useful and this also has to be respected.

    There is another challenge: using statistically a ROC and resulting in thresholds. This was, is and always be “a clinical decision only” and not the decision of the statistician. The clinician tells the statistician, what decision, he wants to make – the responsibility is enormous. Getting back to the roots:
    After the main results of the Munich-group had been published 2001 (Ann Surg) and 2004 (J Clin Oncol):

    http://www.ncbi.nlm.nih.gov/pubmed/11224616

    http://www.ncbi.nlm.nih.gov/pubmed/14990646

    the first reaction in the community was: to difficult, can’t be, not re-evaluated, etc.. However, all evaluated cut-offs / thresholds had been later proven to be the real and best ones by the MD Anderson Cancer Center in Houston, Texas. Jaffer Ajani – a great and critical oncologist – pushed that together with Steve Swisher and they found the same results. Than the upper GI stakeholders went an uncommon way in science: they re-scrutinized their findings. Meanwhile the Goldstandard using histopathology as the basis-criterion had been published in Cancer 2006.

    http://www.ncbi.nlm.nih.gov/pubmed/16607651

    Not every author, who was at the authorlist in 2001 and 2004 wanted to be a part of this analysis and publication ! Why ? Everyone should judge that by himself.

    The data of this analysis had been submitted to the New England Journal of Medicine. In the 2nd review stage process, the manuscript was rejected. The Ann Surg Oncol accepted the publication: the re-scrutinized data resulted in another interesting finding: in the future maybe “one PET-scan” might be appropriate predicting the patient’s response.

    Where are we now ?

    The level of evidence using the response criteria is very low: Miller’s (Cancer 1981) publication belonged to ”one single” experiment from Moertel (Cancer 1976). During that time, there was no definition of “experiences” rather than “oncologists”. These terms had not been in use during that time.

    Additionally they resulted in a (scientifically weak) change of the classification, published by Therasse (J Natl Cancer Inst 2000). Targeted therapy did not result in a change so far. In 2009, the international upper GI experts sent their publication of the Ann Surg Oncol 2009 to the WHO but without any kind of reaction.

    Using molecular biological predictive markers within the last 10years all seem to have potential.

    http://www.ncbi.nlm.nih.gov/pubmed/20012971

    http://www.ncbi.nlm.nih.gov/pubmed/18704459

    http://www.ncbi.nlm.nih.gov/pubmed/17940507

    http://www.ncbi.nlm.nih.gov/pubmed/17354029

    But, experts are aware: the real step breaking barriers had not been performed so far. Additionally, it is very important in trying to evaluate and / predict response, that not different tumor entities with different survival and tumor biology are mixed together. Those data are from my perspective not helpful, but maybe that is my own Bias (!) of my view.

    INCORE, the International Consortium of Research Excellence of the Theodor-Billroth-Academy, was invited publishing the Editorial in Future Oncology 2012. The consortium pointed out, that living within an area of ‘prove of principle’ and also trying to work out level of evidence in medicine, it is “the duty and responsibility” of every clinician, but also of the societies and institutions, also of the WHO.

    Complete remission is not the only goal, as experts dealing with ‘response-research’ are aware. It is so frustrating for patients and clinicians: there is a rate of those patients with complete remission, who develop early recurrence ! This reflects, that complete remission cannot function as the only criterion describing response !

    Again, my heartly thanks, that Dr.Sanexa discussed this issue in detail.
    I hope, I found the way explaining the way of development and evaluating response criteria properly and in a differentiated way of view. From the perspective of INCORE:

    “an interdisciplinary initiative with all key stake¬holders and disciplines represented is imperative to make predictive and prognostic individualized tumor response assessment a modern-day reality. The integrated multidisciplinary panel of international experts need to define how to leverage existing data, tissue and testing platforms in order to predict individual patient treatment response and prognosis.”

  4. Dr. Brucher,

    First of all thanks for expressing your views on the ‘tumor response’ in a comprehensive way. You are the first author of the editorial review one of the prominent people who has taken part in the process of defining tumor response and I am glad that you decided to write a comment on the writeup.
    The topic has been explained well in an immaculate manner and that it further clarifies the need for the perfect markers that would be able to evaluate and predict tumor response. There are, as you mentioned, some molecular markers available including VEGF, cyclins, that have been brought to focus in the context of squamous cell carcinoma.

    It would be great if you could be the guest author for our blog and we could publish your opinion (comment on this blog post) as a separate post. Please let us know if it is OK with you.

    Thanks again for your comment
    Ritu

  5. Thank you all to the compelling discussions, above.

    Please review the two sources on the topic I placed at the bottom of the post, above as post on this Scientific Journal,

    All comments made to both entries are part of thisvdiscussion, I am referring to Dr. Nir’s post on size of tumor, to BB comment to Nir’s post, to Larry’ Pathologist view on Tumors and my post on remission and minimally invasive surgery (MIS).

    Great comments by Dr. Williams, BB and wonderful topic exposition by Dr. Ritu.

  6. Aviva,
    Thats a great idea. I will combine all sources referred by you, the post on tumor imaging by Dr. Nir and the comments made on the these posts including Dr. Brucher’s comments in a new posts.
    Thanks
    Ritu

    • Great idea, ask Larry, he has written two very long important comments on this topic, one on Nir’s post and another one, ask him where, if it is not on MIS post. GREAT work, Ritu, integration is very important. Dr, Williams is one of our Gems.

    • Assessing tumour response it is not an easy task!Because tumours don’t change,but happilly our knowlege(about them) does really change,is everchanging(thans god!).In the past we had the Recist Criteria,then the Modified Recist Criteria,becausa of Gist and other tumors.At this very moment,these are clearly insuficient.We do need more ,new validated facing the reality of nowadays.A great,enormoust post Dr Ritu!Congratulations!

 

 

 

 

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