KRAS Isoforms Research Articles

ES28.01 Biology of KRAS Targeting Agents

ES28.01 Biology of KRAS Targeting Agents

The Prevalence and Concurrent Pathogenic Mutations of KRAS G12C in Northeast Chinese Non-small-cell Lung Cancer Patients.

ObjectiveKRAS mutation is one of important driver genes in non-small-cell lung cancer (NSCLC) and the patients with KRASG12C mutations benefit from the inhibitor AMG510. However, the frequency, concurrent pathogenic mutations, and clinical characteristic of KRASG12C is unknown in the NSCLC population of Northeast China.MethodsThe retrospective analysis was derived from 431 NSCLC patients in Jilin Cancer Hospital between January 2018 and June 2019. The mutation frequency and concurrent mutations of KRASG12C in tumor or peripheral blood was detected by next-generation sequencing (NGS).ResultsThe RAS mutant rate was observed in 10.7% (46/431) of this cohort. All RAS-driver cancers are caused by mutations in the KRAS isoform, while the NRAS and HRAS isoforms were not detected. Among KRAS-mutant patients, 42 (91.3%) showed exon 2 mutation in 12 codon and 13 codon. KRASG12C showed a 4.6% (20/431) mutation rate in this cohort and the highest frequency (43.5%, 20/46) in KRAS-mutant-positive patients. There was no difference between tumor tissue and plasma in terms of either KRAS or KRASG12C mutation. The most frequent co-occurrence mutations with KRASG12C were TP53, followed by PTEN. Furthermore, KRASG12C was exclusive with STK11 mutation. KRASG12C mutation was associated with age, disease stage, and smoking status (P=0.024; P=0.02; P=0.006), smoking remained an independent factor for KRASG12C mutation (P=0.037), and higher mutation frequency in patients older than 60, stage I–III, or smoking in NSCLC (P=0.0151, P=0.0343, P=0.0046, respectively).ConclusionKRAS mutation was the only isoforms of RAS family, of these 43.5% harbored the KRASG12C subtype in northeastern Chinese NSCLC patients. KRASG12C is associated with age, pathological stage and smoking status, more commonly harbored TP53/PTEN mutations, and providing more genome profile for targeted therapy in local clinical practice.

Phase II study of selumetinib, an orally active inhibitor of MEK1 and MEK2 kinases, in KRASG12R-mutant pancreatic ductal adenocarcinoma

SummaryBackground Preclinical evidence has suggested that a subset of pancreatic cancers with the G12R mutational isoform of the KRAS oncogene is more sensitive to MAPK pathway blockade than pancreatic tumors with other KRAS isoforms. We conducted a biomarker-driven trial of selumetinib (KOSELUGO™; ARRY-142886), an orally active, allosteric mitogen-activated protein kinase 1 and 2 (MEK1/2) inhibitor, in pancreas cancer patients with somatic KRASG12R mutations. Methods In this two-stage, phase II study (NCT03040986) patients with advanced pancreas cancer harboring somatic KRASG12R variants who had received at least one standard-of-care systemic therapy regimen received 75 mg selumetinib orally twice a day until disease progression or unacceptable toxicity occurred. The primary outcome of the study was best objective response (BOR). Results From August 2017 to February 2018 a total of 8 patients with confirmed somatic KRASG12R mutations and a median age of 61.5 years were treated with selumetinib. Seven out of eight (87.5%) had received two or more lines of prior systemic chemotherapy. After a median follow-up period of 8.5 months (range 2 to 20), three patients had stable disease for more than 6 months while receiving selumetinib. No patients achieved an objective partial response. Median progression-free survival (PFS) was 3.0 months (95% CI, 0.8–8.2) and median overall survival (OS) 9 months (95% CI, 2.5–20.9). Conclusion This study in heavily pre-treated pancreatic adenocarcinoma patients suggests alternative strategies beyond single agent MEK inhibition are required for this unique, molecular subset of pancreatic cancer patients. The trial was registered on February 2nd, 2017 under identifier NCT03040986 with ClinicalTrials.gov.

Combinations with Allosteric SHP2 Inhibitor TNO155 to Block Receptor Tyrosine Kinase Signaling.

SHP2 inhibitors offer an appealing and novel approach to inhibit receptor tyrosine kinase (RTK) signaling, which is the oncogenic driver in many tumors or is frequently feedback activated in response to targeted therapies including RTK inhibitors and MAPK inhibitors. We seek to evaluate the efficacy and synergistic mechanisms of combinations with a novel SHP2 inhibitor, TNO155, to inform their clinical development. The combinations of TNO155 with EGFR inhibitors (EGFRi), BRAFi, KRASG12Ci, CDK4/6i, and anti-programmed cell death-1 (PD-1) antibody were tested in appropriate cancer models in vitro and in vivo, and their effects on downstream signaling were examined. In EGFR-mutant lung cancer models, combination benefit of TNO155 and the EGFRi nazartinib was observed, coincident with sustained ERK inhibition. In BRAFV600E colorectal cancer models, TNO155 synergized with BRAF plus MEK inhibitors by blocking ERK feedback activation by different RTKs. In KRASG12C cancer cells, TNO155 effectively blocked the feedback activation of wild-type KRAS or other RAS isoforms induced by KRASG12Ci and greatly enhanced efficacy. In addition, TNO155 and the CDK4/6 inhibitor ribociclib showed combination benefit in a large panel of lung and colorectal cancer patient-derived xenografts, including those with KRAS mutations. Finally, TNO155 effectively inhibited RAS activation by colony-stimulating factor 1 receptor, which is critical for the maturation of immunosuppressive tumor-associated macrophages, and showed combination activity with anti-PD-1 antibody. Our findings suggest TNO155 is an effective agent for blocking both tumor-promoting and immune-suppressive RTK signaling in RTK- and MAPK-driven cancers and their tumor microenvironment. Our data provide the rationale for evaluating these combinations clinically.

Oncogenic mutant RAS signaling activity is rescaled by the ERK/MAPK pathway

Activating mutations in RAS are present in ~ 30% of human tumors, and the resulting aberrations in ERK/MAPK signaling play a central role in oncogenesis. However, the form of these signaling changes is uncertain, with activating RAS mutants linked to both increased and decreased ERK activation in vivo. Rationally targeting the kinase activity of this pathway requires clarification of the quantitative effects of RAS mutations. Here, we use live‐cell imaging in cells expressing only one RAS isoform to quantify ERK activity with a new level of accuracy. We find that despite large differences in their biochemical activity, mutant KRAS isoforms within cells have similar ranges of ERK output. We identify roles for pathway‐level effects, including variation in feedback strength and feedforward modulation of phosphatase activity, that act to rescale pathway sensitivity, ultimately resisting changes in the dynamic range of ERK activity while preserving responsiveness to growth factor stimuli. Our results reconcile seemingly inconsistent reports within the literature and imply that the signaling changes induced by RAS mutations early in oncogenesis are subtle.

Abstract LB-122: Combinations of SHP2 inhibitor to overcome RAS activation by receptor tyrosine kinases in response to ERK inhibition

Abstract Introduction: The RTK-RAS-MAPK pathway is frequently activated in cancers due to a variety of mechanisms including mutations or amplifications in RTK, KRAS or BRAF. The effectiveness of inhibitors targeting those oncogenic drivers is often limited by the pathway feedback activation originated at the RTK level in response to ERK inhibition. The non-receptor protein tyrosine phosphatase SHP2 mediates RAS activation downstream of various receptor tyrosine kinases. Potent and selective allosteric SHP2 inhibitors such as TNO155 are in clinical development and offer an appealing one-size-fits-all approach to overcome RTK-mediated feedback activation of RAS and enhance the efficacy of inhibitors targeting RTK, BRAF or KRASG12C. We sought to study the combination efficacy and mechanism in pre-clinical models for prioritization in clinical trials. Experimental procedures: The combination efficacy and synergy of TNO155 with EGF816 (a 3rd generation EGFR inhibitor), dabrafenib+trametinib or a tool KRAS G12C inhibitor (G12Ci) were respectively assessed in a number of EGFR mutant lung cancer models, BRAFV600E colorectal cancer models and KRASG12C lung and colorectal cancer models. The effect of the combinations on ERK inhibition was also studied. Results: TNO155 has varying single agent activity in vitro in EGFR mutant lung cancer cell lines and is not affected by clinically relevant resistance mechanisms to EGFR inhibitors such as secondary mutations in EGFR (T790M and C797S) or MET amplification. In addition, the combination of TNO155 and EGF816 is synergistic across cell lines, coincident with sustained ERK inhibition. In BRAFV600E colorectal cancer cell lines with feedback activation of EGFR, MET or FGFR respectively in response to treatment with dabrafenib+trametinib, TNO155 synergistically enhanced the efficacy of dabrafenib+trametinib in all three cell lines, phenocopying respective RTK inhibitors. In KRASG12C lung cancer cell lines, quick rebound of p-ERK was observed as early as 24 hour post treatment with G12Ci and cannot be blocked by increasing concentrations of G12Ci, suggesting feedback activation of wild-type KRAS or other RAS isoforms. In contrast, TNO155 effectively blocked the p-ERK rebound and enhanced the efficacy of G12Ci. Similar observations were made in KRASG12C colorectal cancer cell lines. Conclusions: Our findings suggest that SHP2 inhibition is an effective strategy to block the feedback activation of wild type and G12C KRAS, as well as NRAS and HRAS, by a variety of RTKs, in response to ERK inhibition. Given the mutant selective properties of those inhibitors we studied, the tolerability of their combinations with TNO155 is highly expected. Our data provide pre-clinical rationale to explore those TNO155 combinations in the corresponding cancer indications in clinic. Citation Format: Huaixiang Hao, Chen Liu, Hongyun Wang, Hengyu Lu, Scott Delach, Matthew LaMarche, Jeffrey Engelman, Peter Hammerman, Giordano Caponigro, Susan Moody, Morvarid Mohseni. Combinations of SHP2 inhibitor to overcome RAS activation by receptor tyrosine kinases in response to ERK inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-122.

Abstract 1453: Specific detection of mutant Ras oncoproteins in cell and tissue lysates by multiplex immunoassay

Abstract Ras proteins (HRAS, KRAS, and NRAS) are small GTPases that function as molecular switches by alternating between inactive GDP-bound and active GTP-bound states. Ras-GTP activates downstream pathways, including MAPK, by binding to Raf kinase and phosphatidylinositol 3-kinase (PI3K) to promote cellular proliferation, survival, growth and differentiation. Oncogenic mutations in Ras are found in approximately 25% of human cancers. Furthermore, 90% of pancreatic cancers harbor KRAS mutations with 80% of KRAS mutations occurring at codon 12 with G12V and G12D being the most prevalent. The Ras-Raf-MEK pathway can also be activated downstream of Ras. BRAF is a well-established oncogene, with the BRAF V600E mutation being prevalent in melanoma. While targeting this BRAF oncoprotein has had therapeutic efficacy, treating cancers driven by Ras oncoproteins remains an urgent unmet clinical need. We developed a novel multiplex immunoassay capable of simultaneously detecting total Ras, RasG12V, RasG12D, phospho-MEK1 (S217/S221), phospho-BRAF (S446) and phospho-CRAF (S338) in a single well. This multiplex assay detects the H-Ras, N-Ras, and K-Ras isoforms for total Ras, RasG12D, and RasG12V. To confirm specific recognition of mutant Ras oncoproteins, this kit was able to detect the known KRAS G12V mutation found in the COR-L23 cell line and the NRAS G12D mutation found in THP-1 cell line, respectively. In addition, the response to BRAF inhibitors vemurafenib and dabrafenib was compared between MDA-MB-435S cells, which harbor the BRAF V600E mutation, and insensitive cell lines, using multiplex immunoassays. While COR-L23 and THP-1 cells were not responsive to vemurafenib and dabrafenib, MDA-MB-435S cells showed a dramatic reduction in pMEK1 and pERK, consistent with inhibition of mutant BRAF protein. We then analyzed commercially-sourced tissue lysates, using tumor and adjacent tissue from a patient with colon cancer harboring a KRAS G12D mutation. While multiplex protein analysis revealed a similar phosphorylation pattern for Raf and MEK proteins, the RasG12D oncoprotein was specifically observed in lysate from the tumor but not the adjacent tissue. Finally, exosomes were enriched from pancreatic cancer serum and tested using the Ras-Raf Oncoprotein panel. These exosomal preparations yielded detectable signal for Total Ras and phospho-BRAF. Our data demonstrate the utility of studying the effects of Ras oncoproteins by multiplex immunoassay in multiple sample types, including cell, tissue, and exosomal lysates. Citation Format: Joseph B. Hwang, Anthony Saporita, Qiang Xiao. Specific detection of mutant Ras oncoproteins in cell and tissue lysates by multiplex immunoassay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1453.

Abstract A26: Precise characterization and comparison of KRAS proteoforms by top-down mass spectrometry

Abstract The two KRAS isoforms, 4A and 4B, exhibit high sequence identity, differing mainly in their C-terminal “hypervariable regions”, which bear post-translational modifications (PTMs) governing their association with the plasma membrane and downstream signaling effectors. Moreover, the KRAS gene bears three “mutation hotspot” residues (Gly12, Gly13, and Gln61) represented in a high percentage of human cancers. The similarity between KRAS isoforms, in addition to HRAS and NRAS, renders linking KRAS mutations and PTMs significantly challenging by standard proteomic methods based on digestion of proteins into short peptides. However, intact modified protein forms, or proteoforms, of KRAS4A and KRAS4B can be identified and characterized by “top-down” (TD) proteomic methods, elucidating crosstalk between N-terminal mutations and C-terminal PTMs with precise molecular detail. Previously, we employed the TD approach as part of a novel workflow to precisely characterize KRAS4B proteoforms isolated from colorectal cancer cell lines and primary tumor samples by immunoprecipitation (IP) and subsequent analysis on a benchtop Orbitrap mass spectrometer (1). Here, we modified our IP-TD protocol to include KRAS4A, then applied our workflow to the characterization, quantification, and direct comparison of KRAS4A and KRAS4B proteoforms within the context of multiple cancer cell lines. We hypothesized that we would be able to identify distinct proteoform populations for each KRAS isoform, which could then be compared both within and between different contexts. We have thus far identified nine novel proteoforms of KRAS4A, which not only exhibits differential PTM to KRAS4B, but varies widely in both abundance and PTM profile across all cell lines evaluated. We have also applied IP-TD to the analysis of endogenous KRAS4A and KRAS4B proteoforms using a more powerful 21 Tesla FT-ICR mass spectrometer at the National High Magnetic Field Laboratory. The high mass resolving power, mass measurement accuracy, and spectral acquisition rate achieved at high field combined with use of efficient dissociation methods and an external fragment ion accumulator to improve signal-to-noise ratios of MS/MS spectra produced unparalleled results with respect to degree of KRAS proteoform characterization. We were able to identify up to ten new KRAS proteoforms per context, several of which appear to be mutation- or context-specific. These latest advances have broadened our understanding of KRAS biology and provided new insights into the potential roles of KRAS4A and KRAS4B proteoforms in colorectal and other cancers. Reference 1. Ntai I, Fornelli L, DeHart CJ, Hutton JE, Doubleday PF, LeDuc RD, van Nispen AJ, Fellers RT, Whiteley G, Boja ES, Rodriguez H, Kelleher NL. Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification crosstalk. Proc Natl Acad Sci U S A 2018 Apr 17;115(16):4140-5. Citation Format: Caroline J. DeHart, Lissa C. Anderson, Lauren M. Adams, Luca Fornelli, Christopher L. Hendrickson, Neil L. Kelleher. Precise characterization and comparison of KRAS proteoforms by top-down mass spectrometry [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A26.

Abstract A06: Biophysical and biochemical characterization of KRAS G12C inhibition through the SMARTTM platform

Abstract RAS proteins are small GTPases involved in cell proliferation, survival, and differentiation, and are mutationally activated in about a third of all human cancers. These mutations drive cancer by impairing GTPase activity so that the RAS protein is found predominantly in its GTP-bound “on” conformation. Most KRAS isoform mutations are located at codon 12, a glycine in the P-loop of the GTPase active site. KRAS mutations in which the glycine is mutated to a cysteine (G12C) are particularly common in lung cancer. Despite its prevalence as an important oncogene and decades of research, the RAS protein remains an unexploited cancer target. We have developed a novel platform, SMART (Small Molecule Assisted Receptor Targeting), to disrupt the protein-protein interactions of so-called “undruggable” targets. Our compounds bind the immunophilin protein Cyclophilin A (CypA), and subsequently form a ternary complex with the target protein, KRAS. An electrophilic moiety on the ligand selectively forms a covalent bond with the cysteine of GTP-KRAS G12C, thus strengthening the stability of the ternary complex, partially occupying the effector face, and thus occluding the binding of downstream effector proteins, such as RAF. In a separate presentation, we describe a wide breadth of data to support the advancement of a small molecule specifically targeting the activated form of KRAS, with a focus on the cellular pharmacology. In this presentation, we highlight biophysical and biochemical findings that support our novel strategy for targeting GTP-KRAS G12C. SPR reveals the CypA-compound binary KD. A novel SPR protocol method was developed to examine CypA-compound-KRAS ternary complex formation, exploiting the A-B-A injection system of the Biacore 8K. LC-MS is used to characterize the crosslinking of our compounds to KRAS G12C in the presence of CypA, and full kinact/KI analysis is conducted with select compounds; in combination with SPR studies, the noncovalent KD of the ternary complex can be determined. Intrinsic warhead reactivity is measured through GSH adduct formation. TR-FRET studies probe the ability of ternary complexes to inhibit the binding of RAF to KRAS G12C. ITC and BLI are used to confirm ternary complex formation for selected compounds. In addition to guiding SAR, these studies characterize the degree of presenter (CypA) dependence, specificity of compound binding to the G12C mutant and to the GTP nucleotide-bound form of KRAS, and the ability to predict cellular efficacy. Our kinetic crosslinking experiments reveal single-digit micromolar KIs and a good kinact/KI ratio for our compounds relative to published literature on compounds targeting GDP-KRAS G12C. When these values are compared to SPR findings, the noncovalent KD contributes largely to KI, and thus noncovalent interactions play a bigger role in our compound’s interaction with RAS as compared to known GDP-KRAS G12C agents. Citation Format: Minyun Zhou, Alexander Yuzhakov, Cindy C Benod, Linlong Xue, Alec D Silver, Ganesh Iyer, Sharon A. Townson, Meizhong Jin, Nicholas R. Perl, Anna Kohlmann, Alan S. Mann, Gizem Akcay, Earl W. May. Biophysical and biochemical characterization of KRAS G12C inhibition through the SMARTTM platform [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A06.

Abstract A37: Mapping KRAS signaling pathways using the Mammalian-Membrane Two-Hybrid (MaMTH) assay to elucidate novel therapeutic targets

Abstract Background: KRAS is a well-established cancer driver. Clinically successful therapeutics have not yet been developed despite its disease burden, suggesting that KRAS-driven cancers are more complicated than previously thought. Aim: Our goal is to identify the global changes in KRAS interaction patterns that occur in disease states. This is accomplished by mapping the dynamic interactome of both WT and oncogenic KRAS isoforms using the Mammalian Membrane Two-Hybrid (MaMTH) assay, which is suitable for identification of protein interactors of virtually any integral membrane or membrane-associated protein. Methods: To probe the protein-protein interactions (PPIs) of KRAS, we used the Mammalian Membrane Two-Hybrid (MaMTH) assay, a split-ubiquitin-based two-hybrid system that can be applied to any cell line. This assay is suited for PPI detection using both low- to-medium throughput array and high-throughput large-scale PPI screening. We generated stably expressing “bait”-tagged KRAS constructs in HEK293T cells that contained the MaMTH reporter system. KRAS baits, namely KRAS-WT, -G12D, -G12V, and -Q61H, were characterized using signaling assays as well as within the MaMTH system using “prey”-tagged CRAF interaction control, Western blotting, and immunofluorescence techniques. Currently, we are performing unbiased screening for PPIs of KRAS using the human ORFeome (hORFeome) library consisting of approximately 13,000 fully sequenced human ORFs. Results: We first validated compatibility of KRAS in the MaMTH assay through several means. Oncogenic KRAS variants showed increased interaction signal with CRAF (a known KRAS binding partner) compared to KRAS-WT. This corresponded with upregulated MAPK pathway activation by oncogenic KRAS, as determined from increased pERK levels via Western blotting. KRAS bait expression levels were similar across all variants, suggesting that these findings were not due to differential expression of KRAS isotypes. Additionally, we confirmed that the KRAS bait correctly localizes to the plasma membrane, consistent with previous literature. After establishing KRAS compatibility with the MaMTH assay, we have begun unbiased screening of the hORFeome against KRAS baits in the WT, G12D, G12V, and Q61H isoforms. Conclusions: We have characterized KRAS compatibility with the MaMTH assay. Currently, we are performing large screening protocols for high-throughput detection of PPIs of KRAS using MaMTH. Citation Format: Ingrid Claudia Grozavu, Jamie Snider, Anna Lyakisheva, Igor Stagljar. Mapping KRAS signaling pathways using the Mammalian-Membrane Two-Hybrid (MaMTH) assay to elucidate novel therapeutic targets [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A37.

Abstract B37: Development of inhibitors of the activated form of KRAS G12C

Abstract Activating mutations in RAS proteins occur in ~1/3 of human cancers. These mutations impair the ability of the protein to hydrolyze GTP to GDP. As a result, mutant RAS proteins exist predominantly in the GTP-bound state, which directly activates aberrant downstream signaling via interaction with effectors such as RAF. Most RAS mutations occur at glycine 12 of the KRAS isoform. One such mutation, KRAS G12C, is particularly common in non-small cell lung cancer where it is found in ~15% of lung adenocarcinomas. Recent efforts have targeted KRAS G12C in the GDP-bound state; however, direct pharmacologic inhibition of active, GTP-bound KRAS G12C has proved challenging. Here, we deployed a novel SMARTTM (Small Molecule Assisted Receptor Targeting) platform to advance covalent compounds that selectively inhibit GTP-bound KRAS G12C. Using a mechanism reminiscent of the natural products rapamycin and cyclosporine, these compounds promote formation of a novel inhibitory ternary complex consisting of cyclophilin A (CypA, an abundant immunophilin present in all human cells), the SMART inhibitor, and GTP-KRAS G12C. Structure-based design of the SMART inhibitor yielded potent covalent inhibitors of GTP-KRAS G12C that exhibit >100-fold selectivity for mutant KRAS G12C over WT KRAS. Structural analysis of the ternary complex revealed that the covalent linkage between the SMART inhibitor and the mutant cysteine of KRAS occurred in the context of extensive interactions between CypA, the SMART inhibitor, and GTP-KRAS G12C that provide significant binding affinity (KI = 2.5 μM). The GTP-KRAS G12C|Inhibitor|CypA complex directly occluded effector binding, and as such, the compounds disrupted the KRAS-RAF interaction in biochemical assays. This activity was dependent on CypA, underlining the importance of the KRAS G12C| CypA protein-protein interaction in driving target engagement. In cell-based studies, SMART inhibitors crosslinked KRAS G12C and potently inhibited ERK phosphorylation and cell growth in G12C mutant tumor cell lines but had no effect on non-G12C bearing tumor cells. CRISPR knockout of cellular CypA confirmed that these activities were dependent on the presence of endogenous CypA. Importantly, SMART inhibitors bind directly to active, GTP-KRAS G12C and thus, their activity does not rely on trapping KRAS G12C in the inactive GDP-bound state. As a result, the cellular potency of SMART inhibitors with respect to crosslinking, pERK inhibition, and growth inhibition was maintained in the presence of growth factor treatments that reduce the cellular GDP-KRAS G12C pool. In contrast, we found that the activity of a previously described GDP-KRAS G12C targeting inhibitor was attenuated by growth factor treatment. To our knowledge, these are the first examples of mutant-selective KRAS inhibitors that target the active, GTP-bound state of KRAS G12C. We are currently optimizing the drug-like properties of these SMART inhibitors and evaluating their activity in in vivo models. Citation Format: Michelle L. Stewart, Nicholas R. Perl, Seung-Joo Lee, Linlong Xue, Minyun Zhou, Jonah Simon, Kathryn M. Luly, Siminia Grigoriu, Alex Yuzhakov, Alec Silver, Jason T. Lowe, Cindy C. Benod, Alan S. Mann, Gregory L. Verdine, Alan C. Rigby, Mark J. Mulvihill, Earl W. May, Anna Kohlmann, Sharon A. Townson, Roy M. Pollock, Meizhong Jin. Development of inhibitors of the activated form of KRAS G12C [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B37.

Abstract C27: Association of mutant KRAS isoforms with weight loss in pancreatic cancer

Abstract Background: Patients with pancreatic cancer often develop progressive weight loss and sarcopenia, described as cancer cachexia syndrome. Despite evidence showing that pancreatic cancer cachexia is associated with a poor prognosis, reduced tolerance to therapy, and diminished quality of life, no predictive biomarkers have been identified to date. Certain mutant KRAS isoforms are associated with significant alterations to tumor metabolism and have been correlated with changes in weight and survival. However, the clinical implications resulting from specific KRAS mutations in pancreatic cancer have yet to be fully explored. Methods: A retrospective analysis was performed to compare weight changes and survival in 150 pancreatic cancer patients with various KRAS alterations identified by next-generation sequencing (Foundation Medicine, Inc., Cambridge, MA). Survival data and weights documented between 6 months prior to diagnosis and the date of death or the last follow-up visit were abstracted from the medical record and evaluated using statistical analyses, including functional principal component analysis for sparse functional data, Ward cluster, logistic regression, analysis of variance, and Cox regression. Using derivative principal component scores, we separated patients into two groups demonstrating either constant or decreasing weight over time. Results: 77% of evaluated patients had a KRAS mutation, and 21% had KRAS wild-type. Of patients with mutant KRAS, 84% had an alteration at G12, 10% at Q61, and 3% at G13. While there was no significant difference in weight changes between KRAS wild-type patients and patients with a Q61 mutation, patients with non-Q61 mutations were 2.5 times more likely (p = 0.05) than wild-type patients to demonstrate weight loss over time. Furthermore, KRAS mutants had worse survival when compared to KRAS wild-type patients. Irrespective of mutational status, patients whose weight did not decrease were 69% less likely to die (p = 0.039). Conclusion: We demonstrated that mutant KRAS isoforms G12 and G13 mutations correlate with weight loss, whereas Q61 KRAS mutations and wild-type KRAS do not. Furthermore, weight loss in all patients increased their risk of death. These findings suggest that KRAS subtypes may have unique genotype-to-phenotype features, particularly with weight loss that could warrant tailored therapeutic strategies in at-risk subgroups. Future studies of larger scope, and ideally prospective in nature, are warranted in patients stratified by KRAS isoform. Citation Format: Haesoo Kim, Camille Ng, Marcio A. Diniz, Michel H. Montoril, Michelle Guan, Victor Brodsky, Eric Vail, Richard Tuli, Edik Blais, Sungjin Kim, Emanuel Petricoin III, Jun Gong, Veronica R. Placencio-Hickok, Andrew E. Hendifar. Association of mutant KRAS isoforms with weight loss in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C27.

Integrin \u03b17 expression is increased in asthmatic patients and its inhibition reduces Kras protein abundance in airway smooth muscle cells

Airway smooth muscle (ASM) cells exhibit plastic phenotypic behavior marked by reversible modulation and maturation between contractile and proliferative phenotypic states. Integrins are a class of transmembrane proteins that have been implicated as novel therapeutic targets for asthma treatment. We previously showed that integrin α7 is a novel marker of the contractile ASM phenotype suggesting that targeting this protein may offer new avenues to counter the increase in ASM cell mass that underlies airways hyperresponsiveness (AHR) in asthma. We now determine whether inhibition of integrin α7 expression would revert ASM cells back to a proliferative phenotype to cause an increase in ASM cell mass. This would be detrimental to asthmatic patients who already exhibit increased ASM mass in their airways. Using immunohistochemical analysis of the Melbourne Epidemiological Study of Childhood Asthma (MESCA) cohort, we show for the first time that integrin α7 expression in patients with severe asthma is increased, supporting a clinically relevant role for this protein in asthma pathophysiology. Moreover, inhibition of the laminin-integrin α7 signaling axis results in a reduction in smooth muscle-alpha actin abundance and does not revert ASM cells back to a proliferative phenotype. We determined that integrin α7-induced Kras isoform of p21 Ras acts as a point of convergence between contractile and proliferative ASM phenotypic states. Our study provides further support for targeting integrin α7 for the development of novel anti-asthma therapies.

KRAS-specific inhibition using a DARPin binding to a site in the allosteric lobe

Inhibiting the RAS oncogenic protein has largely been through targeting the switch regions that interact with signalling effector proteins. Here, we report designed ankyrin repeat proteins (DARPins) macromolecules that specifically inhibit the KRAS isoform by binding to an allosteric site encompassing the region around KRAS-specific residue histidine 95 at the helix α3/loop 7/helix α4 interface. We show that these DARPins specifically inhibit KRAS/effector interactions and the dependent downstream signalling pathways in cancer cells. Binding by the DARPins at that region influences KRAS/effector interactions in different ways, including KRAS nucleotide exchange and inhibiting KRAS dimerization at the plasma membrane. These results highlight the importance of targeting the α3/loop 7/α4 interface, a previously untargeted site in RAS, for specifically inhibiting KRAS function.

Isoform-specific Ras signaling is growth factor dependent.

HRAS, NRAS, and KRAS isoforms are almost identical proteins that are ubiquitously expressed and activate a common set of effectors. In vivo studies have revealed that they are not biologically redundant; however, the isoform specificity of Ras signaling remains poorly understood. Using a novel panel of isogenic SW48 cell lines endogenously expressing wild-type or G12V-mutated activated Ras isoforms, we have performed a detailed characterization of endogenous isoform-specific mutant Ras signaling. We find that despite displaying significant Ras activation, the downstream outputs of oncogenic Ras mutants are minimal in the absence of growth factor inputs. The lack of mutant KRAS-induced effector activation observed in SW48 cells appears to be representative of a broad panel of colon cancer cell lines harboring mutant KRAS. For MAP kinase pathway activation in KRAS-mutant cells, the requirement for coincident growth factor stimulation occurs at an early point in the Raf activation cycle. Finally, we find that Ras isoform-specific signaling was highly context dependent and did not conform to the dogma derived from ectopic expression studies.

Neuroblastoma RAS Viral Oncogene Homolog (NRAS) Is a Novel Prognostic Marker and Contributes to Sorafenib Resistance in Hepatocellular Carcinoma

Inhibition of the RAS-RAF-ERK-pathway using sorafenib as a first-line and regorafenib as a second-line treatment approach is the only effective therapeutic strategy for advanced hepatocellular carcinoma (HCC). Recent studies suggest that wild-type KRAS and HRAS isoforms could majorly contribute to HCC progression and sorafenib resistance. In contrast, the role of neuroblastoma RAS viral oncogene homolog (NRAS) in HCC remained elusive. In this study, wild-type NRAS was found to be overexpressed in HCC cell lines, preclinical HCC models, and human HCC tissues. Moreover, NRAS overexpression correlated with poor survival and proliferation in vivo. However, si-RNA-pool–mediated NRAS knockdown showed only slight effects on HCC proliferation, clonogenicity, and AKT activity. We determined that KRAS upregulation served as a functional compensatory mechanism in the absence of NRAS, which was overcome by combined inhibition of NRAS and KRAS in HCC cells. Furthermore, NRAS expression was elevated in sorafenib-resistant compared to nonresistant HCC cells, and NRAS knockdown enhanced sorafenib efficacy in resistant cells. In summary, NRAS appears to be a prognostic marker in HCC and contributes to sorafenib resistance. Regarding potential therapeutic strategies, NRAS inhibition in HCC should be combined with KRAS inhibition to prevent KRAS-mediated rescue effects.

K-Ras Populates Conformational States Differently from Its Isoform H-Ras and Oncogenic Mutant K-RasG12D

Structures of wild-type K-Ras from crystals obtained in the presence of guanosine triphosphate (GTP) or its analogs have remained elusive. Of the K-Ras mutants, only K-RasG12D and K-RasQ61H are available in the PDB representing the activated form of the GTPase not in complex with other proteins. We present the crystal structure of wild-type K-Ras bound to the GTP analog GppCH2p, with K-Ras in the state1 conformation. Signatures of conformational states obtained by one-dimensional proton NMR confirm that K-Ras has a more substantial population of state1 in solution than H-Ras, which predominantly favors state 2. The oncogenic mutant K-RasG12D favors state 2, changing the balance of conformational states in favor of interactions with effector proteins. Differences in the population of conformational states between K-Ras and H-Ras, as well as between K-Ras and its mutants, can provide a structural basis for focused targeting of the K-Ras isoform in cancer-specific strategies.

Role of epigenetic factors in the selection of the alternative splicing isoforms of human KRAS in colorectal cancer cell lines.

Mutation-driven activation of KRAS is crucial to cancer development. The human gene yields four mRNA splicing isoforms, 4A and 4B being translated to protein. Their different properties and oncogenic potential have been studied, but the mechanisms deciding the ratio 4A/4B are not known. To address this issue, the expression of the four KRAS isoforms was determined in 9 human colorectal cancer cell lines. HCT116 and SW48 were further selected because they present the highest difference in the ratio 4A/4B (twice as much in HCT116 than in SW48). Chromatin structure was analysed at the exon 4A, characteristic of isoform 4A, at its intronic borders and at the two flanking exons. The low nucleosome occupancy at exon 4A in both cell lines may result in a fast transcriptional rate, which would explain the general lower abundance of isoform 4A, also found in cells and tissues by other authors, but due to its similarity between both cell lines, chromatin structure does not influence alternative splicing. DNA methylation downstream exon 4A significantly differs in HCT116 and SW48 cells, but the CCCTC-binding factor, which affects the processivity of RNA polymerase and the alternative splicing, does not bind the differentially methylated sequences. Quantitative epigenetic analysis at mononucleosomal level revealed significant differences between both cell lines in H3K4me3, H3K27me3, H3K36me3, H3K9ac, H3K27ac and H4K20me1, and the inhibition of some histone-modifying enzymes alters the ratio 4A/4B. It can be concluded that the epigenetic modification of histones has an influence on the selection of isoforms 4A and 4B.

Allosteric Activation of GDP-Bound Ras Isoforms by Bisphenol Derivative Plasticisers.

The protein family of small GTPases controls cellular processes by acting as a binary switch between an active and an inactive state. The most prominent family members are H-Ras, N-Ras, and K-Ras isoforms, which are highly related and frequently mutated in cancer. Bisphenols are widespread in modern life because of their industrial application as plasticisers. Bisphenol A (BPA) is the best-known member and has gained significant scientific as well as public attention as an endocrine disrupting chemical, a fact that eventually led to its replacement. However, compounds used to replace BPA still contain the molecular scaffold of bisphenols. BPA, BPAF, BPB, BPE, BPF, and an amine-substituted BPAF-derivate all interact with all GDP-bound Ras-Isoforms through binding to a common site on these proteins. NMR-, SOScat-, and GDI- assay-based data revealed a new bisphenol-induced, allosterically activated GDP-bound Ras conformation that define these plasticisers as Ras allosteric agonists.

Isoform specific gene expression analysis of KRAS in the prognosis of lung adenocarcinoma patients

BackgroundAberrant mutations in KRAS play a critical role in tumor initiation and progression, and are a negative prognosis factor in lung adenocarcinoma (LUAD).ResultsUsing genomic analysis for K-Ras isoforms (K-Ras4A and K-Ras4B) and large-scale multi-omics data, we inspected the overall survival (OS) and disease-free survival (DFS) of LUAD patients based on the abundance of transcript variants by analyzing RNA expression and somatic mutation data from The Cancer Genome Atlas (n = 516). The expression of the minor transcript K-Ras4A and its proportion were positively correlated with the presence of KRAS mutations in LUAD. We found that both K-Ras4A abundance measures (expression and proportion) have a strong association with poor OS (p = 0.0149 and p = 3.18E-3, respectively) and DFS (p = 3.03E-4 and p = 0.0237, respectively), but only in patients harboring KRAS mutations. A Cox regression analysis showed significant results in groups with low expression (hazard ratio (HR) = 2.533, 95% confidence interval (CI) = 1.380−4.651, p = 2.72E-3) and low proportion (HR = 2.549, 95% CI = 1.387−4.684, p = 2.58E-3) of K-Ras4A.ConclusionsBased on the above results, we report the possible use of abundance measures for K-Ras4A for predicting the survival of LUAD patients with KRAS mutations.