Ras Alleles Research Articles

Abstract PR-16: Evaluating direct KRASQ61H inhibition in pancreatic cancer models

Abstract KRASQ61 mutations are associated with the worst prognosis among the various KRAS mutational subtypes of pancreatic ductal adenocarcinoma (PDAC). RMC-7977 is a RAS(ON) multi-selective noncovalent inhibitor with broad spectrum activity against oncogenic RAS mutants and wild-type RAS. The related investigational agent RMC-6236 is currently undergoing clinical trials. Binary complexes of CypA: RMC-7977 bind equipotently to multiple KRAS mutational subtypes however RMC-7977 is less potent at inhibiting growth in KRASQ61H-mutant cell lines. In contrast, RM-047 is a preclinical RAS(ON) Q61H-selective inhibitor tool compound. We evaluated direct KRASQ61H inhibition using both RMC-7977 and RM-047 in cell culture models. We found that in KRASQ61H-rescued RASless mouse embryonic fibroblasts (MEFs), RM-047 inhibited ERK activity and suppressed growth more potently than RMC-7977. Conversely, in a panel of KRASQ61H-mutant PDAC cell lines, RM-047 was less potent than RMC-7977 in two out of three KRASQ61H-mutant PDAC cell lines. Furthermore, we discovered that ERK activity rapidly rebounded with treatment in KRASQ61H-mutant PDAC cell lines, whereas ERK activity remained suppressed in KRASQ61H-rescued RASless MEFS lacking WT RAS alleles. Our analysis of DepMap data indicated that among the KRAS mutational subtypes frequently found in pancreatic cancer, KRASQ61H is the least dependent on KRAS for survival. This suggests that in a KRASQ61H-mutant PDAC setting, additional factors such as WT RAS expression may mediate growth. From our KRAS drug-anchored CRISPR loss-of-function screens, we found that the loss of EGFR sensitized cells to KRAS inhibition, particularly in a KRASQ61H-dependent context. Therefore, we combined erlotinib, an FDA-approved EGFR inhibitor, with either RM-047 or RMC-7977, and found that the addition of erlotinib prevented ERK rebound activity and synergized with RM-047 or RMC-7977 to reduce growth in KRASQ61H-mutant PDAC cell lines. These data provide insights into mechanisms by which KRASQ61H-mutant PDAC may be less dependent on KRAS for survival and further support the clinical evaluation of combined EGFR and RAS(ON) inhibition as a potential therapeutic strategy in the treatment of PDAC patients harboring KRASQ61H mutations. Citation Format: Szu-Aun Long, Amber M Amparo, Haley Todd, Grace Goodhart, Syed A Ahmad, Andrew M Waters. Evaluating direct KRAS inhibition in pancreatic cancer models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-16.

Mutant N- and K- Ras Show Distinct Effects on Growth, Signaling and Drug Response in CRISPR-Cas Edited Alleles of a Myeloma Cell Line

Myeloma is well known to have significant genetic heterogeneity among patients' tumors, affecting both progression as well as therapeutic responses. Moreover, subclonal diversity can lead to selective outgrowths that are influenced by new mutations, as well as the bone marrow microenvironment and therapeutic exposures. The ras family of genes (N -and K-) are the most common mutations (40-50% ) in myeloma. Notably, ras mutations occur among a variety of other co-existing chromosomal abnormalities. Ras mutations were found to be more commonly associated with cyclin D1 high expression (e.g., resulting from 11;14 translocations). We and others have also shown that rasmutations may not be disease-initiating but may arise during disease progression and relapse; or are subclonal events that impart selective growth advantage and emerging drug resistance. There is evidence that activating mutations (codons 12,13, 61) in K- ras may have different clinical impact than activating mutations in N- ras yet there are conflicting clinical outcomes that may be due to therapies analyzed or differences in other co-existing genetic mutations. Previous studies using transfection of genes with targeted mutations show random integration and often in multiple copies, thus, limiting interpretations. We chose to create N ras61 and K ras12 mutations by CRISPR-cas directed edits in a single endogenous ras allele in a myeloma cell line (FLAM76) that initially had two wild type alleles (and an 11;14 translocation). We then compared the impact of these edits on growth, signaling, gene expression, and drug responses. We find both the mutN ras and mutK ras edited cells have increased proliferation compared to wild type. However, we find that the N ras mutation increases MAPK signaling and downstream gene activation to a greater extent than the K rasmutation. Based on RNAseq analysis, a MAPK gene activation score was higher in mutN ras edited cells than mutK ras edited cells. Further, we see distinct gene expression patterns between mutN ras versus mutK ras edited cells (See heat map Fig) impacting distinct pathways. Notably, mutNras cells have much higher NOXA expression, increasing sensitivity to apoptosis-inducing drugs (e.g., proteasome inhibitors); and Kras induces anti-apoptotic Bcl-xL, reducing sensitivity. We have conducted a high throughput drug screen with 84 drugs, and while some drugs are active against both mutN ras and mutK ras edits (Proteasome inhibitors), we have identified drugs with differential response to K versus N ras mutations. For example, mutK ras FLAM76 cells are more resistant to proteasome inhibitors; and particularly more resistant to HSP inhibitors and HDAC inhibitors compared to wild type or mutN ras cells. In contrast, mutK ras cells are more sensitive to PikFyve and IL-12 inhibitors than mutNras or wild type cells. Gene expression analysis shows that mutKras cells result in more downregulation of apoptotic genes and higher upregulation of cell survival genes, as well as ER stress pathway genes. Our results suggest that treating myelomas containing K versus N ras activating mutations may benefit from understanding distinct pathway activations and targeting selective drug therapies. We are currently testing new pan-Ras inhibitors to determine if there are distinct responses between our N- and K- ras edited cells, and we are expanding testing to a wider panel of myeloma cell lines containing ras mutations in the context of other genetic abnormalities. Details of gene expression distinctions, pathways, and drug responses that distinguish mutN ras and mutK ras in myeloma will be presented.

The success rates of clinical cancer next-generation sequencing based on pathologic diagnosis: Experience from a single academic laboratory.

This article aims to establish the relationship between pathologic diagnosis and the rate of success in cancer next-generation sequencing testing. Clinical next-generation sequencing results performed for solid tumors were reviewed. The rate of success was analyzed in the context of tumor type and accompanying variables. Out of 683 total specimens, 533 (78.0%) underwent successful sequencing. The rate of success was 91.8% for ovarian carcinomas, 87.5% for lung non-small cell carcinomas, 82.0% for colorectal adenocarcinomas, 78.3% for melanomas, 75.9% for breast carcinomas, and 64.7% for pancreatic adenocarcinomas. For specimens that successfully underwent sequencing, pancreatic adenocarcinomas had the lowest median tumor proportion and somatic RAS and TP53 mutation allele fractions compared with other tumor types. Cytology specimens had a 33.3% success rate for pancreatic adenocarcinomas (5 of 15) and a 93.3% success rate for lung carcinomas (14 of 15). Compared with tissue from primary sites, tissue from metastatic sites showed a higher success rate for pancreatic adenocarcinomas and lower success rates for colorectal adenocarcinomas and melanomas. The success rate of cancer next-generation sequencing testing is dependent on pathologic diagnosis, tissue site, and diagnostic procedure. Understanding which specimens are at higher risk for failing molecular testing may help pathologists and clinical care teams optimize tissue acquisition and usage for patient care.

Abstract B022: A non-conserved histidine residue on KRAS drives paralog selectivity of the KRAS G12D inhibitor MRTX1133

Abstract The discovery of small molecule inhibitors against mutant KRAS protein has been a breakthrough in targeted therapy. MRTX1133 is the first non-covalent inhibitor with selectivity for the mutant KRAS G12D protein. Using isogenic cell lines expressing single Ras allele to test the selectivity of this compound, we found that in addition to KRAS G12D, MRTX1133 shows significant activity against several other KRAS mutants as well as wildtype KRAS protein. In contrast, MRTX1133 exhibits no activity against both G12D and wildtype forms of HRAS and NRAS proteins. Functional analysis revealed that the selectivity of MRTX1133 towards KRAS is associated with its binding to histidine 95 on KRAS, a residue that is not conserved in HRAS and NRAS. Reciprocal mutation of amino acid 95 among the three Ras paralogs resulted in reciprocal change in their sensitivity towards MRTX1133. Thus, histidine 95 is an essential selectivity handle for MRTX1133 towards KRAS. This knowledge could aid the development of other KRAS-selective inhibitors. (Funding: this research was supported by the Intramural Research Program of the National Cancer Institute, NIH) Citation Format: Miles Keats, John Han, Yeon-Hwa Lee, Chih-Shia Lee, Ji Luo. A non-conserved histidine residue on KRAS drives paralog selectivity of the KRAS G12D inhibitor MRTX1133 [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B022.

Abstract 5328: Novel KRAS-specific in-well lysis ELISA for high throughput screening

Abstract RAS is one of the most frequently mutated genes in cancer. Gain–of–function missense mutations promoting oncogenesis cluster at codons 12, 13, and 61 of KRAS, NRAS and HRAS. These result in enhanced GTP binding due to fast exchange of nucleotide and/or impairment of GAP (GTPase Activating Protein) binding. Although these mutants are all activating, they are not equal in their oncogenic potential and differences in patient survival are associated with different RAS mutants and which RAS allele is affected, with KRAS being the most prevalent. Current RAS therapeutic approaches have migrated to be isoform specific. With this in mind, we have developed an activation state specific high throughput screening compatible (HTS) assay that is specific for KRAS. A novel rabbit anti–KRAS recombinant antibody was isolated via cloning of immunoglobulin variable regions from antigen positive B cells isolated from rabbits immunized with a peptide immunogen targeting a region of low homology between the RAS isoforms. Antibody specificity was validated with recombinant HRAS, KRAS and NRAS proteins over a wide concentration range by both immunoblotting and by a bead–based multiplex ELISA where all three RAS isoforms were present. The new assay allows for direct in–well lysis of cells cultured in 96–well plate format, either directly in media or following a PBS wash step. Activated KRAS in the lysate is captured on the assay plate and detected via chemiluminescence using the KRAS specific antibody. Preliminary assay validation has included EGF treatment of HeLa cells to show activation in cells expressing wildtype KRAS and inhibition with the selective non–covalent KRASG12D inhibitor MRTX1133 in Aspc1 cells known to express KRASG12D over a wide range of cell seeding densities. We anticipate that this assay will further the discovery of novel KRAS specific therapeutics. Citation Format: Hyun Min Park, Jason Poole, Mary Anne Jelinek. Novel KRAS-specific in-well lysis ELISA for high throughput screening. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5328.

RAS oncogene signal strength regulates matrisomal gene expression and tumorigenicity of mouse keratinocytes.

Environmental and molecular carcinogenesis are linked by the discovery that chemical carcinogen induced-mutations in the Hras or Kras genes drives tumor development in mouse skin. Importantly, enhanced expression or allele amplification of the mutant Ras gene contributes to selection of initiated cells, tumor persistence, and progression. To explore the consequences of Ras oncogene signal strength, primary keratinocytes were isolated and cultured from the LSL-HrasG12D and LSL-KrasG12D C57BL/6J mouse models and the mutant allele was activated by adeno-Cre recombinase. Keratinocytes expressing one (H) or two (HH) mutant alleles of HrasG12D, one KrasG12D allele (K), or one of each (HK) were studied. All combinations of activated Ras alleles stimulated proliferation and drove transformation marker expression, but only HH and HK formed tumors. HH, HK, and K sustained long-term keratinocyte growth in vitro, while H and WT could not. RNA-Seq yielded two distinct gene expression profiles; HH, HK, and K formed one cluster while H clustered with WT. Weak MAPK activation was seen in H keratinocytes but treatment with a BRAF inhibitor enhanced MAPK signaling and facilitated tumor formation. K keratinocytes became tumorigenic when they were isolated from mice where the LSL-KrasG12D allele was backcrossed from the C57BL/6 onto the FVB/N background. All tumorigenic keratinocytes but not the non-tumorigenic precursors shared a common remodeling of matrisomal gene expression that is associated with tumor formation. Thus, RAS oncogene signal strength determines cell-autonomous changes in initiated cells that are critical for their tumor-forming potential.

Circulating tumor DNA to guide rechallenge with panitumumab in metastatic colorectal cancer: the phase 2 CHRONOS trial

Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies are approved for the treatment of RAS wild-type (WT) metastatic colorectal cancer (mCRC), but the emergence of resistance mutations restricts their efficacy. We previously showed that RAS, BRAF and EGFR mutant alleles, which appear in circulating tumor DNA (ctDNA) during EGFR blockade, decline upon therapy withdrawal. We hypothesized that monitoring resistance mutations in blood could rationally guide subsequent therapy with anti-EGFR antibodies. We report here the results of CHRONOS, an open-label, single-arm phase 2 clinical trial exploiting blood-based identification of RAS/BRAF/EGFR mutations levels to tailor a chemotherapy-free anti-EGFR rechallenge with panitumumab (ClinicalTrials.gov: NCT03227926; EudraCT 2016-002597-12). The primary endpoint was objective response rate. Secondary endpoints were progression-free survival, overall survival, safety and tolerability of this strategy. In CHRONOS, patients with tissue-RAS WT tumors after a previous treatment with anti-EGFR-based regimens underwent an interventional ctDNA-based screening. Of 52 patients, 16 (31%) carried at least one mutation conferring resistance to anti-EGFR therapy and were excluded. The primary endpoint of the trial was met; and, of 27 enrolled patients, eight (30%) achieved partial response and 17 (63%) disease control, including two unconfirmed responses. These clinical results favorably compare with standard third-line treatments and show that interventional liquid biopsies can be effectively and safely exploited in a timely manner to guide anti-EGFR rechallenge therapy with panitumumab in patients with mCRC. Further larger and randomized trials are warranted to formally compare panitumumab rechallenge with standard-of-care therapies in this patient setting.

Development and extensive analytical validation of deep amplicon sequencing for detecting KRAS and NRAS mutations in metastatic colorectal cancer samples.

The presence of wild-type RAS alleles, as determined by genotyping codons 12, 13, 59, 61, 117, and 146, is a prerequisite for personalized anti-EGFR treatment of metastatic colorectal cancer (mCRC) patients. Here we describe analytical validation of in-house developed massively parallel sequencing technology (MPS) in comparison to the in vitro diagnostics (IVD) certified qPCR method. DNA extracted from FFPE samples from CRC patients (n=703) and reference standards (n=33) were tested for KRAS and NRAS mutations in 6 codons of exons 2, 3, and 4 using deep amplicon sequencing (DAS) on a MiSeq benchtop sequencer (Illumina). Two different amplicon lengths and two different library preparation methods (long-RAS and short-RAS) were tested in order to evaluate their impact on DAS performance. In parallel, identical tumor DNA was tested by the following IVD assays: therascreen KRAS RGQ PCR Kit (Qiagen), cobas® KRAS Mutation Test (Roche Diagnostics), and SNaPshot assay (Thermo Fisher Scientific). Both DAS assays detected all the mutations present in reference standards and external quality control samples, except for the artificially generated KRAS codon 146 mutation. The DAS assays performed sufficient analytical specificity and sensitivity (≥0.95). The use of shorter amplicons prolonged the preparation steps but significantly improved the sequencing success rate of FFPE-derived DNA. RAS mutation frequencies in the Czech CRC patients were similar to previous reports, although rare mutations were also detected. DAS with short amplicons is a good strategy for routine assessment of somatic mutations in low-quality FFPE-derived DNA.

P-290 Mutant allele specific imbalance in RAS as prognostic marker in colorectal cancer

All colorectal cancer patients who progress to metastatic stage have their tumor molecularly analyzed for mutations in RAS. The NGS results reveal the ratio between the mutant allele and the wild-type allele. RAS mutant allele specific imbalance (MASI) is defined as when the number of copies of the mutant RAS allele is higher than that of the wild-type RAS allele strain. The objectives of the present study are to estimate the rate of the RAS MASI phenomenon among patients with metastatic colorectal cancer and to examine the relationship between this phenomenon and survival rate of the patients.

The Role of Wild-Type RAS in Oncogenic RAS Transformation.

The RAS family of oncogenes (HRAS, NRAS, and KRAS) are among the most frequently mutated protein families in cancers. RAS-mutated tumors were originally thought to proliferate independently of upstream signaling inputs, but we now know that non-mutated wild-type (WT) RAS proteins play an important role in modulating downstream effector signaling and driving therapeutic resistance in RAS-mutated cancers. This modulation is complex as different WT RAS family members have opposing functions. The protein product of the WT RAS allele of the same isoform as mutated RAS is often tumor-suppressive and lost during tumor progression. In contrast, RTK-dependent activation of the WT RAS proteins from the two non-mutated WT RAS family members is tumor-promoting. Further, rebound activation of RTK–WT RAS signaling underlies therapeutic resistance to targeted therapeutics in RAS-mutated cancers. The contributions of WT RAS to proliferation and transformation in RAS-mutated cancer cells places renewed interest in upstream signaling molecules, including the phosphatase/adaptor SHP2 and the RasGEFs SOS1 and SOS2, as potential therapeutic targets in RAS-mutated cancers.

Abstract 6425: Sensitivity of oncogenic KRAS-expressing cells to CDK9 inhibition identified by a phenotypic compound screen

Abstract The normal RAS signaling pathway provides a primary switch whereby cell proliferation is controlled through activation of RAS proteins and downstream signaling. Oncogenic mutants of RAS genes often drive hyper-activation of cell proliferation and signaling resulting in tumorigenesis. Oncogenic forms of KRAS protein are known to be drivers of pancreatic, colorectal and lung cancers. These cancers are particularly lethal and are associated with poor prognosis. The goal of this study is to identify chemical leads that inhibit oncogenic KRAS signaling with phenotypic screen. We first developed an isogenic panel of mouse embryonic fibroblast (MEF) cell lines that carry wild-type and oncogenic RAS. We validated these cell lines by screening against a tool compound library of 1402 annotated inhibitors in the CellTiter-Glo® cell viability assay. We identified previously known specific RAS allele vulnerabilities, confirming the biological properties of these cell lines and their utility in future screens. Subsequently, MEFs from this panel expressing KRASG12D, KRASG12V, KRASG13D, KRASQ61R, HRASWT, and NRASWT, were used to conduct a high throughput screen with a proprietary compound library of about 931,000 compounds in the CellTiter-Glo® cell viability assay. 126 compounds (29 chemical clusters and 10 singletons) showed specific activity against mutant KRAS (IC50: RASWT/mutant KRAS ≥ 5), and were selected for secondary assays including a series of cell-based, biophysical and biochemical assays, in order to prioritize and eliminate compounds with undesired properties. Finally, five chemical clusters were chosen. They have specific activity against SW620 (KRASG12V) and LS513 (KRASG12D) over Colo320 (RASWT) colon cancer cell lines. In addition, they have no effects on BRAFV600E, MEK1, ERK2, PI3K alpha, AKT1, or mTOR as tested in in vitro enzymatic activity assays. Biophysical assays, SPR and DSF, demonstrated that these compounds do not bind directly to KRAS. We further identified the mechanism of action of these five chemical clusters, with three of them having CDK9 inhibitory activity. In conclusion, we have developed and validated an isogenic MEF panel that can be used successfully to identify specific RAS oncogenic or wild-type allele vulnerabilities. Furthermore, we identified sensitivity of oncogenic KRAS-expressing cell to CDK9 inhibitors, which warrants future studies of potential use of CDK9 inhibitors in treating KRAS-driven cancers. Citation Format: Lick P. Lai, Viviane Brel, Kanika Sharma, Julia Frappier, Nadia Le-Henanf, Bertrand Vivet, Nicolas Muzet, Emilie Schell, Renaud Morales, Eamonn Rooney, Matthew Holderfield, Frederic Lacroix, Walter Englaro, Christophe Marcireau, Laurent Debussche, Dwight V. Nissley, Frank Mccormick. Sensitivity of oncogenic KRAS-expressing cells to CDK9 inhibition identified by a phenotypic compound screen [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 6425.

The Emerging Role of the FGF/FGFR Pathway in Gastrointestinal Stromal Tumor.

Gastrointestinal stromal tumors (GIST) are rare neoplasms of mesenchymal origin arising in the gastrointestinal tract. The vast majority are characterized by mutually exclusive activating mutations in KIT or Platelet-derived growth factor alpha (PDGFRA) receptors, or less frequently by succinate dehydrogenase complex (SDH) or NF1 inactivation, with very rare cases harboring mutant BRAF or RAS alleles. Approximately 5% of GISTs lack any of such mutations and are called quadruple wild-type (WT) GISTs. Recently, deregulated Fibroblast Growth Factor (FGF)/FGF-receptor (FGFR) signaling emerged as a relevant pathway driving oncogenic activity in different molecular subgroups of GISTs. This review summarizes all the current evidences supporting the key role of the FGF/FGFR pathway activation in GISTs, whereby either activating mutations, oncogenic gene fusions, or autocrine/paracrine signaling have been detected in quadruple WT, SDH-deficient, or KIT-mutant GISTs.

Abstract IA31: Genetic approaches for testing candidate drug targets for Ras-induced tumorigenesis in vivo

Abstract Despite remarkable progress over the past few years, developing chemical inhibitors that directly and selectively target mutant Ras proteins remains a fundamental challenge. Given this, inhibitors of proteins that function upstream or downstream of oncogenic Ras are a promising alternative therapeutic strategy for RAS mutant tumors. However, the complexity of Ras signaling networks and adaptive mechanisms that result in pathway “rewiring” complicates identifying genes and proteins that are essential for the growth and survival of these cancers. Genetically engineered mice that express mutant Ras alleles from the endogenous locus are a robust system for uncovering pathway dependencies and potential therapeutic vulnerabilities. We have generated novel strains of mice to address two specific questions. First, we collaborated with Kevin Haigis to generate a “second site” C181S substitution within a conditional NrasLSL-G12D mutant allele (Nat Genet 2008;40:600) to ask if palmitoylation is essential for myeloid transformation in vivo. We intercrossed mice carrying this mutation with the Mx1-Cre strain and induced Cre recombinase expression in the hematopoietic compartment by injecting compound mutant pups with polyI:polyC. As previously reported, homozygous NrasLSL-G12D mice uniformly developed an aggressive myeloproliferative disorder (MPD) characterized by markedly elevated blood leukocyte counts, splenomegaly, and death by age 9 months. By contrast, homozygous NrasLSL-G12D,C181S mice remained healthy with no evidence of hematologic disease despite constitutively elevated levels of RasGTP in bone marrow cells that were similar to those observed in NrasLSL-G12D mice. Whereas normal NRas and N-RasG12D proteins are predominantly in the plasma membrane, N-RasG12D, C181S was largely mis-localized in the cytosol. We also generated Mx1-Cre, NrasG12D,C181S/G12D compound heterozygous mutant mice to investigate potential interactions between oncogenic NrasG12D alleles with and without the C181S substitution. Over half of these mice developed aggressive hematologic malignancies, albeit with prolonged latency compared to homozygous NrasG12D mice. Importantly, genetic analysis of diseased tissues revealed reduced frequency or complete absence of the NrasG12D,C181S allele due to secondary somatic genetic events, which indicates strong selective pressure to overcome the growth-suppressive properties of unpalmitoylated N-Ras. Together, these studies validate the palmitoylation/depalmitoylation cycle as a therapeutic target in NRAS mutant hematologic malignancies and establish a novel model system for addressing this question in other cancers. In another ongoing project, we collaborated with Pedro Perez-Macera and David Tuveson to modify a LSL-KrasG12D knock-in allele to introduce a second-site amino acid Y64G substitution that severely impairs binding to PI3 kinase. Surprisingly, mice expressing KrasG12D,Y64G in the germline are viable and phenotypically normal, although they are born at a lower than expected Mendelian frequency. Whereas Mx1-Cre, KrasG12D/+ mice uniformly develop aggressive MPD and die by 4 months of age, KrasG12D,Y64G animals have normal blood counts at 12-18 months of age. However, 100% of KrasG12D,Y64G mice develop lung lesions by 1 year of age, and ultimately succumb from lung tumors with a median survival of 496 days. The majority of these lesions are classified pathologically as atypical lymphoid proliferation or papillary adenomas, with a few mice developing adenocarcinomas. These studies demonstrating that expressing a Y64G amino acid substitution in the context of oncogenic KrasG12D rescues embryonic lethality, abrogates myeloid disease, and attenuates lung tumorigenesis are generally consistent with and also extend elegant studies of Pi3kca mutant mice from the Downward lab (Cell 2007;129:957; Cancer Cell 2013;24:617). Beyond the bone marrow and lung, KrasG12D,Y64G mice are a potent genetic tool for dissecting the role of aberrant PI3 kinase signaling in lung, pancreatic, colon, and other tissues characterized by tumors driven by somatic KRAS mutations, and these data have implications for treating human cancers with KRAS mutations. Citation Format: Kevin M. Shannon. Genetic approaches for testing candidate drug targets for Ras-induced tumorigenesis in vivo [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 IA31.

Targeted Deep Sequencing Uncovers Cryptic KIT Mutations in KIT/PDGFRA/SDH/RAS-P Wild-Type GIST.

Background: Gastrointestinal stromal tumors (GIST) are known to carry oncogenic KIT or PDGFRA mutations, or less commonly SDH or NF1 gene inactivation, with very rare cases harboring mutant BRAF or RAS alleles. Approximately 10% of GISTs are devoid of any of such mutations and are characterized by very limited therapeutic opportunities and poor response to standard treatments.Methods: Twenty-six sporadic KIT/PDGFRA/SDH/RAS-pathway wild type GIST were profiled for the molecular status of genes frequently altered in GIST by a targeted next generation sequencing (NGS) approach. Molecular findings were validated by alternative amplicon-based targeted sequencing, immunohistochemistry, gene expression profiling and Sanger sequencing.Results: Three patients harboring NF1 inactivating mutations were identified and excluded from further analysis. Intriguingly, five patients carried cryptic KIT alterations, mainly represented by low-allele-fraction mutations (12–16% allele ratio). These mutations were confirmed by another targeted NGS approaches and supported by CD117 immuno-staining, gene expression profiling, Sanger sequencing, with peak signals at the level of background noise, and by the patients' clinical course assessment.Conclusion: This study indicates that ~20% patients diagnosed with a KIT/PDGFRA/SDH/RAS-pathway wild-type GIST are bona-fide carriers of pathogenic KIT mutations, thus expected to be eligible for and responsive to the various therapeutic lines of TK-inhibitors in use for KIT/PDGFRA-mutant GIST. The centralization for a second level molecular analysis of GIST samples diagnosed as wild-type for KIT and PDGFRA is once again strongly recommended.

Prenatal growth of live weight and coat color of the mini pigs of ICG SB RAS

The article considers the possibility of connection of KIT and MC1R loci with prenatal growth of mini pigs of the breeding group of Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences (ICG SB RAS). It is assumed that this relationship may be the result of increased variability of the sign – the mass of the newborn pig in heterozygous according to the loci of individuals. Since prenatal growth is affected by a number of factors that affect the weight of the newborn pig, instead of the absolute value of the sign – the mass of the individual in kilograms, a relative one was used, which is the ratio of the mass of a particular individual to the average mass of the individual in the nest in which it was born. The study showed that mini-pigs of ICG SB RAS have a relationship between heterozygosity at MC1R and KIT loci and increased concentration of individuals in the modal class of relative weight distribution at birth. The discovered relationship is the result of the interaction of increased variability of mass at birth in individuals’ heterozygous loci, controlling intracellular metabolism, with the vector of phenotypic realization of the genetic potential of growth of individuals in the prenatal period. The decomposition of the specified vector into components showed that optimizes forming the kurtosis variation of the number of components dominate in individuals with domestication coat color, while in individuals with pseudoagouti coat color – biasing, forming the asymmetry of the distribution. The results of the study also showed that the genotypes at the loci MC1R and KIT do not affect the competitive relationship between the fetuses during the prenatal period . In addition, the analysis of the consistency of the estimates of individual classes of variational series of individuals of different races to a normal distribution showed that the mini pigs of ICG SB RAS alleles present low weight at birth, inherited from the Vietnamese breeds , and alleles of the high mass at birth, inherited from the Large White and/or Danish Landrace breed.

Retrospective analysis of overall survival (OS) by subsequent therapy in patients (pts) with RAS wild-type (wt) metastatic colorectal cancer (mCRC) receiving irinotecan ± cetuximab in the EPIC study.

3580 Background: The multicenter, open-label, randomized, phase 3 EPIC study (EMR 062202-025) investigated cetuximab + irinotecan vs irinotecan as a second-line (2L) therapy in pts with EGFR-detectable mCRC. A retrospective analysis in the EPIC RAS wt population showed improved overall response rate (29.4% vs 5.0%) and progression-free survival (5.4 vs 2.6 mo) upon the addition of cetuximab to irinotecan. Median OS (mOS) was similar in both treatment arms, possibly due to differences in subsequent therapies. We present OS by post-study therapy in the RAS wt population. Methods: 1298 RAS-unselected pts were enrolled from May 2003 to February 2006. The primary endpoint was OS. RAS status was determined retrospectively in 2018 from existing DNA samples using BEAMing technology; wt status was defined as having a sum of mutated RAS allele frequencies of ≤5%, with all relevant alleles being analyzable. Results: Among the 452 pts with RAS wt mCRC, 231 received cetuximab + irinotecan and 221 received irinotecan. Baseline characteristics were similar in both arms. OS data by post-study therapy are summarized in the Table. No new or unexpected safety signals were observed. Conclusions: Post-study cetuximab was associated with improved OS in both treatment arms compared with post-study therapy without cetuximab and no subsequent therapy, suggesting that cetuximab-based therapy may be suitable as a standard treatment for pts with RAS wt mCRC in the rechallenge setting. Study limitations include a potential bias due to the differences in proportion of subsequent therapies with and without cetuximab between arms (almost 50% of pts in the irinotecan arm received post-study cetuximab) as well as the likelihood for pts who live longer to receive cetuximab in any subsequent therapy line. [Table: see text]

Anti-EGFR-resistant clones decay exponentially after progression: implications for anti-EGFR re-challenge.

Colorectal cancer (CRC) has been shown to acquire RAS and EGFR ectodomain mutations as mechanisms of resistance to epidermal growth factor receptor (EGFR) inhibition (anti-EGFR). After anti-EGFR withdrawal, RAS and EGFR mutant clones lack a growth advantage relative to other clones and decay; however, the kinetics of decay remain unclear. We sought to determine the kinetics of acquired RAS/EGFR mutations after discontinuation of anti-EGFR therapy. We present the post-progression circulating tumor DNA (ctDNA) profiles of 135 patients with RAS/BRAF wild-type metastatic CRC treated with anti-EGFR who acquired RAS and/or EGFR mutations during therapy. Our validation cohort consisted of an external dataset of 73 patients with a ctDNA profile suggestive of prior anti-EGFR exposure and serial sampling. A separate retrospective cohort of 80 patients was used to evaluate overall response rate and progression free survival during re-challenge therapies. Our analysis showed that RAS and EGFR relative mutant allele frequency decays exponentially (r2=0.93 for RAS; r2=0.94 for EGFR) with a cumulative half-life of 4.4 months. We validated our findings using an external dataset of 73 patients with a ctDNA profile suggestive of prior anti-EGFR exposure and serial sampling, confirming exponential decay with an estimated half-life of 4.3 months. A separate retrospective cohort of 80 patients showed that patients had a higher overall response rate during re-challenge therapies after increasing time intervals, as predicted by our model. These results provide scientific support for anti-EGFR re-challenge and guide the optimal timing of re-challenge initiation.

The Spacing Effect for Structural Synaptic Plasticity Provides Specificity and Precision in Plastic Changes.

In contrast to trials of training without intervals (massed training), training trials spaced over time (spaced training) induce a more persistent memory identified as long-term memory (LTM). This phenomenon, known as the spacing effect for memory, is poorly understood. LTM is supported by structural synaptic plasticity; however, how synapses integrate spaced stimuli remains elusive. Here, we analyzed events of structural synaptic plasticity at the single-synapse level after distinct patterns of stimulation in motoneurons of Drosophila We found that the spacing effect is a phenomenon detected at synaptic level, which determines the specificity and the precision in structural synaptic plasticity. Whereas a single pulse of stimulation (massed) induced structural synaptic plasticity, the same amount of stimulation divided in three spaced stimuli completely prevented it. This inhibitory effect was determined by the length of the interstimulus intervals. The inhibitory effect of the spacing was lost by suppressing the activity of Ras or mitogen-activated protein kinase, whereas the overexpression of Ras-WT enhanced it. Moreover, dividing the same total time of stimulation into five or more stimuli produced a higher precision in the number of events of plasticity. Ras mutations associated with intellectual disability abolished the spacing effect and led neurons to decode distinct stimulation patterns as massed stimulation. This evidence suggests that the spacing effect for memory may result from the effect of the spacing in synaptic plasticity, which appears to be a property not limited to neurons involved in learning and memory. We propose a model of spacing-dependent structural synaptic plasticity.SIGNIFICANCE STATEMENT Long-term memory (LTM) induced by repeated trials spaced over time is known as the spacing effect, a common property in the animal kingdom. Altered mechanisms in the spacing effect have been found in animal models of disorders with intellectual disability, such as Noonan syndrome. Although LTM is sustained by structural synaptic plasticity, how synapses integrate spaced stimuli and decode them into specific plastic changes remains elusive. Here, we show that the spacing effect is a phenomenon detected at the synaptic level, which determines the properties of the response in structural plasticity, including precision of such response. Whereas suppressing or enhancing Ras/mitogen-activated protein kinase signaling changed how synapses decode a pattern of stimuli, a disease-related Ras allele abolished the spacing effect for plastic changes.

MLL-ENL-mediated leukemia initiation at the interface of lymphoid commitment.

Translocations involving the mixed lineage leukemia-1 are recurrent events in acute leukemia and associate with lymphoid (ALL), myeloid (AML) or mixed lineage (MLL) subtypes. Despite an association with ALL in humans, murine MLL fusion models are persistently restricted to AML. We here explored this issue using an inducible mixed lineage leukemia-eleven nineteen leukemia (MLL-ENL) mouse model. Although multiple progenitor cell types with myeloid potential are potent AML leukemia-initiating cells, also the earliest lymphoid progenitors were capable of initiating AML. This ability to evoke a latent myeloid potential in the earliest lymphoid progenitors was lost upon further lymphoid commitment. At the same time, more downstream/committed lymphoid precursors also failed to initiate lymphoid leukemia. Co-expression of MLL-ENL with a constitutively active RAS allele, the most common co-mutation in MLL fusion leukemias, could influence on both disease latency and lineage assignment of developing leukemia in what appears to be a mutation-order-dependent manner. Finally, CEBPB-mediated transdifferentation of committed and otherwise leukemia-incompetent B-cell progenitors imbued these cells with leukemic competence for AML. Therefore, apart from providing detailed insight into the differential responsiveness of candidate target cells to a first-hit MLL fusion event, our data warrants caution to therapeutic approaches based on the concept of transdifferentiation.

Abstract IA20: Targeting hyperactive Ras signaling in acute leukemia

Abstract Ras proteins are molecular switches that cycle between active guanosine triphosphate (GTP) and inactive guanosine diphosphate (GDP)-bound conformations (Ras-GTP and Ras-GDP). Guanine nucleotide exchange factors (GNEFs) stimulate nucleotide exchange on Ras, resulting in increased Ras-GTP levels. Upon GTP binding, the switch I and switch II domains of Ras undergo a conformational change that allows them to interact productively with downstream effectors including Raf, phosphatidylinositol 3-kinase (PI3K), and Ral-GDS to activate kinase signaling cascades that regulate cell proliferation, differentiation, and survival. Mutational, biochemical, and cell biologic studies of human cancers and experiments in mouse models strongly implicate deregulated signaling through the PI3K/Akt/mTOR and Raf/MEK/ERK cascades in cancer initiation and maintenance. Ras-GTP is hydrolyzed to Ras-GDP through an intrinsic GTPase activity. This slow “off” reaction is greatly augmented by GTPase activating proteins (GAPs). Thus, the competing activities of GNEFs and GAPs regulate Ras-GTP levels in vivo. Neurofibromin, the protein encoded by NF1, and p120 GAP are the predominant Ras GAPs in mammalian cells. Codons 12, 13, and 61 of RAS genes are the most common targets of dominant mutations in human cancer, with NRAS codon 12 mutations predominating in AML. Substitutions in these residues result in constitutively elevated levels of Ras-GTP due to reduced intrinsic GTP hydrolysis and resistance to GAPs. RAS and NF1 mutations occur in pediatric patients with a spectrum of hematologic malignancies including juvenile myelomonocytic leukemia (JMML), acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL). Because oncogenic Ras proteins are exceedingly challenging biochemical targets, most recent drug discovery efforts have focused on inhibiting downstream signaling molecules such as Raf, MEK, and Akt with many compounds. We have engineered and exploited strains of mice that recapitulate endogenous expression of cancer-associated RAS alleles or NF1 inactivation to model human acute leukemias and to conduct biologic and preclinical studies. In particular, we have deployed the MOL4070LTR retrovirus as an insertional mutagen to generate genetically heterogeneous transplantable acute leuekmias characterized by hyperactive Ras signaling. This system provides a novel in vivo forward genetic strategy for introducing cooperating mutations and for generating clonal heterogeneity. Leukemia cells can be manipulated ex vivo and transplanted into congenic recipients. This experimental flexibility provides an opportunity to establish cohorts of mice that are engrafted with the same primary cancers for conducting preclinical testing and for investigating mechanisms of intrinsic and acquired drug resistance. I will discuss biologic and preclinical therapeutic studies in which we treated primary Nras, Kras, and Nf1 mutant AMLs characterized by hyperactive Ras signaling with targeted agents in vivo, isolated drug resistant clones at relapse, and compared drug sensitive and resistant clones to discover genes that modulate resistance. This latter strategy represents a potent and unbiased strategy to address mechanisms of resistance to kinase inhibitors, which has emerged as a major clinical problem in cancer therapeutics, and for investigating therapeutic strategies that might be effective in human cancers in which oncogenic RAS is a driver mutation. Our results in these robust AML models support testing drug combinations that include Raf/MEK/ERK pathway inhibitors and other targeted and conventional agents. Citation Format: Kevin M. Shannon. Targeting hyperactive Ras signaling in acute leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA20.