G12D Mutation Research Articles

AMG-510 and cisplatin combination increases antitumor effect in lung adenocarcinoma with mutation of KRAS G12C: a preclinical and translational research

BackgroundThe efficacy of monotherapy of AMG-510 is limited. This study explored whether the AMG-510 and cisplatin combination increases the anti-tumor effect in lung adenocarcinoma with the mutation of Kirsten rat sarcoma viral oncogene (KRAS) G12C.MethodsPatients’ data were used to analyze the proportion of KRAS G12C mutation. Besides, the next-generation sequencing data was used to uncover information about co-mutations. The cell viability assay, the concentration inhibiting 50% of cell viability (IC50) determination, colony formation, and cell-derived xenografts were conducted to explore the anti-tumor effect of AMG-510, Cisplatin, and their combination in vivo. The bioinformatic analysis was conducted to reveal the potential mechanism of drug combination with improved anticancer effect.ResultsThe proportion of KRAS mutation was 2.2% (11/495). In this cohort with KRAS mutation, the proportion of G12D was higher than others. Besides, KRAS G12A mutated tumors had the likelihood of concurrent serine/threonine kinase 11 (STK11) and kelch-like ECH-associated protein 1 (KEAP1) mutations. KRAS G12C and tumor protein p53 (TP53) mutations could appear at the same time. In addition, KRAS G12D mutations and C-Ros oncogene 1 (ROS1) rearrangement were likely to be present in one tumor simultaneously. When the two drugs were combined, the respective IC50 values were lower than when used alone. In addition, there was a minimum number of clones among all wells in the drug combination. In in vivo experiments, the tumor size reduction in the drug combination group was more than twice that of the single drug group (p < 0.05). The differential expression genes were enriched in the pathways of phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt) signaling and extracellular matrix (ECM) proteoglycans compared the combination group to the control group.ConclusionsThe anticancer effect of the drug combination was confirmed to be better than monotherapy in vitro and in vivo. The results of this study may provide some information for the plan of neoadjuvant therapy and the design of clinical trials for lung adenocarcinoma patients with KRAS G12C mutation.

The clinical and genomic characteristics of KRAS G12D mutated cancers.

e15151 Background: KRAS mutations are relatively common in solid tumors. It is once considered to be “non-druggable” due to its high affinity to GTP/GDP and lack of other binding sites. Recently, irreversible inhibitors targeting specific KRAS mutations have been developed. We aimed to investigate the correlation of clinical and genomic factors with KRAS G12D mutation in pancreatic cancer (PC), colorectal cancer (CRC) and non-small-cell lung cancer (NSCLC). Methods: We retrospectively reviewed the clinical and genomic data of patients with PC (n = 300), CRC (n = 300), and NSCLC (n = 1800) who provided tumor tissues and matched peripheral blood samples (served as control to eliminate germline variants) for targeted sequencing of 450 cancer-related genes. Experiments were carried out in OrigiMed’s laboratory following the CAP/CLIA standards. Tumor mutation burden (TMB)-high was defined as TMB &gt; 10 muts/Mb. Results: The prevalence of KRAS G12D mutation in PC, CRC and NSCLC was 38.3% (115/300), 15.0% (45/300) and 2.0% (36/1800), respectively. In PC and CRC, G12D was the major type of KRAS mutations, accounting for 44.9% (115/256) and 30.8% (45/146) of all the KRAS mutations, respectively. In contrast, G12C was the most common KRAS mutation in NSCLC (35.9% [79/220]), followed by G12D (16.4% [36/220]). Patients were divided into three groups according to the KRAS mutation status: the G12D (mutated in KRAS G12D), non-G12D (mutated in KRAS, but not G12D), and wild-type (WT; no KRAS mutation) groups. Clinical features including age, sex and disease stage were not associated with the KRAS grouping in PC and CRC. However, the KRAS grouping was significantly correlated with sex in NSCLC (Female vs Male: 1.9% vs 2.1% in G12D, 4.4% vs 13.9% in non-G12D, 93.7% vs 84.1% in WT, P &lt; 0.001). In PC, KRAS G12D and BRAF mutations were mutually exclusive (P &lt; 0.01), while in CRC, KRAS G12D mutation was found to be co-occurring with PIK3CA mutation (P &lt; 0.05). For NSCLC, KRAS G12D mutation was mutually exclusive with EGFR mutation (P &lt; 0.01) and co-occurred with STK11 mutation (P &lt; 0.05). TMB was comparable between the KRAS grouping in PC and CRC. In contrast, TMB was significantly higher in the non-G12D group (median 11.7 [range 0.7-89.8] muts/Mb) compared to the G12D (median 6.9 [range 0-30.9] muts/Mb; P &lt; 0.001) and WT (median 4.6 [range 0-215.2] muts/Mb; P &lt; 0.001) groups in NSCLC. A higher proportion of TMB-high was also observed in the non-G12D group (58.5%) compared to the G12D (22.2%) and WT (22.6%) groups in NSCLC. Conclusions: Patients with KRAS G12D mutation possessed unique clinical and genomic characteristics. It was more prevalent in PC and CRC and was correlated with sex in NSCLC. KRAS G12D mutation co-occurred with STK11 mutation and showed a relatively low TMB value in NSCLC, suggesting that immunotherapy might be ineffective. While KRAS non-G12D mutated NSCLC patients may be favorable to ICI. Our results provide insights into the precision medicine of KRAS G12D-mutated cancers.

Molecular characteristics of KRAS mutations in Chinese patients with cancer.

e15070 Background: Sotorasib, the first KRAS G12C inhibitor, has been approved for the treatment of non-small cell lung cancer (NSCLC) patients with KRAS G12C mutation-positive. Understanding the molecular characteristics of KRAS in Chinese patients with cancer is imperative, which is conducive to screening the beneficiary groups. Here, we focus on the molecular characterization of KRAS in NSCLC, colorectal cancer (CRC), pancreatic cancer (PC), and gastric cancer (GC). Methods: This study enrolled 17,775 pan-cancer patients detected by next-generation sequencing (NGS) from June 2021 to February 2022, in which NSCLC, CRC, PC and GC were 48.67% (8651/17775), 10.87% (1933/17775), 1.92% (342/17775) and 5.37% (955/17775), respectively. Results: The KRAS mutation rate in pan-cancer is 16.28% (2893/17775). In NSCLC, CRC, PC and GC cohorts, the mutation rates of KRAS were 13.28% (1149/8651), 50.34% (973/1933), 66.67% (228/342) and 7.64% (73/955), respectively. In NSCLC cohort, 1274 KRAS mutations were detected, and the subtypes were concentrated in G12C (346;27.16%), G12D (205;16.10%), G12V (194;15.23%), G12A (86;6.75) and Q61H (57;4.47%). In CRC cohort, 1094 KRAS mutations were detected. The subtypes were mainly distributed in G12D (350;31.40%), G13D (184;16.82%), G12V (180;16.45%), A146P (66;6.03%) and G12C (49;4.48%). In PC cohort, 251 KRAS mutations were detected, and the subtypes were concentrated in G12D (109;43.43%), G12V (70;27.89%), G12R (32;12.75%), G12C (9;3.59%) and Q61H (9;3.59%). In GC cohort, 82 KRAS mutations were detected. The subtypes of these mutations were concentrated in G12D (23;28.05%), G13D (12;14.63%), Q61H (7;8.54%), G12V (5;6.10%) and Q61R (5;6.10%). Conclusions: In NSCLC, CRC, PC and GC cohorts, K RAS mutations were concentrated in exon 2, accounting for 78.41% (999/1274), 81.63% (893/1094), 91.63% (230/251) and 64.63% (53/82), respectively. In NSCLC cohort, KRAS G12C was the molecular subtype with the highest mutation rate. However, in other cohorts, KRAS G12D was the predominant molecular subtype. G12D mutation will become the next research hotspot, and the breakthrough will help to further expand the beneficiary population of KRAS mutations.

Genomic characterization of somatic mutations by race and ethnicity in pancreatic cancer defined through AACR project GENIE.

4138 Background: KRAS, TP53, SMAD4, and CDKN2A are widely recognized as the most common somatic mutations amongst patients with pancreatic ductal adenocarcinoma (PDAC); however, previous genomic studies have disproportionately included non-Hispanic White (NHW) patients with little to no inclusion of racial and ethnic minorities, particularly non-Hispanic Black (NHB) and Hispanic patients. Additionally, little is known about the distribution of KRAS point mutations in PDAC in specific racial and ethnic groups. Here, we describe somatic mutation differences in a larger and more racially and ethnically representative PDAC cohort than previously characterized. Methods: PDAC mutational data was downloaded from AACR Project GENIE (Genomics Evidence Neoplasia Information Exchange) v13.0, an international data registry of sequenced tumor samples from nearly 150,000 patients, on cBioPortal. PDAC cancer type was identified using OncoTree code. NHW, NHB, Hispanic (of any race), and Asian patients were then selected. Mutational frequency was calculated for all mutations. The primary objective was to compare the mutation frequencies between NHW and other races with respect to the most common mutations. To maximize power, we included any mutation with a frequency ≥5.0% where testing for mutation of that gene had been performed in at least 150 patients. Specific point mutations in KRAS were also extracted with stratification for racial and ethnic group. Fisher’s Exact Test was used to calculate the statistical significance of the differences in mutation frequency between NHW and minority groups. Results: Patients in the PDAC cohort included 5,292 individuals (NHW N = 4296, 80.7%; NHB N = 264, 5.0%; Hispanic N = 460, 8.7%; Asian N = 299, 5.7%). As compared to the NHW group, NHB patients had higher rates of TP53 (78.7% vs 69.0%, P = 0.0006) and PTPRT (5.3% vs 1.8%, P = 0.0095) mutations but fewer GNAS (0.4% vs 2.8%, P = 0.0097) mutations while Asian patients had higher frequency of ARID1A mutations (13.0% vs 8.8%, P = 0.024). KRAS G12D mutation was more prevalent in Hispanic patients (47.0% Hispanic vs 41.1% NHW, P = 0.0238), although rate of KRAS mutation overall was the same. Prevalence of KRAS G12C mutation was equivalent amongst all groups with an overall 1.6% frequency. Conclusions: Understanding the genomic landscape of PDAC is critical as we move towards increased use of targeted therapies to treat this disease. In this study, TP53, PTPRT, GNAS, and ARID1A mutations were shown to occur at different frequencies in specific racial and ethnic groups. G12D mutation of KRAS was disproportionately increased in Hispanics. These differences in molecular landscape amongst racial and ethnic groups could contribute to precision medicine strategies used to address this deadly disease.

Not all treated KRAS-mutant pancreatic adenocarcinomas are equal: KRAS G12D and survival outcome.

4020 Background: KRAS is an oncogenic driver in pancreatic ductal adenocarcinoma (PDAC) with mutations identified in &gt; 90% of cases. G12D is the most frequent variant, followed by G12V and G12R. We recently reported on the prognostic impact of distinct KRAS mutations. The current study utilized a large clinical and genomic database, to further explore and characterize the prognostic and molecular differences between KRAS variants, focusing on KRAS G12D and G12R. Methods: PDAC samples were tested using whole transcriptome sequencing (WTS; Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) at Caris Life Sciences (Phoenix, AZ). Transcriptomic signatures including MPAS (MAPK activation score), T-cell inflamed score and tumor micro environment (TME) characterization were calculated on WTS data. Significance was determined by X2 and Fisher-Exact and p-value was adjusted for multiple comparisons (q). Real-world overall survival (rwOS) obtained from insurance claims data was calculated from tissue collection to last contact (comparison done by Kaplan-Meier test). Results: 5,555 PDAC patients harboring either KRAS G12D (n = 2,671), G12V (n=1,871) G12R (n = 904) or G12C (n = 109) variants were identified. Patients with KRAS G12R mutant tumors had significantly longer OS compared to G12D (396 vs 311 days, HR 0.81, CI 0.74-0.88, p&lt;0.0001). There was no difference among KRAS variants in the rate of TP53, CDKN2A and SMAD4 mutations. ARID1A and KMT2D were more frequently mutated in KRAS G12D vs. G12R. The MPAS gene signature reflecting MAPK pathway activation trended lower in G12R vs. G12D. Expression of multiple genes in this pathway was statistically lower in the G12R cohort. Immune profiling suggested that: PDL1 expression is significantly lower in G12R vs G12D (13% vs 19%), TMB-H and dMMR were comparable in G12D vs G12R and several glucose and glutamine metabolism genes were significantly lower in expression when comparing G12R vs G12D - table. OS was improved within the G12R cohort for those patients on metformin (n=273 patients) (416 vs 388 days, HR 0.84, CI 0.72-0.99, p =0.037) while no difference was seen in those with KRAS G12D based on metformin use. Conclusions: Patients with G12D mutations have significantly lower survival compared to G12R. Significant molecular differences were seen in MAPK pathway gene expression, markers of immune activation, and genes involved in glucose and glutamine metabolism. Intriguingly, metformin use appeared to impact survival in the KRAS G12R subgroup. We aim to further explore distinct vulnerabilities based on MAPK pathway activation and dysregulated metabolism. Based on this data, future studies should address the KRAS mutation status and explore distinct therapeutic vulnerabilities. [Table: see text]

KRAS G12C-mutated pancreatic cancer: Clinical outcomes based on chemotherapeutic regimen.

4150 Background: Frontline treatment for advanced pancreatic ductal adenocarcinoma (PDAC) has been either 5-fluorouracil, oxaliplatin and irinotecan (FOLFIRINOX) or gemcitabine and nab-paclitaxel (GP) for the past decade. While the NAPOLI-3 trial, utilizing liposomal irinotecan, highlighted the superiority of a triplet regimen over GP, the question remains whether certain subgroups may derive particular benefit from GP. Pre-clinical data in lung cancer suggest that KRAS G12C may facilitate enhanced DNA adduct removal after platinum chemotherapy and confer resistance to this drug class. While multiple KRAS G12C inhibitors have shown early promise in PDAC, multi-agent chemotherapy remains the frontline standard and will likely remain an important therapeutic tool. This study aimed to investigate clinical outcomes after platinum and non-platinum-based chemotherapy in patients with advanced KRAS G12C-mutated PDAC. Methods: PDAC samples were tested using whole transcriptome sequencing (WTS; Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) at Caris Life Sciences (Phoenix, AZ). Significance was determined by X2 and Fisher-Exact and p adjusted for multiple comparisons (q). Real-world overall survival (rwOS) was obtained from insurance claims data and calculated from first of treatment to last contact with comparison done by Kaplan-Meier test. Results: A total of 5,555 PDAC patients harboring KRAS pathogenic variants were identified, including 109 with a G12C mutation. For KRAS G12C mutants, median overall survival was 470 days for those who received GP (n=22) compared to 240 days for patients who received FOLFIRINOX (n=9) with a median difference of 230 days (HR 0.32, CI 0.12-0.82, p=0.013). In contrast, median overall survival was higher for patients harboring G12D (392 vs 297 days, p=0.003), G12V (500 vs 356 days, p=0.012) or G12R (469 vs 391 days, p=0.095) mutations who received FOLFIRINOX compared to GP. KRAS G12C mutated PDAC showed the highest rate of PDL1+ staining (27%) compared to other variants (G12R 13% and G12D 19%, q&lt;0.01). TMB-H and MSI-H prevalence were highest in G12C compared to other variants (no statistical significance observed). Conclusions: In patients with advanced PDAC and a G12C mutation, median overall survival appears significantly longer in those treated with GP compared to FOLFIRINOX. The opposite trend was seen in patients with other KRAS variants including G12D, G12V, and G12R, consistent with the recently presented NAPOLI-3 trial. PDL1 staining was also highest in the KRAS G12C cohort. While this is the largest reported analysis of outcomes to frontline chemotherapy in KRAS G12C-mutated PDAC, the sample size is small and needs validation in additional datasets. Next steps include evaluating DNA mutational status and RNA expression of genes involved in DNA repair in G12C vs other KRAS variants to better understand the outcome data presented here.

Impact of novel pan-RAS inhibitors on efficacy and resistance to AMG-510 and MRTX-1133 in pancreatic cancer cell lines.

4155 Background: Mutant-activated RAS genes are the most frequently mutated gene family associated with cancer (almost 30% of all cancers contain a mutant RAS gene). KRAS is the predominant isoform mutated in cancer and is the isoform exclusively mutated in pancreatic ductal carcinoma (PDAC). Since almost all PDAC cases harbor a mutant RAS, it is arguably the most RAS-addicted tumor type. There is now considerable evidence implicating mutant KRAS as a driver of PDAC. Recently, several mutant KRAS-targeted therapies (sotorasib and adagrasib) have been developed and show promise in PDAC patients. We have developed a direct inhibitor of RAS with a predicted unique interaction region capable of directly binding to wild-type H- and K-RAS, but which shows preferential binding for KRAS G12D and G12C mutants. This novel inhibitor disrupts the RAS effector domain and blocks the ability of RAS to signal through its effectors. Methods: We used in silico virtual library screening to identify an initial candidate inhibitor which was effective at inhibiting the 3D growth of PDAC cells without affecting their growth in 2D. Subsequent iterative rounds of medicinal chemistry was then performed to identify a series of derivatives with enhanced activity, as determined by 3D growth inhibition assays and effects on Ras signaling, as determined by Western blot analysis of phosphor-ERK, phosphor-Akt and activation of Ral A. Results: Our series of RAS inhibitors effectively block PDAC cell growth in 3D without impacting their 2D proliferation and suppress the interaction of KRAS with its effector cRAF. The inhibitors also effectively inhibit RAS signaling in mutant RAS containing PDAC cells. Since our novel compounds are predicted to bind to RAS at a different site to either AMG-510 (G12C specific inhibitor) and MRTX-1133 (G12D specific inhibitor), we tested the co-operativity of our compounds with these existing agents. Our compounds enhanced the anti-proliferative effects of both MRTX-113 and AMG-510 in mutant KRAS G12D and G12C PDAC cells, respectively. Conclusions: We have developed a series of novel RAS inhibitors that directly bind preferentially to mutant KRAS that may serve as new mutant KRAS-targeted therapeutics, and that may also have the potential to enhance the efficacy or suppress the resistance of AMG-510 and MRTX-1133.

AMPLIFY-201, a first-in-human safety and efficacy trial of adjuvant ELI-002 2P immunotherapy for patients with high-relapse risk with KRAS G12D- or G12R-mutated pancreatic and colorectal cancer.

2528 Background: RAS mutations occur in 25% of solid tumors with G12D being the most frequent variant. ELI-002 2P is a vaccine comprised of lymph-node targeted Amphiphile (Amph)-modified G12D and G12R mutant KRAS peptides with an Amph-modified CpG oligonucleotide adjuvant designed to expand polyfunctional mutant KRAS-specific T cells. Preclinical amphiphile data (relative to non-amphiphile controls) showed increased immunogenicity, tumor clearance and survival in mouse models. Methods: This first-in-human multicenter phase I trial assessed safety, immunogenicity and antitumor activity using a novel adjuvant trial design in patients (pts) with minimal residual disease (MRD) following standard locoregional treatment. Eligibility: elevated circulating tumor DNA (ctDNA) and/or serum tumor biomarker elevation (CA19-9/CEA), and KRAS/NRAS mutation. In a 3+3 dose escalation, n=22 received a subcutaneous course of 6 prime and 4 booster injections of fixed dose Amph-peptides (1.4 mg), admixed with escalating Amph-CpG-7909 (Table). Dose escalation based on observed safety in first 28 days. Safety, antitumor activity including biomarker reduction/clearance and relapse free survival using immune Response Evaluation Criteria in Solid Tumors (iRECIST) were assessed. Immunogenicity assessed by direct ex vivo Fluorospot and intracellular cytokine staining of peripheral blood mononuclear cells (T-cell responders = ≥ 2-fold increase over baseline) and by tumor CD3 immunohistochemistry in subset who underwent a biopsy. Results: No dose-limiting toxicities, treatment related SAEs or cytokine release syndrome were observed, and no maximum tolerated dose was identified. Safety: all grade 1, fatigue (19%), headache (19%), and injection site reaction (9.5%). Biomarker reduction was observed in 15/19 (79%) and in 5/5 at highest doses; clearance of MRD was observed in 4/19 (21% - n=2 pancreas, n=2 colorectal). Polyfunctional mKRAS-specific T cell responses observed in 80% of pts (n=12/15), with both CD8+ and CD4+ T cell responses for most, and CD3+ T cells observed on biopsies. The recommended phase 2 dose (RP2D) is 10.0 mg Amph-CpG-7909. Conclusions: ELI-002 2P, evaluated in a novel MRD+ trial, was safe with ctDNA and serum tumor biomarker reduction and clearance and notable immune responses. The RP2D is the start dose for a phase 1/randomized phase 2 study evaluating a new seven peptide formulation for G12 D, R, V, S, A, C, and G13D mutated solid tumors (NCT05726864). Clinical trial information: NCT04853017 . [Table: see text]

Liquid and tissue profiling of targetable co-mutations with KRAS in non-small cell lung cancer.

e21201 Background: The primary objective of this study was to understand the co-occurrence of KRAS with other actionable drivers in Indian lung cancers patients, and to assess the utility of liquid biopsy in this cohort. Methods: We evaluated single nucleotide alterations, copy number alterations, and fusions in 507 tumor tissue and 1271 blood specimens with targeted NGS panels in NSCLC patients. Results: In tissue-based analysis (n = 507), KRAS mutations were detected in 68 (13.4%) samples. Of these, 63 (92.6%) samples did not show the presence of any targetable co-mutations. Targetable co-mutations were detected in 5 (7.4%), with concomitant EGFR mutations in all 5 samples(100%) and BRAF mutations in 2 samples (33.3%) In the liquid biopsy (n = 1271) analysis, KRAS mutations were observed in 94 (7.4%) samples. The liquid biopsy analysis showed higher presence of targetable co-mutations in 11.7% (n = 11). EGFR was the most common co-occurring targetable gene observed in 9 samples (81.8%). Mutations in BRAF were detected in 3 (27.3%) samples. ERBB2 Exon 20 insertion was detected in 1 sample (9.1%). No ALK, RET or ROS1 fusions were seen in our KRAS positive cohort. In the tissue cohort, one newly diagnosed adenocarcinoma case showed primary co-mutations in EGFR (Exon 19del VAF 76.8%) and KRAS (G13D VAF 10.5%), impacting utility of EGFR TKI as the first line therapy. Another EGFR Exon 19 del patient developed KRAS G12D mutation as an acquired resistance mutation post Osimertinib therapy. In the liquid biopsy cohort (n = 11), the KRAS positive cohort had four newly diagnosed patients with 3 having EGFR and 1 with BRAF co-mutation. Among the patients with prior therapy, one patient at presentations had EGFR Exon 19 deletion and developed EGFR T790M at progression on Afatinib. The baseline tissue did not have coexisting KRAS mutation. Fresh tissue biopsy was performed at the progression on Osimertinib. The noteworthy finding was the KRAS G12C (VAF 0.36%) presence in liquid biopsy but absences in the simultaneous fresh tissue sample. The EGFR Exon 19 was present at lesser frequency than KRAS in the liquid (VAF 0.23%). In the patients from liquid biopsy cohort with Osimertinib therapy (n = 4), all presented with KRAS as acquired resistance mutation. One among these also showed concomitant EGFR C797S mutation. Conclusions: KRAS mutation virtually makes it unlikely for other targetable mutations to coexist. It makes it thus compelling to further study the role of liquid biopsy with KRAS first and decide if chemotherapy with IO can be started considering the standard of care is to wait for the entire targeted panel before initiating IO. Equally its important to include KRAS in the lung panel and although small the concomitant KRAS may compromise the efficacy to TKI. Liquid biopsy is a promising alternative for detection of actionable alterations, including KRAS and can be harnessed for longitudinal profiling, monitoring tumor evolution and treatment outcome.

Combination of 5-FU plus KRAS G12D inhibitor MRTX1133 against human colorectal and pancreatic cancer cells and the affects on inhibition of pERK and immune-stimulatory cytokine patterns in in KRAS G12D and KRAS G12V tumor cells.

e16301 Background: Colorectal cancer (CRC) &amp; pancreatic cancer are among deadliest cancers with great disease burden. The most commonly mutated oncogenic alteration in human cancers is KRAS, which is prevalent in pancreatic, colorectal malignancies. KRAS is considered an especially difficult, if not "undruggable" target. The KRAS G12D is present in more than 40% of PDAC &amp; 34.2% of CRC cases. KRAS mutation subtypes are associated with different downstream pathways and varied clinical outcomes in cancer. MRTX1133 has been identified as a noncovalent, potent, and selective inhibitor of KRASG12D which has been shown to suppress KRASG12D signaling in cells and in vivo. We hypothesized that combination of MRTX1133 with 5-FU will be more effective at slowing or stopping tumor growth, and prolonging overall survival time for patients while allowing lower dose 5-FU with less side effects. Methods: We investigated synergistic effects on pERK inhibition and changes in immune tumor killing with 5-FU plus KRAS G12D inhibitor MRTX1133 in pancreatic and colon cancer cell lines. Human CRC (N=6) and pancreatic (N=4) cancer cell lines with different KRAS mutations were used in experiments evaluating drug effects on pERK at 6 and 24 hours. We evaluated changes at 48 hours in cytokine profiles in treated cells using a custom panel of 62 cytokines, chemokines, and growth factors associated with tumor growth, immune stimulation or immune suppression. Results: Colorectal and pancreatic cancer cell lines had IC50 sensitivities ranging from 7 to 12 microM 5-FU, and &gt;100 nM to &gt;5 microM for MRTX1133 (G12D N=4: LS513, 120 nM; HPAF-II, 1.8 microM; SNUC2B, 5.7 microM; PANC-1, 2.8 microM). For non-G12D, the range of IC50 for MRTX1133 was in the range of 2 to 9 microM for CRC lines with G12V, G13D, or wild- type KRAS (N=7). Synergies between 5-FU and MRTX1133 were observed regardless of presence of KRAS G12D mutation with combination indices of &lt;0.5 indicating strong synergy. pERK was suppressed by MRTX1133 treatment of tumor cells regardless of KRAS G12D mutation. IL8/CXCL8, MICA, Angiopoietin 2, VEGF and TNF-alpha were reduced while IL-18/IL-1F4 increased. Conclusions: Our studies reveal strong synergy between MRTX1133 &amp; 5-FU in human pancreatic &amp; colon cancer models at much lower than IC50 dosage which is important for avoiding side effects. This is the first description that effect of KRAS G12D inhibitor MRTX1133 is active on KRAS G12V. The surprising synergy in KRAS G12V samples with combination therapy and the important synergistic change in cytokine patterns suggests potential strong immune stimulatory anti-cancer effects of MRTX1133 &amp; 5FU against mCRC and pancreatic cancer cells regardless of KRAS G12D mutation which should be considered when including patients with respective mutations in clinical trials.

Abstract B002: Development of bifunctional CRBN-SOS1 degraders for treatment of mutant KRAS cancers

Abstract Although oncogenic Kirsten rat sarcoma virus (KRAS) was once deemed undruggable, recent advances have revealed promising anti-tumor effects with pan-KRAS and KRAS-mutant specific inhibitors in both preclinical and clinical settings. Despite the success of these inhibitors, the diversity of tumors as well as tumor sensitivity results in acquired drug resistance suggesting that combinational therapeutic approaches would be necessary to avoid this problem. An attractive combination strategy would be to couple KRAS inhibition with a blockade of its guanine exchange factor, Son of Sevenless (SOS1). This would keep KRAS in its inactive state as well as mitigate upstream MAPK pathway reactivation notoriously associated with inhibition in this pathway. Utilizing our PRODEGY platform, we developed bifunctional SOS1 degraders for the treatment of patients with KRAS mutant cancers. These degraders have shown rapid and potent CRBN-mediated degradation which exhibit DC50s &amp;lt;10nM in a H358 SOS1-HiBiT cell line. Our high-throughput HiBiT data translated into effective SOS1 degradation in KRAS mutant cells harboring G12C, G12D, G12V, G12S, or G13D mutations. SOS1 degradation was validated to be dependent on the proteasome and CRBN as cotreatment with proteasomal inhibitors (MG132 or MLN4924) or treatment in a CRBN KO cells abolished degradation. Treatment of our SOS1 degraders resulted in the suppression of multiple signaling markers downstream of SOS1. Given its role in RAS activation, we observed a reduction in active KRAS, HRAS and MRAS protein upon treatment of SOS1 degraders in KRAS G12C MIA PaCa-2 cells. Furthermore, pERK and pS6 levels were decreased in a more potent or comparable manner compared to the SOS1 inhibitor, BI3406, in multiple KRAS mutant cell lines. Out competing SOS1 degraders with other CRBN binders highlighted that inhibition of pERK and pS6 was due to CRBN-mediated degradation. To determine functional activity of our SOS1 degraders, we developed and optimized 3D proliferation assays in multiple KRAS mutant (G12C, G12V, G12S, G13D) cell lines which yielded IC50s ranging from 0.5 – 70nM. Similarly, 100- to 300-fold IC50 value shifts were observed in SOS1 KO and CRBN KO H358 cell lines validating that the anti-proliferative activity of SOS1 degraders is specific to its targets. Treatment of SOS1 degraders in KRAS-mutant xenograft models resulted in &amp;gt;90% degradation of SOS1 in tumors and subsequently led to significant tumor growth inhibition as a single agent. Combination treatment of the KRAS G12C inhibitor AMG510 and SOS1 degraders produced strong synergistic antiproliferative effects in H358 and MIA PaCa-2 cell lines. Additionally, synergy was observed with SOS1 degraders and the EGFR inhibitor, Erlotinib, as well as the MEK inhibitor, Trametinib, in KRAS G13D mutant LoVo cells. The ability of our SOS1 degraders to have potency alone as well as in combination with multiple types of different RTK/RAS/MAPK pathway inhibitor highlights the value of SOS1 degraders as a potential therapeutic option for a range of KRAS mutant cancers. Citation Format: Kyle Begovich, Angela Schoolmeesters, Navin Rajapakse, Maneesh Kumar, Elena Martinez-Terroba, Arvind Shakya, Brandon Whitefield, Nataraj Pagadala, Aparajita Chourasia, Leah Fung. Development of bifunctional CRBN-SOS1 degraders for treatment of mutant KRAS cancers [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 B002.

Abstract A033: KRAS codon 12 oncogenic mutations modulate protein conformation within the Switch II/Helix3 pocket

Abstract Up to 30% of cancers harbor activating mutations in one of three RAS genes, HRAS, NRAS, or KRAS, that drive uncontrolled cellular proliferation, alter cellular metabolism, movement, differentiation, and promote cell survival and tumor immune evasion. The frequency of mutation varies across alleles and specific substitutions, with KRAS mutations occurring in roughly 81% of patients, and KRAS G12D, KRAS G12V, and KRAS G12C substitutions constituting 33%, 23%, and 12% of these cases, respectively. Cancer types exhibit characteristic preferences for RAS substitutions, the reason for which is still unknown. Oncogenic amino acid substitutions have been found to endow subtle differences in biochemical properties which cannot be explained by existing X-ray structural models. In addition, it is unclear whether these translate to distinctions in downstream signaling potential. We have initiated this study with the overall goal of understanding the effect of oncogenic mutations on the structure and dynamic properties of KRAS4b proteins in solution, and to identify any ensuing differences in effector binding behavior. Here, we present the data that identifies the structural differences between the common mutants KRAS G12D and KRAS G12C and the wildtype protein in the active conformation using solution-state NMR and Molecular Dynamics (MD) simulations, that could likely be coupled with the differing capacities to engage downstream effectors. Chemical shift perturbation (CSP) analysis demonstrates that oncogenic mutations in codon 12 of KRAS4b induce significant conformational differences when compared to the wildtype protein. These observations are localized to the Switch 2 region. We validate these observations in three-dimensional structures and reveal critical differences that are conferred by these oncogenic mutants. The solved three-dimensional solution structures of KRAS G12C/T35S/C118S-GppNHp and KRAS G12D/T35S/C118S-GppNHp reveal remarkable conformational differences within the Switch II regions, with the G12D-mutant protein adopting a relatively more flexible Switch II conformation than the G12C-mutant. Our MD simulations at a 2 µs timescale support these observations. Furthermore, we observed downstream signaling differences between KRAS G12C and KRAS G12D mutants in several cell line systems. If observed structural differences between KRAS mutants are found to directly translate to effector binding affinity, it would provide critical insight into why cancer types display preferences for RAS substitutions, and would inform RAS-targeted therapeutics. Citation Format: Brian Smith, Alok K. Sharma, Roger Ma, Megan Rigby, Vandana Kumari, Dominic Esposito, Dwight Nissley, Frank McCormick, Anna E. Maciag. KRAS codon 12 oncogenic mutations modulate protein conformation within the Switch II/Helix3 pocket [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 A033.

Abstract B013: Exploring the switch II pocket of KRAS(G12D) with mutant-selective monobody inhibitors

Abstract The G12D mutation is among the most common KRAS mutations associated with cancer, in particular pancreatic cancer. Here, we have developed monobodies, small synthetic binding proteins, that are highly selective to KRAS(G12D) over KRAS(wild type) and other oncogenic KRAS mutations, as well as over HRAS(G12D). Crystallographic studies revealed that, similar to other KRAS mutant-selective inhibitors, the initial monobody bound to the S-II pocket, the groove between switch II and alpha 3 helix. Strikingly, the monobody captured a shape of the S-II pocket distinct from previously reported structures. Unlike other G12D-selective polypeptides reported to date, the monobody used its backbone NH group to directly recognize the side chain of RAS Asp12. This feature closely resembles that of a small-molecule KRAS(G12D) inhibitor, MRTX1133. The monobody also directly interacted with H95, a residue not conserved in HRAS. These features rationalize the high selectivity toward the G12D mutant and the KRAS isoform. Structure-guided affinity maturation resulted in monobodies with low nM KD values. Deep mutational scanning of a G12D-specific monobody generated hundreds of functional and nonfunctional single-point mutants, from which we identified crucial residues for binding and those that contributed to the selectivity toward the GTP- and GDP-bound states. When expressed in cells as genetically encoded reagents, these monobodies engaged selectively with KRAS(G12D) and inhibited KRAS(G12D)-mediated signaling and tumorigenesis. These results demonstrate the feasibility of targeting KRAS(G12D) functions using a mutant selective inhibitor and will guide the development of next-generation KRAS(G12D)-selective inhibitors. Citation Format: Takamitsu Hattori, Padma Akkapeddi, Imran Khan, Akiko Koide, Eliezra Glasser, Gayatri Ketavarapu, Michael Whaby, Mariyam Zuberi, Kai Wen Teng, Julia Lefler, Lorenzo Maso, Injin Bang, Michael C. Ostrowski, John P. O’Bryan, Shohei Koide. Exploring the switch II pocket of KRAS(G12D) with mutant-selective monobody inhibitors [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 B013.

Abstract A037: Oncogenic RAS signals from lysosomes to activate mTORC1 in multiple myeloma

Abstract Oncogenic mutations of both NRAS and KRAS are common in multiple myeloma (MM), an incurable malignancy of plasma cells. However, the mechanisms of pathogenic RAS signaling in MM have remained enigmatic, and there is little evidence to support a dominant role for classical MAPK signaling downstream of RAS in this disease. We recently used an unbiased proteogenomic approach to dissect RAS signaling in MM and discover that mutant isoforms of RAS organized a signaling complex with the amino acid transporter, SLC3A2, and MTOR on lysosomes, which directly activated mTORC1 by co-opting amino acid sensing pathways (PMID: 36115844). To gain further insights into this novel mode of RAS signaling located away from the plasma membrane, we have employed mass spectrometry to characterize proteins associated with lysosomes isolated from MM cell lines. We observed that RAS isoforms, SLC3A2 and several components of the mTORC1 signalosome were purified with lysosomes, consistent with RAS activation of mTORC1 on lysosomes. To identify novel regulators of oncogenic RAS signaling at the lysosome, we used phospho-proteomics to identify proteins with significantly reduced levels of phosphorylation following acute inhibition of KRAS activity with the KRAS G12D inhibitor, MRTX1133, in a MM cell line bearing a KRAS G12D mutation. Many of these phospho-proteins were also associated with lysosomes, including MTOR, which had substantially reduced phosphorylation at the amino-acid dependent phosphorylation site S1261 following KRAS inhibition. Pathway analysis of proteins present in lysosomes and phosphorylated downstream of oncogenic KRAS showed enrichment for proteins that regulate intracellular trafficking. Among these proteins, we found that disruption of a RAB GAP essential for survival in RAS-dependent MM cells substantially reduced associations between RAS and MTOR and nearly abolished RAS-dependent mTORC1 activity. These data suggest that mutant RAS utilizes lysosomal resident proteins to coordinate the colocalization of itself with mTORC1 on lysosomes to drive pathogenic signaling in MM. More generally, these studies suggest that targeting the intracellular localization of RAS may be an alternative route to inhibit oncogenic RAS signaling in MM. Citation Format: Arnold Bolomsky, Ryan M. Young. Oncogenic RAS signals from lysosomes to activate mTORC1 in multiple myeloma [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 A037.

Abstract PR09: KRAS-targeted PROTAC degraders are broadly efficacious against KRAS-dependent tumor models

Abstract KRAS is genomically altered in about one third of all human tumors. Due to its central role in oncogenesis, many attempts have been made in the last four decades to drug mutant KRAS, either directly or indirectly. Despite recent advances in targeting KRAS using small molecule inhibitors, the majority of KRAS alterations do not yet have an existing targeted therapy, and where inhibitors are available, resistance rapidly emerges. Thus, novel approaches to drugging KRAS are needed. Eliminating mutant KRAS using a targeted protein degradation approach may lead to superior efficacy relative to inhibiting the protein. KRAS PROTAC® degraders that selectively target the G12D mutant form of KRAS were identified and profiled in KRAS-dependent cancer models. In vitro, PROTAC degraders targeting the G12D mutant degrade KRAS with picomolar potency, robustly suppress MAPK and PI3K/AKT signaling, induce apoptosis, and have antiproliferative activity that is superior to known inhibitors. These molecules are selective for mutant KRAS G12D, neither degrading wild-type KRAS nor the related isoforms HRAS and NRAS. In vivo, these degraders can eliminate &amp;gt;95% of mutant KRAS from relevant xenograft models, induce apoptosis, and lead to tumor regression. Consistent with the extended pharmacodynamics often observed with PROTAC degraders, a single dose of a G12D PROTAC results in prolonged KRAS degradation and significant pERK suppression up to one week after administration. Combined, these data show that degrading mutant KRAS G12D in tumors is highly efficacious and may have advantages over inhibition, making it an exciting potential new approach for the treatment of KRAS mutant cancers. Citation Format: Kathryn Smith, Andrea Lopez-Arroyo, Jason Berk, Peter Hegan, Peter Nower, Samantha Tice, Aurelie Moutran, Jennifer Pizzano, Amanda Dowtin, Mark Bookbinder, Elizabeth Bortolon, Greg Cadelina, Fazlul Karim, Katie Digianantonio, Miklos Bekes, Jesus Medina. KRAS-targeted PROTAC degraders are broadly efficacious against KRAS-dependent tumor models [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 PR09.

Specific human leukocyte antigen class I genotypes predict prognosis in resected pancreatic adenocarcinoma: a retrospective cohort study.

Patients with resected pancreatic adenocarcinoma (PAAD) often experience short-term relapse and dismal survival, suggesting an urgent need to develop predictive and/or prognostic biomarkers for these populations. Given the potential associations of the human leukocyte antigen class I ( HLA -I) genotype with oncogenic mutational profile and immunotherapy efficacy, we aimed to assess whether differential HLA -I genotype could predict the postoperative outcomes in resected PAAD patients. HLA -I ( A , B , and C ) genotyping and somatic variants of 608 Chinese PAAD patients were determined by targeted next-generation sequencing of matched blood cells and tumor tissues. HLA - A / B alleles were classified with the available definition of 12 supertypes. The Kaplan-Meier curves of disease-free survival (DFS) and multivariable Cox proportional-hazards regression analyses were performed to determine the survival difference in 226 selected patients with radical resection. Early-stage (I-II) patients constituted the majority (82%, 185/226) and some stage I-II individuals with high-quality tumor samples were analyzed by RNA-sequencing to examine immunophenotypes. Patients with HLA-A02 + B62 + B44 - had significantly shorter DFS (median, 239 vs. 410 days; hazard ratio=1.65, P =0.0189) than patients without this genotype. Notably, stage I-II patients carrying HLA-A02 + B62 + B44 - had sharply shorter DFS than those without HLA-A02 + B62 + B44 - (median, 237 vs. 427 days; hazard ratio=1.85, P =0.007). Multivariate analysis revealed that HLA-A02 + B62 + B44 - was associated with significantly inferior DFS ( P =0.014) in stage I-II patients but not in stage III patients. Mechanistically, HLA-A02 + B62 + B44 - patients were associated with a high rate of KRAS G12D and TP53 mutations, lower HLA-A expression, and less inflamed T-cell infiltration. The current results suggest that a specific combination of germline HLA-A02/B62/B44 supertype, HLA-A02 + B62 + B44 - , was a potential predictor for DFS in early-stage PAAD patients after surgery.

Abstract 1072: Combination of 5-Fluorouracil or ONC212 plus KRAS G12D inhibitor MRTX1133 against colorectal and pancreatic cancer cells results in immune-stimulatory cytokine patterns and unexpected synergies independent of G12D mutation

Abstract BACKGROUND: Advanced metastatic colorectal cancer (mCRC) has a 14% 5-year survival rate with little progress made for microsatellite stable tumors. KRAS mutations occur in about 40-50% of mCRC and are associated with more aggressive drug resistance and lack of response to anti-EGFR therapies. Recent advances have led to small molecules targeting KRAS G12C that have been undergoing clinical testing in lung, mCRC, and other tumor types. ONC212 is a fluorinated imipridone with strong anti-cancer activity in nM range and preclinical efficacy against pancreatic and other malignancies. Through extensive structure-based drug design, MRTX1133 was identified as a noncovalent, potent, and selective inhibitor of KRASG12D. MRTX1133 suppresses KRASG12D signaling in cells and in vivo, MRTX1133 binds to the switch II pocket and inhibits the protein-protein interactions necessary for activation of the KRAS pathway. MATERIALS &amp; METHODS: We investigated cell viability and drug synergies of 5-FU or ONC212 plus KRAS G12D inhibitor MRTX1133 in 6 human CRC and 4 human pancreatic cancer cell lines at 72 hours. Experiments evaluated drug effects on pERK using western blot at 6 and 24 hours. We evaluated changes at 48 hours in cytokine profiles in treated cells using a custom panel of 62 cytokines, chemokines, and growth factors associated with tumor growth, immune stimulation or immune suppression. RESULTS: Colorectal and pancreatic cancer cell lines had IC50 sensitivities ranging from 7 to 12 microM for 5-FU 0.2-0.8 microM for ONC212, &amp;gt;100 nM to &amp;gt;5,000 nM for MRTX1133 (G12D N=4: LS513 &amp;gt;100, HPAF-II &amp;gt;1,000, SNUC2B &amp;gt;5,000, PANC-1 &amp;gt;5,000). For non-G12D, the range of IC50 for MRTX1133 was &amp;gt;1,000 to &amp;gt;5,000 nM for CRC lines with G12V, G13D, or WT KRAS (N=7). Synergies between MRTX1133 and either 5-FU or ONC212 were observed across cell lines regardless of the presence of KRAS G12D mutation with combination indices of &amp;lt;0.5 indicating strong synergy. The observed synergy was greater with the combination of MRTX1133 and ONC212 compared to the synergy with 5-FU. Among the cytokine alterations, IL8/CXCL8, MICA, Angiopoietin 2, VEGF and TNF-alpha were reduced while IL-18/IL-1F4 increased with all treatments. CONCLUSIONS: Our ongoing studies reveal the potential activity of MRTX1133 against mCRC and pancreatic cancer cells regardless of KRAS G12D mutation and synergies were observed with 5-FU and ONC212 regardless of KRAS G12D mutation. Immune stimulatory cytokine profiles were observed with 5-FU, MRTX1133 and combination. The results suggest that KRAS G12D, KRAS G13D and WT KRAS should not be excluded from clinical trials especially with combination therapies involving MRTX1133 and standard-of-care 5-FU. Citation Format: Vida Tajiknia, Wafik El-Deiry, Maximilian Pinho Schwermann, Lanlan Zhou, Kelsey Huntington. Combination of 5-Fluorouracil or ONC212 plus KRAS G12D inhibitor MRTX1133 against colorectal and pancreatic cancer cells results in immune-stimulatory cytokine patterns and unexpected synergies independent of G12D mutation [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 1072.

Abstract 1578: Novel Cereblon mediated bifunctional degraders of SOS1 for treatment of pan-KRAS mutant tumors

Abstract KRAS, a GTPase that regulates cellular growth, is the most frequently mutated oncogene with implications in non-small cell lung, pancreatic and colorectal cancers. Hyperactivation of KRAS in mutant alleles favors KRAS in its active GTP-bound state over its inactive GDP-bound state. Once deemed undruggable, recent advances have revealed promising anti-tumor effects with pan-KRAS and KRAS-mutant specific inhibitors in both preclinical and clinical settings. Despite the success of these inhibitors, the diversity of tumors as well as tumor sensitivity results in acquired drug resistance suggesting that combinational therapeutic approaches would be necessary to avoid this problem. An attractive dual approach would be to couple KRAS inhibition with a blockade of its guanine exchange factor (GEF), SOS1, to keep KRAS in its inactive state as well as prevent upstream pathway reactivation. Utilizing our PRODEGY platform, we developed bifunctional SOS1 degraders for the treatment of patients with KRAS mutant cancers. These degraders have shown rapid and potent Cereblon (CRBN)-mediated degradation which exhibit DC50s &amp;lt;10nM in a H358 SOS1-HiBiT cell line. Our high- throughput HiBiT data translated into effective SOS1 degradation in KRAS mutant cells harboring G12C, G12D, G12V, G12S, or G13D mutations. SOS1 degradation was validated to be dependent on the proteasome and CRBN as cotreatment with proteasomal inhibitors (MG132 or MLN4924) or treatment in a CRBN KO cells abolished degradation. Additionally, treatment of SOS1 degraders resulted in suppression of downstream signaling markers, pERK and pS6, in both 3D immunoblots and high-throughput assays with pERK IC50s &amp;lt;10nM. Chemical competitor assays also revealed that inhibition of pERK and pS6 was dependent on CRBN-mediated competition. To determine functional activity of our SOS1 degraders, we developed and optimized 3D proliferation assays in multiple KRAS mutant (G12C, G12D, G12V, G12S, G13D) cell lines which yielded IC50s ranging from 0.5 - 70nM. Similarly, 100- to 300-fold IC50 value shifts were observed in SOS1 KO and CRBN KO H358 cell lines validating that the anti-proliferative activity of SOS1 degraders is specific to its targets. Treatment of SOS1 degraders in KRAS-mutant xenograft models resulted in &amp;gt;75% degradation of SOS1 in tumors and subsequently led to significant tumor growth inhibition as a single agent. SOS1 degradation also provided synergistic anti-proliferation effects with other RAS/MAPK/AKT inhibition in in vitro studies. For example, combination treatment of AMG510 and SOS1 degraders resulted in strong synergistic effects in KRAS G12C H358 cells. Together, our data SOS1 degradation could be a valuable potential treatment option for a range of KRAS mutant cancers. Citation Format: Kyle Begovich, Angela Schoolmeesters, Navin Rajapakse, Maneesh Kumar, Arvind Shakya, Brandon Whitefield, Elena Martinez-Terroba, Nataraj Pagadala, Shenlin Huang, Aparajita H. Chourasia, Leah Fung. Novel Cereblon mediated bifunctional degraders of SOS1 for treatment of pan-KRAS mutant tumors [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 1578.

Abstract 530: Preclinical evaluation of avutometinib (VS-6766; RAF/MEK clamp) in combination with EGFR inhibition in colorectal cancer patient-derived xenograft (PDX) models harboring KRAS mutations

Abstract Background KRAS mutation (mt) occurs in ~43% of patients with colorectal cancer (CRC) with KRAS G12D, G12V and G13D being most prevalent. Avutometinib (avuto, VS-6766) is a unique RAF/MEK clamp that inhibits MEK kinase activity and induces dominant negative RAF/MEK complexes preventing the re-activation of MEK by RAF. Preclinically, avuto inhibited proliferation of KRAS mt CRC cell lines. Clinically, avuto monotherapy produced clinical responses in gynecological cancers and KRAS mt non-small cell lung cancer. Methods 3D proliferation assays were used to quantify synergy between avuto and agents targeting other nodes in the MAPK pathway or parallel signaling pathways. We evaluated the antitumor effects of avuto alone or in combination with panitumumab in CRC PDX models with KRASG12V, concurrent KRASG12V and PIK3CA, or KRASG12D mutations. Mice bearing PDX tumors were randomized into groups of 6 mice treated with avuto alone or in combination with panitumumab for 21 days with tumor volume measured twice weekly. Results In 3D proliferation assays with a panel of KRAS mt CRC cell lines (including G12D, G12V and G13D mutations), the EGFR/ErbB family inhibitor afatinib showed the strongest synergy score with avuto relative to inhibitors of SHP2, ERK1/2, mTOR or CDK4/6. Based on the synergy of avuto with afatinib, the combination of avuto with the anti-EGFR antibody panitumumab was tested in KRAS mt CRC PDX models. In a KRASG12V PDX model, tumor regression (11%) was seen in 1/6 mice treated with avuto alone. No tumor regression was seen in mice treated with panitumumab monotherapy. The combination of avuto and panitumumab led to significant tumor regression (&amp;gt;40%) in 5/6 mice following 21 days of treatment. In a KRASG12V PDX model with concurrent PIK3CA mutations, no tumor regression was seen in mice treated with either avuto or panitumumab alone. The combination of avuto and panitumumab led to initial tumor stabilization followed by tumor growth in 3/6 mice. In a KRASG12D PDX model, tumor regression (17%) was seen in 1/6 mice treated with avuto and 0/6 with panitumumab. Tumor regression (13% and 18%) was noted in 2/6 mice treated with the combination of avuto and panitumumab. Conclusion In KRAS mt CRC cell lines, strong synergy was observed between avuto and afatinib. In KRAS mt CRC PDX models in vivo, avuto monotherapy showed tumor growth inhibition but limited tumor regression, and panitmumab monotherapy failed to show tumor regressions. The combination of avuto and panitumumab showed significant anti-tumor activity in a CRC PDX model harboring KRASG12V with less tumor regression observed in a KRASG12D PDX model or in a KRASG12V PDX model with concurrent PIK3CA mutation. These results support the ongoing clinical evaluation of avutometinib in combination with the anti-EGFR antibody cetuximab for treatment of KRAS mt CRC (NCT05200442). Citation Format: Chongkai Wang, Silvia Coma, Jonathan Pachter, Marwan Fakih. Preclinical evaluation of avutometinib (VS-6766; RAF/MEK clamp) in combination with EGFR inhibition in colorectal cancer patient-derived xenograft (PDX) models harboring KRAS mutations [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 530.

Abstract 491: Tankyrase-selective inhibitor STP1002 reverses resistance to MEK inhibitors in colorectal cancer with KRAS mutations

Abstract Mitogen-activated protein kinase (MEK) inhibitors have shown promising results in KRAS-mutated cancers with constitutive activation of the RAS/RAF/MEK pathway. However, the intrinsic and acquired resistance to MEK inhibitors is frequently observed in clinical trials. Wnt/β-catenin signaling hyper-activation is reported to be responsible for such resistance in colorectal cancer (CRC). Herein, we introduce a novel tankyrase inhibitor STP1002 to revert the intrinsic and acquired resistance to MEK inhibitors in KRAS-mutated CRC. Dual combination of STP1002 and MEK inhibitor synergistically reduced the oncogenic activity of KRAS (G12V or G12D)-mutated CRC cell lines. Data also showed that concomitant treatment with STP1002 and MEK inhibitor dramatically inhibited tumor growth of KRAS (G12V)-mutated CRC xenograft animal models. Moreover, the combination treatment sensitized the acquired MEK inhibitor-resistant CRC cells and suppressed the Wnt/YAP pathway which is the bypass mechanism of MEK inhibitor resistance. Dual combination of STP1002 and MEK inhibitor is a promising candidate to overcome resistance to MEK inhibitors in colorectal cancer with KRAS-G12V/G12D mutations. The phase 1 clinical trial study is ongoing in order to show safety margin. Citation Format: Young-Ju Kwon, Dong Young Kim, Uk-Il Kim, Xue Meng, Ho Kyun Lee, Hyung Tae Bang, Jae-Sung Kim, Kyungjin Kim. Tankyrase-selective inhibitor STP1002 reverses resistance to MEK inhibitors in colorectal cancer with KRAS mutations [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 491.