KRAS G12D Research Articles

KRAS G12D Mutation Subtype in Pancreatic Ductal Adenocarcinoma: Does It Influence Prognosis or Stage of Disease at Presentation?

Background: KRAS G12D mutation subtype is present in over 40% of pancreatic ductal adenocarcinoma (PDAC), one of the leading global causes of cancer death. This retrospective cohort study aims to investigate whether detection of the KRAS G12D mutation subtype in PDAC patients is a determinant of prognosis across all stages of disease. Methods: We reviewed the medical records of 231 patients presenting with PDAC at a large tertiary hospital, and compared survival using the Kaplan Meier, log-rank test and Cox proportional hazards regression model. Results: KRAS G12D mutation subtype was not significantly associated with poorer survival compared across the whole population of PDAC patients (p = 0.107; HR 1.293 95% CI (0.946–1.767)). However, KRAS G12D patients who were resectable had a shorter median survival time of 356 days compared to all other genotypes (median survival 810 days) (p = 0.019; HR 1.991 95% CI (1.121–3.537)). Conclusions: KRAS G12D patients who were resectable at diagnosis had shorter survival compared to all other PDAC patients. These data suggest that KRAS G12D may be a clinically useful prognostic biomarker of PDAC.

54 (PB044) - Non-clinical identification and characterization of KRAS G12D inhibitors

KRAS G12D is the most prevalent KRAS oncogenic mutant and occurs frequently in pancreatic ductal adenocarcinoma (PDAC), colorectal adenocarcinoma (CRC), and non-small cell lung cancer (NSCLC; 37%, 12% and 4%, respectively). KRAS G12D cycles between inactive (GDP) and active (GTP) states to regulate the RAS/MAPK pathway. Although the rate of intrinsic and GAP (GTPase activating protein) mediated hydrolysis is significantly slower in KRAS G12D than in KRAS wild-type (KRAS WT), we were able to build upon success of targeting the GDP state of KRAS G12C and designed novel KRAS G12D lead compounds with promising in vitro and in vivo activity. Our KRAS G12D inhibitors preferentially bind to KRAS G12D GDP as indicated by differential scanning fluorimetry (DSF), block KRAS G12D SOS1-mediated nucleotide exchange, and inhibit KRAS G12D binding to the RAS binding domain of C-RAF (RAF-RBD) with single digit nanomolar potency. Our leads effectively inhibit ERK1/2 phosphorylation (pERK) in AsPC-1 PDAC cells and inhibit cell proliferation in 3-dimensional Cell-Titer Glo assays in a panel of KRAS G12D mutant PDAC, CRC, and NSCLC cell lines. This in vivo activity translates in vitro where robust pharmacodynamic modulation in the PDAC AsPC-1 CDX model was induced. Tumor growth inhibition studies in this model revealed robust dose-dependent tumor growth inhibition and tumor regression in the absence of overt toxicity. In summary, we have identified potent and selective KRAS G12D inhibitors with robust dose-dependent tumor growth inhibition and regression activity in a PDAC CDX model. Lead optimization is ongoing with the aim of identifying a development candidate. No conflict of interest.

85 (PB075) - ASP3082, a First-in-class novel KRAS G12D degrader, exhibits remarkable anti-tumor activity in KRAS G12D mutated cancer models

Background: KRAS is one of the most frequently mutated oncogenes in various cancers. Among KRAS mutations, KRAS G12D is the most frequent driver mutation and is found in approximately 34% of pancreatic ductal adenocarcinoma (PDAC), 10% to 12% of colorectal cancer, 4% of lung adenocarcinoma and also in a subset of other solid tumors. However, there is no direct KRAS G12D-targeted inhibitors used in the clinical setting. Here, we identified ASP3082, a novel KRAS G12D degrader with high potency and selectivity. Methods: We investigated (1) binding affinity of ASP3082 to KRAS G12D protein and an E3 ligase in the conditions of binary complex and ternary complex formation using surface plasmon resonance assay, (2) degradation activity of ASP3082 on KRAS G12D protein using western blotting or in-cell western method, (3) inhibitory effect of ASP3082 on the growth of human cancer cells harboring KRAS G12D mutation and KRAS wild-type, (4) specificity for ASP3082-mediated degradation using proteomics approach, (5) antitumor activity of ASP3082 in mice subcutaneously xenografted with KRAS G12D-mutated pancreatic cancer cells, (6) PK-PD profile of ASP3082 after a single intravenous administration in the xenograft model. Results: ASP3082 directly bound to KRAS G12D-mutated protein and the E3 protein to form a ternary complex. Also, ASP3082 potently degraded KRAS G12D-mutated protein and inhibited phosphorylation of ERK in KRAS G12D-mutated pancreatic cancer cells. In addition, ASP3082 showed the growth inhibitory activity of KRAS G12D-mutated pancreatic cancer cells, but not of KRAS wildtype cancer cells. Furthermore, the quantitative proteomics analysis suggested that ASP3082 selectively degraded the KRAS G12D-mutated protein over other (>9000) proteins. in vivo experiment revealed that once-weekly intravenous administration of ASP3082 induced dose-dependent and significant growth inhibition of KRAS G12D PDAC tumors, resulting in profound tumor regression without body weight loss. ASP3082 showed sustained concentrations in the xenograft tumors after a single intravenous administration and decreased KRAS G12D-mutated protein levels according to the duration of the ASP3082 concentrations. Conclusions: ASP3082 is a potential therapeutic agent for patients with tumors harboring the KRAS G12D mutation. Currently, Phase I clinical trial is underway (NCT05382559). Conflict of interest: Corporate-sponsored Research: All authors are employees of Astellas Pharma Inc.

S2163 Understanding Prolonged Survival in Advanced Colorectal Cancer Using Genomics: A Case Series

Introduction: Recent progress in molecular biology and cancer genome research had opened a new venue for clinical research and changed treatment paradigm for metastatic colorectal cancer. Case Description/Methods: Case 1: A 61-year -old Hispanic male underwent colonoscopy which revealed an mass in the proximal ascending colon with biopsy showing moderately differentiated adenocarcinoma. The patient underwent a right hemicolectomy in 2012. He subsequently completed chemotherapy with mFOLFOX-6. Further treatment was declined by patient. Follow up chest CT scan in April 2018, uncovered progression of pulmonary nodules (Figure, Panel 1). Patient was put on FOLFIRI/bevacizumab chemotherapy. NGS study showed following results; MS stable; TMB 4 mutations/Mb (low); APCR1450, BRAF D594N, FAM 123B K761, SOX9 G225fs, and TP53 R175H.The patient has stable disease on follow up. Case 2: A 73-year-old Hispanic male underwent colonoscopy showing a obstructing mass in the sigmoid colon. Biopsy showed moderately differentiated adenocarcinoma in December 2013. Patient underwent sigmoidectomy. Liver masses were found on repeat CT abdomen. The NGS reported as follows; MS-Stable, TMB 1 muts/Mb(low), APC T282fs*12/P1453fs*20, CDK8 amplification, fms like tyrosine kinase 3 (FLT3) amplification, KRAS amplification and TP53 V147D. It also detected wild type KRAS mutations in exons 2, 3 and 4. The patient was started on chemotherapy with mFOLFOX. Follow- up studies show stable disease. Case 3: A 73-year-old Hispanic male underwent colonoscopy showed a completely obstructing mass in the sigmoid colon. Biopsy showed moderately differentiated adenocarcinoma in June 2012. The tumor cells have KRAS G12D mutation in in codon 12 of KRAS. He underwent a left hemicolectomy. He completed mFOLFOX6.PET scan was done and showed a right upper lobe mass consistent with metastatic cancer. He was started on FOLFIRI/ bevacizumab. NGS reported as follows; MS-Stable, TMB 1 muts/Mb(low), APC V1452fs*21, and ATM S47fs*11. Bone Scan intense uptake in the right distal tibia (Figure, Panel 2). MRI of the right lower extremity showed a lesion in the distal tibia. He was then switched to irinotecan and bevacizumab regimen. He has stable disease. Discussion: Identification of genomic signature is key to understanding the molecular mechanism of CRC and the development of novel therapeutics.Figure 1.: Panel 1 (left) shows CT Chest for Case 1 with multiple pulmonary nodules few with cavitation, largest 9mm. Panel 2 (right) shows Bone Scan for Case 3 in February 2021 with intense uptake in the right distal tibia.

Abstract PR001: Oncogene-induced matrix reorganization controls CD8+ T cell immunity in the UPS microenvironment

Abstract CD8+ T cell dysfunction, characterized by reduced effector function, impaired proliferation, and inhibitory receptor upregulation, is a fundamental barrier to anti-tumor immunity. However, molecular mechanisms underlying the regulation of CD8+ T cell dysfunction in the tumor microenvironment (TME) are incompletely understood. In solid cancers, the extracellular matrix (ECM) facilitates tumor progression in part by inhibiting T cell migration/infiltration, but the impact of individual tumor-associated ECM molecules on T cell function remains unclear. Therefore, we investigated the regulation and impact of ECM composition on CD8+ T cell function in muscle-derived undifferentiated pleomorphic sarcoma (UPS). UPS exhibits durable responses to checkpoint therapy in a subset of human patients, potentially offering valuable insights into strategies for ameliorating T cell function and improving patient responses to immunotherapy. Using the autochthonous Kras G12D/+; Trp53 fl/fl (KP) murine model of UPS, we previously showed that deletion of the central Hippo pathway effector Yap1 (Kras G12D/+; Trp53 fl/fl; Yap1 fl/fl; KPY) suppressed UPS cell proliferation and tumor progression. Given the well-established role of Yap1 in mechanotransduction, we leveraged this system to investigate the effects of Yap1 on the ECM and CD8+ T cell function in UPS. We discovered that loss of UPS-cell intrinsic Yap1 reduced the proportion of dysfunctional CD8+ T cells in the TME and enhanced T cell cytolytic capacity. Yap1 loss also downregulated expression of multiple collagen genes in UPS cells and bulk tumors, including those that encode collagen type VI (ColVI). ColVI is a beaded microfilament collagen that binds to fibril-forming collagens in the ECM, such as collagen type I (ColI), and has been implicated in the pathogenesis of skeletal muscle myopathies. These data suggest that proper ColVI structure and function are critical for normal skeletal muscle physiology, with important implications for muscle-derived tumors such as UPS. Accordingly, COL6A1 was upregulated in human UPS relative to normal skeletal muscle, and inversely associated with UPS patient survival. Moreover, loss of UPS cell-intrinsic Col6a1 suppressed tumor progression, enhanced T cell cytolytic function, and attenuated CD8+ T cell exhaustion, phenocopying the effects of Yap1 deletion. Mechanistically, Yap1-mediated ColVI deposition promoted CD8+ T cell dysfunction by remodeling ColI networks in the UPS TME, and inhibiting T cell autophagic flux. Furthermore, ColI depletion dramatically increased tumor growth in an immunocompetent setting. Our findings reveal a novel role for UPS cell-intrinsic Yap1 in immune activation, and demonstrate that ColVI and ColI have opposing functions downstream of Yap in this context. These results underscore the need to systematically evaluate the roles of individual ECM components in the regulation of immune cell function, and implicate YAP1 and/or COLVI targeting as potential strategies for improving the efficacy of immunotherapy in human patients. Citation Format: Ashley M. Fuller, Hawley C. Pruitt, Hoogeun Song, Ying Liu, Ann Devine, Rohan S. Katti, Samir Devalaraja, Gabrielle E. Ciotti, Michael Gonzalez, Erik F. Williams, Ileana Murazzi, Nicolas Skuli, Hakon Hakonarson, Kristy Weber, Malay Haldar, Joseph A. Fraietta, Sharon Gerecht, T. S. Karin Eisinger-Mathason. Oncogene-induced matrix reorganization controls CD8+ T cell immunity in the UPS microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR001.

The clinical landscape of cell-free DNA alterations in 1671 patients with advanced biliary tract cancer

Targeted therapies have transformed clinical management of advanced biliary tract cancer (BTC). Cell-free DNA (cfDNA) analysis is an attractive approach for cancer genomic profiling that overcomes many limitations of traditional tissue-based analysis. We examined cfDNA as a tool to inform clinical management of patients with advanced BTC and generate novel insights into BTC tumor biology. We analyzed next-generation sequencing data of 2068 cfDNA samples from 1671 patients with advanced BTC generated with Guardant360. We carried out clinical annotation on a multi-institutional subset (n= 225) to assess intra-patient cfDNA-tumor concordance and the association of cfDNA variant allele fraction (VAF) with clinical outcomes. Genetic alterations were detected in cfDNA in 84% of patients, with targetable alterations detected in 44% of patients. Fibroblast growth factor receptor 2 (FGFR2) fusions, isocitrate dehydrogenase 1 (IDH1) mutations, and BRAF V600E were clonal in the majority of cases, affirming these targetable alterations as early driver events in BTC. Concordance between cfDNA and tissue for mutation detection was high for IDH1 mutations (87%) and BRAF V600E (100%), and low for FGFR2 fusions (18%). cfDNA analysis uncovered novel putative mechanisms of resistance to targeted therapies, including mutation of the cysteine residue (FGFR2 C492F) to which covalent FGFR inhibitors bind. High pre-treatment cfDNA VAF was associated with poor prognosis and shorter response to chemotherapy and targeted therapy. Finally, we report the frequency of promising targets in advanced BTC currently under investigation in other advanced solid tumors, including KRAS G12C (1.0%), KRAS G12D (5.1%), PIK3CA mutations (6.8%), and ERBB2 amplifications (4.9%). These findings from the largest and most comprehensive study to date of cfDNA from patients with advanced BTC highlight the utility of cfDNA analysis in current management of this disease. Characterization of oncogenic drivers and mechanisms of therapeutic resistance in this study will inform drug development efforts to reduce mortality for patients with BTC.

A modular XNAzyme cleaves long, structured RNAs under physiological conditions and enables allele-specific gene silencing.

Nucleic-acid catalysts (ribozymes, DNA- and XNAzymes) cleave target (m)RNAs with high specificity but have shown limited efficacy in clinical applications. Here we report on the in vitro evolution and engineering of a highly specific modular RNA endonuclease XNAzyme, FR6_1, composed of 2'-deoxy-2'-fluoro-β-D-arabino nucleic acid (FANA). FR6_1 overcomes the activity limitations of previous DNA- and XNAzymes and can be retargeted to cleave highly structured full-length (>5 kb) BRAF and KRAS mRNAs at physiological Mg2+ concentrations with allelic selectivity for tumour-associated (BRAF V600E and KRAS G12D) mutations. Phosphorothioate-FANA modification enhances FR6_1 biostability and enables rapid KRAS mRNA knockdown in cultured human adenocarcinoma cells with a G12D-allele-specific component provided by in vivo XNAzyme cleavage activity. These results provide a starting point for the development of improved gene-silencing agents based on FANA or other XNA chemistries.

Pixantrone confers radiosensitization in KRAS mutated cancer cells by suppression of radiation-induced prosurvival pathways

Radioresistance towards radiation therapy has generated the need for the development of radiosensitizers as a potential drug. KRAS mutation brings radioresistance in tumor cells. The present work proves sensitization of cancer cells towards radiotherapy through inhibition of KRAS activation. Acquiring a drug repurposing approach, the in-silico screening revealed that pixantrone, an antineoplastic drug, possesses a high affinity towards KRAS G12C and G12D subtypes. The SPR study suggests that maximum affinity of pixantrone was observed with KRAS G12C>WT>G12D and G12S. Pixantrone potentially inhibited the KRAS activation in stable transfectants G12C and G12D cell lines and radiosensitized distinct KRAS mutant subtype cells. The combination of pixantrone with radiation causes enhanced dsDNA breaks along with enhanced ATM expression, and increased late apoptosis. The preclinical studies on NCr-fox1nu xenograft mice showed potent inhibition of tumor progression and prolonged survival of mcie due to the radiosensitizing effect of pixantrone. Radiation-induced activation of key effector proteins of RAS downstream pathways, like MAPK and PI3K/Akt/mTOR pathways, were downregulated in tumor cells upon combination treatment. Interestingly, a robust upregulation of senescence marker p21 was observed in the tumor cells in combination treatment. These findings reveal a convergence between KRAS signaling, pixantrone treatment, and radiation conferring tumor cell death.

Cytotoxicity of combinations of the pan-KRAS SOS1 inhibitor BAY-293 against pancreatic cancer cell lines

KRAS is mutated in approximately 25% of cancer patients and first KRAS G12C-specific inhibitors showed promising responses. Pancreatic cancer has the highest frequency of KRAS mutations but the prevailing KRAS G12D mutation is difficult to target. Inhibition of the GTP exchange factor (GEF) SOS1—KRAS interaction impairs oncogenic signaling independently of the specific KRAS mutations. In general, cell lines exhibiting KRAS mutations show specific alterations in respect to glucose utilization, signal transduction and stress survival. The aim of this investigation was to check the putative synergy of the SOS1 inhibitor BAY-293 with modulators targeting specific vulnerabilities of KRAS-mutated cell lines in vitro. The cytotoxicity of BAY-293 combinations was tested against MIA PaCa-2 (G12C), AsPC1 (G12D) and BxPC3 (KRAS wildtype) cell lines using MTT tests and calculation of the combination indices (CI) according to the Chou-Talalay method. The results show that BAY-293 synergizes with modulators of glucose utilization, inhibitors of the downstream MAPK pathway and several chemotherapeutics in dependence of the specific KRAS status of the cell lines. In particular, divergent responses for BAY-293 combinations between pancreatic and NSCLC cell lines were observed for linsitinib, superior inhibitory effects of trametinib and PD98059 in NSCLC, and lack of activity with doxorubicin in case of the pancreatic cell lines. Phosphoproteome analysis revealed inhibition of distinct signaling pathways by BAY-293 for MIA PaCa-2 on the one hand and for Aspc1 and BH1362 on the other hand. In conclusion, BAY-293 exhibits synergy with drugs in dependence of the tumor type and specific KRAS mutation.

DNA Polymerase Theta Plays a Critical Role in Pancreatic Cancer Development and Metastasis.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers worldwide. The occurrence of oncogenic KRAS mutations is considered a signature event in PDAC, leading to genomic instability. The aim of our study was to evaluate the impact of the oncogenic KRAS G12D mutation on the activity of the error-prone alt-EJ repair mechanism, and to investigate the potential role of Polθ in the development of pancreatic cancer. We found that oncogenic KRAS increases the expression of key alt-EJ proteins in a mouse and human PDAC model. Using TLR assay, we also found increased alt-EJ activity in mouse and human cell lines upon the expression of KRAS D12D. The inactivation/impairment of alt-EJ by polymerase theta (Polθ) depletion delays the development of pancreatic cancer and prolongs the survival of experimental mice, though it does not prevent the PDAC development, which leads to full-blown PDAC with disseminated metastasis. Our studies provide a high-value target as a novel therapeutic candidate for the treatment of pancreatic and other cancers.Pancreatic ductal adenocarcinoma (PDAC), due to its genomic heterogeneity and lack of effective treatment, despite decades of intensive research, will become the second leading cause of cancer-related deaths by 2030. Step-wise acquisition of mutations, due to genomic instability, is considered to drive the development of PDAC; the KRAS mutation occurs in 95 to 100% of human PDAC, and is already detectable in early premalignant lesions designated as pancreatic intraepithelial neoplasia (PanIN). This mutation is possibly the key event leading to genomic instability and PDAC development. Our study aimed to investigate the role of the error-prone DNA double-strand breaks (DSBs) repair pathway, alt-EJ, in the presence of the KRAS G12D mutation in pancreatic cancer development. Our findings show that oncogenic KRAS contributes to increasing the expression of Polθ, Lig3, and Mre11, key components of alt-EJ in both mouse and human PDAC models. We further confirm increased catalytic activity of alt-EJ in a mouse and human model of PDAC bearing the KRAS G12D mutation. Subsequently, we focused on estimating the impact of alt-EJ inactivation by polymerase theta (Polθ) deletion on pancreatic cancer development, and survival in genetically engineered mouse models (GEMMs) and cancer patients. Here, we show that even though Polθ deficiency does not fully prevent the development of pancreatic cancer, it significantly delays the onset of PanIN formation, prolongs the overall survival of experimental mice, and correlates with the overall survival of pancreatic cancer patients in the TCGA database. Our study clearly demonstrates the role of alt-EJ in the development of PDAC, and alt-EJ may be an attractive therapeutic target for pancreatic cancer patients.

EHMT2 methyltransferase governs cell identity in the lung and is required for KRAS G12D tumor development and propagation.

Lung development, integrity and repair rely on precise Wnt signaling, which is corrupted in diverse diseases, including cancer. Here, we discover that EHMT2 methyltransferase regulates Wnt signaling in the lung by controlling the transcriptional activity of chromatin-bound β-catenin, through a non-histone substrate in mouse lung. Inhibition of EHMT2 induces transcriptional, morphologic, and molecular changes consistent with alveolar type 2 (AT2) lineage commitment. Mechanistically, EHMT2 activity functions to support regenerative properties of KrasG12D tumors and normal AT2 cells-the predominant cell of origin of this cancer. Consequently, EHMT2 inhibition prevents KrasG12D lung adenocarcinoma (LUAD) tumor formation and propagation and disrupts normal AT2 cell differentiation. Consistent with these findings, low gene EHMT2 expression in human LUAD correlates with enhanced AT2 gene expression and improved prognosis. These data reveal EHMT2 as a critical regulator of Wnt signaling, implicating Ehmt2 as a potential target in lung cancer and other AT2-mediated lung pathologies.

Clinicopathologic Characteristics and Outcomes for Patients With KRAS G12D-Mutant NSCLC

IntroductionCo-occurring mutations in KRAS-mutant NSCLC are associated with discrete biological properties and modulate therapeutic susceptibilities. As G12D-specific inhibitors are expected to enter the clinic, we sought to investigate the characteristics and outcomes of patients with KRAS G12D-mutant NSCLC. MethodsThis was a retrospective single-institution study. Patients with NSCLC and KRAS G12D mutations detected by the Massachusetts General Hospital SNaPshot next-generation sequencing assay were identified. Clinical and pathologic characteristics were collected by chart review. ResultsA total of 107 patients with KRAS G12D-mutant NSCLC were identified. Most patients were former smokers (80, 74.8%) and had tumors with adenocarcinoma pathologic subtype (93, 86.9%). Among 56 patients evaluated for programmed death-ligand 1 expression, tumor proportion score was less than 50% in 43 (76.8%). Concomitant mutations were identified in STK11 (17 of 107, 15.9%), KEAP1 (10 of 58, 17.2%), TP53 (36 of 107, 33.6%), and SMARCA4 (11 of 107, 10.3%). Among 57 patients treated with first-line therapy, patients with STK11 co-mutations had shorter progression-free survival (1.2 mo, 95% confidence interval [CI]: 0.6–2.9 versus 4.1 mo, 95% CI: 2.5–6.0, p = 0.0235) and overall survival (4.3 mo, 95% CI: 1.2–10.6 versus 17.9 mo, 95% CI: 8.6–31.1, p = 0.0018) compared with wild type. Patients with KEAP1 co-mutations had shorter overall survival (4.6 mo, 95% CI: 1.2–10.6 versus 17.9 mo, 95% CI: 7.1–30.1, p = 0.0125) than those without. TP53 co-mutations exerted no influence on survival. ConclusionsCo-occurring mutations were common in patients with KRAS G12D-mutant NSCLC. STK11 and KEAP1 co-mutations were associated with worse clinical outcomes, whereas co-occurring TP53 did not affect survival.

Somatic mutation analysis of Mesonephric-Like adenocarcinoma and associated putative precursor Lesions: Insight into pathogenesis and potential molecular treatment targets

AimsMesonephric-like adenocarcinoma (MLA) is a recently described histologic tumor subtype of the Müllerian tract. MLA can arise in association with Müllerian lesions that share common mutations. We report three MLAs and hypothesize that concurrent endometriosis and cystadenofibroma with focal borderline changes might also carry common mutations. Methods and resultsWe searched “mesonephric” in our database from 2015 to mid-2021 to retrieve MLA cases. Somatic mutation analysis was performed on tumors and on associated benign proliferative lesions. All MLAs (2 ovarian and 1 uterine) harbored KRAS G12D or G12 V mutations. A PIK3CA alteration (H1047Q) was detected in one MLA and in the associated cystadenofibroma with focal borderline changes. The molecular profile of MLA-associated Müllerian lesions (endometriosis and seromucinous cystadenofibroma with focal borderline changes) was similar to concurrent adenocarcinoma. However, tumor contamination could not be excluded in the endometriotic lesion. Patients presented at various stages, with no evidence of post-operative recurrence after 15 months (FIGO IC) and 33 months (FIGO IIA2). One patient (FIGO IIIA1) died of disease 32 months after surgery. ConclusionsKRAS mutations commonly characterize MLA. At least some MLA-associated Müllerian lesions show MLA-like genetic profiles, suggesting a precursor role. As far as we are aware, we describe for the first time in MLA the potentially actionable H1047Q variant of PIK3CA.

Identification of potential inhibitors, conformational dynamics, and mechanistic insights into mutant Kirsten rat sarcoma virus (G13D) driven cancers.

The mutations at the hotspot region of K-Ras result in the progression of cancer types. Our study aimed to explore the small molecule inhibitors against the G13D mutant K-Ras model with anti-cancerous activity fromfood and drug administration (FDA)-approved drug compounds. We implemented several computational strategies such as pharmacophore-based virtual screening, molecular docking, absorption, distribution, metabolism and excretion features, and molecular simulation to ensure the identified hit compounds have potential efficacy against G13D K-Ras. We found that the FDA-approved compounds, namely, azelastine, dihydrocodeine, paroxetine, and tramadol, are potential candidates to inhibit the action of G13D mutant K-Ras. All four compounds exhibited similar binding patterns of sotorasib, and a structural binding mechanism with significant hydrophobic contacts. The descriptor features from the QikProp of all four compounds are within allowable limits compared to sotorasib drug. Consequently, a molecular simulation result emphasized that the dihydrocodeine and tramadol exhibited less fluctuation, minimal basin, significant h-bonds, and potent inhibition against G13D K-Ras. As a result, the current research identifies prospective K-Ras inhibitors that could be further improved with biochemical analysis for precision medicine against K-Ras-driven cancers.

Impact of KRAS Mutation Subtypes and Co-Occurring Mutations on Response and Outcome in Advanced NSCLC Patients following First-Line Treatment.

(1) Background: The purpose was to systematically assess the impact of KRAS subtypes and co-mutations on responses of first-line treatment and outcomes by genetic classification in advanced KRAS mutant NSCLC. (2) Methods: Molecular pathology was confirmed with NGS; Kaplan–Meier analysis and Cox multivariate model were used to analyze the efficacy of first-line treatment and prognosis in KRAS subgroups. (3) Results: Advanced KRAS mutant NSCLC was confirmed among 183 patients, who received first-line therapy. The most common KRAS subtype and co-mutation were G12C (29.5%) and TP53 (59.6%). ICIs/CHE group prolonged PFS to 16.9 m, vs. (CHE)4.6 m vs. (CHE/BEV)7.0 m (p < 0.0001); mOS (ICIs/CHE)37.1 m vs. (CHE)19.8 m vs. [CHE/BEV] 20.7 m (p = 0.024). PFS benefited to different degrees after first-line ICI-based treatment in each genetic classification. KRAS G12D even benefited from OS (p = 0.045). CHE/BEV prolonged mPFS of KRAS/STK11 co-mutation (p = 0.043), but decreased mPFS in G12A subtype (p = 0.026). Multivariate analysis indicated that heavy smoking history (≥20 pack-years) (HR = 0.45, p = 0.039) predicts optimistic prognosis; PS score 1 (HR = 3.604, p = 0.002) and KRAS/SMAD4 co-mutation (HR = 4.293, p = 0.027) remained as independent predictors of shorter OS. (4) Conclusions: First-line treatment with ICI benefited KRAS-mutant-NSCLC patients and resulted in non-negative predictive value for any genetic classification. Bevacizumab should be cautiously chosen for patients with KRAS G12A subtype but is recommended for KRAS/STK11 patients. KRAS/SMAD4 is a new co-mutation genotype that displayed independent risk prognostic factors in patients with advanced KRAS-mutant NSCLC.

Prognostic Value of KRAS Mutations in Colorectal Cancer Patients.

Simple SummaryKirsten rat sarcoma viral oncogene homolog (KRAS)-mutated colorectal cancer (CRC) seems to have a different biological behavior and therapeutic approach compared with non-KRAS mutated CRC. Except for the proven predictive significance of KRAS mutations in CRC patients, their prognostic significance is still under evaluation. Our study shows that 93.2% of RAS-mutated patients were KRAS mutants, with G12D being the most common subtype. KRAS G12D mutation is correlated with better overall survival (OS). KRAS G12C mutations may indicate worse prognosis regarding progression free (PFS) and OS, as well as exon 4 and exon 3 KRAS mutations for PFS and OS, respectively. Further studies are warranted to confirm these results.Colorectal cancer (CRC) remains a major public health issue. The detection of parameters that affect CRC prognosis is of great significance. KRAS mutations, play a crucial role in tumorigenesis with a strong predictive value. KRAS-mutated stage-IV CRC patients gain no benefit of the anti-EGFR therapy. The KRAS G12C mutation subtype is under investigation for treatment regimens. The present study aimed to detect various RAS mutations in a cohort of 578 RAS-mutated CRC patients; 49% of them had de novo metastatic disease; 60% were male; 71.4% had left-sided tumors; and 94.6% had a good performance status. KRAS mutations were detected in 93.2% of patients, with KRAS G12D being the most common subtype (30.1%). KRAS mutations presented shorter progression-free (PFS) and overall survival (OS), compared with NRAS mutations, although not significantly (PFS: 13.8 vs. 18.5 months; p = 0.552; OS: 53.1 vs. 60.9 months; p = 0.249). KRAS G12D mutations presented better OS rates (p = 0.04). KRAS G12C mutation, even though not significantly, presented worse PFS and OS rates. KRAS exon 3 and 4 mutations presented different PFS and OS rates, although these were not significant. Concluding, KRAS G12D and G12C mutations lead to better and worst prognosis, respectively. Further studies are warranted to validate such findings and their possible therapeutic implication.

Conformational Dynamics Allows Sampling of an “Active-like” State by Oncogenic K-Ras-GDP

Mutations in K-Ras GTPase replacing Gly12 with either Asp or Val are common in cancer. These mutations decelerate intrinsic and catalyzed GTP hydrolysis, leading to accumulation of K-Ras-GTP in cells. Signaling cascades initiated by K-Ras-GTP promote cell proliferation, survival, and invasion. Despite functional differences between the most frequent G12D mutation and the most aggressive and chemotherapy resistant G12V mutation, their long-suspected distinct structural features remain elusive. Using NMR, X-ray structures, and computational methods, we found that oncogenic mutants of K-Ras4B, the predominant splice variant of K-Ras, exhibit distinct conformational dynamics when GDP-bound, visiting the “active-like” conformational state similar to the one observed in GTP-bound K-Ras. This behavior distinguishes G12V from wild type and G12D K-Ras4B-GDP. The likely reason is interactions between the aliphatic sidechain of V12 and the Switch II region of K-Ras4BG12V-GDP, which are distinct in K-Ras4BG12D-GDP. In the X-ray structures, crystal contacts reduce the dynamics of the sidechain at position 12 by stabilizing the Switch I region of the protein. This explains why structural differences between G12V and G12D K-Ras have yet not been reported. Together, our results suggest a previously unknown mechanism of K-Ras activation. This mechanism relies on conformational dynamics caused by specific oncogenic mutations in the GDP-bound state. Our findings also imply that the therapeutic strategies decreasing the level of K-Ras-GTP by interfering with nucleotide exchange or by expediting GTP hydrolysis may work differently in different oncogenic mutants.

SEMA3C Supports Pancreatic Cancer Progression by Regulating the Autophagy Process and Tumor Immune Microenvironment.

To date, driver genes for pancreatic cancer treatment are difficult to pursue therapeutically. Targeting mutated KRAS, the most renowned driver gene in pancreatic cancer, is an active area of study. We discovered a gene named SEMA3C was highly expressed in pancreatic cancer cell lines and patients with a G12D mutation in KRAS. High expression of SEMA3C in patients was significantly associated with the decreased survival of pancreatic cancer patients based on the TCGA database. In pancreatic cancer cells, SEMA3C knockdown or inhibition exhibited growth/colony inhibition and cell cycle arrest. In addition, SEMA3C inhibition sensitized KRAS or MEK1/2 inhibition in pancreatic cancer cells. Overexpression of SEMA3C resulted in the induction of autophagy, whereas depletion of SEMA3C compromised induction of autophagy. SEMA3C modified the PD-L1 expression in tumor and immune cells and is correlated with the M2-like macrophage marker ARG1/CD163 expression, which could reshape the tumor microenvironment. Inhibition of SEMA3C decreased tumor formation in the xenograft model in vivo. Taken together, our data suggest that SEMA3C plays a substantial role in promoting cancer cell survival by regulating the autophagy process and impacting the tumor environment immune response. SEMA3C can be used as a novel target or marker with therapeutic or diagnostic potential in pancreatic cancer especially in tumors harboring the specific KRAS G12D mutation.

Abstract 1038: Novel therapy targeting mutant-KRASG12D and galectin-1 in pancreatic cancer

Abstract Introduction: In pancreatic ductal adenocarcinoma (PDAC), low patient survival rate remains a problem. The activating point mutation of KRAS on codon-12 is present in 70-95% of PDAC cases and so far, no success has been achieved to inhibit KRAS. KRASG12D regulates cell proliferation, differentiation, apoptosis; recent preliminary and published studies show high Galectin-1 (Gal-1) levels in both PDAC and stromal cells, which modulate tumor microenvironment and metastasis. Therefore, we have developed a novel combination therapy for PDAC by targeting mutated KRASG12D and Gal-1 to target both proliferation and metastasis in PDAC. This includes the delivery of KRASG12D inhibiting siRNA (siKRASG12D) using a superparamagnetic iron oxide nanoparticle (SPION) and a galectin inhibitor. Methods: Our patented SPION nano-formulation was used to deliver siKRASG12D and investigated in conjunction with Gal-1 inhibitor for its anticancer efficacy. Particles were investigated for size, physico-chemical characterization (Dynamic light scattering), hemocompatibility (hemolysis assay) and the complexation of siKRAS (gel retardation assay). Cellular internalization and uptake of the particles were investigated. Anti-cancer efficacy was determined using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), clonogenicity, tumor spheroid formation, and in a mouse model. Results: Our results demonstrate optimal particle size/zeta potential of SP-siKRAS formulation. SP-siKRAS efficiently internalized in PDAC cells and suppressed KRASG12D as well as its downstream targets, YAP and PDL-1. Combined targeting of siKRAS and Gal-1 inhibited cell proliferation. It inhibited cell proliferation, clonogenicity, migration, and invasion of PDAC cells. This resulted in activation of death related mechanisms, such as Bax, bcl-2, PARP cleavage in KRASG12D cells. Interestingly, the formulation was highly effective in inhibiting KRASG12D and growth of tumor spheroid in 3D cell models, which recapitulate the heterogeneity and pathophysiology of PDAC. This further provides a clinical validation demonstrating potential of SP-siKRAS particles to efficiently silence KRAS expression. SP-siKRAS also exhibited hemocompatibility and stability suggesting its potential of silencing KRAS without being toxic to the body. The formulation efficiently exhibited KRasG12D silencing and inhibited tumor growth and metastasis in nude mice. Conclusion: This gene therapy targeting KRAS G12D mutation with a Gal-1 inhibition has a potential to modulate the oncogenic network and tumor microenvironment resulting in the repression of growth, metastasis, chemoresistance, and improvement in patient survival. This study will develop a novel sustainable therapeutic approach to target PDAC growth and improve patient survivability. Citation Format: Ana I. Martinez Bulnes, Poornima Shaji, Nirnoy Dan, Melida Cantu, Shabnam Malik, Stephen Behrman, Swathi Holla, Murali Mohan Yallapu, Meena Jaggi, Subhash C. Chauhan, Sheema Khan. Novel therapy targeting mutant-KRASG12D and galectin-1 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1038.

Abstract 2975: RAS precision medicine transatlantic partnership: Exploration of RAS and NF1 co-mutations in NSCLC

Abstract Background: RAS is the most commonly mutated oncogene in cancer, with KRAS mutated in ~30% of non-small cell lung cancer (NSCLC). RAS is a small GTPase cycling between GTP-bound ‘ON’ state and GDP-bound ‘OFF’ state. KRAS oncoproteins cycle between these states via hydrolysis and nucleotide exchange with the aid of GAPs, including NF1, and GEFs. Given the ’inactive state’ inhibition of recently developed KRAS G12C inhibitors on the GDP-bound state, this has highlighted the continued reliance of KRAS-mutants upon cycling, and the potential for mutants to retain dependence upon upstream influences, including NF1, for pathogenicity. Methods: 474 patients with advanced RAS- and/or NF1-mutant NSCLC were retrospectively identified from four tertiary cancer centers between 2008 -2021. DNA from archival FFPE samples, serum or combination underwent targeted NGS panels to identify mutations. Molecular, clinical, pathological and treatment outcome data were collected. Online resources including cBioPortal and Project Achilles were used to assess the functional role of any findings. Results: KRAS mutations were identified in 416/474 patients and NF1-mutations in 63/474 patients, eight of whom harbored two NF1-mutations. 24/63 (38%) of NF1-mutant cancers had a concomitant KRAS-mutation. We identified that KRAS G13D was more prevalent in NF1 mutant cancers vs. NF1 wildtype (NF1 MT: 6/24, 25%; vs. NF1 WT: 4/281, 1.4%; p&amp;lt;0.0001). KRAS G12C was identified in 11/24 (45.8%) of the double mutants vs. 109/282 (38.7%) of the NF1 WT patients (p=0.52). Those with G13D/NF1 co-mutation had a predicted pathogenic NF1-mutation in 5/6 (83%) of cases. Functional analysis of NF1 KO in G13D mutant lung cancer cell lines identified that NF1 was more essential in G13D mutant cell lines, with median Chronos score -0.26 G13D vs. -0.04 G12C (p=0.02). mRNA expression data identified a range of mRNA expression Z-scores relative to all samples for NF1-mutant cancers, ranging from lowest expression at -4.54 to +1.63, median -0.51. A higher mRNA expression was identified for NF1 missense mutations of unknown significance compared to truncating mutations with likely pathogenic, loss of function. Conclusions: These results highlight the co-mutational landscape of KRAS G13D with NF1 in NSCLC, suggesting functional importance conferred by NF1 loss, and highlight NF1 mutations as an additional pathogenic even when combined with KRAS mutation. The genomic landscape of KRAS and NF1 mutant NSCLC will be explored further through Whole Genome Sequencing data from the 100,000 Genomes Project (Genomics England). Citation Format: Helen M. Adderley, Mihaela Aldea, Jaqueline Aredo, Mathew Carter, Matthew Church, Pantelis Nicola, Jamie Weaver, Aisha Ghaus, Damien Vasseur, Matthew Krebs, Nicola Steele, Fiona Blackhall, Heather Wakelee, Benjamin Besse, Colin Lindsay. RAS precision medicine transatlantic partnership: Exploration of RAS and NF1 co-mutations in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2975.