KRAS G13D Mutation Research Articles

Exploring the Role of Hyperinsulinemia in Obesity-Associated Tumor Development.

Although there has been a long-standing connection between hyperinsulinemia and cancer development, there is a lack of understanding of the role of the insulin receptor on cells that can become cancerous. In a recent issue of Cell Metabolism, Zhang and colleagues, using a diet-induced obesity mouse model, identified a direct function of insulin receptors on pancreatic acinar cells expressing a KRASG12D mutation in promoting obesity-associated pancreatic cancer. Furthermore, insulin receptor signaling from hyperinsulinemia promoted the secretion of digestive enzymes that contributed to acinar to ductal metaplasia. These findings highlight an important connection between obesity, diabetes, and pancreatic tumor development and suggest potential strategies for obesity-associated cancer prevention targeting the insulin receptor signaling pathways.

A case description of pulmonary enteric adenocarcinoma with KRAS G12D mutation.

A case description of pulmonary enteric adenocarcinoma with KRAS G12D mutation.

NLRP3 inflammasome activation and symptom burden in KRAS-mutated CMML patients is reverted by IL-1 blocking therapy

Chronic myelomonocytic leukemia (CMML) is frequently associated with mutations in the rat sarcoma gene (RAS), leading to worse prognosis. RAS mutations result in active RAS-GTP proteins, favoring myeloid cell proliferation and survival and inducing the NLRP3 inflammasome together with the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which promote caspase-1 activation and interleukin (IL)-1β release. Here, we report, in a cohort of CMML patients with mutations in KRAS, a constitutive activation of the NLRP3 inflammasome in monocytes, evidenced by ASC oligomerization and IL-1β release, as well as a specific inflammatory cytokine signature. Treatment of a CMML patient with a KRASG12D mutation using the IL-1 receptor blocker anakinra inhibits NLRP3 inflammasome activation, reduces monocyte count, and improves the patient's clinical status, enabling a stem cell transplant. This reveals a basal inflammasome activation in RAS-mutated CMML patients and suggests potential therapeutic applications of NLRP3 and IL-1 blockers.

Abstract B116: Preclinical characterization of orally bioavailable, highly potent pan-KRAS inhibitors with selectivity over HRAS and NRAS

Abstract KRAS is altered in ~16% of all cancers and is an oncogenic driver in non-small cell lung, pancreatic, colorectal, and other cancers. Next generation KRAS inhibitors designed to target multiple oncogenic KRAS mutations, while simultaneously sparing wild-type (WT) HRAS and NRAS inhibition, are expected to offer expanded activity and favorable safety. We have discovered a series of highly potent and selective pan-KRAS inhibitors with activity against KRAS G12C, G12D, and G12V mutants, that also display high selectivity over WT HRAS and NRAS, thus providing an expanded therapeutic index. Here, we describe the preclinical profile of these pan-KRAS inhibitors. Compound potency and selectivity were measured using surface plasmon resonance (SPR) assays and cell-based assays measuring inhibition of p-ERK and 3D cell growth of KRAS-mutant tumor cell lines. These pan-KRAS inhibitors have IC50 values ranging from 3-14 nM for KRAS G12C, G12D, G12V, and WT KRAS in phospho-ERK cell-based assays and selectivity of >200-fold over NRAS WT and >100-fold over HRAS WT. These pan-KRAS inhibitors show a clean safety profile in a 133 off-target panel screen. These pan-KRAS inhibitors demonstrate favorable in vitro ADME properties and oral bioavailability in preclinical species. Tumor growth inhibition and PK/PD studies were performed in mice. In vivo, the pan-KRAS inhibitors administered orally demonstrated dose-dependent target inhibition in KRAS-mutant xenograft models. These data demonstrate that our pan-KRAS inhibitors potently and selectively inhibit KRAS G12D, G12C, and G12V mutations and WT KRAS, while sparing HRAS, NRAS, and other off-targets. We hypothesize that this potency and selectivity profile, along with high oral bioavailability, will provide efficacy and tolerability for patients with KRAS-mutant-driven cancers. An IND submission is planned in 2024. Citation Format: Lourdes Prieto Vallejo, Chandrasekar Iyer, Noelle Goggin, Binghui Li, Peiyi Yang, Huimin Bian, Jessica Podoll, Stefan Grotegut, Manuj Tandon, Bryan D Anderson, Andrew Capen, Min Xiao, Tao Wang, Trent R Stewart, Sean Aronow, Desta Bume, Isabel Rojo Garcia, Chong Si, Andrew Cooke, Robert Bondi, Lakshmi Kelamangalath, Ross Wallace, Gabrielle Kolakowski, Lauire LeBrun, James R Henry, Tim Kercher. Preclinical characterization of orally bioavailable, highly potent pan-KRAS inhibitors with selectivity over HRAS and NRAS [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B116.

Abstract A094: Preclinical characterization of HMPL-415, a second-generation SHP2 inhibitor

Abstract Background: Oncogenic activation of the RAS/MAPK signaling pathway is one of the leading causes for driving a variety of cancers. Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) functions downstream of multiple RTKs, and integrates growth factor signals to promote RAS activation, making it an attractive drug target for cancers harboring oncogenic alterations in RAS/MAPK pathway. Herein, we present the preclinical characterization of HMPL-415, a highly potent, selective and non-competitive SHP2 inhibitor, discovered and being currently developed in phase I clinical trial (NCT05886374) by HUTCHMED. Methods: The inhibition of HMPL-415 on the full length and catalytic domain of SHP2 was detected by a biochemical DIFMUP pseudosubstracte-fluorgenic assay. For selectivity, HMPL-415 was assessed against a panel of 413 kinases and 21 phosphatases by Eurofins. The modulation of HMPL-415 on RAS/MAPK signaling cascades was evaluated by western blot. In vitro anti-proliferation activity was measured by CellTiter-Glo luminescent assay. Multiple human xenograft tumors with RAS/MAPK pathway activation were applied in immune-deficient mice to investigate the anti-tumor activity of HMPL-415. Results: In vitro, HMPL-415 displayed higher potency in enzyme (5 fold) and cellular target inhibition (> 20 fold) compared to first-generation SHP inhibitor TNO155. It strongly inhibited full length of human SHP2 with an IC50 of 0.4 nM, while had no inhibitory activity up to 10 µM against the catalytic domain of SHP2. In NCI-H358 (KrasG12C), NCI-H508 (class Ⅲ BRAFG596R), and NCI-H1838 (NF1N184fs) cells, HMPL-415 inhibited p-ERK with IC50s of 1~3 nM, and significantly suppressed downstream molecules p-RSK and p-S6. Through inhibition of RAS/MAPK signaling pathway, HMPL-415 robustly inhibited cell proliferation in a panel of human cancer cell lines with KRAS G12C/V, BRAF class Ⅲ, NF1 loss of function (LOF), RTK mutations or KRAS amplification (median GI50 =1 nM) compared to cell lines harboring KRAS G12D, G13 or Q61 mutations (median GI50 >1 µM), indicating a dependence on specific KRAS nucleotide-cycling for anti-tumor effect. Furthermore, the selectivity across panels of 413 kinases and 21 phosphatases revealed that HMPL-415 should have a low off-target risk. In vivo, oral administration of HMPL-415 showed prolonged and high tumor exposure and sustained inhibition on RAS/MAPK signaling after repeat dosing. In human xenograft models carrying KRAS, BRAF class Ⅲ, NF1LOF, and EGFR alterations, HMPL-415 dose-dependently induced tumor growth inhibition at continuous daily doses that were well tolerated. Tumor regression was observed in most tumor models tested at the dose of 3 mg/kg/day, which demonstrated much superior anti-tumor activity than that of TNO155 at 30 mg/kg/day. Conclusion: HMPL-415 is a highly potent, selective and non-competitive SHP2 inhibitor that demonstrates strong anti-tumor activity in vitro and in vivo, supporting further clinical evaluation. Citation Format: Jia Hu, Jun Ni, Zhihu Gao, Xiaoqing Liu, Hui Zhang, Pan Wang, Min Cheng, Guanglin Wang, Zeyu Zhong, Jian Wang, Yang Sai, Na Yang, Weiguo Qing, Yongxin Ren, Michael Shi, Weiguo Su. Preclinical characterization of HMPL-415, a second-generation SHP2 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A094.

Abstract LB_C09: QTX3034, a potent and selective multi-KRAS inhibitor, synergizes with EGFR inhibitors and enhances anti-tumor activity

Abstract KRAS, one of the most frequently mutated oncogenes in cancer, was considered undruggable until the development of covalent KRASG12C inhibitors. While these KRASG12C-targeted therapies exhibit clinical benefit, they do not address the most common KRAS mutant variants. QTX3034 is a potent, highly selective, orally bioavailable allosteric multi-KRAS inhibitor that is efficacious against a broad spectrum of the more common KRASG12D- and KRASG12V-mutated cancers in vitro and in vivo. In an effort to enhance anti-tumor activity of QTX3034 and to minimize the development of possible resistance mechanisms, we explored the potential synergy of combining QTX3034 with EGFR inhibitors in multiple KRAS-mutated tumor models. Cell viability assays over a 7-day period revealed that QTX3034 synergized with three different EGFR inhibitors (afatinib, cetuximab, erlotinib) to profoundly inhibit proliferation across multiple KRASG12D, KRASG12V, and KRASWT Amp mutant cell lines with strong ZIP synergy scores (>10). Combination of QTX3034 and afatinib exhibited a markedly greater inhibitory effect on KRAS-mutant colony formation compared to the respective single agents. Analysis of RAS downstream signaling revealed that the combination of QTX3034 and afatinib produced a more potent suppression of the MAPK pathway than either single agent alone in KRAS-mutant cells. Moreover, QTX3034 monotherapy induced a dose-dependent increase in EGFR phosphorylation in the KRASG12D-mutated cell line GP2D, while co-administration with afatinib robustly inhibited this EGFR activation. Combining QTX3034 and cetuximab elicited durable tumor regressions in KRASG12D-mutant cell line-derived xenograft models. Furthermore, tumor regressions in these combination studies were obtained using meaningfully lower doses of QTX3034 than needed to achieve tumor regression by monotherapy. Similar combinations improved QTX3034 anti-tumor activity in patient-derived xenograft models that exceeded monotherapy with either agent. Taken together, these data support the rationale to co-administer QTX3034 and EGFR inhibitors to optimize therapeutic benefit for patients with KRAS-mutated cancers. Citation Format: Yang J. Zhang, Jillian M. Silva, Yang W. Zhang, Elizabeth Donohue Vo, John M. Micozzi, Douglas Krauth, Arghyotri Sinha, Ben Reid, Brooke McDonough, Gregory M. Lee, Christopher C. Pan, Audrey Hospital, Juan I. Luengo, Cameron Pitt, Hong Lin. QTX3034, a potent and selective multi-KRAS inhibitor, synergizes with EGFR inhibitors and enhances anti-tumor activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C09.

Abstract A127: Identification of therapeutic drug combinations targeting KRAS

Abstract KRAS is the most frequently mutated oncogene and increased development efforts targeting KRAS mutations have yielded recent FDA approvals of two KRAS G12C inhibitors. However, for some indications, there are other KRAS mutations more common than G12C. For Pancreatic ductal adenocarcinoma (comprising more than 90% of pancreatic cancer cases), 94% harbor KRAS mutations, where G12D mutations account for around 45% while G12C mutations are rare (1%). Hence, there is a need to develop methods to target a broader range of KRAS mutations. Beyond direct gene mutation, activation of oncogenic KRAS and downstream MAPK signaling through other genomic alterations may benefit from KRAS-specific therapeutic strategies, especially combination therapy. We have developed a platform, Optim.AI™, that rationally converges upon optimal drug combinations within a defined drug search space, without any prior knowledge of the background of the models. In this study, we utilized the platform to identify efficacious drug combinations that contain a histone deacetylase 4/6 inhibitor, KYAN-001 in the context of KRAS mutations. 7 cell lines (6 prostate and pancreatic cancer with normal, epithelial THLE-2 cell line) were evaluated with Optim.AI. The cancer cells and THLE-2 were seeded into 384-well plates and treated with 155 different combinations consisting of indication-specific drugs the following day. Post-drug treatment cell viability was used for Optim.AI™ analysis, which maps experimental data points to a second-order quadratic function to predict and rank cancer cell killing efficacies for all other permutations of drug combinations. In androgen resistant prostate cancer, Optim.AI identified synergistic interaction between KYAN-001 and a MEK inhibitor. With MEK being downstream of the KRAS pathway, we wanted to evaluate the efficacy of this combination on pancreatic cancer. Interestingly, this combination was demonstrated to be top ranked against PANC-1 and AsPC-1 cell lines, both harboring G12D KRAS mutation, as compared to a cell line with wild-type KRAS, BxPC-3. This was further validated via half-maximal inhibitory curves (IC50), where synergism was also observed for the combination on AsPC-1, but not BxPC-3. KYAN-001 was also shown to exhibit monotherapy sensitivity against pancreatic cancer patient-derived organoids (PDOs), with a mean of 4 µM. This study has successfully demonstrated the ability of Optim.AI™ to identify novel, optimal drug combinations for both prostate and pancreatic cancers, without any prior background knowledge on these cell lines. With MEK being downstream of the KRAS pathway and 94% of those diagnosed with pancreatic cancer have KRAS mutations, the differential sensitivities of this combination across the cell lines presents the utility of the platform in precision medicine, especially for undruggable targets like KRAS. With promising and demonstrated monotherapy efficacy of KYAN-001 on PDOs, further experiments could be done to evaluate the clinical relevance of both KYAN-001 and MEK inhibition on other relevant ex vivo models. Citation Format: Masturah Mohd Abdul Rashid, Lisa Chow, Edward Kai-Hua Chow. Identification of therapeutic drug combinations targeting KRAS [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A127.

Abstract B083: Secondary KRAS mutations lead to acquired resistance to KRASG12D inhibitor in colorectal cancer

Abstract KRAS is the most frequently mutated oncogene, with high prevalence in tumor types with a significant unmet clinical need, such as colorectal cancer (CRC). Activating mutations in the KRAS gene leading to amino acid exchange at position 12 from Glycine to Asparagine, G12D, occur in approximately 12.5% of CRC. The approval of mutant specific KRASG12C inhibitors MRTX849 and AMG510 by the FDA raised the question if KRAS targeted therapy approaches could provide benefit to cancer patients with other mutant alleles. Several KRASG12D inhibitors have recently been described and MRTX1133, a selective KRASG12D inhibitor from Mirati Therapeutics, has now entered clinical trial, recruiting patients with advanced solid tumors harboring a KRAS G12D mutation. Alongside the great hopes placed in KRAS targeting therapy, resistance is likely to occur, as it was observed in patients relapsing in response to KRASG12C inhibitors. We aimed to use this pre-clinical study to predict and understand potential resistance mechanisms that may arise during targeted treatment against KRASG12D in the CRC setting. To this aim, the colorectal cancer cell line GP2d was continuously treated with MRTX1133 to generate resistance and individual outgrowing clones were subsequently profiled in a series of assays to identify potential mechanism(s). These assays included analysis of gene expression changes, mutation calling, as well as qPCR and protein-based assays. Interestingly, we identified two individual secondary on-target mutations in the KRAS gene, which could affect binding of MRTX1133 to the pocket in KRAS. These findings were confirmed in an orthogonal assay, by screening for resistance in Ba/F3 cells transduced with the corresponding KRAS double mutant. Combination assays were then performed in parental and resistant cells to study whether additional vertical or alternative pathway inhibition could address the resistance. Collectively our pre-clinical study identified secondary KRAS mutations in the compound binding pocket as a mechanism of resistance to KRAS G12D inhibition by MRTX1133 in colorectal cancer cells. Citation Format: Simone Lieb, Sabine Jurado, Sergej Nowoshilow, Krzysztof M. Zak, Marco H. Hofmann. Secondary KRAS mutations lead to acquired resistance to KRASG12D inhibitor in colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B083.

Abstract B029: Investigating therapeutic strategies to promote immune rejection of KRAS G12C inhibitor-resistant sub-populations in lung cancer

Abstract Oncogenic KRAS signaling not only instigates aberrant growth in cancer cells but can further induce inhibitory effects on anti-tumor immune responses. Hence, recently developed KRAS G12C inhibitors (G12Ci) have the potential to alleviate immune suppression in the tumor microenvironment (TME) and reshape the therapy responses for patients with a KRAS G12C driver mutation. However, rewiring of oncogenic signaling circuits and acquisition of further mutations allows cancer cells to rapidly evolve drug resistance. In fact, clinical trial data from the recently approved G12Ci, adagrasib and sotorasib, showed a median progression-free survival in non-small cell lung cancer of just 7 months. In this project, we investigate how to combat G12Ci-resistant subpopulations in lung tumors by engaging an anti-tumor immune response. We mimic the development of drug resistance by combining traced isogenic G12C- and G12D-mutant lung cancer cells, where the KRAS G12D mutant cells are inherently resistant to G12Ci. By alleviating RAS-driven immune suppression and potentially inducing immunogenic cell death of G12Ci-responsive cells, we aim to further potentiate an adaptive immune response targeting the drug-resistant population. We found that G12Ci as monotherapy drives therapy resistance by giving the drug-resistant subpopulation a proliferative advantage. On the other hand, combining G12Ci with immunotherapy or a SHP2 inhibitor potentiated an adaptive immune response to induce complete eradication of both G12C and G12D KRAS mutant cancer cell populations and also immune memory towards drug-resistant KRAS G12D cells in mice. However, when repeating the combination treatment in Rag1 −/− mice, which lack B- and T-cells, there were no complete responders. Histopathological analysis of tumor sections in immune-competent mice revealed a strong T-cell infiltration in the TME of tumors treated with combination therapies. Although understanding the mechanistic changes in the TME induced by combination therapies is ongoing, these results indicate a role for the adaptive immune response in targeting G12Ci drug-resistant subpopulations. Understanding these underlying mechanisms will contribute to combating the development of drug resistance in patients by enhancing treatment regimens that promote immune-mediated destruction of drug-resistant tumor cell sub-populations. Citation Format: Mona Tomaschko, Miriam Molina-Arcas, Christopher Moore, Sareena Rana, Sophie de Carne, Panayiotis Anastasiou, Claire Pillsbury, Andrea de Castro, Julian Downward. Investigating therapeutic strategies to promote immune rejection of KRAS G12C inhibitor-resistant sub-populations in lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B029.

Stiffness-induced cancer-associated fibroblasts are responsible for immunosuppression in a platelet-derived growth factor ligand-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is associated with a vast stromal reaction that arises mainly from cancer-associated fibroblasts (CAFs) and promotes both immune escape and tumor growth. Here, we used a mouse model with deletion of the activin A receptor ALK4 in the context of the KrasG12D mutation, which strongly drives collagen deposition that leads to tissue stiffness. By ligand-receptor analysis of single-cell RNA-sequencing data, we identified that, in stiff conditions, neoplastic ductal cells instructed CAFs through sustained platelet-derived growth factor (PDGF) signaling. Tumor-associated tissue rigidity resulted in the emergence of stiffness-induced CAFs (siCAFs) in vitro and in vivo. Similar results were confirmed in human data. siCAFs were able to strongly inhibit CD8+ T-cell responses in vitro and in vivo, promoting local immunosuppression. More importantly, targeting PDGF signaling led to diminished siCAF and reduced tumor growth. Our data show for the first time that early paracrine signaling leads to profound changes in tissue mechanics, impacting immune responses and tumor progression. Our study highlights that PDGF ligand neutralization can normalize the tissue architecture independent of the genetic background, indicating that finely tuned stromal therapy may open new therapeutic avenues in pancreatic cancer.

Abstract A111: Impact of KRAS mutations and co-mutations on clinical outcomes in Pancreatic Ductal Adenocarcinoma

Abstract KRAS is the most frequently mutated gene in pancreatic ductal adenocarcinoma (PDAC). Over the past decade, targeting KRAS has emerged as a promising approach for PDAC treatment. However, the associations between KRAS mutations status and comutation landscape with clinical outcomes remain understudied. We investigated the impact of KRAS mutant allelic status and genomic co-mutations with clinical outcomes. 803 patients with PDAC who underwent CLIA somatic tumor mutation testing at MDACC from 1997 to 2023 were identified after an IRB approval. Tumor genomic alterations, demographics, clinical, molecular, and pathological data were extracted from electronic medical records and tumor registry through the Palantir Foundry platform. An external cohort from PanCAN’s Know Your Tumor® dataset with 408 patients served as the validation cohort. Overall Survival (OS) was calculated from the date of initial diagnosis to death or last follow-up. Pearson’s chi-squared tests (or Fisher’s exact tests) were used to determine the association between groups. The Kaplan-Meier method was used to estimate the median OS. 703 patients had KRAS gene included in the mutation testing at MDACC from 1997 to 2023. KRAS was the top mutated gene (82%), followed by TP53 (69%), then CDKN2A (20%). In KRAS mutant patients, G12D was the most common variant (39%), followed by G12V (31%), then G12R (14%), and Q61 (6%). In patients tested with multi-gene panels, a co-mutation analysis revealed that the most common co-occurring mutations with KRAS mutation was TP53 (75%), followed by CDKN2A (22%), SMAD4 (14%) and ARID1A (10%). The median follow-up was 41 months and median OS was 19 months in the overall population. KRAS mutation status and subtypes was prognostic of OS (p<0.001). Patients with KRAS wildtype had median OS of 38 months and KRASG12R had median OS of 34 months (HR:1.0, 95% CI 0.71-1.5, p=0.88), while patients with KRASQ61 had worst median OS of 20 months (HR: 1.9, 95% CI 1.2-3.0, p=0.006) and KRASG12D had median OS of 22 months (HR: 1.7, 95% CI 1.3-2.3, p<0.001). Similar findings were observed in patients with stage IV disease (n=302), KRASG12D remained associated with significantly worse OS (HR= 1.7, 95% CI 1.1-2.6, p=0.009). There was a significant enrichment of KRASG12D mutation in metastatic disease compared to localized disease (stage I-III) (OR: 1.7, 95% CI 1.2-2.4, p=0.001) and enrichment of KRASG12R mutation in well and moderately differentiated tumors versus poorly differentiated tumors (OR: 1.7, 95% CI 1.05-2.99, p=0.04). In the external cohort (PanCAN’s Know Your Tumor® dataset), similar findings were observed. KRASG12R was associated with longer median OS (32 months), while KRASQ61 (16 months, HR: 2.6, 95% CI 0.88-7.8, p=0.02) and KRASG12D (23 months, HR: 1.68, 95% CI 1.06-2.65, p=0.04) were associated with significantly worse median OS. Allele specific associations of KRAS mutation with clinical outcomes were identified in PDAC. We found better OS with G12R and worse OS with G12D and Q61. Citation Format: Abdelrahman Yousef, Mahmoud Yousef, Saikat Chowdhury, Kawther Abdilleh, Mark Knafl, Paul Edelkamp, Kristin Alfaro-Munoz, Ray Chacko, Jennifer Peterson, Brandon Smaglo, Robert Wolff, Shubham Pant, Michael Lee, Jason Willis, Michael Overman, Sudheer Doss, Lynn Matrisian, Mark Hurd, Rebecca Snyder, Matthew Harold Katz, Huamin Wang, Anirban Maitra, John Paul Shen, Dan Zhao. Impact of KRAS mutations and co-mutations on clinical outcomes in Pancreatic Ductal Adenocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A111.

Abstract B023: The next-generation farnesyltransferase inhibitor KO-2806 constrains compensatory signaling reactivation to deepen responses to KRASG12D inhibition

Abstract The landmark approval of KRASG12C inhibitors toppled the decades-held notion that KRAS was undruggable, fueling the design and development of small molecules targeting other oncogenic KRAS mutants, including the most prevalent, KRASG12D. MRTX1133 is the first KRASG12D-selective inhibitor to reach the clinic, providing a much-needed therapeutic option for patients with KRASG12D-mutant tumors. Nevertheless, both preclinical MRTX1133 data and early clinical results from KRASG12C inhibitor trials suggest that responses to KRASG12D inhibition will vary widely, at least in part due to incomplete and/or transient inhibition of MAPK and mTOR signaling. We previously demonstrated that farnesyltransferase inhibitors (FTIs) can prevent adaptive resistance to PI3K inhibition by blunting compensatory reactivation of these signaling nodes. We therefore hypothesized that farnesyltransferase inhibition may similarly enhance the antitumor activity of MRTX1133 through synergistic blockade of mitogenic and survival signaling. In this study, we leveraged a panel of in vivo cell line (CDX) and patient-derived xenograft (PDX) models, in parallel with in vitro mechanistic studies, to assess the efficacy of combined KO-2806, a FTI, and MRTX1133, in controlling KRASG12D-mutant tumor growth. KO-2806 is a next-generation FTI with increased potency and improved pharmacokinetic properties relative to earlier FTI candidates and is poised to enter a first-in-human clinical trial. Given that reactivation of phospho-ERK and phospho-S6 is linked to diminished response to MRTX1133, we first probed the impact of KO-2806 on cell signaling in vitro. In KRASG12D mutant cell lines, MRTX1133 transiently inhibited both ERK and mTOR, as evidenced by decreased ERK, p90 RSK, p70 S6K, S6, and 4EBP1 phosphorylation. However, phosphorylation of these proteins rebounded partially within one day and completely by 48 hours post treatment. In contrast, when cells were co-exposed to MRTX1133 and KO-2806, robust inhibition of these pathways was maintained at 48 hours and corresponded with greater induction of apoptosis (PARP and caspase cleavage) and cell cycle arrest (ablation of Rb phosphorylation) compared to MRTX1133. These results are consistent with prior reports suggesting that durable MAPK and mTOR inhibition is required for optimal response to KRAS inhibitors. The mechanistic synergy we observed in vitro provided a strong rationale for evaluating combined MRTX1133 and KO-2806 in vivo. Across a panel of KRASG12D-mutant CDX and PDX solid tumor models, the MRTX1133/KO-2806 doublet led to deeper tumor growth inhibition compared to the single agents while remaining well tolerated in mice. We posit that KO-2806 augments the MAPK-mTOR inhibitory effects of MRTX1133 by blocking compensatory pathway reactivation, resulting in superior antitumor efficacy of the combination without amplifying toxicity. Based upon these results, combined farnesyltransferase and KRASG12D inhibition represents a promising therapeutic strategy for the treatment of KRASG12D0-mutant solid tumors. Citation Format: Alison Smith, Hetika Patel, Stacia Chan, Francis Burrows, Shivani Malik. The next-generation farnesyltransferase inhibitor KO-2806 constrains compensatory signaling reactivation to deepen responses to KRASG12D inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B023.

The suppressive efficacy of THZ1 depends on KRAS mutation subtype and is associated with super-enhancer activity and the PI3K/AKT/mTOR signalling in pancreatic ductal adenocarcinoma: A hypothesis-generating study.

Inhibition of CDK7, a potent transcription regulator, may bring new hope for treating pancreatic ductal adenocarcinoma (PDAC), which is featured by large genetic heterogeneity and abundant KRAS mutations. This investigation aimed at exploring the discrepant efficacies of THZ1, a small-molecule covalent CDK7 inhibitor, on PDACs with different KRAS mutations and the underlying mechanisms. Associations of CDK7 expression with survival by KRAS mutations were first assessed. Effects of THZ1 on PDAC by different KRAS mutations were then investigated in vitro and in vivo. Moreover, the effects of THZ1 on gene transcription and phosphorylation of RNA polymerase II (RNAPOLII) in different KRAS mutant PDACs were assessed, and the effect of THZ1 on super-enhancer activity was evaluated using chromatin immunoprecipitation sequencing. Lastly, the effects of THZ1 on the binding of H3K27ac to PIK3CA and on the PI3K/AKT/mTOR signalling were analysed. High CDK7 expression was significantly linked to worse survival within PDAC patients carrying KRAS-G12V mutation but not in those with KRAS-G12D mutation. The apoptosis-inducing effect of THZ1 was markedly stronger in KRAS-G12V PDAC than KRAS-G12D cancer. THZ1 significantly inhibited the growth of xenograft tumour with KRAS-G12V mutation, and the inhibition was markedly stronger than for KRAS-G12D tumour. In mini-cell-derived xenograft (CDX) models, THZ1 significantly suppressed KRAS-G12V PDAC but not KRAS-G12D cancer. THZ1 significantly suppressed the phosphorylation of RNAPOLII, and this effect was stronger in KRAS-G12V PDAC (especially at ser5). KRAS-G12V PDAC had more H3K27ac-binding super-enhancers, and the inhibition of THZ1 on super-enhancer activity was also stronger in KRAS-G12V PDAC. Furthermore, THZ1 significantly weakened the binding of H3K27ac to PIK3CA in KRAS-G12V PDAC. THZ1 significantly suppressed the PI3K/AKT/mTOR pathway and its downstream markers, and this effect was stronger in KRAS-G12V cells. In this hypothesis-generating study, THZ1 might selectively inhibit certain PDACs with KRAS-G12V mutation more potently compared with some other PDACs with KRAS-G12D mutation, which might be associated with its effect on super-enhancer activity and the PI3K/AKT/mTOR signalling. Our findings might offer novel key clues for the precise management of PDAC and important evidence for future targeted trial design. THZ1 had a stronger effect on PDAC-bearing KRAS-G12V mutation than G12D mutation. Suppressive effect of THZ1 on phosphorylation of RNAPOLII was stronger in KRAS-G12V than KRAS-G12D PDAC. Inhibition of THZ1 on super-enhancer activity and H3K27ac binding to PIK3CA was stronger in KRAS-G12V PDAC. Suppressive effect of THZ1 on PI3K/AKT/mTOR pathway was stronger in KRAS-G12V PDAC.

Metastatic Mullerian adenocarcinoma involving the pancreas initially misdiagnosed as an intraductal papillary mucinous neoplasm (IPMN): A case report, and lessons learned

Abstract Introduction/Objective Metastases to the pancreas from Mullerian primaries are rare and can be confused with primary pancreatic malignancies. Methods/Case Report A 49-year-old woman presented with right lower abdominal pain. She denied any prior history of malignancy but had undergone hysterectomy and bilateral salpingo-oophorectomy for unknown reasons. CT scan revealed an 8.9 cm predominantly cystic mass in the pancreas, highly suspicious of an invasive mucinous neoplasm. EUS-guided FNA of the pancreas showed large, complex papillary fronds of mucinous epithelial cells in a background of abundant extracellular mucin, findings that were interpreted as an intraductal papillary mucinous neoplasm (IPMN) with foci of high-grade dysplasia and possible adenocarcinoma. A subsequent PET/CT scan demonstrated a 3.9 cm hepatic mass and a 3.8 cm perihepatic lesion. FNA of the liver was interpreted as metastatic pancreatic adenocarcinoma. Despite aggressive treatment, her disease progressed. Molecular profiling of her liver tumor demonstrated a pathogenic KRAS G12D mutation and a PDGFRA variant of uncertain significance. Genomic (DNA) and transcriptomic (RNA) analysis indicated a 96% probability that the patient had ovarian or fallopian tube adenocarcinoma and a 4% probability of uterine endometrial adenocarcinoma rather than metastatic pancreatic adenocarcinoma. A panel of immunocytochemical stains was performed on the pancreas and liver FNAs. The tumor cells were strongly positive for CK7, CK19, ER, and WT1, focally positive for PAX8 (nuclear) and P53 (wild-type), and negative for CK20, CDX2, and SATB2. Results (if a Case Study enter NA) NA Conclusion These findings corroborated the molecular analysis suggesting that the patient had metastatic Mullerian adenocarcinoma rather than metastatic pancreatic adenocarcinoma. Her oncologic treatment was adjusted accordingly.

PHRF1 Promotes Cell Invasion by Modulating SOX4 Expression in Colorectal Cancer HCT116-p53-/- Cells.

PHD and RING finger domain-containing protein 1 (PHRF1) ubiquitinates TGIP (TG-interacting protein) and redistributes cPML (cytoplasmic variant of PML) to the cytoplasm to enhance TGF-β signaling by. It is unclear whether PHRF1 affects invasion and survival when both mutations of the activated oncogene Kras and inactivation of the tumor suppressor p53 are present. We knockout PHRF1 expression using Crispr-Cas9 editing in HCT116-p53-/- (KrasG13D/p53-/-) cells and analyzed the expression profile in HCT116-p53-/-PHRF1-/- cells. In contrast to lung cancer A549 (KrasG12S/p53wt) cells, the expression of Zeb1, a transcription factor for epidermal-mesenchymal transition (EMT), was not affected in PHRF1-knockout HCT116 p53-/- cells. Instead, SOX4 displayed a significant contribution to the impaired invasion in HCT116-p53-/-PHRF1-/- cells. Mechanistically, the C-terminal SRI domain of PHRF1 was required for both transwell invasion and SOX4 expression. The reintroduction of SOX4 into HCT116-p53-/- PHRF1-/- cells partially restored their invasive capability. This study sheds light on the role of PHRF1 in the invasion of colorectal cancer HCT116-p53-/- cells, which harbor the oncogenic KrasG13D mutation and lack p53. These findings provide novel insights regarding the role of PHRF1 in invasion by modulating SOX4 expression in colorectal cancer HCT116-p53-/- cells.

N/KRAS-Mutant AML LSCs Originate from Committed Myelomonocytic Progenitors and Drive Clinical Resistance to Venetoclax

Driver mutations in acute myeloid leukemia (AML) often exhibit distinct temporal acquisition patterns, but the biological basis for this, if any, remains unknown. RAS mutations occur invariably late in AML, upon progression or relapsed/refractory disease, in contrast to their function as early drivers in solid tumors. We developed synthetic leukemogenesis models in human induced pluripotent stem cell (iPSC)- and primary cord blood (CB)-derived human hematopoietic stem/progenitor cells (HSPCs) by introducing the prototypical NRAS G12D mutation alone or with other mutational combinations, using CRISPR, and engraftment of a transplantable leukemia into NSG mice as the readout. RAS mutations alone were not sufficient for leukemogenesis, but required specific cooperating mutations. Subsequently, by creating temporally controlled engineered models, we found that RAS mutations are obligatory late events that can only drive leukemic transformation when acquired after, but not before, cooperating mutations. We provide the mechanistic explanation for this in a requirement for mutant RAS to specifically transform committed granulocyte-monocyte progenitors (GMPs) harboring previously acquired driver mutations, through sorting and transplantation and multi-omics experiments. In contrast, acquisition of RAS mutations in earlier progenitors (common myeloid progenitors, CMP), with or without cooperating mutations, blocked the emergence of GMPs and abolished engraftment or gave rise to a myeloproliferative neoplasm. These results indicate that advanced RAS-mutant (MT) leukemic clones have a different cell type of origin from the ancestral clone. Next, using single-cell transcriptomics in cells isolated from xenografts of two AML-iPSC lines generated from an AML patient with a subclonal KRAS G12D mutation, one capturing the RAS WT major clone and one the KRAS G12D subclone, we show that KRAS G12D leukemia stem cells (LSCs) give rise to leukemia with a more monocytic immunophenotype, while the ancestral clone within the same patient generates more leukemic cells with primitive and neutrophil markers. These results were corroborated by Genotyping of Transcriptomes (GoT) in another AML patient with subclonal NRAS G12D mutation. Recent studies have linked monocytic AML and, independently, RAS pathway mutations to poor responses to Venetoclax (VEN)-containing regiments. In view of the causal link we found between RAS mutations and monocytic disease, we evaluated the associations between monocytic AML, RAS mutations and response to VEN. We reanalyzed data from a cohort of 118 older/unfit newly diagnosed AML patients treated on a prospective clinical trial with VEN and decitabine (DEC) (NCT03404193). All outcomes were comparable between monocytic and non-monocytic groups, including overall response rate, duration of response (DOR) and overall survival. On the other hand, patients with N/KRAS mutations had significantly shorter DOR. These results support the presence of N/KRAS mutations and not the monocytic stage as predictors of inferior responses to VEN regimens. To definitely test this, we treated defined cell types generated from RAS WT and RAS MT iPSC lines - CD34+ LSC-like and CD11b+/CD68+/CD14+ monocytic cells - with VEN. In agreement with previous studies, monocytes were resistant and RAS WT LSCs were sensitive to VEN. However, strikingly RAS MT LSCs were VEN-resistant. Consistent with these, single-cell transcriptomics in patient-derived AML-iPSC cells in vivo, GoT in primary AML cells and bulk transcriptomics in synthetic iPSC-AML cells showed downregulation of BCL2 and upregulation of MCL1 in the RAS MT LSCs. This study provides the first example of a mechanistic explanation of why the timing of mutational acquisition in AML is so strongly biased. It shows for the first time that interaction between an oncogenic driver and the non-genetic developmental hematopoietic hierarchy imposes a specific LSC cell-of-origin restriction and in turn shapes the hierarchical structure of the resulting AML and critically determines therapeutic outcomes in patients. Importantly, our results have direct implications for clinical practice, as they support that RAS MT LSCs drive clinical resistance to VEN and that treatment with VEN in patients with RAS mutations can accelerate disease progression by selection of RAS MT LSCs (Figure).

Metabolomic, Proteomic, and Single-Cell Proteomic Analysis of Cancer Cells Treated with the KRASG12D Inhibitor MRTX1133.

Mutations in KRAS are common drivers of human cancers and are often those with the poorest overall prognosis for patients. A recently developed compound, MRTX1133, has shown promise in inhibiting the activity of KRASG12D mutant proteins, which is one of the main drivers of pancreatic cancer. To better understand the mechanism of action of this compound, I performed both proteomics and metabolomics on four KRASG12D mutant pancreatic cancer cell lines. To obtain increased granularity in the proteomic observations, single-cell proteomics was successfully performed on two of these lines. Following quality filtering, a total of 1498 single cells were analyzed. From these cells, 3140 total proteins were identified with approximately 953 proteins quantified per cell. At 48 h of treatment, two distinct populations of cells can be observed based on the level of effectiveness of the drug in decreasing the total abundance of the KRAS protein in each respective cell, with results that are effectively masked in the bulk cell analysis. All mass spectrometry data and processed results are publicly available at www.massive.ucsd.edu at accessions PXD039597, PXD039601, and PXD039600.

Deciphering the Role of RAS Pathway Mutations in the Biology of Human Acute Myeloid Leukemia Using In Vivo Models

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy for which the mutational profile of the leukemic cells is the most important prognostic factor for disease-free survival and overall survival. The failure of chemotherapy to completely eradicate the leukemic clones is the most common cause of death in AML. The genetic events that can contribute to chemoresistance are therefore important factors for the clinical outcome, but the relative importance of these events on chemoresistance and persistence of leukemia stem cells (LSC) remains poorly defined. MLL rearranged AMLs account for 5-10% of all AMLs and the RAS pathway is the most frequently mutated pathway in this sub-group of AMLs. The goal of this project is to study the role and biology of RAS pathway mutations in clonal evolution in human MLL-rearranged AML. Methods We have previously shown that human cord blood CD34+ cells transduced with a retrovirus encoding the MLL-AF9 fusion gene with an EGFP maker generate human AML when injected into immunodeficient NSG-SGM3 mice. Using a similar experimental design, we added a second hit to the CD34 transduced cells before their injection into NSG-SGM3 mice. MLL-AF9 cells were electroporated with Cas9 enzyme for the control and with Cas9 enzyme plus a single guide RNA (sgRNA) designed to induce the KRAS G13D or the NRAS G12D mutation. Mice were sacrificed when they showed sign of disease and the bone marrow and spleen were collected and analyzed. Kaplan-Meier curves, flow cytometry analyses, RNA and exome sequencing and secondary transplantation were performed on the samples. Results Mice injected with MLL-AF9 + KRAS G13D got sick around day 80 after injection and around day 60 for MLL-AF9 NRAS G12D, whereas it took ~150 days for control mice (MLL-AF9 KRAS WT) to get sick (fig 1). In multiple experiment, the latency was roughly cut by half by the KRAS G13D and NRAS G12D mutations. The cell surface phenotype and morphology were not significantly different between MLL-AF9 WT and MLL-AF9 KRAS/NRAS mutated. The variant allele frequency (VAF) for the KRAS mutation invariably increased from the day of injection to the day of sacrifice, where the percentages varied between 20-40%, which is typically observed in AML patients. In contrast, the VAF for NRAS mutated cells was lower (3-9%) but seems to be very aggressive into mice. We performed limiting dilution analyses in secondary mice to quantify the LSC frequencies in MLL-AF9 WT and KRAS G13D / NRAS G12D leukemia. The average frequencies of control MLL-AF9 AMLs were between 1/510 000 to 1/1 450 000, while MLL-AF9+KRAS G13D AMLs frequencies were >1/45 000 and 1/7 800 for MLL-AF9+NRAS G12D AMLs, demonstrating that these 2 mutations increase the LSC frequency by >20-100 fold in our modelled MLL-AF9 AML. Using transcriptome analyses, we looked at the gene expression changes induced by the KRAS G13D mutation in comparison to MLL-AF9 leukemias. These experiments are in progress for the NRAS G13D mutation. Many more genes were overexpressed in MLL-AF9 KRAS G13D vs MLL-AF9 KRAS WT than down regulated. Genes from the hedgehog (Hh) pathway such as Patched1 (PTCH1) and Smoothened (SMO) were amongst the genes the most differentially overexpressed by the KRAS mutation. It is particularly interesting because they have been shown important in stem cell biology and could explain the increase in LSC. Some SMO inhibitors have been tested in clinical trial and Glasdegib is FDA approved in combination with low dose cytarabine in elderly patients. Its effect on MLL leukemias or on KRAS mutated AMLs has not been tested/reported in a clinical study. Experiments are currently in progress to test the effect of Glasgebib on engraftment of MLL-AF9 KRAS mutated cells in our experimental setting. Single cell RNA sequencing data on the three conditions will be presented at the meeting. Given the success of generating these two point mutations, we are currently generating more RAS pathway mutations, including other KRAS mutations, PNTP11 and NF1 mutations. Conclusion These experiments showed: 1) It is possible to induce 2 oncogenic hits in human primary cells and get leukemia in vivo; 2) the KRAS G13D and NRAS G12D mutations shorten the latency of the disease and 3) increase the LSC frequency in secondary mice; 4) a possible involvement of the Hh pathway on stemness/LSC in RAS mutated cells; 5) our experimental approach is robust and very promising to decipher the RAS pathway in human MLL leukemias.

Platelets Sequester Extracellular DNA, Capturing Tumour-Derived and Free Fetal DNA

Introduction & Hypothesis Platelets are small, multi-functional cells that lack a nucleus but contain RNA and translational machinery for protein synthesis. Platelet RNA mostly derives from parent megakaryocytes, but they also sense and sequester endogenous and pathogen-derived nucleic acids during circulation (D'Ambrosi, 2021; Koupenova, 2019). Nucleated cells release DNA after cell death or aberrant mitosis, resulting in ‘cell-free’ DNA in plasma (cfDNA). Excess cfDNA is deleterious. cfDNA isolated from platelet-poor plasma is emerging as a major liquid biopsy tool in cancer and antenatal screening, but a major limitation is its low abundance, especially in early-stage disease. Given their role in sensing pathogen-derived nucleic acids, we hypothesized that platelets may clear cfDNA from plasma, and that clinically-relevant insights may be derived from the analysis of DNA fragments contained in platelets. Methods Live/fixed imaging using specific DNA probes and fluorescence in-situ hybridization (FISH), droplet digital PCR (ddPCR), NGS and flow cytometry were applied to platelets isolated from Streck, EDTA tubes or apheresis bags from healthy donors, pregnant women, patients with cancer and murine platelets from cancer models. DNA was extracted from platelet pellets or platelet-depleted plasma (cfDNA) using the Qiagen QIAamp Circulating Nucleic Acid Kit. Results Using live cell imaging and flow cytometry, significant DNA content (Draq5+) was detected in ~8% of platelets from healthy donors, including in platelets with low RNA content (SYTO-13-negative). Overall Draq5+ platelets were larger and primed for agonist activation. FISH and ddPCR of platelets from pregnant women carrying male fetuses detected Y-chromosome fragments (n=10), confirming that platelet DNA is not solely derived from parent megakaryocytes but also sequestered during circulation (Fig. 1a). In contrast, Y-chromosome genes were not detected in DNA isolated from mononuclear cells or red blood cells pellets. Acute platelet depletion in an ITP mouse model (n=20) led to a >2-fold increase in cfDNA extracted from platelet-poor plasma, suggesting a role for platelets in plasma DNA clearance. Platelet uptake in vitro was rapid, visible using live cell microscopy within 2 minutes of co-culture with cancer cells labelled with a probe that irreversibly intercalates to nuclear DNA. ddPCR of platelet DNA following co-culture of healthy donor platelets with cancer cell lines detected a range of canonical cancer driver mutations, including in PI3K, BRAF and JAK2. DNase treatment of co-cultured platelets did not reduce mutant allele content, indicating that platelets encapsulate DNA and protect it from degradation. We explored the mechanism of platelet DNA uptake. Platelets internalized DNA contained in extracellular vesicles (EVs). Platelet uptake of “free” DNA was also confirmed, with uptake of synthetic DNA fragments of varying lengths (120 bp to 650 bp). Importantly, platelet DNA detection was enhanced following exocytosis inhibition, confirming that platelet DNA uptake and release is an active process. Whole genome sequencing revealed that platelets contain a repertoire of DNA fragments that map across the nuclear genome, similar to cfDNA, and that the majority of platelet DNA is of nuclear not mitochondrial origin. We also found tumor-derived DNA showing multiple copy number aberrations in a patient with pancreatic carcinoma. In a transgenic mouse model of colorectal cancer, mutant KRASG12D alleles were readily detectable in platelet DNA and, notably, in higher abundance in platelets than platelet-poor plasma (cfDNA) in 11/16 mice (Fig. 1b). Similarly, KRASG12D was detected in platelets of mice with orthotopic pancreatic adenocarcinoma (n=4). Finally, to explore utility in cancer screening, we analysed patients with high-risk, pre-malignant colonic lesions (serrated polyps). Remarkably, the driver mutation BRAFV600E was detected in platelets in 16% patients despite the small-size lesions (5/30, Fig.1b). Conclusions This study establishes a role for platelets in sequestration of cfDNA, an aspect of platelet biology that has not previously been highlighted and is of substantial clinical relevance. Their abundance, ease of isolation, and continuous tissue perfusion make platelets ideal ‘sentinels’ for genetic perturbations including in early stage/pre-malignant disease.

An in vitro-transcribed circular RNA targets the mitochondrial inner membrane cardiolipin to ablate EIF4G2+/PTBP1+ pan-adenocarcinoma.

In vitro-transcribed (IVT) mRNA has arisen as a rapid method for the production of nucleic acid drugs. Here, we have constructed an oncolytic IVT mRNA that utilizes human rhinovirus type 2 (HRV2) internal ribosomal entry sites (IRESs) to selectively trigger translation in cancer cells with high expression of EIF4G2 and PTBP1. The oncolytic effect was provided by a long hGSDMDc .825 T>A/c.884 A>G-F1LCT mutant mRNA sequence with mitochondrial inner membrane cardiolipin targeting toxicity that triggers mitophagy. Utilizing the permuted intron-exon (PIE) splicing circularization strategy and lipid nanoparticle (LNP) encapsulation reduced immunogenicity of the mRNA and enabled delivery to eukaryotic cells in vivo. Engineered HRV2 IRESs-GSDMDp.D275E/E295G-F1LCT circRNA-LNPs (GSDMDENG circRNA) successfully inhibited EIF4G2+/PTBP1+ pan-adenocarcinoma xenografts growth. Importantly, in a spontaneous tumor model with abnormal EIF4G2 and PTBP1 caused by KRAS G12D mutation, GSDMDENG circRNA significantly prevented the occurrence of pancreatic, lung and colon adenocarcinoma, improved the survival rate and induced persistent KRAS G12D tumor antigen-specific cytotoxic T lymphocyte responses.