KRAS-mutant Cancer Research Articles

KRAS G12C inhibitor combination therapies: current evidence and challenge.

Although KRAS G12C inhibitors have proven that KRAS is a "druggable" target of cancer, KRAS G12C inhibitor monotherapies have demonstrated limited clinical efficacy due to primary and acquired resistance mechanisms. Multiple combinations of KRAS G12C inhibitors with other targeted therapies, such as RTK, SHP2, and MEK inhibitors, have been investigated in clinical trials to overcome the resistance. They have demonstrated promising efficacy especially by combining KRAS G12C and EGFR inhibitors for KRAS G12C-mutated colorectal cancer. Many clinical trials of combinations of KRAS G12C inhibitors with other targeted therapies, such as SOS1, ERK, CDK4/6, and wild-type RAS, are ongoing. Furthermore, preclinical data have suggested additional promising KRAS G12C combinations with YAP/TAZ-TEAD inhibitors, FAK inhibitors, and farnesyltransferase inhibitors. The combinations of KRAS G12C inhibitors with immunotherapies and chemotherapies have also been investigated, and the preliminary results were reported. More recently, KRAS-targeted therapies not limited to KRAS G12C are being developed, potentially broadening the treatment landscape of KRAS-mutated cancers. Rationally combining KRAS inhibitors with other therapeutics is likely to play a significant role in future treatment for KRAS-mutated solid tumors.

State-of-the-art and upcoming trends in RAS-directed therapies in gastrointestinal malignancies.

Overall, the review underscores the evolving landscape of KRAS-targeted therapy and the potential for these approaches to improve outcomes for patients with gastrointestinal malignancies. It highlights the importance of ongoing research and clinical trials in advancing precision medicine strategies for KRAS-driven cancers. This review provides a comprehensive overview of the RAS signaling pathway and its significance in gastrointestinal malignancies. The introduction of KRAS inhibitor represents a significant advancement in the treatment landscape for KRAS-mutant cancers. In this review, we discuss upcoming trends in KRAS-targeted therapy, including the development of mutant-specific direct KRAS inhibitors like MRTX1133 and pan-RAS inhibitors such as RMC-6236. It also explores indirect RAS inhibitors targeting upstream and downstream components of the RAS pathway. Additionally, the review examines other upcoming strategies like combination therapies, such as CDK4/6 and ERK MAPK inhibitors, as well as adoptive cell therapy and cancer vaccines targeting KRAS-mutant cancers. Targeting RAS has become an important strategy in treating gastrointestinal cancer. These findings in this review underscore the importance of a multidisciplinary approach, integrating advances in molecular profiling, targeted therapy, immunotherapy, and clinical research to optimize treatment strategies for patients with KRAS-mutant gastrointestinal malignancies.

Metabolomic Approach to Identify Potential Biomarkers in KRAS-Mutant Pancreatic Cancer Cells.

Pancreatic cancer is characterized by its high mortality rate and limited treatment options, often driven by oncogenic RAS mutations. In this study, we investigated the metabolomic profiles of pancreatic cancer cells based on their KRAS genetic status. Utilizing both KRAS-wildtype BxPC3 and KRAS-mutant PANC1 cell lines, we identified 195 metabolites differentially altered by KRAS status through untargeted metabolomics. Principal component analysis and hierarchical condition trees revealed distinct separation between KRAS-wildtype and KRAS-mutant cells. Metabolite set enrichment analysis highlighted significant pathways such as homocysteine degradation and taurine and hypotaurine metabolism. Additionally, lipid enrichment analysis identified pathways including fatty acyl glycosides and sphingoid bases. Mapping of identified metabolites to KEGG pathways identified nine significant metabolic pathways associated with KRAS status, indicating diverse metabolic alterations in pancreatic cancer cells. Furthermore, we explored the impact of TRPML1 inhibition on the metabolomic profile of KRAS-mutant pancreatic cancer cells. TRPML1 inhibition using ML-SI1 significantly altered the metabolomic profile, leading to distinct separation between vehicle-treated and ML-SI1-treated PANC1 cells. Metabolite set enrichment analysis revealed enriched pathways such as arginine and proline metabolism, and mapping to KEGG pathways identified 17 significant metabolic pathways associated with TRPML1 inhibition. Interestingly, some metabolites identified in PANC1 compared to BxPC3 were oppositely regulated by TRPML1 inhibition, suggesting their potential as biomarkers for KRAS-mutant cancer cells. Overall, our findings shed light on the distinct metabolite changes induced by both KRAS status and TRPML1 inhibition in pancreatic cancer cells, providing insights into potential therapeutic targets and biomarkers for this deadly disease.

Abstract LB352: Unraveling therapeutic potentials of mRNA-based cancer vaccine concurrently targeting heterogeneous KRAS mutant cancer

Abstract The utilization of messenger RNA (mRNA)-based cancer vaccines represents a compelling therapeutic strategy, providing distinct advantages over alternative modalities such as peptide vaccines or small-molecule-based cancer therapies. This innovative approach enables the concurrent targeting of heterogeneous cancer populations characterized by multiple mutations while preserving the integrity of normal cells. The Kristen rat sarcoma viral oncogene (KRAS) mutation is prevalent in lung, pancreatic, and colon cancer, and patients harboring KRAS mutations face a particularly dismal prognosis. This study focuses on the optimization of an mRNA-based cancer vaccine designed to target various KRAS mutations, demonstrating its efficacy inducing potent anti-tumor immune responses in both ex vivo immune cell and cancer cell co-culture, as well as in vivo syngeneic mouse cancer models. For the ex vivo evaluation of the multi-targeting capabilities of the vaccine, human peripheral blood mononuclear cells (PBMCs) primed with our mRNA cancer vaccine were subsequently co-incubated with human normal and cancer cell lines harboring KRAS mutations to assess antigen-specific immune responses and the normal cell-sparing effect of the cancer vaccine. Four types of KRAS mutant cancer cells co-cultured with the vaccine-activated PBMCs showed substantially decreased cell viability. We found enrichment of cytotoxic and pro-inflammatory cytokines including ganzyme B, IFNγ, IL-6 and IL-10 in co-culture system with KRAS vaccine transfected monocyte and PBMCs. This suggests that multiple KRAS mutations can be targeted with a single type of mRNA vaccine candidate. In the LL/2 syngeneic model (murine Lewis lung carcinoma, KRAS G12C mutant), the in vivo efficacy of KRAS cancer vaccine candidates was evaluated, revealing a notable reduction in tumor growth in response to the vaccine treatment. Also, during the treatment period, there were no significant clinical symptoms in the mouse between groups indicating the vaccine doses were tolerable. These results indicate that the KRAS vaccine effectively inhibits the growth of an LL/2 tumor which has the immunosuppressive tumor microenvironment. The ability to address the challenges posed by diverse KRAS mutations underscores the promising potential of this multiple-KRAS mutation targeting mRNA-based cancer vaccine in the pursuit of enhanced therapeutic outcomes for patients afflicted with KRAS-mutant cancer. Citation Format: Chang Gyu Lim, Seung-Hyun Shin, Youngjin Han, Yong Ho Heo, Innah Kim, Ji Hee Lee, Miyoung Lee, Seongju Jeong, Yunjae Kim, Jooyun Byun, Daejin Kim, In Young Choi. Unraveling therapeutic potentials of mRNA-based cancer vaccine concurrently targeting heterogeneous KRAS mutant cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB352.

Abstract LB220: Farnesyltransferase inhibitors show synergistic anticancer effects in combination with novel KRAS G12C inhibitors

Abstract Background. Farnesyltransferase inhibitors (FTI) failed as monotherapies for various cancer types until the discovery of their potency in HRAS mutant human head and neck and bladder cancers. On the other hand, RAS mutant cancers have been a great challenge for cancer research until the discoveries and approvals of several KRAS-G12C allele specific inhibitors. However, G12C inhibitors were effective mainly in lung cancer and de novo and acquired resistance prompted the development of various combinational modalities including immunotherapy as well as SOS- and SHP2-inhibitors. Based on in-silico analysis of drug sensitivities of KRAS mutant cancer cell lines treated with various drugs including FTIs, we have explored the possibility of the combination of FTIs with KRAS inhibitors.Methods. Clinically approved farnesyl-transferase inhibitors (tipifarnib and lonafarnib) were combined with, novel KRAS G12C inhibitors (sotorasib and adagrasib) using human lung-, pancreatic and colorectal- adenocarcinoma cells in vitro and in vivo. Antitumoral effects were evaluated for cell proliferation, apoptosis and migratory activity. Mechanisms of action were investigated by immunoblot analyzes of various farnesylated proteins, RAS activation and signaling, videomicroscopy and also by histopathology of xenograft tumors. Results. Synergistic anticancer effects were observed upon the combination of FTIs with G12C inhibitors in KRAS G12C mutant human cancer cell lines in vitro. We found that the combination interfered with the compensatory re-activation of HRAS, farnesylation of RHEB in the PI3K/mTOR pathway and lamin. Furthermore, we observed enhanced efficacy of sotorasib upon combination with tipifarnib in the xenograft models of lung adenocarcinoma affecting mitotic- and apoptotic rates and inducing necrosis.Discussion. Our findings suggest the potential clinical applicability of the combination of KRAS-G12C inhibitors and farnesyl-transferase inhibitors. Furthermore, our preliminary data suggest that the synergistic effect of FTIs on KRAS-G12C inhibitors can be projected to G12D inhibitors as well. Citation Format: Jozsef Timar, Marcell Baranyi, Eszter Molnar, Ivan Randelovic, Mihaly Cserepes, Jozsef Tovari, Balazs Hegedus. Farnesyltransferase inhibitors show synergistic anticancer effects in combination with novel KRAS G12C inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB220.

Abstract LB163: Discovery and characterization of QTX3544, a potent, selective, and orally bioavailable allosteric G12V preferring multi KRAS inhibitor

Abstract Oncogenic KRAS mutations occur in approximately 25% of all human cancers with KRAS G12V being one of the most frequently occurring variants, particularly in pancreatic, colorectal, and lung cancers. These oncogenic mutations cause sustained activation of the mitogen activated protein kinase (MAPK) pathway, which ultimately drives tumorigenesis. While newly approved covalent inhibitors to KRASG12C have demonstrated clinical benefit to patients harboring this mutation, there are no approved drugs targeting mutations beyond KRASG12C, highlighting a significant clinical need. Here we show that QTX3544 is a potent and orally bioavailable KRASG12V preferring multi KRAS inhibitor. QTX3544 exhibited picomolar binding affinity (0.08 nM) and SOS mediated nucleotide inhibition (0.07 nM) to the inactive form of KRASG12V. Furthermore, QTX3544 inhibited “on state” KRAS activity in both biochemical and cellular assays. In vitro, QTX3544 significantly inhibited downstream ERK phosphorylation and cell proliferation in KRASG12V driven cancer cell lines. Similar inhibitory effects were observed across additional mutant KRAS cancer cell lines, including KRAS G12A, KRAS G13D , and KRAS Q61H, among others. Notably, QTX3544 displayed no impact against HRAS, NRAS or BRAF mutant cells. In vivo, oral administration of QTX3544 achieved sustained systemic exposure required for efficacy, indicating potential durable inhibition of KRAS signaling. QTX3544 demonstrated dose dependent anti tumor efficacy and significant tumor regression in various KRASG12V driven xenograft models when administered orally twice daily (BID). The physiochemical profile of QTX3544 is favorable with good measured LogD, solubility, and permeability. QTX3544 exhibits weak Cytochrome P450 inhibition (direct and time dependent) with a favorable in vitro off target selectivity/safety profile. The systemic clearance in rats and dogs was moderate to high, respectively, with good oral bioavailability. QTX3544 was generally well tolerated in rat and dog repeat dose tolerability studies. In addition, QTX3544 showed good brain penetration in mice. Taken together, this preclinical characterization supports the advancement of QTX3544 into IND enabling studies for KRAS G12V mutant cancers. Citation Format: Yang W. Zhang, Elizabeth Donohue Vo, Pei Gan, Dave Rominger, Jillian M. Silva, Yang J. Zhang, Christopher Pan, Greg Lee, John M. Micozzi, Ben Reid, Brooke McDonough, Arghyotri Sinha, Audrey Hospital, Doug Krauth, Juan I. Luengo, Mike Kramer, Cameron Pitt, Hong Lin. Discovery and characterization of QTX3544, a potent, selective, and orally bioavailable allosteric G12V preferring multi KRAS inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB163.

Abstract CT076: AFNT-211: A phase1 study of autologous CD4+ and CD8+ T cells engineered to express a high avidity HLA-A*11:01-restricted, KRAS G12V-specific, transgenic TCR; CD8α/β coreceptor; and a FAS41BB switch receptor in patients with advanced/metastatic solid tumors

Abstract Background: Activating mutations in KRAS (including KRAS G12V) are well-described oncogenic drivers in solid tumors. Patients with KRAS driver mutations have a poor prognosis, and most KRAS-mutated cancers lack effective therapies. T cell receptor (TCR)-T cell therapies targeting mutant KRAS have demonstrated proof of concept in the clinic, but duration of response remains a challenge. AFNT-211 represents a novel strategy to address the immunosuppressive tumor microenvironment and improve response rate and duration in solid tumors. Rationale: AFNT-211 autologous CD4+ and CD8+ T cells are engineered to express a high avidity HLA-A*11:01restricted, KRAS G12V-specific, transgenic TCR; the wildtype CD8α/β coreceptor; and a FAS-41BB switch receptor. The intended mechanism of action is recognition and elimination of KRAS G12V-mutated tumor cells by AFNT-211. AFNT-211 is designed to enhance anti-tumor activity via the following mechanisms of action as shown in preclinical models: (1) the CD8α/β coreceptor enables MHC-I-‍restricted TCR recognition of the KRAS G12V target by CD4+ T cells; (2) engaging CD4+ helper T cell responses drives enhanced CD8+ T cell cytotoxic activity and prevents T cell exhaustion; (3) the FAS-41BB chimeric switch receptor drives increased T cell activity, circumventing native FAS apoptotic signaling. Methods: This ongoing Phase 1, first-in-human, multicenter, open-label study of AFNT-211 (1) evaluates safety/tolerability, (2) determines the optimal biological dose (OBD) and recommended Phase 2 dose (RP2D), and (3) assesses preliminary antitumor activity. The study consists of 2 parts: dose escalation and dose expansion. Dose escalation is guided by the Bayesian optimal interval Phase 1/2 (BOIN12) design to determine the OBD. The BOIN12 design quantifies the desirability of a dose in terms of toxicity-efficacy tradeoff and adaptively allocates patients to the dose with the highest estimated desirability. The RP2D will be selected based on the BOIN12 design and the totality of benefit/risk assessment. Patients enrolling in dose expansion will be treated at the OBD/RP2D in indication-specific cohorts. This study is conducted in patients aged ≥ 18 years who are HLA-A*11:01-positive with advanced/metastatic solid tumors harboring a KRAS G12V mutation, and intolerance of/progression on at least 1 prior systemic therapy. Patients undergo leukapheresis to collect T cells for the manufacturing of AFNT-211 and receive lymphodepleting chemotherapy prior to AFNT-211 infusion. This is followed by a 28-day dose-limiting toxicity observation period (dose escalation only) and a post-treatment follow-up period for 24 months/until disease progression. The study is open for recruitment in the United States (NCT06105021). Citation Format: Shaunica Mitchell, Binaish Khan, Francis Payumo, E. Gabriela Chiorean, Zhubin Gahvari, J. Randolph Hecht, Michael Hurwitz, Rom Leidner, Heinz-Josef Lenz, Meredith Pelster, Salman Punekar, Adam Schoenfeld, Dan Zhao, Markus Vallaster, Dirk Nagorsen. AFNT-211: A phase1 study of autologous CD4+ and CD8+ T cells engineered to express a high avidity HLA-A*11:01-restricted, KRAS G12V-specific, transgenic TCR; CD8α/β coreceptor; and a FAS41BB switch receptor in patients with advanced/metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT076.

Design, synthesis, and evaluation of purine and pyrimidine-based KRAS G12D inhibitors: Towards potential anticancer therapy

Oncogenic RAS mutations, commonly observed in human tumors, affect approximately 30% of cancer cases and pose a significant challenge for effective cancer treatment. Current strategies to inhibit the KRAS G12D mutation have shown limited success, emphasizing the urgent need for new therapeutic approaches. In this study, we designed and synthesized several purine and pyrimidine analogs as inhibitors for the KRAS G12D mutation. Our synthesized compounds demonstrated potent anticancer activity against cell lines with the KRAS G12D mutation, effectively impeding their growth. They also exhibited low toxicity in normal cells, indicating their selective action against cancer cells harboring the KRAS G12D mutation. Notably, the lead compound, PU1-1 induced the programmed cell death of KRAS G12D-mutated cells and reduced the levels of active KRAS and its downstream signaling proteins. Moreover, PU1-1 significantly shrunk the tumor size in a pancreatic xenograft model induced by the KRAS G12D mutation, further validating its potential as a therapeutic agent. These findings highlight the potential of purine-based KRAS G12D inhibitors as candidates for targeted cancer therapy. However, further exploration and optimization of these compounds are essential to meet the unmet clinical needs of patients with KRAS-mutant cancers.

Abstract 6060: Discovery of potent SHP2 protein degraders with strong anti-tumor activities in KRAS mutant cancers

Abstract Background: SHP2 plays a pivotal role in modulating the RTK/RAS/MAPK signaling pathway, which is frequently dysregulated in various cancers. Multiple allosteric SHP2 inhibitors have been under active development for the treatment of RTK/RAS/MAPK-dependent cancers, while certain limitations have come to light, such as their inability to target the active form of SHP2 and narrow therapeutic index necessitating intermittent dosing to mitigate toxicity. To overcome these challenges, we have discovered SHP2 protein degraders as next generation SHP2 targeting agents. Here, we present compelling profiles of our SHP2 degraders. Methods: We employed a variety of cancer cell lines harboring mutations in RTK, KRAS, or SHP2 for assessing in vitro activity of SHP2 degraders. For in vivo assessment of pharmacological profile, we utilized an NCI-H358 KRAS G12C mutant NSCLC xenograft. SHP2 protein levels in cell lysates and tumors were analyzed to assess SHP2 degradation efficiency. Simultaneously, ERK phosphorylation and DUSP6 mRNA levels were analyzed to evaluate downstream effects. Immunoprecipitation assays were used to assess the impact on SHP2's scaffolding roles. Results: A series of SHP2 degraders have been identified, demonstrating highly efficient SHP2 degradation activity with DC50 values in subnanomolar ranges. Robust inhibition of ERK phosphorylation, followed by remarkable anti-cancer effects in multiple KRAS mutant cancer cell lines were observed. Interestingly, in vitro cell growth inhibition by SHP2 degraders are 10 times more potent than SHP2 inhibitors currently under clinical development. Further characterization reveals several advantages of SHP2 degraders compared to SHP2 inhibitors. First, we observed a more efficient disruption of SHP2 scaffolding function, such as the formation of the SHP2-GRB2-GAB1 complex. Second, we detected effective degradation of activating mutants of SHP2, such as E76K and T507K, which are typically not targeted by SHP2 inhibitors. Experiments using a KRAS mutant CDX model demonstrate compelling in vivo profiles. A single administration of lead compounds resulted in Dmax values exceeding 95% in tumors, accompanied by the significant reduction of downstream signaling markers, such as p-ERK and DUSP6 mRNA. Notably, the reduced levels of SHP2 and downstream markers persist for several days. This suggests that utilizing intermittent dosing could be a viable approach with SHP2 degraders. Once-weekly intravenous administration achieved strong tumor growth inhibition without causing significant body weight loss. Conclusion: We have successfully discovered novel SHP2 degraders exhibiting remarkable activities. We demonstrate that SHP2 degraders have potential to overcome the limitations associated with allosteric SHP2 inhibitors and provide a promising therapeutic option for RTK/RAS/MAPK-dependent cancer patients. Citation Format: So Hyun Kim, Jihye Lee, Jiyoung Song, Geon-Tae Park, Jieun Jung, Jungtae Na, Dong Ho Lee, Soohyun Lee, Ji Youn Park, Jiyoung Kim, Onnuri Bae, Jeonghwa Han, Ju Young Kang, Mijin Moon, Yongje Shin, Je Ho Ryu, Song Hee Lee, Sook-Kyung Park. Discovery of potent SHP2 protein degraders with strong anti-tumor activities in KRAS mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6060.

Abstract 5265: Targeting mtDNA dynamics enhances immunogenicity and sensitizes KRAS mutant cancers to PD-1 blockade

Abstract Mitochondrial DNA (mtDNA) can stimulate an innate immune response that potentiates the efficacy of cancer immunotherapy. However, mtDNA-targeting therapy has not been developed to enhance this innate immune system in specific cancer subtypes efficiently. Here we discover that mtDNA is enriched in KRAS mutant tumors where the DNA sensor machinery cGAS-STING signaling is primed for innate immune activation. Intriguingly, MEK inhibitor induces cytosolic mtDNA release to potently activate the cGAS-STING signaling, which in turn induces PRKN-mediated mitophagy in response to mitochondrial stress in KRAS mutant cancer cells. During the process of mitophagy, PRKN, as an E3 ubiquitin ligase, degrades BAX protein and attenuates mitochondrial pore formation, which is essential for mtDNA efflux upon MEK inhibition. Combined MEK and mitophagy inhibitors efficiently stimulate type I IFNs in both mtDNA- and STING-dependent manners, and this combination enhances the efficacy of PD-1 blockade in Kras mutant murine syngeneic tumors. In the clinic, PRKN is associated with a ‘cold tumor’ phenotype among KRAS mutant tumors. Together, targeting mtDNA dynamics by co-inhibition of MEK and mitophagy represents a promising strategy to convert ‘cold to hot tumor’ in mtDNA-enriched KRAS mutant cancers. Citation Format: Kosuke Tanaka, Yasuki Adachi, Miyuki Yoshiya, Takuya Owari, Tomoko Yamamori-Morita, Akihiro Ohashi, Susumu Kobayashi, Shohei Koyama, Hiroyoshi Nishikawa. Targeting mtDNA dynamics enhances immunogenicity and sensitizes KRAS mutant cancers to PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5265.

Abstract 1023: Whole-genome methylation analysis using liquid biopsy for pancreatic cancer diagnosis

Abstract Objective: Pancreatic cancer is one of the highly malignant tumors. Its diagnosis requires invasive ultrasound endoscopic biopsy or endoscopic retrograde cholangiopancreatography (ERCP). However, these methods are limited by false-positive results and complications. Liquid biopsy, which uses blood to detect KRAS mutations in cancer, can be used to overcome these hurdles. However, only about 30% of cases can be detected as a diagnostic marker by KRAS mutations using plasma from patients with stage I-II pancreatic cancer, and its clinical application remains a challenge. Therefore, in this study, we focused on diagnosing pancreatic cancer using methylated DNA in plasma cell-free DNA (cfDNA) obtained from pancreatic cancer and non-cancer patients. Subjects: Plasma was collected before the start of the treatment from six patients diagnosed with pancreatic cancer from 2019 to 2023 at our hospital, and six non-cancer patients simultaneously. Methods: Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing (NGS) was performed using the cfDNA obtained from the plasma of pancreatic cancer and non-cancer patients. Results: The cfDNA fragments obtained by WGBS revealed that promoter regions were mostly deleted, and no difference was observed between cancer and non-cancer patients. The methylation rate of cfDNA fragments by WGBS differed between cancer and non-cancer patients, with multiple methylation sites (>100 sites) in intergenic regions. Discussion: Although attempts have been made to diagnose cancer using genetic mutations and liquid biopsy, only a few studies are available on using methylation markers in the plasma cfDNA of pancreatic cancer patients, and the effectiveness of this method remains unclear. In the present study, we found that promoter regions were deleted by WGBS, and several distinct hypermethylated regions were present in the intergenic region between pancreatic cancer and non-cancer samples, suggesting the presence of certain markers that can distinguish between pancreatic cancer and non-cancer patients. Conclusion: This small number of cases suggests that hypermethylation of the intergenic regions could be helpful in the diagnosis of pancreatic cancer. We will need more cases in the future to verify the usefulness of hypermethylation of intergenic regions in the diagnosis of pancreatic cancer. Citation Format: Tomoaki Ito, Takuya Iwasawa, Satoshi Takeda, Chikako Okamoto, Tomoko Motohashi, Shuhei Ueda, Hisaki Kato, Riku Yamamoto, Akihiro Koizumi, Yuta Murai, Kosaku Nihei, Moe Itakura, Ryunosuke Akima, Mutsumi Sakurada, Kenichiro Tanaka, Tomoyuki Kushida, Koichi Sato. Whole-genome methylation analysis using liquid biopsy for pancreatic cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1023.

Abstract 4574: Next generation nuclear export blocker KPT8602 synergizes with KRASG12D inhibitor MRTX1133 resulting in improved antitumor effects against pancreatic ductal adenocarcinoma

Abstract Introduction: A novel KRAS inhibitor, MRTX1133, specifically targeting the G12D isoform, prevalent in pancreatic ductal adenocarcinoma (PDAC), holds promise for a substantial PDAC patient population. Nevertheless, given the clinical experience with sotorasib (a KRASG12C inhibitor) in lung cancer, it's anticipated that resistance to the KRASG12D inhibitor will eventually emerge in most PDAC patients. This underscores the need to identify strategies that can enhance the efficacy of KRAS inhibitors in PDAC. Emerging evidence suggests that the nuclear export protein exportin 1 (XPO1) is a druggable vulnerability in KRAS mutant cancers. XPO1 plays a pivotal role in shuttling various critical tumor suppressors, genome surveillance proteins, and transcription factors out of the nucleus and is frequently overexpressed in various cancers, including PDAC. In this study, we employed both in vitro and in vivo models to assess the potential of KPT8602, an investigational and next-generation selective XPO1 inhibitor, to synergize with the KRASG12D inhibitor MRTX1133 and sensitize MRTX1133-resistant PDAC cells. Methods: We examined the cytotoxic and molecular effects of KRASG12D inhibitor MRTX1133 in combination with nuclear export blocker KPT8602 in KRASG12D mutant 2D and 3D cellular/spheroid models. The preclinical antitumor efficacy of the combination was evaluated in a KRASG12D mutant PDAC cell derived xenograft (CDX) model. Results: KPT8602 synergized with MRTX1133 in vitro yielding suppressed proliferation of KRASG12D mutant PDAC cells. Similar synergistic effects were also seen in 3D cultures of KRASG12D mutant PDAC cancer cell lines and patient derived primary tumor cells where the combination suppressed spheroid formation and reduced spheroid viability. Also, the combination inhibited the clonogenic potential of KRASG12D mutant PDAC cells. In addition, we developed a PDAC cell line resistant to MRTX1133 and observed that such MRTX1133-resistant cell line was acutely sensitive to treatment with KPT8602. Mechanistically, KPT8602 and MRTX1133 combination led to reduction in protein expression of KRAS downstream effectors and cell cycle markers. Furthermore, a combined administration of KPT8602 and MRTX1133 at sub-optimal doses resulted in remarkable tumor growth inhibition in a KRASG12D mutant HPAC CDX mice model. Moreover, KPT8602 as a maintenance therapy also prevented tumor relapse. Conclusion: This is the first study demonstrating that nuclear export blocker KPT8602 can synergize with KRASG12D inhibitor MRTX1133 and has the potential to target MRTX1133-resistant PDAC cells. This study provides a rationale for combining MRTX1133 with KPT8602 for the treatment of PDAC patients with KRASG12D mutant tumors. Citation Format: Husain Yar Khan, Amro Aboukameel, Md Hafiz Uddin, Sahar F. Bannoura, Khaled Keffri, Medha Jasti, Rachael Virga, Sarah Motorwala, Khalil Choucair, Tarik Hadid, Mohammad Najeeb Al-Hallak, Eliza Beal, Miguel Tobon, Rafic Beydoun, Greg Dyson, Steve Kim, Misako Nagasaka, Philip A. Philip, Basel El-Rayes, Herbert Chen, Anthony Shields, Ramzi Mohammad, Boris Pasche, Asfar Azmi. Next generation nuclear export blocker KPT8602 synergizes with KRASG12D inhibitor MRTX1133 resulting in improved antitumor effects against pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4574.

Abstract 7618: Genomic pathogenic alterations of the SWI/SNF complex genes compromise the outcomes to immunotherapy in Chinese KRAS-mutant non-small cell lung cancer by downregulated STING

Abstract Background: Immune checkpoint inhibitors (ICIs) have been standard of care in non-small cell lung cancer (NSCLC), but lack of response to ICIs remains a major hurdle in cancer immunotherapy. Switch/sucrose nonfermentable (SWI/SNF) chromatin-remodeling complex mutations have been reported in KRAS-mutant cancers with debated influence to clinical outcome of ICIs, which needs further investigation. Methods: According to the OncoKB, COSMIC, and PolyPhen-2 databases, genomic pathogenic alterations (GPAs) of SWI/SNF are defined. Lung tumors were classified according to the presence or absence of mutations in six SWI/SNF genes (ARID1A, ARID1B, ARID2, PBRM1, SMARCA4, and SMARCB1). Lung cancer cell lines with different genotypes of SWI/SNF were used for differentially expressed genes analysis by RNA sequencing. Protein expression of STING was detected by immunohistochemistry (IHC) in tumor specimens. Results: Out of 2660 NSCLC patients (NSCLCs), the GPAs of SWI/SNF were detected in 15.0% (401/2660) patients. 23.1% (69/299) EGFRwtALKwt and 27.8% (25/90) STK11wtKEAP1wtKRASmut NSCLCs who received ICIs had SWI/SNF GPAs. Progression-free survival (mPFS) of SWI/SNF-wild type (wt) compared to SWI/SNF-mutant (mut) NSCLCs was not significantly different. However, GPAs of SWI/SNF in KRAS-mut ICI-treated NSCLCs associated with poorer clinical outcome. Patients with ARID1A, ARID1B, ARID2 or PBRM1 mutant tumors had a significantly shorter PFS (2.7 vs 6.5 months, HR=2.44 [95%CI: 1.31-4.54], P=0.004) as compared to SWI/SNF-wt patients. In STK11wtKEAP1wtKRASmut NSCLCs, all SWI/SNF mutations (4.9 vs 9.1 months, HR=2.03 [95%CI: 1.07-3.86], P=0.029) or ARID1A/ARID1B/ARID2/PBRM1 alterations (3.2 vs 9.1 months, HR=2.68 [95%CI: 1.33-5.41], P=0.004) significantly shortened the mPFS. In cell lines, SWI/SNF-mut cells had downregulated STING expression compared to SWI/SNF-wt cells. Downregulated STING protein were observed in KRAS-mut NSCLCs without benefit from the treatment of ICIs. Conclusions: In KRAS-mut NSCLCs with or without STK11/KEAP1 mutations, the presence of GPAs of DNA binding genes ARID1A/ARID1B/ARID2 could compromise the clinical outcome of immunotherapy, which might be due to the downregulated STING. Citation Format: Lingling Gao, Jie Jiang, Zhi Xie, Linlin Zhu, Yu Chen, Zhiyi Lv, Yuqing Chen, Wenbin Zhou, Ji Chen, Yumin Zhong, Li Zhang, Penghui Zeng, Xiaodan Huang, Wenqing Yan, Danxia Lu, Shuilian Zhang, Weibang Guo, Xuchao Zhang. Genomic pathogenic alterations of the SWI/SNF complex genes compromise the outcomes to immunotherapy in Chinese KRAS-mutant non-small cell lung cancer by downregulated STING [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7618.

Abstract 4048: Comparative effects of SOS1 inhibitor with IL-6 blockade and their combination on Kras mutant lung cancer

Abstract Despite recent advances in treatment, lung adenocarcinoma (LUAD), particularly harboring Kras mutations, is still the leading cause of cancer mortality. We and others have previously shown that Kras mutant LUAD development and progression is associated with intrinsic inflammatory characteristics driven by epithelial NF-κB activation, increased levels of IL-6 (which is transcriptionally regulated by NF-κB), and activation of the IL-6-responsive transcription factor STAT3. We further demonstrated that IL-6 blockade suppresses lung tumorigenesis by reducing tumor-associated immunosuppressive myeloid populations and induction of a T cell-mediated anti-tumor response. The recent discovery of a novel SOS1-targeting compound (BI-3406) that selectively inhibits the KRAS-SOS1 interaction and leads to anti-proliferative activity has opened up the possibility to more efficiently inhibit Kras mutant lung cancer. Accordingly, in this study we used a mouse model of KRAS mutant LUAD (CC-LR) to compare the therapeutic effects of BI-3406, IL-6 blockade, and MEK inhibitor (trametinib). We also tested whether inhibiting IL-6 driven pro-tumor immune response would provide a synergistic/additive response and increase the tumor inhibitory effect of either BI-3406 or Trametinib. Eight cohorts of 14-week-old female mice were treated with either anti-IL-6 antibody, trametinib, or BI-3406, or their combinations for 4 weeks, and their tumor burden was compared at the end of study. We observed a significant reduction in tumor multiplicity and tumor area in response to all monotherapies, with the highest effect in the trametinib treated cohort. Neither combination therapy led to decreased tumor burden over either of the monotherapy regimens in our model. However, we observed an increase in T helper 1 and CD8 T expressing IFNγ cell populations in the cohort treated with Trametinib and anti-IL-6 Ab combination. In addition, using qPCR analysis of RNA extracted from the whole lung we found a higher expression of IFNγ along with a reduced expression of IL-10 in the cohort treated with BI-3406 and anti-IL-6 Ab combination. This suggests that these combinations may have the potential to improve the anti-tumor immune responses. Hence, we conclude that further adjustment of the treatment regimen (doses, interval, duration) may allow to obtain improved anti-tumor efficacy with these combinations. Overall, this study provides a new research perspective for the improvement of currently available treatment modalities for patients with Kras mutant lung cancer that could be further explored preclinically. Citation Format: Katherine E. Larsen, Maria J. Arredondo Sancristobal, Melody Zarghooni, Avantika Krishna, Maria T. Grimaldo, Nastaran Karimi, Arnav Gaitonde, Michael J. Clowers, Seyed Javad Moghaddam. Comparative effects of SOS1 inhibitor with IL-6 blockade and their combination on Kras mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4048.

Abstract 1931: Targeting YAP1/TEAD signaling re-sensitizes MAPK/ERK pathway inhibitors in KRAS- driven cancer cells

Abstract Many TEAD small-molecule inhibitors (SMIs) have recently reported initial clinical evaluations on Hippo-mutated cancer types. Several studies have demonstrated that activation of the YAP1/TEAD transcriptional complex via a Hippo-independent manner can drive resistance to MAPK/ERK pathway inhibitors. Here, we elucidated the potential mechanism of TEAD inhibition overcoming MAPK/ERK pathway resistance with a TEAD SMI (hereafter abbreviated as TEADi). Firstly, the MoA of TEADi was validated in Hippo-mutated cells. TEADi inhibited cell growth of Hippo-mutated cancer cell lines NCI-H226 and NCI-H2052 but not that of MKN45, a YAP1-deletion cell line. Co-IP and qRT-PCR results demonstrated that TEADi disrupted the interaction between YAP1 and TEAD, and thus markedly repressed the expression of CTGF and CYR61, two downstream targets of YAP1/TEAD, in NCI-H226 cells. Secondly, we hypothesized that hyper-activation of TEAD confers resistance to KRAS mutant cancer types. To test this hypothesis, we generated two resistant cell lines. One is KARS G12C inhibitor Sotorasib-resistant NCI-H358 (NCI-H358-R). The other is MEK inhibitor Trametinib-resistant HCT116 (HCT116-R). Immunofluoresence assay showed that YAP1 nucleus translocation was enhanced in both resistant cells (NCI-H358-R and HCT116-R), but not in their parental counterparts (NCI-H358-P and HCT116-P). The enhanced YAP1 nucleus translocation resulted in increased transcription activities of TEAD in both resistant cells, as illustrated by luciferase reporter assay. Accordingly, CTGF and CYR61 were observed upregulated in both resistant cells. The above data indicate that YAP1/TEAD mediated-transactivation plays a role in MAPK pathway resistance. Indeed, TEADi treatment alone displayed substantial difference in cell growth inhibition between parental and resistant cell lines in both NCI-H358 and HCT116, further confirming that the resistant cells are more dependent on YAP1/TEAD signaling. Finally, we evaluated the combinational efficacies of TEADi and MAPK/ERK pathway inhibitors in the resistant cells. It was observed that addition of TEADi could significantly restore the response of NCI-H358-R and HCT116-R to Sotorasib and Trametinib, respectively. Mechanistically, TEADi efficiently suppressed TEAD transcriptional activities and subsequently the expression of CTGF and CYR61 in both resistant cells. Moreover, TEADi had almost no impact on ERK phosphorylation in either of the resistant cells, suggesting that re-sensitization of MAPK/ERK pathway inhibitors by TEADi is independent of primary onco-genetic signaling pathway. Taken together, our study demonstrates that inhibition of YAP1/TEAD signaling would be an efficient approach to overcome resistance to MAPK/ERK pathway inhibitors in the patients carrying KRAS mutations, and provides the scientific basis for development of combination therapy strategies. Citation Format: Xianwen Yang, Guiping Li, Peiling Ren, Xiaoyun Shi, Ying Yu, Baoyu Hao, Pan Wang, Min Cheng, Guangxiu Dai. Targeting YAP1/TEAD signaling re-sensitizes MAPK/ERK pathway inhibitors in KRAS- driven cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1931.

Abstract 6061: Development of target cell-penetrating scFv conjugate degrader for the selective inhibition of mutant, resistant KRAS cancer

Abstract The activation of mutations in the RAS gene, which is frequently observed in human cancers, induces downstream critical effectors, contributing to tumorigenesis. While attempts to directly target the GTP-bound, activated RAS have shown some success in certain cancer treatments, resistance remains a challenge. Our objective is to develop a cell-penetrating single-chain fragment variable (scFv) degrader that targets mutant KRAS for degradation. The scFv, binding specifically to antigens, can mitigate the side effects of non-specific binding seen with chemical drugs. However, due to the limited cell-penetrating capability of scFv, a delivery carrier is often necessary. In this study, a cancer targeting, and cell-penetrating synthetic peptide (CPP) was developed as a drug delivery carrier and added to the sequence of scFv. To this conjugate, a von Hippel-Lindau (VHL) expressing sequence was added to enable the scFv binding to mutant KRAS, enabling its proteasomal degradation. These conjugates were engineered as a plasmid DNA to be expressed as a protein conjugate in CHO cells. The purity of the scFv-VHL-CPP degrader was confirmed by SDS PAGE. In SPR results, the degrader not only exhibited high binding ability to mutant KRAS but also influenced the proliferation of KRAS mutant cells in cell viability tests. Mutant GTP-bound KRAS was reduced by the KRAS degrader in KRAS mutant cancer cells. The in vivo study further confirmed the degradation of the KRAS, as reflected by the significantly reduced tumor growth. In conclusion, a degrader protein binding to mutant KRAS that consists of cell penetrating peptide, scFv, and VHL is expected to become a new protein anticancer drug especially effective against mutant, resistant KRAS-mediated cancer cells. Especially, the design of the target cell penetrating peptide-scFv-VHL would be served as a platform for the target protein degrader development. Citation Format: Gookjin Yoon, Beom Soo Jo, Dong Woo Lee, Jinwook Yang, Min-Ho Park, Sanghui Seok, Jue Yeon Lee, Chong Pyoung Chung, Yoon Jeong Park. Development of target cell-penetrating scFv conjugate degrader for the selective inhibition of mutant, resistant KRAS cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6061.

Abstract 1542: Epithelial cell adhesion molecule induces canonical Wnt pathway to promote stemness in colorectal cancer

Abstract Epithelial cell adhesion molecule (EpCAM) is a pleiotropic type-1 transmembrane glycoprotein and known cancer stem cell marker, yet its tumorigenic mechanisms remain elusive. Cancer stem cells (CSC) have been implicated in cancer progression and recurrence, but targeting them with therapeutics is a major challenge. It is known that EpCAM is highly expressed in CSCs of different cancer types. Here, we studied the role of EpCAM in colorectal cancer (CRC) stemness. First, we show that the extracellular domain of EpCAM (EpEX) functions as a ligand for Wnt receptors, frizzled6/7 and LRP5/6, while the intracellular domain (EpICD) upregulates transcription of these receptors. Further, EpEX-induced Wnt signaling activates TACE and γ-secretase enzymes to augment shedding of EpEX and EpICD, establishing a positive feedback loop. Importantly, we show that the EpCAM-neutralizing antibody, EpAb2-6, targets the Wnt pathway to attenuate stemness characteristics in colorectal cancer (CRC). Our EpCAM-neutralizing antibody (EpAb2-6) and a porcupine inhibitor (LGK974) each partially attenuated cancer stemness, while their combination abolished stemness-related endpoints, induced apoptosis in vitro and markedly diminished tumor progression in animal models of human CRC. From these findings, we conclude that EpCAM stimulates Wnt signaling to promote cancer stemness, and the combination of EpAb2-6 and porcupine inhibitors may represent an effective CRC treatment, including for KRAS-mutant cancers. Our findings reveal an opportunity to combat drug resistance in tumor stem cells. Thus, the mechanistic insights gained from our study may be useful to improve existing treatments or to develop novel anticancer therapeutics. Citation Format: Han-Chung Wu, Sushree Shankar Panda, Kai-Chi Chen, Chi-Chiu Lee, Hsing-Pang Hsieh, Yi-Jen Su. Epithelial cell adhesion molecule induces canonical Wnt pathway to promote stemness in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1542.

Screening Metabolic Biomarkers in KRAS Mutated Mouse Acinar and Human Pancreatic Cancer Cells via Single-Cell Mass Spectrometry.

Pancreatic cancer is a highly aggressive and rapidly progressing disease, often diagnosed in advanced stages due to the absence of early noticeable symptoms. The KRAS mutation is a hallmark of pancreatic cancer, yet the underlying mechanisms driving pancreatic carcinogenesis remain elusive. Cancer cells display significant metabolic heterogeneity, which is relevant to the pathogenesis of cancer. Population measurements may obscure information about the metabolic heterogeneity among cancer cells. Therefore, it is crucial to analyze metabolites at the single-cell level to gain a more comprehensive understanding of metabolic heterogeneity. In this study, we employed a 3D-printed ionization source for metabolite analysis in both mice and human pancreatic cancer cells at the single-cell level. Using advanced machine learning algorithms and mass spectral feature selection, we successfully identified 23 distinct metabolites that are statistically significantly different in KRAS mutant human pancreatic cancer cells and mouse acinar cells bearing the oncogenic KRAS mutation. These metabolites encompass a variety of chemical classes, including organic nitrogen compounds, organic acids and derivatives, organoheterocyclic compounds, benzenoids, and lipids. These findings shed light on the metabolic remodeling associated with KRAS-driven pancreatic cancer initiation and indicate that the identified metabolites hold promise as potential diagnostic markers for early detection in pancreatic cancer patients.

An atlas of epithelial cell states and plasticity in lung adenocarcinoma.

Understanding the cellular processes that underlie early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies1. Here we studied 246,102 single epithelial cells from 16 early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal and cancer cell states, and diversity among cancer cells was strongly linked to LUAD-specific oncogenic drivers. KRAS mutant cancer cells showed distinct transcriptional features, reduced differentiation and low levels of aneuploidy. Non-malignant areas surrounding human LUAD samples were enriched withalveolar intermediate cells that displayed elevated KRT8 expression (termed KRT8+ alveolar intermediate cells (KACs) here), reduced differentiation, increased plasticity and driver KRAS mutations. Expression profiles of KACs were enriched in lung precancer cells and in LUAD cells and signified poor survival. In mice exposed to tobacco carcinogen, KACs emerged before lung tumours and persisted for months after cessation of carcinogen exposure. Moreover, they acquired Kras mutations and conveyed sensitivity to targeted KRAS inhibition in KAC-enriched organoids derived from alveolar type 2 (AT2) cells. Last, lineage-labelling of AT2 cells or KRT8+ cells following carcinogen exposure showed that KACs are possible intermediates in AT2-to-tumour cell transformation. This study provides new insights into epithelial cell states at the root of LUAD development, and such states could harbour potential targets for prevention or intervention.

Gluing GAP to RAS Mutants: A New Approach to an Old Problem in Cancer Drug Development.

Mutated genes may lead to cancer development in numerous tissues. While more than 600 cancer-causing genes are known today, some of the most widespread mutations are connected to the RAS gene; RAS mutations are found in approximately 25% of all human tumors. Specifically, KRAS mutations are involved in the three most lethal cancers in the U.S., namely pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and lung adenocarcinoma. These cancers are among the most difficult to treat, and they are frequently excluded from chemotherapeutic attacks as hopeless cases. The mutated KRAS proteins have specific three-dimensional conformations, which perturb functional interaction with the GAP protein on the GAP-RAS complex surface, leading to a signaling cascade and uncontrolled cell growth. Here, we describe a gluing docking method for finding small molecules that bind to both the GAP and the mutated KRAS molecules. These small molecules glue together the GAP and the mutated KRAS molecules and may serve as new cancer drugs for the most lethal, most difficult-to-treat, carcinomas. As a proof of concept, we identify two new, drug-like small molecules with the new method; these compounds specifically inhibit the growth of the PANC-1 cell line with KRAS mutation G12D in vitro and in vivo. Importantly, the two new compounds show significantly lower IC50 and higher specificity against the G12D KRAS mutant human pancreatic cancer cell line PANC-1, as compared to the recently described selective G12D KRAS inhibitor MRTX-1133.