KRAS G12C Mutation Research Articles

Prognostic Value of KRAS Mutations in Relation to PDL1 Expression and Immunotherapy Treatment in Adenocarcinoma and Squamous Cell Carcinoma Patients: A Greek Cohort Study.

Factors that could predict which patients will benefit from Immune Checkpoint Inhibitors (ICIs) are not fully understood. This study aimed to investigate the prognostic value of KRAS biomarker in patients with advanced non-small cell lung cancer (NSCLC) in relation to clinical characteristics, treatment response and PDL1 expression. The study included 100 patients with NSCLC who received immunotherapy with or without chemotherapy as 1st line treatment. In biopsy samples, the PDL1 biomarker expression rate and somatic mutations of KRAS gene were determined. The mean age of the patients was 67 ± 8 years. Patients were all male and 66% were found with adenocarcinoma whereas 34% with squamous cell carcinoma. The KRAS G12C mutation was found with the highest percentage (73%). In the Kaplan-Meier survival analysis, patients with PDL1 > 49% in combination with a negative KRAS result had a median overall survival of 40 months compared to patients with a positive KRAS result (9 months, p < 0.05). In addition, patients diagnosed with adenocarcinoma, PDL1 < 49% and negative KRAS result had a median overall survival of 39 months compared to patients with a positive result (28 months, p < 0.05). Our study suggests that the presence of KRAS mutations in advanced NSCLC patients has a poor prognostic value, regardless of their PDL1 expression values, after receiving immunotherapy as first-line treatment.

Flavonoids as potential KRAS inhibitors: DFT, molecular docking, molecular dynamics simulation and ADMET analyses

KRAS mutations linked with cancer. Flavonoids were docked against KRAS G12C and G12D receptors. Abyssinone III, alpha naphthoflavone, beta naphthoflavone, abyssinone I, abyssinone II and beta naphthoflavone, genistin, daidzin showed good docking scores against KRAS G12C and G12D receptors, respectively. The MD simulation data revealed that Rg, RMSD, RMSF, and SASA values were within acceptable limits. Alpha and beta naphthoflavone showed good binding energies with KRAS G12C and G12D receptors. DFT and MEP analysis highlighted the nucleophilic and electrophilic zones of best-docked flavonoids. A novel avenue for the control of KRAS G12C and G12D mutations is made possible by flavonoids.

Exploration of efficacy of different therapy regimens for advanced NSCLC patients with KRAS mutation in the first-line treatment.

The treatment of the advanced non-small cell lung cancer (NSCLC) with KRAS mutation has been closely paid more attention. The aim of this study is to investigate the efficacy of different first-line regimens in advanced KRAS-mutated non-small cell lung cancer. In our retrospective study, we collected patients with advanced NSCLC with KRAS mutation in Zhejiang Cancer Hospital between January 2015 and May 2023. We analyzed the benefit of different first-line therapy according to theraputic methods and the differential effect of the same treatment method among KRAS-mutated subtypes. We divided the patients into group A (A1, chemotherapy alone; A2, immunotherapy alone) and group B (B1, chemotherapy plus immunotherapy; B2, chemotherapy combined with antiangiogenic therapy; B3, chemotherapy combined with immunotherapy plus antiangiogenic therapy). The Kaplan-Meier survival curve was used to reflect the PFS and OS of different methods. The objective response rate (ORR) and the disease control rate (DCR) were used to evaluated the response. We enrolled 227 patients including eighty-two with KRAS G12C mutation. The ORR and DCR of first-line treatment in the overall population were 32.2% and 80.6% respectively. The median PFS was 6.7months and the median OS was 17.4months for the overall population. The PFS of the Group B was significantly better than that of the Group A (7.7months vs 5.4months, P = 0.003), while no significant difference in OS was observed (19.4months vs 15.0months, P = 0.077). In the Group B, chemotherapy combined immunotherapy with antiangiogenic therapy showed better PFS than chemotherapy plus immunotherapy (14.1months vs 7.7months, P = 0.049), and OS also showed that tendency of difference (31.9months vs 19.3months, P = 0.158). There was no statistically significant difference between KRAS G12C and non-G12C mutation according to first-line treatment methods, whereas patients with TP53 co-mutation showed a better survival benefit (OS, 23.7 vs 12.5months, P = 0.023). In the first-line treatment, combination regimen has advantages over single regimen. Among them, chemotherapy combined with immunotherapy plus antiangiogenic therapy can achieve significant efficacy benefits.

Efficacy of Regorafenib and Trifluridine/Tipiracil According to Extended RAS Evaluation in Advanced Metastatic Colorectal Cancer Patients: A Multicenter Retrospective Analysis.

There are few molecular markers driving treatment selection in later lines of treatment for advanced colorectal cancer patients. The vast majority of patients who progress after first- and second-line therapy undergo chemotherapy regardless of molecular data. We aimed to assess the prognostic and predictive effects of specific RAS mutations on overall survival of patients receiving regorafenib (rego), trifluridine/tipiracil (TFD/TPI), or both. This was a retrospective observational study based on data from a previous study of our research network, involving nine Italian institutions over a 10-year timeframe (2012-2022). Extended RAS analysis, involving KRAS exon 2-4 and NRAS exon 2-4, and BRAF were the main criteria for inclusion in this retrospective evaluation. Patients with BRAF mutation were excluded. Patients were classified according to treatment (rego- or TFD/TPI-treated) and RAS mutational status (wild-type [WT], KRAS codon 12 mutations, KRAS codon 13 mutations, KRAS rare mutations and NRAS mutations, KRAS G12C mutation and KRAS G12D mutation). Overall, 582 patients were included in the present analysis. Overall survival did not significantly differ in rego-treated patients according to RAS extended analysis, although a trend toward a better median survival in patients carrying G12D mutation (12.0 months), Codon 13 mutation (8.0 months), and Codon 12 mutation (7.0 months) has been observed, when compared with WT patients (6.0 months). Overall survival did not significantly differ in TFD/TPI-treated patients according to RAS extended analysis, although a trend toward a better median survival in WT patients had been observed (9.0 months) in comparison with the entire population (7.0 months). Patients receiving both drugs displayed a longer survival when compared with the population of patients receiving rego alone (p=0.005) as well as the population receiving TFD/TPI alone (p<0.001), suggesting a group enriched for favorable prognostic factors. However, when each group was analyzed separately, the addition of TFD/TPI therapy to the rego-treated group improved survival only in all-RAS WT patients (p=0.003). Differently, the addition of rego therapy to TFD/TPI-treated patients significantly improved OS in the Codon 12 group (p=0.0004), G12D group (p=0.003), and the rare mutations group (p=0.02), in addition to all-RAS WT patients (p=0.002). The rego-TFD/TPI sequence, compared with the reverse sequence, significantly improved OS only in the KRAS codon 12 group (p=0.003). Our data demonstrate that RAS mutations do not affect outcome in rego-treated patients as well as TFD/TPI-treated patients. Nevertheless, a trend toward a higher efficacy of rego in RAS-mutated (in particular codon 12, rare RAS mutations, and G12D) patients has been recorded. The rego-TFD/TPI sequence seems to be superior to the reverse sequence in patients carrying an RAS codon 12 mutation, although the impact of other factors as disease burden or performance status cannot be excluded.

Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy.

RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 611. Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selectiveinhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRASG12X). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRASG12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selectiveinhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selectiveinhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).

Abstract CT119: Safety and efficacy of HS-10370 in KRAS G12C-mutated solid tumors including non-small cell lung cancer (NSCLC)

Abstract Background: KRAS G12C mutation occurs in ~13% of NSCLC and up to 4% of other solid tumors. HS-10370 is a selective, covalent, orally bioavailable KRAS G12C inhibitor. We conducted a first-in-human phase I trial to evaluate the dose-limiting toxicity (DLT), safety, tolerability, pharmacokinetics, and efficacy of HS-10370 in patients (pts) with advanced solid tumors bearing KRAS G12C mutation. Methods: In a phase I study HS-10370-101 (NCT05367778), patients of advanced or metastatic solid tumors with measurable disease were recruited. HS-10370 monotherapy was given at oral doses of 200~1600 mg QD until disease progression, intolerance, or withdrawal from consent. Rolling 6 was applied as the dose escalation schedule in this phase I dose escalation trial. The primary objectives of the dose escalation part was safety and tolerability. The secondary objectives included pharmacokinetics (PK) profile and efficacy. Results: As of Dec 15th, 2023, 55 patients with advanced solid tumors were enrolled in this study, including 49 patients with KRAS G12C mutation. Of the 49 patients, 43 were with NSCLC, 5 with colon cancer, and 1 with pancreatic cancer. Safety: No dose-limiting toxicity was observed, neither maximum tolerated dose (MTD) been identified. Treatment-emergent adverse events(TEAEs) occurred in 87.3% of the treated patients (48/55), the most common TEAEs (≥10%) were AST increase (29.1%, 16/55), ALT increase (29.1%, 16/55), anemia(27.3%, 15/55) , diarrhea (25.5%, 14/55), weight gain (18.2%, 10/55), decreased appetite (14.5%, 8/55), hypoproteinemia (12.7%,7/55), nausea(12.7%,7/55), fatigue(14.5%, 8/55) and rash (12.7%, 7/55). The TEAEs ≥Grade 3 occurred in 27.3% patients (15/55). TEAEs leading to dose interruptions and dose reductions were reported in 18.2% patients (10/55) and 7.3% patients (4/55), respectively. Efficacy: Of the 49 patients with KRAS G12C mutant status, 24 had partial response (PR) and 20 had stable disease (SD). The confirmed objective response rate (ORR) and disease control rate (DCR) were 49.0% and 89.8% respectively. Objective responses were observed in patients received 400mg QD or above. In patients with KRAS G12C mutant NSCLC who received more than or equal to 400mg daily dose, the confirmed ORR and DCR were 51.2% (21/41) and 95.1% (39/41), respectively. Of the same subpopulation, the median duration of response was not reached (5.4, NA). Meanwhile, the median progression-free-survival (PFS) was 11.3 month (6.1, NA), with the 6-months PFS rate 75% and the 9-months PFS rate 63.6%. Responses were also observed in patients with colon cancer and pancreatic cancer. Conclusions: HS-10370 was well tolerated and showed inspiring antitumor activities in KRAS G12C mutated advanced solid tumors, especially in NSCLC. Citation Format: Xiaorong Dong, Xiangjiao Meng, Yan Zhang, Yan Wang, Jianhua Chen, Liang Han, Qiming Wang, Zhong Lin, Hongying Wei, Siheng Lin, Chuan Li, Qiong Wu. Safety and efficacy of HS-10370 in KRAS G12C-mutated solid tumors including non-small cell lung cancer (NSCLC) [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 CT119.

Abstract CT246: Open-label, single-arm, multicenter, phase 2 trial of garsorasib in KRAS G12C-mutated non-small-cell lung cancer

Abstract Background: Garsorasib (D-1553), a potent KRAS G12C inhibitor, has previously demonstrated promising anti-tumor activity in KRAS G12C-mutated non-small-cell lung cancer (NSCLC) in a phase 1 study. Here we present the efficacy and safety of garsorasib in a pivotal phase 2 study. Methods: In this open-label, multicenter, single-arm phase 2 study, patients received garsorasib 600 mg twice daily in 21-day cycles. Eligible criteria included patients with locally advanced or metastatic NSCLC harboring KRAS G12C mutation, who had disease progression after prior anti-PD-(L)1 therapy and platinum-based chemotherapy or intolerance to the above regimens due to toxicity, and had measurable tumor lesions according to the RECIST, v1.1. The primary end point was objective response rate (ORR) evaluated by independent review committee (IRC) per RECIST v1.1. The secondary end points included duration of response (DOR), disease control rate (DCR), time to response (TTR), progression-free survival (PFS), overall survival (OS), and safety. Results: As of November 17, 2023, 123 patients (108 [87.8%] male, median age 64 [range: 33-80], and 11.4%/88.6% ECOG PS 0/1) were enrolled and treated, with a median follow-up of 7.92 months (range, 0.7, 16.5) and a median duration of treatment of 6.24 months (range, 0.7, 15.9). At data cutoff date, 82 patients (66.7%) discontinued treatment, with disease progression (46 patients [37.4%]) being the most common reason. Among 123 patients, IRC confirmed-ORR was 49.6% (61/123, 95% CI, 40.5 to 58.8) and DCR was 88.6% (109/123, 95% CI, 81.6 to 93.6). The median TTR was 1.38 months (range, 1.12 to 5.55). The median DOR was 12.78 months (95% CI, 6.21 to NE). The median PFS was 7.56 months (95% CI, 5.55 to 9.69), and the median OS was not reached. A total of 117 (95.1%) patients reported treatment-related adverse events (TRAEs) with 61 (49.6%) patients experiencing grade 3 or higher events. The most commonly reported (≥25%) TRAEs (any grade) were increased AST, ALT and γ-GGT, anemia, increased blood bilirubin, and increased blood ALP. No new safety signals were identified, and most of the adverse events were well managed. No TRAE resulted in permanent discontinuation of garsorasib. Conclusions: Garsorasib has shown a high tumor response rate and long response duration in patients with KRAS G12C mutated NSCLC that have progressed after prior anti-PD-(L)1 therapy and platinum-based chemotherapy. Its safety profile was also shown to be well-tolerated and manageable. Garsorasib could be considered as a promising treatment option for KRAS G12C-mutant NSCLC patients with a high unmet medical need. Citation Format: Ziming Li, Xiaomin Dang, Dingzhi Huang, Shi Jin, Weiwei Li, Jianhua Shi, Xicheng Wang, Yiping Zhang, Zhengbo Song, Junping Zhang, Wu Zhuang, Xuewen Liu, Liyan Jiang, Xiangjiao Meng, Mingfang Zhao, Jianying Zhou, Liangming Zhang, Pingli Wang, Hui Luo, Junquan Yang, Shundong Cang, Xiang Wang, Jing Wang, Jiuwei Cui, Yan Yu, Zhihong Zhang, Junguo Lu, Weihua Yang, Gaofeng Li, Jifeng Feng, Dongqing Lv, Lin Wu, Yong Fang, Yan Wang, Yanqiu Zhao, Baoshan Cao, Wei Zhu, Zhixiang Zhuang, Qingshan Li, Mingxi Wang, Huan Zhou, Xiaorong Dong, Sheng Hu, Jian Fang, Yihong Zhang, Wenjia Wang, Ziyong Xiang, Zhe Shi, Ling Zhang, Shun Lu. Open-label, single-arm, multicenter, phase 2 trial of garsorasib in KRAS G12C-mutated non-small-cell lung 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 CT246.

Abstract ND07: BBO-8520, a first-in-class, direct inhibitor of KRASG12C (ON), locks GTP-bound KRASG12C in the state 1 conformation resulting in rapid and complete blockade of effector binding

Abstract The glycine to cysteine mutation on codon 12 of KRAS (KRASG12C) is found in ~15% of non-small cell lung cancers and in a low percentage of colorectal and pancreatic adenocarcinomas. This activating mutation pushes the balance of cellular KRAS towards its active, GTP-bound (ON) state that signals downstream and drives cellular proliferation. Recently approved inhibitors of KRASG12C that bind and sequester the oncogenic protein in its inactive GDP-bound (OFF) state, have demonstrated clinical efficacy; however, median duration of response has been &amp;lt; 9 months. Rapid tumor cell adaptation to KRASG12C (OFF)-only inhibitors like sotorasib and adagrasib has been attributed to reactivation of MAPK signaling through increased RTK flux and KRASG12C gene amplification that result in increased KRASG12C (ON) presence and activity. To block rapid KRASG12C (ON)-driven adaptation to KRASG12C (OFF) inhibitors, we have developed a first-in-class, direct, small molecule covalent inhibitor of both KRASG12C (ON) and (OFF) states. BBO-8520 binds in the switch II pocket and covalently modifies both the (ON) and (OFF) forms of KRASG12C independently of any other partner proteins. Mass spectrometry analysis of KRASG12C covalent engagement by BBO-8520 shows complete modification of both KRASG12C states within 15 minutes, while sotorasib and adagrasib, only modify the (OFF) state. 31P NMR studies demonstrate that BBO-8520 inhibits KRASG12C (ON) by locking the GTP-bound protein in state 1, a conformation where it is unable to bind effectors. The ability to directly bind KRASG12C (ON) leads to potent (~30 nM) inhibition in an effector (Raf1) binding assay where inactive (OFF) inhibitors demonstrate no measurable potency. BBO-8520 displays highly significant binding to KRASG12C in a global cysteine proteome analysis and is 100x more selective for KRASG12C than for WT KRAS and other mutant isoforms, with no measurable activity against N- or H-RAS. Cellular signaling and viability assays show that BBO-8520 has sub-nanomolar potency against KRASG12C mutant cell lines. In effector binding assays, BBO-8520 rapidly and completely blocks the RAS-RAF1 interaction, clearly differentiating it from inactive (OFF) inhibitors that require longer times to allow for cycling to the inactive (OFF) state. Additionally, long-term clonogenic assays that detect the emergence of resistance in the presence of inhibitors show that BBO-8520 is at least 30x more potent than sotorasib and adagrasib at preventing outgrowth. Importantly, the presence of growth factors (e.g., EGF) that push KRASG12C into its (ON) state, and significantly lower the potency of inactive (OFF) inhibitors (&amp;gt;20x), have only minor effects on BBO-8520’s potency. Drug-like pharmacokinetic properties allow BBO-8520 to achieve strong dose- and time-dependent pharmacodynamic effects (&amp;gt;80% inhibition of pERK) following a single, oral dose in KRASG12C mutant tumor bearing mice. In vivo target engagement and pERK inhibition in the MIAPaCa-2 and H358 KRASG12C mutant tumor models resulted in durable tumor regressions at 10 mg/kg. Similarly, daily dosing of 10 mg/kg of BBO-8520 in the KrasG12C-p53 driven GEMM model for 6 weeks resulted in better than 50% lung tumor volume regression. BBO-8520’s potent activity against KRASG12C (ON) presents, for the first time, the potential opportunity to directly target all mutant KRASG12C in cells, including the active form of KRASG12C, enabling efficacy following complete or near-complete target inhibition. Citation Format: Anna E. Maciag, James Stice, Bin Wang, Alok Sharma, Albert Chan, Ken Lin, Devansh Singh, Marcin Dyba, Yue Yang, Saman Setoodeh, Brian P. Smith, Dana Rabara, Zuhui Zhang, Erik K. Larsen, Dom Esposito, John Paul Denson, Michela Ranieri, Mary Meynardie, Sadaf Mehdizadeh, Patrick Alexander, Maria Abreu Blanco, David Turner, Rui Xu, Felice C. Lightstone, Kwok Kin Wong, Dhirendra Simanshu, Keshi Wang, Andrew G. Stephen, Kerstin Sinkevicius, Dwight V. Nissley, Eli Wallace, Frank McCormick, Pedro J. Beltran. BBO-8520, a first-in-class, direct inhibitor of KRASG12C (ON), locks GTP-bound KRASG12C in the state 1 conformation resulting in rapid and complete blockade of effector binding [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 ND07.

Abstract CT117: A phase 1 study of D3S-001, a second-generation GDP-bound KRAS G12C inhibitor, as monotherapy in patients with KRAS G12C-mutated solid tumors

Abstract Background: D3S-001 is a second-generation (2G) GDP-bound KRAS G12C inhibitor (G12Ci). Its high covalent potency and rapid target engagement kinetics correlated with robust anti-tumor activity preclinically and translated into promising clinical activity in a phase 1 first-in-human trial (FIH). Here we report clinical data from D3S-001 FIH trial (NCT05410145). Methods: D3S-001 was administered as monotherapy to patients (pts) with KRAS G12C mutated advanced solid tumors at 6 planned dose levels (50-900mg QD) from 3 countries. The key objectives were safety, PK, antitumor activity, and ctDNA kinetics. Results: As of 6 Feb 2024, a total of 41 pts (25 non-small cell lung cancer [NSCLC], 12 colorectal cancer [CRC] and 4 pancreatic cancer [PDAC]) were dosed. Median follow-up was 5.1 months (m, range 0.2-15.4m) and 28 pts (68.3%) remain on-study treatment. No treatment-related DLT or deaths were observed. Treatment-related AEs (TRAE) of any grade occurred in 30 pts (73.2%). Of which, 6 (14.6%) were Grade 3 (no Grade 4 or higher). D3S-001 dose was modified in 11 pts (26.8%) due to TRAE, and no pts had dose discontinuation due to TRAE. PK analysis indicates dose/exposure linearity and terminal plasma T1/2 of ~11 hours. Of 37 pts with post-baseline tumor assessments, 33 were naïve to G12Ci and 4 were G12Ci pre-treated. In 33 G12Ci naïve pts, PRs were achieved in 25/33 overall population (75.8%, 95% CI 57.7, 88.9%), in 14/20 NSCLC pts (70.0%, 95% CI 45.7, 87.2%), in 7/9 CRC pts (77.8%, 95% CI 40.2, 96.1%), and in 4/4 PDAC pts (100.0%, 95%CI 39.6, 100.0%). CNS efficacy was observed in 2/6 NSCLC pts. Of all G12Ci naive pts (n=36), 6-month PFS rate is 69.1% (95% CI 46.3, 83.7%), but median PFS and DoR are not yet mature. Deeper tumor reductions were observed in 5 out of 9 G12Ci naïve pts after intra-subject dose escalation. All 4 G12Ci pre-treated pts (2 NSCLC, 2 CRC) showed stable disease at the first post-baseline assessment. Serial assessment of ctDNA showed significant declines in G12C VAF as early as on Day 8 of treatment. 600mg QD was selected as RP2D. Conclusions: D3S-001, a 2G G12Ci, is well tolerated and delivers promising anti-tumor activity in pts with NSCLC, CRC, and PDAC carrying KRAS G12C mutation across all dose levels in FIH trial. Citation Format: Byoung Chul Cho, Shun Lu, Myung Ah Lee, Zhengbo Song, John Park, Sun Min Lim, Ziming Li, Jun Zhao, Gary Richardson, Yanqiao Zhang, Jun Zhang, Anwen Liu, Cheng Chen, Jia Wang, Jingtao Lu, Haopeng Rui, Qian Chen, Hui Wang, Jing Zhang, Zhi Jian Chen, Tony Mok. A phase 1 study of D3S-001, a second-generation GDP-bound KRAS G12C inhibitor, as monotherapy in patients with KRAS G12C-mutated 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 CT117.

Abstract LB266: AMG510-mediated inactivation of BCL-xL protein promotes Gasdermin-E-dependent pyroptosis in KRAS-G12C mutant non-small cell lung cancer

Abstract Introduction: Studies have shown that molecular targeted therapy can mediate Gasdermin-E (GSDME) dependent pyroptosis. However, GSDME expression is suppressed in many mutated patients, including those with KRAS-G12C-mutated non-small-cell lung cancer (NSCLC). The efficacy of AMG510 against KRAS-G12C inhibition (KRAS-G12Ci) is constrained by tumor recurrence. Pyroptosis is widely involved in the occurrence and development of tumors. However, the role and molecular mechanism of AMG510 in pyroptosis have not been well identified. Here, we demonstrate that inactivation of anti-apoptotic Bcl-2 family members during KRAS-G12Ci treatment enhances pyroptosis, which then promotes the sensitivity of AMG510 in NSCLC. Methods: We assessed pyroptosis using live cells imaging, LDH release assays, ELISA, Western blotting, and flow cytometry. In vitro assays were used to investigate the function of GSDME. Co-culture between peripheral blood mononuclear cells (PBMCs) and KRAS-G12C mutant lung cancer cell lines explored the anti-tumor immunity of cytotoxic T lymphocytes affected by knockdown of GSDME. The molecular mechanism was determined using bulk RNA sequencing, molecular cloning technology, and Western blotting. Results: We demonstrated that KRAS G12Ci treatment promotes cleavage of GSDME, release of high mobility group protein B1 (HMGB1), and LDH, markers of pyroptotic cell death. Additionally, GSDME was significantly downregulated in KRAS-mutated NSCLC compared with patients of KRAS wild type in TCGA cohort, while significantly upregulated after AMG510 treatment and downregulated after AMG510 resistance in vitro assay. We identified that KRAS-G12Ci resulted in the inactivation of BCL-xL, leading to caspase-3-dependent cleavage of gasdermin E. Conversely, amplification of BCL-xL downregulated the expression of GSDME, therefore suppressing the process of pyroptosis. GSDME knockdown in KRAS-G12C mutant lung cancer cell lines showed defective HMGB1 release, reduced caspase-3 viability, and decreased granzyme B and IFN-γ secretion by CD8+ T cells in response to KRAS-G12Ci, and more frequent tumor cell growth. Conclusion: Our findings indicate that GSDME-dependent pyroptosis is a critical determinant of KRAS-G12C-targeted inhibitor AMG510, and through the modulation exerted by the Bcl-2 family protein BCL-xL upon GSDME. These findings suggest that activation of GSDME may provide an effective strategy to sensitize cancer cells to KRAS-G12Ci treatment. Citation Format: Linfeng Luo, Anlin Li, Wei Du, Maojian Chen, JIng Li, Yixin Zhou, Li Zhang, Shaodong Hong. AMG510-mediated inactivation of BCL-xL protein promotes Gasdermin-E-dependent pyroptosis in KRAS-G12C mutant non-small cell lung 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 LB266.

Abstract LB031: Discovery and Characterization of HH100937, a potent and selective SOS1 inhibitor demonstrates synergistic efficacy in combination with KRAS/MAPK therapies

Abstract Besides marketed inhibitors targeting KRAS G12C, novel therapies are needed to effectively treat cancer patients harboring other KRAS mutations. SOS1, a guanine nucleotide-exchange factors (GEFs), plays a critical role in catalyzing the conversion of KRAS from its GDP- to GTP-bound form, regardless of KRAS mutation status and represents a promising therapeutic target for all KRAS-driven tumors. Up to date, several SOS1 inhibitors have entered early phase clinical trials. But a more potent SOS1 inhibitor with improved drug-like properties is still needed to prove efficacy as monotherapy or in combination with other drugs targeting the KRAS/MAPK pathway.We discovered a potent SOS1 inhibitor, HH100937 that effectively disrupted the interaction between SOS1 and wild-type KRAS or with G12C, G12D or G12V mutations. HH100937 exhibited lower IC50s than BI-3406 and MTRX0902 in blocking ERK phosphorylation and cancer cell proliferation, leading to better anti-tumor efficacies in xenografts than reference compounds at same doses. HH100937 also showed synergistic effect with AMG510 in cancer cells with KRAS G12C mutation but refractory to AMG510. The combination of HH100937 and AMG510 in xenografts led to tumor regression in all mice, while some tumors did not response well to AMG510 monotherapy. Our SOS1 inhibitor also exhibited synergistic effect with HH2710, a clinical-stage ERK inhibitor, in a broad panel of cancer cells with KRAS mutations.The pharmacological potency of HH100937 was highly selective over SOS2, other kinases tested or other targets in a safety panel of 47 targets. HH100937 was well tolerated in 28-day DRF study in rats and dogs up to 250 mg/kg QD and 30 mg/kg QD, respectively. The AUC of drug exposure in DRF studies was approximately three to six times higher than that at the efficacious dose. HH100937 also showed favorable DMPK properties in mice, rats and dogs, and acceptable safety profiles in vitro.Taken together, HH100937 is a highly potent and selective SOS1 inhibitor with better anti-tumor activities than some SOS1 inhibitors at clinical stage. It can be developed as mono therapy or in combination with other KRAS/MAPK targeting therapies. Citation Format: Qing Xie, Dongsheng Li, Shujuan Jiang, Jie Zhu, Songda Yu, Maozhi Yang, Yalei Cai, Lei Liu, Bing Yu, Wangyang Tu, Yixiang Zhang, Leping Li. Discovery and Characterization of HH100937, a potent and selective SOS1 inhibitor demonstrates synergistic efficacy in combination with KRAS/MAPK therapies [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 LB031.

Abstract ND10: NST-628 is a novel, potent, fully brain-penetrant MAPK pathway molecular glue that inhibits RAS- and RAF-driven cancers

Abstract Alterations in the RAS-MAPK signaling cascade are common across multiple solid tumor types and are drivers for many cancers. Apart from approved mutation-selective inhibitors for BRAF Class I and KRAS G12C mutations, other mutations of RAS and RAF are not directly addressable by currently approved inhibitors and there is a need for therapeutic strategies having superior efficacy, durability and tolerability for the MAPK pathway. NST-628 is a potent pan-RAF-MEK molecular glue that prevents phosphorylation and activation of MEK by RAF. In this way, NST-628 overcomes the limitations of traditional RAS-MAPK inhibitors and drives deep, durable inhibition of the RAS-MAPK pathway. Cellular, biochemical, and structural analysis of RAF-MEK complexes show that NST-628 engages all isoforms of RAF and prevents the formation of BRAF-CRAF and BRAF-ARAF heterodimers, a differentiated mechanism from all current RAF-directed inhibitors. With its potent and durable inhibition of the RAF-MEK signaling complex, NST-628 demonstrates broad efficacy in cellular and patient-derived, in vivo tumor models harboring KRAS, NRAS, BRAF class II/III, and NF1 mutations. In addition, high intrinsic permeability across the blood-brain-barrier results in NST-628-mediated intracranial anti-tumor activity in orthotopic tumor models. Furthermore, NST-628 pharmacokinetic and metabolic profiles have been optimized for daily dosing in the clinic to maximize therapeutic index. Given its differentiated molecular mechanism, best-in-class drug-like properties, and superior safety profile in IND-enabling GLP studies, NST-628 is positioned to make an impact clinically to address unmet patient need. First-in-human trials of NST-628 have been initiated in early 2024 Citation Format: Meagan B. Ryan, Bradley Quade, Natasha Schenk, Zhong Fang, Marshall Zingg, Steven E. Cohen, Chun Li, Brooke M. Swalm, Aysegul Ozen, Chaoyang Ye, Maria Stella Ritorto, Xin Huang, Arvin C. Dar, Yongxin Han, Michael Hale, Margit Hagel, Klaus P. Hoeflich. NST-628 is a novel, potent, fully brain-penetrant MAPK pathway molecular glue that inhibits RAS- and RAF-driven cancers [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 ND10.

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 LB056: IBI334, a novel ADCC-enhanced B7-H3/EGFR bispecific antibody, demonstrated potent pre-clinical efficacy in solid tumors

Abstract Many solid tumors, such as colorectal cancer (CRC) and lung cancer, are dependent on epidermal growth factor receptor (EGFR) pathway for tumor growth. Antibodies that inhibit EGFR signaling, such as Cetuximab, show clinical efficacy but are associated with on-target toxicities that potentially limit the drug efficacy. To enhance the EGFR signaling inhibition and reduce on-target toxicities, we performed bioinformatics analysis for EGFR/TAA co-expression. We found that B7-H3, a member of the B7 family of immune checkpoint proteins, is broadly expressed in many solid tumors and their expression correlates with EGFR expression in multiple cancers, including lung cancer, head and neck cancer, pancreatic cancer and esophageal cancer. Using Innovent’s proprietary Innobody platform, we developed a bispecific antibody, IBI334, against B7-H3 and EGFR. IBI334 has an EGFR arm for signal blockage and is coupled with a fine-tuned B7-H3 arm with optimal affinity and binding domain. With the aid of B7-H3, IBI334 showed enhanced EGFR signal inhibition than Zalutumumab (EGFR mAb) and Amivantamab (c-met/EGFR bsAb). In addition, IBI334 is afucosylated to enhance its antibody-mediated cell cytotoxicity (ADCC) effects as compared to wild type antibody. IBI334 showed significantly better in vivo efficacy than Amivantamab in NCI-H358 (bronchioalveolar carcinoma with KRAS-G12C mutation), NCI-H292 (lymph node metastasis of a pulmonary mucoepidermoid carcinoma), SK-MES1 (lung cancer) and SK-MES1-LTC (lung cancer with EGFR L858R, T790M and C797S mutations) xenograft models and comparable efficacy in NCI-H322-EGFRexon20ins (lung cancer) model. Cetuximab, an EGFR monoclonal antibody, had high on-target toxicity with 50% early mortalities in cynomolgus monkeys at 120/75 mg/kg/week. With the careful design of B7-H3/EGFR bispecific antibody, IBI334 ameliorated the on-target toxicities and showed only slight hardening of the skin at the injection site at 120 mg/kg/week. As a result, IBI334 has a large therapeutic window of &amp;gt;200 folds. In conclusion, IBI334 is a potent B7-H3/EGFR bispecific antibody with broad application in many EGFR-driven solid tumors. The large therapeutic window enables safe and effective cancer treatment at high exposure levels. Citation Format: Jian Guan, Shuaixiang Zhou, Weiwei Wu, Tianyu Zhu, Lei Cao, Ming Wu, Ninghuan Li, Fenggen Fu, Zhengguan Liao, Shuming Lin, Zeyu Liu, Mengjia Zhu, Nana Luo, Ying Zhang, Tiong Sun Chia, Bingliang Chen, Kaijie He. IBI334, a novel ADCC-enhanced B7-H3/EGFR bispecific antibody, demonstrated potent pre-clinical efficacy in 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 LB056.

Abstract CT163: FIT-001: A phase 1 clinical trial of the farnesyl transferase inhibitor KO-2806 alone or as part of combination therapy for advanced solid tumors

Abstract Background: Farnesyl transferase inhibitors (FTIs) block post-translational modification of RAS and other farnesylated proteins. HRAS-driven tumors are highly sensitive to FTI treatment. Recent clinical trials (NCT03719690, NCT02383927) of the FTI, tipifarnib, in patients with HRAS-mutant (HRAS-m) head and neck squamous cell carcinoma harboring high variant allele frequency mutations (VAF ≥20%), showed objective response rates of up to 50% and favorable long-term outcomes. KO-2806 is a next-generation FTI that has increased potency and improved pharmacokinetic properties. In preclinical studies, KO-2806 has been shown to: (1) enhance tumor growth inhibition of tyrosine kinase inhibitors, including cabozantinib, in multiple clear cell renal cell carcinoma (ccRCC) cell line- and patient-derived xenograft models; and (2) enhance activity of KRAS inhibitors, including adagrasib, in KRAS mutant non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and pancreatic ductal adenocarcinoma (PDAC) mouse models. These preclinical data support clinical investigation of KO-2806 alone and in combination therapy. Study Design: FIT-001 is a first-in-human, multicenter, open-label clinical trial that will evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics (Pd), and preliminary antitumor activity of KO-2806 as monotherapy or in combination therapy in advanced solid tumors (KO-2806-001; NCT06026410). Up to 270 patients will be enrolled in phase 1a and phase 1b combined across 50 sites. Phase 1a will have separate monotherapy and combination dose-escalation arms. As monotherapy, the study will enroll patients with N/K/HRAS alterations in specific solid tumor types, such as NSCLC, CRC, and PDAC, who are refractory to standard-of-care therapies. KO-2806 and cabozantinib will be combined in patients with advanced or metastatic ccRCC who progressed on ≥1 prior line of immunotherapy-based systemic therapy; KO-2806 and adagrasib will be combined in patients with KRAS-G12C mutant locally advanced or metastatic NSCLC who received ≥1 prior systemic therapy. On the basis of emerging data from phase 1a, two Pd cohorts (n≤12) with mandatory pre- and on-treatment tumor biopsies may be enrolled. In each Phase 1b dose expansion, patients will receive the recommended phase 2 dose [RP2D] of KO-2806 with cabozantinib (in ccRCC) or adagrasib (in NSCLC), or will be randomized by dose if 2 potential KO-2806 RP2Ds are identified for a combination. Other combination arms may also be considered. The study began accrual in October 2023. Citation Format: Glenn J. Hanna, Douglas R. Adkins, Jacob S. Thomas, Justine Y. Bruce, Manish R. Patel, Guru Sonpavde, Jason Henry, Nawal Bendris, Zijing Zhang, Amitava Mitra, Amaya Gascó, Andrew Saunders, Stephen Dale. FIT-001: A phase 1 clinical trial of the farnesyl transferase inhibitor KO-2806 alone or as part of combination therapy for advanced 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 CT163.

KRASG12C Inhibitor as a Treatment Option for Non-Small-Cell Lung Cancer with Comorbid Interstitial Pneumonia.

Non-small-cell lung cancer (NSCLC) with comorbid interstitial pneumonia (IP) is a population with limited treatment options and a poor prognosis. Patients with comorbid IP are at high risk of developing fatal drug-induced pneumonitis, and data on the safety and efficacy of molecularly targeted therapies are lacking. KRAS mutations have been frequently detected in patients with NSCLC with comorbid IP. However, the low detection rate of common driver gene mutations, such as epidermal growth factor receptor and anaplastic lymphoma kinase, in patients with comorbid IP frequently results in inadequate screening for driver mutations, and KRAS mutations may be overlooked. Recently, sotorasib and adagrasib were approved as treatment options for advanced NSCLC with KRASG12C mutations. Although patients with comorbid IP were not excluded from clinical trials of these KRASG12C inhibitors, the incidence of drug-induced pneumonitis was low. Therefore, KRASG12C inhibitors may be a safe and effective treatment option for NSCLC with comorbid IP. This review article discusses the promise and prospects of molecular-targeted therapies, especially KRASG12C inhibitors, for NSCLC with comorbid IP, along with our own clinical experience.

Targeted therapeutic options in early and metastatic NSCLC-overview.

The complex therapeutic strategy of non-small cell lung cancer (NSCLC) has changed significantly in recent years. Disease-free survival increased significantly with immunotherapy and chemotherapy registered in perioperative treatments, as well as adjuvant registered immunotherapy and targeted therapy (osimertinib) in case of EGFR mutation. In oncogenic-addictive metastatic NSCLC, primarily in adenocarcinoma, the range of targeted therapies is expanding, with which the expected overall survival increases significantly, measured in years. By 2021, the FDA and EMA have approved targeted agents to inhibit EGFR activating mutations, T790 M resistance mutation, BRAF V600E mutation, ALK, ROS1, NTRK and RET fusion. In 2022, the range of authorized target therapies was expanded. With therapies that inhibit KRASG12C, EGFR exon 20, HER2 and MET. Until now, there was no registered targeted therapy for the KRAS mutations, which affect 30% of adenocarcinomas. Thus, the greatest expectation surrounded the inhibition of the KRAS G12C mutation, which occurs in ∼15% of NSCLC, mainly in smokers and is characterized by a poor prognosis. Sotorasib and adagrasib are approved as second-line agents after at least one prior course of chemotherapy and/or immunotherapy. Adagrasib in first-line combination with pembrolizumab immunotherapy proved more beneficial, especially in patients with high expression of PD-L1. In EGFR exon 20 insertion mutation of lung adenocarcinoma, amivantanab was registered for progression after platinum-based chemotherapy. Lung adenocarcinoma carries an EGFR exon 20, HER2 insertion mutation in 2%, for which the first targeted therapy is trastuzumab deruxtecan, in patients already treated with platinum-based chemotherapy. Two orally administered selective c-MET inhibitors, capmatinib and tepotinib, were also approved after chemotherapy in adenocarcinoma carrying MET exon 14 skipping mutations of about 3%. Incorporating reflex testing with next-generation sequencing (NGS) expands personalized therapies by identifying guideline-recommended molecular alterations.

Prospective virtual screening combined with bio-molecular simulation enabled identification of new inhibitors for the KRAS drug target

Lung cancer is a disease with a high mortality rate and it is the number one cause of cancer death globally. Approximately 12–14% of non-small cell lung cancers are caused by mutations in KRASG12C. The KRASG12C is one of the most prevalent mutants in lung cancer patients. KRAS was first considered undruggable. The sotorasib and adagrasib are the recently approved drugs that selectively target KRASG12C, and offer new treatment approaches to enhance patient outcomes however drug resistance frequently arises. Drug development is a challenging, expensive, and time-consuming process. Recently, machine-learning-based virtual screening are used for the development of new drugs. In this study, we performed machine-learning-based virtual screening followed by molecular docking, all atoms molecular dynamics simulation, and binding energy calculations for the identifications of new inhibitors against the KRASG12C mutant. In this study, four machine learning models including, random forest, k-nearest neighbors, Gaussian naïve Bayes, and support vector machine were used. By using an external dataset and 5-fold cross-validation, the developed models were validated. Among all the models the performance of the random forest (RF) model was best on the train/test dataset and external dataset. The random forest model was further used for the virtual screening of the ZINC15 database, in-house database, Pakistani phytochemicals, and South African Natural Products database. A total of 100 ns MD simulation was performed for the four best docking score complexes as well as the standard compound in complex with KRASG12C. Furthermore, the top four hits revealed greater stability and greater binding affinities for KRASG12C compared to the standard drug. These new hits have the potential to inhibit KRASG12C and may help to prevent KRAS-associated lung cancer. All the datasets used in this study can be freely available at (https://github.com/Amar-Ajmal/Datasets-for-KRAS).

Abstract 5264: KRASG12C inhibition synergizes with checkpoint blockade in KRASG12C-mutated tumor model

Abstract Introduction: The KRASG12C mutation is found in 14% of non-small cell lung cancer (NSCLC) and 3% of colorectal cancer (CRC). Sotorasib (AMG510), which covalently binds to the mutated cysteine 12 of KRASG12C protein, became the first KRASG12C inhibitors approved by the FDA for KRASG12C-mutated NSCLC treatment. AMG510 not only increased apoptosis in cancer cells harboring KRASG12C mutation, but also re-modelled tumor microenvironment. Thus, to evaluate novel therapeutic strategies for treating KRASG12C-mutated tumors, we established a CT26-KRASG12C tumor model and explored the combination of AMG510 with anti-PD1 therapy. Methods: We knocked in KRASG12C mutation in murine CT26 colorectal cancer cells using CRISPR/Cas9 to generate CT26-KRASG12C cells. The mutation was confirmed by PCR sequencing. In vitro, CT26-KRASG12C cells were treated with AMG510 for 2h or 72h, and then p-ERK and cell viability were analyzed. In vivo, 3×105 CT26-KRASG12C cells were subcutaneously injected into BALB/c mice, and the tumor bearing mice were treated with AMG510 (30 mg/kg or 100 mg/kg, QD×21), anti-PD-1 (10 mg/kg, BIW×3) and combination of AMG510 and anti-PD-1. On day 5 after randomization, the composition of immune cells, including CD4+ T cells, CD8+ T cells, dendritic cells (DC), and natural killer (NK) cells in tumors were analyzed by flow cytometry. The mice with complete tumor regression were re-challenged with bilateral tumors of CT26-KRASG12C (3×105) and unrelated mouse breast tumor cells, 4T1 (3×105) to evaluate the effect on tumor growth. Results: Following confirmation of KRASG12C mutation, AMG510 treatment decreased p-ERK expression by ~50% and impaired the cell growth with IC50 value of 0.1802 μM in CT26-KRASG12C cells. In vivo, single treatment of AMG510 at 30 mg/kg or 100 mg/kg significantly inhibited tumor growth with TGI of 60% or 99% on day 15 after randomization, respectively (both p&amp;lt;0.001). Combined treatment of AMG510 (30 mg/kg or 100 mg/kg) and anti-PD-1 also led to significant tumor inhibition with TGI of 64% or 99% on the same day, respectively (both p&amp;lt;0.001), although PD-1 blockade didn’t show anti-tumor effect alone. Noticeably, combined treatment of AMG510 (100 mg/kg) and anti-PD-1 induced complete tumor regression in 3 of 10 mice, which remained cured 124 days later. Moreover, the combined treatment significantly improved survival compared to the AMG510 treatment alone. Notably, all 4T1 tumors grew, but none of the CT26-KRASG12C tumors were palpable in the cured mice after the re-challenge. In the control group, all tumors were well established. Additionally, combined treatment of AMG510 (100mg/kg) and anti-PD-1 markedly increased the infiltration of CD4+ T cells, DC, and NK cells in the tumors. Conclusion: The successfully established CT26-KRASG12C model is a useful tool for investigating therapeutic strategies to combine KRASG12C inhibitors with immunotherapy. Citation Format: Li Hua, Shuang Li, Chenpan Nie, Chenfei Zhang, Ludovic Bourre, Jingjing Wang. KRASG12C inhibition synergizes with checkpoint blockade in KRASG12C-mutated tumor model [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 5264.

Abstract 1942: Characterization of a panel of CRISPR/Cas9 engineered KRAS G12C inhibitor-resistant tumor models

Abstract Introduction: KRAS is one of the most frequent mutated oncogenes and has been recognized as undruggable for many years. AMG510, the first therapy to directly target KRAS, was approved by the FDA to treat non-small cell lung cancer (NSCLC) bearing KRAS G12C mutation. However, the emergence of resistance in patients remains a challenge and limits its clinical benefits, which calls for next-generation targeted therapy or combination strategies to overcome the resistance to KRAS G12C inhibitors. A variety of secondary mutations in KRAS attributing to the resistance have been identified, which requires robust in vitro and in vivo preclinical models to validate potential therapeutics targeting these mutations. Therefore, we generated a panel of KRAS G12C inhibitor-resistant tumor models to facilitate the development of possible strategies to overcome such resistance. Methods: First, a secondary KRAS mutation, including H95D, H95Q, H95R, Q61H and R68S, was introduced by using CRISPR/Cas9 technology in MIA PaCa cell line, which harbors a homozygous KRAS G12C mutation in addition to Y96D, Y96C and Y96S previously published by us. Point mutation knock-in was validated by sanger sequencing, and cell identity was confirmed by SNP assay. In vitro, the parental and mutated cells were treated with either AMG510 and MRTX849 and cell viability was measured by CellTiter-Glo. In vivo efficacy of KRAS G12C inhibitors, AMG510 and MRTX849, SOS1 inhibitor, BI-3406 and MEK inhibitor, Trametinib were evaluated in the Y96D mutated MIA PaCa subcutaneous xenograft. Results: Homozygous secondary point mutation knock-in in MIA PaCa cells was confirmed by sanger sequencing. Similar growth rate and morphology was observed in selected clones compared to the parental line. Similar to Y96D mutation previously published, R68S mutation was highly resistant to both KRAS G12C inhibitors, with IC50 increased more than 100 fold for both MRTX849 and AMG510, whereas H95D, H95Q and H95R mutation was more resistant to MRTX849, while Q61H had a minimal effect on either one of the inhibitors. In addition, cells expressing KRAS G12C/Y96D were also resistant to AMG510 and MRTX849 in in vivo study, whereas combination of BI-3406 (single treatment: TGI of 27%) and Trametinib (single treatment: TGI of 76%) suppressed tumor growth significantly with TGI of 93% on day 17 after randomization compared to control group (p&amp;lt;0.001). Also, the combination showed significant improvement compared to BI-3406 single treatment (p&amp;lt;0.001) whereas no significant improvement was observed compared to Trametinib single treatment (p&amp;gt;0.05). Conclusion: CRISPR/Cas9 engineered second site KRAS mutations in cells harboring KRAS G12C mutation displayed a differentially resistant profile to KRAS G12C inhibitors. Thus, H95D/Q/R, R68S and Y96D can be used as preclinical inhibitor-resistant models to evaluate clinical strategies to overcome resistance to KRAS-targeted therapies. Citation Format: Jun Zhou, Li Hua, Jian Feng, Ning Bao, Dan Zhang, Jingjing Wang, Marrit Putker, Ludovic Bourre. Characterization of a panel of CRISPR/Cas9 engineered KRAS G12C inhibitor-resistant tumor models [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 1942.