KRAS gene alterations are prevalent in various cancers, with the G12C mutant being significant in NSCLC (13%) and CRC (3%). KRAS G12C inhibitors (KRAS G12C i), like sotorasib and adagrasib, show clinical benefit but often lead to resistance. KRAS cycles between inactive (GDP-bound) and active (GTP-bound) states; GEFs regulate the transition to the GTP-bound state, while GAPs enhance GTPase activity. Active KRAS drives cell growth via MAPK and PI3K signaling; activating mutations maintain the GTP-bound state. KRAS G12C i bind to GDP-bound KRAS G12C, inactivating it. However, resistance mechanisms such as RAS-MAPK signaling re-establishment through mutant or WT RAS activation, secondary KRAS mutations, or upstream/downstream bypass events can emerge. Combination therapies targeting regulators of RAS GTP loading (RTKs) and downstream signaling (SHP2) have shown promise in preclinical models by shifting the balance toward inactive KRAS. KRAS G12C i plus SHP2 inhibitors (SHP2i) and KRAS G12C i plus EGFR antagonists (EGFRi) are in clinical trials. This research hypothesizes that SOS1 inhibition, by maintaining KRAS in the inactive state, would synergize with KRAS G12C i. Co-targeting SOS1, a common node for multiple RTKs, may provide more durable responses than single RTK targeting. The study aimed to evaluate the efficacy and durability of KRAS G12C i plus BI-3406 (SOS1i) compared to KRAS G12C i alone or with SHP2i (TNO155) or EGFRi (cetuximab) in KRAS G12C -driven NSCLC and CRC models.

The literature extensively covers KRAS mutations in cancer and the development of KRAS G12C inhibitors. Studies highlight the clinical success of these inhibitors, such as sotorasib and adagrasib, but also their limitations due to the rapid development of resistance. Preclinical research has explored various combination strategies to overcome this resistance, focusing on targeting upstream activators of KRAS, including RTKs, and downstream signaling effectors like SHP2. The success of these combinations in preclinical models and the initiation of clinical trials with such combinations were highlighted to provide context for the current study. The mechanism of action of KRAS G12C inhibitors and how they are affected by different feedback loops are discussed.

The study employed a multi-pronged approach involving in vitro and in vivo experiments. In vitro studies utilized high-throughput screening to identify synergistic combinations of KRAS G12C i (sotorasib or adagrasib) with a panel of small molecule inhibitors in KRAS G12C -driven NSCLC (NCI-H2122) and CRC (SW837) cell lines. The combination scores (cScore) assessed synergy. IncuCyte kinetic cell confluence assays and western blots analyzed the effects on cell growth, apoptosis, and RAS-MAPK pathway signaling. In vivo studies used cell line-derived xenografts (CDX) and patient-derived xenografts (PDX) models of NSCLC and CRC to evaluate tumor growth inhibition (TGI) in response to single-agent and combination therapies. Adagrasib, BI-3406, TNO155, and cetuximab were administered according to clinically relevant dosing schedules. Tumor volume, body weight, and pharmacodynamic biomarkers were monitored. RNA sequencing (RNA-seq), MPAS analysis, and immunohistochemistry (IHC) assessed the effects on gene expression and MAPK pathway activity. Studies were conducted on adagrasib-resistant cell lines and xenograft models to investigate the potential of combination therapy to overcome acquired resistance. Statistical analyses included ANOVA, t-tests, and Wilcoxon rank sum tests, with appropriate multiple comparisons corrections.

High-throughput screening identified synergistic anti-proliferative effects in KRAS G12C -driven cell lines when combining KRAS G12C i with SOS1i (BI-3406), SHP2i (TNO155, SHP099), or ErbB inhibitors. In vivo studies showed adagrasib plus BI-3406, TNO155, or cetuximab enhanced anti-tumor responses compared to adagrasib alone in NSCLC and CRC models (CDX and PDX). Adagrasib combined with BI-3406 or TNO155 achieved greater RAS-MAPK pathway inhibition and durable anti-proliferative effects in vitro and in vivo, as measured by reduced RAS-GTP levels, pERK, p-S6, and increased cleaved PARP. Combination treatments induced stronger overall transcriptomic changes and more pronounced MAPK pathway downregulation compared to monotherapy, sustained over 48 hours. In adagrasib-resistant models, BI-3406 or TNO155 combination with adagrasib reversed resistance, showing tumor regression. Analysis of adagrasib-resistant tumors revealed no secondary KRAS mutations but identified upregulation of MRAS and RasGRP1, suggesting alternative mechanisms for resistance.

The study demonstrates that combining KRAS G12C i with SOS1 inhibition enhances and prolongs anti-tumor responses in both KRAS G12C i-naïve and resistant tumors. The superior performance of the BI-3406/adagrasib combination compared to other tested combinations suggests a promising therapeutic strategy. The mechanism appears to involve enhanced suppression of RAS-MAPK signaling, potentially through more effective blockage of KRAS activation. The ability to overcome acquired resistance, possibly through alternative pathways rather than solely secondary KRAS mutations, is notable. The findings support the clinical evaluation of SOS1i and KRAS G12C i combinations. The study highlights the complexity of acquired resistance to KRAS G12C i and suggests that targeting multiple nodes in the RAS-MAPK pathway may be crucial for durable responses. The findings support further investigation into the roles of MRAS and RasGRP1 in acquired resistance.

This preclinical study strongly supports the potential of combining SOS1 inhibitors with KRAS G12C inhibitors as a novel strategy for treating KRAS G12C -driven cancers. The combination therapy demonstrated enhanced anti-tumor efficacy and the ability to overcome acquired resistance, surpassing monotherapy and some other combination approaches. Future research should focus on clarifying the mechanisms of acquired resistance, investigating other combination partners, and extending these findings to clinical trials.

The study is limited to preclinical models, and results may not fully translate to the clinical setting. The mechanisms of acquired resistance in the models require further investigation to definitively identify the key driver genes beyond the increased expression of MRAS and RasGRP1. The study focused primarily on adagrasib and did not assess the impact of the combination on other KRAS G12C inhibitors or “KRAS G12C-on” inhibitors.