G12Ci DTPs show reduced G12Ci engagement after washout.

SOS1i synergizes with G12Ci under both 2D and 3D culture conditions.

SOS1i prevents G12Ci-induced TIC outgrowth.

KRASG12C-mutated cell lines show universal G12Ci:SHP2i synergy.

SOS1i synergizes with G12Ci to drive transcriptional changes regulating MAPK signaling.

SOS2 expression determines the extent of G12Ci:SOS1i synergy.

SOS2 expression determines the extent of G12Ci:SOS1i synergy.

SOS1 inhibition limits the development of acquired G12Ci resistance.

KRAS mutations are common oncogenic drivers in human cancers, with the KRASG12C variant being a key target in lung adenocarcinoma (LUAD). Despite the FDA approval of KRASG12C-selective inhibitors, their clinical efficacy has been limited, as evidenced by trials showing modest response rates and resistance development through the selection of acquired genomic alterations. Our study explores the mechanisms underlying acquired EML4-ALK fusion in KRASG12C-driven tumors developing resistance to KRASG12C inhibitors and potential therapeutic strategies to overcome it. Our findings reveal that combined ALK/KRASG12C inhibition is an effective therapeutic approach in this context. Moreover, we observed that KRASG12C/EML4-ALK tumor cells kept under constant pressure with KRASG12C inhibitors exhibit sensitivity to single-agent ALK inhibitors, suggesting a potential for rationally designed sequential treatments. Mechanistically, EML4-ALK bypasses KRASG12C inhibition by activating wild-type RAS, highlighting an additional therapeutic opportunity for multi-selective RAS inhibitors under clinical investigation.

SOSi prevents MEKi-induced TIC outgrowth.

SHP2i prevents G12Ci-induced TIC outgrowth.

G12Ci DTPs show increased CD133+ / ALDHhigh cells.

Disease persistence and resistance remain major challenges in chronic myeloid leukemia (CML). We evaluated pharmacological SOS1 inhibition with BI-3406, alone or in combination with BCR::ABL1 tyrosine-kinase inhibitors (TKI), across complementary preclinical models. In p210BCR/ABL transgenic mice, BI-3406 plus imatinib was well-tolerated, markedly prolonged survival, improved hematologic indices, reduced splenomegaly, and decreased bone-marrow LSK/hematopoietic stem and progenitor cells compared to either agent alone. In human CML cell lines and primary patient-derived bone-marrow cells, combined therapeutic treatments with BI-3406 and imatinib (standard of care BCR-ABL TKI) or next-generation TKIs showed strong synergistic suppression of cellular proliferation and increased apoptosis relative to single-agent treatment. Remarkably, drug combinations containing BI-3406 also restored TKI sensitivity to primary bone-marrow cell samples derived from a variety of CML patients that had been initially identified as clinically resistant to imatinib. Mechanistically, BI-3406 attenuated RAS/RAC-ERK/AKT signaling, while SOS1 knockdown phenocopied BI-3406-mediated imatinib sensitization, supporting an on-target SOS1-dependent therapeutic effect. Upregulation of the membrane transport protein SLC22A4 following combination treatment may contribute to the rescued TKI sensitivity but is probably not the sole underlying mechanism as BI-3406+imatinib retained strong synergistic activity despite SLC22A4 knockdown. Comparison of the transcriptomic profiles revealed that the combined BI-3406+imatinib drug regimen triggered the strongest repression of oncogenic, metabolic and stemness-associated transcriptional programs in treated CML cells. Consistently, serial replating MethoCult assays functionally confirmed the impairment of clonogenic self-renewal capacity and the depletion of murine LSK and human Lin⁻CD34⁺CD38⁻ stem/progenitor-enriched compartments after dual SOS1 and BCR::ABL1 inhibition. Our data support SOS1 targeting as a rational therapeutic strategy to potentiate TKI efficacy, impair CML stem/progenitor self-renewal, and restore drug responsiveness in clinically relevant TKI-resistant settings.

Abstract KRAS is the most frequently mutated oncogene, with high prevalence in indications with significant unmet clinical needs, such as lung, pancreatic and colorectal cancer. The most frequent alterations result in an amino acid exchange at position 12 from Glycine to Aspartic Acid (G12D), Valine (G12V) or Cysteine (G12C). The approval of mutant specific KRAS G12C inhibitors by the FDA opened a new field of treatment options for patients with KRAS G12C mutation and additional KRAS targeted therapy approaches are currently being clinically tested in the hope to provide benefit to cancer patients with cancers harboring other KRAS mutant alleles. These therapies include pan-RAS, pan-KRAS and allele selective inhibitors as well as degraders. For patients with advanced solid tumors harboring a KRAS G12D mutation, several selective inhibitors have entered clinical trials (MRTX1133, RMC-9805, QTX3034/46, LY3962673...) and RMC-6236, a pan-RAS inhibitor from Revolution Medicines, has recently shown promising early clinical data. However, alongside the great hopes placed in (K)RAS targeting therapy, resistance is likely to occur, as it was already observed in patients relapsing in response to KRAS G12C inhibitors. We aimed to use this pre-clinical study to predict and understand potential resistance mechanisms that may arise during targeted treatment against (K)RAS in the CRC setting. To this aim, the colorectal cancer cell line GP2d, expressing KRAS G12D, was continuously treated with a pan-RAS inhibitor to generate resistance. Once the resistance was confirmed, individual outgrowing clones were subsequently profiled in a series of assays to identify potential mechanism(s) and cross-tested with other inhibitors. Interestingly, cells were cross resistant to both pan-RAS (RMC-6236) and KRAS G12D (RMC-9805) inhibitors from Revolution Medicine, highlighting a common mechanism of action, while remaining sensitive to other KRAS G12D inhibitors. These findings were then confirmed in an orthogonal assay. Collectively our in vitro preclinical study identified a resistance mechanism to Tri-complex inhibitors in colorectal cancer cells. This resistance can still be addressed by other KRAS G12D inhibitors, opening options for patients harboring this characteristic post RMC-therapy. Future investigations may focus on characterizing tumors from patients who relapse on RMC-6236 and RMC-9805 to validate these findings clinically. Citation Format: Sabine Jurado, Simone Lieb, Marco H. Hofmann, Mark Pearson, Phillipp Schmalhorst, Krzysztof Zak. Acquired resistance to Tri-complex inhibitors in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1877.

KEAP1 and STK11 co-mutations alter basal ERK and AKT phosphorylation.

Oncogenic signaling in cancer cells is essential for proliferation, and its disruption, either through inhibition or overactivation, can provide therapeutic opportunities. Dual specificity phosphatase 4 (DUSP4), a negative regulator of the MAPK pathway that dephosphorylates ERK, has been proposed as a potential target; however, its therapeutic relevance has not been evaluated in vivo. In this study, we show that DUSP4 knock-down induces G1 cell cycle arrest and reduces proliferation in BRAFV600E-mutant and BRAF inhibitor-resistant colorectal cancer models, both in vitro and in vivo, and induces a rapid DUSP5-mediated adaptive response. While treatment achieved tumor stasis indicating disease control, it did not yield tumor regression, suggesting that DUSP4 may have limited efficacy as a monotherapy target in cancer. ### Competing Interest Statement All authors are full time employees at Boehringer Ingelheim RCV.

G12Ci DTPs show reduced sensitivity to G12Ci that is restored by SOS1i or ALDHi treatment.

G12Ci treatment increases aldehyde dehydrogenase activity in LUAD cells.

SOS1i synergizes with G12Ci to limit MAPK pathway signaling under 2D and 3D conditions.

KRASG12C-mutated cell lines show universal G12Ci:SOS1i synergy in the absence of SOS2.