Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer-related mortality. KRAS mutations, present in approximately 40% of CRC cases, drive aggressive tumor behavior and remain a major therapeutic challenge. While FDA-approved inhibitors exist for KRAS G12C, this mutation represents only a small subset of CRC, and resistance frequently develops through MAPK pathway reactivation or compensation via alternative survival pathways. KRAS G12D is the most prevalent allele in CRC; however, there are currently no FDA-approved inhibitors and few investigational agents in advanced development. To address KRAS G12D resistance, we developed a high-throughput combinatorial screening platform using both cell lines and patient-derived organoids (PDOs) with fully annotated molecular profiles to identify synergistic drug combinations capable of overcoming resistance. Method: We performed a large-scale combinatorial drug screen in both parental and resistant KRAS G12D mutant CRC cell lines. The screening library consisted of approximately 2, 600 kinase inhibitors and 3, 500 FDA-approved or clinical-stage compounds, tested both as single agents and in combination with MRTX1133, a preclinical KRAS G12D inhibitor. Viability was assessed using the ATP-based luminescence assay CellTiter-Glo. Compounds demonstrating robust synergy were prioritized for secondary validation using 8×8 dose-response matrices. Subsequently, the most promising compounds were validated in KRAS G12D PDOs to confirm translational potential. Result: Multiple compounds displayed synergistic activity with MRTX1133. Notably, "NT-1, " a novel analog of the FDA-approved EGFR inhibitor osimertinib, demonstrated significant synergy (synergy score 10) at nanomolar concentrations across both sensitive and resistant CRC models Compared with other EGFR inhibitors (osimertinib, erlotinib, afatinib), NT-1 achieved greater inhibitory effects at lower doses. These findings were extended to combinations with emerging pan-KRAS inhibitors, where NT-1 consistently outperformed approved EGFR inhibitors and cetuximab, producing deeper suppression of p-EGFR and p-ERK, and stronger induction of apoptosis. Ex vivo validation using KRAS G12D PDOs confirmed the translational potential of these combinations, demonstrating that NT-1 induces stronger effects than current EGFR inhibitors and can mitigate both intrinsic and acquired resistance to G12Di. Ongoing in vivo studies are assessing NT-1 in combination with G12D selective and pan-KRAS inhibitors to delineate mutation-specific versus pathway-level vulnerabilities. Conclusion: Our combinatorial screening platform provides novel biological insights into KRAS G12D resistance mechanisms in CRC and effectively identifies drug combinations with immediate clinical utility, particularly among approved or clinical-grade drugs. The combination of NT-1 with G12D selective or pan-KRAS inhibitors represents a promising therapeutic strategy to overcome KRAS G12D resistance. Citation Format: Natalie Thielen, Chaoyuan Kuang, Seiya Kitamura, Emiko Nagai, Ning Wei. A high-throughput combination screen identifies NT-1 as a superior compound to overcome KRASG12D inhibitor resistance abstract. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (5Suppl₁): Abstract nr B031.
Thielen et al. (Thu,) studied this question.
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