Supplementary Figure S10. Effects of co-inhibition of KRAS and wild-type RAS in KRASmutant PDAC tumor models.

Supplementary Figure S4. Expression of EMT at baseline.

PURPOSE:Oncogenic KRAS mutations are present in >90% of pancreatic ductal adenocarcinoma (PDAC), with KRASG12D being the most common. Mutant-selective KRASG12D inhibitors (KRASiG12D) have demonstrated promising initial clinical activity in KRASG12D-mutant PDAC. However, adaptive resistance to KRASi constrains efficacy in some tumor types, such as colorectal cancer, in which EGFR-mediated RAS-MAPK pathway reactivation can be targeted to improve response. Some studies have suggested a similar role for EGFR in PDAC, but the mechanisms of adaptive resistance to KRAS inhibition are unclear. EXPERIMENTAL DESIGN:Mechanisms of adaptive resistance to KRASiG12D were investigated in a panel of KRASG12D-mutant PDAC models. RESULTS:We observed receptor tyrosine kinase (RTK)-driven adaptive reactivation of RAS pathway signaling following KRASiG12D in PDAC models. EGFR was a primary driver of adaptive RAS-MAPK reactivation in some models but was limited to those with epithelial differentiation. Conversely, adaptive RAS-MAPK reactivation in models with mesenchymal differentiation was primarily driven by FGFR signaling. In clinical PDAC specimens from The Cancer Genome Atlas, EGFR and ERBB3 expression was highly correlated with the expression of epithelial markers, whereas the expression of FGFR1 and mesenchymal markers was correlated. Notably, a RAS(ON) multi-selective inhibitor, which inhibits both wild-type and mutant RAS, abrogated RAS-MAPK reactivation in combination with KRASi in both epithelial and mesenchymal models and led to more consistent antitumor activity compared with combinations of KRASi and EGFR blockade. CONCLUSIONS:In PDAC, adaptive RAS-MAPK reactivation following KRASG12D inhibition can be mediated by different RTKs and influenced by cell state. Combinations of mutant-selective KRASi and RAS(ON) multi-selective inhibitors may represent a promising universal strategy to surmount adaptive resistance in patients with PDAC.

Supplementary Figure S2. Effect of KRASG12D inhibition on KRASG12D-mutant PDAC cell lines.

Kaplan–Meier curves for survival outcomes in patients with MSS/pMMR mCRC receiving ICI-based therapies, stratified by liver metastasis status at the time of ICI initiation. A, PFS: Patients without liver metastases had significantly higher 12-month PFS rates (12.8% vs. 1.1%; P = 0.034). The median PFS was 2.1 months (95% CI, 1.59–2.62) in those with liver metastases and 2.5 months (95% CI, 2.22–2.71) in those without liver metastases (HR, 1.68; 95% CI, 1.13–2.5; P = 0.009). B, OS: Median OS was 11.53 months (95% CI, 3.01–20.06) in those without liver metastases and 6.17 months (95% CI, 2.87–9.46) in those with liver metastases (HR, 2.03; 95% CI, 1.35–3.06; P < 0.001).

Supplementary Table S6. Statistical comparison of median survival for KP-4 model.

Supplementary Figure S9. Western blot analysis of combined MRTX1133+RMC-7977 in epithelial lines SU86.86 and HPAF-II.

Supplementary Table S3. Antibodies employed in the studies.

Cancer evolution is a complex and dynamic process, yet most treatment strategies remain static. Infrequent tumor sampling has limited our ability to counteract the transient adaptive states that precede resistance. To address this gap, ARPA-H launched the ADAPT program, an initiative aimed at transforming cancer care by aligning therapies with real-time tumor evolution. Within this framework, the ASCEND-CRC trial aims to uncover early adaptive mechanisms and identify biomarkers to guide therapeutic decision-making in metastatic colorectal cancer (CRC). The study moves beyond single pre-treatment biomarkers by integrating multimodal profiling to longitudinally track tumor evolution and define an actionable set of dynamic biomarkers that inform treatment decisions. Together with other ADAPT initiatives, ASCEND-CRC represents a paradigm shift in precision oncology, establishing a scalable platform to intercept resistance.

Supplementary Figure S3. Effect of MAPK reactivation over 0-96h exposure of MRTX1133 on KRASG12D-mutant PDAC cell lines.

Demographic and clinical characteristics of patients with MSS/pMMR mCRC by liver metastasis status.

Background:Comprehensive genomic profiling (CGP) is critical for optimal clinical care for gastrointestinal (GI) carcinomas. Outside of biliary tract cancers (BTCs), actionable fusions are uncommon and the utility of routine RNA-based fusion testing in GI cancers is not well established. We therefore evaluated routinely conducted RNA-based fusion testing in GI cancers at a single center. Methods:Patients with GI carcinomas who underwent RNA-based tissue fusion testing at Massachusetts General Brigham Cancer Institute were included. Tumors with fusions were characterized for molecular and histopathologic associations. RSPO2/3 fusions in lower GI cancers were further characterized for survival and benefit from targeted therapy. Results:A total of 2,300 patients with GI carcinomas were included; 139 (6.0%) had a detected fusion. BTCs had the highest fusion incidence (13.7%), mostly FGFR2. Fusions were detected in 3.5-7.7% of colorectal cancer (CRC), pancreatic, esophagogastric adenocarcinoma (EGA), and small bowel carcinomas. Fusions of BRAF, FGFR1-3, NTRK1/3, RSPO2/3, and NRG1 were rare (excepting FGFR2 in BTCs) but observed in 3 or more cancer types. RSPO2/3 fusions were the second most frequent overall (1.4%) and most frequent in CRC (2.6%) and small bowel cancers (5.8%). RSPO2/3 fusions were associated with poorly differentiated and mucinous histology, preserved mismatch repair, wild-type (WT) APC, and either KRAS or BRAF V600E mutations, however were not associated with differential survival in CRC patients receiving BRAF targeted therapy. Fifteen of 20 (75%) pancreatic tumors with fusions were KRAS WT; in KRAS-mutated pancreatic cancer, most fusions were of unclear significance. Receipt of fusion targeted therapy was associated with longer survival in fusion positive pancreatic cancers and FGFR2-fusion positive BTCs. 20 fusions detected in EGA were heterogenous, including FGFR, CLDN18, and RSPO2. Conclusions:RNA-based testing identifies potentially actionable fusions in a clinically significant fraction of GI carcinomas. Patients who receive fusion targeted therapy during their clinical course demonstrate longer survival, supporting CGP including fusion testing for all patients. Lower GI cancers harboring RSPO2/3 fusions have a distinct molecular and pathologic phenotype, but demonstrate few clinical differences. Several detected fusions had novel gene partners, highlighting the utility of partner-agnostic fusion testing.

Regimens of immune checkpoint inhibitor-based therapies

Supplementary Figure S7. EGFR blockade did not inhibit MAPK rebound mediated by KRAS inhibition in certain KRAS-mutant PDAC cell lines.

Immune checkpoint inhibitors (ICI) have limited efficacy in microsatellite-stable (MSS) metastatic colorectal cancer (mCRC), potentially because of immunosuppressive mechanisms associated with liver metastases. The impact of liver metastases on survival outcomes was evaluated in a retrospective cohort of patients with MSS mCRC treated with ICI-based therapies at Mass General Brigham between January 2015 and December 2022. Patients were stratified by liver metastasis status at ICI initiation. The primary endpoint was progression-free survival (PFS); the secondary endpoint was overall survival (OS). A total of 132 patients were included, of whom 93 (70.5%) had liver metastases at ICI initiation. Most patients in both groups had received ≥2 prior lines of therapy. No significant differences were observed between groups for RAS/BRAF mutation status or tumor mutational burden. Patients without liver metastases demonstrated higher clinical benefit rates (46.2% vs. 16.1%; P = 0.001), longer median PFS (2.5 vs. 2.1 months; HR, 1.68; P = 0.009), and higher 12-month PFS rates (12.8% vs. 1.1%; P = 0.034). The median OS was also prolonged in patients without liver metastases (11.5 vs. 6.2 months; HR, 2.03; P < 0.001). No history of liver metastases was an independent favorable risk factor for PFS and OS in univariable and multivariable analyses. These findings indicate that liver metastases are associated with inferior survival outcomes in patients with MSS mCRC treated with ICI-based therapies, supporting the immunosuppressive role of liver metastases and underscoring the importance of stratifying patients by liver metastasis status to guide patient selection and optimize therapeutic strategies. SIGNIFICANCE:The limited efficacy of ICIs in MSS mCRC remains a major challenge. The association between liver metastases and inferior outcomes supports the liver's immunosuppressive role and suggests that liver metastasis status may guide patient selection and treatment optimization.

Alterations in KRAS, NRAS, and HRAS occur in roughly 20% of patients with cancer, making RAS one of the most intensively studied oncogenic targets. The discovery of mutant-selective KRASG12C inhibitors has provided a proof-of-concept for RAS-directed therapies, heralding a new era in the treatment of RAS-driven cancers. Yet, the efficacy of first-generation KRASG12C inhibitors is limited by the rapid emergence of resistance. Novel classes of (K)RAS inhibitors with distinct mechanisms of action and broader target coverage hold promise to overcome resistance and extend the benefits of RAS-targeted therapies to a wider patient population. In this review, we summarize clinical evidence for KRASG12C inhibitors across tumor types and delineate key mechanisms of resistance. We further discuss the rapidly evolving landscape of next-generation (K)RAS inhibitors, with particular emphasis on their target selectivity, mechanisms of action, preliminary clinical efficacy, and the therapeutic opportunities and challenges inherent to each class.

Abstract KRAS mutations are among the most common oncogenic drivers across human cancers. Although RAS was long considered undruggable, recent advances have led to multiple classes of direct RAS inhibitors. KRAS(OFF) inhibitors preferentially target the inactive GDP-bound state whereas RAS(ON) tri-complex inhibitors selectively engage the active, GTP-bound form. In addition to suppressing RAS signaling by disrupting interactions with downstream effectors via steric occlusion, the RAS(ON) multi-selective inhibitor daraxonrasib (RMC-6236) and the preclinical tool compound RMC-7977 also activate RAS(ON) GTPase activity and promote the conversion of RAS(ON) to RAS(OFF). By stimulating formation of the inactive, GDP-bound state the RAS(ON) inhibitor enables potent target engagement by a KRAS(OFF) inhibitor and synergistic inhibition of oncogenic KRAS G12 mutant signaling. The inhibitory activity of the combination of daraxonrasib or RMC-7977 with representative mutant-selective KRAS(OFF) and pan-KRAS(OFF) inhibitors was evaluated in a series of KRAS G12 mutant cancer cell lines. In addition, real-time RAS-RAF complex disruption assays were used to monitor the kinetics of target engagement. As predicted, the RAS(ON) multi-selective inhibitors accelerated the rate of RAS-RAF disruption for KRAS(OFF) inhibitors. Consistent with this, a synergistic increase in potency of inhibition of KRAS signaling and cell viability was observed with the combinations. Synergy was dependent on the ability of the RAS(ON) inhibitors to convert RAS(ON) to RAS(OFF) and was abolished with the use of RAS(ON) inhibitors that do not stimulate GTP hydrolysis or the introduction of mutations in RAS that block GTP hydrolysis. In vivo, the combination of daraxonrasib and KRAS(OFF) inhibitors at well-tolerated doses drove deep and durable RAS pathway suppression and tumor regressions in various KRAS G12 mutant xenograft models, including models with reduced sensitivity to either single agent, e.g., those with mutant KRAS amplification. Overall, these preclinical findings provide a mechanistic rationale for, and experimental evidence in support of, the clinical evaluation of the combination of a RAS(ON) multi-selective inhibitor that stimulates the GTPase activity of oncogenic KRAS mutants with a KRAS(OFF) inhibitor as a potential therapeutic strategy to maximize RAS inhibition and enhance antitumor activity in RAS-addicted cancers. Citation Format: Hiroyuki Matsubara, Alec Millner, Yu C. Yang, Jun Sun, Stephanie Change, Shelby L. Steele, Marini Thian, Miguel Sandoval, Zhican Wang, Mike Flagella, Mallika Singh, Jingjing Jiang, Jacqueline A. Smith, Ryan B. Corcoran, Kyle J. Seamon. RAS(ON) multi-selective inhibitors stimulate the hydrolysis of RAS-GTP to RAS-GDP and drive synergistic combination benefit with KRAS(OFF) inhibitors in G12 mutant tumors [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 5696.

Supplementary Figure S6. Quantitative expression of RAS activity in mesenchymal and epithelial KRAS-mutant PDAC cell lines treated with combinations of RTK inhibitors and KRASi.

Supplementary Figure S8. Combination effects of KRASi+EGFRi vs. KRASi+FGFRi.

Subsequent treatments after immune checkpoint inhibitor-based therapy