Abstract B024: KRAS inhibition is an effective therapy for appendiceal adenocarcinoma: Single cell profiling identifies tumor intrinsic and microenvironmental mechanisms unique to appendix cancer

Abstract Appendiceal adenocarcinoma (AA) is a rare, orphan disease with no FDA approved systemic therapy. KRAS is the most frequent mutated gene in AA, occurring in nearly 80% of mucinous adenocarcinoma. Here we report the results of the first 5 AA patients treated with KRAS inhibitors as well as results of a G12D specific (MRTX1133) and a pan-KRAS (RMC-6236) inhibitor tested in our novel KRAS mutant organoid and PDX models and profiled with high-content imaging, IHC, RNAseq, and RPPA. All 5 patients treated with KRAS inhibition (1 G12D, 2 G12C, 2 pan-KRAS) had biochemical response based on CEA/Ca19-9 or ctDNA and clinical benefit by RECIST criteria (1 CR, 2 PR, 2 SD). Patient 1 with a KRAS G12D, GNAS R201C grade 3 tumor treated with G12D inhibitor had a CR that is ongoing. Patient 5 with a KRAS G12V, GNAS R201C, PIK3CA E545K grade 1 tumor treated with a pan-Ras inhibitor as 7th line therapy had a 66% drop in Ca19-9 and PR after just 2 months of treatment and remains on treatment. MRTX1133 was highly effective for KRAS G12D but not KRAS G12V organoids (IC50 4. 1 nM vs 1. 8 uM, sum-of-squares F test p 0. 0001). As expected both KRAS G12D and KRAS G12V organoids were sensitive to RMC-6236 (IC50 4. 4 nM vs 0. 5 nM, respectively). In orthotopic PDX models of peritoneal carcinomatosis from AA MRTX1133 significantly decreased tumor growth relative to control as measured by MRI in the KRAS G12D PDX model TM00351. Pathologic evaluation showed markedly reduced cellularity in tumor tissues, increased expression of Caspase3, with few tumor cells positive for Ki-67 post-treatment and marked reduction in pERK expression specifically in tumor cells. In a survival experiment all MRTX1133 treated mice remained alive at 60 days whereas all of control mice were dead (HR 13. 4, p=0. 001). Similar results were seen with RMC-6236 treatment of the KRAS G12V mutant model AAPDX-16 with 52. 5% reduction in tumor volume vs vehicle control, p = 0. 01 at 7 days. GSEA revealed significant downregulation of hallmark gene sets ‘E2F targets (NES = -1. 9, p-adj = 0. 06) ’ and the newly developed ‘RAS/ERK upregulated (NES = -2. 3, p-adj = 0. 06) ’ after MRTX1133 or RMC-6236 treatment consistent with the observed decrease in cell proliferation. Additionally, downregulations of RAS-MEK-ERK pathway proteins, such as pMEK (logFC = -3. 3, p-adj = 2. 9 X 10-5) in RPPA, confirmed robust inhibition of this pathway. There was marked up regulation of EMT (NES ≈ 2. 7, FDR 0. 001) and TGF-β signaling (NES ≈ 2. 3, FDR = 0. 004) in the few remaining tumor cells suggesting these could be resistance pathways, seen in both bulk and scRNAseq. Pseudo-time analysis identified that KRAS inhibition prevented the transition of Transit Amplifying cells to Serpine1high EMT cells. scRNA-seq analysis of the TME showed dramatic shifts in CAFs with KRAS inhibition driving normal resident fibroblasts to inflammatory CAFs. Related, analysis of mouse reads from bulk RNAseq showed upregulation of interferon alpha 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (5Suppl₁): Abstract nr B024.