Pathway-selective strategies reveal specific PI3K–mTOR inhibitors as key partners in KRAS G12D-targeted therapy

KRAS G12D is the most common oncogenic mutation in pancreatic ductal adenocarcinoma (PDAC), driving resistance and heterogeneity. Using robotic high-throughput drug screening with live-cell imaging, we evaluated normalized organoid growth rate (NOGR) in KRAS G12D-mutant PDAC organoids treated with the inhibitor MRTX1133. Monotherapy induced predominantly cytostatic, dose-dependent effects, reflecting heterogeneity observed in single-cell transcriptomics, where compensatory MAPK/ERK and PI3K–mTOR activation emerged. Drug response profiles were variable, and synergy screening revealed patient- and compound-specific interactions. The most consistent cytotoxic synergy was achieved with PI3K–mTOR inhibitors. Isoform-specific PI3Kα inhibitors (inavolisib, alpelisib) demonstrated robust synergy with MRTX1133 and favorable tumor selectivity, whereas dual PI3K/mTOR inhibitors (e.g., VS-5584) were more cytotoxic but lacked specificity. These results indicate that KRAS G12D inhibition alone is insufficient due to underlying transcriptional diversity, and highlight isoform-specific PI3Kα inhibitors as promising partners for combination therapy in PDAC.