Abstract Direct KRAS inhibitors are reshaping the therapeutic landscape of pancreatic ductal adenocarcinoma (PDAC), yet responses are frequently transient and the spatiotemporal microenvironmental programs unleashed by oncogene suppression remain incompletely defined. Here, we identify widespread intrinsic resistance to KRASG12D inhibition across pancreatic cancer models, in which subclonal tumor populations persist despite oncogene suppression. Using genetic barcoding coupled with single-cell RNA sequencing (scRNA-seq), we show that neither pan-RAS inhibition nor genetic deletion of mutant KRAS eliminates all tumor subclones, implicating genomic copy-number alterations and cell-state/identity shifts as key drivers of persistence and highlighting the need for rational combination strategies. To resolve how resistant subclones shape local tumor microenvironments, we integrated scRNA-seq with high-resolution spatial transcriptomics and developed an autochthonous dual-recombinase PDAC model enabling inducible, irreversible KrasG12D deletion. Mutation-specific detection of KrasG12D transcripts in single-cell and spatial data revealed a multi-phase response: early cancer-cell cycle arrest and interferon signaling followed by complement activation and a coordinated stromal transition from myofibroblastic CAFs to inflammatory–like states. This fibroblast rewiring establishes lymphoid chemokine niches, and drives marked T and B cell recruitment. Exploiting intra-tumoral genetic mosaicism, we further show that residual KrasG12D-high pockets act as short-range organizers of an immune-excluded microenvironment, maintaining myofibroblastic stroma and immunosuppressive macrophages while excluding CD8+ T cells. In contrast, KRAS-depleted regions recruit T cells, yet spatial scoring and pseudotime analyses indicate rapid acquisition of terminal exhaustion upon tumor-core entry. Together, our data define KRAS as a spatial gatekeeper of stromal–immune architecture and uncover a transient window of immune priming after oncogene depletion that is constrained by cancer cell–mediated T cell dysfunction, providing a mechanistic rationale and spatial biomarkers for combining RAS inhibitors with therapies that sustain immune priming and restore cytotoxic T cell fitness. Citation Format: Dieter Saur. Spatiotemporal determinants of KRAS effector signalling in PDAC: From subclonal evolution to stromal–immune niches 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 IA010.
Dieter Saur (2026) studied this question.