Adaptive and reversible resistance to Kras inhibition in pancreatic cancer cells

Activating Kras mutations are the hallmark genetic alterations in pancreatic ductal adenocarcinoma (PDAC) and key drivers of PDAC initiation and progression. Despite increased efforts to develop novel Kras inhibitors, the degree of Kras oncogene addiction in PDAC cells remains unclear. Here, we analyzed the requirement of endogenous Kras for the maintenance of murine PDAC cells using an inducible shRNA-based system that enables temporal control of endogenous Kras expression. Surprisingly, the majority of murine PDAC cells analyzed tolerated acute and sustained Kras knockdown by adapting to a reversible cell state, characterized by differences in cell morphology, proliferative kinetics, and tumor-initiating capacity. While significant mutational or transcriptional changes were not observed in the Kras-inhibited state, global phosphoproteomic profiling revealed alterations in cell signaling, including increased phosphorylation of focal adhesion pathway components. Accordingly, Kras-inhibited cells displayed prominent focal adhesion plaque structures, enhanced adherence properties, and increased dependency on adhesion for viability in vitro. Our analyses highlight the possibility of adaptive non-genetic and non-transcriptional mechanisms of resistance to Kras inhibition. Furthermore, we have identified candidate proteins whose signaling activities are altered in the Kras-inhibited state, providing a basis for the rational design of combination therapeutic strategies with novel Kras inhibitors. Overall design: 6 DOX-inducible shKras-expressing clones derived from 3 parental pancreatic tumor cell lines (A, B, and D; 2 clones per cell line) were treated with doxycycline (DOX) to induce Kras knockdown. One control DOX-inducible shLuc-expressing line was used as a control. RNA-sequencing was performed on long-term DOX-treated (LT DOX) or untreated (No DOX) cells. Technical replicates for specific samples are noted.