KRAS silencing impacts chromatin organization and transcriptional activity in colorectal cancer cells.

Preclinical and clinical data have highlighted the challenges in targeting KRAS mutant tumors, revealing that cancer cells initially sensitive to treatment circumvent KRAS dependence and become tolerant. However, the exact mechanisms governing the transition from a drug-sensitive to a drug-tolerant state remain unclear. Herein, we used 3D culture models of mutant KRAS colorectal cancer cells with distinct KRAS dependencies to show that sensitive and resistant cells undergo distinct chromatin and transcriptional adaptations upon acute KRAS loss. KRAS-silenced sensitive cells presented G0/G1 cell cycle arrest and exhibited a transcriptional signature characteristic of quiescent cells. Moreover, proteomic profiling underscored common biological processes up-regulated in sensitive cells, including nucleosome assembly, gene expression regulation, and mRNA splicing. A detailed analysis of chromatin features revealed that sensitive cells altered eu/heterochromatin states and gained TADs after KRAS silencing whereas the eu/heterochromatin states of resistant cells remained unchanged, and the number of TADs decreased. Strikingly, only KRAS-silenced sensitive cells displayed alterations in how chromatin organizes into packing domains, displaying a lower domain packing scaling. Chromatin packing scaling regulates the interaction between macromolecular complexes and DNA, thus controlling transcriptional malleability and plasticity. Accordingly, although KRAS-silenced sensitive and resistant cells displayed a transcriptomic profile distinct from their controls, only sensitive cells presented higher variability over time, thus suggesting higher transcriptional plasticity. Overall, our findings provide compelling evidence supporting chromatin 3D reorganization and transcriptional variability in KRAS-silenced sensitive cells. This epigenetic mechanism is likely to underlie the remarkable ability of cancer cells to adapt, persist, and sustain malignancy without oncogenic KRAS.