Mariachiara Gani, Francesca Agostini, Luca Secco, Lavinia De Pino, Raffaella Picco, Danilo Licastro, Vanessa Tolotto, Francesca D'Este, Himanshi Choudhary, Annalisa Ferino, Valentina Rapozzi, Leonard Barasa, Yuhei Ohta, Hidehiko Nakagawa, Riccardo Sgarra, Paul R Thompson, Luigi E Xodo, Eros Di Giorgio
KRAS(ON) and KRAS(OFF) inhibitors have improved the treatment of KRAS-driven tumors, yet resistance remains a major challenge. Here, we identify PADI1 and PADI3 as negative prognostic markers in KRAS-mutant colorectal and pancreatic cancers. KRAS-driven metabolic rewiring sustains their expression through an enhancer within the PADI1 locus. Although KRAS inhibition suppresses PADI1/3 expression in sensitive cells, resistant models maintain elevated PADI1/3 expression and accumulate intracellular calcium, sustaining PADI-dependent adaptive survival. Pharmacological inhibition of PADIs synergizes with KRAS(ON) and KRAS(OFF) inhibitors in two- and three-dimensional cancer models, restores sensitivity in resistant cells, and enhances antitumor activity in vivo. Integrated transcriptomic and proteomic analyses identify HSPA9/Mortalin as a critical citrullinated effector. PADI3-mediated citrullination enhances Mortalin ATPase activity and ATP/ADP cycling, whereas loss of citrullination correlates with apoptosis. Disruption of this adaptive circuitry triggers mitochondrial dysfunction, caspase activation, and non-lytic apoptosis without detectable DAMP release, revealing a therapeutic vulnerability of KRAS-driven tumors.