Updated project metadata. Acute myeloid leukemia (AML) pathogenesis often involves a mutation in the NPM1 nucleolar chaperone, but the bases for its transforming properties and association with favorable outcome remain incompletely understood. Here we demonstrate that an oncogenic mutant form of NPM1 (NPM1c) hampers formation of PML nuclear bodies (NBs), key senescence effectors, and impairs mitochondrial function to drive an integrated stress response. Actinomycin D (ActD), an antibiotic with unambiguous clinical efficacy in relapsed/refractory NPM1c-AMLs, preferentially targets these primed mitochondria, activating cGAS signaling and boosting ROS production. The later restores PML NB formation to drive senescence of NPM1c-AMLs cells. Dual targeting of mitochondria by Venetoclax and ActD synergized for AML elimination. Our studies reveal a central role of mitochondria downstream of NPM1c and implicate a mitochondrial/ROS/PML/TP53 senescence pathway as a key effector of ActD-based, and possibly others, chemotherapies.