Replication fork plasticity is a therapeutic vulnerability in acute myeloid leukemia

<jats:p>Uncontrolled proliferation of myeloid progenitor cells in acute myeloid leukemia (AML) is counteracted in most patients by toxic and often ineffective systemic treatments. Poly (ADP-ribose) polymerase inhibitors (PARPi) show subtype-restricted activity - potent in RUNX1-RUNX1T1 and PML-RARɑ fusions, limited in KMT2A-rearranged (KMT2A-r) disease - but the lack of molecular understanding has hampered their clinical implementation. We combined single-cell and single-molecule assays on DNA replication intermediates and DNA damage signalling with therapy response readouts to investigate the role of fork plasticity factors in response to PARPi and AML standard-of-care (cytarabine, araC). In PARPi-sensitive AML models, PARP inhibition deregulates RECQ1-mediated fork restart, initially triggering fork acceleration and later fork breakage within the same S phase. Conversely, PARPi resistant KMT2A-r AML lines are protected by PrimPol-dependent DNA synthesis and its inactivation promptly induces fork breakage and PARPi sensitivity. Strikingly, PrimPol overexpression in PARPi-sensitive AML models prevents fork collapse and PARPi/araC therapy response, both in vitro and in vivo, identifying PrimPol as novel predictive biomarker and therapeutic target in AML. Our data uncover novel tissue-specific mechanisms of action for PARPi and pinpoint replication fork plasticity as key molecular determinant of AML therapy response.</jats:p>