Ruxolitinib-Induced Defects in DNA Repair Cause Sensitivity to PARP Inhibitors in Myeloproliferative Neoplasms

Philadelphia chromosome-negative (Ph-) myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), which are associated with mutations in JAK2, CALR and MPL genes. Current treatment options for Ph- MPNs include cytoreductive therapy with hydroxyurea, and the JAK2 kinase inhibitors (e.g., ruxolitinib), which produce durable reductions in splenomegaly and improvement of symptoms and probably of survival, but do not eliminate the disease-initiating cell population. All three disease "driver" mutations, [JAK2(V617F), CALR(del52), and MPL(W515L)] have been detected not only in mature MPN cells, but also in MPN stem cells, and therefore these cells must be eliminated to eradicate the disease. Since JAK2 kinase inhibitors did not eliminate the disease-initiating population novel therapeutic approaches are needed. We show here that cell lines expressing JAK2(V617F), MPL(W515L) or CALR(del52) accumulated reactive oxygen species-induced lethal DNA double-strand breaks (DSBs), which were efficiently repaired thus preventing MPN cell death. Inhibition of JAK2 kinase by ruxolitinib caused downregulation of BRCA1, RAD51 and LIG4, the key proteins in two major DSB repair mechanisms, BRCA-mediated homologous recombination (HR) and DNA-PK -mediated non-homologous end-joining (NHEJ). We recently reported (Nieborowska-Skorska et al., J.Clin.Invest., 2017) that NHEJ-deficient acute myeloid leukemia (AML) quiescent stem cells and HR/NHEJ-deficient AML proliefrating stem and progenitor cells were extremely sensitive to synthetic lethality triggered by poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673 (talazoparib). Therefore, we hypothesized that PARP inhibitors should be very effective against ruxolitinib-treated HR/NHEJ-deficient MPN quiescent and proliferating cells. Olaparib, when combined with ruxolitinib caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of samples. Importantly, combination of BMN673, ruxolitinib and hydroxyurea was highly effective in vivo against JAK2(V617F)-positive murine MPN-like disease and also against pre-selected JAK2(V617F), CALR(del52), and MPL(W515L)-positive primary MPN xenografts. However, MPN samples from individual patients displayed high degree of variability in the sensitivity to JAK2 inhibitor combined with PARP inhibitor and/or hydroxyurea. The differences between susceptibility of individual MPNs to these drugs may depend on additional mutations accompanying the "driver" mutations (e.g., TET2 mutation) and on homo/heterozygosity of the "driver" mutation allele. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized pre-selected MPN cells to synthetic lethality triggered by PARP inhibitors.