Parg Is Essential for Polθ-Mediated Dna End-Joining by Removing Repressive Poly-Adp-Ribose Marks

DNA polymerase theta (Polθ)-mediated end-joining (TMEJ) repairs DNA double-strand breaks and confers resistance to genotoxic agents. How Polθ is regulated at the molecular level to exert TMEJ remains poorly characterized. We find that Polθ interacts with and is PARylated by PARP1 in a HPF1- independent manner. PARP1 recruits Polθ to the vicinity of DNA damage via PARylation dependent liquid demixing, however, PARylated Polθ cannot perform TMEJ due to its inability to bind DNA. PARG-mediated de-PARylation of Polθ reactivates its DNA binding and end-joining activities. Consistent with this, PARG is essential for TMEJ and the temporal recruitment of PARG to DNA damage corresponds with TMEJ activation and dissipation of PARP1 and PAR. In conclusion, we show a two-step spatiotemporal mechanism of TMEJ regulation. First, PARP1 PARylates Polθ and facilitates its recruitment to DNA damage sites in an inactivated state. PARG subsequently activates TMEJ by removing repressive PAR marks on Polθ

Ruxolitinib-induced defects in DNA repair cause sensitivity to PARP inhibitors in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) often carry JAK2(V617F), MPL(W515L), or CALR(del52) mutations. Current treatment options for MPNs include cytoreduction by hydroxyurea and JAK1/2 inhibition by ruxolitinib, both of which are not curative. We show here that cell lines expressing JAK2(V617F), MPL(W515L), or CALR(del52) accumulated reactive oxygen species-induced DNA double-strand breaks (DSBs) and were modestly sensitive to poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673. At the same time, primary MPN cell samples from individual patients displayed a high degree of variability in sensitivity to these drugs. Ruxolitinib inhibited 2 major DSB repair mechanisms, BRCA-mediated homologous recombination and DNA-dependent protein kinase-mediated nonhomologous end-joining, and, when combined with olaparib, caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of patients. Moreover, the combination of BMN673, ruxolitinib, and hydroxyurea was highly effective in vivo against JAK2(V617F)+ murine MPN-like disease and also against JAK2(V617F)+, CALR(del52)+, and MPL(W515L)+ primary MPN xenografts. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized MPN cells to synthetic lethality triggered by PARP inhibitors

BCR/ABL kinase induces self-mutagenesis via reactive oxygen species to encode imatinib resistance

Mutations in the BCR/ABL kinase domain play a major role in resistance to imatinib mesylate (IM). We report here that BCR/ABL kinase stimulates reactive oxygen species (ROS), which causes oxidative DNA damage, resulting in mutations in the kinase domain. The majority of mutations involved A/T→G/C and G/C→A/T transitions, a phenotype detected previously in patients, which encoded clinically relevant amino acid substitutions, causing IM resistance. This effect was reduced in cells expressing BCR/ABL(Y177F) mutant, which does not elevate ROS. Inhibition of ROS in leukemia cells by the antioxidants pyrrolidine dithiocarbamate (PDTC), N-acetylcysteine (NAC), and vitamin E (VE) decreased the mutagenesis rate and frequency of IM resistance. Simultaneous administration of IM and an antioxidant exerted better antimutagenic effect than an antioxidant alone. Therefore, inhibition of ROS should diminish mutagenesis and enhance the effectiveness of IM. (Blood. 2006;108:319-327