DNA Ligase III Alpha and (Poly-ADP) Ribose Polymerase (PARP1) Are Therapeutic Targets in Imatinib-Resistant (IR) Chronic Myeloid Leukemia (CML).

Abstract 853

Therapy with the tyrosine kinase inhibitor imatinib, targeting the constitutively active BCR-ABL kinase has been remarkably successful in Philadelphia chromosome-positive (Ph+) CML, but resistance to tyrosine kinase inhibitors is a growing clinical problem, prompting the search for new therapeutic targets. BCR-ABL expression leads to increased reactive oxygen species (ROS), repair errors and genomic instability. We have previously shown that an error-prone alternative non-homologous end-joining (ALT NHEJ) pathway involving PARP1 and DNA ligase IIIa/XRCC1 is upregulated in Ph+ CML, providing a mechanism for the repair errors and genomic instability. To determine whether ALT NHEJ components may be novel therapeutic targets in IR CML, we characterized two IR cell lines (P210Mo7eIR, Baf3P210IR) for DSB repair abnormalities. Both IR cell lines demonstrate significantly higher levels of DSBs and NHEJ abnormalities (P<0.05) compared with their imatinib-sensitive (IS) counterparts. Notably, whereas steady state levels of the ALT NHEJ components DNA ligase IIIa and PARP1 are increased in IS P210Mo7e and Baf3P210 cells, compared with parental Mo7e and Baf3, the levels of these proteins are increased even further in the IR cells. Presence of increased DNA ligase IIIa and PARP1 levels in the IR cell lines suggests that these enzymes may be targets for therapy using the DNA ligase inhibitors that we have previously identified and PARP1 inhibitors, which have been used successfully in the treatment of cancers with DSB repair defects. Initial tests for cytotoxicity in BCR-ABL-positive cell lines and parental controls showed that the DNA ligase inhibitor L67, which specifically inhibits DNA ligase I and IIIα, is cytotoxic in BCR-ABL-positive cells and parental controls at concentrations of >10 μM, and that cytotoxicity is not influenced by BCR-ABL1 expression. Therefore, we examined the effect of a subtoxic concentration of L67 (0.3 μM) in the presence or absence of the PARP1 inhibitor Nu1025 (Calbiochem) at 50 μM in IR versus IS and parental cells. Combined treatment with L67 and Nu1025 significantly (p<0.001) reduces survival of IR cells compared with IS and parental controls, which were not significantly affected. To determine whether cells from CML patients that are resistant to imatinib are also sensitive to the combination of DNA ligase and PARP inhibitors, we next tested primary bone marrow mononuclear cells (BM MNC) from 6 CML patients with IR disease, compared with normal BM MNC. Cells from 3 of the 6 patients demonstrated a significant decrease in colony survival in response to the combination of DNA repair inhibitors, similar to the sensitivity demonstrated by the two IR cell lines studied. Interestingly, the patient demonstrating the highest sensitivity to the combination of DNA repair inhibitors had significantly increased levels of both DNA ligase IIIa and PARP1, whereas patients demonstrating less sensitivity had increased levels of either DNA ligase IIIa or PARP1, compared with normal BM MNC. Importantly, sensitivity to the DNA repair inhibitors is not correlated with mutations in BCR-ABL because the BCR-ABL mutation T315I that is found in Baf3P210IR cells when overexpressed in Baf3 cells has no effect on colony survival following drug treatment. Together, our results suggest that the process of acquiring IR may select for cells with high levels of PARP1 and DNA ligase IIIa and/or may upregulate ALT NHEJ pathways. Thus, patients with high levels of these proteins are likely to benefit from therapy using inhibitors of ALT NHEJ.