Abstract 5454: Inhibition of Bcr-Abl tyrosine kinase sensitizes cells to sapacitabine

Abstract

Abstract CNDAC, the active form of sapacitabine, is a nucleoside analog which kills cells by a unique mechanism of action. Incorporation of CNDAC triphosphate into DNA results in a single-strand break, which is converted to a one-ended double-strand break (DSB) upon subsequent collapse of the DNA replication fork. CNDAC-induced DSBs are repaired predominantly through the homologous recombination pathway which consists the key recombinase Rad51 and its associated proteins. Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm; its central pathogenesis is a reciprocal chromosomal translocation between chromosomes 9 and 22. This abnormal chromosomal fusion results in the formation of a chimeric gene that encodes a constitutively active tyrosine kinase, Bcr-Abl, which promotes CML through stimulating downstream signaling cascades for cell survival and deregulated cell growth. Bcr-Abl tyrosine kinase modulates DNA repair mechanisms to protect leukemic cells from DNA damage induced cell death. In response to DSBs, it phosphorylates Rad51 at Tyr315 which enhance the damage repair process. Based on this evidence, we hypothesized that inhibition of Bcr-Abl will further sensitize cells to CNDAC. To test this postulate, we used the CML cell line K562 as well as murine Ba/F3 cells transfected with wild-type Bcr-Abl (Ba/F3p210wt) or the Bcr-Abl mutant (Ba/F3p210T315I) as model systems. Two tyrosine kinase inhibitors (TKI) were utilized to inhibit Bcr-Abl. Imatinib, the first generation TKI, is only effective inhibiting wild type Bcr-Abl. Ponatinib, a subsequent TKI, is potent in inhibiting both wild type and mutant Bcr-Abl tyrosine kinases. First, these cells were treated with CNDAC, imatinib or ponatinib alone to determine the IC50 values for each drug using PrestoBlue cell viability assay. Based on the single drug IC50s, combinations of CNDAC with imatinib or ponatinib were applied to these cell lines and their combination indexes were calculated by CalcuSyn software. We found that CNDAC and imatinib were synergistic in K562 and Ba/F3 wild type Bcr-Abl cells while they were additive in the Bcr-Abl T315I mutant cells. In contrast, ponatinib sensitized CNDAC treatment as their combination was synergistic in all these cell lines. Finally, to investigate the mechanism underlying the observed synergism, K562 cells were treated with either equitoxic concentrations of CNDAC, imatinib or ponatinib alone or in combinations for 48 hours when lysates were collected for immunoblot analysis. It was found that when Bcr-Abl was inhibited, the phosphoryation of Rad51 at Tyr315 was reduced, phospho H2AX and cleaved caspase 3 were increased in response to CNDAC. Thus further sensitization of CNDAC was a result of deficient damage repair due to Bcr-Abl inhibition. Together, our results link the Bcr-Abl tyrosine kinase to CNDAC-induced DNA damage repair process and provide rationale for CNDAC/sapacitabine combination therapy in diseases such as CML. Citation Format: Yingjun Jiang, Xiaojun Liu, Adrienne Chestang, William Plunkett. Inhibition of Bcr-Abl tyrosine kinase sensitizes cells to sapacitabine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5454. doi:10.1158/1538-7445.AM2014-5454