Cancer Therapy: Preclinical Temozolomide-MediatedDNAMethylation inHumanMyeloid Precursor Cells: Differential Involvement of Intrinsic and Extrinsic Apoptotic Pathways

Purpose:Anunderstandingof howhematopoietic cells respond to therapy that causesmyelosuppression will help develop approaches to prevent this potentially life-threatening toxicity. The goal of this study was to determine how humanmyeloid precursor cells respond to temozolomide (TMZ)-induced DNA damage. Experimental Design:Wedeveloped an ex vivo primary humanmyeloid precursor cells model system to investigate the involvement of cell-death pathways using a known myelosuppressive regimen of O -benzylguanine (6BG) and TMZ. Results: Exposure to 6BG/TMZ led to increases in p53, p21, g-H2AX, and mitochondrial DNA damage. Increases in mitochondrial membrane depolarization correlated with increased caspase-9 and -3 activities following 6BG/TMZ treatment. These events correlated with decreases in activated AKT, downregulation of the DNA repair protein O-methylguanine–DNA methyltransferase (MGMT), and increased cell death. During myeloid precursor cell expansion, FAS/CD95/APO1(FAS) expression increased over time and was present on approximately 100% of the cells following exposure to 6BG/TMZ. Although c-flipshort, an endogenous inhibitor of FAS-mediated signaling, was decreased in 6BG/TMZ–treated versus control, 6BG-, or TMZalone–treated cells, therewere no changes in caspase-8 activity. In addition, therewere no changes in the extent of cell death in myeloid precursor cells exposed to 6BG/TMZ in the presence of neutralizing or agonistic anti-FAS antibodies, indicating that FAS-mediated signaling was not operative. Conclusions: In human myeloid precursor cells, 6BG/TMZ–initiated apoptosis occurred by intrinsic, mitochondrial-mediated and not extrinsic, FAS-mediated apoptosis. Human myeloid precursor cells represent a clinically relevant model system for gaining insight into how hematopoietic cells respond to chemotherapeutics and offer an approach for selecting effective chemotherapeutic regimens with limited hematopoietic toxicity. Clin Cancer Res; 19(10); 2699–709. 2013 AACR.