Oncogenic BCR-ABL1 tyrosine kinase transforms hematopoietic stem cells (HSCs) to leukemia stem cells (LSCs) to induce chronic myeloid leukemia in chronic phase (CML-CP) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL). CML-CP may progress to more advanced accelerated phase (CML-AP), and subsequently to a very aggressive blast phase (CML-BP). Most CML/Ph+ALL patients are currently treated with tyrosine kinase inhibitors (TKis) such as imatinib, dasatinib and nilotinib. However, it is unlikely that TKis will “cure” CML/Ph+ALL patients due to the presence of TKi-refractory cells (e.g., quiescent LSCs), TKi-resistant cells (e.g., proliferating LSCs carrying BCR-ABL1 kinase T315I mutant) and LSCs carrying additional somatic mutations. Therefore, novel treatment modalities are needed to eradicate TKi-refractory/resistant CML/Ph+ALL cells in the responding patients and to treat patients who do not respond favorably to TKis. CML/Ph+ALL cells accumulate more DNA double strand breaks (DSBs), the most lethal DNA lesions, than normal counterparts. Leukemia cells can tolerate high numbers of DSBs because the repair mechanisms are altered and hyper-activated. Therefore, CML/Ph+ALL cells are “addicted” to these pathways to survive pro-apoptotic challenge from high numbers of lethal DSBs. There are critical differences between DSB repair in normal and BCR-ABL1 leukemia cells. Proliferating LSCs usually employ RAD52-dependent DSB repairs and PARP1 –dependent alternative non-homologous end-joining (Alt-NHEJ), whereas normal counterparts use BRCA1/2-mediated homologous recombination (HR) and DNA-PKcs –dependent NHEJ (D-NHEJ). Quiescent LSCs use PARP1-mediated Alt-NHEJ instead of DNA-PKcs –dependent D-NHEJ, which is predominant in normal quiescent HSCs. Research supported by previous award demonstrated that genetic and pharmacological targeting of PARP1 and/or RAD52 exerted synthetic lethal effect against BCR-ABL1 –positive leukemias. However, somatic mutations often detected in CMLs/Ph+ALLs not responding favorably to TKi and/or progressing to more malignant stages can modulate the response to PARP1 and/or RAD52 inhibition. We have discovered that DNA polymerase theta (Polθ, encoded by POLQ) plays a vital role in microhomology-mediated end-joining (MMEJ), a branch of Alt-NHEJ. Our preliminary data indicate that Polθ is essential for BCR-ABL1 –mediated leukemogenesis and that targeting of Polθ eliminated CML/Ph+ALL cells. Aim #1 is designed to determine if/how BCR-ABL1 –mediated signaling modifies Polθ to regulate its biological activities and to pinpoint the role of Polθ in CML and Ph+ALL stem cells. Aim #2 will optimize Polθ inhibitor (Polθi) to be suitable for in vivo use. Aim #3 is focused on genetic and pharmacological targeting of Polθ and/or PARP1 and RAD52 against TKi-naive and TKi-treated CMLs/Ph+ALLs in in vitro conditions mimicking peripheral blood and bone marrow microenvironment and also in vivo...