Targeting DNA repair to eradicate TKi-refractory/resistant CML and Ph+ALL

致癌BCR-ABL1酪氨酸激酶将造血干细胞（HSC）转化为白血病干细胞 细胞（LSC）诱导慢性期慢性髓性白血病（CML-CP），以及 费城染色体阳性急性淋巴细胞白血病（Ph + ALL）。CML-CP可以 进展到更高级的加速阶段（CML-AP），随后进入非常 侵袭性急变期（CML-BP）。大多数CML/Ph + ALL患者目前接受以下治疗： 酪氨酸激酶抑制剂（TK），例如伊马替尼、达沙替尼和尼洛替尼。但 由于存在TKI难治性，TKI不太可能"治愈" CML/Ph + ALL患者 细胞（例如，静止的LSC），TKi抗性细胞（例如，增殖携带BCR-ABL1的LSC 激酶T315I突变体）和携带另外的体细胞突变的LSC。因此，新的治疗 需要在应答中根除TKi难治性/耐药性CML/Ph + ALL细胞的方式 患者，并治疗对TKis反应不佳的患者。 CML/Ph + ALL细胞积累更多的DNA双链断裂（DSB），最致命的DNA 病变，比正常同行。白血病细胞可以耐受大量的DSB， 修复机制被改变和过度激活。因此，CML/Ph + ALL细胞是“上瘾”的。 这些途径以在来自大量致死DSB的促凋亡挑战中存活。那里 是正常和BCR-ABL1白血病细胞中DSB修复之间的关键差异。 LSC的修复通常采用依赖于RAD52的DSB修复和依赖于PARP1的DSB修复。 替代性非同源末端连接（Alt-NHEJ），而正常对应物使用 BRCA1/2介导的同源重组（HR）和DNA-PKcs依赖性NHEJ（D-NHEJ）。静态 LSC使用PARP1介导的Alt-NHEJ而不是DNA-PKcs依赖的D-NHEJ，这在LSC中占主导地位。 正常静止期HSC。 由以前的奖项支持的研究表明，遗传和药理学 靶向PARP1和/或RAD52对BCR-ABL1阳性细胞产生合成致死作用。 白血病然而，在CML/Ph + ALL中经常检测到体细胞突变， TKi和/或进展到更恶性的阶段可以调节对PARP1的反应和/或 RAD52抑制。 我们已经发现DNA聚合酶θ（Pol θ，由POLQ编码）在以下方面起着至关重要的作用： 微同源介导的末端连接（MMEJ），Alt-NHEJ的一个分支。我们的初步数据显示 Pol θ在BCR-ABL 1介导白血病发生中是必需的，Pol θ的靶向作用被消除 CML/Ph + ALL细胞。目的#1旨在确定BCR-ABL1介导的信号传导是否/如何改变Pol θ 探讨Pol θ在CML和Ph + ALL中的作用 细胞目标#2将优化Pol θ抑制剂（Pol θ i），使其适合体内使用。目标#3 专注于Pol θ和/或PARP1和RAD 52针对TKi初治患者的遗传和药理学靶向 和模拟外周血和骨的体外条件下经TKi处理的CML/Ph + ALL 骨髓微环境以及在携带原发性白血病的人源化免疫缺陷小鼠中的体内 异种移植

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 in humanized immunodeficient mice bearing primary leukemia xenografts.