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转化为白血病干细胞 诱导慢性粒细胞白血病慢性期的细胞(LSCs)和 费城染色体阳性急性淋巴细胞白血病(Ph ALL)。CML-CP可能 进展到更高级的加速阶段(CML-AP)，随后进入非常 攻击性爆炸阶段(CML-BP)。大多数CML/Ph患者目前都在接受治疗 酪氨酸激酶抑制剂(TKI)，如伊马替尼、达沙替尼和尼洛替尼。然而，它 由于TKI难治性的存在，TKI不太可能治愈所有的CML/Ph患者 细胞(例如，静止的LSCs)、抗TKI的细胞(例如，携带BCR-ABL1的增殖LSCs 激酶T315I突变)和携带其他体细胞突变的LSCs。因此，新的治疗方法 需要药物来根除TKI耐药/耐药的CML/Ph应答中的所有细胞 并用于治疗对TKIs反应不佳的患者。 CML/Ph ALL细胞积累更多的DNA双链断裂(DSB)，这是最致命的DNA 损伤，比正常的对应物。白血病细胞可以耐受大量的DSB，因为 修复机制被改变和过度激活。因此，CML/Ph所有细胞都成瘾了 这些途径可以在大量致死性双链球菌的促凋亡攻击中存活下来。那里 是正常和bcr-abl1白血病细胞中DSB修复的关键区别。 增殖的LSC通常采用依赖RAD52的DSB修复和依赖PARP1的修复 另一种非同源末端连接(Alt-NHEJ)，而正常对应的使用 BRCA1/2介导的同源重组(HR)和依赖DNA-PKcs的NHEJ(D-NHEJ)。静止的 LSC使用PARP1介导的Alt-NHEJ而不是DNA-PKcs依赖的D-NHEJ，后者在 正常的静止的HSC。 由先前奖项支持的研究表明，遗传学和药理学 靶向PARP1和/或RAD52对bcr-abl1阳性的综合致死作用 白血病。然而，在CMLS/Ph细胞中检测到的体细胞突变往往反应不佳 TKI和/或进展到更恶性阶段可以调节对PARP1和/或 RAD52抑制作用。 我们发现DNA聚合酶theta(POLθ，由POLQ编码)在 微同源基因介导的末端连接(MMEJ)是Alt-NHEJ的一个分支。我们的初步数据显示 Polθ在bcr-abl 1介导白血病发生中是必需的，并消除了Polθ的靶向 CML/Ph ALL细胞。AIM#1旨在确定bcr-abl1介导的信号是否/如何修改POLθ 调节其生物学活性，明确Polθ在慢性粒细胞白血病和Ph ALL干细胞中的作用 细胞。目的2优化POLθ抑制剂(POLθI)，使其适合体内应用。目标3是 Polθ和/或PARP1RAD52针对TKI-naive的遗传和药理学靶向 和TKI处理的CMLS/Ph细胞在模拟外周血和骨的体外条件下 原发白血病人源化免疫缺陷小鼠的骨髓微环境及体内研究 异种移植物。

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.