Targeting DNA repair to eradicate TKI-refractory/resistant CML

描述(申请人提供)：慢性粒细胞白血病慢性期(CML-CP)由t(9；22)编码p210BCR-ABL1酪氨酸激酶启动，可转化造血干细胞(HSCs)。慢性粒细胞白血病(CML-CP)是白血病干细胞(LSCs)来源的疾病，但LSCs来源的白血病祖细胞(LPCs)的生长失调导致了该病的出现。CML-CP可能进展到更晚期的加速期(CML-AP)，随后发展到非常侵袭性的急变期(CML-BP)。目前，大多数CML-CP患者使用酪氨酸激酶抑制剂(TKI)治疗，如伊马替尼、达沙替尼和尼洛替尼。然而，由于存在对TKI不敏感的细胞(例如，静止的LSCs)和对TKI有抵抗性的细胞(例如，携带BCR-ABL1激酶T315I突变体的增殖LSCs/LPCs)，TKI不太可能“治愈”CML患者。此外，人群研究显示，总体上只有51%的CML-CP患者对TKI治疗反应良好。因此，需要新的治疗方式来根除有反应的患者中的TKI耐药/耐药CML细胞，并治疗对TKI反应不佳的患者。为了治愈CML，这些策略应该同时针对两个根本不同的白血病细胞群体：TKI耐药的静止期LSCs和TKI耐药/难治性增殖的LSCs/LPCs。我们发现，CML LSCs和LPCs，包括静止的LSCs，积累的活性氧(ROS)诱导的DNA双链断裂(DSB)是正常同行的2-4倍(Cramer等人，癌症研究，2008；Nieborowska-Skorska等人，布拉德，2012；Bolton-Gillesbie等人，布拉德，2013)。DSB是最致命的DNA损伤。我们报道了CML细胞能够耐受大量的DSB，因为两种主要的修复机制，同源重组修复(HRR)和非同源末端连接(NHEJ)是高度激活的(Slupianek等人，Mol.细胞，2001年；癌基因，2005年；DNA修复，2006年；癌症研究，2011年；布拉德，2011年；诺维基等人，布拉德，2005年)。慢性粒细胞白血病细胞对这些途径“上瘾”，以求在大量致命性双链断裂的促凋亡挑战中生存下来。然而，在正常细胞和CML细胞中，DSB修复之间存在着重要的差异。增殖的LSCs/LPCs使用RAD52依赖的HRR，与正常LSCs/LPCs的BRCA1介导的HRR相反。静止期LSC使用PARP1介导的NHEJ，而不是DNA-PKcs依赖的NHEJ，后者在正常静止期HSC中占主导地位。我们将探索这些差异，以同时靶向静止的LSCs和增殖的LSCs/LPCs中的白血病特异性DNA修复机制，以实现“双重合成致死性”，而对正常细胞和组织的影响可以忽略不计。据我们所知， “双重合成致命性”的概念以前没有被测试过。在TKI处理的CML-CP/AP细胞中，通过使用最近发现的干扰RAD52和PARP1关键功能的候选小分子抑制物同时靶向RAD52和/或PARP1：RAD52 DNA结合活性和组蛋白4刺激PARP1，将在TKI处理的CML-CP/AP细胞中诱导“双重合成致死性”。此外，使用CML-CP类转基因小鼠、Rad52-/-PARP1-//-双敲除小鼠、诱变方法、肽适配子和CML-CP/AP原代细胞，我们将确定是否可以靶向其他RAD52和/或PARP1活性来同时在TKI耐药静止期LSCs和TKI抗药LSCs/LPCS中触发更有效的“双重合成致死性”。我们的长期计划是进行一项临床试验，测试通过诱导TKI治疗患者的“双重合成致死性”来根除CML-CP/AP的可能性。

DESCRIPTION (provided by applicant): Chronic myeloid leukemia in chronic phase (CML-CP) is initiated by t(9;22) encoding for p210BCR-ABL1 tyrosine kinase that transforms hematopoietic stem cells (HSCs). CML-CP is leukemia stem cells (LSCs) - derived disease, but deregulated growth of LSCs-derived leukemia progenitor cells (LPCs) leads to the manifestation of the disease. CML-CP may progress to more advanced accelerated phase (CML-AP), and subsequently to a very aggressive blast phase (CML-BP). Most CML-CP patients are currently treated with tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib and nilotinib. However, it is unlikely that TKIs will "cure" CML patients due to the presence of TKI- refractory cells (e.g, quiescent LSCs) and TKI-resistant cells (e.g., proliferating LSCs/LPCs carrying BCR- ABL1 kinase T315I mutant). In addition, population studies revealed that overall only 51% of CML-CP patients respond favorably to TKI treatment. Therefore, novel treatment modalities are needed to eradicate TKI- refractory/resistant CML cells in the responding patients and also to treat patients who do not respond favorably to TKIs. To cure CML these strategies should simultaneously target two fundamentally different leukemia cell populations: TKI-refractory quiescent LSCs and TKI-resistant/refractory proliferating LSCs/LPCs. We found that CML LSCs and LPCs, including quiescent LSCs accumulate 2-4 times more reactive oxygen species (ROS)-induced DNA double strand breaks (DSBs) than normal counterparts (Cramer et al., Cancer Res., 2008; Nieborowska-Skorska et al., Blood, 2012; Bolton-Gillespie et al., Blood, 2013). DSBs are the most lethal DNA lesions. We reported that CML cells can tolerate high numbers of DSBs because two major repair mechanisms, homologous recombination repair (HRR) and non-homologous end-joining (NHEJ) are hyper- activated (Slupianek et al., Mol. Cell, 2001; Oncogene, 2005; DNA Repair, 2006; Cancer Res., 2011; Blood, 2011; Nowicki et al., Blood, 2005). CML cells are "addicted" to these pathways to survive pro-apoptotic challenge from high numbers of lethal DSBs. However, there are critical differences between DSB repair in normal and CML cells. Proliferating LSCs/LPCs employ RAD52-dependent HRR, in contrast to BRCA1-mediated HRR in normal counterparts. Quiescent LSCs use PARP1-mediated NHEJ instead of DNA-PKcs -dependent NHEJ, which is predominant in normal quiescent HSCs. We will explore these differences to target leukemia-specific DNA repair mechanisms simultaneously in quiescent LSCs and proliferating LSCs/LPCs to achieve "dual synthetic lethality", with negligible effect on normal cells and tissues. According to our best knowledge the concept of "dual synthetic lethality" was not tested before. "Dual synthetic lethality" will be induced in TKI-treated CML-CP/AP cells by simultaneous targeting of RAD52 and PARP1 using recently identified candidate small molecule inhibitors interrupting key functions of RAD52 and PARP1: RAD52 DNA binding activity and stimulation of PARP1 by histone 4. In addition, using CML-CP -like transgenic mice, Rad52-/-Parp1-/- double knockout mice, mutagenic approach, peptide aptamers, and CML-CP/AP primary cells we will determine if other RAD52 and/or PARP1 activities could be targeted to trigger more efficient "dual synthetic lethality" simultaneously in TKI-refractory quiescent LSCs and TKI-resistant proliferating LSCs/LPCs. Our long-term plan is to run a clinical trial testing the possibility to eradicate CML-CP/AP by induction of "dual synthetic lethality" in TKI-treated patients.