Targeting DNA repair to eradicate TKI-refractory/resistant CML

描述（由申请人提供）：慢性期慢性髓性白血病（CML-CP）由t（9;22）启动，t（9;22）编码转化造血干细胞（HSC）的p210 BCR-ABL 1酪氨酸激酶。CML-CP是白血病干细胞（LSC）衍生的疾病，但LSC衍生的白血病祖细胞（LPC）的生长失调导致疾病的表现。CML-CP可能进展到更晚期的加速期（CML-AP），随后进展到非常侵袭性的急变期（CML-BP）。大多数CML-CP患者目前接受酪氨酸激酶抑制剂（TKI）治疗，如伊马替尼、达沙替尼和尼洛替尼。然而，TKI不太可能“治愈”CML患者，因为存在TKI难治性细胞（例如，静止LSC）和TKI抗性细胞（例如，增殖携带BCR-ABL 1激酶T315 I突变体的LSC/LPC）。此外，人群研究显示，总体上只有51%的CML-CP患者对TKI治疗有良好反应。因此，需要新的治疗方式来根除应答患者中的TKI难治性/耐药性CML细胞，并且还治疗对TKI没有有利应答的患者。为了治愈CML，这些策略应同时靶向两种根本不同的白血病细胞群：TKI难治性静止LSC和TKI耐药/难治性增殖LSC/LPC。我们发现CML LSC和LPC，包括静止LSC，比正常对应物积累2-4倍的活性氧（ROS）诱导的DNA双链断裂（DSB）（Cramer et al.，癌症研究所，2008; Nieborowska-Skorska等人，Blood，2012; Bolton-Gillespie等人，Blood，2013）。DSB是最致命的DNA损伤。我们报道了CML细胞可以耐受大量的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）是超活化的（Slupianek等人，摩尔Cell，2001; Oncogene，2005; DNA Repair，2006; Cancer Res.，2011; Blood，2011; Nowicki等人，Blood，2005）。CML细胞对这些途径“上瘾”，以在来自大量致死DSB的促凋亡挑战中存活。然而，正常细胞和CML细胞中的DSB修复之间存在关键差异。与正常对应物中BRCA 1介导的HRR相反，活化的LSC/LPC采用RAD 52依赖的HRR。静止期LSC使用PARP 1介导的NHEJ而不是DNA-PKcs依赖的NHEJ，这在正常静止期HSC中占主导地位。我们将探索这些差异，同时在静止LSC和增殖LSC/LPC中靶向白血病特异性DNA修复机制，以实现“双重合成致死性”，对正常细胞和组织的影响可忽略不计。据我们所知， “双重合成杀伤力”的概念以前没有得到检验。通过使用最近鉴定的候选小分子抑制剂同时靶向RAD 52和PARP 1，在TKI处理的CML-CP/AP细胞中诱导“双重合成致死性”，所述候选小分子抑制剂中断RAD 52和PARP 1的关键功能：RAD 52 DNA结合活性和组蛋白4对PARP 1的刺激。此外，使用CML-CP样转基因小鼠、Rad 52-/-Parp 1-/-双敲除小鼠、诱变方法、肽适体和CML-CP/AP原代细胞，我们将确定是否可以靶向其他RAD 52和/或PARP 1活性，以在TKI难治性静止LSC和TKI耐药增殖LSC/LPC中同时触发更有效的“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.