Targeting DNA repair to eradicate TKI-refractory/resistant CML

描述（由申请人提供）：慢性髓性白血病慢性期（CML-CP）是由t（9;22）编码转化造血干细胞（hsc）的p210BCR-ABL1酪氨酸激酶引发的。CML-CP是白血病干细胞（LSCs）衍生的疾病，但LSCs衍生的白血病祖细胞（LPCs）生长失调导致疾病的表现。CML-CP可能发展到更高级的加速期（CML-AP），随后发展到极具侵袭性的爆炸期（CML-BP）。大多数CML-CP患者目前使用酪氨酸激酶抑制剂（TKIs）治疗，如伊马替尼、达沙替尼和尼洛替尼。然而，由于TKI难治性细胞（如静止LSCs）和TKI耐药细胞（如携带BCR- ABL1激酶T315I突变体的增殖性LSCs/LPCs）的存在，TKIs不太可能“治愈”CML患者。此外，人群研究显示，总体而言，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应答患者中的TKI难治性/耐药CML细胞，并治疗对TKI反应不佳的患者。为了治疗CML，这些策略应该同时针对两种根本不同的白血病细胞群：tki难治性静止LSCs和tki耐药/难治性增殖LSCs/LPCs。我们发现，CML LSCs和LPCs，包括静止LSCs积累的活性氧（ROS）诱导的DNA双链断裂（dsb）比正常LSCs多2-4倍（Cramer等人，Cancer Res., 2008； Nieborowska-Skorska等人，Blood, 2012； Bolton-Gillespie等人，Blood, 2013）。dsb是最致命的DNA病变。我们报道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）被超激活（Slupianek等，Mol. Cell, 2001; Oncogene, 2005； DNA修复，2006;Cancer Res, 2011; Blood, 2011； Nowicki等，Blood, 2005）。CML细胞对这些途径“上瘾”，以在大量致死性dsb的促凋亡挑战中存活。然而，在正常细胞和CML细胞中，DSB修复存在关键差异。与正常LSCs/LPCs中brca1介导的HRR相比，增殖的LSCs/LPCs使用rad52依赖的HRR。静止的LSCs使用parp1介导的NHEJ，而不是DNA-PKcs依赖的NHEJ，后者在正常的静止hsc中占主导地位。我们将探索这些差异，在静止LSCs和增殖LSCs/LPCs中同时靶向白血病特异性DNA修复机制，以实现“双重合成致死”，而对正常细胞和组织的影响可以忽略不计。据我们所知

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.