Targeting transcription-coupled DNA damage responses in CLL

我们评估了转录阻断DNA病变在CLL细胞靶向治疗中非常有效的假设。Björn Schumacher的DNA修复小组与Marco Herling的翻译小组合作，利用转录偶联核苷酸切除修复（TC-NER）机制来改善CLL的治疗选择。这个假设来源于三个具体的特征。(1) CLL细胞通常处于G0/G1期，因此可能不太容易受到常规化疗的影响，常规化疗通常依赖于造成损害DNA复制的DNA损伤。相比之下，TC-NER独立于细胞周期发挥作用，以维持活性转录基因的完整性。(2)在目前的一线CLL治疗中，核苷类似物氟达拉滨通过毒害完成NER反应的间隙填充步骤来发挥细胞毒性作用。(3)细胞对转录阻断病变的反应独立于p53或ATM，因此可能为有效靶向临床有问题的化疗耐药CLL病例提供机会。在第一个资助期间，我们系统地进行了概念验证研究。IlludinM及其衍生物铁- im和trabectedin被测试为“TC-NER活性”物质。IlludinM和铁- im造成DNA损伤被TC-NER识别。Trabectedin已被证明，一旦其加合物被TC-NER作用，导致不可修复的链断裂，就会导致高度细胞毒性病变。证实了我们的假设，这些TC-NER活性物质在不依赖于p53或ATM的CLL细胞中诱导细胞死亡，并在治疗耐药的CLL细胞中有效诱发。此外，我们确定了氟达拉滨和TC-NER活性物质之间的协同作用。作为一种已经被临床批准的物质，我们在tcl1驱动的CLL小鼠模型中验证了trabectedin，并确定了其优越的疗效。我们进一步在tc - ner缺陷秀丽隐杆线虫中进行了小分子化合物筛选，允许有效选择触发体内复制和细胞周期无关的DNA损伤反应的物质。我们分离了拓扑异构酶II （Topo II）和DNA聚合酶α -引物酶（DNA pola）的抑制剂。来自线虫的遗传数据支持这些类型的复制相关因子在有丝分裂后细胞类型的DNA修复中的作用。我们计划在二期项目中(1)研究TC-NER活性物质诱导CLL细胞p53/ atm不依赖凋亡的机制，(2)建立Topo II和DNA pola抑制介导的复制不依赖DNA损伤反应机制，(3)评估联合TC-NER活性物质对CLL细胞和小鼠模型中Topo II和DNA pola抑制的潜在临床意义。我们期望揭示新的见解，将改善CLL治疗，特别是那些对目前可用的治疗方案有耐药性的CLL病例。

We evaluate the hypothesis that transcription-blocking DNA lesions are highly effective in the therapeutic targeting of CLL cells. The DNA repair group of Björn Schumacher teamed up with the translational group of Marco Herling to exploit transcription-coupled nucleotide excision repair (TC-NER) mechanisms to improve the therapeutic options for CLL. The hypothesis was derived from three specific characteristics. (1) CLL cells are typically residing in the G0/G1 phase and might therefore be less susceptible to conventional chemotherapy that often relies on inflicting DNA lesions that impair DNA replication. In contrast, TC-NER functions independently of the cell cycle to maintain integrity of actively transcribed genes. (2) In the current front-line CLL therapy the nucleoside analogue fludarabine exerts cytotoxic effects by poisoning the gap-filling step that completes the NER reaction. (3) Cellular responses to transcription-blocking lesions are independent of p53 or ATM and might therefore offer opportunities to effectively target clinically problematic chemo-resistant CLL cases. During the first funding period, we systematically conducted proof-of-concept studies. IlludinM, its derivative ferrocen-IM, and trabectedin were tested as 'TC-NER active' substances. IlludinM and ferrocen-IM inflict DNA lesions that are recognized by TC-NER. Trabectedin has been demonstrated to lead to highly cytotoxic lesions once its adducts are acted upon by TC-NER resulting in irreparable strand breaks. Confirming our hypothesis, those TC-NER active substances induced cell death in CLL cells that was independent of p53 or ATM and also effectively evoked in therapy-resistant CLL cells. Moreover, we determined synergies between fludarabine and TC-NER active substances. As an already clinically approved substance, we validated trabectedin in TCL1-driven murine models of CLL and determined its superior efficacy. We further performed small-molecule compound screens in TC-NER-deficient C. elegans that allows for the efficient selection of substances that trigger replication- and cell cycle-independent DNA damage responses, in vivo. We isolated inhibitors of Topoisomerase II (Topo II) and DNA polymerase alpha-primase (DNA pola). Genetic data from the nematode support the role of such types of thus far replication-associated factors in DNA repair in postmitotic cell types. We propose in phase-II of the project to (1) investigate the mechanisms of the p53/ATM-independent apoptosis in CLL cells induced by the TC-NER active compounds, (2) establish the mechanisms of Topo II and DNA pola inhibition-mediated replication-independent DNA damage response, and to (3) assess the potential clinical relevance of Topo II and DNA pola inhibition in CLL cells and mouse models in combination with TC-NER active substances. We expect to reveal new insights that will improve CLL therapy particularly of those CLL cases that are resistant to currently available treatment options.