Targeting transcription-coupled DNA damage responses in CLL

我们评估了转录阻断DNA损伤在治疗靶向CLL细胞方面高度有效的假设。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)机制来改进CLL的治疗方案。这一假设是从三个具体特征中得出的。(1)CLL细胞通常处于G0/G1期，因此可能不太容易受到传统化疗的影响，因为传统化疗往往依赖于造成DNA损伤，从而损害DNA复制。相反，TC-NER的功能独立于细胞周期，以维持活跃转录基因的完整性。(2)在目前的一线CLL治疗中，核苷类似物氟达拉滨通过毒化完成NER反应的缝隙填充步骤而发挥细胞毒作用。(3)细胞对转录阻断损伤的反应独立于P53或ATM，因此可能提供有效靶向临床有问题的化疗耐药CLL病例的机会。在第一个资助期，我们系统地进行了概念验证研究。伊卢丁M、其衍生物二茂铁-IM和曲贝替丁被测试为具有TC-NER活性的物质。伊鲁丁M和二茂铁-IM造成TC-NER识别的DNA损伤。已经证明，一旦其加合物被TC-NER作用，Trabectedin就会导致高度的细胞毒性损伤，导致不可修复的链断裂。证实了我们的假设，这些TC-NER活性物质诱导了CLL细胞的细胞死亡，这种死亡不依赖于P53或ATM，也有效地诱导了耐药CLL细胞的死亡。此外，我们还确定了氟达拉滨和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小鼠模型中验证了曲贝替丁，并确定了其优越的疗效。我们进一步在TC-NER缺陷的线虫中进行了小分子化合物筛选，允许在体内有效地选择触发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种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。在项目的第二阶段，我们建议(1)研究TC-NER活性化合物诱导CLL细胞P53/ATM非依赖性凋亡的机制，(2)建立Topo II和DNA Pola抑制介导的复制非依赖DNA损伤反应的机制，以及(3)评估Topo II和DNA Pola抑制在CLL细胞和小鼠模型中与TC-NER活性物质联合使用的潜在临床意义。我们希望揭示新的见解，将改善CLL治疗，特别是那些对当前可用的治疗方案产生抗药性的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.