Reversing intrinsic cancer cell resistance to alkylating agents by histone deacetylase inhibition

烷基化剂是研究DNA损伤引发的细胞死亡的理想选择，因为DNA损伤负责激活细胞死亡以及激活的修复和损伤反应途径已被详细阐明。然而，这一知识并不能解释为什么胶质瘤细胞在诱导关键DNA损伤后的存活率与未转化细胞相比有如此显著的差异。这个问题的答案可能是在组蛋白去乙酰化酶（HDAC）依赖的DNA损伤反应、死亡执行和/或DNA修复途径组分的解除管制中找到的。由于在胶质母细胞瘤的恶性转化过程中确实会发生I类hdac HDAC1和HDAC3的失调控，HDAC1和HDAC3可能导致抗癌耐药。然而，I类hdac的抑制是否对胶质瘤细胞固有的烷基化剂敏感性有任何影响，仍然是一个有争议的问题。在之前的DFG资助期间，我研究了I类hdac对黑色素瘤耐药性的影响。数据显示，HDAC2刺激同源重组修复蛋白RAD51和范可尼贫血蛋白FANCD2的表达，导致黑素瘤对烷基化剂产生耐药性（Krumm et al. 2016, Roos and Krumm 2016）。这种耐药性可以通过I类hdac的小分子抑制来克服。对于胶质瘤，我的初步数据显示，恩替诺他（MS-275）抑制HDAC1和HDAC2使胶质瘤细胞对烷基化剂敏感，尽管其致敏机制与我们报道的黑色素瘤细胞致敏机制不同。在胶质瘤中，初步数据指出了一种可能的机制，即HDAC1和/或HDAC3过表达导致对烷基化剂替莫唑胺（TMZ）产生耐药性，这是由于刺激了TMZ诱导的长时间DNA损伤反应。这导致暴露于TMZ的胶质瘤细胞进入一种类似衰老的状态，从而保护它们免于启动细胞凋亡。与HDACi MS-275和TMZ共同作用后，TMZ诱导的DNA损伤反应下调，胶质瘤不再进入这种类似衰老的状态，并发生凋亡。在这次dfg拨款中，我希望重点研究HDAC1/2/3介导的胶质瘤细胞对烷基化剂的抗性机制，并解决以下具体问题。(1)哪一类hdac导致胶质瘤对烷基化剂产生抗性？(2) HDAC1/2/3在胶质瘤中tmz触发的DNA损伤反应异常激活中起什么作用？本文将讨论HDAC1/2/3对组蛋白乙酰转移酶TIP60、磷酸酶PP2A和WIP1以及表观遗传标记H3K56Ac和H4K91Ac的作用。(3) HDAC1/2/3在烷基化剂引发衰老中起什么作用？

Alkylating agents are ideal for the study of DNA damage triggered cell death, as the DNA lesions responsible for activating cell death along with the activated repair and damage response pathways have been elucidated in detail. This knowledge does, however, not explain why glioma cells differ so significantly in their survival following the induction of critical DNA lesions compared to non-transformed cells. The answer to this may be found in the histone deacetylase (HDAC) dependent deregulation of components of the DNA damage response, death execution and/or DNA repair pathways. As deregulation of the class I HDACs HDAC1 and HDAC3 do occur during the malignant transformation of glioblastoma, HDAC1 and HDAC3 may lead to anticancer drug resistance. However, whether or not the inhibition of class I HDACs has any effect on the intrinsic alkylating agent sensitivity of glioma cells is still a matter of debate. During the previous DFG funding period, I addressed the influence of class I HDACs on melanoma resistance. The data showed that HDAC2 stimulates the expression of the homologous recombination repair protein RAD51 and the Fanconi anaemia protein FANCD2, which causes resistance in melanomas to alkylating agents (Krumm et al. 2016, Roos and Krumm 2016). This resistance could be overcome by small molecule inhibition of class I HDACs. For gliomas, my preliminary data show that the inhibition of HDAC1 and HDAC2 with Entinostat (MS-275) sensitises glioma cells to alkylating agents, although the mechanism of sensitization differs from the mechanism we reported for melanoma cells. The preliminary data, in gliomas, points to a possible mechanism where overexpressed HDAC1 and/or HDAC3 causes resistance to the alkylating agent temozolomide (TMZ), due to the stimulation of a prolonged TMZ-induced DNA damage response. This causes the TMZ exposed glioma cells to enter a senescence-like state that protects them from initiating apoptosis. Upon co-treatment with the HDACi MS-275 and TMZ, the TMZ-induced DNA damage response is downregulated, gliomas no longer enter this senescence-like state and they become apoptotic. In this DFG-grant, I wish to focus on the mechanism of HDAC1/2/3 mediated resistance of glioma cells to alkylating agents and address the following specific questions. (1) Which of the class I HDACs causes the resistance of gliomas to alkylating agents? (2) What roles do HDAC1/2/3 play in the aberrant activation of the TMZ-triggered DNA damage response in glioma? Here the roles of HDAC1/2/3 on the histone acetyltransferase TIP60, the phosphatases PP2A and WIP1 as well as the epigenetic marks H3K56Ac and H4K91Ac will be addressed. (3) What roles do HDAC1/2/3 play in alkylating agent triggered senescence?