The role of a histone H4 phosphorylation in drug resistance

描述(申请人提供)：多形性胶质母细胞瘤(GBM)是人类最具侵袭性的原发脑瘤。替莫唑胺(TMZ)是治疗新诊断的GBM的关键药物。TMZ是一种DNA烷化剂，将鸟嘌呤的N7和O6位甲基化，已被用于治疗GBM和黑色素瘤。TMZ的治疗效果取决于其损伤DNA和引发细胞死亡的能力。除了细胞死亡外，TMZ诱导的DNA损伤也可以修复，导致细胞存活。后一种结果导致TMZ疗效降低和TMZ耐药性的产生。事实上，几乎所有的GBM患者都对这种药物产生了抗药性。因此，TMZ耐药是脑肿瘤治疗的一大障碍，确定获得性TMZ耐药的分子机制至关重要。众所周知，O6-甲基鸟嘌呤损伤是由O6-甲基鸟嘌呤-DNA-甲基转移酶(MGMT)修复的，因此，MGMT的表达使其对TMZ产生抗性。支持这一观点的多项临床研究表明，MGMT启动子上的DNA甲基化导致MGMT沉默，与接受放射治疗和TMZ治疗的患者的生存时间延长有关。然而，即使在有利的MGMT启动子超甲基化的情况下，超过40%的患者在TMZ治疗期间发生肿瘤进展，这表明除了MGMT表达之外的机制也有助于TMZ耐药。我们的初步结果表明，组蛋白H4丝氨酸47(H4S47P)的磷酸化通过调节MGMT和其他与TMZ抗性有关的基因的表达，促进了TMZ耐药性的发展。这种新的表观遗传机制，即H4S47P和pak2介导的基因调控，以前从未在任何形式的癌症中被研究过。因此，在这项研究中，我们将确定在TMZ诱导的应激下H4S47的磷酸化是如何调节的，阐明Pak2和H4S47P参与TMZ耐药的分子机制，并确定Pak2和H4S47P水平与原发性脑肿瘤的预后有多大的相关性。总之，这些研究将揭示一种新的表观遗传学机制，通过这种机制来调节获得性TMZ耐药性，并验证Ak2作为克服TMZ耐药性的潜在治疗靶点。 公共卫生相关性：多形性胶质母细胞瘤(GBM)是最具侵袭性的脑肿瘤类型，占所有原发脑肿瘤病例的52%。替莫唑胺(TMZ)是治疗GBM的标准药物之一。然而，TMZ的疗效受到这样一个事实的限制，即大多数患者对该药物产生抗药性。因此，有一个迫切的需要 以解决TMZ抗性是如何产生的。我们的初步结果支持组蛋白H4丝氨酸47(H4S47P)的磷酸化与脑肿瘤对TMZ耐药性的形成有关的假说。在这个提案中，我们将使用GBM异种移植模型、原始GBM样本和分子生物学技术来验证这一假说。这些研究将揭示一种新的表观遗传学机制，从而产生TMZ耐药性，并可能确定一种新的药物靶点来对抗TMZ耐药性，TMZ耐药性是癌症化疗成功的一个巨大障碍。

DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumor in humans. Temozolomide (TMZ) is a critical component of therapy for newly-diagnosed GBM. TMZ is a DNA alkylating agent, methylating the N7 and O6 positions of guanine, and has been used for the treatment of GBM and melanoma. The therapeutic benefit of TMZ depends on its ability to damage DNA and trigger cell death. In addition to cell death, TMZ induced DNA damage can also be repaired, leading to cell survival. The latter outcome results in reduced TMZ efficacy and the development of TMZ resistance. Indeed, almost all GBM patients develop resistance to this drug. Therefore, TMZ resistance is a giant obstacle for the treatment of brain tumors, and it is critically important to determine the molecular mechanisms of acquired TMZ resistance. It is known that O6- methylguanine lesions are repaired by O6-methylguanine-DNA-methytransferase (MGMT); therefore, expression of MGMT confers TMZ resistance. Supporting this idea, multiple clinical studies have indicated that DNA methylation at the MGMT promoter, which results in silencing of MGMT, is associated with prolonged survival of patents receiving both radiation and TMZ treatment. However, even with favorable MGMT promoter hypermethylation, over 40% of patients suffer tumor progression during TMZ therapy, suggesting that mechanisms other than MGMT expression also contribute to TMZ resistance. Our preliminary results indicate that phosphorylation of histone H4 serine 47 (H4S47P) by the Pak2 kinase contributes to the development of TMZ resistance by regulating the expression of MGMT and other genes that confer TMZ resistance. This novel epigenetic mechanism, H4S47P and Pak2-mediated gene regulation, has not been studied in any form of cancer before. Therefore, in this proposal, we will determine how phosphorylation of H4S47 is regulated under TMZ-induced stress, elucidate the molecular mechanisms by which Pak2 and H4S47P contribute to TMZ resistance; and determine to what extent Pak2 and H4S47P levels correlate with the prognosis of primary brain tumors. Together, these studies will reveal a novel epigenetic mechanism by which acquired TMZ resistance is regulated and validate Pak2 as a potential therapeutic target for overcoming TMZ resistance. PUBLIC HEALTH RELEVANCE: Glioblastoma multiforme (GBM), accounting for 52% of all primary brain tumor cases, is the most aggressive type of primary brain tumor. Temozolomide (TMZ) is one of the standard drugs used to treat GBM. However, the efficacy of TMZ is limited by the fact that most patients develop resistance to this drug. Therefore, there is a critical need to address how TMZ resistance is developed. Our preliminary results support the hypothesis that phosphorylation of histone H4 serine 47 (H4S47P) catalyzed by the Pak2 kinase contributes to the development of TMZ resistance in brain tumors. In this proposal, we will employ GBM xenograft models, primary GBM samples and molecular biology techniques to test this hypothesis. These studies will reveal a novel epigenetic mechanism whereby TMZ resistance is developed and potentially identify a novel drug target to combat TMZ resistance, a giant obstacle to successful cancer chemotherapy.