The role of a histone H4 phosphorylation in drug resistance

描述（由申请人提供）：多形性胶质母细胞瘤（GBM）是人类原发性脑肿瘤中最具侵袭性的形式。替莫唑胺（TMZ）是治疗新诊断GBM的关键成分。TMZ是一种DNA烷基化剂，甲基化鸟嘌呤的N7和O6位置，已被用于治疗GBM和黑色素瘤。TMZ的治疗效果取决于它破坏DNA和引发细胞死亡的能力。除了细胞死亡外，TMZ诱导的DNA损伤也可以被修复，从而导致细胞存活。后一种结果导致TMZ疗效降低和TMZ耐药性的发展。事实上，几乎所有GBM患者都会对这种药物产生耐药性。因此，TMZ耐药是脑肿瘤治疗的巨大障碍，确定获得性TMZ耐药的分子机制至关重要。已知O6-甲基鸟嘌呤损伤可通过O6-甲基鸟嘌呤- dna甲基转移酶（MGMT）修复；因此，MGMT的表达赋予TMZ抗性。支持这一观点，多项临床研究表明，MGMT启动子的DNA甲基化导致MGMT沉默，与接受放射和TMZ治疗的患者存活时间延长有关。然而，即使MGMT启动子超甲基化有利，超过40%的患者在TMZ治疗期间出现肿瘤进展，这表明MGMT表达以外的机制也有助于TMZ耐药。我们的初步结果表明，Pak2激酶磷酸化组蛋白H4丝氨酸47 (H4S47P)，通过调节MGMT和其他赋予TMZ抗性的基因的表达，有助于TMZ抗性的发展。这种新的表观遗传机制，即H4S47P和pak2介导的基因调控，此前尚未在任何形式的癌症中进行过研究。因此，在本课题中，我们将确定在TMZ诱导的胁迫下H4S47的磷酸化是如何调控的，阐明Pak2和H4S47P参与TMZ抗性的分子机制；并确定Pak2和H4S47P水平与原发性脑肿瘤预后的关联程度。总之，这些研究将揭示一种新的表观遗传机制，通过该机制调节获得性TMZ抗性，并验证Pak2作为克服TMZ抗性的潜在治疗靶点。

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.