Molecular Mechanisms Underlying the Prevention of BCC Resistance

摘要 基底细胞癌是美国最常见的人类恶性肿瘤，影响着300多万美国人 每年一次。Hedgehog(HH)信号调节缺陷，通常是由于肿瘤功能丧失所致 抑制者补丁(PTCH)导致SMO的致癌激活，被认为是SMO的主要驱动因素 BCC增长。与PTCH的配体结合解除SMO抑制，触发其迁移到初级纤毛 激活GLI转录因子，推动细胞增殖/肿瘤生长。异常的HH信号是基础 Gorlin-Goltz综合征，也称为基底细胞痣综合征(BCNS)，是一种以遗传性为主的疾病 哪些受影响的个体出生时带有一个功能性PTCH等位基因，并在一生中获得了 在这些患者中，第二个等位基因加速HH信号转导并驱动BCC的生长 在他们的一生中，肿瘤负担需要多次昂贵的破坏性外科手术。此外，HH 在接受治疗的个体中，抑制剂(HHI)与无法忍受的副作用有关，因此一半的患者 尽管有显著的抗肿瘤疗效，但仍需停止治疗。我们的组织和世界各地的其他人 利用针对HH信号的药物促进了床边临床试验，2012年，这些努力产生了 FDA批准Vismodegib，一种口服治疗局部晚期SMO的有效抑制剂， 无法手术且有可能致命的基底细胞癌。尽管它们的有效性是不可否认的，但目前可用的 HH信号抑制物在肿瘤耐药和肿瘤复发的快速发展中受到阻碍。而当 未被抑制的HH信号明显促进了基底细胞癌的抵抗和复发，许多基底细胞癌不表现为SMO 突变表明参与了额外的致癌机制。我们发现vismodegib 抗性涉及含溴结构域蛋白BRD7和BRD9的失调 Switch/蔗糖不可发酵(SWI/SNF)核小体重塑复合体。利用好基因- 定义体外和体内小鼠BCC模型，以及患者来源的人BCC细胞，我们的初步数据 令人信服地证明：(I)HHI抗性与组蛋白乙酰化和组蛋白乙酰化的全球减少有关 染色质的可及性，以及(Ii)BRD7的遗传消融使药物幼稚的BCC细胞对HHI产生抗药性。基座 根据我们的初步数据，这个应用程序将检验BRD7-BRD9联结驱动HHI抵抗的假设 而BRD9的封锁防止了HHI抵抗的出现。目标一号将探测染色质 与HHI抗性相关的修饰和基因表达特征及其与BRD7- BRD9轴。AIM 2将在体内测试BRD7和BRD9连接的遗传操作的后果 基因定义的模型(即BRD7[Brd7 KO]或Akt1[Akt1/-]中的表皮特异性缺失。AIM 3将测试 选择BRD9和Akt抑制剂在体内克服/预防HHI耐药性的潜在效用 在PI的实验室开发的BCC模型系统，忠实地模仿人类BCNS，以前使用过 目的：验证HHI的有效性和安全性。

SUMMARY BCCs are the most common type of human malignancy in the US, affecting more than 3 million Americans annually. Defective regulation of Hedgehog (Hh) signaling, typically through loss of function of the tumor suppressor Patched (PTCH) leading to oncogenic activation of SMO, are thought to be the primary drivers of BCC growth. Ligand binding to PTCH relieves SMO repression, triggering its migration to the primary cilium with activation of GLI transcription factors that drive cell proliferation/tumor growth. Aberrant HH signaling underlies the Gorlin-Goltz syndrome, also known as basal cell nevus syndrome(BCNS), a dominantly inherited disorder in which affected individuals are born with one functional PTCH allele and during life acquire mutations in the second allele that accelerate HH signaling and drive the growth of BCCs in these patients whose inordinate tumor burden necessitates multiple costly mutilating surgical procedures over their lifetime. Furthermore, Hh inhibitors (HHi) are associated with intolerable side-effects in treated individuals such that half the patients discontinue treatment despite substantial anti-tumor efficacy. Our group and others around the world have fostered bench-to-bedside clinical trials with drugs that target HH signaling and in 2012 these efforts resulted in FDA approval of vismodegib, a potent orally administered SMO inhibitor for the treatment of locally advanced, surgically inoperable and potentially fatal BCCs. Despite their undeniable efficacy, the utility of currently available HH signaling inhibitors is hampered by rapid development of tumor resistance and tumor recurrence. While uninhibited Hh signaling clearly drives BCC resistance and recurrence, many BCCs do not manifest SMO mutations indicating involvement of additional tumorigenic mechanisms. We have discovered that vismodegib resistance involves dysregulation of the bromodomain-containing proteins BRD7 and BRD9 of the SWItch/Sucrose NonFermentable (SWI/SNF) nucleosome remodeling complexes. Utilizing genetically well- defined in vitro and in vivo murine models of BCC, and patient-derived human BCC cells, our preliminary data compellingly demonstrate that (i) HHi resistance is associated with global decreases in histone acetylation and chromatin accessibility, and (ii) genetic ablation of BRD7 renders drug-naïve BCC cells resistant to HHi. Based on our preliminary data, this application will test the hypothesis that the BRD7-BRD9 nexus drives HHi resistance and that the BRD9 blockade prevents the emergence of HHi resistance. Aim 1 will probe the chromatin modifications and gene expression signatures associated with HHi resistance, and their relevance to the BRD7- BRD9 axis. Aim 2 will test in vivo consequences of genetic manipulation of the BRD7 and BRD9 nexus in genetically-defined models (i.e., epidermis-specific deletions in BRD7 [Brd7 KO] or Akt1 [Akt1+/-]. Aim 3 will test the potential utility of select BRD9 and Akt inhibitors for overcoming/preventing HHi resistance using an in vivo BCC model system developed in the PI’s laboratory that faithfully mimics human BCNS and previously was used to verify the efficacy and safety of HHi.