Molecular Mechanisms Underlying the Prevention of BCC Resistance

总结 BCC是美国最常见的人类恶性肿瘤类型，影响超过300万美国人 每年。Hedgehog（Hh）信号传导调节缺陷，通常通过肿瘤功能丧失 导致SMO致癌激活的抑制子修补（PTCH）被认为是SMO的主要驱动因素。 BCC增长。与PTCH结合的配体解除SMO抑制，触发其迁移到初级纤毛， 激活GLI转录因子，驱动细胞增殖/肿瘤生长。异常HH信号传导是 Gorlin-Goltz综合征，也称为基底细胞痣综合征（BCNS），是一种显性遗传性疾病， 受影响的个体出生时具有一个功能性PTCH等位基因，并在生命过程中获得PTCH基因突变。 第二个等位基因，加速HH信号传导，并驱动这些患者的BCC生长， 肿瘤负荷在其一生中需要多次昂贵的毁损外科手术。此外，Hh 抑制剂（HHi）与治疗个体中不可耐受的副作用相关， 尽管有显著的抗肿瘤疗效，仍停止治疗。我们的团队和世界各地的其他团队 促进了针对HH信号传导的药物的临床试验，2012年，这些努力导致 FDA批准vismodegib，一种有效的口服SMO抑制剂，用于治疗局部晚期， 无法手术且可能致命的BCC尽管它们的功效不可否认，但目前可用的 HH信号传导抑制剂受到肿瘤抗性和肿瘤复发的快速发展的阻碍。而 不受抑制的Hh信号传导明显驱动BCC耐药和复发，许多BCC不表现SMO 突变表明涉及额外的致瘤机制。我们发现维莫德吉 耐药性涉及细胞中含溴结构域蛋白BRD 7和BRD 9的调节异常。 SWITCH/蔗糖不可发酵（SWI/SNF）核小体重塑复合物。利用基因优势- 定义的体外和体内BCC小鼠模型，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的整体降低相关， 染色质可及性，和（ii）BRD 7的基因消融使得药物初始BCC细胞对HHi具有抗性。基于 根据我们的初步数据，本申请将检验BRD 7-BRD 9关系驱动HHi抗性的假设 并且BRD 9阻断防止HHi抗性的出现。AIM 1将探测染色质 与HHi抗性相关的修饰和基因表达特征，以及它们与BRD 7- BRD 9轴。目的2将测试在体内对BRD 7和BRD 9关系进行遗传操作的结果， 遗传定义的模型（即，BRD 7 [Brd 7 KO]或Akt 1 [Akt 1 +/-]中的表皮特异性缺失。目标3将测试 选择BRD 9和Akt抑制剂用于克服/预防HHi抗性的潜在效用使用体内 在PI的实验室中开发的BCC模型系统忠实地模拟了人类BCNS， 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.