Molecular Mechanisms Underlying the Prevention of BCC Resistance

摘要 BCC 是美国最常见的人类恶性肿瘤，影响超过 300 万美国人 每年。 Hedgehog (Hh) 信号传导存在缺陷，通常是由于肿瘤功能丧失所致 抑制子补丁 (PTCH) 导致 SMO 致癌激活，被认为是 SMO 致癌的主要驱动因素 BCC 增长。配体与 PTCH 结合可缓解 SMO 抑制，从而触发其迁移至初级纤毛 激活驱动细胞增殖/肿瘤生长的 GLI 转录因子。异常 HH 信号传导的基础 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就带有一个功能性 PTCH 等位基因，并在一生中获得了 PTCH 等位基因突变 第二个等位基因加速 HH 信号传导并驱动这些患有异常疾病的患者的 BCC 生长 肿瘤负担使得在其一生中需要进行多次昂贵的残肢外科手术。此外，Hh 抑制剂（HHi）与接受治疗的个体中无法忍受的副作用相关，因此一半的患者 尽管具有显着的抗肿瘤功效，但仍停止治疗。我们的团队和世界各地的其他人已经 促进了针对 HH 信号传导的药物的从实验室到临床的临床试验，这些努力在 2012 年取得了成果 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。尽管它们具有不可否认的功效，但目前可用的实用性 HH 信号抑制剂受到肿瘤耐药性和肿瘤复发快速发展的阻碍。同时 不受抑制的 Hh 信号明显导致 BCC 耐药和复发，许多 BCC 并不表现出 SMO 表明涉及其他致瘤机制的突变。我们发现 vismodegib 耐药性涉及含溴结构域蛋白 BRD7 和 BRD9 的失调。 SWIitch/蔗糖不可发酵 (SWI/SNF) 核小体重塑复合物。充分利用基因—— 定义了 BCC 的体外和体内小鼠模型以及源自患者的人类 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下降相关 染色质可及性，以及 (ii) BRD7 的基因消除使未接受过药物治疗的 BCC 细胞对 HHi 产生耐药性。基于 根据我们的初步数据，该应用程序将测试 BRD7-BRD9 连接驱动 HHi 抵抗的假设 并且 BRD9 阻断可以防止 HHi 耐药性的出现。目标 1 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-的相关性 BRD9 轴。目标 2 将测试 BRD7 和 BRD9 连接的基因操作的体内后果 基因定义的模型（即 BRD7 [Brd7 KO] 或 Akt1 [Akt1 /-] 中表皮特异性缺失。目标 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 PI实验室开发的BCC模型系统，忠实模仿人类BCNS，之前曾使用过 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.