Augmenting Melanoma Response to B-Raf V600E Targeting

描述(申请人提供)：罗博士于2008年7月建立了他的独立研究计划，在加州大学洛杉矶分校内科/皮肤科和Jonsson综合癌症中心的支持下，专注于黑色素瘤的发病机制和靶向治疗。转移性皮肤黑色素瘤是一种侵袭性的恶性肿瘤，缺乏有效的治疗，但包含一个常见的和可药物的靶点，V600EB-RAF突变激酶。使用新型V600EB-RAF靶向小分子PLX4032治疗黑色素瘤患者的早期临床经验显示，在预先选择的V600EB-RAF突变肿瘤患者中，肿瘤反应无与伦比。然而，PLX4032单一治疗的戏剧性反应总是伴随着肿瘤逃脱和复发。 拟议的项目旨在建立一个知识库，用于设计组合疗法和解决耐药性问题。我们正在采用互补的临床前基因组方法(目标1和2)，以剖析接受PLX4032治疗的黑色素瘤患者的敏感性和耐药性模式(目标3)。首先(目标1)，我们试图确定与V600EB-RAF一起缓冲黑色素瘤生长停滞/死亡(即共同依赖的生长/存活基因)的黑色素瘤蛋白。为了构建这样一个维持V600EB-RAF阳性黑色素瘤生存的细胞“接线图”，我们建立了一个基于siRNA的强大和高通量的功能筛选，以识别其敲除使黑色素瘤细胞对V600EB-RAF抑制敏感的黑色素瘤蛋白。第二(目标2)，我们正在通过产生逃避慢性PLX4032暴露的V600EB-RAF阳性黑色素瘤细胞株来模拟PLX4032的体外耐药性。通过比较亲本PLX4032敏感细胞系及其相应的PLX4032抗性亚系，我们提出了一种完整的基因组学方法来发现V600EB-RAF靶向抑制的肿瘤逃逸机制。我们相信，用这种方法获得的知识武装自己，对于以活检-治疗-复发-活组织检查方案对珍贵的、成对的黑色素瘤样本和细胞系进行系统的假设驱动研究(AIM 3)至关重要。 PLX4032可能是针对仅在癌症中发现的激活的、突变的分子的一系列药物中的第一个。我们提出的工作可能会为理解对此类治疗剂的反应/耐药模式和设计克服耐药性的联合疗法提供关键的理性基础。所提出的以黑色素瘤为模型的全面和客观的方法也可能为其他恶性肿瘤，如结肠癌和卵巢癌的V600EB-RAF阳性亚群提供见解。此外，这笔拨款将为申请者提供关键资金，以竞争RO1和其他既定的调查员奖项。 公共卫生相关性：皮肤黑色素瘤是年发病率上升最快的人类恶性肿瘤之一，在晚期被发现时具有高度致命性。针对常见黑色素瘤突变V600EB-RAF的小分子疗法正显示出前所未有的前景，但它面临着所有靶向治疗、癌症耐药性和临床复发共同的巨大挑战。通过了解决定药物敏感性的因素和获得性耐药的关键机制，我们可以设计出更好的治疗这种致命皮肤癌的方法。

DESCRIPTION (provided by applicant): Dr. Lo established his independent research program in July of 2008, focusing on melanoma pathogenesis and targeted therapy with support from the UCLA Department of Medicine/Dermatology and the Jonsson Comprehensive Cancer Center. Metastatic cutaneous melanoma is an aggressive malignancy, which lacks effective treatment but harbors a common and druggable target, the V600EB-RAF mutant kinase. Early clinical experience with melanoma patients treated with a novel V600EB-RAF-targeting small molecule, PLX4032, is demonstrating unparalleled tumor response in pre-selected patients with V600EB-RAF-mutant tumors. However, dramatic responses to PLX4032 monotherapy are invariably followed by tumor escape and relapse. The proposed project aims to build a knowledge base for designing combinatorial therapies and tackling drug resistance. We are taking complementary pre-clinical genomic approaches (Aims 1 & 2) in order to dissect the pattern of sensitivity and resistance in melanoma patients treated with PLX4032 (Aim 3). First (Aim 1), we seek to identify melanoma kinases that, together withV600EB-RAF, buffer melanoma against growth arrest/death (i.e., co-dependent growth/survival genes). To construct such a cellular "wiring diagram" sustaining the survival of V600EB-RAF-positive melanomas, we have established a robust and high-throughput siRNA-based functional screen to identify melanoma kinases whose knockdown sensitizes melanoma cells toV600EB-RAF inhibition. Second (Aim 2), we are modeling PLX4032 resistance in vitro by generating V600EB-RAF-positive melanoma cell lines escaping chronic PLX4032 exposure. Comparing parental PLX4032-sensitive cell lines and their corresponding PLX4032-resistant sub-lines, we propose an integrated genomic approach to discover tumor escape mechanisms to V600EB-RAF targeted inhibition. We believe arming ourselves with knowledge derived from such approaches is critical for a systematic hypothesis-driven study (Aim 3) of precious, paired melanoma samples and cell lines from patients who relapse on PLX4032 in a biopsy-treat-relapse-biopsy protocol. PLX4032 will likely be the first in a series of drugs targeting activated, mutated molecules found specifically only in cancer. Our proposed work will likely provide a critical rational basis to understand the pattern of response/resistance to such therapeutic agents and to design combinatorial therapy to overcome resistance. The proposed comprehensive and objective approaches using melanoma as a model may also offer insights into V600EB-RAF-positive subsets of other malignancies such as colon and ovarian cancers. Furthermore, this grant will provide the applicant with crucial funding to compete for RO1 and other established investigator awards. PUBLIC HEALTH RELEVANCE: Cutaneous melanoma ranks among the fastest rising human malignancies in annual incidence and is highly lethal when detected at advanced stages. Small molecule therapy targeting a common melanoma mutation, V600EB-RAF, is showing unprecedented promise but meets a formidable challenge common to all targeted therapy, cancer resistance and clinical relapse. By understanding the factors determining drug sensitivity and key mechanisms of acquired resistance, we can design better therapies to treat this deadly skin cancer.