Augmenting Melanoma Response to B-Raf V600E Targeting

描述（由申请人提供）：Lo博士于2008年7月建立了他的独立研究项目，专注于黑色素瘤发病机制和靶向治疗，并得到了UCLA医学/皮肤科和Jonsson综合癌症中心的支持。转移性皮肤黑色素瘤是一种侵袭性恶性肿瘤，缺乏有效的治疗，但具有一个共同的和可药用的目标，V600 EB-RAF突变激酶。用新型V600 EB-RAF靶向小分子PLX 4032治疗黑色素瘤患者的早期临床经验表明，在预选的V600 EB-RAF突变型肿瘤患者中，肿瘤缓解无与伦比。然而，对PLX 4032单一疗法的显著反应总是伴随着肿瘤逃逸和复发。 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。我们正在采取补充的临床前基因组方法（目的1和2），以剖析用PLX 4032治疗的黑色素瘤患者的敏感性和耐药性模式（目的3）。首先（目的1），我们试图鉴定与V600 EB-RAF一起缓冲黑素瘤免于生长停滞/死亡（即，共依赖生长/存活基因）。为了构建维持V600 EB-RAF阳性黑色素瘤存活的细胞“接线图”，我们建立了一种稳健的高通量siRNA功能筛选，以鉴定黑色素瘤激酶，其敲低可使黑色素瘤细胞对V600 EB-RAF抑制敏感。第二（目的2），我们通过产生逃避长期PLX 4032暴露的V600 EB-RAF阳性黑素瘤细胞系来体外建模PLX 4032抗性。比较亲本PLX 4032敏感细胞系及其相应的PLX 4032耐药亚系，我们提出了一种整合的基因组方法来发现V600 EB-RAF靶向抑制的肿瘤逃逸机制。我们相信，用来自这些方法的知识武装自己对于在活检-治疗-复发-活检方案中对PLX 4032复发的患者的珍贵配对黑色素瘤样本和细胞系进行系统性假设驱动研究（目标3）至关重要。 PLX 4032可能是一系列针对仅在癌症中发现的活化突变分子的药物中的第一种。我们提出的工作可能会提供一个重要的理性基础，了解这种治疗药物的反应/耐药模式，并设计组合治疗，以克服阻力。所提出的使用黑色素瘤作为模型的全面和客观的方法也可以提供对其他恶性肿瘤如结肠癌和卵巢癌的V600 EB-RAF阳性子集的见解。此外，这笔赠款将为申请人提供关键的资金，以竞争RO 1和其他既定的研究者奖项。 公共卫生相关性：皮肤黑色素瘤是每年发病率上升最快的人类恶性肿瘤之一，在晚期发现时具有高度致命性。靶向常见黑色素瘤突变V600 EB-RAF的小分子治疗显示出前所未有的前景，但也面临着所有靶向治疗、癌症耐药性和临床复发共同面临的巨大挑战。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，我们可以设计更好的疗法来治疗这种致命的皮肤癌。

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.