A NOVEL DRUGGABLE GENETIC VULNERABILITY PATHWAY IN MELANOMA

 描述(申请人提供)：仅今年一年，就有超过76,000名美国人被诊断出患有黑色素瘤，令人遗憾的是，其中10,000名患者将死于这种疾病。NRAS和BRAF基因的致癌突变存在于超过75%的黑色素瘤中。然而，目前还没有针对NRAS突变型黑色素瘤的有效治疗方法，而针对BRAF突变型黑色素瘤的靶向治疗由于耐药性的迅速出现而未能提供长期的临床益处。我们推测，识别新的可药物遗传易损性(DGV)途径将导致针对NRAS突变和靶向治疗耐药的BRAF突变黑色素瘤的替代和有效治疗的发展。为此，通过采用体内可药物基因组RNAi筛选的创新方法，我们确定LIM结构域激酶2(LIMK2)是黑色素瘤的一个新的药物靶点。基于RNAi和CRISPR/Cas9的LIMK2抑制抑制NRAS和BRAF突变的黑色素瘤的肿瘤和转移生长，LIMK2的过度表达使BRAF和MEK靶向治疗产生抵抗。我们的中心假设是LIMK2调节NRAS/BRAF突变黑色素瘤的可药物遗传易损性途径，这对于靶向治疗的耐药性也是重要的。总体目标是确定LIMK2在黑色素瘤发生和发展中的作用，了解其作用机制，并评估LIMK2作为黑色素瘤治疗的药物靶点。在目标1中，我们将确定LIMK2在黑色素瘤的启动和转移进展中的作用。为此，我们将首先确定LIMK2是否是致癌NRAS/BRAF诱导转化所必需的。此外，基于我们的研究结果，LIMK2通过诱导失巢凋亡抵抗促进循环中黑色素瘤细胞的侵袭和存活，我们将使用基于异种移植的器官特异性和自发性黑色素瘤转移小鼠模型来确定LIMK2在黑色素瘤转移中的体内作用。在目标2中，我们将确定LIMK2维持肿瘤和NRAS/BRAF突变黑色素瘤转移生长的机制。为此，基于我们的无偏高通量转录组mRNA测序和串联亲和纯化/质谱分析的结果，我们将评估一种新的LIMK2底物双特异性磷酸酶1(DUSP1)作为LIMK2功能的潜在下游介质在黑色素瘤中的作用。在目标3中，我们将确定LIMK2在诱导对BRAF和MEK靶向治疗的耐药性中的作用，并评估其作为黑色素瘤药物靶点的有效性。为此，我们将确定LIMK2驱动的DGV通路抑制是否防止或延缓了对BRAF和MEK靶向治疗的耐药性的出现，以及LIMK2的系统药理抑制是否在体内治疗转移性黑色素瘤和BRAF和MEK靶向治疗耐药黑色素瘤的基于异种移植的和遗传的黑色素瘤生长和转移模型中有效。综上所述，我们的提议将揭示一种新的DGV途径，该途径可以在药理学上靶向治疗转移性和耐药黑色素瘤。

 DESCRIPTION (provided by applicant): Over 76,000 Americans will be diagnosed with melanoma this year alone and regrettably 10,000 of these patients will die of their disease. Oncogenic mutations in NRAS and BRAF genes are present in over 75% of melanoma. However, currently there is no effective treatment for NRAS mutant melanoma and targeted therapies against BRAF mutant melanoma fail to provide long-term clinical benefits due to rapid emergence of drug resistance. We postulated that identifying novel Druggable Genetic Vulnerability (DGV) pathways will lead to the development of alternative and effective therapies for NRAS mutant and targeted therapy resistant BRAF mutant melanoma. Towards this end, by employing an innovative approach of an in vivo druggable genome RNAi screen, we identified LIM-domain Kinase 2 ( LIMK2) as a novel drug target in melanoma. RNAi and CRISPR/CAS9-based inhibition of LIMK2 blocked the tumor and metastatic growth of NRAS- and BRAF-mutant melanoma and LIMK2 overexpression conferred resistance to BRAF- and MEK- targeted therapies. Our central hypothesis is that LIMK2 regulates a druggable genetic vulnerability pathway in NRAS/BRAF mutant melanoma, which is also important for conferring resistance to targeted therapies. The overall objective is to determine the role of LIMK2 in melanoma initiation and progression, understand its mechanism-of-action and evaluate LIMK2 as a drug target for melanoma therapy. In Aim 1, we will determine the role of LIMK2 in melanoma initiation and metastatic progression. Towards this end, we will first determine if LIMK2 is necessary for oncogenic NRAS/BRAF-induced transformation. Additionally, based on our results that LIMK2 promotes invasion and survival of circulating melanoma cells by conferring anoikis resistance, we will determine the in vivo role of LIMK2 in melanoma metastasis using xenograft- based organ-specific and spontaneous mouse model of melanoma metastasis. In Aim 2, we will determine the mechanism by which LIMK2 sustain tumor and metastatic growth of NRAS/BRAF mutant melanoma. Towards this end, based on the results of our unbiased high-throughput approaches of transcriptome-wide mRNA sequencing and Tandem Affinity Purification/Mass spectrometry analysis, we will evaluate the role of a novel LIMK2 substrate Dual Specificity Phosphatase 1 (DUSP1) as a potential downstream mediator of LIMK2 function in melanoma. In Aim 3, we will determine the role of LIMK2 in conferring resistance to BRAF- and MEK-targeted therapies and evaluate its utility as a drug target in melanoma. Towards this end, we will determine if LIMK2-driven DGV pathway inhibition prevents or delay emergence of resistance to BRAF- and MEK-targeted therapies and if systemic pharmacological inhibition of LIMK2 is effective in treating metastatic and BRAF- and MEK-targeted therapy resistant melanoma in vivo in xenograft-based and genetic mouse models of melanoma growth and metastasis. Taken together, our proposal will uncover a novel DGV pathway that can be pharmacologically targeted for treating metastatic and drug resistant melanoma.