A NOVEL DRUGGABLE GENETIC VULNERABILITY PATHWAY IN MELANOMA

黑色素瘤中一种新的可药物遗传脆弱性途径

• 批准号: 9920866
• 负责人: Narendra Wajapeyee
• 金额: $ 19.86万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9920866
• 关键词: Affinity Chromatography; Alternative Therapies; American; Anoikis; BRAF gene; Biochemical; Biology; CRISPR/Cas technology; Cell Culture Techniques; Cessation of life; Clinical; Combined Modality Therapy; Development; Diagnosis; Disease; Drug Targeting; Drug resistance; Effectiveness; Epigenetic Process; Genes; Genetic; Genetic Models; Genetic Predisposition to Disease; Genome; Growth; Human; LIM Domain; LIM Domain Kinase 1; MEKs; Mass Spectrum Analysis; Mediator of activation protein; Melanoma Cell; Metastatic Melanoma; Mitogen-Activated Protein Kinases; Modeling; Molecular; Mutation; NRAS gene; Neoplasm Metastasis; Oncogenic; Organ; Pathway interactions; Patients; Peptide Hydrolases; Pharmacology; Phosphoric Monoester Hydrolases; Phosphotransferases; Protein-Serine-Threonine Kinases; Proteins; RNA Interference; RNA interference screen; Resistance; Role; Sampling; Skin Cancer; Specificity; Testing; TimeLine; Ubiquitin; United States Food and Drug Administration; Xenograft procedure; base; clinically significant; effective therapy; experimental study; improved; in vivo; inhibitor/antagonist; innovation; mRNA sequencing; melanoma; mouse model; mutant; new therapeutic target; novel; novel therapeutics; overexpression; pre-clinical; prevent; public health relevance; response; targeted treatment; therapy resistant; transcriptome; transcriptome sequencing; tumor

 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.

 描述（由适用提供）：仅今年，超过76,000名美国人将被诊断出患有黑色素瘤，其中10,000名患者将死于其疾病。超过75％的黑色素瘤中，NRA和BRAF基因中的致癌突变呈现。但是，目前尚无对NRAS突变黑色素瘤和针对BRAF突变黑色素瘤的靶向疗法的有效治疗方法，由于耐药性的迅速出现，无法提供长期的临床益处。我们假设确定新型的可毒遗传脆弱性（DGV）途径将导致开发NRAS突变体和靶向疗法耐药的BRAF突变体黑色素瘤的替代和有效疗法。为此，通过采用可吸毒的基因组RNAi筛查的创新方法，我们确定了Lim-domomain激酶2（Limk2）是黑色素瘤中的新药物靶标。基于RNAi和基于CRISPR/CAS9的limk2抑制阻止了NRAS和BRAF突变药物黑色素瘤的肿瘤和转移性生长，以及limk2的过表达对BRAF和MEK靶向疗法的耐药性。我们的中心假设是，LIMK2调节NRAS/BRAF突变体黑色素瘤中可吸毒的遗传脆弱性途径，这对于赋予对靶向疗法的耐药性也很重要。总体目标是确定Limk2在黑色素瘤倡议和进展中的作用，了解其行动机理，并评估Limk2作为黑色素瘤治疗的药物靶标。在AIM 1中，我们将确定Limk2在黑色素瘤倡议和转移进展中的作用。为此，我们将首先确定limk2是否对于致癌NRAS/BRAF诱导的转化是否需要。此外，基于我们的结果，即通过会议的抗原抗性促进循环黑色素瘤细胞的侵袭和存活，我们将使用基于基于Xenographogrogroticon的器官特异性和赞助的黑色素瘤转移模型来确定Limk2在黑色素瘤转移中的体内作用。在AIM 2中，我们将确定LIMK2维持NRAS/BRAF突变体黑色素瘤的转移性生长的机制。为此，基于我们无偏向转录组mRNA测序和串联亲和力纯化/质谱分析的无偏高通量方法的结果，我们将评估新型LIMK2底物双重特异性磷酸酶1（DUSP1）的作用，作为在糖浆瘤中limk2功能的潜在下游介质。在AIM 3中，我们将确定LIMK2在对BRAF和MEK靶向疗法的抗性中的作用，并评估其作为黑色素瘤药物靶标的效用。 Towards this end, we will determine if LIMK2-driven DGV pathway inhibition prevents or delay emergency of resistance to BRAF- and MEK-targeted therapies and if systemic pharmaceutical inhibition of LIMK2 is effective in treating metastatic and BRAF- and MEK-targeted therapy resistant melanoma in vivo in xenographic-based and genetic mouse models of melanoma growth and metastasis.综上所述，我们的建议将发现一种新型的DGV途径，该途径可以针对治疗转移性和耐药性黑色素瘤的药理目标。