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（由申请人提供）：仅今年就有超过76，000名美国人被诊断患有黑色素瘤，令人遗憾的是，其中10，000名患者将死于这种疾病。超过75%的黑色素瘤中存在NRAS和BRAF基因的致癌突变。然而，目前还没有针对NRAS突变型黑色素瘤的有效治疗方法，并且针对BRAF突变型黑色素瘤的靶向治疗由于快速出现耐药性而未能提供长期临床益处。我们假设，确定新的药物遗传脆弱性（DGV）途径将导致NRAS突变和靶向治疗耐药性BRAF突变黑色素瘤的替代和有效的治疗方法的发展。为此，通过采用体内可药物化基因组RNAi筛选的创新方法，我们鉴定了LIM结构域激酶2（LIMK 2）作为黑色素瘤中的新药物靶点。基于RNAi和CRISPR/CAS9的LIMK 2抑制阻断了NRAS和BRAF突变型黑色素瘤的肿瘤和转移性生长，并且LIMK 2过表达赋予了对BRAF和MEK靶向疗法的抗性。我们的中心假设是LIMK 2调节NRAS/BRAF突变型黑色素瘤中的可药物化遗传脆弱性途径，这对于赋予靶向治疗抗性也很重要。总体目标是确定LIMK 2在黑色素瘤发生和进展中的作用，了解其作用机制，并评估LIMK 2作为黑色素瘤治疗的药物靶点。在目标1中，我们将确定LIMK 2在黑色素瘤发生和转移进展中的作用。为此，我们将首先确定LIMK 2是否是致癌NRAS/BRAF诱导转化所必需的。此外，基于我们的LIMK 2通过赋予失巢凋亡抗性来促进循环黑素瘤细胞的侵袭和存活的结果，我们将使用基于异种移植物的器官特异性和自发性黑素瘤转移小鼠模型来确定LIMK 2在黑素瘤转移中的体内作用。在目标2中，我们将确定LIMK 2维持NRAS/BRAF突变型黑色素瘤的肿瘤和转移生长的机制。为此，基于我们无偏高通量方法的全转录组mRNA测序和串联亲和纯化/质谱分析的结果，我们将评估新型LIMK 2底物双特异性磷酸酶1（DUSP 1）作为黑素瘤中LIMK 2功能的潜在下游介体的作用。在目标3中，我们将确定LIMK 2在赋予BRAF和MEK靶向治疗抗性中的作用，并评估其作为黑色素瘤药物靶标的效用。为此，我们将确定LIMK 2驱动的DGV途径抑制是否预防或延迟对BRAF和MEK靶向治疗的抗性的出现，以及LIMK 2的全身药理学抑制是否有效治疗转移性和BRAF和MEK靶向治疗抗性黑素瘤，在黑素瘤生长和转移的异种移植物和遗传小鼠模型中。总而言之，我们的提案将揭示一种新型的DGV途径，可以在药理学上靶向治疗转移性和耐药性黑色素瘤。