Regulation of oncogenic KIT by LMTK3

 描述(由申请人提供)： KIT的功能获得突变在许多人类恶性肿瘤中被发现，包括胃肠道间质瘤(GIST)、肥大细胞肿瘤、黑色素瘤、精原细胞瘤和急性髓系白血病。胃肠道肿瘤对放射和化疗都有抵抗力。KIT突变是大多数GIST的致癌驱动因素。酪氨酸激酶抑制剂(TKI)，如伊马替尼，使晚期GIST的治疗发生了革命性的变化。在肥大细胞肿瘤(SM)中，这种疾病是由含有激活KIT突变的肥大细胞引起的。SM患者是化疗的候选对象，但这些疗法毒性大，效果不佳。带有激活试剂盒突变的黑色素瘤具有侵袭性，对化疗没有反应。在所有这三种疾病中，KIT TKIs的有效性都受到原发或继发耐药的限制。我们的建议旨在通过确定LMTK3在正向调节致癌KIT蛋白表达中的作用来改进KIT突变恶性肿瘤的治疗。计划：我们的建议有三个具体目标。在SA1中，我们将确定LMTK3调节致癌试剂盒异构体转录的机制。在SA2中，我们将确定LMTK3的功能结构域，以调控致癌试剂盒的表达。在SA3中，我们将识别与LMTK3相互作用的蛋白质，并确定它们在促进KIT转录方面的作用。方法：在SA1中，我们将使用KIT启动子-报告结构来确定KIT启动子受LMTK3敲除调控的区域。此外，我们还将确定通过LMTK3间接调节KIT转录的候选转录因子。在SA2中，我们将创建一系列具有特定结构域(例如LMTK3激动域)突变的LMTK3突变体，并确定哪些结构域是调节KIT启动子所必需的。此外，我们还将利用质谱仪鉴定LMTK3的磷酸化位点。在SA3中，我们将使用质谱学和蛋白质下拉分析来确定与LMTK3相互作用并可能调节KIT转录的蛋白质。临床相关性：对患有癌症的退伍军人的护理占退伍军人事务部卫生保健预算的很大一部分。针对肿瘤生物学重要方面的新型癌症治疗药物将极大地提高患有癌症的退伍军人的生活质量。我们对LMTK3调节致癌KIT蛋白的机制的研究有可能发现KIT突变癌症的新治疗方法。这些新疗法可能比传统疗法更有效，而且治疗毒性明显较小。

 DESCRIPTION (provided by applicant): Gain-of-function mutations of KIT are found in a number of human malignancies, including gastrointestinal stromal tumors (GIST), mast cell neoplasms, melanoma, seminoma, and acute myeloid leukemia. GIST tumors are resistant to both radiation and chemotherapy. KIT mutations are the oncogenic driver in the case of most GISTs. Tyrosine kinase inhibitors (TKIs) such as imatinib have revolutionized the treatment of advanced GIST. In mast cell neoplasms (SM), the disease is caused by mast cells harboring activating KIT mutations. SM patients are candidates for chemotherapy, but these therapies are toxic and ineffective. Melanomas with activating KIT mutations are aggressive and do not respond to chemotherapy. In all three diseases, the effectiveness of KIT TKIs is limited by primary or secondary drug resistance. Our proposal seeks to improve therapy for KIT-mutant malignancies by identifying the role LMTK3 has in positively regulating the expression of oncogenic KIT protein. Plan: Our proposal has three specific aims. In SA1, we will determine the mechanisms by which LMTK3 regulates the transcription of oncogenic KIT isoforms. In SA2, we will determine the functional domains of LMTK3 required for regulated expression of oncogenic KIT. In SA3, we will identify proteins that interact with LMTK3 and determine their role in promoting KIT transcription. Methods: In SA1, we will use a KIT promoter-reporter construct to identify regions of the KIT promoter that are regulated by LMTK3 knockdown. In addition, we will identify candidate transcription factors that mediate indirect regulation of KIT transcription by LMTK3. In SA2, we will create a series of LMTK3 mutants with mutation of specific domains (e.g. LMTK3 kinase domain) and determine which domains are required for regulation of the KIT promoter. In addition, we will identify LMTK3 phosphorylation sites using mass spectrometry. In SA3 we will use mass spectrometry and protein pull down assays to identify proteins that interact with LMTK3 and potentially regulate KIT transcription. Clinical Relevance: The care of veterans with cancer represents a significant portion of the overall Veterans Affairs Health Care budget. New cancer treatment agents that target significant aspects of tumor biology will greatly increase the quality of life o veterans with cancer. Our study of the mechanisms by which LMTK3 regulates oncogenic KIT proteins has the potential to identify new treatments for KIT-mutant cancers. These new treatments are likely to be more effective than conventional treatments and be associated with significantly less treatment toxicity.