Molecular mechanisms of BRAF inhibitor induced UPR and autophagy

 描述(申请人提供)：在接受BRAF抑制剂治疗的BRAF突变黑色素瘤患者中观察到的戏剧性反应反映了黑色素瘤治疗的重大突破。然而，在患者肿瘤中出现的多种耐药机制限制了BRAF抑制剂的有效性。我们发现，在接受BRAF抑制剂治疗的患者肿瘤和细胞系中，自噬被激活，这种与治疗相关的自噬保护黑色素瘤细胞，并有助于肿瘤的再生。我们以前的工作证实，BRAF抑制剂通过激活早期的内质网应激反应来激活自噬，而内质网应激反应反过来又引起细胞保护性自噬。在这项提案中，PI将与合作研究人员密切合作，结合他们在自噬、黑色素瘤生物学和治疗以及内质网应激反应方面的各自专业知识，以阐明突变的BRAF-ER应激-自噬信号的机制基础。我们的假设是，内质网应激途径的某些组成部分对于BRAF抑制剂诱导的自噬至关重要，因此可以作为联合疗法的新靶点。我们的策略将是首先确定突变的BRAF与内质网应激反应和自噬联系的分子机制(目标1)；然后在2D和3D黑色素瘤培养中阐明靶向ER应激反应或自噬的特定成分在BRAFi诱导的黑色素瘤细胞死亡中的生物学效应(目标2)；以及通过遗传和药物抑制ER应激或自噬并结合BRAF抑制来表征BRAFi诱导的内质网应激和自噬在体内的作用(目标3)。这些研究的重点是发展对这三个途径之间的机制联系的复杂理解。从这些研究中获得的知识将增加我们对BRAF突变黑色素瘤的基础生物学，突变BRAF与ER应激反应之间的相互作用，细胞质GRP78在靶向治疗反应中的作用，以及ER应激反应调节自噬的机制的理解。除了这些根本性的进展，该项目还具有翻译潜力，因为它将在BRAF突变黑色素瘤患者的BRAF和自噬抑制的登记试验中研究这些途径，并将确定潜在的新组合，这些组合可能对未来的临床试验更有效。

 DESCRIPTION (provided by applicant): The dramatic responses observed in patients with BRAF mutant melanoma treated with BRAF inhibitors reflect a major therapeutic breakthrough in melanoma. However, there are multiple resistance mechanisms that arise in patient tumors that limit the effectiveness of BRAF inhibitors. We have found that autophagy is activated in patient tumors and cell lines treated with BRAF inhibitors, and this therapy-associated autophagy protects melanoma cells and contributes to regrowth of tumors. Our previous work establishes that BRAF inhibitors activate autophagy by activating an early ER stress response which in turn gives rise to cytoprotective autophagy. In this proposal, the PI will work closely with the co-investigator, combining their respective expertise in autophagy, melanoma biology and treatment, and the ER stress response to elucidate the mechanistic underpinnings of the mutant BRAF-ER stress-autophagy signaling. Our hypothesis is that certain components of the ER stress pathway are critical for BRAF inhibitor-induced autophagy, and therefore could serve as novel targets for combinations regimens. Our strategy will be to first determine the molecular mechanism that links mutant BRAF with the ER stress response and autophagy (aim 1); then elucidate the biological effects of targeting specific components of the ER stress response or autophagy in BRAFi-induced cell death in 2D and 3D melanoma culture (aim 2); and to characterize the role of BRAFi-induced ER stress and autophagy in vivo using genetic and pharmacological inhibition of ER stress or autophagy in combination with BRAF inhibition (aim 3). The focus of these studies is on the development of a sophisticated understanding of the mechanistic links between these three pathways. The knowledge gained from these studies will increase our understanding about the fundamental biology of BRAF mutant melanoma, the interaction between mutant BRAF and the ER stress response, the role of cytoplasmic GRP78 in the response to targeted therapy, and the mechanisms by which the ER stress response regulates autophagy. Besides these fundamental advances, this project has translational potential because it will investigate these pathways in an enrolling trial of BRAF and autophagy inhibition in BRAF mutant melanoma patients, and it will identify potential new combinations that may be even more effective for future clinical trials.