Combinatorial Approaches to Overcoming Resistance to BRAF(V600E) Targeted Thera

项目3将侧重于努力了解和克服对BRAF(V600E)抑制的严重阻力 通过了解BRAF(V600E)是如何调节死亡信号的，我们发现了BRAF在黑色素瘤中的作用。多项研究表明， 抑制BRAF(V600E)可强烈诱导促凋亡因子BIM。然而，很少有细胞凋亡 发生。相反，MCL1或BCL2A1绑定和隔离BIM。我们报告BCL2A1是一种新发现的 约三分之一的患者存在基因扩增的黑色素瘤癌基因。它在转录水平上也受 MITF是一种谱系特异的主要转录因子，在约20%的黑色素瘤中也有扩增。BCL2A1 因此，MITF是谱系受限的抗细胞凋亡调节因子。我们发现BRAF-MEK-MAPK 使MITF磷酸化，引发泛素依赖的降解。相反，BRAF(V600E)抑制 阻断MITF降解，从而上调MITF。反过来，MITF的目标被有力地诱导了一些 它们是抗凋亡的，而其他的则是免疫抗原。免疫系统可能对 BRAF(V600E)抑制剂的疗效，因为1)治疗在10天内诱导T细胞瘤侵袭，2) MITF能强烈诱导黑素细胞抗原的表达，3)BRAF(600e)靶向性显著 免疫缺陷背景下的黑色素瘤较弱。为了追问这些问题，Aim1将研究 MITF‘s和BCL2A1’S作为BRAF抑制剂的细胞凋亡拮抗剂。我们将使用临床注解的 患者样本以确定MITF或BCL2A1中的基因组扩增是否可预测预后 对BRAF(V600E)目标的反应。此外，基因靶向(基因敲除)的使用表明MITF和 BCL2A1是抑制BRAF敏感性的谱系特异性拮抗剂。因此有几个小分子 哪些可拮抗MITF或BCL2A1，哪些可用于临床，将单独或与BRAF一起测试 在体外抑制，在小鼠中，和(对于MITF拮抗)在最近开放的临床试验中。《目标2》将剖析 免疫完整小鼠中BRAF(V600E)、MITF和黑色素瘤免疫反应之间的相互作用。 BRAF、MEK或ERK的抑制剂将与免疫检查点阻断抗体(抗CTLA4)联合使用 抗PDLI，或抗PDI)，并在小鼠身上测试，以及在人体试验中进行的相关研究 维莫拉非尼+ipilimumab。将针对拷贝数和拷贝数分析“抗性”行中的明显漏洞 深度测序数据(项目1、2和核心A、B)和shRNA功能筛选(项目1和2)以确定 临床前和临床发展的可用药组合途径。

Project 3 will focus on efforts to understand and overcome acute resistance to BRAF(V600E) suppression in melanoma by understanding how BRAF(V600E) modulates death signals. Multiple studies show that BRAF(V600E) suppression strongly induces BIM, a pro-apoptotic factor. However very little apoptosis occurs. Instead either MCL1 or BCL2A1 bind & sequester BIM. We report BCL2A1 as a newly recognized genomically amplified melanoma oncogene in about 1/3 of patients. It is also transcriptionally regulated by MITF, a lineage specific master transcription factor which is also amplified in ~20% of melanomas. BCL2A1 and MITF are thus lineage-restricted anti-apoptotic regulators. We found that BRAF-MEK-MAPK phosphorylates MITF, triggering ubiquitin-dependent degradation. Conversely BRAF(V600E) suppression blocks MITF degradation, thereby upregulating MITF. In turn MITF's targets are potently induced¿some of which are anti-apoptotic while others serve as immune antigens. The immune system may contribute to efficacy of BRAF(V600E) inhibitors because 1) treatment induces T cell tumor infiltrates within 10 days, 2) melanocytic antigen expression is strongly induced via MITF, and 3) BRAF(\/600E) targeting is significantly weaker for melanomas in an immunodeficient background. To pursue these questions Aim1 will examine MITF's and BCL2A1's roles as apoptosis antagonists to BRAF inhibitors. We will utilize clinically annotated patient specimens to determine whether genomic amplifications in MITF or BCL2A1 are prognostic for response to BRAF(V600E) targeting. Further, the use of gene targeting (knockdown) suggests that MITF and BCL2A1 are lineage specific antagonists to inhibition of BRAF sensitivity. Therefore several small molecules which antagonize MITF or BCL2A1, and which are clinically available, will be tested alone or with BRAF suppression in vitro, in mice, and (for MITF antagonism) in a recently opened clinical trial. Aim 2 will dissect interactions between BRAF(V600E), MITF, and melanoma immune responses in immune-intact mice. Inhibitors to BRAF, MEK, or ERK will be combined with immune checkpoint blocking antibodies (anti-CTLA4 anti-PDLI, or anti-PDI) and tested in mice, and in correlative studies for a human trial for vemurafenib+ipilimumab. Distinct vulnerabilities in "resistant" lines will be analyzed against copy number and deep sequencing data (Project 1, 2 & Cores A, B) and shRNA functional screens (Projects 1 & 2) to identify drugable combination pathways for preclinical and clinical development.