Combinatorial Targeting of Epigenomic and Microenvironmental Pathways to Suppress MAPK Inhibitor Resistance in Melanoma

摘要 突变靶向小分子抑制剂和免疫检查点抗体延长了质量和 晚期MUTBRAF黑色素瘤患者的生活质量以此为基础的组合开发 基础疗法在不久的将来应该会产生更多的生存益处。BRAF和MEK的组合 抑制剂特异性地抑制一种主要由导致MAPK的基因改变驱动的耐药性- MAPK成瘾的重新激活和恢复。然而，这种经临床验证的抑制BRAF的方法 抑制剂耐药性不能解决通过MAPK驱动耐药性的表观基因组和免疫学变化- 冗余和CD8T细胞耗尽的肿瘤状态。我们提出了在高级应用中发现组合目标 了解MAPK抑制剂诱导的表观基因组和免疫抑制的V600BRAF突变型黑色素瘤 机制及其相互作用。我们假设MAPK抑制剂诱导的肿瘤细胞--内源性和外源性 与先天的抗PD-1抗性有关的适应，集中在以下几个方面： 肿瘤免疫微环境中转化生长因子β、骨形态发生蛋白或血管内皮生长因子的表达。 Lo Lab有整合临床和实验耐药性演变分析的记录，以得出 抑制耐药性的可翻译的临床前策略。我们建议(目标1)创造一个景观 通过与人类的比较分析展望表观基因组指导的肿瘤细胞本征耐药进化 黑色素瘤细胞系、患者来源的异种移植和免疫活性小鼠黑色素瘤模型。这一分析 寻求确定调节表型转换的主要转录因子，以提供对 抗性调节生长因子和信号通路。在目标2中，我们将检验MAPK 抑制剂通过调控特异性转化生长因子βS诱导适应性表观基因组和免疫抑制过程 BMPS。分析这些因素对肿瘤细胞和肿瘤微环境的作用， 分别通过鉴定涉及转化生长因子β/骨形态发生蛋白的顺式调控增强子或超级增强子模块 受体调节的smads和分离的肿瘤的单细胞rna-seq。在目标3中，我们将评估 阻断转化生长因子β、骨形态发生蛋白或血管内皮生长因子在MAPKIAPDL1基础上的疗效和机制 解剖最有效的组合对单个T细胞克隆型和表观遗传状态的影响 精力充沛或精疲力竭。综上所述，这些研究将推进我们对翻译的多方面理解 晚期黑色素瘤下一代联合疗法的耐药机制。

ABSTRACT Mutation-targeted small molecular inhibitors and immune checkpoint antibodies have extended the quality and quantity of life for patients with advanced MUTBRAF melanoma. Development of combinations based on these foundational therapies should yield further survival benefits in the near future. The combo of BRAF and MEK inhibitors specifically suppresses a form of resistance driven mainly by genetic alterations that result in MAPK- reactivation and restoration of MAPK-addiction. However, this clinically validated approach to suppress BRAF inhibitor resistance does not address epigenomic and immunologic alterations that drive resistance via MAPK- redundant and CD8 T-cell-depleted tumor states. We propose to uncover combinatorial targets in advanced V600BRAF mutant melanoma by understanding MAPK inhibitor-induced epigenomic and immune-suppressive mechanisms and their interplay. We hypothesize that MAPK inhibitor-induced tumor cell-intrinsic and extrinsic adaptations, which have been linked to innate anti-PD-1 resistance, converge on the elaboration and actions of TGFβ, BMP, or VEGFA in the tumor immune microenvironment. The Lo Lab has a track record of integrating analysis of clinical and experimental resistance evolution to derive translatable preclinical strategies to suppress resistance. We propose (Aim 1) to generate a landscape perspective of epigenome-directed tumor cell-intrinsic resistance evolution by comparative analysis of human melanoma cell lines, patient-derived xenografts and immune-competent murine melanoma models. This analysis seeks to identify master transcriptional factors regulating phenotypic transitions to provide insights into resistance-regulatory growth factors and signaling pathways. In Aim 2, we will test the hypothesis that MAPK inhibitors induce adaptive epigenomic and immune-suppressive processes via elaboration of specific TGFβs or BMPs. The action of these factors on the tumor cells and the tumor microenvironment will be analyzed, respectively, by characterization of cis-regulatory enhancer or super-enhancer modules involving TGFβ/BMP receptor-regulated SMADs and by single-cell RNA-seq of dissociated tumors. In Aim 3, we will evaluate the efficacy and mechanisms of blocking TGFβ, BMP, or VEGFA on top of the MAPKi+aPD-L1 foundation and dissect the influence of the most efficacious combination on single T-cell clonotypes and epigenetic states of activation or exhaustion. Together, these studies will advance translation of our understanding of multi-faceted resistance mechanisms into next-generation combinatorial therapies for advanced melanoma.