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

摘要 靶向突变的小分子抑制剂和免疫检查点抗体已经延长了质量， 晚期MUTBRAF黑色素瘤患者的生活质量。在此基础上开发组合 基础治疗在不久的将来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主要由导致MAPK的遗传改变驱动的抗性形式， MAPK成瘾的再激活和恢复。然而，这种临床验证的抑制BRAF的方法， 抑制剂耐药性并不解决通过MAPK驱动耐药性的表观基因组和免疫学改变， 冗余和CD 8 T细胞耗尽的肿瘤状态。我们建议在先进的技术中发现组合目标， V600 BRAF突变黑色素瘤通过理解MAPK通路诱导的表观基因组和免疫抑制 机制及其相互作用。我们假设MAPK通路诱导的肿瘤细胞内源性和外源性 与先天性抗PD-1抗性有关的适应，集中在以下方面的阐述和行动上： TGFβ、BMP或VEGFA在肿瘤免疫微环境中的作用。 Lo Lab在整合临床和实验耐药性演变分析方面有着良好的记录， 可转化的临床前策略来抑制耐药性。我们建议（目标1）生成一个景观 通过比较分析人类表观基因组指导的肿瘤细胞内在抗性进化的前景 黑色素瘤细胞系、患者来源的异种移植物和免疫活性鼠黑色素瘤模型。该分析 旨在确定调节表型转换的主转录因子， 抗性调节生长因子和信号通路。在目标2中，我们将检验MAPK 抑制剂诱导适应性表观基因组和免疫抑制过程，通过加工特定的TGFβs或 BMP。将分析这些因素对肿瘤细胞和肿瘤微环境的作用， 分别通过表征涉及TGFβ/BMP的顺式调节增强子或超级增强子模块 受体调节的SMAD和解离肿瘤的单细胞RNA-seq。在目标3中，我们将评估 在MAPKi+aPD-L1基础上阻断TGFβ、BMP或VEGFA的疗效和机制， 剖析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， 激活或耗尽。这些研究将共同推进我们对翻译的多方面认识 耐药机制的新一代组合疗法用于晚期黑色素瘤。

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.