Defining and targeting epigenetic plasticity-driven drug resistance and immune escape in melanoma

项目总结 黑色素瘤是一种异质性肿瘤，具有高度的表型可塑性，可以快速适应。 BRAF-MEK抑制剂治疗。最初的治疗反应通常之后是一段时间的静止期 这些信号被重新连接以逃避治疗，随后出现了真正的抵抗状态。小才是 已知最初的持久细胞状态如何转变为不可逆转的抵抗状态，免疫的作用 系统中这些血统的出现，以及这种抵抗状态是否可以被靶向。在预赛中 研究表明，I型组蛋白去乙酰基酶HDAC8是一种谱系转换的主要调节因子 耐药相关黑色素瘤细胞状态。HDAC8的激活改变黑色素瘤细胞的表型 MITF等谱系识别基因功能丧失，AP-1/TEAD转录因子活性增强 以及对治疗的抗拒。即使在去除靶向治疗后，耐药细胞状态仍然存在，并且 与突变负荷增加有关。HDAC8的过表达也导致HSP70的表达降低。 黑色素瘤抗原，免疫检查点蛋白水平增加，T细胞渗透减少。我们 我认为这种状态的稳定性可能至少部分是由于乙酰化和功能的改变而导致的表观遗传 转录因子、辅因子以及参与染色质环的粘附素复合体的功能改变， 和基因表达。我们假设HDAC8是耐药相关黑色素瘤的主要调节者。 细胞状态，并代表防止谱系转换的目标。在这项提案中，我们将使用多组学 定义HDAC8重新编程机制的方法(ATAC-Seq、scRNA-Seq、Hi-C、蛋白质组学) 并将确定HDAC8是否影响粘连蛋白复合体，从而导致固定的耐药 遗传或表观遗传变化。我们将利用BRAF/PTEN-HDAC8驱动的黑色素瘤小鼠模型 单细胞RNA-Seq研究HDAC8如何调节免疫微环境导致 出现耐药黑色素瘤谱系。我们将具体确定HDAC8是否会驱动 转录程序与MHC-I类分子和抗原表达减少相关 肿瘤-T细胞识别。进一步的研究将确定Jun介导的IL-11转录是否会重新编程 髓系隔间导致抑制黑色素瘤免疫环境，从而根除肿瘤反应 CD8T细胞。将进行CRISPR筛查，以确定防止出现抗药性的目标 小鼠模型中的血统。然后，我们将评估这些目标和HDAC8抑制剂作为改进策略 BRAF-MEK抑制剂和免疫检查点抑制剂对同基因小鼠的治疗反应 黑色素瘤模型。我们期待着对支撑这一现象的机制的新见解 黑色素瘤耐药谱系的维持将导致新的治疗策略的发展 改善对黑色素瘤免疫治疗和靶向治疗的反应深度和持续时间。我们的 这一发现将对多种癌症类型和许多不同的治疗方法产生重要影响。

Project summary Melanoma is a heterogeneous tumor, with a high degree of phenotypic plasticity that allows for rapid adaptation to BRAF-MEK inhibitor therapy. Initial therapeutic responses are typically followed by a period of quiescence in which signaling is rewired to evade therapy, followed by the emergence of a bona fide resistant state. Little is known about how the initial persister cell state transitions to an irreversibly resistant state, the role of the immune system in the emergence of these lineages, and whether this resistant state can be targeted. In preliminary studies we identified the type I histone deacetylase HDAC8 as a master regulator of a lineage switch to a resistance-associated melanoma cell state. Activation of HDAC8 alters the phenotype of melanoma cells with loss of function of lineage identity genes such as MITF, increased activity of AP-1/TEAD transcription factors and resistance to therapy. The resistant cell state persisted even after removal of targeted therapy, and was associated with increased mutational load. HDAC8 overexpression also led to the reduced expression of melanoma antigens, increased levels of the immune checkpoint proteins and reduced T cell infiltration. We believe the stability of this state may be at least in part be epigenetic due to altered acetylation and function of transcription factors, cofactors as well as altered function of the cohesin complex involved in chromatin looping, and gene expression. We hypothesize that HDAC8 is a master regulator of a resistance-associated melanoma cell state and represents a target for the prevention of lineage switching. In this proposal, we will use multi-omics approaches (ATAC-Seq, scRNA-Seq, Hi-C, proteomics) to define the mechanisms by which HDAC8 reprograms melanoma cells, and will determine if HDAC8 affects the cohesin complex, leading to fixed resistance-conferring genetic or epigenetic changes. We will utilize a mouse model of BRAF/PTEN-HDAC8-driven melanoma and single cell RNA-Seq to investigate how the HDAC8 modulates the immune microenvironment leading to the emergence of drug resistant melanoma lineages. We will specifically determine whether HDAC8 drives a transcriptional program associated with decreased MHC class I and antigen expression leading to decreased tumor-T cell recognition. Further studies will define whether JUN-mediated transcription of IL-11 reprograms the myeloid compartment leading to a suppressive melanoma immune environment that eradicates tumor-reactive CD8 T cells. CRISPR screens will be performed to identify targets to prevent the emergence of drug resistant lineages in mouse models. We will then evaluate these targets and HDAC8 inhibitors as strategies to improve therapeutic responses to BRAF-MEK inhibitor and immune checkpoint inhibitor therapy in syngeneic mouse melanoma models. We expect that new insights into the mechanisms underpinning the emergence and maintenance of drug resistant lineages in melanoma will lead to the development of new therapeutic strategies to improve the depth and duration of responses to immunotherapy and targeted therapy in melanoma. Our findings will have important implications across multiple cancer types and many different therapies.