Project 1: Remodeling Chromatin and the Tumor Microenvironment: Direct Oncogenesis and Therapeutic Targeting

项目摘要 进行了一项综合致死性筛选，以确定黑色素瘤中的高度依赖性， ARID基因缺陷-SWI/SNF染色质重塑的组成部分-发生在20-30%的 人类癌症ARID突变型黑色素瘤表现出对H3 K9-二甲基酶G9 a的极端依赖性， 药物酶这导致发现了6例黑色素瘤病例， G9 a催化结构域中相同残基。该突变过度激活G9 a并与致癌基因 EZH 2中的突变，EZH 2是一种先前已知的致癌甲基转移酶，靶向不同的组蛋白-3赖氨酸 （K27）。除了这些罕见的激活G9 a突变，我们还发现了更多常见的基因组G9 a拷贝增加。 27.8%的黑色素瘤在TCGA中发现G9 a依赖性（通过遗传或药理学手段） 只有一个额外的G9 a拷贝的黑色素瘤细胞。为了证实我们的结果，阿基里斯项目（基因组- 在许多深度注释的癌细胞系中进行的大规模功能丧失活力筛选）证实了G9 a 具有G9 a扩增或ARID缺陷的黑素瘤细胞系之间的依赖性。相同的 依赖性也见于除跟腱黑色素瘤以外的多种癌症中。此外，G9 a拷贝增益 伴随着H3 K9二甲基化的全球升高，这与G9 a的生物标志物相关 G9 a依赖性获得/扩增的黑素瘤细胞。机制研究揭示了典型的激活 Wnt通路作为重要的依赖性靶点，其由G9 a抑制Wnt拮抗剂DKK 1诱导。 肿瘤内源性Wnt激活在黑色素瘤中很常见，尽管通常没有途径内源性突变， 这一现象现在主要由G9 a激活来解释。重要的是，Wnt活性与 被其他人认为是肿瘤诱导的免疫逃避的触发因素，这对免疫治疗的功效非常感兴趣。在这里， 目的1将验证G9 a拷贝获得和ARID功能丧失的人黑色素瘤细胞系对以下的依赖性： G9 a、GLP、MITF、DKK 1和Wnt信号传导。将使用全局ATAC-seq、RNA-seq和H3 K9 me 2-ChIP-seq 以确定这些黑色素瘤中的其他G9 a靶点和存活介质。目标2将测试药理学 在含有G9 a或GLP或ARID 2拷贝增益的多种小鼠和斑马鱼模型中的G9 a抑制 缺乏，与BRAF靶向治疗或抗PD 1免疫治疗相结合。后一项研究将 利用含有或缺乏UV-新抗原的新的同基因工程化的同基因鼠黑素瘤， 允许测量G9 a靶向作为钝化Wnt途径免疫抑制的策略， 活性，以及直接靶向G9 a的细胞内在致癌功能。人类黑色素瘤活组织检查 也被询问以证实临床前模型。这些研究鉴定了一种新的癌基因（G9 a）， 通过与Zon博士（斑马鱼黑色素瘤模型）、Liu博士和Zon博士（表观遗传学）的重要合作， 机制），Wucherpfennig和Rodig博士（免疫学分析），将揭示机制，新的 生物标志物和黑色素瘤的治疗机会，与其他耐药癌症相关。

PROJECT SUMMARY A synthetic lethality screen was performed to identify hyper-dependencies in melanomas harboring deficiencies in ARID genes—components of the SWI/SNF chromatin remodelers—which occur in 20-30% of human cancers. ARID mutant melanomas exhibited extreme dependency on the H3K9-dimethylase G9a, a druggable enzyme. This led to discovery of 6 melanoma cases harboring recurrent point mutation at an identical residue in G9a’s catalytic domain. The mutation hyper-activates G9a and aligns to oncogenic mutations in EZH2, a previously known oncogenic methyltransferase that targets a different histone-3 lysine (K27). Beyond these rare activating G9a mutations we identified more common genomic G9a copy gains in 27.8% of melanomas in TCGA and found G9a dependency (via genetic or pharmacologic means) in human melanoma lines with as little as one extra G9a copy. Corroborating our results, the Achilles Project (genome- scale loss-of-function viability screening in many deeply annotated cancer cell lines) confirmed G9a dependency among melanoma lines harboring either G9a amplification or ARID deficiency. The same dependencies were also seen in multiple cancers beyond melanoma in Achilles. Furthermore, G9a copy gain was accompanied by global elevations in H3K9 dimethylation, which correlated as a biomarker of G9a dependency in G9a gained/amplified melanoma cells. Mechanistic studies revealed activation of the canonical Wnt pathway as a vital dependency target, which is induced by G9a’s suppression of Wnt antagonist DKK1. Tumor intrinsic Wnt activation is common in melanomas, though usually without pathway-intrinsic mutations—a phenomenon now largely explained by G9a activation. Importantly, Wnt activity has been strongly implicated by others as a trigger of tumor-induced immune evasion—of keen interest for efficacy of immunotherapy. Here, Aim 1 will validate dependencies of G9a copy-gained and ARID loss-of-function human melanoma lines on G9a, GLP, MITF, DKK1, and Wnt signaling. Global ATAC-seq, RNA-seq, and H3K9me2-ChIP-seq will be used to identify additional G9a targets and mediators of viability in these melanomas. Aim 2 will test pharmacologic G9a inhibition in multiple mouse and zebrafish models containing copy gains of G9a or GLP, or ARID2 deficiency, combined with either BRAF-targeted therapy or anti-PD1 immunotherapy. The latter study will utilize novel isogenic engineered syngeneic murine melanomas containing or lacking UV-neoantigens, permitting measurements of G9a targeting as a strategy to blunt the Wnt pathway’s immunosuppressive activity, together with direct targeting of G9a’s cell-intrinsic oncogenic function. Human melanoma biopsies will also be interrogated to corroborate preclinical models. These studies identify a novel oncogene (G9a) and, through vital collaborations with Dr. Zon (zebrafish melanoma model), Drs. Liu and Zon (epigenetic mechanisms), and Drs. Wucherpfennig and Rodig (immunological profiling), will reveal mechanisms, novel biomarkers, and therapeutic opportunities in melanoma, with relevance to other treatment-resistant cancers.