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

项目总结 进行了一项合成致命性筛查，以确定黑色素瘤的高度依赖性。 干旱基因的缺陷--SWI/SNF染色质重构体的组成部分--出现在20%-30%的 人类癌症。干旱突变黑色素瘤对H3K9-二甲基酶G9a，a 可下药的酶。这导致发现6例黑色素瘤病例存在复发性点突变 G9a催化区有相同的残基。突变使G9a过度激活，并与致癌基因相一致 EZH2是一种已知的致癌甲基转移酶，以不同的组蛋白-3赖氨酸为靶标 (K27)。除了这些罕见的激活G9a突变外，我们还发现了更常见的基因组G9a拷贝增加 TCGA中27.8%的黑色素瘤，并在人类中发现G9a依赖(通过遗传或药物手段) 黑色素瘤株只有一个额外的G9a拷贝。为了证实我们的结果，阿喀琉斯计划(基因组- 在许多深度注释的癌细胞系中进行规模功能丧失活性筛查)证实了G9a 存在G9a扩增或干旱缺陷的黑色素瘤细胞系之间的相关性。相同 在跟腱黑色素瘤以外的多种癌症中也发现了依赖性。此外，G9a的复制收益 伴随着H3K9二甲基化的全球升高，这作为G9a的生物标志物相关 G9a获得/扩增的黑色素瘤细胞的依赖性。机械论研究揭示了正则的 WNT通路是一个重要的依赖靶点，这是由G9a抑制Wnt拮抗剂Dkk1诱导的。 肿瘤固有的Wnt激活在黑色素瘤中很常见，尽管通常没有途径固有的突变-a 这一现象现在主要由G9a激活来解释。重要的是，WNT的活动被强烈地牵连起来 被其他人作为肿瘤诱导的免疫逃避的触发器--人们对免疫疗法的疗效非常感兴趣。这里, AIM 1将验证G9a拷贝获得和功能丧失的人黑色素瘤细胞系对 G9a、GLP、MITF、Dkk1和WNT信令。将使用全局ATAC-seq、RNA-seq和H3K9me2-Chip-seq 以确定这些黑色素瘤的其他G9a靶点和生存能力的介体。AIM 2将测试药理 包含G9a、GLP或ARID2复制增益的多个小鼠和斑马鱼模型中的G9a抑制 缺乏，结合BRAF靶向治疗或抗PD1免疫治疗。后一项研究将 利用含或不含紫外线新抗原的新型同基因工程化小鼠黑色素瘤， 允许测量G9a靶向作为钝化Wnt途径免疫抑制的策略 活性，以及直接靶向G9a的细胞内在致癌功能。人类黑色素瘤活检将 也被审问以证实临床前模型。这些研究发现了一种新的癌基因(G9a)， 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)，以及Wucherpfennig和RoDigg博士(免疫图谱)，将揭示机制，新颖 黑色素瘤的生物标记物和治疗机会，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.