P2: CTNNB1 Mutation and Wnt Pathway Activation Define Clinically Aggressive Endometrioid Endometrial Carcinoma

摘要 子宫内膜样癌(EEC)约占所有子宫内膜的75% 癌症是美国女性第四常见的癌症。许多早期和低血压病患者 级别EEC仅通过手术即可治愈，但对于出现较高级别、晚期EEC的女性， 需要更积极的治疗方法来控制这种疾病。来自TCGA数据(并从我们的 我们已经确定了四种不同临床病理类型的EEC转录组亚型 特征和突变谱。II组由患有低度EEC的较年轻、肥胖的患者组成 存活率下降。虽然簇II肿瘤的总体突变率最低，但CTNNB1外显子3 突变是非常常见的。这些突变与Wnt/β-连环蛋白信号的激活有关 路径。我们假设CTNNB1外显子3的突变重新编程了导致 临床上具有侵袭性的EEC。对这些突变的了解将更好地为针对 WNT途径包括细胞周期蛋白D和CDK4。为了检验这一假设，我们提出了以下具体目标。 具体目标1.检验CTNNB1外显子3突变改变细胞表观遗传程序的假设，从而 抑制激素基因表达程序并激活间充质/神经元和 免疫抑制基因表达计划。1.1.通过对7个热点突变体进行功能鉴定 建立表达每个突变体的等基因稳定系。1.2.描述监管网络的特点 通过转录组和miRNA对稳定品系的突变CTNNB1进行分析，并初步鉴定目标基因 关注ESR1、PGR、N-钙粘蛋白、PDGFA、WNT5A、WNT5B、IL-10和TGFB2。1.3.确定 N-钙粘蛋白、WNT5A和WNT5B可能通过siRNA获得或丧失功能而发挥驱动作用 (单独或组合)，并检查相关的体外细胞终点。具体目标2.确立 验证CTNNB1激活突变是重要驱动因素假说的临床前和临床模型 关于肿瘤发生的研究。2.1.评价CTNNB1基因突变的体内效应。2.2.利用3D体外系统 测试针对Wnt/β-连环蛋白信号的替代疗法。2.3.确定CTNNB1突变是否促进 子宫切除标本中免疫抑制微环境的形成。具体目标3. 进行核糖核酸库(诺华CDK4/6抑制剂)、来曲唑和 维拉莫司治疗晚期/复发性食道癌。我们先前已经证明来曲唑+伊波利莫斯对 这些患者的子集。细胞周期蛋白D1是CTNNB1突变子宫内膜中诱导率最高的蛋白之一 它与CDK4/6相互作用，促进细胞周期进程。我们假设患有这种疾病的患者 携带CTNNB1基因突变的肿瘤会有更高的Cyclin D1表达，因此反应更灵敏 用这种组合进行治疗。这项试验将丰富CTNNB1突变肿瘤患者的研究。

Abstract Endometrioid-type endometrial carcinoma (EEC) accounts for approximately 75% of all endometrial carcinomas, the fourth most common cancer in women in the US. Many patients with early stage and low grade EEC will be cured by surgery alone but for women who present with higher grade, advanced stage EEC, more aggressive therapeutics are needed to control the disease. From TCGA data (and validated from our own patients) we have identified four transcriptome subtypes of EEC with distinct clinicopathologic characteristics and mutation spectra. Cluster II consists of younger, obese patients with low grade EEC yet diminished survival. Although the Cluster II tumors had the lowest overall mutation rate, CTNNB1 exon 3 mutations were very common. These mutations were associated with activation of the Wnt/β-catenin signaling pathway. We hypothesize that mutations in exon 3 of CTNNB1 reprogram the molecular landscape leading to clinically aggressive EEC. Understanding of these mutations will better inform specific strategies targeting the Wnt pathway including cyclin D and CDK4. The following specific aims are proposed to test this hypothesis. Specific Aim 1. Test the hypothesis that exon 3 mutations of CTNNB1 alter cellular epigenetic programs, thus suppressing the hormonal gene expression program and activating a mesenchymal/neuronal and immunosuppressive gene expression programs. 1.1. Functionally characterize the 7 hot spot mutants by establishing isogenic stable lines expressing each of the mutants. 1.2. Characterize the regulatory network of mutant CTNNB1 by transcriptome and miRNA profiling of the stable lines and identify the target genes, initially focusing on ESR1, PGR, N-cadherin, PDGFA, WNT5A, WNT5B, IL-10, and TGFB2. 1.3. Determine the possible driver effect of N-cadherin, WNT5A, and WNT5B by gain of function or loss of function via siRNA (individually or in combinations) and examining relevant in vitro cellular endpoints. Specific Aim 2. Establish preclinical and clinical models to test the hypothesis that activating mutations of CTNNB1 are important drivers of tumorigenesis. 2.1. Evaluate the effects of CTNNB1 mutation in vivo. 2.2. Utilize a 3D in vitro system to test alternative therapeutics targeting Wnt/β-catenin signaling. 2.3. Determine if CTNNB1 mutation promotes the formation of an immunosuppressive microenvironment in hysterectomy specimens. Specific Aim 3. Conduct a phase II, single arm therapeutic trial of ribociclib (Novartis CDK4/6 inhibitor), letrozole, and everolimus for advanced/recurrent EEC. We have previously shown that letrozole+everolimus is effective in a subset of these patients. Cyclin D1 is one of the highest induced proteins in CTNNB1-mutated endometrial carcinomas, and it interacts with CDK4/6 to promote cell cycle progression. We hypothesize that patients with carcinomas with CTNNB1 mutation will have higher expression of Cyclin D1 and therefore be more responsive to treatment with this combination. The trial will be enriched for patients with tumors with CTNNB1 mutations.