Estrogen receptor fusions genes as drivers of endocrine resistance in breast cancer

抗雌激素治疗是防治雌激素受体(ER、ESR1)阳性乳腺的关键 癌症，但在多达一半的病例中会出现治疗抵抗，从而导致无法治愈的晚期疾病。我们 最近报道的伴有配体丢失的晚期内分泌抵抗乳腺癌患者复发的ESR1融合 结合代表了耐药的典型模型。有趣的是，同时发生了ESR1融合的病例 ESR1碱基对突变，可能指向长期抗雌激素下的趋同进化 心理治疗。我们小组和其他人的证据表明，在~10%-20%的晚期患者中发现了ESR1融合 然而，许多ESR1融合基因含有不同的3‘伴侣基因，这使得检测具有挑战性。 为了确定患病率，我们将全面重新分析转移数据中的ESR1基因组位点 乳腺癌。此外，我们还开发和优化了一种靶捕获测序方法，该方法能够 在肿瘤活检和ctDNA中综合检测ESR1融合。我们已经确定了内源性 在两个患者来源的异种移植(PDX)(ESR1-LPP和ESR1-NFkB)中表达的ESR1融合 ER阳性乳腺癌细胞系MDA-MB-435(ESR1-SYNE1)。我们将在最近表达ESR1融合 生成的患者衍生的有机化合物(PDO)，显示ER表达和对雌激素的反应相等 或者比传统的细胞系更大。有趣的是，不同的ESR1融合显示出明显的活性差异， 暗示3‘基因伴侣在调节ESR1 N末端结构域(NTD)的活性中起作用。NTD 是反式激活区域内的一种本质上无序的蛋白质，并使用了一种新的整合生物物理学 方法，我们最近报告说，虽然它大多是无序的，但它可以采用意想不到的紧凑 构象，进而指导与协调控子的相互作用。在这项提议中，我们试图检验这一假设 ESR1融合是内分泌抵抗和乳腺癌死亡率的关键媒介。我们将聘用 丰富以确定ESR1融合(和点突变)在国内和国际临床中的流行率 晚期内分泌抵抗型乳腺癌的队列和在治疗期间出现时的监测 纵向收集血浆和ctDNA分析。我们将使用患者衍生的乳房器官模型 癌症以比较和对比ESR1融合和点突变的活性和功能相对于野生- 键入ER并将其转化为体内患者衍生的异种器官移植(PDOX)和(PDX)模型以评估 内分泌抵抗。我们将研究其作用机制，包括对DNA结合和 转录活性，以及3‘伴侣基因在任何功能活动获得中的作用。最后，我们会 研究ESR1融合的结构-功能，重点放在NTD上，并确定在 在评估特定NTD氨基酸的功能之前，融合伙伴的背景以及它们如何 改变结构和随后的靶基因表达。我们相信我们的多学科研究将提供 帮助开发ESR1融合乳腺癌新疗法的机械论洞察力。

Anti-estrogen therapy is key to the prevention and treatment of estrogen receptor (ER, ESR1) positive breast cancer, but therapy resistance occurs in up to a half of cases resulting in incurable advanced disease. We recently reported recurrent ESR1 fusions in advanced endocrine-resistant breast cancers, with loss of ligand binding representing a canonical model of drug resistance. Intriguingly, cases with ESR1 fusions co-occurred with ESR1 base-pair mutations, likely pointing towards convergent evolution under prolonged anti-estrogen therapy. Evidence from our group and others suggest that ESR1 fusions are found in ~10-20% of advanced breast cancers, however, many ESR1 fusions harbor different 3’ partner genes, making detection challenging. To determine prevalence, we will comprehensively re-analyze the ESR1 genomic locus in data from metastatic breast cancers. Further, we have developed and optimized a target capture sequencing assay capable of comprehensively detecting ESR1 fusions in both tumor biopsies and ctDNA. We have identified endogenous ESR1 fusions that are expressed in two patient derived xenografts (PDX) (ESR1-LPP and ESR1-NFkB) and in an ER-positive breast cancer cell line MDA-MB-435 (ESR1-SYNE1). We will express ESR1 fusions in recently generated patient-derived organoids (PDO) which show ER expression and response to estrogen that is equal or greater than traditional cell lines. Intriguingly, different ESR1 fusions show stark differences in activity, implicating a role for the 3’ gene partner in modulating activity of the ESR1 N terminal domain (NTD). The NTD is an intrinsically disordered protein within the transactivation domain, and using a novel integrative-biophysics approach, we recently reported that while it is mostly disordered, it can adopt an unexpectedly compact conformation which in turn directs interaction with coregulators. In this proposal we seek to test the hypothesis that ESR1 fusions are key mediators of endocrine-resistance and breast cancer mortality. We will employ EnRich to determine the prevalence of ESR1 fusions (and point mutations) in national and international clinical cohorts of advanced endocrine resistant breast cancer and monitor when they arise during therapy using longitudinal collection of plasma and ctDNA analyses. We will use patient-derived organoid models of breast cancer to compare and contrast the activity and function of ESR1 fusions and point mutations relative to wild- type ER and translate this into in vivo patient derived organoid xenograft (PDOX) and (PDX) models to assess endocrine resistance. We will study mechanism of action, including the effects on DNA binding and transcriptional activity, and the role of the 3’ partner gene in any gain of function activities. Finally, we will examine the structure-function of ESR1 fusions focusing on the NTD and determining the altered structure in the context of the fusion partner before assessing the function of specific NTD amino acids and how they change structure and subsequent target gene expression. We believe our multi-disciplinary studies will provide mechanistic insight to aid in the development of new therapies against breast cancers with ESR1 fusions.