Targeting APOBEC3A-induced genetic heterogeneity and drug resistance in bladder cancer

项目概要/摘要 在美国，每五分钟就有一名新患者被诊断出患有尿路上皮癌（UC）， 每年有18,000名患者死亡。几乎所有的晚期UC患者都会对全身化疗产生耐药性。 治疗肿瘤内异质性（ITH）是治疗抵抗的主要原因，因为它增加了肿瘤的生长速度。 有机会出现耐药的亚克隆。然而，遗传性ITH目前是不可药物化的，并且不被考虑。 在治疗决策中，从而使耐药患者表型恶化。基础知识 关于ITH遗传驱动因素的差距阻碍了有效治疗策略的发展， 消除耐药性。我们的长期目标是确定ITH和治疗抵抗的靶向机制 制定有效的精确策略，以治愈晚期UC患者。总体目标 目的是确定APOBEC 3A介导的ITH驱动耐药性的靶向机制， 消除具有APOBEC 3A活性的UC细胞的策略。我们的中心假设是APOBEC 3A诱导的 胞苷脱氨驱动遗传性ITH，导致治疗抗性UC克隆的出现， 这样做，同时创造独特的有针对性的漏洞。这一假设是基于 基于我们已发表的工作和强有力的初步数据，显示APOBEC 3A在同基因UC细胞系中的表达 患者源性类器官驱动遗传性ITH。我们发现，APOBEC 3A诱导的，从头突变， PIKC 3A-AKT-MTOR信号传导中心驱动对Erdafitinib的耐药性，Erdafitinib是一种FGFR 3抑制剂（FGFR 3 i）， UC治疗。我们的初步数据还显示，APOBEC 3A诱导的双链DNA断裂是一个重要的机制。 优先修复的微同源介导的末端连接（MMEJ）途径和靶向 关键的MMEJ介体聚合酶θ（Polθ）在表达APOBEC 3A的克隆中是合成致死的。的 基本原理是，确定APOBEC 3A诱导的ITH驱动耐药性的靶向机制， 开发消除表达APOBEC 3A的UC细胞的策略将提高患者的治愈率。我们将 通过追求两个具体目标来验证我们的假设。目的1：确定APOBEC 3A- 诱导的突变ITH驱动UC中的治疗抗性。目标2：确定针对UC的合成致死策略 具有APOBEC 3A诱导的DNA双链断裂的肿瘤。Aim 1将使用纵向克隆条形码， 鉴定介导FGFR 3 i的靶向APOBEC 3A驱动的激酶枢纽的体外和体内实验室进展 并在来自FGFR 3 i临床试验的患者样品中验证它们。目标2将使用遗传和 Polθ在表达APOBEC 3A的UC模型中的药理学抑制作用和一项患者源性 UC类器官和异种移植物，以鉴定对APOBEC 3A-MMEJ合成致死性反应的临床生物标志物。 这种方法在概念和技术上都是创新的，为消除治疗创造了新的范例- 耐药性癌症影响：完成拟议的研究将建立APOBEC 3A作为遗传驱动因素， 治疗抗性，并使合成致命的方法，以提高治愈率。

PROJECT SUMMARY/ ABSTRACT Every five minutes, a new patient is diagnosed with urothelial carcinoma (UC) in the United States, resulting in the death of 18,000 patients annually. Nearly all patients with advanced UC will develop resistance to systemic treatment. Intratumoral heterogeneity (ITH) is a major contributor to treatment resistance by increasing the chance for resistant subclones to emerge. However, genetic ITH is currently not druggable and not considered in therapeutic decision-making, thus worsening drug-resistant patient phenotypes. The fundamental knowledge gap regarding genetic drivers of ITH impedes the development of effective therapeutic strategies to prevent and eliminate drug resistance. Our long-term goal is to define targetable mechanisms of ITH and treatment resistance to develop an effective precision strategy to achieve cures in patients with advanced UC. The overall objective is to define targetable mechanisms by which APOBEC3A-mediated ITH drives drug resistance and identify strategies to eliminate UC cells with APOBEC3A activity. Our central hypothesis is that APOBEC3A-induced cytidine deamination drives genetic ITH leading to the emergence of therapy-resistant UC clones and, in so doing, simultaneously creating unique targetable vulnerabilities. This hypothesis was formulated based on our published work and strong preliminary data showing that APOBEC3A expression in isogenic UC cell lines and patient-derived organoids drives genetic ITH. We found that APOBEC3A-induced, de novo mutations in the PIKC3A-AKT-MTOR signaling hub drive the resistance to erdafitinib, an FGFR3-inhibitor (FGFR3i) approved for UC treatment. Our preliminary data also revealed that APOBEC3A-induced double-stranded DNA breaks are preferentially repaired by the microhomology-mediated end-joining (MMEJ) pathway and that targeting the critical MMEJ mediator, polymerase theta (Polθ), is synthetically lethal in APOBEC3A-expressing clones. The rationale is that identifying targetable mechanisms by which APOBEC3A-induced ITH drives drug resistance and developing strategies to eliminate APOBEC3A-expressing UC cells will improve cure rates for patients. We will test our hypothesis by pursuing two specific Aims. Aim 1: Identify targetable mechanisms by which APOBEC3A- induced mutational ITH drives treatment resistance in UC. Aim 2: Identify synthetic lethal strategies to target UC tumors with APOBEC3A-induced DNA double-strand breaks. Aim 1 will use longitudinal clonal barcoding and in vitro and in vivo laboratory evolution to identify targetable APOBEC3A-driven kinase hubs that mediate FGFR3i resistance and validate them in patient samples from FGFR3i clinical trials. Aim 2 will use genetic and pharmacologic inhibition of Polθ in APOBEC3A-expressing UC models and a co-clinical trial of patient-derived UC organoids and xenografts to identify clinical biomarkers of response to APOBEC3A-MMEJ synthetic lethality. The approach is conceptually and technically innovative, creating a new paradigm for eliminating treatment- resistant cancers. Impact: Completion of the proposed research will establish APOBEC3A as a genetic driver of treatment resistance and enable synthetic lethal approaches to increase cure rates.