NPEPPS is a novel and druggable determinant of chemotherapy resistance in bladder cancer

项目摘要 顺铂（Cis）为基础的新辅助化疗（NAC）是化疗前的标准治疗， 肌肉浸润性膀胱癌（MIBC）高达30%的患者反应，并显示无残留肿瘤， 在一些实施方案中，“无应答者”的存活率> 80%，但“无应答者”的存活率<30%。从而改善 基于CIS的NAC的有效性将大大改善BC的结果。通过全基因组CRISPR-Cas9 在顺式抗人BC细胞系的合成致死筛选中，我们发现NPEPPS是一种新的可药用的 其表达决定对顺式的敏感性的靶标。NPEPPS是13种M1氨基肽酶中唯一的一种 被发现与顺式异构体合成致死NPEPPS的耗尽增强了Cis治疗并降低了动物的生长 模型为了找到NPEPPS如何驱动这两种表型，我们使用质谱法（MS）来鉴定NPEPPS。 在BC细胞中与NPEPPS复合的蛋白质。我们发现NPEPPS与亚基（LRRC8A-1）复合。 E）容量调节阴离子通道（VRAC），最近确定的铂（Pt）细胞的机制， 导入.在BC细胞中，LRRC8A/D缺失增加了对顺铂和卡铂的耐药性，而NPEPPS缺失增加了对顺铂和卡铂的耐药性。 消耗具有相反的效果。支持在人类BC生长中的作用，NPEPPS表达与 无论是否使用化疗，患者结局均较差。利用我们的MS结果，我们开发了一种方法， 优先考虑与NPEPPS复合的候选基因，这些基因最有可能影响生长，与 侵袭性疾病，并且是预后标志物。因此，我们提出了指导假设，即NPEPPS 通过抑制VRAC活性并与调控基因相互作用， 细胞增殖。具体目的检验这一假设的目的是为 改善BC患者预后的新方法。在目标1中，我们将检验NPEPPS 氨肽酶活性是Pt抗性和使用酶促死亡的NPEPPS突变体生长所必需的。 体外和体内。接下来，我们评估我们的首选候选基因CHD2，一个染色质调节因子和假定的肿瘤 NPEPPS抑制剂在NPEPPS驱动的肿瘤生长中的作用以及这种作用对NPEPPS酶的依赖性 活动在目的2中，我们将确定LRRC8A/D在NPEPPS介导的Pt抗性中的作用。我们已经使用 分子建模以鉴定NPEPPS上与LRRC8A/D相互作用的残基。的定点诱变 这些残基将检验NPEPPS与LRRC8A/D的直接相互作用降低了NPEPPS与LRRC8A/D的相互作用的能力的假设。 VRAC正常发挥功能，并有助于体外和体内NPEPPS介导的Pt耐药性。建立 避免铂耐药性的新型抗肿瘤药物的临床前原理，目标3将 检验Tosedostat（Tose），一种临床耐受性良好的氨肽酶抑制剂， BC对Pt的敏感性，并且NPEPPS表达为此是必需的。Tose对BC细胞± NPEPPS的影响 将在局部和局部的人BC模型中检查表达的生长、Pt敏感性和体内输入， 转移性疾病将使用Pt ± Tose检测在基于顺式NAC之前来源于患者肿瘤的类器官。

PROJECT SUMMARY Cisplatin (Cis)-based Neoadjuvant chemotherapy (NAC) is the standard of care prior to cystectomy, for patients with muscle-invasive bladder cancer (MIBC). Up to 30% of patients respond and show no residual tumor at cystectomy with >80% survival, but “non responders” have <30% chance of surviving 5 years. Thus, improving the effectiveness of Cis-based NAC will greatly improve outcomes in BC. Through whole-genome CRISPR-Cas9 synthetic lethal screens in Cis-resistant human BC cell lines, we discovered NPEPPS as a novel and druggable target whose expression determines sensitivity to Cis. NPEPPS was the only one of 13 M1 aminopeptidases found to be synthetic lethal with Cis. Depletion of NPEPPS enhanced Cis therapy and reduced growth in animal models. To find how NPEPPS drives these two phenotypes, we used mass spectrometry (MS) to identify the proteins that are in complex with NPEPPS in BC cells. We found NPEPPS in complex with subunits (LRRC8A- E) of the volume regulated anion channel (VRAC), a recently identified mechanism of platinum (Pt) cellular import. In BC cells, LRRC8A/D depletion increases resistance to both cisplatin and carboplatin, while NPEPPS depletion had the opposite effect. Supporting a role in human BC growth, NPEPPS expression is associated with poor patient outcome regardless of chemotherapy use. Leveraging our MS results, we developed an approach to prioritize candidate genes found in complex with NPEPPS that most likely affect growth, are associated with aggressive disease, and are prognostic markers. Thus, we propose the Guiding Hypothesis that NPEPPS drives Pt resistance and tumor growth in BC by inhibiting VRAC activity and interacting with genes regulating cell proliferation respectively. Specific Aims test this hypothesis with the Objective to lay the foundation for novel approaches to improve the outcomes for BC patients. In Aim 1 we will test the hypothesis that NPEPPS aminopeptidase activity is required for Pt resistance and growth using enzymatically dead NPEPPS mutants in vitro and in vivo. Next, we evaluate our top candidate gene CHD2, a chromatin regulator and putative tumor suppressor for its role in NPEPPS-driven tumor growth and the dependency of this role on NPEPPS enzymatic activity. In Aim 2 we will determine the role of LRRC8A/D in NPEPPS-mediated Pt resistance. We have used molecular modeling to identify residues on NPEPPS that interact with LRRC8A/D. Site-directed mutagenesis of these residues will test the hypothesis that direct interaction of NPEPPS with LRRC8A/D reduces the ability of VRACs to properly function and contributes to NPEPPS-mediated Pt resistance in vitro and in vivo. To establish the preclinical rationale for the effect of novel, anti-neoplastic agents that circumvent Pt resistance, Aim 3 will test the hypothesis that Tosedostat (Tose), a clinically well-tolerated aminopeptidase inhibitor, enhances the sensitivity of BC to Pt, and NPEPPS expression is required for this. The impact of Tose on BC cells ± NPEPPS expression will be examined for growth, Pt sensitivity and import in vivo in human BC models of localized and metastatic disease. Organoids derived from patient tumors before Cis-based NAC, will be tested with Pt ± Tose.