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

项目总结 以顺铂(Cis)为基础的新辅助化疗(Nac)是膀胱切除术前患者的标准护理。 患有肌肉浸润性膀胱癌(MIBC)。高达30%的患者有反应，并显示无肿瘤残留 膀胱切除术的存活率为80%，但无反应者有30%的机会存活5年。因此，改善 基于独联体的NAC的有效性将极大地改善卑诗省的成果。通过全基因组CRISPR-Cas9 在顺铂耐药的人BC细胞系中合成致死筛选，我们发现NPEPPS是一种新的可药物 其表达式确定对顺式的敏感度的目标。NPEPPS是13种M1氨基肽酶中唯一的一种 被发现是顺式合成致命性的。NPEPPS的耗竭增强顺式治疗和动物生长抑制 模特们。为了找出NPEPPS是如何驱动这两种表型的，我们使用质谱仪(MS)来鉴定 BC细胞中与NPEPPS形成复合体的蛋白质。我们在含有亚基的复合体(LRRC8A-)中发现了NPEPPS E)容量调节阴离子通道(Vrac)，这是最近发现的铂(铂)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 而耗尽则会产生相反的效果。支持人类BC生长的NPEPPS的表达与 无论是否使用化疗，患者的预后都很差。利用我们的MS结果，我们开发了一种方法 对在NPEPP复合体中发现的最可能影响生长的候选基因进行优先排序，这些基因与 侵袭性疾病，是预后的标志。因此，我们提出了NPEPPS的指导性假设 通过抑制vrac活性和与调控基因的相互作用促进BC的铂耐药和肿瘤生长 分别进行细胞增殖实验。具体的目的是检验这一假设，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的假设 利用酶促死亡的NPEPPS突变体进行铂抗性和生长所需的氨基肽酶活性 体外和体内。接下来，我们评估我们的头号候选基因CHD2，它是一种染色质调节基因，也是一种可能的肿瘤 抑制子在NPEPPS诱导的肿瘤生长中的作用及其对NPEPPS酶的依赖性 活动。在目标2中，我们将确定LRRC8A/D在NPEPPS介导的铂电阻中的作用。我们已经使用了 分子模拟鉴定NPEPP上与LRRC8A/D相互作用的残基 这些残基将检验NPEPPS与LRRC8A/D直接相互作用降低 在体外和体内，VRAC功能正常，并参与NPEPPS介导的铂耐药。建立 避开铂耐药的新型抗肿瘤药物效果的临床前理论基础，目标3将 测试Tosedostat(Tose)，一种临床上耐受性良好的氨基肽酶抑制剂，增强 BC对铂的敏感性，这需要NPEPPS表达。Tose对BC细胞±NPEPPS的影响 将检查在人BC模型中的生长、铂敏感性和体内导入情况 转移性疾病。在以顺式为基础的NAC之前，从患者肿瘤中提取的有机类物质将用铂±Tose进行测试。

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.