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

NPEPPS 是膀胱癌化疗耐药的新型可药物决定因素

• 批准号: 10662535
• 负责人: James Christopher Costello
• 金额: $ 49.16万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10662535
• 关键词: Adjuvant Chemotherapy; Affect; Aminopeptidase; Animal Model; Anions; Antineoplastic Agents; Automobile Driving; Back; Binding; Biochemical; Biological Assay; Biophysics; Bladder; CRISPR screen; CRISPR/Cas technology; Cancer Model; Cancer Patient; Cancer cell line; Candidate Disease Gene; Carboplatin; Cell Proliferation; Cells; Chemoresistance; Chromatin; Cisplatin; Clinical; Combination Drug Therapy; Complex; Coupled; Cystectomy; Data; Dependence; Disease; Drug resistance; Effectiveness; Excision; Foundations; Future; Genes; Growth; Human; Impairment; In Vitro; Kidney; Malignant neoplasm of urinary bladder; Mass Spectrum Analysis; Mediating; Mediator; Mutate; Neoadjuvant Therapy; Organoids; Outcome; Patient-Focused Outcomes; Patients; Pharmaceutical Preparations; Phenotype; Physiological; Platinum; Pre-Clinical Model; Prognostic Factor; Prognostic Marker; Proteins; Regulation; Residual Neoplasm; Resistance; Role; Site; Site-Directed Mutagenesis; Testing; Translations; Tumor Suppressor Proteins; cancer cell; cell growth; cellular imaging; chemotherapy; design; druggable target; improved; improved outcome; in vivo; inhibitor; knock-down; member; molecular modeling; muscle invasive bladder cancer; mutant; novel; novel strategies; pharmacologic; pre-clinical; preclinical evaluation; puromycin-sensitive aminopeptidase; reconstitution; response; standard of care; therapeutic evaluation; therapeutic target; translational therapeutics; tumor; tumor growth; whole genome

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.

项目概要 基于顺铂 (Cis) 的新辅助化疗 (NAC) 是膀胱切除术之前患者的标准治疗 患有肌层浸润性膀胱癌（MIBC）。高达 30% 的患者有反应并且在 膀胱切除术的存活率 >80%，但“无反应者”5 年存活率 <30%。因此，改善 基于顺式 NAC 的有效性将大大改善 BC 的治疗结果。通过全基因组 CRISPR-Cas9 在顺式抗性人 BC 细胞系中进行合成致死筛选，我们发现 NPEPPS 是一种新型且可药物化的药物 其表达决定对顺式敏感性的靶标。 NPEPPS 是 13 种 M1 氨肽酶中唯一的一种 发现与顺式合成致死。 NPEPPS 的消耗增强了顺式治疗并减少了动物的生长 模型。为了了解 NPEPPS 如何驱动这两种表型，我们使用质谱 (MS) 来识别 BC 细胞中与 NPEPPS 复合的蛋白质。我们发现 NPEPPS 与亚基 (LRRC8A- E) 容量调节阴离子通道 (VRAC)，这是最近发现的铂 (Pt) 细胞机制 进口。在 BC 细胞中，LRRC8A/D 缺失会增加对顺铂和卡铂的耐药性，而 NPEPPS 耗尽会产生相反的效果。 NPEPPS 表达与人类 BC 生长相关 无论使用何种化疗，患者预后均不佳。利用我们的 MS 结果，我们开发了一种方法 优先考虑与 NPEPPS 复合物中发现的最有可能影响生长的候选基因，这些基因与 侵袭性疾病，并且是预后标志物。因此，我们提出指导性假设：NPEPPS 通过抑制 VRAC 活性并与调节基因相互作用来驱动 BC 中的 Pt 抗性和肿瘤生长 分别是细胞增殖。具体目标检验这一假设，目的是为以下目标奠定基础： 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 氨肽酶活性是 Pt 抗性和使用酶促死亡 NPEPPS 突变体生长所必需的 体外和体内。接下来，我们评估我们的顶级候选基因 CHD2，一种染色质调节因子和假定的肿瘤 抑制其在 NPEPPS 驱动的肿瘤生长中的作用以及该作用对 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 Pt 耐药性中的作用。我们已经用过 分子建模来识别 NPEPPS 上与 LRRC8A/D 相互作用的残基。定点诱变 这些残基将检验以下假设：NPEPPS 与 LRRC8A/D 的直接相互作用会降低 VRAC 在体外和体内正常发挥作用并有助于 NPEPPS 介导的 Pt 耐药性。建立 规避 Pt 耐药性的新型抗肿瘤药物的临床前基本原理，目标 3 将 检验托塞多司他 (Tose)（一种临床耐受性良好的氨肽酶抑制剂）可增强 BC 对 Pt 的敏感性，为此需要 NPEPPS 表达。 Tose 对 BC 细胞的影响 ± NPEPPS 将在局部和局部的人类 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。在使用顺式 NAC 之前，源自患者肿瘤的类器官将使用 Pt ± Tose 进行测试。