Therapeutic targeting of p53 reactivation-induced OXPHOS dependency and stress responses to overcome resistance to venetoclax/HMA in AML

治疗靶向 p53 重新激活诱导的 OXPHOS 依赖性和应激反应，以克服 AML 中对 Venetoclax/HMA 的耐药性

• 批准号: 10550265
• 负责人: MICHAEL ANDREEFF
• 金额: $ 22.27万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-13 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10550265
• 关键词: Acute Myelocytic Leukemia; Address; Apoptosis; Apoptosis Regulation Gene; Apoptotic; Azacitidine; BCL1 Oncogene; BCL2L1 gene; Biological Assay; Cell Death; Cell Death Induction; Cell Line; Cell Nucleus; Cells; Characteristics; Chronic; Clinical; Complex; Cytogenetics; Cytometry; Cytoplasm; Dacogen; Decitabine; Dependence; Diagnostic; Disease; Disease remission; Disease-Free Survival; Exhibits; FDA approved; FLT3 gene; Flow Cytometry; Genes; Hematopoietic Neoplasms; Hematopoietic System; In Vitro; Induction of Apoptosis; Intervention; MCL1 gene; MDM2 gene; Malignant Neoplasms; Measures; Mediating; Metabolic; Mitochondria; Modality; Modeling; Monitor; Nuclear; Nuclear Proteins; Oncogenes; Oxidative Phosphorylation; Pathway interactions; Patients; Prognosis; Protein Family; Proteins; Proteomics; Refractory; Regimen; Relapse; Reporting; Research; Residual state; Resistance; Respiratory Chain; Role; Sampling; Signal Transduction; Suppressor Mutations; TP53 gene; Therapeutic; Time; Translations; Transplantation; Tumor Suppressor Genes; Tumor Suppressor Proteins; Up-Regulation; activating transcription factor 4; acute myeloid leukemia cell; age related; biological adaptation to stress; clinical practice; combinatorial; demethylation; exportin 1 protein; high risk; improved; in vivo; inhibitor; leukemia; leukemia treatment; leukemic stem cell; metabolic profile; novel; overexpression; patient derived xenograft model; prognostic; relapse patients; relapse prevention; resistance factors; resistance mechanism; response; survival outcome; targeted treatment; therapeutic target; treatment strategy; ubiquitin-protein ligase

Project Summary/Abstract Given the persistently poor prognosis of acute myeloid leukemia (AML), diagnostic and therapeutic strategies need to be developed to achieve significantly improved cure rates. While initial response rates and event-free- survival have increased, most patients relapse and succumb to the disease. The regimen consisting of BCL-2 inhibitor venetoclax (VEN) in combination with a hypomethylating agent (HMA) (VEN/HMA) has revolutionized AML therapy with complete remission rates, now ranging from 45 to 90%, accompanied by prolonged survival. Notably, the underlying mechanism of action of VEN/HMA therapy resides in the inhibition of oxidative phosphorylation (OXPHOS), especially in AML leukemia stem cells (LSCs). However, the majority of patients receiving VEN/HMA eventually relapse, especially patients with high-risk characteristics including complex cytogenetics and aberrant RAS and FLT3 signaling. The role of p53 in AML cell death is poorly understood. We recently we reported that the inhibition of nuclear exporter XPO1 (CRM1) causes accumulation of p53 in the nuclei of AML cells, and that dual inhibition of the ubiquitin E3 ligase MDM2 and XPO1 substantially amplifies this activity leading to synergistic p53-mediated killing of AML cells, even of VEN/HMA resistant cells, in vitro and in vivo. After dual MDM2 and XPO1 inhibition, a small fraction of surviving AML cells expressed high levels of p21, a p53 target, LC3B, and the key integrated stress response (ISR) factor activated transcription factor 4 (ATF4), which should render the residual AML cells vulnerable to BCL-2 inhibition. Indeed, the triple combination of a MDM2, XPO1, and BCL-2 inhibitor resulted in the highest ATF4 protein levels and the deepest cytoreduction. Interestingly, we found highly increased protein levels of OXPHOS complexes in AML cells with acquired resistance to dual MDM2 and XPO1 inhibition in vivo suggesting OXPHOS activation. This finding provided the rationale for overcoming this resistance mechanism with VEN/HMA in combination. We hypothesized that 1) chronic p53 reactivation confers AML OXPHOS dependency thereby restoring sensitivity to VEN-based therapy; and 2) the concomitant combinatorial treatment of MDM2, XPO1 inhibitors with VEN/HMA efficiently suppresses AML and AML LSCs. The hypothesis will be examined with the following Specific Aims (SAs). In SA 1, we will investigate the functional dependency on OXPHOS in AML cells resistant to dual MDM2 and XPO1 inhibition. We will characterize the cellular responses and cell fates, at the single-cell level, using high-parametric flow cytometry and mass cytometry (CyTOF) for LSCs and blasts upon maximal p53 activation by dual inhibition of MDM2 and XPO1 with or without VEN/HMA, to assess apoptosis and other modes of regulated cell death at multiple time points. In SA 2, we will examine the anti- leukemia effects of dual MDM2 and XPO1 inhibition combined with VEN/HMA in AML and AML LSCs with wild-type p53. The successful completion of the proposed research should provide a novel treatment approach for VEN/HMA-resistant AML with non-genotoxic, targeted therapeutics.

项目摘要/摘要 鉴于急性髓系白血病(AML)预后持续不良，诊断和治疗策略 需要开发，以实现显著提高的治愈率。虽然初始响应率和无事件- 存活率增加了，大多数患者复发并死于这种疾病。Bc1-2为主的化疗方案 抑制剂万乃馨(VEN)与去甲基化药物(HMA)(VEN/HMA)的结合使其发生了革命性的变化 AML治疗的完全缓解率现在在45%到90%之间，并伴随着更长的生存时间。 值得注意的是，VEN/HMA治疗的潜在作用机制在于抑制氧化 磷酸化(OXPHOS)，特别是在急性髓系白血病干细胞(LSCs)中。然而，大多数患者 接受VEN/HMA的患者最终会复发，特别是具有包括复杂性在内的高危特征的患者 细胞遗传学与RAS和Flt3信号的异常。P53在AML细胞死亡中的作用还知之甚少。 我们最近报道，核出口蛋白XPO1(CRM1)的抑制导致细胞中P53的积聚。 AML细胞的细胞核，以及泛素E3连接酶MDM2和XPO1的双重抑制基本上 放大这种活性，导致协同P53介导的杀伤AML细胞，甚至是对VEN/HMA耐药细胞， 在体外和体内。在MDM2和XPO1双重抑制后，一小部分存活的AML细胞表达 高水平的p21、P53靶标、LC3B和关键的综合应激反应(ISR)因子被激活 转录因子4(ATF4)，这应该使残留的AML细胞容易受到bcl2的抑制。的确， MDM2、XPO1和bcl2抑制剂的三重组合导致最高的ATF4蛋白水平和 最深的细胞减少。有趣的是，我们发现OXPHOS复合体的蛋白质水平在 AML细胞在体内对MDM2和XPO1双重抑制具有获得性抗性，提示OXPHOS激活。 这一发现为联合使用VEN/HMA克服这种耐药机制提供了理论基础。 我们假设1)慢性P53的重新激活会导致AML OXPHOS依赖，从而恢复 对基于VEN的治疗的敏感性；以及2)MDM2、XPO1抑制剂的联合治疗 使用VEN/HMA可有效抑制AML和AML LSCs。这一假设将通过 遵循特定目标(SA)。在SA 1中，我们将研究AML中对OXPHOS的函数依赖 耐受MDM2和XPO1双重抑制的细胞。我们将描述细胞反应和细胞命运，在 单细胞水平，使用高参数流式细胞术和质量细胞术(CyTOF)检测LSC和原始细胞 在有或没有VEN/HMA的情况下，通过双重抑制MDM2和XPO1来最大限度地激活P53，以评估 多个时间点的细胞凋亡和其他调节细胞死亡的方式。在SA 2中，我们将检查反- MDM2和XPO1双重抑制联合VEN/HMA对AML和AML LSCs的白血病作用 野生型P53。拟议研究的成功完成应提供一种新的治疗方法 对于VEN/HMA耐药的AML，采用无遗传毒性的靶向治疗。