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: 10356325
• 负责人: MICHAEL ANDREEFF
• 金额: $ 18.93万
• 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 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10356325
• 关键词: Acute Myelocytic Leukemia; Address; Apoptosis; Apoptosis Regulation Gene; Apoptotic; Azacitidine; BCL1 Oncogene; BCL2 gene; BCL2L1 gene; Biological Assay; Cell Death; 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; Intervention; MCL1 gene; MDM2 gene; Malignant Neoplasms; Measures; Mediating; Metabolic; Mitochondria; Modality; Modeling; Monitor; Nuclear; Nuclear Proteins; Oncogenes; Oxidative Phosphorylation; PF4 Gene; Pathway interactions; Patients; Prognosis; Protein Family; Proteins; Proteomics; Refractory; Regimen; Relapse; Reporting; Research; Residual state; Resistance; Respiratory Chain; Role; Sampling; Signal Transduction; Stress; Suppressor Mutations; TP53 gene; Therapeutic; Time; Translations; Transplantation; Tumor Suppressor Genes; Tumor Suppressor Proteins; Up-Regulation; acute myeloid leukemia cell; age related; base; biological adaptation to stress; clinical practice; combinatorial; exportin 1 protein; high risk; improved; in vivo; inhibitor; leukemia; leukemic stem cell; metabolic profile; novel; overexpression; patient derived xenograft model; prevent; prognostic; relapse patients; resistance factors; resistance mechanism; response; survival outcome; targeted treatment; therapeutic target; transcription factor; 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 鉴于急性髓样白血病（AML）的预后持续不佳，诊断和治疗策略 需要开发以实现明显提高的治愈率。而初始响应率和无事件 生存增加了，大多数患者复发并屈服于该疾病。由BCL-2组成的方案 抑制剂Venetoclax（VEN）与降压剂（HMA）（VEN/HMA）结合使用了 AML治疗的完全缓解率，目前为45％至90％，伴随着长时间的生存率。 值得注意的是，VEN/HMA治疗的基本作用机理在于抑制氧化 磷酸化（OXPHOS），特别是在AML白血病干细胞（LSC）中。但是，大多数患者 接受VEN/HMA最终复发，尤其是具有高风险特征的患者，包括复合 细胞遗传学和异常RAS和FLT3信号传导。 p53在AML细胞死亡中的作用知之甚少。 最近，我们报道了核出口XPO1（CRM1）的抑制作用导致p53的积累 AML细胞的核，以及对泛素E3连接酶MDM2和XPO1的双重抑制作用。 扩增这种活性，导致p53介导的AML细胞的杀伤，甚至是VEN/HMA抗性细胞， 体外和体内。双重MDM2和XPO1抑制后，一小部分存活的AML细胞表达 高水平的p21，p53靶标，LC3B和关键的集成应力响应（ISR）因子激活 转录因子4（ATF4），它应使残留的AML细胞容易受到Bcl-2抑制作用。的确， MDM2，XPO1和Bcl-2抑制剂的三组合导致ATF4蛋白水平最高，并且 最深的细胞量。有趣的是，我们发现高度升高的Oxphos复合物水平 具有对双MDM2的抗性的AML细胞在体内抑制双重MDM2和XPO1抑制作用表明OXPHOS激活。 这一发现提供了与VEN/HMA联合克服这种抗性机制的基本原理。 我们假设1）慢性P53重新激活赋予AML OXPHOS依赖性，从而恢复 对基于VEN的治疗的敏感性； 2）MDM2，XPO1抑制剂的伴随组合处理 使用VEN/HMA有效抑制AML和AML LSC。该假设将通过 遵循特定目标（SAS）。在SA 1中，我们将研究AML中OXPHOS的功能依赖性 细胞抗双重MDM2和XPO1抑制。我们将表征细胞反应和细胞命运，在 使用高参数流式细胞仪和LSC和BLAST的单细胞水平（Cytof） 最大p53通过双重抑制MDM2和XPO1在有或没有VEN/HMA的情况下进行评估，以评估 多个时间点的细胞凋亡和其他调节细胞死亡模式。在SA 2中，我们将检查抗 双重MDM2和XPO1抑制的白血病效应与AML和AML LSC中的VEN/HMA结合 野生型P53。拟议研究的成功完成应提供一种新颖的治疗方法 用于具有非生物毒性的靶向治疗剂的VEN/HMA抗AML。