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.

项目概要/摘要 鉴于急性髓系白血病（AML）的持续不良预后，诊断和治疗策略 需要开发以显着提高治愈率。虽然初始响应率和无事件- 生存率有所提高，但大多数患者会复发并死于该疾病。该方案由 BCL-2 组成 抑制剂维奈托克 (VEN) 与低甲基化剂 (HMA) 组合 (VEN/HMA) 彻底改变了 AML 治疗的完全缓解率目前为 45% 至 90%，并且生存期延长。 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)，尤其是在 AML 白血病干细胞 (LSC) 中。然而，大多数患者 接受 VEN/HMA 最终会复发，尤其是具有复杂特征的高危患者 细胞遗传学以及异常的 RAS 和 FLT3 信号传导。 p53 在 AML 细胞死亡中的作用尚不清楚。 我们最近报道了核输出蛋白 XPO1 (CRM1) 的抑制导致 p53 的积累 AML 细胞的细胞核，并且对泛素 E3 连接酶 MDM2 和 XPO1 具有双重抑制作用 放大这种活性，导致 p53 介导的协同杀伤 AML 细胞，甚至 VEN/HMA 耐药细胞， 体外和体内。 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)，这将使残留的 AML 细胞容易受到 BCL-2 抑制的影响。的确， MDM2、XPO1 和 BCL-2 抑制剂的三重组合产生了最高的 ATF4 蛋白水平 最深的细胞减灭术。有趣的是，我们发现 OXPHOS 复合物的蛋白质水平显着增加 AML 细胞对体内 MDM2 和 XPO1 双重抑制具有获得性抗性，表明 OXPHOS 激活。 这一发现为结合 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 重新激活会导致 AML OXPHOS 依赖性，从而恢复 对基于 VEN 的治疗敏感； 2) MDM2、XPO1抑制剂的联合联合治疗 与 VEN/HMA 一起有效抑制 AML 和 AML LSC。该假设将通过 遵循具体目标 (SA)。在 SA 1 中，我们将研究 AML 中对 OXPHOS 的功能依赖性 细胞对 MDM2 和 XPO1 双重抑制具有抵抗力。我们将描述细胞反应和细胞命运的特征， 单细胞水平，使用高参数流式细胞术和质谱细胞术 (CyTOF) 检测 LSC 和母细胞 在有或没有 VEN/HMA 的情况下，通过双重抑制 MDM2 和 XPO1 来最大程度地激活 p53，以评估 细胞凋亡和其他在多个时间点调节的细胞死亡模式。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 LSC 的白血病影响 野生型p53。拟议研究的成功完成应提供一种新的治疗方法 使用非基因毒性的靶向治疗治疗 VEN/HMA 耐药的 AML。