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

项目概要/摘要 鉴于急性髓细胞白血病（AML）的持续不良预后，诊断和治疗策略 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，大多数患者复发并死于疾病。由BCL-2组成的方案 抑制剂venetoclax（VEN）与低甲基化剂（HMA）（VEN/HMA）的组合彻底改变了 AML治疗的完全缓解率目前在45%至90%之间，并伴有生存期延长。 值得注意的是，VEN/HMA疗法的潜在作用机制在于抑制氧化应激。 在某些实施方案中，OXPHOS可被抑制磷酸化（OXPHOS），特别是在AML白血病干细胞（LSC）中。然而，大多数患者 接受VEN/HMA的患者最终复发，特别是具有高危特征的患者，包括复杂的 细胞遗传学和异常RAS和FLT 3信号传导。p53在AML细胞死亡中的作用知之甚少。 我们最近报道了核输出蛋白XPO 1（CRM 1）的抑制导致p53在细胞中的积累。 AML细胞核，以及泛素E3连接酶MDM 2和XPO 1的双重抑制 放大了这种活性，导致协同p53介导的AML细胞杀伤，甚至VEN/HMA抗性细胞， 在体外和体内。在MDM 2和XPO 1双重抑制后，一小部分存活的AML细胞表达 高水平的p21、p53靶点、LC 3B和关键的综合应激反应（ISR）因子激活 转录因子4（ATF 4），这将使残留的AML细胞易受BCL-2抑制的影响。的确， MDM 2、XPO 1和BCL-2抑制剂的三重组合导致最高的ATF 4蛋白水平， 最深的细胞减少。有趣的是，我们发现在小鼠中OXPHOS复合物的蛋白水平高度增加， AML细胞对体内MDM 2和XPO 1双重抑制具有获得性抗性，表明OXPHOS活化。 这一发现为VEN/HMA联合治疗克服这种耐药机制提供了依据。 我们假设：1）慢性p53再激活赋予AML OXPHOS依赖性，从而恢复 对基于VEN的治疗的敏感性;和2）MDM 2、XPO 1抑制剂的伴随组合治疗 VEN/HMA有效抑制AML和AML LSC。该假设将被检查与 具体目标（SA）。在SA 1中，我们将研究AML中对OXPHOS的功能依赖性， 对MDM 2和XPO 1双重抑制具有抗性的细胞。我们将描述细胞反应和细胞命运， 单细胞水平，使用LSC和原始细胞的高参数流式细胞术和质谱细胞术（CyTOF） 在有或没有VEN/HMA的情况下，通过MDM 2和XPO 1的双重抑制实现最大p53活化，以评估 在多个时间点的细胞凋亡和其他调节的细胞死亡模式。在SA 2中，我们将检查反- MDM 2和XPO 1双重抑制联合VEN/HMA在AML和AML LSC中的白血病作用 野生型p53。拟议研究的成功完成应提供一种新的治疗方法 用于VEN/HMA耐药AML的非遗传毒性靶向治疗。

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.