Targeting the Metabolic Regulator SIRT5 in Acute Myeloid Leukemia

项目总结 靶向代谢调节基因SIRT5治疗急性髓系白血病AML是一种咄咄逼人的 长期存活率为30%的恶性血液病。目前的治疗标准，单独化疗或 结合异基因干细胞移植，几十年来没有改变。尽管最初的反应是，大多数 患者最终复发，这表明白血病启动细胞持续存在于保护性的壁龛中。抗肿瘤药物 Flt3或突变的异柠檬酸脱氢酶1/2(IDH1/2)扩大了治疗选择并验证了 基因导向治疗的范例。然而，即使使用这些新药，复发也很常见和频繁。 由于选择了药物靶标中具有耐药性突变的亚克隆。与Flt3和IDH1/2抑制剂不同， BCL2抑制剂ventoclax在多种AML基因型中都是活跃的，这表明靶向共同的漏洞 以一种与基因无关的方式可能是有效的。不幸的是，许多万乃馨诱导的反应并不是 白血病细胞通过激活替代的抗凋亡机制或通过重新编程来适应 线粒体代谢。微环境保护、瘤内异质性和代谢灵活性 限制目前急性髓系白血病治疗的实用性。为了确定急性髓细胞白血病的新治疗靶点，我们采用了shRNA筛查 用于在类似骨髓微环境的条件下检测原代AML细胞。我们发现很多人 AML患者样本高度依赖SIRT5，而正常CD34+细胞不依赖SIRT5。SIRT5是一种赖氨酸 脱酰酶参与能量代谢途径的调节，包括氧化磷酸化 (OXPHOS)、脂肪酸β氧化和糖酵解。SIRT5基因敲除(KD)会降低生长速度并增加 大多数AML细胞系中的细胞凋亡，使用CRISPR/Cas9或 NRD167，一种新型的细胞渗透性SIRT5抑制剂。SIRT5基因缺失对人卵巢癌体外转化的影响 包括MLL-AF9在内的几种髓系癌基因对小鼠造血细胞的抑制作用 在活体内。在生化水平上，SIRT5kD或用NRD167抑制与OXPHOS的减少有关， 谷胱甘肽水平降低和线粒体超氧化物歧化，提示AML细胞依赖SIRT5 以维持氧化还原动态平衡。SIRT5-/-小鼠可以存活，但有轻微的代谢异常，这表明在 对SIRT5的体内抑制是可以容忍的。我们假设SIRT5是AML的治疗靶点，并将测试 具体目的有三个：(1)确定和验证SIRT5调节的正常和 急性髓系白血病干细胞和祖细胞。(2)确定原发AML对SIRT5抑制敏感的生物标志物 细胞。(3)从NRD167工具开始，确定一种有效的、选择性的和生物可用的SIRT5抑制剂 化合物。我们的工作将严格测试SIRT5是否是AML的治疗靶点，阐明其机制 潜在的SIRT5依赖，并为未来的临床开发寻找有效和选择性的SIRT5抑制剂。

PROJECT SUMMARY TARGETING THE METABOLIC REGULATOR SIRT5 IN ACUTE MYELOID LEUKEMIA. AML is an aggressive hematologic malignancy with <30% long-term survival. The current therapy standard, chemotherapy alone or combined with allogeneic stem cell transplant, has not changed for decades. Despite initial responses, most patients eventually relapse, suggesting persistence of leukemia initiating cells in protective niches. Inhibitors of FLT3 or mutant isocitrate dehydrogenase 1/2 (IDH1/2) have expanded therapy options and validated the paradigm of genotype-directed therapy. However, even with these new drugs, relapse is common and frequently due to selection of subclones with resistance mutations in the drug target. Unlike FLT3 and IDH1/2 inhibitors, the BCL2 inhibitor venetoclax is active in multiple AML genotypes, indicating that targeting shared vulnerabilities in a genotype-agnostic manner can be effective. Unfortunately many venetoclax-induced responses are not durable as leukemia cells adapt by activating alternative anti-apoptosis mechanisms or by reprogramming mitochondrial metabolism. Microenvironmental protection, intra-tumoral heterogeneity and metabolic flexibility limit the utility of current AML therapies. To identify new therapy targets in AML, we adapted an shRNA screen for testing primary AML cells under bone marrow microenvironment-like conditions. We discovered that many AML patient samples are highly dependent on SIRT5, while normal CD34+ cells are not. SIRT5 is a lysine deacylase implicated in the regulation of energy metabolic pathways, including oxidative phosphorylation (OXPHOS), fatty acid β-oxidation and glycolysis. SIRT5 knockdown (KD) reduces growth and increases apoptosis in most AML cell lines, with consistent results upon disruption of SIRT5 using CRISPR/Cas9 or NRD167, a novel cell-permeable SIRT5 inhibitor. Genetic absence of Sirt5 impairs in vitro transformation of mouse hematopoietic cells by several myeloid oncogenes, including MLL-AF9, and attenuates leukemogenesis in vivo. At a biochemical level, SIRT5 KD or inhibition with NRD167 is associated with reduced OXPHOS, reduced glutathione levels and increased mitochondrial superoxide, suggesting that AML cells depend on SIRT5 to maintain redox homeostasis. Sirt5-/- mice are viable with minor metabolic abnormalities, suggesting that in vivo inhibition of SIRT5 would be tolerated. We hypothesize that SIRT5 is a therapy target in AML and will test this in three Specific Aims: (1) Identify and validate SIRT5-regulated metabolic pathways in normal and AML stem and progenitor cells. (2) Identify biomarkers of sensitivity to SIRT5 inhibition in primary AML cells. (3) Identify a potent, selective, and bioavailable SIRT5 inhibitor, starting from the NRD167 tool compound. Our work will rigorously test whether SIRT5 is a therapy target in AML, clarify the mechanisms underlying SIRT5 dependence, and identify potent and selective SIRT5 inhibitors for future clinical development.