Targeting the Metabolic Regulator SIRT5 in Acute Myeloid Leukemia

项目摘要 靶向代谢调节因子SIRT 5治疗急性髓性白血病AML是一种侵袭性的 血液恶性肿瘤，长期存活率<30%。目前的治疗标准，单独化疗或 与异基因干细胞移植相结合，几十年来一直没有改变。尽管初步反应，大多数 患者最终复发，表明白血病起始细胞在保护性龛中的持续存在。的抑制剂 FLT 3或突变型异柠檬酸脱氢酶1/2（IDH 1/2）扩大了治疗选择， 基因型导向治疗的范例。然而，即使使用这些新药，复发也是常见的， 这是由于选择了在药物靶点中具有抗性突变的亚克隆。与FLT 3和IDH 1/2抑制剂不同， BCL 2抑制剂venetoclax在多种AML基因型中具有活性，表明针对共有的脆弱性 以基因型不可知的方式进行的研究可能是有效的。不幸的是，许多维奈托克引起的反应不是 白血病细胞通过激活替代性抗凋亡机制或通过重编程来适应 线粒体代谢微环境保护、肿瘤内异质性和代谢灵活性 限制了当前AML疗法的实用性。为了确定AML的新治疗靶点，我们采用了shRNA筛选， 用于在骨髓微环境样条件下检测原代AML细胞。我们发现， AML患者样本高度依赖SIRT 5，而正常CD 34+细胞则不依赖SIRT 5。SIRT 5是赖氨酸 脱酰基酶，参与调节能量代谢途径，包括氧化磷酸化 （OXPHOS）、脂肪酸β-氧化和糖酵解。SIRT 5敲低（KD）降低生长并增加 在大多数AML细胞系中的细胞凋亡，在使用CRISPR/Cas9或 NRD 167，一种新型的细胞渗透性SIRT 5抑制剂。Sirt 5的遗传缺失损害了体外转化 小鼠造血细胞通过几种骨髓癌基因，包括MLL-AF 9，并减弱白血病发生 in vivo.在生物化学水平上，SIRT 5 KD或NRD 167的抑制与OXPHOS减少相关， 谷胱甘肽水平降低和线粒体超氧化物增加，表明AML细胞依赖于SIRT 5 来维持氧化还原平衡Sirt 5-/-小鼠是存活的，具有轻微的代谢异常，这表明， SIRT 5的体内抑制将是耐受的。我们假设SIRT 5是AML的一个治疗靶点， 这在三个具体目标：（1）确定和验证SIRT 5调节的代谢途径，在正常和 AML干细胞和祖细胞。(2)鉴定原发性AML中对SIRT 5抑制敏感的生物标志物 细胞(3)从NRD 167工具开始，确定一种有效的、选择性的和生物可利用的SIRT 5抑制剂 化合物.我们的工作将严格测试SIRT 5是否是AML的治疗靶点，阐明其机制， 潜在的SIRT 5依赖性，并为未来的临床开发确定有效和选择性SIRT 5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.