Targeting Acid Ceramidase and Bcl-2 in Acute Myeloid Leukemia

项目总结/摘要 急性髓系白血病（AML）是一种异质性疾病，其特征是骨髓细胞不受控制地增殖， 导致骨髓衰竭诊断时的中位年龄为68岁， 60岁以上的人占13%。因此，迫切需要改进的AML治疗剂。鞘脂 是一类重要的调节增殖、耐药性和凋亡的两亲性脂质。的 促凋亡神经酰胺和促存活鞘氨醇-1-磷酸（S1 P）的平衡构成了生物活性 鞘脂信号的核心。酸性神经酰胺酶（AC）是一种脂质水解酶，其分解代谢神经酰胺， 产生S1 P合成的关键底物鞘氨醇。我们之前证明了AML原始细胞 依靠AC生存。转录组学分析和AC活性测定显示AC基因表达和活性 在原发性AML样品中上调。用shRNA基因敲低AC降低细胞活力， 在人类AML细胞系和患者样本中下调促存活蛋白Mcl-1。药理 用神经酰胺类似物SACLAC抑制AC，导致多个人中的半胱天冬酶依赖性细胞凋亡。 AML细胞系和原代AML患者样品。SACLAC的质谱鞘脂组学分析 治疗的AML细胞系显示S1 P减少和神经酰胺水平广泛增加。有趣的是， SACLAC处理导致Mcl-1选择性剪接成其较短的促凋亡同种型Mcl-1 S， Mcl-1 S与全长Mcl-1的比率决定细胞存活。重要的是，全长Mcl-1过表达或Mcl-1 S 在AML中敲减SACLAC毒性。维奈托克（ABT-199），一种高选择性促生存抑制剂 Bcl-2被批准用于复发性AML，并正在几项临床试验中进行测试。然而，对ABT的反应- 199作为单一药剂是短暂的，并且抗性通过Bcl-2同源物Mcl-1的上调而发展。 在这里，我们提出了AC和Bcl-2在AML中的双重靶向作用。我们的核心假设是，针对AC将 在AML中与ABT-199协同作用并克服ABT-199抗性。由于遗传和药理抑制 AC调节Mcl-1，我们假设靶向AC将与ABT-199协同作用以克服ABT-199 通过Mcl-1介导的机制在AML中产生耐药性（Aim 1）。因为鞘脂失调 参与癌症进展和耐药性，我们将表征是否AML获得性耐药 与ABT-199共享特定的鞘脂谱以及某些鞘脂种类是否有助于ABT-199 阻力（目标2）。总之，这些实验旨在确定一种新的AML治疗方法， 结合AC抑制和Bcl-2家族抑制剂，为进一步的临床前研究提供理论基础 靶向AML中的AC和Bcl-2家族蛋白。

PROJECT SUMMARY/ABSTRACT Acute myeloid leukemia (AML) is a heterogeneous disease characterized by uncontrolled proliferation of myeloid blasts resulting in bone marrow failure. The median age at diagnosis is 68 and the 5-year survival rate for individuals over 60 is 13%. Thus, there is an urgent unmet need for improved therapeutics for AML. Sphingolipids are an important class of amphipathic lipids that regulate proliferation, drug resistance, and apoptosis. The balance of pro-apoptotic ceramides and pro-survival sphingosine-1-phosphate (S1P) constitutes the bioactive core of sphingolipid signaling. Acid ceramidase (AC) is a lipid hydrolase that catabolizes ceramides and generates the critical substrate for S1P synthesis, sphingosine. We previously demonstrated that AML blasts rely on AC for survival. Transcriptomic analyses and AC activity assays showed AC gene expression and activity upregulated in primary AML samples. Genetic knockdown of AC with shRNA reduced cell viability and downregulated the pro-survival protein, Mcl-1, in human AML cell lines and patient samples. Pharmacological inhibition of AC with the ceramide analog, SACLAC, resulted in caspase-dependent apoptosis in multiple human AML cell lines and primary AML patient samples. Mass spectrometric sphingolipidomic profiling of SACLAC treated AML cell lines showed a reduction of S1P and a widespread increase in ceramide levels. Intriguingly, SACLAC treatment led to alternative splicing of Mcl-1 to its shorter pro-apoptotic isoform, Mcl-1S, and the ratio of Mcl-1S to full-length Mcl-1 determined cell survival. Importantly, full-length Mcl-1 overexpression or Mcl-1S knockdown attenuated SACLAC toxicity in AML. Venetoclax (ABT-199), a highly selective inhibitor of pro-survival Bcl-2, is approved for relapsed AML and is being tested in several clinical trials. However, responses to ABT- 199 as a single agent are short-lived and resistance develops through the upregulation of Mcl-1, a Bcl-2 homolog. Here, we propose dual targeting of AC and Bcl-2 in AML. Our central hypothesis is that targeting AC will synergize with ABT-199 and overcome ABT-199 resistance in AML. Since genetic and pharmacological inhibition of AC modulates Mcl-1, we hypothesize that targeting AC will synergize with ABT-199 to overcome ABT-199 resistance in AML through an Mcl-1-mediated mechanism (Aim 1). Because sphingolipid dysregulations are involved in cancer progression and drug resistance, we will characterize whether AML with acquired resistance to ABT-199 share a specific sphingolipid profile and whether certain sphingolipid species contribute to ABT-199 resistance (Aim 2). Overall, these experiments aim to identify a novel therapeutic approach for AML that combines AC inhibition and Bcl-2 family inhibitors in order to provide the rationale for further preclinical studies targeting AC and Bcl-2 family proteins in AML.