Targeting Acid Ceramidase and Bcl-2 in Acute Myeloid Leukemia

靶向酸性神经酰胺酶和 Bcl-2 治疗急性髓系白血病

• 批准号: 10651649
• 负责人: Johnson Ung
• 金额: $ 1.01万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2023-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10651649
• 关键词: ABCB1 gene; ANXA5 gene; Acute Myelocytic Leukemia; Affect; Age; Alternative Splicing; Anemia; Apoptosis; Apoptosis Regulation Gene; Apoptotic; Attenuated; BCL2 gene; BCL2L1 gene; Biological Assay; Bone Marrow Cells; Bone marrow failure; CD34 gene; Caspase; Cell Line; Cell Membrane Permeability; Cell Survival; Cells; Ceramides; Chemoresistance; Chronic; Clinical; Clinical Trials; Combined Modality Therapy; Competitive Binding; Complex; Data; Diagnosis; Disease; Drug Metabolic Detoxication; Drug resistance; Enzymes; Equilibrium; Exposure to; Family; Family member; Fingerprint; Flow Cytometry; Gene Expression; Genetic; Glucosylceramides; Heterogeneity; Homologous Gene; Human; Hydrolase; Immunologic Deficiency Syndromes; Individual; Lactosylceramides; Length; Lipids; MCL1 gene; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Metabolism; Molecular; Myelogenous; Normal Cell; Outer Mitochondrial Membrane; Pathway interactions; Patients; Production; Proliferating; Protein Family; Protein Isoforms; Proteins; RNA Splicing; Relapse; Reporting; Resistance; Resistance development; Role; Sampling; Signal Transduction; Small Interfering RNA; Specificity; Sphingolipids; Sphingomyelins; Sphingosine; Survival Rate; Testing; Therapeutic; Toxic effect; Up-Regulation; acute myeloid leukemia cell; analog; cancer drug resistance; cytochrome c; effective therapy; effectiveness evaluation; experimental study; galactosylgalactosylglucosylceramidase; genetic manipulation; glycosylation; improved; inhibitor; knock-down; member; novel therapeutic intervention; overexpression; pharmacologic; preclinical study; response; small hairpin RNA; sphingosine 1-phosphate; standard of care; synergism; therapeutic target; transcriptomics; tumor progression

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.

项目概要/摘要 急性髓系白血病 (AML) 是一种异质性疾病，其特征是髓系细胞增殖失控 原始细胞导致骨髓衰竭。诊断时的中位年龄为 68 岁，5 年生存率为 60岁以上的人为13%。因此，对于改进的 AML 治疗方法存在迫切的未满足需求。鞘脂类 是一类重要的两亲性脂质，可调节增殖、耐药性和细胞凋亡。这 促凋亡神经酰胺和促存活 1-磷酸鞘氨醇 (S1P) 的平衡构成了生物活性 鞘脂信号传导的核心。酸性神经酰胺酶 (AC) 是一种脂质水解酶，可分解神经酰胺并 生成 S1P 合成的关键底物——鞘氨醇。我们之前证明了 AML 爆炸 依靠交流电生存。转录组分析和 AC 活性测定显示 AC 基因表达和活性 在原始 AML 样本中上调。使用 shRNA 基因敲低 AC 会降低细胞活力 下调人类 AML 细胞系和患者样本中的促生存蛋白 Mcl-1。药理作用 用神经酰胺类似物 SACLAC 抑制 AC，导致多种人类细胞发生 caspase 依赖性细胞凋亡 AML 细胞系和原发性 AML 患者样本。 SACLAC 的质谱鞘脂组分析 治疗后的 AML 细胞系显示 S1P 减少，神经酰胺水平普遍增加。有趣的是， SACLAC 处理导致 Mcl-1 选择性剪接为其较短的促凋亡异构体 Mcl-1S，并且比率 Mcl-1S 到全长 Mcl-1 的变化决定了细胞的存活。重要的是，全长 Mcl-1 过表达或 Mcl-1S 敲低可减轻 AML 中的 SACLAC 毒性。 Venetoclax (ABT-199)，一种高度选择性的促生存抑制剂 Bcl-2 被批准用于治疗复发性 AML，并正在多项临床试验中进行测试。然而，对 ABT 的反应 199 作为单一药物的寿命很短，并且通过上调 Mcl-1（Bcl-2 同源物）产生耐药性。 在这里，我们建议在 AML 中双重靶向 AC 和 Bcl-2。我们的中心假设是，针对 AC 将 与 ABT-199 协同作用并克服 AML 中的 ABT-199 耐药性。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 介导的机制对 AML 产生耐药性（目标 1）。因为鞘脂失调 参与癌症进展和耐药性，我们将表征 AML 是否具有获得性耐药性 ABT-199 具有特定的鞘脂谱，以及某些鞘脂种类是否有助于 ABT-199 阻力（目标 2）。总的来说，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依据 靶向 AML 中的 AC 和 Bcl-2 家族蛋白。