Inhibition of Acyl-CoA Synthetase Long Chain (ACSL) Isozymes Decreases Multiple Myeloma Cell Proliferation, Mitochondrial Function and Affects Key Survival Pathways

Multiple myeloma (MM) is an incurable cancer of plasma cells that will cause ~12,590 deaths in the USA in 2023. Dysregulation of fatty acid (FA) metabolism is associated with MM development and progression but the underlying mechanisms remains unknown. The acyl-CoA synthetase long-chain family members (ACSLs) convert free long-chain fatty acids into fatty acyl-CoA esters, and play a key role in catabolic and anabolic fatty acid metabolism. Cancer Dependency Map data suggested that ACSL4 and ACSL3 are among the top 25% Hallmark Fatty Acid Metabolism genes that support MM fitness. Here, we show inhibition of the ACSLs in human myeloma cell lines using the pharmacological inhibitor Triascin C (TriC) caused apoptosis, and decreased proliferation in a dose- and time-dependent manner. As KRAS mutants are the most common mutation among MM patients, we used MM.1S cells to study the mechanisms of TriC toxicity. RNA-seq of MM.1S cells treated with 1.00 µM TriC for 24 hours had gene expression profiles significantly enriched in apoptosis, ferroptosis, and ER stress compared to vehicle treated cells. Proteomics of MM.1S cells treated with either 1.00 µM or 2.00 µM TriC for 48 hours revealed that mitochondrial dysfunction and oxidative phosphorylation were significantly enriched pathways of interest relative to vehicle treated cells. Indeed, metabolic flux analysis showed MM.1S cells treated with TriC after 24 hours had decreased mitochondrial ATP production rate. Flow cytometric analyses revealed concomitant decreases in number and mitochondrial membrane potential, and increases in mitochondrial superoxide. Implications: Overall, our data support the hypothesis that suppression of ACSL in human MM cells inhibited their growth and viability, potentially indicating that ACSL proteins may be promising therapeutic targets in treating myeloma progression. Overall design: To understand the transcriptional changes associated with triacsin C inhibition of the ACSL family, we treated the human multiple myeloma cell line, MM.1S cells that constitutively express GFP and luciferase (MM.1S GFP(+), Luc(+) ) with either vehicle (dimethyl sulfoxide, DMSO) or 1.00 µM triacsin C for 24 hours. We then performed gene expression profiling analysis using paired-end RNA seqencing for three biological replicates per treatment condition.

多发性骨髓瘤（Multiple myeloma, MM）是一种无法治愈的浆细胞恶性肿瘤，2023年美国预计将有约12590例患者因该病死亡。脂肪酸（fatty acid, FA）代谢失调与MM的发生发展密切相关，但其潜在分子机制尚未阐明。长链脂酰辅酶A合成酶家族（acyl-CoA synthetase long-chain family members, ACSLs）可将游离长链脂肪酸转化为脂酰辅酶A酯，在脂肪酸的分解与合成代谢过程中发挥关键调控作用。癌症依赖图谱（Cancer Dependency Map）数据显示，ACSL4与ACSL3属于支持MM细胞生存能力的前25%标志性脂肪酸代谢基因。本研究通过药理学抑制剂三叠霉素C（Triascin C, TriC）抑制人骨髓瘤细胞系中的ACSL活性，结果可诱导细胞凋亡，并以剂量与时间依赖的方式抑制细胞增殖。由于KRAS突变是MM患者中最常见的突变类型，本研究选用MM.1S细胞探究TriC的细胞毒性机制。对经1.00 μM TriC处理24小时的MM.1S细胞进行RNA测序，结果显示与溶剂对照组相比，其基因表达谱显著富集于凋亡、铁死亡与内质网应激通路。对分别经1.00 μM、2.00 μM TriC处理48小时的MM.1S细胞进行蛋白质组学分析，结果显示相较于溶剂对照组，线粒体功能障碍与氧化磷酸化是显著富集的目标通路。代谢流分析进一步证实，经TriC处理24小时的MM.1S细胞线粒体ATP生成速率降低。流式细胞术分析显示，细胞数量与线粒体膜电位同步下降，而线粒体超氧化物水平升高。研究意义：综上，本研究数据支持"抑制人MM细胞中的ACSL可抑制其生长与存活"这一假说，提示ACSL蛋白或可成为治疗骨髓瘤进展的潜在治疗靶点。实验整体设计：为探究三叠霉素C抑制ACSL家族所引发的转录组变化，本研究将组成型表达绿色荧光蛋白（GFP）与荧光素酶（luciferase）的人多发性骨髓瘤细胞系MM.1S（MM.1S GFP(+), Luc(+)）分别用溶剂（二甲基亚砜, DMSO）或1.00 μM三叠霉素C处理24小时，随后对每个处理组的3个生物学重复样本进行双端RNA测序，以开展基因表达谱分析。