Role of Branched Chain Amino Acid Transaminase 1 (BCAT1) in Acute Myeloid Leukemia [hydroxymethylation_HL60_BCAT1-OE_12weeks]

The branched chain amino acid (BCAA) pathway and high levels of BCAA transaminase 1 (BCAT1) have recently been associated with aggressiveness in several cancer entities. However, the mechanistic role of BCAT1 in this process remains largely uncertain. By performing high-resolution proteomic analysis of human acute myeloid leukaemia (AML) stem cell (LSC) and non-LSC populations, we found the BCAA pathway enriched and BCAT1 overexpressed in LSCs. We show that BCAT1, which transfers α-amino groups from BCAAs to α-ketoglutarate (αKG), is a critical regulator of intracellular αKG homeostasis. Next to its role in the tricarboxylic acid (TCA) cycle αKG is an essential co-factor for αKG-dependent dioxygenases such as EGLN1 and the TET family of DNA demethylases. Knockdown of BCAT1 in leukaemia cells caused accumulation of αKG leading to HIF1a protein degradation mediated by EGLN1. This resulted in a growth and survival defect and abrogated leukaemia-initiating potential. In contrast, overexpression (OE) of BCAT1 in leukaemia cells decreased intracellular αKG levels and caused DNA hypermethylation via altered TET activity. BCAT1high AMLs displayed a DNA hypermethylation phenotype similar to cases carrying mutant isocitrate dehydrogenase (IDHmut), in which TET2 is inhibited by the oncometabolite 2-hydroxyglutarate. High levels of BCAT1 strongly correlate with shorter overall survival in IDHwtTET2wt, but not IDHmut or TET2mut AMLs. Gene sets characteristic for IDHmut AMLs were enriched in IDHwtTETwtBCAT1high patient samples. BCAT1high AMLs showed robust enrichment for LSC signatures and paired sample analysis revealed a significant increase of BCAT1 levels upon disease relapse. In summary, by limiting intracellular αKG, BCAT1 links BCAA catabolism to HIF1a stability and regulation of the epigenomic landscape. Our results suggest the BCAA-BCAT1-αKG pathway as a therapeutic target to compromise LSC function in IDHwtTET2wt AML patients. HL-60 cell lines were lentivirally transduced with an inducible BCAT1 overexpression construct and selected for transduced cells. Overexpression was induced with Doxycyclin for 12 weeks and cells were lysed and RNA was extracted.

支链氨基酸（branched chain amino acid, BCAA）代谢通路与高表达的支链氨基酸转氨酶1（BCAT1）近期被证实与多种癌种的侵袭性密切相关。然而，BCAT1在该过程中的具体机制仍未明确。本研究通过对人急性髓系白血病（acute myeloid leukaemia, AML）干细胞（leukaemia stem cell, LSC）与非LSC群体开展高分辨率蛋白质组学分析，发现BCAA通路在LSC中富集，且BCAT1在LSC中呈过表达状态。我们证实，负责将支链氨基酸的α-氨基转移至α-酮戊二酸（α-ketoglutarate, αKG）的BCAT1，是细胞内αKG稳态的关键调控因子。除参与三羧酸循环（tricarboxylic acid cycle, TCA循环）外，αKG还是αKG依赖性双加氧酶（如EGLN1与DNA去甲基化酶TET家族）的必需辅因子。对白血病细胞进行BCAT1敲低会导致αKG积累，进而通过EGLN1介导缺氧诱导因子1α（hypoxia-inducible factor 1α, HIF1α）的蛋白质降解。这会引发细胞生长与存活缺陷，并消除白血病起始潜能。与之相反，在白血病细胞中过表达（overexpression, OE）BCAT1会降低细胞内αKG水平，并通过改变TET活性引发DNA高甲基化。高表达BCAT1的AML样本呈现出与携带异柠檬酸脱氢酶突变体（isocitrate dehydrogenase mutant, IDHmut）的病例相似的DNA高甲基化表型——此类病例中，癌代谢物2-羟基戊二酸会抑制TET2的活性。在野生型IDH且野生型TET2（IDHwtTET2wt）的AML患者中，高BCAT1水平与更短的总生存期显著相关，但在IDHmut或TET2mut的AML患者中则无此关联。IDHmut AML的特征基因集在IDHwtTETwtBCAT1high的患者样本中显著富集。高表达BCAT1的AML样本显著富集LSC特征基因，配对样本分析显示，疾病复发时BCAT1水平显著升高。综上，通过限制细胞内αKG水平，BCAT1将BCAA分解代谢与HIF1α稳定性及表观基因组调控联系起来。本研究结果表明，BCAA-BCAT1-αKG通路可作为治疗靶点，以削弱IDHwtTET2wt AML患者体内LSC的功能。研究人员使用慢病毒将可诱导BCAT1过表达载体转导至HL-60细胞系，并筛选得到转导阳性细胞。用多西环素（Doxycyclin）诱导过表达12周后，收集细胞并提取RNA。