Role of Branched Chain Amino Acid Transaminase 1 (BCAT1) in Acute Myeloid Leukemia [methylation_HL60_BCAT1-OE_10weeks]

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 10 weeks and cells were lysed and RNA was extracted.

支链氨基酸（branched chain amino acid, BCAA）通路与高表达BCAA转氨酶1（BCAA transaminase 1, BCAT1）近期在多项癌症实体中被发现与肿瘤侵袭性相关。然而，BCAT1在该过程中的具体分子机制仍未完全明确。通过对人类急性髓系白血病（acute myeloid leukaemia, AML）干细胞（leukaemia stem cell, LSC）及非LSC群体进行高分辨率蛋白质组学分析，我们发现LSC中BCAA通路富集且BCAT1过表达。我们证实，BCAT1可将支链氨基酸上的α-氨基转移至α-酮戊二酸（α-ketoglutarate, αKG），是细胞内αKG稳态的关键调控因子。除在三羧酸（tricarboxylic acid, TCA）循环中发挥作用外，αKG还是αKG依赖性双加氧酶（如EGLN1及DNA去甲基化酶TET家族）的必需辅因子。对白血病细胞进行BCAT1基因敲低后，会导致αKG积累，进而引发由EGLN1介导的缺氧诱导因子1α（hypoxia-inducible factor 1α, HIF1α）蛋白降解。这会造成细胞增殖与存活缺陷，并消除白血病起始细胞潜能。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。BCAT1高表达的AML患者样本呈现出与异柠檬酸脱氢酶突变（mutant isocitrate dehydrogenase, IDHmut）携带者相似的DNA高甲基化表型，此类样本中癌代谢物2-羟基戊二酸（2-hydroxyglutarate）会抑制TET2活性。在异柠檬酸脱氢酶野生型（IDHwt）且TET2野生型（TET2wt）的AML患者中，高BCAT1表达与较短总生存期显著相关，但在IDH突变型（IDHmut）或TET2突变型（TET2mut）AML患者中无此关联。IDHmut AML特有的基因集在IDHwtTETwtBCAT1高表达的患者样本中显著富集。BCAT1高表达的AML样本显著富集LSC特征基因集，配对样本分析显示疾病复发时BCAT1表达水平显著升高。综上，BCAT1通过限制细胞内αKG水平，将BCAA分解代谢与HIF1α稳定性及表观基因组图谱调控联系起来。本研究结果表明，BCAA-BCAT1-αKG通路可作为靶向干预IDHwtTET2wt AML患者LSC功能的治疗靶点。本研究采用慢病毒将可诱导BCAT1过表达载体转导至HL-60细胞系，并筛选获得转导阳性细胞。使用多西环素（Doxycyclin）诱导过表达10周后，裂解细胞并提取RNA。