Human Acute Leukemia Is Addicted to Branched-Chain Amino Acid Metabolism to Maintain Leukemia Stemness

Hematopoietic stem cells (HSCs) have capabilities to self-renew, maintaining an undifferentiated status, as well as to proliferate and mature into blood cells. Similarly, cancer stem cells (CSCs) self-renew and propagate to form cancer tissues. In human acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), CSC-like populations that can reconstitute human leukemia in immuno-deficient mice have been found, and are called leukemic stem cells or leukemia-initiating cells (LICs). In order to obtain "cure" by eradicating LICs, it should be critical to understand molecular machineries as to how LICs self-renew and expand to exert their cancer stemness properties. Recent studies revealed that several specific metabolism pathways actively contribute to the maintenance of stemness in several types of stem cells including ES/iPS cells. However, little is known about the LICs-specific metabolic activity. To clarify metabolic features of human LICs, we comprehensively analyzed 116 cellular metabolites of human CD34+ normal hematopoietic stem progenitor cells (HSPCs) (n=10), CD34+ AML (n=30) and CD34+ ALL cells (n=18). The metabolome analysis revealed that CD34+ AML and ALL cells commonly contain extremely high levels of branched chain amino acids (BCAA) as compared to normal CD34+ HSPCs. AML and ALL cells, but not normal HSCsexpressed BCAA-metabolism related enzymes as well as BCAA-transporters at high levels, and actively transport BCAAs into cytoplasm. Enzymatic inhibition of BCAA metabolism induced apoptosis in LICs but not in normal HSCs. Furthermore, deprivation of BCAA from daily diet in mice xeno-transplanted with human LICs caused significant inhibition of their reconstitution activities in vivo. Serial transplantation experiments revealed that hCD34⁺AML/ALL cells from BCAA-free-dieted mice exhibited extremely impaired reconstitution of human AML/ALL in secondary recipients fed with conventional diet, but those from mice with control diet developed AML/ALL with a high leukemia burden.

To clarify how BCAA metabolism pathway regulates the leukemia-initiating activity, the global gene expression of primary CD34⁺AML /ALL cells and cell lines including THP-1(AML), Kasumi-9(B-ALL), Jurkat cells (T-ALL) were compared before and after pharmacological BCAA-metabolism inhibition and BCAA-deprivation. Gene set enrichment analysis (GSEA) revealed BCAA metabolism inhibition induced the expression of ES-related PRC2 target genes, which should be suppressed in ES cells via H3K27me3 histone modification, and ChIP-Seq analysis confirmed the significantly decreased H3K27me3 level around thetranscription start site ofPRC2 target genes both in CD34+ AML and ALL cells. Furthermore, BCAA metabolism inhibition or BCAA-deprivation resulted in the down regulation of EZH2 and EED, critical components of PRC2, at mRNA and protein level in human AML/ALL cells. Thus, BCAA-metabolism activity is a common metabolic machinery to maintain PRC2 function through potentiating EZH2 and EED expression in AML/ALL.

We also found that human AML/ALL cells, but not normal HSPCs are dependent on BCAA metabolism for maintaining energy production through TCA cycle activity, leading to the adequate supply of alpha ketoglutarate (α-KG), an intermediate metabolite of TCA cycle. Since BCAT1, a catalytic enzyme for BCAA, requires α-KGas a substrate, the active energy production driven by BCAA metabolism contributes to prevent α-KG exhaustion to maintain the high BCAA catabolism activity in human AML/ALL. Consistent with persistent α-KG supply through enhanced BCAA metabolism, primary CD34+AML/ALL cells, which highly express BCAT1 did not exhibit decreased cellular α-KG level as compared to normal HSPCs in our metabolome analysis. Thus, the energy production and regulation of PRC2 function driven by enhanced BCAA metabolism constitute a vicious molecular cycle to maintain the leukemia stemness through the maintaining α-KG level in human acute leukemia.

In summary, the current study demonstrates that human LICs are addicted to the activated BCAA metabolism to maintain their leukemia stemness irrespective of their lineage origin, and that the BCAA metabolic pathway is a generic therapeutic target in human acute leukemias.