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Cancer progression by reprogrammed BCAA metabolism in myeloid leukaemia

Abstract

Reprogrammed cellular metabolism is a common characteristic observed in various cancers1,2. However, whether metabolic changes directly regulate cancer development and progression remains poorly understood. Here we show that BCAT1, a cytosolic aminotransferase for branched-chain amino acids (BCAAs), is aberrantly activated and functionally required for chronic myeloid leukaemia (CML) in humans and in mouse models of CML. BCAT1 is upregulated during progression of CML and promotes BCAA production in leukaemia cells by aminating the branched-chain keto acids. Blocking BCAT1 gene expression or enzymatic activity induces cellular differentiation and impairs the propagation of blast crisis CML both in vitro and in vivo. Stable-isotope tracer experiments combined with nuclear magnetic resonance-based metabolic analysis demonstrate the intracellular production of BCAAs by BCAT1. Direct supplementation with BCAAs ameliorates the defects caused by BCAT1 knockdown, indicating that BCAT1 exerts its oncogenic function through BCAA production in blast crisis CML cells. Importantly, BCAT1 expression not only is activated in human blast crisis CML and de novo acute myeloid leukaemia, but also predicts disease outcome in patients. As an upstream regulator of BCAT1 expression, we identified Musashi2 (MSI2), an oncogenic RNA binding protein that is required for blast crisis CML. MSI2 is physically associated with the BCAT1 transcript and positively regulates its protein expression in leukaemia. Taken together, this work reveals that altered BCAA metabolism activated through the MSI2–BCAT1 axis drives cancer progression in myeloid leukaemia.

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Figure 1: Activated BCAA production by BCAT1 in BC-CML.
Figure 2: Bcat1 is essential for BC-CML propagation and differentiation arrest.
Figure 3: BCAT1 activation and requirement in human myeloid leukaemia.
Figure 4: RNA binding protein MSI2 mediates BCAT1 activation in BC-CML.

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Acknowledgements

We thank W. Pear, D. Baltimore and S. Lowe for plasmids, and S. Dalton, C. Jordan, B. Zimdahl, J. Ninomiya-Tsuji, K. Sai, K. Matsumoto, H. Hanafusa, T. Mizuno, Y. Kuwatsuka, Y. Minami and M. Merritt for discussions and comments on the manuscript. We also thank J. Nelson at the CTEGD Cytometry Shared Resource Laboratory, University of Georgia, for assistance in cell sorting, K. Sekimizu, C. West, M. Mandalasi and H. van der Wel for advice on radioisotope use, and K. MacKeil, J. Nist and K. Ogata for technical help. This work was supported by grants from the University of Georgia Research Foundation and the Heather Wright Cancer Research Fund (T.I.); by the Japan Society for the Promotion of Science Bilateral Open Partnership Joint Research Projects Program (M.T.); A.S.E. and the Complex Carbohydrate Research Center NMR facility were partly supported by the Southeast Center for Integrated Metabolomics, National Institutes of Health U24DK097209 and the Georgia Research Alliance.

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Authors

Contributions

A.H. designed the studies, performed all experiments, analysed the data and wrote the manuscript. M.T. designed and performed experiments related to quantitative analysis of amino and keto acids. T.K., M.K. and A.T. provided and performed experiments with human primary samples. T.N. performed histological and cytological analysis. J.G., F.T. and A.S.E. designed and conducted NMR-based metabolic analysis. D.M. and N.K. performed bioinformatics analysis of gene expression datasets. T.I. conceived and supervised the project and wrote the manuscript.

Corresponding author

Correspondence to Takahiro Ito.

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Competing interests

T.I. and A.H. are named inventors of a provisional patent application number 62/413,028.

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Reviewer Information Nature thanks B. Huntly, D. Sabatini and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Change in the amino acid metabolism in leukaemic mice.

ad, Representative chromatograms of (a, c) CP-CML and (b, d) BC-CML plasma samples derivatized with the amine-specific fluorescent labelling agent NBD-F and analysed in mobile phases at (a, b) pH 6.2 or (c, d) pH 4.4. Each NBD–amino acid peak was assigned as indicated. IS, internal standard (NBD–6-aminocaproic acid). e, Plasma amino acid levels in mice with CP- and BC-CML. Blood plasma samples were prepared from mice with CP- and BC-CML, methanol-extracted and dried under a vacuum. The dried extracts were analysed for quantification. Open and closed bars indicate CP-CML (n = 5) and BC-CML (n = 7) specimens, respectively. Two-tailed t-test. †P < 0.06, *P < 0.05, **P < 0.01. f, Leucine transport in primary CP- and BC-CML cells. BCRABL1–YFP+PI live leukaemia cells (5 × 105) were sorted from premorbid animals and placed in a pre-warmed uptake media containing 10 μM [(U)-14C]l-leucine. After incubation at 37 °C for the indicated times, the cells were washed with cold HBSS and lysed with 0.1 M sodium hydroxide, and the radioactivity was measured using a scintillation counter. The grey and blue lines indicate the average leucine uptake in CP- and BC-CML samples (n = 5 and 3, respectively). Error bars, s.e.m. *P < 0.05. NS, not statistically significant (P > 0.05). g, RT–qPCR analysis of Bcat1 and Bcat2 expression in CP- and BC-CML cells (n = 4 each). The expression levels are normalized and displayed relative to the control β-2-microglobulin gene expression. Error bars, s.e.m.; ***P < 0.001, NS, not statistically significant (P > 0.05). h, BCAT1 protein expression in mouse primary CP- and BC-CML cells. This graph shows BCAT1 protein expression levels normalized relative to the HSP90 loading control (CP-CML, n = 7; BC-CML, n = 9). Error bars, s.e.m. *P < 0.05. i, Tissue-specific expression of mouse Bcat1. The expression was detectable in the myeloid cell line M1, primary mouse BC-CML cells, olfactory bulb (Olf bulb), whole brain and testis. B2m, β-2-microglobulin. j, Schematics of the structures of human and mouse BCAT1 proteins. The shaded boxes represent aminotransferase domains. K, a Lys residue for the binding of the pyridoxal phosphate cofactor. CVVC, a conserved redox-sensitive CXXC motif. Regions targeted with shRNAs in this study are shown as thick bars (shBcat1-a and -b, and shBCAT1-c and -d). k, l, Alanine and aspartate transaminase gene expression in CP- and BC-CML. RT–qPCR analysis of (k) Gpt1 and Gpt2, and (l) Got1 and Got2 expression in CP- and BC-CML samples (n = 4 each). The expression levels are normalized and displayed relative to the expression of the B2m control. Error bars, s.e.m.; NS, not statistically significant (P > 0.05).

Extended Data Figure 2 Keto acid metabolism in leukaemic mice.

a, b, Representative chromatograms of (a) CP-CML and (b) BC-CML plasma samples derivatized with the keto acid-reactive OPD. Each OPD–keto acid peak was assigned as indicated. KG, α-ketoglutarate; PYR, pyruvate; KIV, keto-isovalerate; KIC, keto-isocaproate; KMV, keto-methylvalerate; IS, internal standard for keto acid analysis (OPD–α-ketovalerate). c, d, Plasma and intracellular branched-chain keto acid levels in CP- and BC-CML. c, d, Blood plasma fractions from leukaemic mice (c) or 5 × 106 live leukaemia cells purified by fluorescence-activated cell sorting (d) were methanol-extracted and dried under a vacuum. The dried extracts were labelled with OPD, extracted with ethyl acetate and analysed using an HPLC system equipped with a fluorescence detector. Open and closed bars indicate CP-CML (plasma, n = 9; intracellular, n = 5) and BC-CML (plasma, n = 10; intracellular, n = 6) specimens, respectively. *P < 0.05. Error bars, s.e.m. e, Molar amount of intracellular BCAAs and BCKAs in primary mouse BC-CML cells. The amount of each organic acid per one million cells was estimated using calibration curves obtained with reference standards for each compound. ‘%KA/AA’ shows the amount of a BCKA relative to the corresponding BCAA species.

Extended Data Figure 3 Intracellular BCAA production from BCKA in human K562 BC-CML cells.

af, Regions of HSQC spectra of 13C-labelled metabolites. K562 cells were cultured in media supplemented with (a, c) 170 μM [13C]valine and 30 μM non-labelled KIV, or (b, d) 170 μM non-labelled valine and 30 μM [13C]KIV. After labelling for 15 min, the cells were collected, washed with PBS and methanol-extracted for HSQC analysis by high-field NMR spectroscopy. Each panel shows the regions of one- and two-dimensional HSQC spectra for the (a, b) intracellular fraction, (c, d) culture supernatant and (e, f) labelling media alone, respectively. Panels a and b are the same as shown in Fig. 1e and f, respectively. gi, Absence of detectable intracellular KIC generation by leucine breakdown. K562 cells were cultured in the labelling medium supplemented with 170 μM [13C]leucine and 30 μM non-labelled KIC for 15 min, and the intracellular 13C-labelled metabolites were analysed by HSQC analysis. Each panel shows region of the two-dimensional spectrum showing 1H–13C HSQC signals for β, γ and δ carbons of Leu and KIC. g, Intracellular fraction, (h) KIC reference standard (HSQC signals derived from natural abundance [13C]KIC), (i) overlay of the spectra in g (black) and h (red). Note the absence of KIC signals in g.

Extended Data Figure 4 Intracellular BCAA production via transamination.

ac, Regions of 600 MHz two-dimensional heteronuclear multiple-bond correlation spectra showing cross-peaks between the amine nitrogen and the β carbon protons. Only those amino acids that incorporated a significant amount of [15N]amine show cross-peak signals. df, Regions of 600 MHz one-dimensional 1H spectra. Each proton peak is assigned as indicated. DSS, 2,2-dimethyl-2-silapentane-5-sulfonate. a, d, Mixture of reference standards of the indicated amino acids. b, e, K562 cell sample cultured in the medium containing (amine-15N)-glutamine. c, f, K562 cell sample cultured in the non-labelled standard medium. g, Percentage of newly synthesized 15N-labelled BCAAs within total intracellular pool at 72 h after post-labelling for each amino acid indicated. ‘Total BCAAs’ shows the percentage including all three BCAA species.

Extended Data Figure 5 Roles of Bcat1 in differentiation, proliferation and leukaemia development in vivo.

a, RT–qPCR analysis of Bcat1 expression. Lin cells from NUP98–HOXA9/BCR–ABL-induced BC-CML were infected with shCtrl or Bcat1 shRNA (shBcat1-a and shBcat1-b) for three days and resorted for analysis of Bcat1 expression. The expression levels are normalized to the level of B2m expression and displayed relative to the control, which was arbitrarily set at 1. Error bars, s.e.m. of triplicate PCRs. **P < 0.01. b, RT–qPCR analysis of Bcat1 expression in leukaemia cells isolated from diseased mice transplanted with shCtrl- or shBcat1-expressing BC-CML cells. The expression levels are normalized and displayed relative to the B2m control. ***P < 0.001. c, Bcat2 expression in shBcat1-expressing cells. Lin cells from NUP98–HOXA9/BCR–ABL-induced BC-CML were infected with shCtrl or Bcat1 shRNA (shBcat1-a and shBcat1-b) for three days and resorted for analysis of Bcat2 expression. The expression levels are normalized to the level of B2m and are displayed relative to the control arbitrarily set at 1. Error bars, s.e.m. of triplicate PCRs. NS, not statistically significant (P > 0.05). d, Functional rescue of the shBcat1-induced reduction in colony-forming ability with the expression of shRNA-resistant mutant Bcat1 cDNA. Primary Lin BC-CML cells transduced with the vector or shRNA-resistant Bcat1 gene together with the indicated shRNA constructs. **P < 0.01 compared with the vector and shBcat1-b. e, f, Colony-forming ability of primary HSPCs. e, Normal HSPCs purified from bone marrow on the basis of their LSK phenotype were transduced with the Bcat1 shRNAs (shBcat1-a and shBcat1-b) and plated for colony formation. NS, not statistically significant (P > 0.05). f, Normal HSPCs were plated for colony formation with the indicated concentrations of gabapentin or PBS (−). NS, not statistically significant (P > 0.05). **P < 0.01 compared with the PBS control. Photomicrographs showing representative colonies formed under each condition. Scale bars, 500 μm. Three hundred LSK cells were plated per well in triplicate for the evaluation of colony-forming activity. Error bars, s.e.m. g, Haematoxylin and eosin staining of sections of the liver, lung and spleen at the time of onset of clinical signs (top six rows) and of tissue sections from a disease-free survivor (bottom two rows). White arrows indicate immature myeloid cells. Portal triad (PT), haemorrhagic necrosis (N), central veins (CV), arteriolar profiles (A), bile ducts (B), veins (V), white pulp (WP) and red pulp (RP) are indicated. Scale bars, 100 μm for images at ×10 and 20 μm for images at ×40 magnification. h, Representative flow cytometry plots showing lineage marker expression in leukaemia cells from mice transplanted with the shRNA-infected BC-CML cells. Leukaemia cells were analysed for their frequency of the Lin population. ik, Effect of conditional Bcat1 knockdown on BC-CML maintenance in vivo. i, Lin BC-CML cells were infected with doxycycline-inducible shRNAs against shBcat1-b or a control (shCtrl) and transplanted into recipients (1,500 cells per recipient). After 10 days of the transplantation with leukaemia cells expressing the indicated constructs, (j) donor-derived chimaerisms were analysed. Mice were then fed with chow containing doxycycline to induce shRNA expression, and (k) survival was monitored. The data shown are from two independent experiments. n = 4 for shCtrl with no Dox (DOX); n = 7 for shBcat1-b, DOX; and n = 9 each for shCtrl with Dox (DOX+) and shBcat1-b, DOX+. **P < 0.01 (shCtrl versus shBcat1-b, DOX+). NS, not statistically significant (P > 0.05). l, Cell cycle distribution of the shRNA-infected leukaemia cells. Live leukaemia cells were isolated from mice transplanted with shRNA-infected BC-CML cells, fixed and stained with propidium iodide for analysis of cell cycle distribution via flow cytometry. m, Apoptotic cells from leukaemic mice transplanted with shRNA-infected BC-CML cells were analysed via flow cytometry using Annexin V and 7-aminoactinomycin D (7-AAD) staining.

Extended Data Figure 6 BCAT1 cooperates with BCRABL1 in blastic transformation in vivo.

a, RT–qPCR analysis of Bcat1 expression in normal LSK or Lin c-Kit+ HSPCs transduced with either the vector or Bcat1 retroviruses. The expression levels are normalized and displayed relative to the control B2m expression. ***P < 0.001. b, Normal LSK or Lin c-Kit+ HSPCs were purified from healthy bone marrow and transduced with the indicated retroviruses, and the infected cells were plated in triplicate to assess colony formation after 10 days. Error bars, s.e.m. NS, not statistically significant (P > 0.05). c, Colony-forming ability of normal HSPCs expressing BCRABL1 and Bcat1. LSK cells were purified from healthy bone marrow and transduced with either the control vector or Bcat1 together with BCRABL1 (B/A) retroviruses, and double-positive cells were plated in triplicate to assess colony formation after 10 days (plated at a density of 150 cells per well). Photomicrographs show representative colonies formed in each group. Scale bar, 500 μm. Error bars, s.e.m. ***P < 0.001. d, Chimaerism of donor-derived cells after transplantation with LSK cells expressing the indicated constructs. n = 15 for each group. *P < 0.05. e, Haematoxylin and eosin staining of liver, lung and spleen sections from mice transplanted with LSK cells expressing BCRABL1 and vector or Bcat1. White arrows indicate immature myeloid cells. Scale bars, 100 μm for ×10 images and 20 μm for ×40 images. f, Plasma α-amino acid levels in mice transplanted with LSK cells infected with BCRABL1 and the vector or Bcat1. Blood plasma fractions were prepared from peripheral blood samples, methanol-extracted and dried under a vacuum. The dried extracts were labelled with NBD-F and analysed using an HPLC equipped with a fluorescence detector. Open and closed bars indicate vector control (n = 17) and Bcat1 (n = 18) specimens, respectively. *P < 0.05, **P < 0.01. g, Representative flow cytometry plots showing lineage marker expression in leukaemia cells from mice transplanted with LSK cells infected with either the control vector or Bcat1 together with BCRABL1. Leukaemia cells were analysed for their frequency of the Lin population.

Extended Data Figure 7 BCAT1 is required for human myeloid leukaemia.

a, RT–qPCR analysis of BCAT1 expression in the human K562 BC-CML cell line transduced with lentiviral shCtrl or BCAT1 shRNA (shBCAT1-c and shBCAT1-d). The expression levels are normalized and displayed relative to the expression of the B2M control. **P < 0.01. b, Western blot analysis of BCAT1 protein levels in K562 cells infected with the indicated lentiviral shRNA constructs. Human β-tubulin protein (β-Tub) was used as the loading control. β-Tubulin image is the same as shown in Fig. 3j. c, d, Colony-forming ability of (c) K562 cells transduced with control (shCtrl) or BCAT1 shRNAs (shBCAT1-c and shBCAT1-d) and (d) K562 cells cultured with the indicated concentrations of Gbp. One hundred cells were plated per well in triplicate. Photomicrographs show representative colonies formed. Scale bar, 200 μm. Error bars, s.e.m. **P < 0.01, ***P < 0.001. e, RT–qPCR analysis of BCAT1 expression in the samples from the patient with BC-CML used in the data presented in Fig. 3d that were transduced with control (shCtrl) or BCAT1 shRNA (shBCAT1-d). ***P < 0.001. f, Colony-forming ability of primary human CD34+ BC-CML cells from another specimen from a patient treated with Gbp. Error bars, s.e.m. **P < 0.01. gi, Colony-forming ability of (g) MV4-11, (h) U937 and (i) HL60 human AML cells treated with the indicated concentrations of Gbp. MV4-11, HL60 cells (300 per well) or U937 (100 per well) were plated in triplicate. Photomicrographs show representative colonies formed. Scale bars, 200 μm. Error bars, s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. j, BCAT1 expression in human patients with de novo AML. Data for BCAT1 expression levels from the TCGA AML dataset were divided into quartiles and compared. On average, the top quartile cohort showed 1.6-fold higher expression level than the bottom quartile. **P < 0.01.

Extended Data Figure 8 Impact of BCAT1 knockdown in K562 cells.

a, b, Effect of (a) BCAT1 knockdown or (b) Gbp treatment on the intracellular concentrations of glutamate and BCAAs in K562 cells. The shCtrl or PBS control values were set as 100%. Error bars, s.e.m. n = 10 each for (a) and n = 3 each for (b). *P < 0.05, **P < 0.01. NS, not statistically significant (P > 0.05). c, AKT activation status in BCAT1- or MSI2-knockdown K562 cells. K562 cells treated with shCtrl, shBCAT1 or shMSI2 were analysed by western blotting for pAKT (at Thr308 or Ser473), total AKT, hBCAT1, hMSI2 and HSP90 levels. d, Effect of α-ketoglutarate supplementation on the colony-forming ability of BCAT1-knockdown cells. K562 cells transduced with shCtrl (−) or shBCAT1-d (+) were plated in triplicate with or without 1 mM dimethyl-α-ketoglutarate (KG) and/or 4 mM BCAAs as indicated. n = 3 technical replicates. Error bars, s.e.m. *P < 0.05, **P < 0.01. NS, not statistically significant (P > 0.05).

Extended Data Figure 9 MSI2 and BCAT1 expression in human cancer.

a, Microarray data analysis of MSI2 expression in human CML. Gene expression data of chronic (grey, n = 57), accelerated (pink, n = 15) and blast crisis (blue, n = 41) phase patients were retrieved from the GEO database (accession number GSE4170). Bar, the normalized expression value in each specimen. b, Co-expression analysis of the BCAT1 and MSI2 genes in human cancer. Pearson correlation coefficients were used to evaluate the extent of co-expression patterns. c, Schematic of the human BCAT1 transcript. Bars, the putative MBEs (r(G/A)U1–3AGU). Forty MBEs were identified within the 3′ UTR of BCAT1. CDS, coding sequence for hBCAT1 protein. d, K562 cells infected with lentiviral shRNA against MSI2 (shMSI2) or shCtrl (−) were analysed by western blotting for pS6 kinase (at Thr389), total S6K, hMSI2 and HSP90 levels. Note that MSI2 knockdown reduced the levels of BCAT1 protein and pS6K.

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Hattori, A., Tsunoda, M., Konuma, T. et al. Cancer progression by reprogrammed BCAA metabolism in myeloid leukaemia. Nature 545, 500–504 (2017). https://doi.org/10.1038/nature22314

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