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1 doi: /nature22314 Supplementary Discussion In mammals, BCAAs are essential amino acids that must be supplied from food. These dietary BCAAs are subsequently used for protein synthesis or are catabolized to BCKAs via deamination, primarily in muscle tissues 1,2. BCKAs that are generated in muscle are further oxidized to form acetyl- and succinyl-coas for ATP and fatty acid synthesis, or they are released into the circulation for reamination to BCAAs in the liver and other tissues under normal healthy conditions 3-5. Consistent with these previous reports, we found that BCKAs are present in leukemic mice at levels equivalent to 20-50% of the corresponding BCAA species (Extended Data Fig. 2c-e). As demonstrated under normal conditions, muscle tissues may serve as a major source of the circulating BCKAs in plasma. Collectively, these findings suggest the presence of an oncogenic metabolic cycle that enables BCAA production in cancer cells by recycling BCKAs in the circulation. Enhanced intracellular BCAA production by activated may also contribute to muscle wasting, atrophy and cancer cachexia by continuously utilizing plasma BCKAs, which in turn promote the breakdown of BCAAs in muscles 6. The systemic metabolic changes induced by cancer cells and non-cell autonomous alterations of cellular metabolism in nonhematopoietic tissues 7, in addition to the increased BCAA production in leukemia cells, may collectively contribute to the significant changes in the plasma BCAA levels (Extended Data Fig. 1e). Upon knockdown, we observed decreased levels of glutamate in K562 cells (Extended Data Fig. 8a and 8b). Glutamate plays a crucial role in diverse intracellular processes and cellular functions Glutamate is generated from either alpha-ketoglutarate or glutamine and is utilized in a number of intracellular reactions catalyzed by various enzymes, including glutamine synthetase (GLUL), glutaminases (GLS), glutamate dehydrogenases (GLUD), alanine aminotransferases (GPT), aspartate aminotransferases (GOT) and BCAT. In addition, cancer cells are known to be glutamine-dependent due to the activation of glutaminolysis, which significantly contributes to an increase in intracellular glutamate flux 9,10. Consistently, we have found that some of the genes that are responsible for Glu 1

2 RESEARCH metabolism are in fact differentially expressed in BC-CML (data not shown). Thus, the overall glutamate level is determined as a result of all these metabolic reactions and cellular usages, and the contribution of BCAT transamination is likely to represent only a fraction of the total Glu pool. Mammalian genomes encode two paralogous BCAA transaminase genes: cytoplasmic and mitochondrial BCAT2. Importantly,, but not BCAT2, is activated in BC-CML, suggesting a requirement for an increased BCAA availability in the cytoplasm. Previous studies have identified several amino acid sensors upstream of mtorc1 in the cytoplasm. The SLC3A2-SLC7A5 amino acid transporter complex mediates the uptake of cytoplasmic BCAA into lysosomes, which is essential for the amino acid-induced mtorc1 activation triggered by vacuolar H + - ATPase Recently, Wolfson and Chantranupong et al. demonstrated that cytosolic leucine directly binds to Sestrin2 and disrupts the Sestrin2-GATOR2 interaction, which subsequently activates the Rag GTPases for mtorc1 activation 15. Cytosolic leucyl-trna synthetase (LRS), which catalyzes the ligation of leucine to trna Leu, also mediates nutrient-induced mtor signals 16,17. Thus, the - mediated increase of BCAA in the cytoplasm, but not in the mitochondria, is required for mtorc1 activation in leukemia cells. The mtorc1 pathway may serve as an alternative therapeutic target in leukemia. We showed that rapamycin treatment reversed the BCAA suppression of the knockdown phenotype in K562 BC-CML cells (Fig. 3i and 3k). Everolimus/RAD001, a rapamycin analog, acts synergistically with TKIs and is effective for the treatment of imatinib-resistant CP-CML and acute lymphoblastic leukemia Consistently, we found that the drug inhibits colony formation of K562 cells and phenocopies knockdown (data not shown). Intracellular AAs are essential intermediates for protein synthesis and energy production and serve as precursors for biosynthetic reactions in rapidly proliferating cells because of their roles as anaplerotic substrates. In addition to BCAAs, the reaction also generates alpha-ketoglutarate (KG), which can enter the Krebs cycle and replenish intermediates. We observed that KG enhanced the effects of BCAAs on colony formation and on mtorc1 activation in K562 BC- 2

3 RESEARCH CML cells, although KG alone did not exhibit a significant enhancement (Extended Data Fig. 8d). A recent study showed that intracellular KG promotes self-renewal and inhibits differentiation in embryonic stem cells through epigenetic modifications regulated by the KG-dependent histone demethylases UTX and JMJD3 21. It is necessary to determine whether activation affects self-renewal and differentiation potential by altering the leukemia epigenome during cancer progression. Supplementary References 1. Odessey, R., Khairallah, E. A. & Goldberg, A. L. Origin and possible significance of alanine production by skeletal muscle. J. Biol. Chem. 249, (1974). 2. Hutson, S. M., Sweatt, A. J. & Lanoue, K. F. Branched-chain amino acid metabolism: implications for establishing safe intakes. J. Nutr. 135, 1557S 64S (2005). 3. Walser, M., Lund, P., Ruderman, N. B. & Coulter, A. W. Synthesis of essential amino acids from their alpha-keto analogues by perfused rat liver and muscle. J Clin Invest 52, (1973). 4. Livesey, G. & Lund, P. Enzymic determination of branched-chain amino acids and 2-oxoacids in rat tissues. Transfer of 2-oxoacids from skeletal muscle to liver in vivo. Biochem. J. 188, (1980). 5. Hutson, S. M., Lieth, E. & LaNoue, K. F. Function of leucine in excitatory neurotransmitter metabolism in the central nervous system. J. Nutr. 131, 846S 850S (2001). 6. Argilés, J. M., Busquets, S., Stemmler, B. & López-Soriano, F. J. Cancer cachexia: understanding the molecular basis. Nat. Rev. Cancer 14, (2014). 7. Mayers, J. R. et al. Elevation of circulating branched-chain amino acids is an early event in human pancreatic adenocarcinoma development. Nat Med 20, (2014). 8. Marsboom, G. et al. Glutamine Metabolism Regulates the Pluripotency Transcription Factor OCT4. Cell Rep 16, (2016). 9. DeNicola, G. M. & Cantley, L. C. Cancer's Fuel Choice: New Flavors for a Picky Eater. Mol Cell 60, (2015). 10. DeBerardinis, R. J. & Thompson, C. B. Cellular metabolism and disease: what do metabolic outliers teach us? Cell 148, (2012). 11. Zoncu, R. et al. mtorc1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase. Science 334, (2011). 12. Wang, S. et al. Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mtorc1. Science 347, (2015). 3

4 RESEARCH 13. Rebsamen, M. et al. SLC38A9 is a component of the lysosomal amino acid sensing machinery that controls mtorc1. Nature 519, (2015). 14. Milkereit, R. et al. LAPTM4b recruits the LAT1-4F2hc Leu transporter to lysosomes and promotes mtorc1 activation. Nat Commun 6, 7250 (2015). 15. Wolfson, R. L. et al. Sestrin2 is a leucine sensor for the mtorc1 pathway. Science 351, (2016). 16. Bonfils, G. et al. Leucyl-tRNA synthetase controls TORC1 via the EGO complex. Mol Cell 46, (2012). 17. Han, J. M. et al. Leucyl-tRNA synthetase is an intracellular leucine sensor for the mtorc1-signaling pathway. Cell 149, (2012). 18. Mohi, M. G. et al. Combination of rapamycin and protein tyrosine kinase (PTK) inhibitors for the treatment of leukemias caused by oncogenic PTKs. Proc. Natl. Acad. Sci. U.S.A. 101, (2004). 19. Mancini, M. et al. mtor inhibitor RAD001 (Everolimus) enhances the effects of imatinib in chronic myeloid leukemia by raising the nuclear expression of c-abl protein. Leuk. Res. 34, (2010). 20. Kuwatsuka, Y. et al. The mtor inhibitor, everolimus (RAD001), overcomes resistance to imatinib in quiescent Ph-positive acute lymphoblastic leukemia cells. Blood Cancer J 1, e17 (2011). 21. Carey, B. W., Finley, L. W. S., Cross, J. R., Allis, C. D. & Thompson, C. B. Intracellular α-ketoglutarate maintains the pluripotency of embryonic stem cells. Nature 518, (2015). 4

5 Figure 1b Figure 1c RESEARCH Bcat1 Bcat1 20 kda B2m 1 Hsp90 Figure 3j Figure 3k β-tub β-tub 1 Figure 4e 1 Msi

6 RESEARCH Ex. 1i Ex. 7b Bcat1 B2m β-tub Ex. 8c Ex. 9d pakt (T308) MSI2 20 kda 1 MSI2 pakt (S473) 1 AKT 6