Hematopoietic stem cells (HSCs) are used clinically in bone marrow (BM) transplantation due to their unique ability to reform the entire hematopoietic system. Recently, we reported that HSCs are highly sensitive to valine, one of the three branched-chain amino acids (BCAAs) in addition to isoleucine and leucine. Dietary depletion of valine could even be used as a conditioning regimen for HSC transplantation. Here, we report that HSCs are highly sensitive to the balance of BCAAs, with both proliferation and survival reduced by BCAA imbalance. However, low but balanced BCAA levels failed to rescue HSC maintenance. Importantly, in vivo depletion of all three BCAAs was significantly less toxic than depletion of valine only. We demonstrate that BCAA depletion can replace valine depletion as a safer alternative to BM conditioning. In summary, by determining HSC metabolic requirements, we can improve metabolic approaches to BM conditioning

Morita, Maiko

Nakauchi, Hiromitsu

Wilkinson, Adam C

Yamazaki, Satoshi

English

Apollo (Cambridge)

Experimental Hematology 2018;63:1216BRIEF COMMUNICATIONBranched-chain amino acid depletion conditions bone marrow forhematopoietic stem cell transplantation avoiding amino acidimbalance-associated toxicityTaggedPAdam C. Wilkinsona,b,c, Maiko Moritad, Hiromitsu Nakauchia,b,d, and Satoshi YamazakidTaggedPaInstitute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA; bDepartment ofGenetics, Stanford University, Stanford, CA, USA; cDepartment of Haematology, University of Cambridge, Cambridge, UK; dDivision of StemCell Therapy, Center for Stem Cell Biology and Regeneration Medicine, Institute of Medical Science, University of Tokyo, Tokyo 108-8639,Japan(Received 9 January 2018; revised 6 April 2018; accepted 18 April 2018)Offprint requests to:Biology and Regeneratof Tokyo, Tokyo 108-8Supplementary matonline at doi: 10.100301-472X/© 2018 IS(http://creativecommhttps://doi.org/10.101Hematopoietic stem cells (HSCs) are used clinically in bone marrow (BM) transplantation dueto their unique ability to reform the entire hematopoietic system. Recently, we reported thatHSCs are highly sensitive to valine, one of the three branched-chain amino acids (BCAAs) inaddition to isoleucine and leucine. Dietary depletion of valine could even be used as a condition-ing regimen for HSC transplantation. Here, we report that HSCs are highly sensitive to the bal-ance of BCAAs, with both proliferation and survival reduced by BCAA imbalance. However,low but balanced BCAA levels failed to rescue HSC maintenance. Importantly, in vivo depletionof all three BCAAs was significantly less toxic than depletion of valine only. We demonstratethat BCAA depletion can replace valine depletion as a safer alternative to BM conditioning. Insummary, by determining HSC metabolic requirements, we can improve metabolic approachesto BM conditioning. © 2018 ISEH – Society for Hematology and Stem Cells. Published by Elsev-ier Inc. This is an open access article under the CC BY license. (http://creativecommons.org/licenses/by/4.0/)TaggedPBone marrow (BM) or hematopoietic stem cell (HSC)transplantation (HSCT) is a potentially curative treat-ment for a range of hematological disorders, such asimmunodeficiency diseases [1]. However, “space”within the recipient’s HSC niche [2,3] must be firstmade to allow donor HSCs to engraft [4]. Unfortu-nately, the morbidity and mortality associated with tra-ditional BM conditioning regimens, radiation and/orchemotherapy, currently limit the application of HSCT[5]. Recently, we reported that dietary valine depletioncould be used to condition mice for HSCT [6]. Impor-tantly, this type of metabolic BM conditioning wasDivision of Stem Cell Therapy, Center for Stem Cellion Medicine, Institute of Medical Science, University639, Japan; E-mail: y-sato4@ims.u-tokyo.ac.jperial associated with this article can be found16/j.exphem.2018.04.004.EH – Society for Hematology and Stem Cells. Publishedons.org/licenses/by/4.0/).6/j.exphem.2018.04.004entirely reversible, with mice returning to full healthand fertility after transplantation and return to a com-plete diet. Here, we describe important optimization ofthis novel conditioning approach for improved safetyand tolerance based on further characterization of themetabolic sensitivity of HSCs.TaggedPNearly 60 years ago, Harper [7] proposed that aminoacid imbalance could be a mechanism of disease. Valineis a one of three branched-chain amino acids (BCAAs) inaddition to isoleucine and leucine. BCAA imbalance hasbeen suggested to cause cellular toxicity, including neuro-toxicity [8]. Stimulated by these reports, we investigatedthe sensitivity of HSCs to BCAA imbalance using ex vivoHSC expansion cultures. Expansion of mouse CD34/loKit+Sca1+Lineage (CD34-KSL) HSCs [9] was deter-mined after a 7-day culture in Dulbecco’s modifiedEagle’s medium (DMEM)/F12-based self-renewal condi-tions [10]. DMEM/F12 medium was used because it con-tains approximately equimolar concentrations of BCAAs:by Elsevier Inc. This is an open access article under the CC BY license.A.C. Wilkinson et al. / Experimental Hematology 2018;63:1216 13451 mmol/L valine, 415 mmol/L leucine, and 451 mmol/Lisoleucine.TaggedPWhen the concentration of valine was reduced to »10%in this context (valine-low), we only mildly inhibitedHSC expansion (Figures 1A and 1B). However, whenvaline was reduced to »10% while the concentration ofisoleucine/leucine (I/L) was simultaneously increased five-fold (valine-low, I/L-high), HSC expansion was blockedcompletely (Figures 1A and 1B). These results reproducedour initial screening [6], which used medium containing asimilar BCAA imbalance. In contrast, increasing I/L con-centrations fivefold in complete conditions (I/L-high)increased HSC expansion (Supplementary Figure E1,online only, available at www.exphem.org). Notably,reducing all three BCAAs to »10% in BCAA-low condi-tions (Figures 1A and 1B) only resulted in a modest, non-statistically significant reduction in HSC expansion.Through single-cell assays, we found that BCAA imbal-ance (valine-low, I/L-high) blocked HSC expansionthrough a combination of increasing cell death and inhibit-ing proliferation (Figures 1C and 1D). However, whereasBCAA-low conditions did not influence HSC proliferationor survival significantly, it was not able to sustain in vivofunction of HSCs activity, as indicated by decreasedreconstitution capability of these cultured HSCs(Figure 1E). We therefore conclude that BCAA imbalancereduces HSC proliferation and survival, whereas lowvaline results in poor HSC maintenance.TaggedP o further investigate the translational potential ofthese findings, we investigated the consequences ofBCAA imbalance on human cord blood-derivedCD34+CD38Lineage hematopoietic stem and progen-itor cells (HPSCs). As seen with mouse HSCs, BCAAimbalance (valine-low, I/L-high) caused greater toxic-ity, with essentially no cells surviving the 7-day culture(Figures 1F and 1G). However, whereas BCAA-lowconditions rescued mouse HSC expansion, it failed torescue human HSPC expansion (Figures 1F and 1G).This is likely due to our previous finding that humanHSPCs are also sensitive to leucine depletion [6].TaggedPOne concern with our initial proof-of-concept dem-onstration of metabolic BM conditioning [6] was thepotential toxicity of dietary valine depletion. Althoughthe effects of this metabolic conditioning approachwere entirely reversible in mice after treatment (unlikeradiation conditioning), the conditioning process didcause on-treatment side effects. Given that the imbal-ance of amino acids (and particularly BCAAs) is asso-ciated with systemic toxicities [8], we wonderedwhether our new understanding that HSCs were depen-dent of valine irrespective of BCAA balance could beused to improve non-chemoirradiative HSCT. We ini-tially compared the consequences of dietary valine andBCAA depletion on hematopoiesis in mice. Consistentwith our in vitro data, a BCAA-free (BCAA) dietlargely mirror a valine-free (valine) diet, displayingreduced phenotypic HSC frequencies within the BM(Figure 2A) and peripheral blood (PB) whole bloodcell counts (Figure 2B).TaggedPInterestingly, although we observed a reduction inred cell blood counts after a valine diet, this anemiawas milder with a BCAA diet (Figure 2C). This sug-gests erythropoiesis may also be sensitive to BCAAimbalance and implies that the BCAA diet is bettertolerated. Our previous valine dieting experimentshad also been hampered by mortality after 4 weeks [6].We therefore investigated the survival of mice onvaline and BCAA diets directly (Figure 2D). Micesurvived for a median of 49 days on the valine diet.In contrast, a BCAA diet was much better toleratedby mice, with all mice surviving >7 weeks and 70%surviving 14 weeks. These data are also consistent withthe report that a valine diet caused neurotoxicity inrats, but this was not observed after a BCAA diet[8]. Combined, these data suggest that the use of aBCAA diet displays better safety and tolerance com-pared with a valine diet.TaggedPFinally, we wanted to confirm that a BCAA dietcould be used as efficiently as a valine diet to condi-tion the BM for HSCT. We conditioned NOD.Cg-Prkdcscid Il2rgtm1Sug/ShiJic (NOG) mice [11] for 2weeks and then transplanted 104 C57BL/6 (C57BL/6NCrSlc) KSL cells before returning the diet to nor-mal. Both BCAA and valine diets resulted in closeto 100% PB chimerism in the long term (Figure 2E). ABCAA diet therefore affords comparable BM condi-tioning for HSCT, but with reduced nonspecific toxic-ity compared with a valine diet.TaggedPIn summary, we have demonstrated that BCAA bal-ance can resolve the sensitivity of HSCs to valine viaits influence on cell cycle progression, survival, andinfluence on HSC maintenance. From these insights,we have developed a second-generation metabolic BM-conditioning regimen using BCAA depletion, whichdisplays reduced in vivo systemic toxicity comparedwith valine depletion. These data provide an importantstep toward optimizing safe and well-tolerated meta-bolic BM-conditioning approaches for HSCT.TaggedPWe hope this further understanding of the metabolicrequirements of HSCs will lead to successful transla-tion of safe metabolic BM-conditioning regimens toHSCT [12]. Metabolic BM conditioning may also beuseful for basic HSC research, particularly for modelanimals in which radiation conditioning is challenging[13]. Through understanding the roles of BCAA-sensing mechanisms [1416] and BCAA metabolism[17,18] in HSC maintenance, we may also be able todevelop pharmacological strategies for BM condition-ing. Finally, it has been suggested recently that BCAAmetabolism regulates leukemic progression [19,20],Figure 1. BCAA imbalance caused by low valine and high I/L blocks HSC expansion through reducing survival and inhibiting proliferation.(A,B) Mouse BM CD34-KSL HSCs were expanded (40 cells/well) for 7 days in DMEM/F12-based media supplemented with 0.1% human serumalbumin, stem cell factor (SCF, 50 ng/mL), thrombopoietin (TPO, 50 ng/mL), and 1% S-clone SF-O3 medium supplement. Representative colonyimages (4£magnification) are shown in (A) and average cell numbers per well in (B). (C,D) Single HSCs were monitored over 5 days in themedia described above. The percentage of empty wells in shown in (C). Estimated average number of hours per cell division event based ontotal number of cells at day 5 in (D). Forty-eight cells were analyzed per condition. (E) Average donor PB chimerism § SEM from competitivetransplantation assays using 7-day cultured HSCs from (A) at 16 weeks after transplantation. C57BL/6 Ly5.1 HSCs were injected into irradiatedC57BL/6 Ly5.2 mice (5 mice/condition) alongside 106 Ly5.1/Ly5.2 whole BM competitor cells, as described previously [10]. All animal experi-ments described herein followed guidance and approval from the Animal Care and Use Committee, Institute of Medical Science, University ofTokyo, or the Administrative Panel on Laboratory Animal Care, Stanford University. (F,G) Human CD34+CD38Lineage HSPCs from umbili-cal cord blood (kindly provided the Stanford Binns Cord Blood Program) were expanded (300 cells/well) for 7 days in DMEM/F12-based mediasupplemented with 1% bovine serum albumin, SCF (50 ng/mL), TPO (50 ng/mL), FLT3L (50 ng/mL), interleukin-6 (IL-6, 20 ng/mL), IL-3(20ng/mL), and 1% S-clone SF-O3 medium supplement, as described previously [6]. Representative colony images are shown in (F) and averagecell numbers per well § SEM (n = 5) are shown in (G). Statistically significant differences (one-way ANOVA) are denoted by asterisks: *p > 0.05,**p > 0.01, ***p > 0.001. n.s.=not significant.14 A.C. Wilkinson et al. / Experimental Hematology 2018;63:1216A BDComplete-Valine-BCAA050100% PB chimerism MyeloidT cellB cellCComplete-Valine-BCAA0.0000.0020.0040.0060.0080.0100.012CD34-KSL BM frequency ***n.s******0 20 40 60 80 100020406080100Days% SurvivalComplete-Valine-BCAAMetabolic BM conditioningEComplete-Valine-BCAA******n.sComplete-Valine-BCAA020406080100WBC x10^3/ul******n.sComplete-Valine-BCAA60080010001200RBC x10^6/ul**n.sn.sComplete-Valine-BCAA****n.s2 weeksWBC counts4 weeks 2 weeksRBC counts4 weeksFigure 2. Dietary BCAA depletion is a less toxic metabolic conditioning regimen for HSCT. (AC) C57BL/6 mice were fed a complete, valine, orBCAA diet (Research Diet, Inc.). Average BM and PB parameters were determined (n = 5 mice/condition). (A) BM CD34-KSL frequency after 4 weeks.(B) PB whole blood cell counts after 2 and 4 weeks. (C) PB red blood cell counts after 2 and 4 weeks. Statistically significant differences (one-wayANOVA) are denoted by asterisks: *p > 0.05, **p > 0.01, ***p > 0.001. n.s.=not significant. (D) Survival of C57BL/6 mice on complete, valine, andBCAA diets (10 mice/condition). Statistical testing was done using the logrank test (***p > 0.001). (E) NOG mice were fed complete, valine, orBCAA diets for 2 weeks after an initial 48-hour fast and then injected with 104 (C57BL/6) BM KSL cells and the complete diet was restored gradually.No mortality was observed during transplantation. Average donor PB chimerism § SEM at 16 weeks after transplantation (n = 45 mice per condition) isshown.A.C. Wilkinson et al. / Experimental Hematology 2018;63:1216 15which means that targeting BCAAs may also have ther-apeutic value in other contexts.AcknowledgementsTaggedP he authors thank Y. Yamazaki, R. Ishida, R. Yama-moto, and Y. Nakauchi for excellent technical supportand the Stanford Binns Cord Blood Program for humancord blood samples.TaggedP his work was supported by the Japan Society forthe Promotion of Science, the Japan Science and Tech-nology Agency, Ministry of Education, Culture, Sport,Science, and Technology, the California Institute ofRegenerative Medicine, the Ludwig Foundation, theNational Institutes of Health National Center forAdvancing Translational Science Clinical and Transla-tional Science Award (UL1TR001085), and a GlennCenter Seed Grant. ACW is a Bloodwise Visiting Fel-low (15050). The content is solely the responsibility ofthe authors and does not necessarily represent the offi-cial views of the National Institutes of Health.Conflict-of-interest disclosureTaggedPHN is a cofounder and scientific advisor of ReproC-ELL, Inc. The remaining authors declare no competingfinancial interests.ReferencesTaggedP 1. Copelan EA. Hematopoietic stem-cell transplantation. N Engl JMed. 2006;354:1813–1826.TaggedP 2. Morrison SJ, Scadden DT. The bone marrow niche for haemato-poietic stem cells. Nature. 2014;505:327–334.TaggedP 3. Boulais PE, Frenette PS. Making sense of hematopoietic stemcell niches. Blood. 2015;125:2621–2629.TaggedP 4. Czechowicz A, Kraft D, Weissman IL, Bhattacharya D. Efficienttransplantation via antibody-based clearance of hematopoieticstem cell niches. Science. 2007;318:1296–1299.TaggedP 5. Gyurkocza B, Sandmaier BM. Conditioning regimens forhematopoietic cell transplantation: one size does not fit all.Blood. 2014;124:344–353.TaggedP 6. Taya Y, Ota Y, Wilkinson AC, et al. Depleting dietary valinepermits nonmyeloablative mouse hematopoietic stem cell trans-plantation. Science. 2016;354:1152–1155.TaggedP 7. Harper AE. Amino acid balance and imbalance. I. Dietary levelof protein and amino acid imbalance. J Nutr.. 1959;68:405–418.16 A.C. Wilkinson et al. / Experimental Hematology 2018;63:1216TaggedP 8. Cusick PK, Koehler KM, Ferrier B, Haskell BE. The neurotoxicityof valine deficiency in rats. J Nutr. 1978;108:1200–1206.TaggedP 9. Osaw M, Hanada K, Hamada H, Nakauchi H. Long-term lym-phohematopoietic reconstitution by a single CD34-low/negativehematopoietic stem cell. Science. 1996;273:242–245.TaggedP10. Ieyasu A, Ishida R, Kimura T, et al. An all-recombinant protein-based culture system specifically identifies hematopoieticstem cell maintenance factors. Stem Cell Rep. 2017;8:500–508.TaggedP11. Ito M, Hiramatsu H, Kobayashi K, et al. NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraft-ment of human cells. Blood. 2002;100:3175–3182.TaggedP12. Wilkinson AC, Yamazaki S. The hematopoietic stem cell diet.Int J Hematol (in press).TaggedP13. Stolfi JL, Pai CC, Murphy WJ. Preclinical modeling of hemato-poietic stem cell transplantation: advantages and limitations.FEBS J. 2016;283:1595–1606.TaggedP14. Wolfson RL, Chantranupong L, Saxton RA, et al. Sestrin2 is a leu-cine sensor for the mTORC1 pathway. Science. 2016;351:43–48.TaggedP15. Kalaitzidis D, Lee D, Efeyan A, et al. Amino acid-insensitivemTORC1 regulation enables nutritional stress resilience inhematopoietic stem cells. J Clin Invest. 2017;127:1405–1413.TaggedP16. Efeyan A, Comb WC, Sabatini DM. Nutrient-sensing mecha-nisms and pathways. Nature. 2015;517:302–310.TaggedP17. Brosnan JT, Brosnan ME. Branched-chain amino acids: enzymeand substrate regulation. J Nutr. 2006;136:207S–211S.TaggedP18. Ananieva EA, Wilkinson AC. Branched-chain amino acid metabo-lism in cancer. Curr Opin Clin Nutr Metab Care. 2018;21:64–70.TaggedP19. Hattori A, Tsunoda M, Konuma T, et al. Cancer progression byreprogrammed BCAA metabolism in myeloid leukaemia. Nature.2017;545:500–504.TaggedP20. Raffel S, Falcone M, Kneisel N, et al. BCAT1 restricts aKG lev-els in AML stem cells leading to IDHmut-like DNA hyperme-thylation. Nature. 2017;551:384–388.A.C. Wilkinson et al. / Experimental Hematology 2018;63:1216 16.e1Supplementary materialsTaggedPBA Mouse (CD34-/lo Kit+ Sca1+ Lin-) HSCs Human (CD34+ CD38- Lin-) HSPCsCompleteIL-highValine-lowValine-low IL-highBCAA-low0200040006000800010000Cell numberCompleteIL-highValine-lowValine-low IL-highBCAA-low01000020000300004000050000Cell numberSupplementary Figure E1. (A) Mouse BM CD34¡KSL HSCs were expanded (40 cell/well) for seven days in DMEM/F12-based medias supple-mented with 0.1% human serum albumin, SCF (50ng/mL), TPO (50ng/mL), and 1% S-clone SF-O3 medium supplement. (B) HumanCD34+CD38¡Lineage¡ HSPCs from umbilical cord blood were expanded (300 cells/well) for seven days in DMEM/F12-based medias supple-mented with 1% BSA, SCF (50ng/mL), TPO (50ng/mL), FLT3L (50ng/mL) IL-6 (20ng/ml), IL-3 (20ng/ml), and 1% S-clone SF-O3 medium sup-plement.

Branched-chain amino acid depletion conditions bone marrow for hematopoietic stem cell transplantation avoiding amino acid imbalance-associated toxicity.

https://www.repository.cam.ac.uk/bitstream/handle/1810/278510/1-s2.0-S0301472X18301735-main.pdf?sequence=4&isAllowed=y

Branched-chain amino acid depletion conditions bone marrow for hematopoietic stem cell transplantation avoiding amino acid imbalance-associated toxicity.

Abstract

Similar works

Full text

Available Versions

Apollo (Cambridge)