Garcia, Elaine G

Garcia, Sara P

Iyer, Sowmya

Langenau, David M

Loontiens, Siebe

Sadreyev, Ruslan I

Speleman, Franki

English

Elaine G. Garcia

Sowmya Iyer

Sara P. Garcia

Siebe Loontiens

Ruslan I. Sadreyev

Frank Speleman

David M. Langenau

Crossref

Leukemia

Cell of origin dictates aggression and stem cell number in acute lymphoblastic leukemia

Ghent University Academic Bibliography

Leukemiahttps://doi.org/10.1038/s41375-018-0130-0BRIEF COMMUNICATIONAcute lymphoblastic leukemiaCell of origin dictates aggression and stem cell number in acutelymphoblastic leukemiaElaine G. Garcia1,2,3,4 ● Sowmya Iyer1,2,3,4 ● Sara P. Garcia 1,2,3,4 ● Siebe Loontiens5 ● Ruslan I. Sadreyev1,4 ●Frank Speleman5 ● David M. Langenau1,2,3,4Received: 6 January 2018 / Revised: 5 March 2018 / Accepted: 29 March 2018© Macmillan Publishers Limited, part of Springer Nature 2018Acute lymphoblastic leukemia (ALL) comprises a highlyheterogeneous set of diseases that are deﬁned by their cellsof origin, stage of maturation arrest, and the underlyingoncogenic driver pathways. Subclassiﬁcation of lymphoidneoplasms is often based on the presumed cell of originbased on T and B progenitor gene expression. In general, T-ALL has a worse prognosis and requires more intensivetherapies to achieve similar remission rates as their B-ALLcounterparts [1, 2]. Given the common origin of these cellsfrom committed lymphoid progenitors, investigators havesuggested that the differences in clinical presentation of T-ALL and B-ALL are likely accounted for by the underlyingdifferences in the oncogenic drivers of transformation. Forexample, T-ALLs often express oncogenic transcriptionfactors and acquire activating NOTCH1 mutations thatelevate MYC activity to drive tumor initiation and main-tenance [3, 4]. By contrast, B-ALLs are more frequentlydriven by chromosomal rearrangements that create fusiononcogenes and have recurrent chromosomal aberrations [5].Despite these differences, MYC is also commonly activatedin human B-ALL [6] and drives leukemia initiation andgrowth in mouse models [7], suggesting important roles forthis pathway in both T-ALL and B-ALL. It is clear thatunderlying oncogenic drivers exert important roles in reg-ulating the functional characteristics of leukemia cells,including regulating the overall fraction of leukemia stemcells (LSCs) that drive continued tumor growth and relapsefollowing therapy resistance. Yet, to date, the effect of celllineage on inﬂuencing LSC number and self-renewal islargely unknown, accounted for in part, by lack of experi-mental models to address these questions.Here, we have used a zebraﬁsh transgenic model of Myc-induced ALL to investigate the roles of cell lineage onmodulating growth, aggression, and LSC frequency in T, B,and biphenotypic ALL. Previously, the rag2-Myc trans-genic zebraﬁsh model has been exploited to develop robustT-ALL models when introduced into AB strain zebraﬁsh[8]. Moreover, ﬁndings in this model have led to signiﬁcantnew insights into human T-ALL [9, 10]. Using this sametransgenic approach, we had previously generated leuke-mias in syngeneic CG1 strain zebraﬁsh and performedlarge-scale cell transplantation assays to assess latency andLSC frequency differences between intra-tumoral and inter-tumoral clones [9]. These experiments required implantingsingle LSCs into large cohorts of transplant animals, thuscreating monoclonal tumors (schematic shown in Fig. 1a).Each monoclonal leukemia was then assessed for latency ofregrowth and LSC frequency using large-scale limitingdilution cell transplantation [9]. From these previouslypublished studies, we uncovered that clonal heterogeneity iscommon in T-ALL, with individual leukemia clones exhi-biting striking differences in latency, aggression, and LSCfrequency, and also identiﬁed the PI3-kinase/AKT pathwayas a driver of elevated growth and LSC frequency in bothzebraﬁsh and human disease [9].Using this previously generated library of well-annotatedmonoclonal ALLs deﬁned by Blackburn et al. [9], weselected leukemias that harbored high (>1%) and lownumbers of LSCs (<1%, Supplemental Table 1) [9]. Theseleukemias are reported using the same nomenclature fromBlackburn et al [9] and were subjected to bulk RNAsequencing. Principal Component Analysis (PCA) wasthen performed to identify molecular differences betweenclones. Principal Component 1 (PC1) and PC2 represent thetop two gene expression proﬁles derived as dimensionsfrom Principal Component Analysis. This analysisThese authors contributed equally: Sowmya Iyer, Sara P. Garcia.* David M. Langenaudlangenau@mgh.harvard.eduExtended author information available on the last page of the articleElectronic supplementary material The online version of this article(https://doi.org/10.1038/s41375-018-0130-0) contains supplementarymaterial, which is available to authorized users.1234567890();,:1234567890();,:conﬁrmed that replicate samples always segregated withone another, and more importantly, identiﬁed two easilydiscernable molecular subgroups of leukemia, labeled group1 (in blue) and group 2 (in red, Fig. 1b). Interestingly, thetwo clusters in the PCA corresponded to signiﬁcant differ-ences in tumor behavior: group 1 leukemias hadE. G. Garcia et al.signiﬁcantly shorter latency of leukemia regrowth in trans-planted ﬁsh and had more LSCs when compared with group2 (Fig. 1c, Wilcoxin Rank Order Test). Hierarchical clus-tering using the 100 most highly variable, positively andnegatively correlated transcripts identiﬁed from PC1,afﬁrmed the classiﬁcation of two molecularly distinct ALLgroups (Fig. 1d, Supplemental Table 2). Unexpectedly,GSEA analysis uncovered that PC1 was deﬁned by T and Bcell lineage genes, with T cell genes being highly expressedin the fast growing and high LSC containing leukemiaswhile B cell genes were conﬁned to leukemias with longerlatency and low LSCs (Fig. 1d, e). PC2 genes comprisedproliferation genes (Supplemental Table 3), identifying T-ALL clone 8.3 as having lower overall proliferativepotential and likely accounting for its low percentage ofLSCs.Given that the rag2-Myc transgenic model had nevergenerated B-ALL in other strains of zebraﬁsh [8–11], wenext sought to independently conﬁrm cell lineage byassessing T and B cell receptor expression. As expected, allgroup 1 leukemias expressed tcr-beta, with most expressinga single unique variable region, independently conﬁrming(1) derivation from a single LSC, (2) monoclonality of eachALL, and (3) correct classiﬁcation as T-ALL. In contrast towhat is seen in human, the tcrbc1 and c2 constant regionsare not excised from the zebraﬁsh genomic locus followingreceptor recombination and hence each can be used tocreate a functional receptor following splicing. As reportedin human, a small subset of T cells can also express twoTCR-beta chains [12], likely accounting for the expressionfrom both recombined alleles in T-ALL clone 15.2.Meanwhile, clones 9.2 and 10.2 failed to express tcr-beta orother T cell receptors including alpha, delta, and gamma,but rather expressed the constant region of IgHC (Fig. 2a).Importantly, only the IgHC constant regions for IgM, IgD,and IgZ were expressed while the variable gene segmentswere not, suggesting that the IgHC locus is open but thatrag1/2-mediated receptor recombination had not yetoccurred in these B-ALLs (Supplemental Table 4). Addi-tional gene expression analysis showed that T-ALLs werearrested at a CD4+/CD8+cortical thymocyte stage, as pre-viously described [13], while B-ALLs 9.2 and 10.2 expressgenes pro-B marker genes including rag1/2, ebf1, pax5, dntt(tdt), and cd79a (Fig. 2b). Remarkably, Myc expressionlevels were not signiﬁcantly different between T-ALL andB-ALLs (Supplemental Figure 1), failing to account for theobserved differences in LSC frequency and latency differ-ences between leukemias. Altogether, these results showthat CG1 strain zebraﬁsh develop both Myc-induced T-ALLand B-ALL and that cell of origin likely has important rolesin regulating LSC frequency and overall aggression. Giventhat all previous leukemia generated in this model weresuggested to be T-ALLs, our reﬁned analysis has led to theunexpected ﬁnding of B-ALL in a small fraction ofCG1 strain ﬁsh and suggest mis-classiﬁcation of a subset ofleukemias in our previous work [9].In completing our receptor gene expression studies, wealso observed that leukemia 12.2 had both T and B cellcharacteristics. This leukemia recombined and expressedtcr-beta, failed to express IG heavy chain, and exhibitedexpression of both T and B lineage markers along withunique genes not found in the other leukemias (Fig. 2b).These data raised the interesting possibility that leukemia12.2 was a mixed phenotypic acute leukemia (MPAL). Infact, the World Health Organization recently published arevision for acute leukemia classiﬁcation that now includesnew criteria for classiﬁcation and diagnosis of MPAL,uncovering that MPALs occur in ~2–5% of pediatric andadult acute leukemia [14]. Because clinical MPAL casescan be further subclassiﬁed into either bilineal, a leukemiathat contains two distinct blast populations, or biphenotypicALL, a leukemia that contains a single blast population thatexpresses multiple lineage markers [14], we next usedsingle-cell gene expression analysis to clarify if leukemia12.2 was a mixture of two distinct blast types or if they werebiphenotypic [13]. Principal component analysis showedthat tumor 12.2 was molecularly distinct and separatedcleanly into its own PCA grouping when compared withnon-transformed thymic T cells, marrow-derived B cellsand three independent T-ALL clones (Fig. 2c). Remarkably,gene expression correlation analysis revealed that individualleukemia cells from 12.2 indeed expressed both T and Bcell genes, conﬁrming assignment as biphenotypic B/T-ALL (Fig. 2d). Human biphenotypic B/T-ALL is exceedingrare and to date has been characterized by CD3ε T cellmarker expression in combination with CD19 and eitherCD79a, CD22, or CD10 B cell marker expression [15].Zebraﬁsh orthologs to many of these genes have yet to befully described and antibodies are lacking. Moreover,detailed transcriptional analysis of human B/T-ALL has yetto be completed, obviating direct comparison of human andFig. 1 Myc-induced ALLs comprise two distinct molecular subtypesthat differ in latency and leukemia stem cell number. a A schematic ofthe cell transplantation screen previously used to identify differencesin latency and leukemia stem cell number (LSCs) between ALL clones(Blackburn et al. [9]). ** denotes p= 0.0065, by Wilcoxon rank ordertest. b RNA sequencing and principal component analysis of ALLswith high and low LSCs. All samples, except for 14.1.1.1 and 8.4.1.1,were assessed as technical replicates. Group 1 leukemias are demar-cated by blue shading, while group 2 leukemias have been shaded inred in panels b, c, and d. c The two molecularly-deﬁned subgroupsdiffer in latency and LSC frequency when assessed by Wilcoxon RankSum Test. d Heat map and hierarchical clustering of group 1 and group2 leukemias showing the top 100 genes positively and negativelycorrelated with PC1. e Gene set enrichment analysis using genespositively and negatively correlated with PC1. Genes positively cor-related with PC1 are associated with T cells while those found to benegatively correlated with PC1 were B cell associated genesCell of origin dictates aggression and stem cell number in acute lymphoblastic leukemiazebraﬁsh disease at this time. Future experiments will assessif there are species-speciﬁc differences in this disease entityor, more importantly, if common molecular programs drivetheir growth.In summary, our results have uncovered unexpectedzebraﬁsh strain differences in driving cell fate decisions inthe genesis of ALL and provide new and exciting avenuesfor modeling B-ALL and biphenotypic B/T-ALL usingFig. 2 B cell molecular programs drive lower aggression and stem cellactivity in acute lymphoblastic leukemia. a T cell receptor beta and Igheavy chain expression within individual ALL clones. Left plot showsonly the tcrb variable regions that were expressed within each clone.Right plot displays expression for each tcrb and IgHC constant regionfor clones in the same order as presented on the left. b Heatmapshowing gene expression of T and B cell associated genes and genesspeciﬁcally expressed in biphenotypic B/T-ALL clone 12.2. c Single-cell quantitative real-time PCR and principal component analysisvisualization showing that 12.2 represents a clearly distinct molecularsubtype when compared with T-ALL clones 8.4.1.1, 13.1.1.1, and14.1.1.1, and normal rag2-dsRed+thymic T cells and marrow-derivedB cells. d Gene expression correlation comparing T vs B cell geneswithin individual ALL cells (x-axis vs. y-axis, respectively), con-ﬁrming that clone 12.2 is a bi-phenotypic B/T-ALLE. G. Garcia et al.zebraﬁsh models. To date only a single report has char-acterized the development of a B cell leukemia model in thezebraﬁsh and no robust models of biphenotypic B/T-ALLhave been reported [16]. Perhaps more importantly, wehave used our zebraﬁsh model to show that cell lineage is amajor determinant of oncogenic phenotypes in ALL, with Tcell pathways driving elevated growth, aggression, and LSCself-renewal. By contrast, intrinsic B cell pathway activa-tion leads to reduced growth and leukemia stem cells. Theseresults provide new insights into the clinical differencesbetween T-ALL vs. B-ALL and suggest that underlyingdifferences in cell of origin likely account for superiorclinical outcome in B-ALL patients, with the prediction thatB cell lineage leukemias will have lower aggression andoverall numbers of relapse driving LSCs.Acknowledgements We thank Drs. Jessica Blackburn and FinolaMoore for supplying zebraﬁsh leukemias for RNA sequencing andFluidigm single cell PCR analysis; the MGH Flow Cytometry Core forhelp with single cell sorting; Na Qu and Dr. Toshi Shioda for help withNextGen sequencing; and Dr. Antony Anselmo for superior bioin-formatics analysis. This work is supported by NIH grantR24OD016761 and R01CA211734 (DML) and by the Fund for Sci-entiﬁc Research Flanders (FWO Vlaanderen, doctoral grant SL).Compliance with ethical standardsConﬂict of interest The authors declare that they have no conﬂict ofinterest.References1. Cortelazzo S, Ponzoni M, Ferreri AJ, Hoelzer D. Lymphoblasticlymphoma. Crit Rev Oncol Hematol. 2011;79:330–43.2. Goldberg JM, Silverman LB, Levy DE, Dalton VK, Gelber RD,Lehmann L, et al. Childhood T-cell acute lymphoblastic leukemia:the Dana-Farber Cancer Institute acute lymphoblastic leukemiaconsortium experience. J Clin Oncol. 2003;21:3616–22.3. 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Langenau DM, Traver D, Ferrando AA, Kutok JL, Aster JC,Kanki JP, et al. Myc-induced T cell leukemia in transgenic zeb-raﬁsh. Science. 2003;299:887–90.9. Blackburn JS, Liu S, Wilder JL, Dobrinski KP, Lobbardi R,Moore FE, et al. Clonal evolution enhances leukemia-propagatingcell frequency in T cell acute lymphoblastic leukemia throughAkt/mTORC1 pathway activation. Cancer Cell. 2014;25:366–78.10. Lobbardi R, Pinder J, Martinez-Pastor B, Theodorou M, Black-burn JS, Abraham BJ, et al. TOX regulates growth, DNA repair,and genomic instability in T-cell acute lymphoblastic leukemia.Cancer Discov. 2017;7:1336–53.11. Gutierrez A, Feng H, Stevenson K, Neuberg DS, Calzada O, ZhouY, et al. Loss of function tp53 mutations do not accelerate theonset of myc-induced T-cell acute lymphoblastic leukaemia in thezebraﬁsh. Br J Haematol. 2014;166:84–90.12. Padovan E, Giachino C, Cella M, Valitutti S, Acuto O, Lanza-vecchia A. Normal T lymphocytes can express two different T cellreceptor beta chains: implications for the mechanism of allelicexclusion. J Exp Med. 1995;181:1587–91.13. Moore FE, Garcia EG, Lobbardi R, Jain E, Tang Q, Moore JC,et al. Single-cell transcriptional analysis of normal, aberrant, andmalignant hematopoiesis in zebraﬁsh. J Exp Med.2016;213:979–92.14. Weinberg OK, Arber DA. Mixed-phenotype acute leukemia:historical overview and a new deﬁnition. Leukemia.2010;24:1844–51.15. Arber DA, Orazi A, Hasserjian R, Thiele J, Borowitz MJ, Le BeauMM, et al. The 2016 revision to the World Health Organizationclassiﬁcation of myeloid neoplasms and acute leukemia. Blood.2016;127:2391–405.16. Sabaawy HE, Azuma M, Embree LJ, Tsai H-J, Starost MF,Hickstein DD. TEL-AML1 transgenic zebraﬁsh model of pre-cursor B cell acute lymphoblastic leukemia. Proc Natl Acad SciUSA. 2006;103:15166–71.AfﬁliationsElaine G. Garcia1,2,3,4 ● Sowmya Iyer1,2,3,4 ● Sara P. Garcia 1,2,3,4 ● Siebe Loontiens5 ● Ruslan I. Sadreyev1,4 ●Frank Speleman5 ● David M. Langenau1,2,3,41 Department of Pathology, Massachusetts General HospitalResearch Institute, Boston, MA 02114, USA2 Center for Cancer Research, Massachusetts General Hospital,Charlestown, MA 02129, USA3 Harvard Stem Cell Institute, Boston, MA 02114, USA4 Center for Regenerative Medicine, Massachusetts GeneralHospital, Boston, MA 02114, USA5 Cancer Research Institute Ghent (CRIG) and Center for MedicalGenetics, Ghent University, Ghent, BelgiumCell of origin dictates aggression and stem cell number in acute lymphoblastic leukemia

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Cell of origin dictates aggression and stem cell number in acute lymphoblastic leukemia

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Ghent University Academic Bibliography