Acute Lymphoblastic Leukemia ARTICLE Enhanced hemato-endothelial specification Ferrata Storti Foundation during human embryonic differentiation through developmental cooperation between AF4-MLL and MLL-AF4 fusions Clara Bueno,1,2 Fernando J Calero-Nieto,3 Xiaonan Wang,3 Rafael Valdés-Mas,4 Francisco Gutiérrez-Agüera,1 Heleia Roca-Ho,1 Veronica Ayllon,5 Pedro J Real,5 David Arambilet,6 Lluis Espinosa,6,2 Raul Torres-Ruiz,1 Antonio Agraz-Doblas,1,7 Ignacio Varela,7 Jasper de Boer,8 Anna Bigas,6,2 Bertie Gottgens,3 Rolf Marschalek9 and Pablo Menendez1,2,10 Haematologica 2019 Josep Carreras Leukemia Research Institute and Department of Biomedicine, School 1 Volume 104(6):1189-1201 of Medicine, University of Barcelona, Spain; 2Centro de Investigación Biomédica en Red de Cáncer (CIBER-ONC), ISCIII, Barcelona, Spain; 3Department of Hematology, Cambridge Institute for Medical Research and Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, UK; 4Dreamgenics S.L. Oviedo. Spain; 5GENyO, Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regional Government and University of Granada, Department of Biochemistry and Molecular Biology, Granada, Spain; 6 Programa de Cáncer, Instituto Hospital del Mar de Investigaciones Médicas. Barcelona. Spain; 7Instituto de Biomedicina y Biotecnología de Cantabria (CSIC-UC-Sodercan), Departamento de Biología Molecular, Universidad de Cantabria, Santander, Spain; 8 Cancer Section, UCL Great Ormond Street Institute of Child Health, London, UK; 9 Institute of Pharmaceutical Biology, Goethe-University, Frankfurt, Germany and 10 Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain ABSTRACT Correspondence: T he t(4;11)(q21;q23) translocation is associated with high-risk infant CLARA BUENO pro-B-cell acute lymphoblastic leukemia and arises prenatally during cbueno@carrerasresearch.org embryonic/fetal hematopoiesis. The developmental/pathogenic con- tribution of the t(4;11)-resulting MLL-AF4 (MA4) and AF4-MLL (A4M) PABLO MENENDEZ pmenendez@carrerasresearch.org fusions remains unclear; MA4 is always expressed in patients with t(4;11)+ B-cell acute lymphoblastic leukemia, but the reciprocal fusion A4M is Received: July 18, 2018. expressed in only half of the patients. Because prenatal leukemogenesis Accepted: January 21, 2019. manifests as impaired early hematopoietic differentiation, we took advan- tage of well-established human embryonic stem cell-based hematopoietic Pre-published: January 24, 2019. differentiation models to study whether the A4M fusion cooperates with MA4 during early human hematopoietic development. Co-expression of doi:10.3324/haematol.2018.202044 A4M and MA4 strongly promoted the emergence of hemato-endothelial Check the online version for the most updated precursors, both endothelial- and hemogenic-primed. Double fusion- information on this article, online supplements, expressing hemato-endothelial precursors specified into significantly higher and information on authorship & disclosures: numbers of both hematopoietic and endothelial-committed cells, irrespec- www.haematologica.org/content/104/6/1189 tive of the differentiation protocol used and without hijacking survival/pro- ©2019 Ferrata Storti Foundation liferation. Functional analysis of differentially expressed genes and differen- Material published in Haematologica is covered by copyright. tially enriched H3K79me3 genomic regions by RNA-sequencing and All rights are reserved to the Ferrata Storti Foundation. Use of H3K79me3 chromatin immunoprecipitation-sequencing, respectively, con- published material is allowed under the following terms and firmed a hematopoietic/endothelial cell differentiation signature in double conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode. fusion-expressing hemato-endothelial precursors. Importantly, chromatin Copies of published material are allowed for personal or inter- immunoprecipitation-sequencing analysis revealed a significant enrichment nal use. Sharing published material for non-commercial pur- of H3K79 methylated regions specifically associated with HOX-A cluster poses is subject to the following conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode, genes in double fusion-expressing differentiating hematopoietic cells. sect. 3. Reproducing and sharing published material for com- Overall, these results establish a functional and molecular cooperation mercial purposes is not allowed without permission in writing between MA4 and A4M fusions during human hematopoietic develop- from the publisher. ment. haematologica | 2019; 104(6) 1189 C. Bueno et al. Introduction ment. We report a functional and molecular cooperation between MA4 and A4M fusions, which results in The mixed-lineage leukemia (MLL) gene encodes for an enhanced hemato-endothelial output during human H3K4 histone methyltransferase important in hematopoi- embryonic development. etic development.1 The human MLL gene is frequently rearranged in acute leukemia and typically confers a dis- mal outcome.2,3 Of particular interest is the Methods t(4;11)(q21;q23) translocation, which encodes the fusion proteins MLL-AF4 (MA4) and AF4-MLL (A4M), and is Vector construction and lentiviral transduction associated with infant B-cell acute lymphoblastic The cDNA for MA4 and A4M were subcloned into the pRRL- leukemia (B-ALL). This t(4;11)+ infant leukemia is charac- EF1α-PGK-NEO vector.11,16 Both fusions have been described pre- terized by a very brief latency, raising the question of how viously (Online Supplementary Figure S1A).11,23 We used the follow- it evolves so quickly.4 Moreover, the exceptionally high ing lentivectors containing either neomycin or dTo for cell selec- concordance rate of t(4;11)+ B-ALL in monozygotic twin tion: pRRL-EF1α-PGK-NEO (empty vector; EV), pRRL-EF1α- infants5,6 suggests that all the necessary (epi)genetic events MA4-PGK-NEO (MA4) and pRRL-EF1α-A4M-PGK-dTo (A4M). required for leukemogenesis are accomplished prenatally, VSV-G-pseudotyped lentiviral particles were generated in 293T during embryonic/fetal hematopoietic development.7 cells using standard transfection protocols and concentrated by However, our understanding of t(4;11)-mediated develop- ultracentrifugation.33 hESC were infected overnight with concen- mental effects is limited due, at least in part, to the variety trated EV or MA4 lentivirus plus 8 μg/mL polybrene. Viral super- duced hESC were then selected with G418 (50-100 μg/mL) for 3 of phenotypes and long latency observed in currently natants were washed away the next day, and EV- and MA4-trans- available t(4;11) mouse models.2,8-17 These different pheno- types likely result from targeting a cell in the wrong devel- weeks. For dual transduction of MA4 and A4M fusions, G418- opmental stage, or not addressing the impact of secondary resistant MA4-expressing hESC were infected with A4M-express- hits, leaving open questions about the developmental ing viruses. EV/G418-selected hESC were also transduced with impact of the t(4;11) translocation during early human A4M alone. Transgene expression was confirmed for all the geno- development. types (Figure 1). The functional and molecular contribution of the recip- rocal fusion genes resulting from the derivative translocat- Human embryonic stem cell culture and ed chromosomes remains obscure in cancer. The MA4 characterization of transgenic human embryonic fusion is always expressed in t(4;11)+ B-ALL patients, stem cell lines whereas the reciprocal fusion A4M is expressed in only hESC (AND1 cell line) were maintained undifferentiated on a half of the patients.18-20 Importantly, t(4;11)+ cell lines dis- layer of irradiated human mesenchymal stem cells in complete play addiction to MA4 but not to A4M,21,22 and although knockout Dulbecco modified Eagle medium containing 20% A4M was not sufficient to initiate leukemia in cord blood- knockout serum replacement and 8 ng/mL basic fibroblast growth derived CD34+ cells,23 it was nevertheless capable of initi- factor.34,35 The medium was changed daily, and cells were passaged ating B-ALL in mice without the requirement of MA4, weekly by dissociation with 1:1 collagenase IV:dispase. Cultures indicating that it contributes to t(4;11)-driven leukemoge- were visualized daily by phase contrast microscopy. Approval for nesis.11,24,25 Strikingly, a very recent clinical study has unrav- hESC work was obtained from the Spanish National Embryo eled an independent prognostic value for MA4 expression Ethical Committee. The pluripotency of transgenic hESC was in t(4;11)+ infant B-ALL, thus adding a new piece to the characterized by flow cytometry using antibodies against SSEA-3, puzzle.19 Thus, the developmental/pathogenic contribu- SSEA-4 TRA-1-60 and TRA-1-81 (BD Biosciences).36 Expression of tion of the t(4;11)– resulting reciprocal fusion A4M remains the pluripotency-associated transcription factors OCT4, NANOG, enigmatic. SOX2, CRIPTO, and DNMT3B as well as transgene expression Human embryonic stem cells (hESC) represent a power- (MA4 and A4M) were analyzed by quantitative real-time poly- ful tool for modeling different developmental aspects of merase chain reaction (PCR) (Online Supplementary Table S1 shows human disease that cannot otherwise be addressed by the primers and PCR conditions used).23,37,38 analyses of patients’ samples or mouse models.7,26,27 Given that prenatal leukemogenesis manifests as impaired early Hematopoietic differentiation from human embryonic hematopoietic differentiation, modeling hematopoietic stem cells by embryoid body formation differentiation in hESC may represent a promising in vitro Undifferentiated hESC were treated with collagenase IV:dispase approach to study the onset of hematopoiesis and the for 1 h at 37ºC. To examine embryoid body (EB) formation, cells mechanisms underlying early human hematopoietic were transferred to low-attachment plates and incubated development.7 During hESC differentiation, a primitive overnight in differentiation medium (knockout Dulbecco modi- population of CD45– hemato-endothelial precursors (HEP) fied Eagle medium supplemented with 20% fetal bovine serum, arises and further differentiates into CD45+ hematopoietic 1% non-essential amino acids, 1 mmol/L L-glutamine, and 0.1 and mature endothelial cells.28-30 Beyond its pathogenic mmol/L b-mercaptoethanol). The medium was changed the next role in acute leukemias, the MLL gene has also been impli- day to the same differentiation medium supplemented with the cated in endothelial cell maturation,31 and endothelial dys- following hematopoietic cytokines: 300 ng/mL stem cell factor, function was recently linked to disease outcomes in child- 300 ng/mL Flt3L, 10 ng/mL interleukin-3, 10 ng/mL interleukin-6, hood leukemias.32 We previously reported that MA4 50 ng/mL granulocyte - colony-stimulating factor and 25 ng/mL favors the emergence of endothelial-primed HEP but not bone morphogenetic protein-4 (all from R&D).9,29,39-41 EB were dis- hemogenic HEP from hESC.10 Here, we took advantage of sociated at different time points during development using colla- well-established hESC-based differentiation systems to genase B and enzyme-free Cell Dissociation Buffer (Invitrogen). study whether the A4M fusion cooperates with MA4 dur- Dissociated cells were stained with anti-CD34-PE, anti-CD31- ing early human hematopoietic and endothelial develop- FITC, anti-CD45-APC or anti-CD34-PE-Cy7, CD31-BV510, anti- 1190 haematologica | 2019; 104(6) AF4-MLL in human embryonic hematopoietic development glycophorin A, anti-CD43-FITC, anti-CD45-APC antibodies (all Cells were then suspended in propidium iodide-containing buffer from BD Biosciences) and 7-actinomycin D, and analyzed using a and acquired and analyzed on a FACS Canto-II using Modfit LT4.0 FACS Canto flow cytometer.9,29,39-41 Colony-forming unit assays software, discriminating between quiescent cells (G0/G1), cycling were performed on days 10 and 15 of EB differentiation by plating cells (S-phase) and G2/M cells.45,46 Apoptosis was assessed with an 60x104 EB cells onto serum-free methylcellulose H4435 (Stem Cell Annexin-V Apoptosis Detection kit (BD Biosciences).16 Technologies). Colonies were scored after 12 days.9,29,42-44 Human embryonic stem cell-OP9 co-cultures Cell cycle and apoptosis analysis hESC-OP9 co-cultures were performed as described else- For cell cycle analysis of hESC-derived HEP and CD45+ cells, where.47,48 OP9 stroma was prepared by plating OP9 cells in gela- day 15 EB were dissociated and harvested cells were fixed tin-coated dishes, and allowing them to overgrow as a monolayer. overnight in 70% ice-cold ethanol. Cells were then washed in hESC were prepared as a suspension of small aggregates using col- phosphate-buffered saline and incubated with anti-CD31-FITC, lagenase IV:dispase. One-tenth of this suspension was plated on anti-CD34-PE-Cy7 and anti-CD45-APC antibodies for 15 min. top of the 8-day overgrown OP9 stroma. Media were replaced on A B C D Figure 1. Characterization of transgenic human embryonic stem cells expressing the reciprocal fusion A4M together with MA4. (A) RNA-sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) valida- tion revealed that ~45% (11/25) of the patients with t(4;11)+ B-cell precursor acute lymphoblastic leukemia do not express the reciprocal fusion A4M.18 (B) Left, Phase-con- trast morphology of representative colonies from each transgenic human embryonic stem cell (hESC) line. Right, Reverse transcriptase polymerase chain reaction analysis (RT-PCR) confirming expression of both fusions in undif- ferentiated hESC. (C) qRT-PCR expression of the pluripotency genes OCT4, SOX2, NANOG, CRIPTO, and DNMT3B. (D) Representative FACS data confirming expression of the pluripo- tency surface markers SSEA-3, SSEA-4, TRA-1- 60, and TRA-1-81. BCP-ALL: B-cell precursor acute lymphoblastic leukemia; RNA-seq: RNA- sequencing; pos: positive; neg: negative; EV: empty vector; C+: positive control. haematologica | 2019; 104(6) 1191 C. Bueno et al. the next day and one-half volume media changes were performed Results every other day thereafter. Hematopoietic differentiation was assessed by flow cytometry at day 9 of co-culture. Accordingly, Co-expression of A4M and MA4 does not hijack hESC-OP9 co-cultures were treated with collagenase IV/TrypLE pluripotency and cells were dissociated and filtered through a 70-μm strainer. We showed very recently that only 45% of t(4;11)+ B- Cell suspensions were stained with anti-mouse CD29-FITC and ALL patients express the reciprocal fusion A4M, whereas anti-human CD34-PE and CD45-APC antibodies. The proportion MA4 is consistently expressed in all t(4;11)+ B-ALL of HEP (CD34+CD31+CD45-) and total blood cells (CD45+) were patients (Figure 1A).18-20 Here, we generated transgenic analyzed within the CD29- hESC-derived cell population. hESC lines expressing “MA4 alone”, “A4M alone” or Hemogenic and endothelial HEP were distinguished based on MA4+A4M (double fusion), (Figure 1B and Online CD34 and CD43 expression.40 Supplementary Figure S1B). EV (control)- and MA4-hESC were established by G418 selection.9 G418-resistant EV- or Culture of FACS-isolated hemato-endothelial MA4-expressing hESC were then transduced with precursors in MS5 stroma or liquid culture A4M/dTo-expressing lentiviruses and greater than 90% Day 9 human hESC-OP9 co-cultures were dissociated as above transduction efficiency was achieved. Transgenic hESC and both CD45+ cells and HEP were analyzed. FACS-purified HEP lines were maintained for more than 50 passages and (CD29-CD34+CD31+CD45-) were plated onto MS5 stroma or in retained hESC-like morphology (Figure 1B, left), transgene liquid culture for 30 or 16 days, respectively, in differentiation expression (Figure 1B, right), and expression of pluripoten- medium with hematopoietic cytokines (50 ng/mL stem cell factor, cy-associated transcription factors (Figure 1C) and surface 50 ng/mL Flt3L, 10 ng/mL interleukin-3, 20 ng/mL interleukin-7). markers (Figure 1D). All hESC genotypes formed ter- The medium was changed every 7 days, and the emergence of atomas in NSG mice (data not shown).9,50 Thus, (co-)expres- CD45+ hematopoietic cells was analyzed by FACS. sion of A4M and/or MA4 is compatible with hESC pluripotency. Endothelial differentiation of hemato-endothelial precursors A4M and MA4 co-operate to promote emergence HEP (2×104) from day 9 human hESC-OP9 co-cultures were of hemato-endothelial precursors and enhance seeded onto 0.1% gelatin-coated plates in complete EGM-2 medi- blood production um with microvasculature supplements (Lonza) for 7 days. Cells Hematopoietic differentiation was assessed using two were then fixed, permeabilized and stained with rabbit anti- distinct and well-established differentiation systems: EB human VE-cadherin (Cayman), mouse anti-human endothelial formation43,47 (Figure 2) and OP9 co-culture47,48 (Figure 3). nitric oxide synthase (BD Biosciences), and mouse anti-human During differentiation, a population of primitive HEP aris- von Willebrand factor (DAKO) followed by Alexa 488-conjugated es, which is responsible for further hematopoietic and anti-rabbit or Cy3-conjugated anti-mouse (Jackson endothelial commitment10,30 (Figures 2A and 3A). We Immunoresearch) antibodies. Nuclei were counterstained with investigated whether co-expression of A4M and MA4 4′,6-diamidino-2-phenylindole. Images were obtained using an affects hESC-derived hematopoiesis by analyzing the inverted fluorescence microscope. Day 7 differentiating cells were emergence of HEP during EB development in hESC indi- trypsinized and cell suspensions were stained with anti-human vidually expressing the single fusions or the double fusion. CD31-FITC and CD144-PerCP-Cy5.5 antibodies. We observed a pronounced (~5- to 10-fold; P<0.05) increase in HEP at days 7 and 10 of development in EB Mouse transplantation and analysis of expressing the double A4M and MA4 fusion over those hematopoietic-endothelial engraftment expressing single fusions (Figure 2B, upper-left panel). We NOD/LtSz-scid IL-2Rγ−/− (NSG) mice were housed under sterile next assessed whether co-expression of A4M and MA4 conditions. The Animal Care Committee approved all mouse pro- influences subsequent hematopoietic commitment of tocols. Briefly, cord blood-derived CD34+ hematopoietic stem and HEP. The kinetics of emergence and output of both total progenitor cells (3×104 cells) or cells from day 15 EB (5×105 cells) CD45+ hematopoietic cells and CD45+CD34+ hematopoi- were transplanted into the bone marrow as described previously.49 etic progenitors was faster (EB day 10) from double Animal health was monitored throughout the entire experiment. fusion-expressing hESC than from equivalent single Mice were killed 10 weeks after transplantation and cell suspen- fusion-expressing cells, achieving a 2- to 3-fold higher sions were analyzed by FACS for human chimerism using anti- hematopoietic output by day 15 of EB development HLA-ABC-FITC, anti-CD31-PE, CD144-PerCP-Cy5.5, and anti- (Figure 2B). Furthermore, double fusion-expressing HEP CD45-APC antibodies. massively accelerated (EB day 10) the emergence of clono- genic hematopoietic progenitors as compared to single RNA- and chromatin immunoprecipitation-sequencing fusion-expressing HEP (Figure 2B, bottom-right panel). Details of the RNA- and chromatin immunoprecipitation- According to our previous work, if the kinetics of human sequencing and analysis are provided in the Online Supplementary EB differentiation is extended, allowing for a continuum Methods. of HEP-to-blood transition, MA4-expressing human EB display enhanced HEP production coupled to impaired Statistical analysis blood output (EB day 20) (Online Supplementary Figure S2A) All data are expressed as mean ± standard error of mean. and clonogenic potential (EB day 15) (Figure 2B). We con- Statistical comparisons were performed using GraphPad Prism firmed stable expression of ectopic MA4 and A4M upon software with the nonparametric Mann-Whitney test, two-tailed EB differentiation, supporting the link between genotype P-value (with 95% confidence interval). Statistical significance and phenotype (Figure 2C). was defined as a P-value <0.05. We also investigated hematopoietic differentiation using the OP9 differentiation system (Figure 3A,B), and by plating FACS-sorted HEP in either hematopoietic liquid 1192 haematologica | 2019; 104(6) AF4-MLL in human embryonic hematopoietic development culture (Figure 3C) or onto MS5 feeders (Figure 3D). After co-cultures and allowed to differentiate into CD45+ blood 10 days on OP9 stroma, double fusion-expressing hESC cells, the yield of CD45+ cells was up to 60-fold higher in yielded a 10-fold higher number of CD45+ hematopoietic double fusion-expressing HEP than in single fusion- cells than did single fusion-expressing hESC (Figure 3B). expressing HEP (Figure 3C,D). Encouraged by these Moreover, when HEP were FACS-sorted from day 9 OP9 results, we next investigated whether ectopic expression A B C D Figure 2. A4M cooperates with MA4 to accelerate human embryonic stem cell/erythroid body specification towards hemato-endothelial precursors and subse- quent hematopoietic differentiation. (A) Schematic of erythroid body hematopoietic differentiation of human embryonic stem cells (hESC) and end-point analyses. (B) Upper left, specification into hemato-endothelial precursors (HEP; CD31+CD34+CD45-) is accelerated in double fusion-expressing hESC. Subsequent differentia- tion of HEP into hematopoietic progenitors (upper right) and mature CD45+ blood cells (bottom left) is enhanced in double fusion-expressing HEP. Bottom right, Colony-forming unit read-out and scoring (pie charts) confirming accelerated and enhanced hematopoietic progenitor potential from double fusion-expressing blood derivatives. (C) Reverse transcriptase polymerase chain reaction analysis confirming stable expression of MA4 and A4M upon erythroid body differentiation. (D) Neither MA4- nor double fusion-expressing blood derivatives display in vivo hematopoietic engraftment potential in irradiated NSG mice. Data are presented as mean ± standard error of mean from at least three independent experiments. *P<0.05. EB: erythroid body; bFGF: basic fibroblast growth factor; BMP4: bone morphogenet- ic protein-4; SCF: stem cell factor; IL: interleukin; G-CSF: granulocyte colony-stimulating factor; CFU: colony-forming unit; EV: empty vector; IBMT; intra-bone marrow transplantation; C+: positive control; CB: cord blood. haematologica | 2019; 104(6) 1193 C. Bueno et al. of both A4M and MA4 confers in vivo engraftment capac- into mature endothelial cells. OP9-hESC co-cultures were ity to hESC-derived hematopoietic derivatives. To do this, dissociated on day 9 of development and HEP were FACS- we transplanted 5×105 hESC hematopoietic derivatives sorted and cultured for 1 week in endothelial-promoting from each genotype into myeloablated NSG mice,4,43,47 conditions (Figure 4A). The expression of the mature finding that, despite regulating hematopoietic develop- endothelial markers VE-cadherin (CD144), von ment in vitro, double fusion-expression did not confer in Willebrand factor, endothelial nitric oxide synthase and vivo engraftment to hESC hematopoietic derivatives CD31 was then analyzed. Irrespective of the genotype, (Figure 2D). HEP cultured in endothelial conditions attached, became The increased hematopoietic output of double fusion- spindle-shaped, and formed VE-cadherin+ endothelial-like expressing hESC might be the consequence of transgene- structures co-expressing endothelial nitric oxide synthase, mediated proliferation/survival of the emerging HEP or von Willebrand factor and CD31 (Figure 4B,C, top panel). CD45+ cells. To address this, we analyzed cell cycle distri- However, double fusion-expressing HEP were more prone bution (Online Supplementary Figure S2B) and apoptosis to differentiate into mature endothelial cells than were (Online Supplementary Figure S2C) within both HEP and the single fusion-expressing HEP. Accordingly, they yielded a CD45+ cell population. No differences in the proportions 20-fold higher number of VE-cadherin+ endothelial-like of either cycling HEP or CD45+ cells were detected structures (Figure 4C, top panel) and CD144+CD31+ between genotypes (25–36% for HEP and 35–41% for endothelial cells (Figure 4C, bottom panel). Interestingly, CD45+ cells) (Online Supplementary Figure S2B). Apoptotic endothelial cells (HLA.ABC+CD31+CD34+CD144+CD45- levels were similarly low in the different genotypes of CD43-) were found in the bone marrow of mice trans- HEP (6–8%) and CD45+ cells (5–7%) (Online Supplementary planted with double fusion-expressing hESC blood deriv- Figure S2C). Collectively, these results show that A4M atives at levels ~4-fold higher than those in mice trans- cooperates with MA4 to induce HEP specification and planted with single fusion-expressing cells (Figure 4D). blood commitment, without hijacking proliferation or sur- Within CD34+CD31+CD45- HEP, two subpopulations of vival of HEP. phenotypically and functionally distinct HEP can be dis- tinguished based on the expression of CD34 and CD43: A4M and MA4 cooperate to enhance endothelial cell hemogenic HEP (CD34low/+CD43+CD45-) and endothelial fate from hemato-endothelial precursors HEP (CD34++CD43-CD45-) (Figure 5A).40,48,51 We thus ana- We next addressed the developmental impact of A4M in lyzed the contribution of both HEP populations to the endothelial maturation from HEP.10,47 We hypothesized superior hematopoietic and endothelial differentiation that co-expression of A4M and MA4 in HEP may con- observed in double fusion-expressing HEP. Co-expression comitantly promote subsequent endothelial and of A4M and MA4, but not single fusions, robustly hematopoietic commitment or skew the hemato- enhanced the emergence of both endothelial and endothelial commitment in favor of hematopoiesis. To hemogenic HEP (Figure 5B,C). The identity of hemogenic test this, we analyzed the ability of HEP to differentiate and endothelial HEP was confirmed by the specific A C D B Figure 3. Co-expression of MA4 and A4M enhances hematopoietic differentiation of human embryonic stem cells in OP9 co-culture. (A) Experimental design of OP9-based human embryonic stem cell (hESC) differentiation towards hemato-endothelial precursors (HEP) and further hematopoietic commitment of HEP main- tained in either liquid culture for 16 days or in MS5 co-culture for 30 days. (B) Frequency of total CD45+ blood cells after 9 days in OP9 co-culture. (C,D) CD45- CD31+CD34+ HEP were FACS-purified at day 9 of OP9 co-culture and allowed to differentiate into CD45+ cells in liquid culture (C) or in MS5 co-culture (D). Data are represented as the mean ± standard error of mean from independent experiments. bFGF: basic fibroblast growth factor; MTG: monothioglycerol; SCF: stem cell factor; IL: interleukin; G-CSF: granulocyte colony-stimulating factor; EV: empty vector. 1194 haematologica | 2019; 104(6) AF4-MLL in human embryonic hematopoietic development expression of early hematopoietic and endothelial master we performed RNA-sequencing analysis on FACS-purified genes (Figure 5D). Thus, A4M cooperates with MA4 to EV-, MA4-, A4M- and double fusion-expressing HEP from promote hematopoietic and endothelial cell fate. day 15 EB. Figure 6A shows a heatmap representation of the hierarchical clustering of the 335 genes differentially Genome-wide transcriptomic and H3K79 methylation expressed between the four genotypes (Online profiles support the developmental cooperation Supplementary Table S2). There is a clear transcriptomic between A4M and MA4 transition towards a hematopoietic/endothelial gene sig- To identify patterns of gene expression that might pro- nature from EV-HEP to double fusion-expressing HEP. vide a molecular explanation of the functional cooperation Single fusion-expressing HEP clustered interspersed between A4M and MA4 in hematopoietic specification, between EV and double-fusion HEP. The biological func- A B C D Figure 4. Enhanced endothelial cell fate from hemato-endothelial precursors co-expressing MA4 and A4M. (A) Scheme of hemato-endothelial precursor (HEP) endothelial differentiation and phenotypic characterization. (B) FACS-sorted HEP from day 9 human ESC-OP9 co-cultures were cultured in EGM2 medium for 5 days and analyzed by immunofluorescence for VE-cadherin, endothelial nitric oxide synthase and von Willebrand factor. (C) Top, Endothelial-like structures were identified and quantified based on VE-cadherin staining (white dotted-lined areas in B, top panel). Bottom, Frequency of CD45-CD31+CD144+ endothelial cells quantified by flow cytometry. (D) In vivo endothelial engraftment potential (HLA.ABC+CD31+CD144+CD45-) analyzed in bone marrow of NSG mice 8 weeks after transplantation of HEP. Data are presented as mean ± standard error of mean from five independent experiments. *P<0.05. bFGF: basic fibroblast growth factor; EV: empty vector; CB: cord blood. haematologica | 2019; 104(6) 1195 C. Bueno et al. tions affected by genes differentially expressed in MA4-, sequencing analysis of the H3K79 trimethylation A4M- and double fusion-expressing HEP relative to EV (H3K79me3) profiles in control, MA4-, A4M- and double were classified by Ingenuity Pathway Analysis soft- fusion-expressing hESC-derived blood derivatives (Figure ware;47,52 the top significantly enriched functional cate- 7, Online Supplementary Figure S3A, Online Supplementary gories included “hematological system development and Table S3). In line with the RNA-sequencing data, function- function”, “cancer” and “hematological disease” (Figure al analysis of the differential H3K79me3 peaks specific for 6B). Statistical (-logp-value) power shows distant effects of double fusion-expressing cells revealed significant gene MA4 and A4M; however, co-expression of both fusions ontology functional categories associated with ”definitive seems to establish a molecular balance/developmental hematopoiesis”, “myeloid and erythroid cooperation in promoting blood-endothelial specification differentiation/homeostasis” and “endothelial cell devel- from hPSC. Strikingly, biofunctions specifically associat- opment” (Figure 7A, Online Supplementary Figure S3B). ed with hematologic lymphoid malignancies (not with This further supports the developmental co-operation non-hematologic cancer) were predicted to be activated between A4M and MA4 in promoting hemato-endothelial (positive z-score) exclusively in double fusion-expressing specification. HEP, further suggesting a molecular cooperation between Finally, we analyzed the H3K79me3 profiles at genomic MA4 and A4M in development and infant leukemia. loci of the classical MLL target genes reported by (Figure 6C). Guenther et al.54 Non-HOX-A classical MLL targets such as The C-terminal partners of MLL fusions normally inter- RUNX1, LMO2, ADMA10, and KDM6A showed a slight act with the histone methyltransferase DOT1L, which is although non-significant, enrichment of H3K79me3 in the sole histone methyltransferase catalyzing histone 3 MA4-expressing hESC, validating our chromatin lysine 79 (H3K79) methylation, a chromatin modification immunoprecipitation-sequencing approach (Figure 7B). widely associated with the dysregulated expression of However, enrichment of H3K79me3 in HOX-A cluster HOX-A cluster genes in MLL leukemias.13,53 We thus per- genes was observed exclusively in A4M-expressing cells formed genome-wide chromatin immunoprecipitation- although it was statistically significant only in double A Figure 5. Co-expression of MA4 and A4M significantly enhances the emer- gence of both endothelial and hemogenic hemato-endothelial precur- sors. (A) Representative flow cytometry analysis of hemato-endothelial precur- sors (HEP) with hemogenic (CD45- CD31+CD43+CD34dim/+) and endothelial (CD45-CD31+CD43-CD34++) potential. (B,C) A4M co-operates with MA4 to boost the emergence of both endothe- lial (B) and hemogenic (C) HEP. Data are presented as the mean ± standard error of mean from three independent experiments. (D) Expression of RUNX1c and Ve-Cad in hemogenic and endothe- B D lial HEP. *P<0.05. EB: erythroid body; EV: empty vector. C 1196 haematologica | 2019; 104(6) AF4-MLL in human embryonic hematopoietic development fusion-expressing differentiating hESC (false discovery S4). Collectively, these data suggest that the deregulated rate <0.1) (Figure 7C). As such, HOX-A genes were upreg- expression of HOX-A genes in MLL leukemias may be ulated in double fusion-expressing hematopoietic clono- imposed by the reciprocal A4M fusion through H3K79 genic progenitors (Online Supplementary Figure S3C). No methyltransferase activity. In support of this, a recent differential enrichment of the repressive H3K4me2/3 RNA-sequencing study performed in 42 infants with mark was observed in either HOX-A or non-HOX genes in t(4;11)+ B-ALL enrolled in the Interfant treatment protocol, double fusion-expressing cells (Online Supplementary Figure revealed that 45% of t(4;11)+ patients express the A4M A B C Figure 6. Transcriptional transition towards a hematopoietic/endothelial gene signature in double fusion-expressing hemato-endothelial precursors. (A) Heatmap representation of hierarchical clustering of genes differentially expressed between empty vector (EV)-, single fusions- and double fusion-expressing hemato-endothe- lial precursors (HEP). Each column represents a technical replicate from three independent experiments. (B,C) Statistically significant functional categories (B) and cancer/leukemia-associated biofunctions (C) identified using Ingenuity Pathway Analysis on genes differentially expressed in single fusions-, and double fusion- expressing HEP relative to EV. They are ranked by z-score. Functional categories associated with “hematological system development and function” and “cardiovas- cular system development” are shown in bold. All significant biofunctions are associated with blood cell differentiation, homeostasis and migration/movement. haematologica | 2019; 104(6) 1197 C. Bueno et al. fusion, and that HOX-A cluster genes are exclusively siveness and short latency, our current understanding expressed in this AF4-MLL-expressing subgroup of t(4;11)+ about its etiology, pathogenesis and cellular origin is still patients, who do in fact have a significantly more favor- limited.2,4,14,16,52 Importantly, a recently developed able clinical outcome.19 xenograft model which represents the most bona fide model for t(4;11)+ B-ALL so far, has revealed the instruc- tive role of MLL-Af4 in cord blood-derived CD34+ cells.14 Discussion Studies using primary cells from t(4;11)+ B-ALL patients are incapable of addressing the developmental genesis of From etiological and pathogenic standpoints, infant the hematopoietic system. Recent data suggest that fetal cancer is distinct from adult cancer and should be studied liver lymphoid-primed multipotent progenitors may pro- as a developmental disease.4,7,16 A biologically and clini- vide the developmental prerequisites for the initiation of cally intriguing infant cancer is the t(4;11)+ B-ALL, which t(4;11)+/MLL-AF4 infant leukemia.56 Because leukemogen- is associated with a dismal outcome.4,21,23 Evidence in sup- esis manifests as a blockage or altered cell differentiation, port of its prenatal origin comes from studies in monozy- the hematopoietic differentiation of hESC may represent gotic twins and archived blood spots, providing com- a promising in vitro model for studying the onset of pelling evidence of a single prenatal cell as the origin for hematopoiesis and the earliest events leading to the spec- t(4;11),5 and also from recent genome-wide studies ification of the hematopoietic cells.36 Previous studies have demonstrating that this infant leukemia has one of the addressed the oncogenic role of leukemic fusion genes in lowest frequencies of somatic mutations of any hESC-derived hematopoiesis.57-59 We previously explored sequenced cancer.55 The stable genome of these patients the developmental impact of the prenatal fusion MA4 in suggests that in infant developmental cancer, one “big- hESC hemato-endothelial development,10 and found that hit” might be sufficient to cause overt disease, support- MA4 expression promotes the emergence of endothelial- ing a key contribution of the prenatal cell-of-origin dur- primed HEP and further endothelial commitment, but ing a critical developmental window of stem cell vulner- hijacks the specification of hemogenic-primed HEP, ability in leukemogenesis. However, despite its aggres- impairing hematopoietic output.10 A B C Figure 7. H3K79 methylation profiles at genomic loci of MLL targets in MA4-, A4M- and double fusion-expressing human embryonic stem cell-derived blood deriv- atives. (A) Gene ontology enrichment of differential H3K79me3 peaks specific for double fusion-expressing cells. (B,C) Representative profiles for chromatin immuno- precipitation-sequencing using anti-H3K79me3 antibody at genomic regions of typical non-HOXA (B) and HOXA MLL targets (C). 1198 haematologica | 2019; 104(6) AF4-MLL in human embryonic hematopoietic development The functional and molecular contribution of the recip- ing differentiating blood cells, MA4 was never individual- rocal fusion genes resulting from the derivative translocat- ly expressed in the absence of A4M. When hematopoietic ed chromosomes remains obscure in cancer.18,60 The MA4 differentiation of hESC was induced by EB formation both fusion is always expressed in t(4;11)+ B-ALL patients, fusions were readily co-expressed, ruling out a biased whereas the reciprocal fusion A4M is expressed in only functional phenotype driven by the sequential expression 45-50% of the patients.18-20,60 Here, we took advantage of of each transgene in distinct developmental windows. well-established hESC-based differentiation systems to Mechanistically, a putative function of A4M is to acti- study whether the A4M fusion cooperates with MA4 dur- vate chromatin, rendering a chromatin landscape similar ing early human hematopoietic and endothelial develop- to that present during stem cell development. It is current- ment. Co-expression of A4M and MA4 strongly promoted ly unknown how A4M is able to reprogram chromatin, the emergence of both endothelial-primed and but it does contain the SET domain disrupted from its hemogenic-primed HEP. Moreover, the double fusion- "specification domain", the N-terminal portion of MLL, expressing HEP specified into significantly higher num- which binds to MEN1 and LEDGF, shaping the gene tar- bers of both hematopoietic and endothelial cells, irrespec- geting module of the MLL gene. When A4M is expressed, tive of the in vitro differentiation protocol used and with- the N-terminal portion is substituted by the AF4 N-termi- out affecting survival or proliferation, indicating a func- nus, which is the crucial domain (AF4N) that binds to and tional (developmental) cooperation between MA4 and strongly activates RNA polymerase II (RNAP II) for tran- A4M fusions during human hematopoietic development. scriptional elongation. Overexpression of either AF4, This notion was confirmed by genome-wide transcrip- AF4N or the fusion protein A4M induces robust RNAP II- tomic analysis of differentiating HEP. These developmen- dependent gene transcription by overwriting the elonga- tal biology studies support previous evidence suggesting tion control process in a dominant fashion.62-64 Since gene that A4M contributes to the pathobiology of t(4;11)+ B- transcription per se and in particular “sterile” transcription ALL. Accordingly, Bursen et al.11 reported that A4M-trans- is a powerful mechanism for chromatin activation, A4M duced murine hematopoietic stem cells developed pro-B- could potentiate MA4 to skew normal and leukemic ALL, whereas co-transduction with MA4 and A4M result- hematopoietic cell fate decisions. This also explains why ed in mixed lineage leukemia. Moreover, studies from MA4 has a more prominent role in the disease than the Milne’s laboratory demonstrated that RUNX1 is directly reciprocal A4M. If A4M functioned to initiate this process activated by MA4 and the RUNX1 protein interacts with only by itself, then it would become obsolete after fulfill- the A4M protein, suggesting a mechanism of cooperation ing the "chromatin opening job". However, transcription between the two fusion genes at the molecular level.25 factors such as MA4, RUNX1 or others then establish the In the embryo, definitive hematopoiesis cannot occur in transcriptional program leading to leukemogenesis. This is the absence of endothelial cell development. Definitive reflected in the enhanced hematopoietic potential of dou- hematopoietic stem cells in both mice and humans ble fusion-expressing hESC and the enriched H3K79me3 emerge from the hemogenic endothelium by a process activation mark in HOX-A cluster genes exclusively when known as endothelial-to-hematopoietic transition.61 MA4 and A4M are co-expressed. Thus, A4M prepares Hematopoietic differentiation of hESC occurs through the other transcription factors to become oncoproteins. generation of HEP, from which both endothelial and Molecularly, C-terminal-partners of MLL fusions (AF4, hematopoietic cells then originate. Here, co-expression of AF9, ENL) interact with DOT1L, which is the sole histone A4M and MA4 in HEP concomitantly promoted endothe- methyltransferase catalyzing H3K79 methylation, a chro- lial and hematopoietic commitment rather than skewing matin modification widely associated with the dysregulat- the hemato-endothelial commitment in favor of one line- ed expression of the HOX-A gene cluster in MLL- age over the other. This finding is important because rearranged leukemias.13,53 Here, chromatin immunoprecip- beyond its pathogenic role in acute leukemias, the MLL itation-sequencing analysis of differentially enriched gene is implicated in endothelial cell maturation, and H3K79me3 genomic regions confirmed a endothelial dysfunction was recently linked to disease hematopoietic/endothelial cell differentiation signature in outcome in childhood leukemias.31,32 Furthermore, other double fusion-expressing HEP, and revealed a significant leukemia fusion oncogenes as well as lymphoma-specific enrichment of H3K79 methylated regions specifically genetic aberrations have been found in endothelial cells associated with HOX-A cluster MLL target genes (but not from patients with chronic myeloid leukemia and B-cell with non-HOX-A MLL targets) in double fusion-express- lymphoma,10,43,44 suggesting that endothelial cells may be ing differentiating hematopoietic cells. This is in line with part of the neoplastic clone. In addition, bone marrow- the recently found significant positive correlation between derived mesenchymal stem cells from infant t(4;11)+ B-cell the upregulation of the HOX-A gene cluster and the ALL were recently found to harbor and express the t(4;11) expression of A4M in primary t(4;11)+ infant B-cell ALL translocation.33 The existence of a common embryonic samples, and with previous studies identifying that precursor for mesenchymal stem cells and endothelial cells approximately one-half of t(4;11)+ patients do not have an has been recently demonstrated by hESC-directed differ- activated HOX-A signature.20,65,66 This may explain why entiation.10,45 The finding of such a common embryonic MA4 failed recently to bind to HOX-A genes to regulate precursor, and the presence of t(4;11) in both leukemic HOXA gene expression.14 Collectively, MA4 and A4M blasts and bone marrow mesenchymal stem cells of infant might cooperate through a complex molecular interaction patients, suggests that the t(4;11) translocation arises and to control HOX-A gene regulation.25 In conclusion, we has a developmental impact on early pre-hematopoietic describe a functional and molecular cooperation between precursors. As a technical caveat, it is important to empha- MA4 and A4M fusions during human hematopoietic size that MA4 and A4M were sequentially transduced to development, and demonstrate how hESC-based allow for antibiotic selection and homogeneously-trans- hematopoietic differentiation represents a promising sys- duced hESC cultures. However, in double fusion-express- tem to explore the developmental impact of the chimeric haematologica | 2019; 104(6) 1199 C. Bueno et al. proteins resulting from chromosomal translocations, III (ISCIII/FEDER, PI17/01028 and PI17/01028) to CB and which remains obscure in human leukemia. PJR, the NIHR GOSH BRC and Great Ormond Street Hospital Children's Charity to J.dB, and Bloodwise and Cancer Research Acknowledgments UK to BG. RM and PM were also supported by the Deutsche Financial support for this work was obtained from the European José Carreras Leukämie Stiftung. PM also acknowledges finan- Research Council (CoG-2014-646903 and PoC-2018-811220) cial support from the Obra Social La Caixa-Fundaciò Josep and the Generalitat de Catalunya (SGR330 and PERIS 2017- Carreras. R-T-R is supported by a fellowship from the Spanish 2019) to PM, the Spanish Ministry of Economy and Association of Cancer Research (AECC). RV-M is supported by Competitiveness (SAF2016-80481-R and SAF2016-76758-R) a Torres Quevedo fellowship from the Spanish Ministry of Science to PM and IV, the Spanish Association against Cancer (AECC- and Innovation (PTQ-16-08623). P.M is an investigator of the CI-2015) and Fero Foudation to CB, the Health Institute Carlos Spanish Cell Therapy cooperative network (TERCEL). leukemia. Cancer Cell. 2016;30(5):737-749. 27. Romero-Moya D, Santos-Ocana C, Castano References 15. Metzler M, Forster A, Pannell R, et al. A con- J, et al. Genetic rescue of mitochondrial and 1. Milne TA. 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