Page 108 - 2019_06-Haematologica-web

C. Bueno et al.
Introduction
The mixed-lineage leukemia (MLL) gene encodes for an H3K4 histone methyltransferase important in hematopoi- 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 t(4;11)(q21;q23) translocation, which encodes the fusion proteins MLL-AF4 (MA4) and AF4-MLL (A4M), and is associated with infant B-cell acute lymphoblastic leukemia (B-ALL). This t(4;11)+ infant leukemia is charac- terized by a very brief latency, raising the question of how it evolves so quickly.4 Moreover, the exceptionally high concordance rate of t(4;11)+ B-ALL in monozygotic twin infants5,6 suggests that all the necessary (epi)genetic events required for leukemogenesis are accomplished prenatally, during embryonic/fetal hematopoietic development.7 However, our understanding of t(4;11)-mediated develop- mental effects is limited due, at least in part, to the variety of phenotypes and long latency observed in currently
2,8-17
available t(4;11) mouse models. These different pheno-
types likely result from targeting a cell in the wrong devel- opmental stage, or not addressing the impact of secondary hits, leaving open questions about the developmental impact of the t(4;11) translocation during early human development.
The functional and molecular contribution of the recip- rocal fusion genes resulting from the derivative translocat- ed chromosomes remains obscure in cancer. The MA4 fusion is always expressed in t(4;11)+ B-ALL patients, whereas the reciprocal fusion A4M is expressed in only half of the patients.18-20 Importantly, t(4;11)+ cell lines dis- play addiction to MA4 but not to A4M,21,22 and although A4M was not sufficient to initiate leukemia in cord blood- derived CD34+ cells,23 it was nevertheless capable of initi- ating B-ALL in mice without the requirement of MA4, indicating that it contributes to t(4;11)-driven leukemoge- nesis.11,24,25 Strikingly, a very recent clinical study has unrav- eled an independent prognostic value for MA4 expression in t(4;11)+ infant B-ALL, thus adding a new piece to the puzzle.19 Thus, the developmental/pathogenic contribu- tion of the t(4;11)– resulting reciprocal fusion A4M remains enigmatic.
Human embryonic stem cells (hESC) represent a power- ful tool for modeling different developmental aspects of human disease that cannot otherwise be addressed by analyses of patients’ samples or mouse models.7,26,27 Given that prenatal leukemogenesis manifests as impaired early hematopoietic differentiation, modeling hematopoietic differentiation in hESC may represent a promising in vitro approach to study the onset of hematopoiesis and the mechanisms underlying early human hematopoietic development.7 During hESC differentiation, a primitive population of CD45– hemato-endothelial precursors (HEP) arises and further differentiates into CD45+ hematopoietic and mature endothelial cells.28-30 Beyond its pathogenic role in acute leukemias, the MLL gene has also been impli- cated in endothelial cell maturation,31 and endothelial dys- function was recently linked to disease outcomes in child- hood leukemias.32 We previously reported that MA4 favors the emergence of endothelial-primed HEP but not hemogenic HEP from hESC.10 Here, we took advantage of well-established hESC-based differentiation systems to study whether the A4M fusion cooperates with MA4 dur- ing early human hematopoietic and endothelial develop-
ment. We report a functional and molecular cooperation between MA4 and A4M fusions, which results in enhanced hemato-endothelial output during human embryonic development.
Methods
Vector construction and lentiviral transduction
The cDNA for MA4 and A4M were subcloned into the pRRL- EF1α-PGK-NEO vector.11,16 Both fusions have been described pre- viously (Online Supplementary Figure S1A).11,23 We used the follow- ing lentivectors containing either neomycin or dTo for cell selec- tion: pRRL-EF1α-PGK-NEO (empty vector; EV), pRRL-EF1α- MA4-PGK-NEO (MA4) and pRRL-EF1α-A4M-PGK-dTo (A4M). VSV-G-pseudotyped lentiviral particles were generated in 293T cells using standard transfection protocols and concentrated by ultracentrifugation.33 hESC were infected overnight with concen- trated EV or MA4 lentivirus plus 8 μg/mL polybrene. Viral super- natants were washed away the next day, and EV- and MA4-trans- duced hESC were then selected with G418 (50-100 μg/mL) for 3 weeks. For dual transduction of MA4 and A4M fusions, G418- resistant MA4-expressing hESC were infected with A4M-express- ing viruses. EV/G418-selected hESC were also transduced with A4M alone. Transgene expression was confirmed for all the geno- types (Figure 1).
Human embryonic stem cell culture and characterization of transgenic human embryonic stem cell lines
hESC (AND1 cell line) were maintained undifferentiated on a layer of irradiated human mesenchymal stem cells in complete knockout Dulbecco modified Eagle medium containing 20% knockout serum replacement and 8 ng/mL basic fibroblast growth factor.34,35 The medium was changed daily, and cells were passaged weekly by dissociation with 1:1 collagenase IV:dispase. Cultures were visualized daily by phase contrast microscopy. Approval for hESC work was obtained from the Spanish National Embryo Ethical Committee. The pluripotency of transgenic hESC was characterized by flow cytometry using antibodies against SSEA-3, SSEA-4 TRA-1-60 and TRA-1-81 (BD Biosciences).36 Expression of the pluripotency-associated transcription factors OCT4, NANOG, SOX2, CRIPTO, and DNMT3B as well as transgene expression (MA4 and A4M) were analyzed by quantitative real-time poly- merase chain reaction (PCR) (Online Supplementary Table S1 shows the primers and PCR conditions used).23,37,38
Hematopoietic differentiation from human embryonic stem cells by embryoid body formation
Undifferentiated hESC were treated with collagenase IV:dispase for 1 h at 37oC. To examine embryoid body (EB) formation, cells were transferred to low-attachment plates and incubated overnight in differentiation medium (knockout Dulbecco modi- fied Eagle medium supplemented with 20% fetal bovine serum, 1% non-essential amino acids, 1 mmol/L L-glutamine, and 0.1 mmol/L b-mercaptoethanol). The medium was changed the next day to the same differentiation medium supplemented with the following hematopoietic cytokines: 300 ng/mL stem cell factor, 300 ng/mL Flt3L, 10 ng/mL interleukin-3, 10 ng/mL interleukin-6, 50 ng/mL granulocyte - colony-stimulating factor and 25 ng/mL bone morphogenetic protein-4 (all from R&D).9,29,39-41 EB were dis- sociated at different time points during development using colla- genase B and enzyme-free Cell Dissociation Buffer (Invitrogen). Dissociated cells were stained with anti-CD34-PE, anti-CD31- FITC, anti-CD45-APC or anti-CD34-PE-Cy7, CD31-BV510, anti-
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haematologica | 2019; 104(6)