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AF4-MLL in human embryonic hematopoietic development
The functional and molecular contribution of the recip- rocal fusion genes resulting from the derivative translocat- ed chromosomes remains obscure in cancer.18,60 The MA4 fusion is always expressed in t(4;11)+ B-ALL patients, whereas the reciprocal fusion A4M is expressed in only 45-50% of the patients.18-20,60 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. Co-expression of A4M and MA4 strongly promoted the emergence of both endothelial-primed and hemogenic-primed HEP. Moreover, the double fusion- expressing HEP specified into significantly higher num- bers of both hematopoietic and endothelial cells, irrespec- tive of the in vitro differentiation protocol used and with- out affecting survival or proliferation, indicating a func- tional (developmental) cooperation between MA4 and A4M fusions during human hematopoietic development. This notion was confirmed by genome-wide transcrip- tomic analysis of differentiating HEP. These developmen- tal biology studies support previous evidence suggesting that A4M contributes to the pathobiology of t(4;11)+ B- ALL. Accordingly, Bursen et al.11 reported that A4M-trans- duced murine hematopoietic stem cells developed pro-B- ALL, whereas co-transduction with MA4 and A4M result- ed in mixed lineage leukemia. Moreover, studies from Milne’s laboratory demonstrated that RUNX1 is directly activated by MA4 and the RUNX1 protein interacts with the A4M protein, suggesting a mechanism of cooperation between the two fusion genes at the molecular level.25
In the embryo, definitive hematopoiesis cannot occur in the absence of endothelial cell development. Definitive hematopoietic stem cells in both mice and humans emerge from the hemogenic endothelium by a process known as endothelial-to-hematopoietic transition.61 Hematopoietic differentiation of hESC occurs through the generation of HEP, from which both endothelial and hematopoietic cells then originate. Here, co-expression of A4M and MA4 in HEP concomitantly promoted endothe- lial and hematopoietic commitment rather than skewing the hemato-endothelial commitment in favor of one line- age over the other. This finding is important because beyond its pathogenic role in acute leukemias, the MLL gene is implicated in endothelial cell maturation, and endothelial dysfunction was recently linked to disease outcome in childhood leukemias.31,32 Furthermore, other leukemia fusion oncogenes as well as lymphoma-specific genetic aberrations have been found in endothelial cells from patients with chronic myeloid leukemia and B-cell lymphoma,10,43,44 suggesting that endothelial cells may be part of the neoplastic clone. In addition, bone marrow- derived mesenchymal stem cells from infant t(4;11)+ B-cell ALL were recently found to harbor and express the t(4;11) translocation.33 The existence of a common embryonic precursor for mesenchymal stem cells and endothelial cells has been recently demonstrated by hESC-directed differ- entiation.10,45 The finding of such a common embryonic precursor, and the presence of t(4;11) in both leukemic blasts and bone marrow mesenchymal stem cells of infant patients, suggests that the t(4;11) translocation arises and has a developmental impact on early pre-hematopoietic precursors. As a technical caveat, it is important to empha- size that MA4 and A4M were sequentially transduced to allow for antibiotic selection and homogeneously-trans- duced hESC cultures. However, in double fusion-express-
ing differentiating blood cells, MA4 was never individual- ly expressed in the absence of A4M. When hematopoietic differentiation of hESC was induced by EB formation both fusions were readily co-expressed, ruling out a biased functional phenotype driven by the sequential expression of each transgene in distinct developmental windows.
Mechanistically, a putative function of A4M is to acti- vate chromatin, rendering a chromatin landscape similar to that present during stem cell development. It is current- ly unknown how A4M is able to reprogram chromatin, but it does contain the SET domain disrupted from its "specification domain", the N-terminal portion of MLL, which binds to MEN1 and LEDGF, shaping the gene tar- geting module of the MLL gene. When A4M is expressed, the N-terminal portion is substituted by the AF4 N-termi- nus, which is the crucial domain (AF4N) that binds to and strongly activates RNA polymerase II (RNAP II) for tran- scriptional elongation. Overexpression of either AF4, AF4N or the fusion protein A4M induces robust RNAP II- dependent gene transcription by overwriting the elonga- tion control process in a dominant fashion.62-64 Since gene transcription per se and in particular “sterile” transcription is a powerful mechanism for chromatin activation, A4M could potentiate MA4 to skew normal and leukemic hematopoietic cell fate decisions. This also explains why MA4 has a more prominent role in the disease than the reciprocal A4M. If A4M functioned to initiate this process only by itself, then it would become obsolete after fulfill- ing the "chromatin opening job". However, transcription factors such as MA4, RUNX1 or others then establish the transcriptional program leading to leukemogenesis. This is reflected in the enhanced hematopoietic potential of dou- ble fusion-expressing hESC and the enriched H3K79me3 activation mark in HOX-A cluster genes exclusively when MA4 and A4M are co-expressed. Thus, A4M prepares other transcription factors to become oncoproteins.
Molecularly, C-terminal-partners of MLL fusions (AF4, AF9, ENL) interact with DOT1L, which is the sole histone methyltransferase catalyzing H3K79 methylation, a chro- matin modification widely associated with the dysregulat- ed expression of the HOX-A gene cluster in MLL- rearranged leukemias.13,53 Here, chromatin immunoprecip- itation-sequencing analysis of differentially enriched H3K79me3 genomic regions confirmed a hematopoietic/endothelial cell differentiation signature in double fusion-expressing HEP, and revealed a significant enrichment of H3K79 methylated regions specifically associated with HOX-A cluster MLL target genes (but not with non-HOX-A MLL targets) in double fusion-express- ing differentiating hematopoietic cells. This is in line with the recently found significant positive correlation between the upregulation of the HOX-A gene cluster and the expression of A4M in primary t(4;11)+ infant B-cell ALL samples, and with previous studies identifying that approximately one-half of t(4;11)+ patients do not have an activated HOX-A signature.20,65,66 This may explain why MA4 failed recently to bind to HOX-A genes to regulate HOXA gene expression.14 Collectively, MA4 and A4M might cooperate through a complex molecular interaction to control HOX-A gene regulation.25 In conclusion, we describe a functional and molecular cooperation between MA4 and A4M fusions during human hematopoietic development, and demonstrate how hESC-based hematopoietic differentiation represents a promising sys- tem to explore the developmental impact of the chimeric
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