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457 Modeling Acute Megakaryoblastic Leukemia of Down Syndrome Using Induced Pluripotent Stem Cells

Program: Oral and Poster Abstracts
Type: Oral
Session: 602. Disordered Gene Expression in Hematologic Malignancy, including Disordered Epigenetic Regulation: Altered Transcription Factor Regulation
Hematology Disease Topics & Pathways:
AML, Diseases, Myeloid Malignancies
Sunday, December 6, 2020: 3:15 PM

Brahim Arkoun1,2*, Virginie Dufour3*, Aurélie Siret4*, Stefania Mazzi1*, Yasmine Mammasse3*, Mathieu Vieira1*, Fabien Boudia4*, Elie Robert4*, Zakia Aid4*, Marie Cambot3*, Rachel Petermann3*, Sylvie Souquere5*, Philippe Rameau6*, Cyril Catelain6*, Romain Diot7*, Gérard Tachdjian7*, Najet Debili1*, Isabelle Plo1,2*, Hana Raslova1*, Sebastien Malinge4,8*, Eric Soler9*, Thomas Mercher4* and William Vainchenker1,2,3*

1INSERM, UMR1287, Gustave Roussy, Université Paris-Saclay, Equipe labellisée Ligue Nationale Contre le Cancer, Equipe GRex, Villejuif, France
2Laboratory of Excellence GReX, Paris, France
3Institut National de la Transfusion Sanguine (INTS), Department of Platelet Immunology, F-75739, Paris, France
4INSERM, UMR1170, Gustave Roussy, Université Paris-Saclay, Equipe labellisée Ligue Nationale Contre le Cancer, Villejuif, France
5Centre National de La Recherche Scientifique, UMR-8122, Gustave Roussy, Villejuif, France
6INSERM US23 AMMICA, Gustave Roussy, Université Paris-Saclay, Villejuif, France
7Assistance Publique-Hôpitaux de Paris Service d'Histologie, Embryologie et Cytogénétique, Université Paris-Saclay, Hôpital Antoine Béclère, Clamart, France
8Telethon Kids Cancer Centre, Telethon Kids Institute, University of Western Australia., Perth, Australia
9Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS., Montpellier, France


Development of Acute megakaryoblastic leukemia in Down syndrome children (DS-AMKL) is a multi-step process. Acquired GATA1s mutation during fetal hematopoiesis is responsible of a transient myeloproliferative disorder (TMD) characterized by an accumulation of megakaryoblasts. Although most of TMD regress around birth, some TMD can progress from the initial GATA1s clone to AMKL through the acquisition of additional mutations, including in (i) the cohesin complex (i.e: SMC3), (ii) the JAK/STAT signaling pathway, such as MPL and (iii) the polycomb repressive complex 2 (EZH2).

How these mutations cooperate to deregulate megakaryocyte (MK) differentiation and to induce a full-blown AMKL, along with the precise role of trisomy 21 (T21) during this transformation process remain unclear. Because modeling of DS-AMKL is particularly difficult in mice, we performed a step-wise introduction of GATA1s, a gain of function mutation of MPL (MPLW515K) and a heterozygous loss of function mutation in a cohesin (SMC3), separately or in combination, in T21 and isogenic disomic 21 (Dis21) human induced Pluripotent Stem Cells (iPSCs).


Trisomy 21 iPSCs were kindly provided by M. Weiss (Memphis, TN). CRISPR/Cas 9 genome editing of GATA1 or SMC3 allowed the generation of GATA1s T21, SMC3+/- T21 and GATA1s SMC3+/- T21 iPSC clones. CRISPR/Cas9-mediated knock-in of MPLW515K was performed in T21 GATA1s iPSCs. The subsequent T21 GATA1s MPLW515K/W515K clones were selected as well as a revertant Dis21 GATA1s MPLW515K/W515K clone. Finally, SMC3 insertion/deletion were obtained in isogenic T21 and Dis21 GATA1s MPLW515K/W515K SMC3+/- iPSCs clones. Hematopoietic differentiation was induced in 2D cultures in presence of a matrix and a cocktail of cytokines followed by a MK differentiation with SCF and TPO. MK differentiation was studied by clonogenic assays, flow cytometry, confocal microscopy and ultrastructural studies. Gene expression analyses were performed by RNA-seq on highly purified MK from all genotypes.


GATA1s alone blocked MK maturation characterized by a persistent CD34 expression, an accumulation of abnormal large granules, a defect in the development of demarcation membranes (DMS), and a marked decrease in proplatelet formation. The typical GATA1s MK were large megakaryoblasts with numerous large granules and rare DMS. However, GATA1s alone had no effect on the clonogenic activity in CFU-MK assays and MK numbers. The introduction of the MPLW515K mutation did not modify this phenotype either in Dis21 or T21 GATA1s MK, but induced a complete TPO independence. SMC3+/- alone enhanced the MK maturation allowing the generation of a higher number of proplatelets-generating MK. Importantly, the combination of GATA1s and SMC3+/- mutations had a marked cooperative effect that worsened the MK maturation defect, led to the generation of abnormal megakaryoblasts with only a pre-DMS and resulted in enhanced proliferation and ploidization both in Dis21 and T21 iPSCs. Interestingly, the proliferation was markedly higher in T21 clones compared to Dis21 counterparts.

RNA-seq and GSEA analyses showed that T21 GATA1s SMC3+/- mutant MK exhibited transcriptional signatures consistent with a dramatic decrease in the expression of maturation genes, including GATA1 target genes, while DNA replication gene markers were increased compared with GATA1s alone. T21 GATA1s MPLW515K/W515K SMC3+/- MK were enriched for AMKL signatures as compared to isogenic Dis21 GATA1s MPLW515K/W515K SMC3+/- MK. Ongoing ATAC-seq analyses will define the consequence of the different mutations on chromatin accessibility.


Using iPSC modeling, we analyzed in a human cell-context the consequences of the different combination of mutations associated with DS-AMKL that would be difficult to model using human primary cells. Our data demonstrate that GATA1s expression cooperates with SMC3+/- to enhance proliferation of megakaryoblasts from T21 iPSCs and isogenic Dis21 iPSCs hence reproducing the abnormalities observed in DS-AMKL. T21 is not directly involved in the MK differentiation defects but rather give a proliferative advantage supporting its role in leukemia development.

Disclosures: No relevant conflicts of interest to declare.

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