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

• Published in: Blood 2020-11, Vol.136 (Supplement 1), p.1-1
• Main Authors: Arkoun, Brahim, Dufour, Virginie, Siret, Aurélie, Mazzi, Stefania, Mammasse, Yasmine, Vieira, Mathieu, Boudia, Fabien, Robert, Elie, Aid, Zakia, Cambot, Marie, Petermann, Rachel, Souquere, Sylvie, Rameau, Philippe, Catelain, Cyril, Diot, Romain, Tachdjian, Gérard, Debili, Najet, Plo, Isabelle, Raslova, Hana, Malinge, Sebastien, Soler, Eric, Mercher, Thomas, Vainchenker, William
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2020-140986

Introduction 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). Methods 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/W515Kclones were selected as well as a revertant Dis21 GATA1s MPLW515K/W515Kclone. 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. Results 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