Stepwise GATA1 and SMC3 mutations alter megakaryocyte differentiation in a Down syndrome leukemia model
Brahim Arkoun, … , Thomas Mercher, William Vainchenker
Brahim Arkoun, … , Thomas Mercher, William Vainchenker
Published May 19, 2022
Citation Information: J Clin Invest. 2022;132(14):e156290. https://doi.org/10.1172/JCI156290.
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Research Article Hematology Oncology

Stepwise GATA1 and SMC3 mutations alter megakaryocyte differentiation in a Down syndrome leukemia model

Abstract

Acute megakaryoblastic leukemia of Down syndrome (DS-AMKL) is a model of clonal evolution from a preleukemic transient myeloproliferative disorder requiring both a trisomy 21 (T21) and a GATA1s mutation to a leukemia driven by additional driver mutations. We modeled the megakaryocyte differentiation defect through stepwise gene editing of GATA1s, SMC3+/–, and MPLW515K, providing 20 different T21 or disomy 21 (D21) induced pluripotent stem cell (iPSC) clones. GATA1s profoundly reshaped iPSC-derived hematopoietic architecture with gradual myeloid-to-megakaryocyte shift and megakaryocyte differentiation alteration upon addition of SMC3 and MPL mutations. Transcriptional, chromatin accessibility, and GATA1-binding data showed alteration of essential megakaryocyte differentiation genes, including NFE2 downregulation that was associated with loss of GATA1s binding and functionally involved in megakaryocyte differentiation blockage. T21 enhanced the proliferative phenotype, reproducing the cellular and molecular abnormalities of DS-AMKL. Our study provides an array of human cell–based models revealing individual contributions of different mutations to DS-AMKL differentiation blockage, a major determinant of leukemic progression.

Authors

Brahim Arkoun, Elie Robert, Fabien Boudia, Stefania Mazzi, Virginie Dufour, Aurélie Siret, Yasmine Mammasse, Zakia Aid, Matthieu Vieira, Aygun Imanci, Marine Aglave, Marie Cambot, Rachel Petermann, Sylvie Souquere, Philippe Rameau, Cyril Catelain, Romain Diot, Gérard Tachdjian, Olivier Hermine, Nathalie Droin, Najet Debili, Isabelle Plo, Sébastien Malinge, Eric Soler, Hana Raslova, Thomas Mercher, William Vainchenker

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Figure 6

NFE2 overexpression in TG MK partially rescues maturation and PPT defects.

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NFE2 overexpression in TG MK partially rescues maturation and PPT defect...
(A) Schematic overview of NFE2 overexpression strategy in T and TG iMKs and of subsequent phenotypic studies. (B) Validation by RT-qPCR of NFE2 overexpression in T and TG iMKs. (C) RT-qPCR analyses of the known NFE2 target gene expression with empty or NFE2 lentiviral vectors. (D) Effect of NFE2 overexpression on the clonogenic potential of T and TG iMKs. (E) Effect of NFE2 overexpression on the ploidization of T and TG iMKs. (F) Confocal analyses of NFE2 overexpressing MK in T and TG for CD41 and vWF expression. Scale bars: 50 μm. (G) Histograms of the percentages of MKs with normal or large-sized vWF (upper panel) and the percentages of MKs with pre-DMS or DMS (lower panel) according to the absence (empty) or presence of NFE2 lentiviral vector. Quantifications were performed on 20 MKs from 3 independent experiments. (H) Confocal analyses of β1-tubulin expression in T- and TG-derived MKs according to the absence (empty) or presence of NFE2 lentiviral vector. Scale bars: 50 μm. (I) Representative microphotographs of CD41+CD42+ MKs under PPT formation assay. Note the presence of PPT-forming MKs (blue arrows) in the absence or presence of NFE2 lentivector for the T, while in TG, the presence of PPT-forming MK was observed only in the presence of NFE2 lentivector (blue arrows). Scale bars: 50 μm. (J) Histogram of the number of PPT-forming MK shown in I according to the compared genotypes. Data are represented as mean ± SEM; n = 3. Statistical significance was determined using 1-tailed Mann-Whitney’s U test. *P < 0.05; **P < 0.01; ***P < 0.001.