Relieving DYRK1A repression of MKL1 confers an adult-like phenotype to human infantile megakaryocytes
Kamaleldin E. Elagib, … , Camelia Iancu-Rubin, Adam N. Goldfarb
Kamaleldin E. Elagib, … , Camelia Iancu-Rubin, Adam N. Goldfarb
Published August 4, 2022
Citation Information: J Clin Invest. 2022;132(19):e154839. https://doi.org/10.1172/JCI154839.
View: Text | PDF
Research Article Development Hematology Article has an altmetric score of 96

Relieving DYRK1A repression of MKL1 confers an adult-like phenotype to human infantile megakaryocytes

Abstract

Infantile (fetal and neonatal) megakaryocytes (Mks) have a distinct phenotype consisting of hyperproliferation, limited morphogenesis, and low platelet production capacity. These properties contribute to clinical problems that include thrombocytopenia in neonates, delayed platelet engraftment in recipients of cord blood stem cell transplants, and inefficient ex vivo platelet production from pluripotent stem cell–derived Mks. The infantile phenotype results from deficiency of the actin-regulated coactivator, MKL1, which programs cytoskeletal changes driving morphogenesis. As a strategy to complement this molecular defect, we screened pathways with the potential to affect MKL1 function and found that DYRK1A inhibition dramatically enhanced Mk morphogenesis in vitro and in vivo. Dyrk1 inhibitors rescued enlargement, polyploidization, and thrombopoiesis in human neonatal Mks. Mks derived from induced pluripotent stem cells responded in a similar manner. Progenitors undergoing Dyrk1 inhibition demonstrated filamentous actin assembly, MKL1 nuclear translocation, and modulation of MKL1 target genes. Loss-of-function studies confirmed MKL1 involvement in this morphogenetic pathway. Expression of Ablim2, a stabilizer of filamentous actin, increased with Dyrk1 inhibition, and Ablim2 knockdown abrogated the actin, MKL1, and morphogenetic responses to Dyrk1 inhibition. These results delineate a pharmacologically tractable morphogenetic pathway whose manipulation may alleviate clinical problems associated with the limited thrombopoietic capacity of infantile Mks.

Authors

Kamaleldin E. Elagib, Ashton Brock, Cara M. Clementelli, Goar Mosoyan, Lorrie L. Delehanty, Ranjit K. Sahu, Alexandra Pacheco-Benichou, Corinne Fruit, Thierry Besson, Stephan W. Morris, Koji Eto, Chintan Jobaliya, Deborah L. French, Paul Gadue, Sandeep Singh, Xinrui Shi, Fujun Qin, Robert Cornelison, Hui Li, Camelia Iancu-Rubin, Adam N. Goldfarb

×

Figure 5

MKL1 involvement in Dyrk control of Mk morphogenesis.

Options: View larger image (or click on image) Download as PowerPoint
MKL1 involvement in Dyrk control of Mk morphogenesis. (A) Effects of Dyr...
(A) Effects of Dyrk1 inhibition on targets of MKL1 and P-TEFb. Cord blood CD34+ cells cultured for 6 days in Mk medium with or without 5 μM inhibitors underwent immunoblot (IB) of whole cell lysates. Arrows indicate Filamin A isoforms. (B) Tubulin-normalized densitometry signals from IBs as in (A). Graph shows mean fold changes with inhibitors ± SEM for 3 independent experiments. *P < 0.05; **P < 0.01, 1-way ANOVA with Tukey’s post hoc test. (C) Transcriptomic effects in human mK precursors of ontogenic stage (adult versus neonatal) and Dyrk inhibition (neonatal with or without inhibitors). CD34+ cells cultured for 4 days in Mk medium underwent purification of CD61+ cells followed by RNA-Seq. Overlapping genes (Sh) with hypergeometric P values are shown; n = 3 independent experiments. See Supplemental Table 1 for gene lists. (D–F) MKL1 requirement for morphogenesis enhancement. Marrow progenitors from indicated strains cultured for 3 days in murine Mk medium with or without 5 μM harmine underwent flow cytometry after costaining with FITC-anti-CD41 and PI. (D) Mk polyploidization (PI). (E) Graph shows relative percent Mk ≥ 8N ± SEM, n = 4/group. ***P < 0.005, 2-way ANOVA. (F) Mk size (FSC). Graph shows mean ± SEM, n = 4/group. *P < 0.05, 2-way ANOVA. (G) MKL1 localization. Human CD34+ progenitors cultured 24 hours in Mk medium with or without 5 μM inhibitors underwent immunofluorescent staining (IF) and confocal microscopy (Zeiss LSM700, original magnification, ×630; scale bar: 10 μm). (H) Graph shows mean ratio nuclear/cytoplasmic MKL1 signal ± SEM for 3 independent experiments. *P < 0.05, 1-way ANOVA with Tukey’s post hoc test.