Megakaryocyte lineage development is controlled by modulation of protein acetylation

Abstract

Treatment with lysine deacetylase inhibitors (KDACi) for haematological malignancies, is accompanied by haematological side effects including thrombocytopenia, suggesting that modulation of protein acetylation affects normal myeloid development, and specifically megakaryocyte development. In the current study, utilising ex-vivo differentiation of human CD34+ haematopoietic progenitor cells, we investigated the effects of two functionally distinct KDACi, valproic acid (VPA), and nicotinamide (NAM), on megakaryocyte differentiation, and lineage choice decisions. Treatment with VPA increased the number of megakaryocyte/erythroid progenitors (MEP), accompanied by inhibition of megakaryocyte differentiation, whereas treatment with NAM accelerated megakaryocyte development, and stimulated polyploidisation. Treatment with both KDACi resulted in no significant effects on erythrocyte differentiation, suggesting that the effects of KDACi primarily affect megakaryocyte lineage development. H3K27Ac ChIP-sequencing analysis revealed that genes involved in myeloid development, as well as megakaryocyte/erythroid (ME)-lineage differentiation are uniquely modulated by specific KDACi treatment. Taken together, our data reveal distinct effects of specific KDACi on megakaryocyte development, and ME-lineage decisions, which can be partially explained by direct effects on promoter acetylation of genes involved in myeloid differentiation.

Fig 1. KDACi treatment differentially modulates megakaryocyte development.

(A) From the peripheral blood the absolute number of platelets was analysed in patients treated with VPA (n = 217) together with plasma VPA concentration, measured at the same day. Data represent linear regression analysis of the absolute number of platelets (dependent variable), and plasma VPA concentration (independent variable). (B) UCB-derived CD34+ cells were differentiated towards megakaryocytes in the absence or presence of 10nM TSA, 250μM SB, 200μM VPA, or 1mM NAM for 11 days. Differentiation was determined based on the surface expression of CD61 and CD42b (percentage of double positive cells compared to the control), and (C) cytospin analysis. Data are representative for 3 independent experiments. Error bars represent SEM, * p<0.05, ** p< 0.01.

Fig 2. VPA and NAM have opposing effects on megakaryocyte differentiation.

UCB-derived CD34+ cells were differentiated towards megakaryocytes for 11 days in the absence or presence of 100–200μM VPA, or 1-5mM NAM. (A) At day 7 and 11 of differentiation surface expression of CD61 and CD42b was analysed by FACS. Data represent the percentage of double positive cells, compared to the control. (B) At day 11 of differentiation, cellular DNA content was analysed by FACS. Data represent the percentage of polyploid cells (>4N), compared to the control within the CD42b positive population (left panel), and FACS histogram plots of DNA profile (right panel). Data are representative for 3 independent experiments. Error bars represent SEM, * p<0.05, ** p< 0.01.

Fig 3. VPA and NAM treatment inhibits megakaryocyte progenitor proliferation.

UCB-derived CD34+ cells were differentiated towards megakaryocytes for 11 days in the absence or presence of 100–200μM VPA, or 1-5mM NAM. (A) At all culture time points, trypan blue negative cells were counted. Data represent the fold expansion of megakaryocyte precursors during development. (B) At day 7 and 11, the percentage of apoptotic cells was determined by FACS. Data represent the percentage of Annexin V-positive cells, compared to the control. (C) Absolute numbers of CD61/CD42b positive megakaryocytes were calculated after 11 days of differentiation. Data are representative for 4 independent experiments. Error bars represent SEM, * p<0.05, ** p< 0.01.

Fig 4. VPA treatment increases the absolute number of MEP.

UCB-derived CD34+ cells were differentiated towards megakaryocytes for 4 days in the absence or presence of 200μM VPA, or 5mM NAM. Myeloid progenitor staining was performed according to Manz et al. (46), and distinct progenitor populations were analysed by FACS. MEP were gated from the Lin- CD34+, CD38+ CD123- and CD45RA- cell population. Data represent (A) the absolute numbers of CD34+ cells and MEP, compared to the control, and (B) the percentage of CD34+ cells and MEP, compared to the control. Data are representative for 3 independent experiments. Error bars represent SEM, * p<0.05, ** p< 0.01.

Fig 5. VPA and NAM treatment modulates H3K27 acetylation at the promoters of myeloid specific genes.

UCB-derived CD34+ cells were differentiated towards megakaryocytes. At day 4 of differentiation, cells were treated overnight with 200μM, or 5mM NAM. Next, lysates were prepared, followed by ChIP-sequencing (see Methods). Data represent a Venn diagram comparing the number of genes identified based on a >2-fold increase or decrease in H3K27 acetylation levels compared to the control (A). Gene ontology analysis of up- and down-regulated genes by VPA (B) and NAM (C) treatment. From the same cells, RNA was isolated, followed by cDNA synthesis and qPCR (see Methods). Data represent the relative mRNA level of GATA1, RUNX1, LMO2 and RUNX2 compared to the control (D). Data are representative for 3 experiments. Error bars represent SEM, * p<0.05, ** p<0.01.