Panobinostat (LBH589)-induced acetylation of tubulin impairs megakaryocyte maturation and platelet formation

Abstract

Drug-induced thrombocytopenia often results from dysregulation of normal megakaryocytopoiesis. In this study, we investigated the mechanisms responsible for thrombocytopenia associated with the use of Panobinostat (LBH589), a histone deacetylase inhibitor with promising anti-cancer activities. The effects of LBH589 were tested on the cellular and molecular aspects of megakaryocytopoiesis by utilizing an ex vivo system in which mature megakaryocytes (MK) and platelets were generated from human primary CD34(+) cells. We demonstrated that LBH589 did not affect MK proliferation or lineage commitment but inhibited MK maturation and platelet formation. Although LBH589 treatment of primary MK resulted in hyperacetylation of histones, it did not interfere with the expression of genes that play important roles during megakaryocytopoiesis. Instead, we found that LBH589 induced post-translational modifications of tubulin, a nonhistone protein that is the major component of the microtubule cytoskeleton. We then demonstrated that LBH589 treatment induced hyperacetylation of tubulin and alteration of microtubule dynamics and organization required for proper MK maturation and platelet formation. This study provides new insights into the mechanisms underlying LBH589-induced thrombocytopenia and provides a rationale for using tubulin as a target for selective histone deacetylase inhibitor therapies to treat thrombocytosis in patients with myeloproliferative neoplasms.

Figure 1. Evaluation of CFU-MK colony formation

A. CFU-MK and CFU-Mix-MK colonies formed by control CD34⁺ cells or by CD34⁺ cells treated with 2.5 nM LBH589 were enumerated after 16 days incubation in collagen-based semisolid media in the presence of TPO, Il-6 and Il-3. Each column represents the average number of colonies ±SD enumerated in three independent experiments. B. Representative microphotographs of CFU-MK colonies in control (a) and LBH589-treated (b) showing GPII/IIIa expression (i.e. red staining) and platelet-like particles (arrows in a and b).

Figure 2. Evaluation of MK maturation in liquid cultures

A. Representative flow cytometric analysis of MK cultures labeled with FITC-conjugated anti-CD41 and APCconjugated anti-CD42b antibodies. The density plot on the left represents analysis of control MK showing three different populations of MK: immature (i.e. assigned in purple), undergoing cytoplasmic maturation (i.e. assigned in green) and fully mature (i.e. assigned in red). The next three density plots represent analyses of MK treated with 2.5 nM LBH589 as it follows: continuously during the two-steps of the 14-day period (7±7 days), only during the first step (7±0 days) and only during the second step. B. An analytical gate was set to include only viable cells (i.e. negative for 7-AAD staining) then CD41⁺/CD42⁺ MK were quantified in Control cultures, in cultures treated with LBH589 during the entire 14-day culture period (Treated) and in cultures treated with LBH589 only during the first step of the culture period (Withdrawn). * p value < 0.05. C. Representative flow cytometric analyses of DNA content of PI-stained CD41⁺ MK grown in the absence (Control) or in the presence of 2.5 nM LBH589 (Treated). The arrows indicate ploidy classes ranging from 2N to 32N.

Figure 3. Evaluation of in vitro platelet production

A. Primary MK cultures visualized by phase contrast light microscopy. Representative exemples of MK in control cultures display cytoplasmic extensions or proplatelets with nascent platelets at their ends (arrows in a) while MK treated with 2.5 nM LBH589 show short cytoplasmic protrusions (arrows in b). B. Flow cytometric analysis of human peripheral blood-derived platelets (PB) and of platelets derived in control cultures or in cultures treated with 2.5 nM LBH589. The upper panels represent forward and light scatter plots displaying size-based analytical gates for platelets (PTL). The lower panels represent analysis of gated platelets based on CD41-PE (y-axis) and thiazole orange (TO) labeling (x axis). CD41⁺/TO⁺ cells in the right upper quadrant of each dot plot represent newly formed reticulated platelets. C. Quantification of culture-derived platelets in control cultures and in cultures treated with 2.5 nM and 5 nM LBH589 in three independent experiments ±SD. * p value < 0.05.

Figure 4. Evaluation of histone acetylation and MK gene expression

A. Flow cytometric analysis of acetylated histone H3 in primary MK from control cultures and in cultures treated with LBH589 (2.5 nM and 5 nM) for 72 hrs. The shift in the mean AcH3-Alexa 488 fluorescence (x axis) indicates increased H3 acetylation. B. Quantification of relative AcH3 levels in three different experiments ±SD. * p value < 0.05. C and D. mRNAs extracted from undifferentiated CD34⁺ cells (CD34), from control mature MK (C) and from mature MK treated with 2.5 nM and 5 nM LBH589 during the maturation step (i.e. final 7 days in liquid culture) were reverse transcribed then amplified by quantitative real-time PCR using human GATA-1 (C) and NF-E2 (D) primers. Each column represents the average ±SD of three independent experiments each performed in duplicate and normalized to GAPDH.

Figure 5. Evaluation of LBH589 on tubulin acetylation and polymerization

A. Immunofluorescence microscopy analysis of control MK and of MK treated with 2.5 nM LBH589 for 72 hours. CD41⁺ MK from each condition were sorted by means of immunomagnetic labeling then stained with anti-acetylated tubulin antibodies to visualize acetylated tubulin (Ac-Tub, green fluorescence) and with Hoechst 33342 to visualize the nuclei (DNA, blue fluorescence). Note, the intense, uneven immunofluorescence in LBH589-treated MK as compared to the dim, uniform labeling in control MK. B. Soluble (S) and polymerized (P) tubulin were isolated from equal numbers of control MK (0) or LBH589-treated MK (2.5 nM, 5 nM and 10 nM) sorted based on CD41 expression then assessed by Western blotting as described in the Materials and Methods. The numbers represent the ratio of polymerized:soluble tubulin (Ratio P:S) calculated from the relative protein levels measured on the autoradiographs using NIH ImageJ software.

Figure 6. Evaluation of microtubule organization

Immunofluorescence microscopy analysis of Meg-01 cells (a, b) and CD41⁺ primary MK (c, d) grown in the absence (a, c) and in the presence (b, d) of 2.5 nM LBH589 for 72 hours. The cells were labeled with anti-α-tubulin antibodies to visualize MT (green fluorescence) and with Hoechst 33342 to visualize the nuclei (blue fluorescence). Note, most of the cells in control cultures have normal network of MT radiating from the perinuclear MT organizing center (arrows in panels a and c) and some cells display MT structures resembling proplatelets and platelets (arrow heads in panels a and c). Most of the cells in LBH589-treated cultures have a marginal band of MT at the cell periphery or perinuclear (arrows in b and d), shorter filaments of MT or aggregates of polymerized tubulin (asterisks in b and d). Magnification 40X/0.60 dry objective in panels a and b; 100X/1.3 oil objective in inserts panels a and b; 63X/1.25 oil objective panels c and d.