Thrombocytopenia induced by the histone deacetylase inhibitor abexinostat involves p53-dependent and -independent mechanisms

Abstract

Abexinostat is a pan histone deacetylase inhibitor (HDACi) that demonstrates efficacy in malignancy treatment. Like other HDACi, this drug induces a profound thrombocytopenia whose mechanism is only partially understood. We have analyzed its effect at doses reached in patient plasma on in vitro megakaryopoiesis derived from human CD34(+) cells. When added at day 0 in culture, abexinostat inhibited CFU-MK growth, megakaryocyte (MK) proliferation and differentiation. These effects required only a short incubation period. Decreased proliferation was due to induction of apoptosis and was not related to a defect in TPO/MPL/JAK2/STAT signaling. When added later (day 8), the compound induced a dose-dependent decrease (up to 10-fold) in proplatelet (PPT) formation. Gene profiling from MK revealed a silencing in the expression of DNA repair genes with a marked RAD51 decrease at protein level. DNA double-strand breaks were increased as attested by elevated γH2AX phosphorylation level. Moreover, ATM was phosphorylated leading to p53 stabilization and increased BAX and p21 expression. The use of a p53 shRNA rescued apoptosis, and only partially the defect in PPT formation. These results suggest that HDACi induces a thrombocytopenia by a p53-dependent mechanism along MK differentiation and a p53-dependent and -independent mechanism for PPT formation.

Figure 1

The pan HDACi abexinostat (A) decreases hematopoietic progenitors growth. Representative experiments are shown to illustrate the number of progenitors generated in a semi-solid assay in the presence of indicated doses of abexinostat, n=3. In each experiment, CD34⁺ cells were seeded in triplicate. Results: mean±S.E.M., ***P<0.0001; **P<0.004; *P<0.05. (a and b) Number of BFU-E, CFU-GM CFU-GEMM and total colonies (total CFU) generated by plating 500 CD34⁺ cells from leukapheresis or bone marrow samples in methylcellulose assays in the presence of SCF+IL-3+EPO+G-CSF+ IL-6 as described in Materials and Methods. (a) Leukapheresis samples; (b) bone marrow exposed all along the culture or only 24 h to abexinostat. (c) Number of CFU-MK colonies generated by plating 1000 CD34⁺ cells in a fibrin clot assay in the presence of TPO+SCF+IL-6. (d and e) Number of CFU-MK colonies generated by plating CD34⁺ cells from leukapheresis (d) exposed 24 h to the indicated concentration of abexinostat, then washed before plating 500 CD34⁺ cells or from bone marrow samples exposed all along the culture or only 24 h to abexinostat (e)

Figure 2

Abexinostat induces a decrease of cellular growth, an increase in apoptosis and a decrease of ploidy. (a) CD34⁺ cells were seeded in liquid culture in the presence of TPO±increasing dose of abexinostat, viable cells were counted using trypan blue exclusion at day 6 and results are the mean±S.E.M. (representative experiments of n=3;*P<0.05; **P<0.004; ***P<0.002). (b and c) CD34⁺ cells were seeded with TPO±increasing dose of abexinostat for 48 h then stained with allophycocyanin-Annexin V-propidium iodide. The percentage of apoptotic cells was determined by flow cytometry. (b) Mean of three independent experiments; (c) representative experiments. *P<0.04; **P<0.001, ***P<0.0001. (d and e) Mean ploidy value of MKs at day 10 of culture with indicated doses of abexinostat (d) mean of three independent experiments: control N: 3.82; 10 nM: 3.66; 50 nM: 2.59; 100 nM: 2.35. *P<0.03; (e) representative ploidy experiment. (f) Mean ploidy value at indicated times, with 100 nM abexinostat added at day 1 or 8. Control at day 8N: 3.80; control at day 10N: 4.7; day 10 analysis for 100 nM abexinostat added either at day 1 (N: 2.51) or at day 8 (N: 4.03)

Figure 3

Abexinostat affects proplatelet formation. CD41⁺-sorted cells were seeded in triplicate in 96 wells with TPO and increasing doses of abexinostat (from 10 to 100 nM). Proplatelets were scored at day 12 under an inverted microscope. (a) Examples of proplatelet bearing MKs after treatment with indicated doses of the pan-HDACi. Proplatelet bearing MKs in control (i) and at 10 nM (ii) were similar; at 20 nM (iii), MK displayed less branching extensions compared with control MKs (i); at 50 nM (iv) and 100 nM (v) of abexinostat most MKs displayed apoptotic morphology and had shorter proplatelets. (b) Typical dose response of abexinostat on proplatelet formation. One representative experiment out of four experiments with increasing dose of abexinostat; ***P<0.0005. (c and d) Effects of a abexinostat short-time exposure on proplatelet formation. MKs were treated 24 h with 100 nM of abexinostat at day 8 (A-100 nM), then washed and seeded in triplicate in serum-free medium plus 10 ng/ml TPO. (d) Proplatelet formation was scored after 3 supplementary days of culture, n=2, P<0.0001. In c, even a short treatment (24 h) induces a proplatelet defect with a few extensions

Figure 4

Representative immunoblot blot analysis of the TPO/MPL/JAK2 signaling pathway in MKs treated or not with abexinostat. CD41⁺ cells were sorted at day 7 of culture, seeded in serum-free medium with TPO with or without 50 nM abexinostat for 4 additional days. Then, MKs were TPO-deprived 12 h in the presence or absence of 50 nM abexinostat and then stimulated for indicated times with or without 50 nM abexinostat. Phosphorylation of STAT5, STAT3 and ERK was evaluated by western blot as well as the JAK2 level. Actin was used as a loading control

Figure 5

Abexinostat modulates expression of genes involved in DNA repair. (a) Gene set enrichment analysis (GSEA) of CD41⁺ cells treated with 100 nM of abexinostat reveals that DNA repair, DNA double-strand break (DSB), DNA recombination and response to DNA damage were the most prominent signatures of abexinostat-treated cells. (b) RAD51 protein level by immunoblot analysis. Day 7 CD41⁺-sorted MK were cultured for 48 h with TPO with or without increasing the dose of abexinostat. HSC70 was used as loading control

Figure 6

Activation of p53 in abexinostat-treated MK. CD41⁺ cells were sorted at day 7 of culture and seeded in serum-free medium with TPO with or without indicated doses of abexinostat for 48 h. (a) Immunoblot analysis of the phosphorylation status of ATM and γH2AX. (b) Immunoblot analysis of caspase 3 in sorted MK treated with or without abexinostat (50 and 100 nM). (c) Immunoblot analysis of the phosphorylated p53 on serine residue 15, BAX and BCL-xL. HSC70 used as loading charge. (d) qRT-PCR studies of p53 target genes. Data are mean±S.E.M., n=3, each experiments performed in triplicate, ***P<0.0007

Figure 7

p53 knockdown rescues MK apoptosis, but not proplatelet formation. (a) Immunoblot analysis of p53 performed on sorted MK transduced with shSCR or shp53. (b) Untreated or treated MKs were stained with Annexin V and analyzed by flow cytometry. Data represent the mean±S.E. of the % of Annexin V⁺ cells, n=2. In these two independent experiments, results show that the shp53 markedly reduced the apoptosis induced by abexinostat in comparison to the SCR. (c and d) Proplatelet formation derived from shp53-treated and non-treated MK. shp53 increased proplatelet formation by MKs in control cultures (P<0.0007), and in 50 nM abexinostat cultures (**P<0.002) (d). At 100 nM abexinostat, MKs kept their round shapes and remained alive but did not form proplatelets. ***P<0.0007