Panobinostat (LBH589)-Mediated Acetylation of Tubulin Plays a Role in Megakaryocyte Maturation and Platelet Release

Abstract 704

Panobinostat (LBH589) is a histone deacetylase inhibitor (HDACi) which has been shown to have therapeutic effects against several types of human cancers. One of the major dose-limiting toxicities associated with the use of LBH589 has been thrombocytopenia. In this study, we investigated the mechanisms underlying thrombocytopenia induced by LBH589. Thrombocytopoiesis is the result of a highly regulated process in which bone marrow (BM) megakaryocyte (MK) progenitors proliferate and acquire lineage-specific markers in the early stages, followed by polyploidization, maturation and platelet formation in the later stages. We evaluated the effects of LBH589 on thrombocytopoiesis, by utilizing an in vitro system in which MK were generated from human BM-derived CD34⁺ hematopoietic progenitor cells. At low nanomolar concentrations, LBH589 did not interfere with either cellular proliferation or the ability of CD34⁺ cells to generate MK: both control and LBH589-treated CD34⁺ cells expanded 2.5 to 5 fold and generated a comparable number of CD61⁺ MK progenitors. This was also confirmed by MK colonies assay and suggested that LBH589 treatment does not affect the proliferation and commitment of CD34⁺ cells towards MK lineage. We next evaluated the effects of LBH589 on MK maturation and platelet production. The percentage of mature CD61⁺/CD42b⁺ MK generated in the presence of LBH589 was 3-fold lower compared to that generated in the absence of LBH589. Interestingly, the fraction of CD61⁺/CD42b⁺ MK generated in cultures in which LBH589 was withdrawn during the maturation step was only 1.4 lower than that found in control cultures, indicating that the negative effects of LBH589 on MK maturation are reversible, confirming similar observations made during clinical trials of LBH589. Culture-derived platelets were analyzed phenotypically and quantitated after dual labeling with anti-CD41 antibodies and thiazole orange (TO). The percentage of CD41⁺/TO⁺ platelets derived from MK generated in the presence of LBH589 was significantly reduced (11%) as compared with MK generated in the absence of LBH589 (18.5%). These findings were consistent with the presence of significant numbers of proplatelet-bearing MKs being observed in control cultures but not in LBH 589-treated cultures. Collectively, these data indicate that LBH589 impairs the ability of MK to mature and release platelets.

To better understand the mechanisms responsible for these later stage effects on thrombocytopoiesis, we investigated the potential molecular targets of LBH589 in MK lineage. MK maturation is highly dependent on microtubule (MT) cytoskeleton which represents the structural scaffold for proplatelet extension and ensures the transport of cytoplasmic organelles into nascent platelets. Acetylation of tubulin, a non-histone protein which is critical for proper MT function, is a hallmark of polymerized MT and is mediated by HDAC6, a target of LBH589 inhibition. Based on this, we hypothesized that LBH589 treatment might induce changes in tubulin acetylation that in turn result in alterations in MT cytoskeleton. To test this hypothesis, we assessed tubulin acetylation and demonstrated that treatment with LBH589 resulted in an increase in the levels of acetylated tubulin. To determine if LBH589-induced changes in tubulin acetylation resulted in alterations in MT dynamics, we performed Western blot analysis of polymerized vs. soluble (depolymerized) tubulin in the presence or absence of LBH589. The ratio of soluble:polymerized tubulin in control MK was up to 2-fold greater than that found in MK treated with LBH589. In other words, LBH589 treatment induced an increase in the levels of polymerized MT which correlated with an increase in tubulin acetylation. These changes in MT dynamics at molecular levels were reflected by morphological alterations of cytoskeleton organization as visualized by microscopic analysis.Based on these findings we suggest that LBH589-induced changes in tubulin acetylation results in aberrant cytoskeletal organization that in turn leads to defective MK maturation and platelet release. In summary, we demonstrate that the negative effects of LBH589 on thrombocytopoiesis are mediated by targeting acetylation of tubulin. Furthermore, these studies suggest that tubulin can serve as a drug target for HDACi such as LBH589 to treat patients with extreme thrombocytosis.