Pathology - Theses
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ItemDeciphering the molecular and biological processes that mediate histone deacetylase inhibitor-induced thrombocytopenia( 2012)Somatic mutations in a variety of genes involved in cell cycle, signalling, differentiation, proliferation and apoptotic pathways can lead to the initiation and progression of malignancy. In addition, multiple ‘epigenetic’ changes have been described, mediated by enzymes linked to DNA packaging and transcription, which are not characterised by changes in the primary sequence of the DNA yet influence the pattern of gene expression. The histone deacetylase inhibitors (HDACi) are new anti-cancer drugs which induce chromatin remodelling by a net increase in histone acetylation, altering gene expression as well as influencing the function of non-histone proteins by direct acetylation. These changes mediate multiple biological responses such as tumour cell apoptosis and cell cycle arrest, as well as inhibition of angiogenesis and modulation of innate and adaptive immunity. Two structurally different HDACi, panobinostat and romidepsin have had success in clinical trials, particularly in the treatment of cutaneous T-cell lymphoma (CTCL). Although very well tolerated by patients, both cause severe reductions in blood platelet numbers, called thrombocytopenia. Platelets are vital for the prevention of bleeding via the closure of any vessel wall defect. Thrombocytopenia therefore compromises the use of these highly effective novel drugs, particularly in combination treatment strategies. This study examined the pathophysiology of HDACi-induced thrombocytopenia and showed this clinical problem could be circumvented, potentially allowing more patients to receive treatment. Following the finding that HDACi induced dose-dependant reductions in platelet number in C57BL/6 mice, susequent platelet production and half life studies demonstrated that this was not due to HDACi reducing platelet half life or inducing platelet apoptosis. These results were in accordance with mice with genetic ablation of pro-apoptotic molecules also becoming thrombocytopenic post HDACi. Pro-platelet assays in primary cultured megakarycytes confirmed a marked reduction in pro-platelet formation following HDACi treatment. Thrombopoietin (TPO)-mimetics are clinically developed growth factors, which increase megakaryocyte production of platelets. TPO-mimetics given to HDACi treated mice prevented thrombocytopenia occurring in both wild type and tumour bearing settings, a result which may allow patients to continue to receive HDACi despite thrombocytopenia in the future. Decreased pro-platelet formation following HDACi was shown associated with an increase in phosphorylated myosin light chain (pMLC) both in-vitro and in vivo. HDACi increase pMLC levels by causing the proteasomal degradation of the cytoskeletal remodelling Rho-GTPase proteins CDC42, Rac1 and RhoA. Immuno-precipitation studies demonstrated HDACi treatment resulted in the the dissociation of GTPases dissociate from their chaperone protein, Rho-GDI, causing their rapid, transient activation and subsequent degradation. Confocal microscopy confirmed the specific degradation of non-plasma membrane bound Rho-GTPases. Ultimately, exhaustion of the available pool of the Rho-GTPases occurs, resulting in a reduction of both the activated and total fractions available to the cell. HDACi are known to acetylate the heat shock protein (HSP)-90, releasing its chaperone proteins for degradation. Using immuno-precipitation studies, this thesis shows HSP90 to co-chaperone the Rho-GTPases with Rho-GDI, explaining why inhibition of HSP90 recapitulates the effects of HDACi, specifically dissociation of Rho-GTPases from Rho-GDI followed by their degradation, increasing pMLC and reducing pro-platelet formation. The data therefore suggests HDACi-induced acetylation of HSP90, and potentially Rho-GDI may be responsible for causing the clinical problem of thrombocytopenia.