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Title: Investigation of pan-HDAC inhibitor Vorinostat and novel microtubule targeting agent PBOX 15 as a potential therapeutic combinational approach for chronic lymphocytic leukaemia
Author: Jameson, Warren Randall
Awarding Body: Ulster University
Current Institution: Ulster University
Date of Award: 2012
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Chronic Lymphocytic Leukaemia (CLL) is the most common form of leukaemia in adults in the western world. It is characterised by an accumulation of CDS, CD19 and CD23 positive lymphocytes in the peripheral blood, lymph node, bone marrow, liver and spleen. Within CLL, two subtypes have been defined by the degree of somatic hypermutation, Unmutated (UM) or Mutated (M) in the variable region of the immunoglobulin heavy chain (IgHV) gene. First line chemotherapy treatment of CLL has demonstrated prolonged first remission; however disease progression and chemotherapy resistance occurs through Microenvironment interactions that have been identified include TH2 helper cells via CD40L with the secretion of Interleukin 4 within the lymph node, nurse like cells (NLCs) within blood and bone-marrow stromal cells. In our study, we demonstrate that pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) treatment of CLL cell lines causes downregulation of c-FLIP mRNA and protein, caspase 8 activation and subsequent apoptosis. Combinations of SAHA and recombinant TRAIL/Ch-ll demonstrated re-sensitivity of CLL cells to death receptor mediated apoptosis. This mechanism was found to be due to c-FLIP downregulation rather than XIAP loss or death receptor expression modulation. Interestingly, combinational treatment of SAHA and chemotherapy agent Fludarabine demonstrated potentiated apoptosis in UM IgHV CLL cell line only. Combinations of SAHA and Chlorambucil demonstrated potentiated apoptosis over single agent treatment in both CLL cell lines. Lenti-viral knockdown of c-FLIP in UM CLL cell line caused re-sensitivity to TRAIL induced apoptosis and increased sensitivity to Fludarabine. CLL patient cells treated with SAHA showed increases in apoptosis within 24hrs with further increase in apoptosis following recombinant-TRAIL treatment. When CLL cells were placed into CD40L/IL-4 co-culture, increases in anti-apoptotic protein expression was found, this included c-FLIP isoforms, XIAP, and Bcl-xL. Primary CLL cells from 18 patients treated whilst in the CD40L/IL-4 system for 24hrs show significant increased levels of apoptosis with inhibited the upregulation of c-FLIP. Previously co-cultured cells (24hrs) before treatment became apoptotic after SAHA treatment. Surprisingly, SAHA treatment alone of patient cells in CD40L/IL-4 show significantly more apoptosis compared to Fludarabine treatment. However, whilst in CD40L/IL-4 combinations of SAHA and TRAIL or SAHA and Ch-ll (Anti-Fas antibody) did not show potentiated apoptosis over single agent treatment. Finally, we were very lucky to have the chance to treat CLL cells derived from the bone marrow of a patient. SAHA was able to also cause downregulation of c-FLIP protein and activation of caspase 8 with subsequent apoptosis. Pyrrolo-l,5-benzoxazepine-15 (PBOX-15) is a novel microtubule depolymerisation agent that induces cell cycle arrest and subsequent apoptosis in a number of cancer cell lines. CLL cell lines treated with PBOX 15 presented increases in apoptosis and Gz/M arrest. PBOX 15 treatment caused increased expression of c-FLIP isoforms even with the activation of caspase 8. Combinational treatments of SAHA and PBOX 15 on CLL cell lines presented synergistic CI calculated by CalcuSyn. This was further confirmed by annexin V /PI. Addition of SAHA with PBOX 15 blocked the PBOX 15 induced upregulation of c-FLIP. PBOX 15 induced apoptosis of CLL cells while in NT-L but CD40L/IL-4 co-culture reduced its apoptotic effect. Furthermore, combinations of SAHA and PBOX 15 caused significant increased apoptosis over single agents of CLL cells while in CD40L/IL-4. We speculate that the addition of SAHA causes downregulation of c-FLIP allowing further apoptosis via caspase 8. The increases in apoptosis observed were not dependent on IgHV status. In summary, this study hypothesis is to identify that CLL cell lines can be sensitised to death receptor mediated apoptosis and chemotherapy with treatment in combination with HDACi SAHA causing the downregulation of c-FLIP. Furthermore, microenvironment interactions such as CD40L/IL-4 can cause upregulation of protective proteins causing resistance to apoptosis. SAHA treatment of CLL cells while in CD40L/IL-4 can cause apoptosis of CLL cells by causing downregulation of c-FLIP. Furthermore, we hypothesize that novel microtubule targeting agent PBOX 15 can cause cell cycle arrest and apoptosis of CLL cell lines and apoptosis of CLL patient cells as early as 24hrs. Resistance is found with CD40L/IL-4; however SAHA will be able to cause sensitisation and potentiation of apoptosis when CLL cells are in CD40L/IL-4. We further theorise that combinations of SAHA and PBOX 15 could be effective against CLL cells in pro-survival microenvironment niche LN which are resistant to standard chemotherapy.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available