Investigation of the Efficacy of PXD101 (Belinostat) on Primary Leukaemic Cells and Cell Lines as a Novel Agent for Childhood Acute Lymphoblastic Leukaemia (ALL).

PXD101 (belinostat) is a novel hydroxamate-type of histone deacetylation inhibitor that has been reported to inhibit the growth of a panel of adult carcinomas and melanoma tumour cell lines both in vitro and as xenografts. Activity was associated with a rapid and sustained increase in acetylation of histone proteins H3 and H4 in vitro, and in some cell lines with the induction of apoptosis as measured by PARP cleavage. We have evaluated the in vitro and ex vivo activity of belinostat in a panel of leukaemic cell lines and in primary childhood acute lymphoblastic leukaemia (ALL) to determine the potential for taking this compound forward into early clinical trials in childhood leukaemia. The cell lines used in this study were CCRF-CEM (T-ALL), including steroid sensitive and resistant derivatives, MOLT4 and Jurkat (T-ALL), NALM6 (B-ALL), Daudi (Burkitt’s lymphoma) and Kasumi (AML). In vitro cytotoxicity was measured using the Sulphorodamine B assay and cell viability and apoptosis was assessed by flow cytometry using annexin V and propidium iodide staining respectively. The effects of belinostat as a single agent and in combination with dexamethasone, vincristine and doxorubicin were evaluated. Belinostat was also studied in primary leukaemia cells from children with ALL and mononuclear cells from normal bone marrow. Western blotting measuring the acetylation of histones H3 and H4 was used to assess belinostat HDAC inhibitory activity. The time course for onset and duration of HDAC inhibition was investigated. The leukaemia cell lines demonstrated a wide range of in vitro sensitivity to belinostat, with LC50s ranging from 0.3 μM in the Jurkat cells to 10 μM in MOLT4 cells. Normal bone marrow was shown to be refractory to belinostat compared to primary leukaemia cells and the majority of leukaemia cells lines. Primary childhood ALL samples were notably more sensitive to belinostat than the cell lines studied. Interestingly, primary ALL samples observed to be resistant dexamethasone were sensitive to belinostat. Inhibition of histone deacetylation (H3 and H4) was demonstrated within 30 minutes of exposure to belinostat in all cell lines and persisted throughout continuous 24 hour exposure. Histone acetylation was restored between 4–8 hours following removal of belinostat. The induction of histone acetylation did not always correlate with induction of cell death. Rapid inhibition of histone deacetylation was observed in the AML cell line Kasumi, which was refractory to belinostat. The cytotoxic activity of belinostat was associated with markers of apoptosis, as demonstrated by PARP cleavage and Annexin V staining In conclusion, belinostat demonstrated dose dependent in vitro cytotoxicity against a panel of leukaemia cell lines and primary childhood ALL samples. Notably, belinostat was effective in steroid resistant cell lines and primary ALL samples. No synergy was observed between belinostat and drugs used in the treatment of ALL. Further investigation of the scheduling of belinostat when combined with conventional cytotoxic agents is on-going. In addition, the in vivo efficacy of belinostat in a xenograft model of childhood leukaemia is currently being evaluated.