Pyrrolo-1,5-benzoxazepines induce apoptosis in chronic B-lymphoid malignancies

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Pyrrolo-1,5-benzoxazepine (PBOX) compounds are novel agents that we have shown induce apoptosis in chronic myelogenous leukemia (CML), breast and prostate cancer cell lines. PBOX-induced apoptosis in CML cells is dependent on early activation of c-Jun N-terminal kinase (JNK) and subsequent phosphorylation of bcl-2 and cell cycle arrest. Importantly, PBOX compounds have been shown to display minimal toxicity against normal blood and bone marrow cells. Here we describe the ability of a potent PBOX compound, PBOX-15, to induce apoptosis in chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) cells. These incurable chronic B-lymphoid malignancies are characterised by the acculumation of mature peripheral B cells with defective apoptotic mechanisms. PBOX-15 induced apoptosis in CLL cells isolated from the peripheral blood of CLL patients (n=30) as demonstrated by flow cytometry analysis, annexinV binding assay and PARP cleavage. PBOX-15-induced apoptosis occurred within 24 hours and was more rapid than fludarabine-induced apoptosis in these cells. In addition to activity in CLL cells from treatment naïve patients, PBOX-15 induced apoptosis in fludarabine resistant CLL cells harbouring a 17p deletion resulting in loss of p53 expression. PBOX-15-induced apoptosis in CLL cells was partially caspase and JNK dependent. Bcl-2 is over-expressed in CLL cells and has been implicated in CLL resistance to apoptosis, however no change in bcl-2 and bcl-xl proteins were observed prior to PBOX-15-induced apoptosis in CLL cells. PBOX-15 arrested two MM cell lines (H929 and U266) in the G₂/M phase of the cell cycle, however only the H929 cells were found to undergo apoptosis as assessed by morphological changes in the cells, DNA laddering and AnnexinV binding assay. Caspase-8 was activated following PBOX-15 treatment of H929 cells and inhibition of caspase-8 completely inhibited PBOX-15-mediated apoptosis in these cells. Treatment of H929 cells with a potent JNK inhibitor had no effect on PBOX-15-induced apoptosis. In addition, expression of Bcl-2 in H929 cells was decreased in a dose- and time-dependent manner following PBOX-15 treatment. In conclusion, PBOX-15 exhibited varied anti-cancer activity against CLL cells ex vivo and MM cell lines. Studies are underway to further elucidate the mechanism of action of PBOX-15-induced apoptosis in these chronic B-lymphoid malignancies.