We are investigating the anticancer potential of a novel compound, Pyrrolo-1,5-benzoxazapines-15 (PBOX-15), in childhood acute lymphoblastic leukemia (cALL). We have previously shown that PBOX compounds induce apoptosis in a wide range of malignancies including chronic myeloid leukemia and chronic lymphocytic leukemia (CLL). We have recently identified tubulin as a molecular target of pro-apoptotic PBOX compounds (Mulligan et al., Mol Pharmacol. 2006 Jul;70).

SD-1 (pre B-ALL) and CCRF-CEM (T-ALL) cell lines were used as representative tumor models. The cytotoxicity of PBOX-15 and its ability to induce apoptosis in ALL cells was examined by MTT assays and flow cytometry. PBOX-15 induced cell cycle arrest at the G2/M phase of the cell cycle and was cytotoxic in both ALL cell lines (IC50= 0.5μM). The level of PBOX-15 induced apoptosis was comparable to the tubulin targeting drugs vincristine and nocodazole, used in the treatment of ALL. As ALL is a malignancy of immune cell origin, the effect of PBOX-15 on both the T- and B-ALL cells was examined using the Applied Biosystems 7900HT Micro Fluidic Card. This immune profiling array analyses 96 genes involved in the immune response and includes a range of growth factors, cytokines and chemokines which may have an important role in sustaining and promoting ALL cell survival. Expression of a number of cytokines was affected by PBOX-15 treatment including interleukin (IL)-1α, IL-3, IL-4, IL-10, IL-13, IL-15 and interferon-γ. Interestingly, there was a >9 fold increase in the expression of the chemokine IL-8 in the SD-1 cells following 1μM PBOX-15 treatment, however there was a >10 fold decrease in the expression of IL-8 in the T-ALL cell line CCRF-CEM. IL-8 has been shown to play a role in other hematological malignancies including CLL, where it is associated with prolonged survival of these cells. The SD-1 cell line was found to produce high levels of IL-8, while CCRF-CEM cells produced only low amounts of IL-8. The ability of IL-8 to act as a survival factor for these cells was examined by serum starvation of ALL cells followed by addition of recombinant IL-8. Cell proliferation was assessed using a BrdU assay. IL-8 did not appear to act as a survival signal for the B cell ALL cell line, however, at lower concentrations of IL-8 there was an increase in cell proliferation in the T cell ALL cell line, suggesting that IL-8 may be a possible survival factor for these T-ALL cells.

These results show that PBOX-15 is a potent inducer of apoptosis in cALL and may be of particular benefit in drug refractory and resistant patients. This work also highlights the importance of examining the effect of chemotherapeutic drugs on the immune system particularly in diseases of immune origin such as leukemia.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA