A Novel Chromene-Based Pan-PI3 Kinase Inhibitor Displays Preclinical Activity in Leukemia and Myeloma.

D-cyclins are universally dysregulated in multiple myeloma and frequently over-expressed in acute leukemia. Therefore, to better understand the regulation of the D-cyclins and identify leads for novel therapeutic agents for the treatment of hematologic malignancies, we conducted a high throughput screen of 50,000 novel chemical compounds to identify inhibitors of D-cyclin transactivation. From this screen, we identified a chromene-based compound 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (pichromene). In secondary assays, pichromene reduced expression of cyclins D1, D2, and D3 in myeloma and leukemia cell lines at low micromolar concentrations. Furthermore, pichromene arrested the cells in the G0/G1 phase of the cell cycle. In myeloma and leukemia cell lines, pichromene decreased levels of phospho-AKT, but did not alter levels of total AKT.

PI3 kinases regulate AKT phosphorylation that, in turn, regulate D-cyclin expression and cell cycle progression. Therefore, we evaluated the effects of pichromene on the enzymatic activity of PI3 kinases. In cell-free enzymatic assays, pichromene inhibited the enzymatic activity of all four isoforms of the PI3 kinase, PI3Kalpha, beta, delta and gamma, with similar efficacy. In contrast it did not markedly inhibit the enzymatic activities of unrelated kinases AKT 1, 2 or 3, PDK 1 or 2, or GSK3β or 3α at concentrations up to 300 μM in a similar cell-free assay. However, in intact cells, due to its inhibition of PI3 kinases, pichomene inhibited AKT activity as noted above.

As inhibitors of PI3 kinases are pro-apoptotic and may have anti-cancer activity, we evaluated the effects of pichromene on the viability of leukemia and myeloma cells. Leukemia and myeloma cell lines were treated with increasing concentrations of pichromene and cell viability was measured after 72 hours by an MTS assay. Pichromene induced cell death in 9/10 leukemia and 9/10 myeloma cell lines with an ED50 < 10 μM. In contrast, it was less cytotoxic to primary normal hematopoietic cells obtained from volunteer donors of stem cells for allotransplant. Apoptosis was confirmed by Annexin V staining. Cell death was associated with caspase activation as demonstrated by the cleavage of caspase-3 and PARP through immunoblotting. Interestingly, U266 was the one myeloma cell line that was resistant to pichromene, and lacked detectable basal levels of phospho-AKT by immunoblotting.

Given the effects of pichromene on malignant cells, we evaluated the efficacy of this compound in a leukemia xenograft mouse model. K562 cells were implanted subcutaneously into sublethally irradiated NOD/SCID mice. Mice were then treated with pichromene (50 mg/kg/day) or buffer control by oral gavage. Pichromene decreased tumor weight and volume by more than 35% as early as 8 days after treatment. No evidence of weight loss or gross organ toxicity was observed even when mice were treated with up to 500mg/kg/day of pichromene by oral gavage or intraperitoneally.

Thus, in summary, we have identified a novel pan-inhibitor of PI3 kinases that displays preclinical efficacy in myeloma and leukemia.