Induction of Bim Facilitates Apoptosis in Leukemia Cells Treated with HDAC Inhibitors.

Abstract

Lymphoblastic leukemias containing chromosomal translocations involving the Mixed Lineage Leukemia (MLL, HRX, ALL-1) gene, as well as most acute myeloid leukemias (AMLs) have relatively poor clinical prognoses due in part to intrinsic drug resistance. Therefore, new avenues are being explored for treatment of MLL-rearranged ALL and AMLs. One possible new therapeutic class currently being investigated is the histone deacetylase (HDAC) inhibitors. We utilized the histone deacetylase inhibitor NVP-LAQ824 (Novartis, Basel, Switzerland) and analyzed its effects on MLL rearranged and other myeloid leukemias. We also made use of an MLL-AF9 expressing myeloid leukemia cell line (AKLG) derived from purified murine leukemia stem cells to perform gene expression analysis on NVP-LAQ824 treated cells in order to further understand the mechanism of action of HDAC inhibitors, and to potentially identify cooperating therapeutics. NVP-LAQ824 inhibits cell growth at similar concentrations for all cell lines and primary patient samples tested (~25–50nM) as determined by MTT assay 48 hours after treatment. NVP-LAQ824 does not appear to induce apoptosis solely through inhibition of the HSP90/FLT3-ITD complex as cell lines possessing FLT3-ITD (a HSP90-chaperoned protein) and cells without this mutation have similar drug sensitivities. In fact, in cells overexpressing FLT3-ITD that are treated with NVP-LAQ824, phospho-FLT3-ITD levels do not diminish. Microarray data indicated that NVP-LAQ824 induces the BH3-only family member bim. This finding was verified by Western blotting in all cell lines and patient samples tested. Further, shRNA-mediated knockdown of Bim induced relative resistance of cells to NVP-LAQ824. The expression profile also showed similarities to gene expression patterns of dexamethasone treated cells, namely, increased bim levels and decreased expression of c-myc, raising the possibility of synergy between the two drugs. Using MTT assays, we discovered that NVP-LAQ824 in low doses (25nM) induces sensitivity to dexamethasone in glucocorticoid resistant cell lines in a glucocorticoid receptor (GR) dependent manner. Therefore, our data indicate that NVP-LAQ824 may reverse glucocorticoid resistance and may provide insight into glucocorticoid resistance in MLL rearranged leukemias. The biochemistry behind HDAC inhibitors merits further study.