alvocidib and Blast-Crisis

Flavopiridol is a cyclin-dependent kinase inhibitor that induces cell cycle arrest, apoptosis, and clinical responses in selected patients with acute myeloid leukemia (AML). A better understanding of the molecular pathways targeted by flavopiridol is needed to design optimal combinatorial therapy. Here, we report that in vivo administration of flavopiridol induced expression of the BCL-2 anti-apoptotic gene in leukemic blasts from adult patients with refractory AML. Moreover, flavopiridol repressed the expression of genes encoding oncogenic transcription factors (HMGA1, STAT3, E2F1) and the major subunit of RNA Polymerase II. Our results provide mechanistic insight into the cellular pathways targeted by flavopiridol. Although further studies are needed, our findings also suggest that blocking anti-apoptotic pathways could enhance cytotoxicity with flavopiridol.

Topics: Adult; Antineoplastic Agents; Apoptosis Regulatory Proteins; Blast Crisis; Female; Flavonoids; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Oncogenes; Piperidines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; RNA Polymerase II; Transcription Factors; Young Adult

The mechanism and functional significance of XIAP and Mcl-1 down-regulation in human leukemia cells exposed to the histone deacetylase inhibitor vorinostat and the cyclin-dependent kinase inhibitor flavopiridol was investigated. Combined exposure of U937 leukemia cells to marginally toxic concentrations of vorinostat and flavopiridol resulted in a marked increase in mitochondrial damage and apoptosis accompanied by pronounced reductions in XIAP and Mcl-1 mRNA and protein. Down-regulation of Mcl-1 and XIAP expression by vorinostat/flavopiridol was associated with enhanced inhibition of phosphorylation of RNA polymerase II and was amplified by caspase-mediated protein degradation. Chromatin immunoprecipitation analysis revealed that XIAP and Mcl-1 down-regulation were also accompanied by both decreased association of nuclear factor-kappaB (XIAP) and increased E2F1 association (Mcl-1) with their promoter regions, respectively. Ectopic expression of Mcl-1 but not XIAP partially protected cells from flavopiridol/vorinostat-mediated mitochondrial injury at 48 h, but both did not significantly restored clonogenic potential. Flavopiridol/vorinostat-mediated transcriptional repression of XIAP, Mcl-1-enhanced apoptosis, and loss of clonogenic potential also occurred in primary acute myelogenous leukemia (AML) blasts. Together, these findings indicate that transcriptional repression of XIAP and Mcl-1 by flavopiridol/vorinostat contributes functionally to apoptosis induction at early exposure intervals and raise the possibility that expression levels may be a useful surrogate marker for activity in current trials.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Blast Crisis; Blotting, Western; Butyrates; Caspases; Chromatin Immunoprecipitation; Cyclin-Dependent Kinases; Cytochromes c; Down-Regulation; Drug Interactions; Flavonoids; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia, Myeloid, Acute; Membrane Potential, Mitochondrial; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Piperidines; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Tumor Stem Cell Assay; U937 Cells; Vorinostat; X-Linked Inhibitor of Apoptosis Protein

Interactions between proteasome and cyclin-dependent kinase inhibitors have been examined in human leukemia cells in relation to induction of apoptosis. Simultaneous exposure (24 h) of U937 myelomonocytic leukemia cells to 100 nM flavopiridol and 300 nM MG-132 resulted in a marked increase in mitochondrial injury (cytochrome c, Smac/DIABLO release, loss of deltaPsi(m)), caspase activation, and synergistic induction of cell death, accompanied by a marked decrease in clonogenic potential. Similar effects were observed with other proteasome inhibitors (e.g., Bortezomib (VELCADE trade mark bortezomib or injection), lactacystin, LLnL) and cyclin-dependent kinase inhibitors (e.g., roscovitine), as well as other leukemia cell types (e.g., HL-60, Jurkat, Raji). In U937 cells, synergistic interactions between MG-132 and flavopiridol were associated with multiple perturbations in expression/activation of signaling- and survival-related proteins, including downregulation of XIAP and Mcl-1, activation of JNK and p34(cdc2), and diminished expression of p21(CIP1). The lethal effects of MG-132/flavopiridol were not reduced in leukemic cells ectopically expressing Bcl-2, but were partially attenuated in cells ectopically expressing dominant-negative caspase-8 or CrmA. Flavopiridol/proteasome inhibitor-mediated lethality was also significantly diminished by agents and siRNA blocking JNK activation. Lastly, coadministration of MG-132 with flavopiridol resulted in diminished DNA binding of NF-kappaB. Notably, pharmacologic interruption of the NF-kappaB pathway (e.g., by BAY 11-7082, PDTC, or SN-50) or molecular dysregulation of NF-kappaB (i.e., in cells ectopically expressing an IkappaBalpha super-repressor) mimicked the actions of proteasome inhibitors in promoting flavopiridol-induced mitochondrial injury, JNK activation, and apoptosis. Together, these findings indicate that proteasome inhibitors strikingly lower the apoptotic threshold of leukemic cells exposed to pharmacologic CDK inhibitors, and suggest that interruption of the NF-kappaB cytoprotective pathway and JNK activation both play key roles in this phenomenon. They also raise the possibility that combining proteasome and CDK inhibitors could represent a novel antileukemic strategy.

Topics: Anisomycin; Antineoplastic Agents; Apoptosis; Blast Crisis; Boronic Acids; Bortezomib; Cyclin-Dependent Kinases; Cysteine Endopeptidases; Enzyme Inhibitors; Flavonoids; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Jurkat Cells; Leukemia, Myeloid, Acute; Leupeptins; Mitogen-Activated Protein Kinases; Multienzyme Complexes; NF-kappa B; Piperidines; Proteasome Endopeptidase Complex; Pyrazines; Tumor Cells, Cultured; U937 Cells