piperidines 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

Approximately 20% of patients with acute myeloid leukaemia (AML) have a mutation in FMS-like-tyrosine-kinase-3 (FLT3). FLT3 is a trans-membrane receptor with a tyrosine kinase domain which, when activated, initiates a cascade of phosphorylated proteins including the SRC family of kinases. Recently our group and others have shown that pharmacologic inhibition and genetic knockdown of Bruton's tyrosine kinase (BTK) blocks AML blast proliferation, leukaemic cell adhesion to bone marrow stromal cells as well as migration of AML blasts. The anti-proliferative effects of BTK inhibition in human AML are mediated via inhibition of downstream NF-κB pro-survival signalling however the upstream drivers of BTK activation in human AML have yet to be fully characterised. Here we place the FLT3-ITD upstream of BTK in AML and show that the BTK inhibitor ibrutinib inhibits the survival and proliferation of FLT3-ITD primary AML blasts and AML cell lines. Furthermore ibrutinib inhibits the activation of downstream kinases including MAPK, AKT and STAT5. In addition we show that BTK RNAi inhibits proliferation of FLT3-ITD AML cells. Finally we report that ibrutinib reverses the cyto-protective role of BMSC on FLT3-ITD AML survival. These results argue for the evaluation of ibrutinib in patients with FLT3-ITD mutated AML.

Topics: Adenine; Agammaglobulinaemia Tyrosine Kinase; Apoptosis; Blast Crisis; Cell Line, Tumor; Cell Survival; Daunorubicin; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Mutation; Piperidines; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; RNA, Small Interfering; Signal Transduction

Inhibitors of nicotinamide phosphoribosyltransferase have recently been validated as therapeutic targets in leukemia, but the mechanism of leukemogenic transformation downstream of this enzyme is unclear.. Here, we evaluated whether nicotinamide phosphoribosyltransferase's effects on aberrant proliferation and survival of myeloid leukemic cells are dependent on sirtuin and delineated the downstream signaling pathways operating during this process.. We identified significant upregulation of sirtuin 2 and nicotinamide phosphoribosyltransferase levels in primary acute myeloid leukemia blasts compared to in hematopoietic progenitor cells from healthy individuals. Importantly, specific inhibition of nicotinamide phosphoribosyltransferase or sirtuin 2 significantly reduced proliferation and induced apoptosis in human acute myeloid leukemia cell lines and primary blasts. Intriguingly, we found that protein kinase B/AKT could be deacetylated by nicotinamide phosphoribosyltransferase and sirtuin 2. The anti-leukemic effects of the inhibition of nicotinamide phosphoribosyltransferase or sirtuin 2 were accompanied by acetylation of protein kinase B/AKT with subsequent inhibition by dephosphorylation. This leads to activation of glycogen synthase kinase-3 β via diminished phosphorylation and, ultimately, inactivation of β-catenin by phosphorylation.. Our results provide strong evidence that nicotinamide phosphoribosyltransferase and sirtuin 2 participate in the aberrant proliferation and survival of leukemic cells, and suggest that the protein kinase B/AKT/ glycogen synthase kinase-3 β/β-catenin pathway is a target for inhibition of nicotinamide phosphoribosyltransferase or sirtuin 2 and, thereby, leukemia cell proliferation.

Topics: Acrylamides; Apoptosis; Benzothiazoles; beta Catenin; Blast Crisis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Leukemic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Leukemia, Myeloid; Models, Biological; Nicotinamide Phosphoribosyltransferase; Piperidines; Proto-Oncogene Proteins c-akt; RNA, Messenger; Signal Transduction; Sirtuin 2

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