alvocidib and Multiple-Myeloma

An increasing number of unique active new chemotherapeutic and biologic agents are currently available for clinical research studies. Nucleoside analogs in development for non-Hodgkin's lymphoma (NHL) include clofarabine, troxacitabine, and bendamustine, a hybrid of an alkylating nitrogen mustard group and a purine-like benzimidazole, with demonstrated activity in NHL. Drugs directed at the cell cycle include flavopiridol and UCN-01. The proteasome plays a pivotal role in cellular protein regulation and activation of NFkappaB, which maintains cell viability through the transcription of inhibitors of apoptosis. PS-341 is a specific, selective inhibitor of the 26S proteasome which induces apoptosis and has activity in cell types characterized by overexpression of Bcl-2. Response rates of 50%, including complete remissions, have been reported using this agent in patients with refractory multiple myeloma. Studies are ongoing in NHL and chronic lymphocytic leukemia. G3139, an antisense oligonucleotide, has shown promise in early studies. Rituximab has revolutionized the treatment of NHL. However, other active antibodies are now available, including alemtuzumab, epratuzumab, and Hu1D10. The radioimmunoconjugates (90)Y-ibritumomab tiuxetan and (131)I-tositumomab may also play an important role in the management of NHL. Future therapeutic strategies should involve rational combinations of new chemotherapy drugs, biologic agents, and antisense compounds to increase the cure rate in patients with lymphoma.

Topics: Adenine Nucleotides; Alemtuzumab; Alkaloids; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibodies, Monoclonal, Murine-Derived; Antibodies, Neoplasm; Antineoplastic Agents; Apoptosis; Arabinonucleosides; Bendamustine Hydrochloride; Boronic Acids; Bortezomib; Cell Cycle; Cell Survival; Clofarabine; Cytosine; Dioxolanes; Flavonoids; Hematologic Neoplasms; Humans; Immunoconjugates; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma; Lymphoma, Non-Hodgkin; Multiple Myeloma; NF-kappa B; Nitrogen Mustard Compounds; Oligonucleotides, Antisense; Peptide Hydrolases; Piperidines; Protease Inhibitors; Proteasome Endopeptidase Complex; Pyrazines; Remission Induction; Rituximab; Staurosporine; Thionucleotides

Flavopiridol is primarily a cyclin-dependent kinase-9 inhibitor, and we performed a dose escalation trial to determine the maximum tolerated dose and safety and generate a pharmacokinetic (PK) profile.. Patients with a diagnosis of relapsed myeloma after at least two prior treatments were included. Flavopiridol was administered as a bolus and then continuous infusion weekly for 4 weeks in a 6-week cycle.. Fifteen patients were treated at three dose levels (30 mg/m(2) bolus, 30 mg/m(2) CIV to 50 mg/m(2) bolus, and 50 mg/m(2) CIV). Cytopenias were significant, and elevated transaminases (grade 4 in 3 patients, grade 3 in 4 patients, and grade 2 in 3 patients) were noted but were transient. Diarrhea (grade 3 in 6 patients and grade 2 in 5 patients) did not lead to hospital admission. There were no confirmed partial responses although one patient with t(4;14) had a decrease in his monoclonal protein >50 % that did not persist. PK properties were similar to prior publications, and immunohistochemical staining for cyclin D1 and phospho-retinoblastoma did not predict response.. Flavopiridol as a single agent given by bolus and then infusion caused significant diarrhea, cytopenias, and transaminase elevation but only achieved marginal responses in relapsed myeloma (ClinicalTrials.gov identifier NCT00112723).

Topics: Aged; Aged, 80 and over; Antineoplastic Agents; Dose-Response Relationship, Drug; Flavonoids; Humans; Middle Aged; Multiple Myeloma; Piperidines; Recurrence

Lenalidomide, an immunomodulatory agent, and flavopiridol, a broad cyclin-dependent kinase inhibitor, are active therapies for clinical use in genomic high-risk chronic lymphocytic leukemia. A high-performance liquid chromatographic assay with tandem mass spectrometric detection has been developed to simultaneously quantify lenalidomide and flavopiridol in human and mouse plasma to facilitate their combined clinical development. Samples were prepared by liquid-liquid extraction with acetonitrile (ACN)-containing internal standard, genistein, followed by evaporation of solvent and reconstitution in 95/5 H2O/ACN. Lenalidomide and internal standard were separated by reversed-phase liquid chromatography on a C-18 column using a gradient of H2O and ACN, each with 0.1% formic acid. Atmospheric pressure chemical ionization in positive ion mode with single reaction monitoring on a triple quadrupole mass spectrometer was applied to detect transitions of lenalidomide (260.06 > 149.10) and flavopiridol (402.09 > 341.02). Lower limits of quantification of lenalidomide and flavopiridol were 1 and 0.3 nM, respectively. Recoveries of lenalidomide and flavopiridol from human plasma ranged from 99% to 116% throughout their linear ranges. Within- and between-run precision and accuracy of replicate samples were all less than 15%. This is the most sensitive analytical method reported to date for both lenalidomide and flavopiridol. This sensitivity will enable late terminal phase concentration measurements and accurate pharmacokinetic parameter estimation in a planned clinical trial with lenalidomide and flavopiridol in patients with chronic lymphocytic leukemia.

Topics: Chromatography, Liquid; Flavonoids; Humans; Lenalidomide; Multiple Myeloma; Piperidines; Recurrence; Spectrometry, Mass, Electrospray Ionization; Thalidomide; Time Factors

Flavopiridol downregulates anti-apoptotic regulators including Mcl-1, upregulates p53, globally attenuates transcription through inhibition of P-TEFb, binds to DNA, and inhibits angiogenesis. Eighteen myeloma patients were treated with 1-hour flavopiridol infusions for 3 consecutive days every 21 days. Immunoblotting for Mcl-1, Bcl-2, p53, cyclin D, phosphoRNA polymerase II and phosphoSTAT 3 was conducted on myeloma cells. Ex vivo flavopiridol treatment of cells resulted in cytotoxicity, but only after longer exposure times at higher flavopiridol concentrations than were anticipated to be achieved in vivo. No anti-myeloma activity was observed in vivo. As administered, flavopiridol has disappointing activity as a single agent in advanced myeloma.

Topics: Aged; Aged, 80 and over; Female; Flavonoids; Humans; Male; Middle Aged; Multiple Myeloma; Neoplasm Recurrence, Local; Piperidines; Treatment Outcome

The BCL-2-specific BH3-mimetic ABT-199 (venetoclax) has been reported to be principally active against favourable-risk multiple myeloma (MM) cells, prompting efforts to extend its activity to include more resistant, higher-risk MM subsets.. Effects of the CDK9 inhibitor flavopiridol (FP; alvocidib) on responses to ABT-199 were examined in MM cells. Cell death and protein expression were evaluated by western blot and immunofluorescence. Xenograft models were used to study combination effects in vivo.. FP synergistically increased ABT-199 lethality in both ABT-199-sensitive and insensitive MM cells. FP blocked CDK9 activation/positive transcription elongation factor B phosphorylation, downregulated MCL-1, increased BCL-2/MCL-1 ratios, and upregulated BIM. MCL-1 ectopic expression or knockdown in MM cells significantly diminished or increased ABT-199 sensitivity, respectively. CDK9 knockdown triggered MCL-1 downregulation and increased ABT-199 activity, whereas BIM knockdown significantly reduced FP/ABT-199 lethality. FP also enhanced ABT-199 lethality in unfavourable prognosis primary MM cells. HS-5 cell co-culture failed to protect MM cells from the FP/ABT-199 regimen, suggesting circumvention of microenvironmental signals. Finally, FP/ABT-199 significantly increased survival in systemic xenograft and immune-competent MM models while exhibiting minimal toxicity.. These findings argue that CDK9 inhibitors, for example, FP may increase the antimyeloma activity of ABT-199, including in unfavourable-risk MM minimally responsive to ABT-199 alone.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bcl-2-Like Protein 11; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cyclin-Dependent Kinase 9; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Flavonoids; Gene Knockdown Techniques; Humans; Mice; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Transplantation; Piperidines; Primary Cell Culture; Proto-Oncogene Proteins c-bcl-2; Risk Assessment; Sulfonamides; Up-Regulation

Multiple Myeloma (MM) is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cell that eventually develops resistance to chemotherapy. Drug resistance, differentiation block and increased survival of the MM tumor cells result from high genomic instability. Chromosomal translocations, the most common genomic alterations in MM, lead to dysregulation of cyclin D, a regulatory protein that governs the activation of key cell cycle regulator--cyclin dependent kinase (CDK). Genomic instability was reported to be affected by over expression of another CDK regulator--cyclin E (CCNE). This occurs early in tumorigenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore sought to investigate the role of cyclin E in MM. CCNE1 expression was found to be heterogeneous in various MM cell lines (hMMCLs). Incubation of hMMCLs with seliciclib, a selective CDK-inhibitor, results in apoptosis which is accompanied by down regulation of MCL1 and p27. Ectopic over expression of CCNE1 resulted in reduced sensitivity of the MM tumor cells in comparison to the paternal cell line, whereas CCNE1 silencing with siRNA increased the cell sensitivity to seliciclib. Adhesion to FN of hMMCLs was prevented by seliciclib, eliminating adhesion-mediated drug resistance of MM cells. Combination of seliciclib with flavopiridol effectively reduced CCNE1 and CCND1 protein levels, increased subG1 apoptotic fraction and promoted MM cell death in BMSCs co-culture conditions, therefore over-coming stroma-mediated protection. We suggest that seliciclib may be considered as essential component of modern anti MM drug combination therapy.

Topics: Apoptosis; Cell Line, Tumor; Cyclin D1; Cyclin E; Flavonoids; Gene Expression Profiling; Gene Expression Regulation; Gene Silencing; Genomic Instability; Humans; Multiple Myeloma; Oncogene Proteins; Piperidines; Protein Kinase Inhibitors; Purines; RNA, Small Interfering; Roscovitine

BH3 mimetic drugs induce cell death by antagonizing the activity of antiapoptotic Bcl-2 family proteins. Cyclin-dependent kinase (CDK) inhibitors that function as transcriptional repressors downregulate the Bcl-2 family member Mcl-1 and increase the activity of selective BH3 mimetics that fail to target this protein. In this study, we determined whether CDK inhibitors potentiate the activity of pan-BH3 mimetics directly neutralizing Mcl-1. Specifically, we evaluated interactions between the prototypical pan-CDK inhibitor flavopiridol and the pan-BH3 mimetic obatoclax in multiple myeloma (MM) cells in which Mcl-1 is critical for survival. Coadministration of flavopiridol and obatoclax synergistically triggered apoptosis in both drug-naïve and drug-resistant MM cells. Mechanistic investigations revealed that flavopiridol inhibited Mcl-1 transcription but increased transcription of Bim and its binding to Bcl-2/Bcl-xL. Obatoclax prevented Mcl-1 recovery and caused release of Bim from Bcl-2/Bcl-xL and Mcl-1, accompanied by activation of Bax/Bak. Whether administered singly or in combination with obatoclax, flavopiridol also induced upregulation of multiple BH3-only proteins, including BimEL, BimL, Noxa, and Bik/NBK. Notably, short hairpin RNA knockdown of Bim or Noxa abrogated lethality triggered by the flavopiridol/obatoclax combination in vitro and in vivo. Together, our findings show that CDK inhibition potentiates pan-BH3 mimetic activity through a cooperative mechanism involving upregulation of BH3-only proteins with coordinate downregulation of their antiapoptotic counterparts. These findings have immediate implications for the clinical trial design of BH3 mimetic-based therapies that are presently being studied intensively for the treatment of diverse hematopoietic malignancies, including lethal multiple myeloma.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; bcl-X Protein; Biomimetic Materials; Cell Line, Tumor; Cyclin-Dependent Kinases; Drug Synergism; Flavonoids; Humans; Indoles; Membrane Proteins; Mice; Mice, Inbred NOD; Mice, Nude; Mice, SCID; Mitochondria; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Peptide Fragments; Piperidines; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Up-Regulation; Xenograft Model Antitumor Assays

Despite significant treatment advances over the past decade, multiple myeloma (MM) remains largely incurable. In this study we found that MM cells were remarkably sensitive to the death-inducing effects of a new class of sangivamycin-like molecules (SLM). A panel of structurally related SLMs selectively induced apoptosis in MM cells but not other tumor or nonmalignant cell lines at submicromolar concentrations. SLM6 was the most active compound in vivo, where it was well tolerated and significantly inhibited growth and induced apoptosis of MM tumors. We determined that the anti-MM activity of SLM6 was mediated by direct inhibition of cyclin-dependent kinase 9 (CDK9), which resulted in transcriptional repression of oncogenes that are known to drive MM progression (MAF, CCND1, MYC, and others). Furthermore, SLM6 showed superior in vivo anti-MM activity more than the CDK inhibitor flavopiridol, which is currently in clinical trials for MM. These findings show that SLM6 is a novel CDK9 inhibitor with promising preclinical activity as an anti-MM agent.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Boronic Acids; Bortezomib; Cell Death; Cell Line, Tumor; Cyclin-Dependent Kinase 9; Drug Screening Assays, Antitumor; Flavonoids; Humans; Mice; Multiple Myeloma; Oncogenes; Piperidines; Protein Kinase Inhibitors; Pyrazines; Pyrimidine Nucleosides; Translocation, Genetic; Treatment Outcome; Tubercidin

Multiple myeloma (MM) is an incurable indolent malignancy with an average lifespan of 3 years, underscoring the need for new therapies. Studies have shown that the receptor MET and its ligand hepatocyte growth factor play an important role in proliferation, migration, adhesion, and survival of MM cells. Hence, an effective way to decrease MET receptor may act as a viable therapeutic option. Since MET mRNA and protein have short half-lives, we hypothesized that transcription inhibitor will reduce MET transcript and protein levels and this will lead to cell death. Pharmacological (flavopiridol) and molecular (shRNA) transcription inhibitor were used to impede formation of MET transcripts. The diminution of global RNA synthesis with flavopiridol was related to phosphorylation status of Ser residues (r (2) = 0.90 and 0.92 for Ser2 and Ser5) on the C-terminal-domain of RNA polymerase II. This was accompanied with a time-dependent decrease in MET transcript, which reached to less than 30% (1 microM) and 10% (3 microM) by 24 h. This decline in transcript level was directly associated with a reduction in MET protein level (r (2) = 0.82) and resulted in cell death. Assessment of MET in MM survival was done by using shRNA targeted towards MET. When cells were infected with shRNA viral construct, there was increased cell death with a decline in MET transcript and protein. Taken together, our study demonstrates that MET plays a critical role in the survival and removal or lowering of MET by flavopiridol or shRNA results in the demise of MM cells.

Topics: Antineoplastic Agents; Apoptosis; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Multiple Myeloma; Phosphorylation; Piperidines; Protein Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-met; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; RNA Polymerase II; RNA, Messenger; RNA, Small Interfering; Transcription, Genetic; Tumor Cells, Cultured

Flavopiridol is known to modulate the transcription of genes. We investigated the effect of flavopiridol pretreatment on TRAIL cytotoxicity and on the expression of FLIP(L) in different TRAIL-resistant cell lines, because FLIP expression is known to confer TRAIL-resistance.. Apoptosis was assessed by PI staining and protein expression by Western blotting. RT-PCR was used for mRNA quantitation. siRNA gene silencing was used to knock down FLIP(L).. Flavopiridol pretreatment synergized TRAIL-induced apoptosis in human myeloma and breast cancer cells. Flavopiridol treatment repressed the transcription of FLIP(L) and downregulated its expression in both myeloma and breast cancer cells. Silencing of FLIP(L) gene by siRNA sensitized myeloma cells to TRAIL. Flavopiridol treatment downregulated the expression of the proapoptotic members of the Bcl-2 family proteins (Bak, Bax and PUMA-alpha). The expression of the antiapoptotic Bcl-2 members (Bcl-2 and Bcl-X(L)) was not altered by flavopiridol treatment in myeloma cells.. Our data indicate that flavopiridol synergizes TRAIL cytotoxicity by downregulation of FLIP(L) and this synergistic effect is Bcl-2 family independent.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Coloring Agents; Down-Regulation; Drug Synergism; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Multiple Myeloma; Piperidines; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; TNF-Related Apoptosis-Inducing Ligand

Interactions between the cyclin-dependent kinase inhibitor flavopiridol (FP) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L), were examined in human leukemia cells (U937 and Jurkat). Coexposure of cells to marginally toxic concentrations of TRAIL and FP (24 h) synergistically increased mitochondrial injury (eg, cytochrome c, AIF, Smac/DIABLO release), cytoplasmic depletion of Bax, activation of Bid as well as caspase-8 and -3, PARP cleavage, and apoptosis. Coadministration of TRAIL markedly increased FP-induced apoptosis in leukemic cells ectopically expressing Bcl-2, Bcl-x(L), or a phosphorylation loop-deleted form of Bcl-2 (DeltaBcl-2), whereas lethality was substantially attenuated in cells ectopically expressing CrmA, dominant-negative-FADD, or dominant-negative-caspase-8. TRAIL/FP induced no discernible changes in FLIP, DR4, DR5, Mcl-1, or survivin expression, modest declines in levels of DcR2 and c-IAP, but resulted in the marked transcriptional downregulation of XIAP. Moreover, cells stably expressing an XIAP-antisense construct exhibited a pronounced increase in TRAIL sensitivity comparable to degrees of apoptosis achieved with TRAIL/FP. Conversely, enforced XIAP expression significantly attenuated caspase activation and TRAIL/FP lethality. Together, these findings suggest that simultaneous activation of the intrinsic and extrinsic apoptotic pathways by TRAIL and FP synergistically induces apoptosis in human leukemia cells through a mechanism that involves FP-mediated XIAP downregulation.

Topics: Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspases; Cell Cycle; Complement Membrane Attack Complex; Complement System Proteins; Cytochromes c; Down-Regulation; Drug Interactions; Drug Synergism; Flavonoids; Flavoproteins; Glycoproteins; HL-60 Cells; Humans; Intracellular Signaling Peptides and Proteins; Jurkat Cells; Leukemia; Membrane Glycoproteins; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Multiple Myeloma; Phosphorylation; Piperidines; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; TNF-Related Apoptosis-Inducing Ligand; Transcription, Genetic; Tumor Necrosis Factor-alpha; U937 Cells; X-Linked Inhibitor of Apoptosis Protein

Interactions between the cyclin-dependent kinase inhibitor flavopiridol and the small-molecule Bcl-2 antagonist HA14-1 were examined in human multiple myeloma cells. Whereas individual treatment of U266 myeloma cells with 10 micromol/L HA14-1 or 100 nmol/L flavopiridol had little effect, exposure of cells to flavopiridol (6 hours) followed by HA14-1 (18 hours) resulted in a striking increase in mitochondrial dysfunction (cytochrome c and Smac/DIABLO release; loss of mitochondrial membrane potential), activation of the caspase cascade, apoptosis, and diminished clonogenic survival. Similar findings were noted in other myeloma cell lines (e.g., MM.1S, RPMI8226, and NCI-H929) as well as in those resistant to dexamethasone and cytotoxic agents (e.g., MM.1R, 8226/Dox40, and 8226/LR5). Combined exposure to flavopiridol and HA14-1 was associated with down-regulation of Mcl-1 and Bcl-xL, Bid cleavage, and mitochondrial translocation of Bax. Flavopiridol/HA14-1-treated cells also exhibited a pronounced activation of Jun NH2-terminal kinase, a modest activation of p38 mitogen-activated protein kinase, and down-regulation of cyclin D1. Flavopiridol/HA14-1-induced apoptosis was associated with a marked increase in reactive oxygen species generation; moreover,both events were attenuated by the antioxidant N-acetyl-l-cysteine. Finally, in contrast to dexamethasone, flavopiridol/HA14-1-induced lethality was unaffected by exogenous interleukin-6 or insulin-like growth factor-I. Together, these findings indicate that flavopiridol and the small-molecule Bcl-2 antagonist HA14-1 cooperate to trigger oxidant injury, mitochondrial dysfunction, caspase activation, and apoptosis in human multiple myeloma cells and suggest that this approach may warrant further evaluation as an antimyeloma strategy.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Benzopyrans; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Cyclin D1; Cyclin-Dependent Kinases; Cytochromes c; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Free Radicals; Humans; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Nitriles; p38 Mitogen-Activated Protein Kinases; Piperidines; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

Multiple myeloma (MM) is a B-cell malignancy characterized by the accumulation of malignant plasma cells with slow proliferative rate but enhanced survival. MM cells express multiple Bcl-2 family members, including Bcl-2, Bcl-XL, and Mcl-1, which are thought to play a key role in the survival and drug resistance of myeloma. The cyclin-dependent kinase inhibitor flavopiridol has antitumor activity against hematopoietic malignancies, including CLL, in which induction of apoptosis was associated with reduced expression of antiapoptotic proteins. Therefore, we sought to characterize the effect of flavopiridol on the proliferation and survival of myeloma cells and to define its mechanisms of action. Treatment of MM cell lines (8226, ANBL-6, ARP1, and OPM-2) with clinically achievable concentrations of flavopiridol resulted in rapid induction of apoptotic cell death that correlated temporally with the decline in Mcl-1 protein and mRNA levels. Levels of other antiapoptotic proteins did not change. Overexpression of Mcl-1 protected MM cells from flavopiridol-induced apoptosis. Additional analysis demonstrated that flavopiridol treatment resulted in a dose-dependent inhibition of phosphorylation of the RNA polymerase II COOH-terminal domain, thus blocking transcription elongation. These data indicate that Mcl-1 is an important target for flavopiridol-induced apoptosis of MM that occurs through inhibition of Mcl-1 mRNA transcription coupled with rapid protein degradation via the ubiquitin-proteasome pathway.

Topics: Apoptosis; bcl-X Protein; Blotting, Northern; Blotting, Western; Caspases; Cell Division; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Down-Regulation; Drug Resistance, Neoplasm; Flavonoids; Humans; In Situ Nick-End Labeling; Membrane Glycoproteins; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Phosphorylation; Piperidines; Protein Structure, Tertiary; Proteoglycans; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Syndecans; Time Factors; Transcription, Genetic; Tumor Cells, Cultured

Although myeloma shows responsiveness in intensive chemotherapy, overall survival remains less than 40% at 2 years. Since myeloma appears to be dependent on cytokines, such as IL-6, we hypothesized that targeting signal transduction molecules could effectively treat myeloma. Two myeloma cell lines U266 and RPMI-8226 and CD38+ myeloma cells were studied by immune complex kinase assay or anti-phosphotyrosine blot for evidence of constitutive activation of tyrosine kinases. Growth arrest and apoptosis were evaluated in these two cell lines following their treatment with specific kinase inhibitors. We found that a variety of Src and Janus kinases were present and constitutively active in U266 and RPMI-8226 cells. Inhibitors of both Src and Janus kinases were inferior to the cyclin-dependent kinase inhibitor, flavopiridol, in inducing both growth arrest with GI50 of 100 nM and apoptosis in both cell lines and CD38+ myeloma cells. Although, flavopiridol did not affect cyclin D1 and cyclin A levels, it inhibited Mcl-1 and Bcl-2 protein levels and cyclin-dependent kinase 2 activity. Flavopiridol is a well-tolerated drug, currently in phase I-II trials for a variety of tumors. A clinical trial using flavopiridol should be performed in patients with myeloma. Its mechanism of action may involve targets other than the cyclin-dependent kinases.

Topics: ADP-ribosyl Cyclase; ADP-ribosyl Cyclase 1; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation; Antineoplastic Agents; Apoptosis; Bone Marrow Cells; Cell Division; Cell Survival; Cyclin-Dependent Kinases; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Humans; JNK Mitogen-Activated Protein Kinases; Membrane Glycoproteins; Mitogen-Activated Protein Kinases; Multiple Myeloma; NAD+ Nucleosidase; Phosphotyrosine; Piperidines; src-Family Kinases; Tumor Cells, Cultured