alvocidib and Leukemia--Myeloid--Acute

Conventional chemotherapy with cytarabine and anthracycline (often referred to as "7+3") has been used for many years in the treatment of acute myeloid leukemia (AML). Despite meaningful advances in areas of supportive care and transplantation, little progress has been made in developing new chemotherapy options. In 2018, The Food and Drug Administration (FDA) of the US approved several novel agents for AML treatment as follows: ivosidenib, an inhibitor of isocitrate dehydrogenase-1; venetoclax, a potent inhibitor of bcl2; and glasdegib, an inhibitor of hedgehog signaling pathway. Moreover, clinical trials of alvocidib (flavopiridol), an inhibitor of the CDK9, pevonedistat, an inhibitor of NEDD8, and APR-246, a reactivator of mutant p53, are in progress. These agents will either be incorporated into the conventional 7+3 regimen or combined with hypomethylating agents to improve the outcome of AML therapy, and the results will guide the next stage of precision medicine in the treatment of AML.

Topics: Benzimidazoles; Bridged Bicyclo Compounds, Heterocyclic; Cyclopentanes; Drug Approval; Flavonoids; Glycine; Humans; Leukemia, Myeloid, Acute; Molecular Targeted Therapy; Phenylurea Compounds; Piperidines; Pyridines; Pyrimidines; Sulfonamides; United States; United States Food and Drug Administration

There have been minimal therapeutic advancements in acute myeloid leukemia (AML) over the past 4 decades and outcomes remain unsatisfactory. Alvocidib (formerly flavopiridol) is a multi-serine threonine cyclin-dependent kinase inhibitor with demonstrable in vitro and clinical activity in AML when combined in a timed sequential chemotherapy regimen, FLAM (alvocidib followed by cytarabine continuous infusion and mitoxantrone). FLAM has been evaluated in sequential phase 1 and phase 2 studies in 149 and 256 relapsed/refractory and newly diagnosed non-favorable risk AML patients, respectively, with encouraging findings in both patient populations warranting further investigation. This review highlights the mechanism of action of alvocidib, pre-clinical studies of alvocidib in AML, and the clinical trials evaluating alvocidib alone and in combination with cytotoxic agents (FLAM) in AML.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Cell Cycle; Cyclin-Dependent Kinases; Drug Interactions; Drug Screening Assays, Antitumor; Flavonoids; Humans; Leukemia, Myeloid, Acute; Molecular Structure; Molecular Targeted Therapy; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Piperidines; Protein Kinase Inhibitors; Salvage Therapy; Tumor Lysis Syndrome

The current treatment of patients with acute myeloid leukaemia yields poor results, with expected cure rates in the order of 30-40% depending on the biological characteristics of the leukaemic clone. Therefore, new agents and schemas are intensively studied in order to improve patients' outcomes. This review summarizes some of these new paradigms, including new questions such as which anthracycline is most effective and at what dose. High doses of daunorubicin have shown better responses in young patients and are well tolerated in elderly patients. Monoclonal antibodies are promising agents in good risk patients. Drugs blocking signalling pathways could be used in combination with chemotherapy or in maintenance with promising results. Epigenetic therapies, particularly after stem cell transplantation, are also discussed. New drugs such as clofarabine and flavopiridol are reviewed and the results of their use discussed. It is clear that many new approaches are under study and hopefully will be able to improve on the outcomes of the commonly used '7+3' regimen of an anthracycline plus cytarabine with daunorubicin, which is clearly an ineffective therapy in the majority of patients.

Topics: Adenine Nucleotides; Antibodies, Monoclonal; Antineoplastic Agents; Arabinonucleosides; Clofarabine; Daunorubicin; Flavonoids; Humans; Leukemia, Myeloid, Acute; Piperidines

Topics: Antineoplastic Combined Chemotherapy Protocols; Bridged Bicyclo Compounds, Heterocyclic; Humans; Leukemia, Myeloid, Acute

Topics: Adolescent; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Cytarabine; Female; Flavonoids; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Mitoxantrone; Myeloid Cell Leukemia Sequence 1 Protein; Piperidines

To improve the outcome of relapsed/refractory acute myeloid leukemia (AML), a randomized phase II trial of three novel regimens was conducted. Ninety patients were enrolled and were in first relapse or were refractory to induction/re-induction chemotherapy. They were randomized to the following regimens: carboplatin-topotecan (CT), each by continuous infusion for 5 days; alvocidib (formerly flavopiridol), cytarabine, and mitoxantrone (FLAM) in a timed sequential regimen; or sirolimus combined with mitoxantrone, etoposide, and cytarabine (S-MEC). The primary objective was attainment of a complete remission (CR). A Simon two-stage design was used for each of the three arms. The median age of the patients in the FLAM arm was older at 62 years compared with 55 years for the CT arm and the S-MEC arm. The overall response was 14% in the CT arm (5/35, 90% CI 7%-35%), 28% in the FLAM arm (10/36, 90% CI, 16%-43%), and 16% in the S-MEC arm (3/19, 90% CI, 4%-36%). There were nine treatment-related deaths, seven of which occurred in the FLAM arm with four of these in elderly patients. We conclude that the FLAM regimen had an encouraging response rate and should be considered for further clinical development but should be used with caution in elderly patients.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Carboplatin; Cytarabine; Disease-Free Survival; Etoposide; Female; Flavonoids; Follow-Up Studies; Gastrointestinal Diseases; Hematologic Diseases; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Mitoxantrone; Piperidines; Recurrence; Remission Induction; Salvage Therapy; Sirolimus; Topotecan; Tumor Lysis Syndrome

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Daunorubicin; Female; Flavonoids; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Mitoxantrone; Piperidines; Risk Factors

Serial studies have demonstrated that induction therapy with FLAM [flavopiridol (alvocidib) 50 mg/m(2) days 1-3, cytarabine 667 mg/m(2)/day continuous infusion days 6-8, and mitoxantrone (FLAM) 40 mg/m(2) day 9] yields complete remission rates of nearly 70% in newly diagnosed poor-risk acute myeloid leukemia. Between May 2011-July 2013, 165 newly diagnosed acute myeloid leukemia patients (age 18-70 years) with intermediate/adverse-risk cytogenetics were randomized 2:1 to receive FLAM or 7+3 (cytarabine 100 mg/m(2)/day continuous infusion days 1-7 and daunorubicin 90 mg/m(2) days 1-3), across 10 institutions. Some patients on 7+3 with residual leukemia on day 14 received 5+2 (cytarabine 100 mg/m(2)/day continuous infusion days 1-5 and daunorubicin 45 mg/m(2) days 1-2), whereas patients on FLAM were not re-treated based on day 14 bone marrow findings. The primary objective was to compare complete remission rates between one cycle of FLAM and one cycle of 7+3. Secondary end points included safety, overall survival and event-free survival. FLAM led to higher complete remission rates than 7+3 alone (70% vs. 46%; P=0.003) without an increase in toxicity, and this improvement persisted after 7+3+/-5+2 (70% vs. 57%; P=0.08). There were no significant differences in overall survival and event-free survival in both arms but post-induction strategies were not standardized. These results substantiate the efficacy of FLAM induction in newly diagnosed AML. A phase III study is currently in development. This study is registered with clinicaltrials.gov identifier: 01349972.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Daunorubicin; Disease-Free Survival; Female; Flavonoids; Follow-Up Studies; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Mitoxantrone; Piperidines; Survival Rate

Flavopiridol is a protein-bound, cytotoxic, cyclin dependent kinase inhibitor. A phase II trial of flavopiridol followed by ara-C and mitoxantrone with flavopiridol given by 1-h bolus for adults with newly-diagnosed, poor-risk acute myelogenous leukemia yielded 67% complete remission with median disease-free survival of 13.6 months.. We compared bolus flavopiridol (50 mg/m(2)/day, Arm A) versus 'hybrid' flavopiridol (30 mg/m(2) over 30 min followed by 40 mg/m(2) over 4 h, Arm B) followed by ara-C and mitoxantrone in 78 patients (39 per arm) with newly diagnosed, poor-risk acute myelogenous leukemia. To mitigate imbalance, patients were stratified by presence or absence of secondary leukemia and therapy for antecedent disorder.. Death at or before Day 60 occurred in 8% of patients per arm. Complete remission plus complete remission with incomplete recovery was 68% (Arm A, 62%; Arm B, 74%) overall, and 65% or over in both arms for patients with secondary leukemia and leukemia with adverse genetics. In Arm A 91% and in Arm B 86% of patients received chemotherapy and/or allogeneic transplantation in complete remission. Median overall survival for all remission patients has not been reached for either arm, with median disease free survival of 13.6 months for Arm A and of 12.0 months for Arm B.. Both flavopiridol schedules produce comparably encouraging results in adults with poor-risk acute myelogenous leukemia. Given the greater ease of bolus administration, we are conducting a randomized phase II study of bolus flavopiridol followed by ara-c and mitoxantrone versus conventional induction therapy for patients aged 70 years and under with intermediate or poor-risk acute myelogenous leukemia. This study is registered at www.clinicaltrials.gov as #NCT 00407966.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Disease-Free Survival; Female; Flavonoids; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Mitoxantrone; Piperidines; Survival Rate

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

Flavopiridol, a cyclin-dependent kinase inhibitor, is cytotoxic to leukemic blasts. In a Phase II study, flavopiridol 50 mg/m(2) was given by 1-h infusion daily x 3 beginning day 1 followed by 2 g/m(2)/72 h ara-C beginning day 6 and 40 mg/m(2) mitoxantrone on day 9 (FLAM) to 45 adults with newly diagnosed acute myelogenous leukemia (AML) with multiple poor-risk features. Thirty patients (67%) achieved complete remission (CR) and 4 (9%) died. Twelve (40%) received myeloablative allogeneic bone marrow transplant (BMT) in first CR. Median OS and DFS are not reached (67% alive 12.5-31 months, 58% in CR 11.4-30 months), with median follow-up 22 months. Sixteen received FLAM in CR, with median OS and DFS 9 and 13.1 months, and 36% alive at 21-31 months. Short OS and DFS correlated with adverse cytogenetics, regardless of age or treatment in CR. The addition of allogeneic BMT in CR translates into long OS and DFS in the majority of eligible patients.

Topics: Adult; Aged; Allopurinol; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Transplantation; Combined Modality Therapy; Cytarabine; Disease-Free Survival; Female; Flavonoids; Follow-Up Studies; Heart Diseases; Humans; Hyperkalemia; Kaplan-Meier Estimate; Leukemia, Myeloid, Acute; Male; Middle Aged; Mitoxantrone; Piperidines; Polyamines; Premedication; Remission Induction; Risk; Sepsis; Sevelamer; Transplantation, Homologous; Treatment Outcome; Tumor Lysis Syndrome; Young Adult

Flavopiridol is a cyclin-dependent kinase inhibitor that is cytotoxic to leukemic blasts. In a phase I study of flavopiridol followed by 1-beta-d-arabinofuranosylcytosine (ara-C) and mitoxantrone, overall response rate for adults with relapsed and refractory acute myelogenous leukemias (AML) was 31%. We have now completed a phase II study of sequential flavopiridol, ara-C, and mitoxantrone in 62 adults with poor-risk AML.. Flavopiridol (50 mg/m(2)) was given by 1-h infusion daily x 3 beginning day 1 followed by 2 gm/m(2)/72 h ara-C beginning day 6 and 40 mg/m(2) mitoxantrone on day 9.. Flavopiridol caused a > or =50% decrease in peripheral blood blasts in 44% by median day 2 and > or =80% decrease in 26% by day 3. Self-limited tumor lysis occurred in 53%. Three (5%) died during therapy (2 multiorgan failure and 1 fungal pneumonia). Complete remissions (CR) were achieved in 12 of 15 (75%) newly diagnosed secondary AML, 18 of 24 (75%) first relapse after short CR (median CR, 9 months, including prior allotransplant), and 2 of 13 (15%) primary refractory but 0 of 10 multiply refractory AML. Disease-free survival for all CR patients is 40% at 2 years, with newly diagnosed patients having a 2-year disease-free survival of 50%.. Flavopiridol has anti-AML activity directly and in combination with ara-C and mitoxantrone. This timed sequential regimen induces durable CRs in a significant proportion of adults with newly diagnosed secondary AML (including complex cytogenetics) and adults with AML in first relapse after short first CR.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Disease-Free Survival; Female; Flavonoids; Humans; Infusions, Intravenous; Leukemia, Myeloid, Acute; Male; Maximum Tolerated Dose; Middle Aged; Mitoxantrone; Neoplasm Recurrence, Local; Piperidines; Prognosis; Remission Induction; Salvage Therapy; Survival Rate

The serine/threonine kinase inhibitor flavopiridol targets multiple cyclin-dependent kinases, induces checkpoint arrest, and interrupts transcriptional elongation. We designed a phase I clinical trial using a timed sequential therapy approach where flavopiridol was given for the dual purpose of initial cytoreduction and enhancing cell cycle progression of the remaining leukemia cell cohort followed by cycle-dependent drugs 1-beta-D-arabinofuranosylcytosine (ara-C) and mitoxantrone.. Flavopiridol was given by 1-hour infusion daily for 3 days beginning day 1 followed by 2 g/m2/72 h ara-C beginning day 6 and 40 mg/m2 mitoxantrone beginning day 9. In vivo correlates included pharmacokinetics, modulation of blast cycle regulators, and serum and marrow supernatant vascular endothelial growth factor levels.. Of 34 adults receiving induction therapy, 16 (47%) evinced direct leukemia cytotoxicity with > or =50% drop in peripheral blast counts and tumor lysis in 9 (26%). Four (12%) died during therapy (two fungal infections and two sudden death). Dose-limiting toxicity occurred at 60 mg/m2/d with profound neutropenia >40 days duration, and maximal tolerated dose was 50 mg/m2/d. Overall response rate was 31% in 26 acute myelogenous leukemia and 12.5% in acute lymphoblastic leukemia. Pharmacokinetics showed that a linear two-compartment model with first-order elimination provided the best fit of the observed concentration versus time data. Flavopiridol down-regulated one or more target proteins in marrow blasts in vivo. Vascular endothelial growth factor was detected in sera and marrow supernatant pretreatment, and sera obtained on day 3 inhibited bovine aortic endothelial cell proliferation by a mean of 32% (range, 10-80%).. Our data suggest that flavopiridol is cytotoxic to leukemic cells and, when followed by ara-C and mitoxantrone, exerts biological and clinical effects in patients with relapsed and refractory acute leukemias. These findings warrant continuing development of flavopiridol at 50 mg/m2/d x 3 days in combination with cytotoxic and biological agents for acute leukemias.

Topics: Adult; Aged; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Cells; Cattle; Cell Proliferation; Cohort Studies; Cytarabine; Endothelium, Vascular; Female; Flavonoids; Humans; Leukemia, Myeloid, Acute; Male; Maximum Tolerated Dose; Middle Aged; Mitoxantrone; Neoplasm Recurrence, Local; Piperidines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Salvage Therapy; U937 Cells; Vascular Endothelial Growth Factor A

For more than 30 years, treatment of acute myeloid leukemia (AML) has remained largely unchanged and reliant on chemotherapeutic drug combinations, specifically cytarabine and daunorubicin (the 7 + 3 regimen). One broad spectrum drug, flavopiridol (also known as Alvocidib) has shown significant activity against AML through the inhibition of cyclin-dependent kinases. Flavopiridol is a semisynthetic flavonoid and our research team recently described methods to formulate another flavonoid, quercetin, through the ability of flavonoids to bind divalent metals. This method relies on use of copper-containing liposomes to enhance the apparent solubility of flavopiridol and to create formulations suitable for intravenous (i.v.) use. Similar to quercetin, flavopiridol is defined as an aqueous-insoluble compound (< 1 mg/mL in water) and this research sought to evaluate whether the copper-binding capabilities of flavopiridol could be used to prepare an injectable formulation that would exhibit enhanced exposure and improved efficacy. Flavopiridol powder was added directly to preformed copper-containing liposomes (DSPC:Chol or DSPC:DSPE-PEG2000) and the resulting formulations were characterized. Pharmacokinetic and efficacy studies were then conducted. The liposomal flavopiridol formulations were well-tolerated in mice following i.v. administration at a dose of 5 mg/kg with no apparent acute or chronic toxicities. In vivo pharmacokinetics of the optimized DSPC/DSPE-PEG2000 liposomal flavopiridol formulation demonstrated a 30-fold increase in AUC (0.804 μg-hr/mL versus 26.92 μg-hr/mL) compared to the free flavopiridol formulation. The resultant liposomal formulation also demonstrated significant therapeutic activity in MV4-11 and MOLM-13 subcutaneous AML models. Additional studies will be required to define whether formulation changes can be made to enhance flavopiridol retention in the selected composition. The results suggest that further increases in flavopiridol retention will result in improved therapeutic activity.

Topics: Animals; Cytarabine; Flavonoids; Leukemia, Myeloid, Acute; Liposomes; Mice; Piperidines

Cytarabine (ara-C) is the major drug for the treatment of acute myeloid leukemia (AML), but cellular resistance to ara-C is a major obstacle to therapeutic success. The present study examined enhanced anti-apoptosis identified in 3 newly established nucleoside analogue-resistant leukemic cell line variants and approaches to overcoming this resistance.. HL-60 human AML cells were used to develop the ara-C- or clofarabine (CAFdA)-resistant variants. The Bcl-2 inhibitor venetoclax and the Mcl-1 inhibitor alvocidib were tested to determine whether they could reverse these cells' resistance.. A 10-fold ara-C-resistant HL-60 variant, a 4-fold CAFdA-resistant HL-60 variant, and a 30-fold CAFdA-resistant HL-60 variant were newly established. The variants demonstrated reduced deoxycytidine kinase and deoxyguanosine kinase expression, but intact expression of surface transporters (hENT1, hENT2, hCNT3). The variants exhibited lower expression of intracellular nucleoside analogue triphosphates compared with non-variant HL-60 cells. The variants also overexpressed Bcl-2 and Mcl-1. Venetoclax as a single agent was not cytotoxic to the resistant variants. Nevertheless, venetoclax with nucleoside analogs demonstrated synergistic cytotoxicity against the variants. Alvocidib as a single agent was cytotoxic to the cells. However, alvocidib induced G1 arrest and suppressed the cytotoxicity of the co-administered nucleoside analogs.. Three new nucleoside analogue-resistant HL-60 cell variants exhibited reduced production of intracellular analogue triphosphates and enhanced Bcl-2 and Mcl-1 expressions. Venetoclax combined with nucleoside analogs showed synergistic anti-leukemic effects and overcame the drug resistance.

Topics: Antineoplastic Combined Chemotherapy Protocols; Bridged Bicyclo Compounds, Heterocyclic; Cell Proliferation; Clofarabine; Cytarabine; Flavonoids; Humans; Leukemia, Myeloid, Acute; Piperidines; Sulfonamides

Topics: Adult; Aged; Antineoplastic Agents; Cohort Studies; Drug Resistance, Neoplasm; Female; Flavonoids; Gene Expression Profiling; Genetic Heterogeneity; Humans; Induction Chemotherapy; Leukemia, Myeloid, Acute; Male; Middle Aged; Nuclear Proteins; Nucleophosmin; Piperidines; United States; Young Adult

Bromodomain and extra-terminal (BET) protein inhibitors have been reported as treatment options for acute myeloid leukemia (AML) in preclinical models and are currently being evaluated in clinical trials. This work presents a novel potent and selective BET inhibitor (BI 894999), which has recently entered clinical trials (NCT02516553). In preclinical studies, this compound is highly active in AML cell lines, primary patient samples, and xenografts. HEXIM1 is described as an excellent pharmacodynamic biomarker for target engagement in tumors as well as in blood. Mechanistic studies show that BI 894999 targets super-enhancer-regulated oncogenes and other lineage-specific factors, which are involved in the maintenance of the disease state. BI 894999 is active as monotherapy in AML xenografts, and in addition leads to strongly enhanced antitumor effects in combination with CDK9 inhibitors. This treatment combination results in a marked decrease of global p-Ser2 RNA polymerase II levels and leads to rapid induction of apoptosis in vitro and in vivo. Together, these data provide a strong rationale for the clinical evaluation of BI 894999 in AML.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase 9; Down-Regulation; Drug Synergism; Drug Therapy, Combination; Enhancer Elements, Genetic; Flavonoids; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mice; Piperidines; Proteins; Pyrazines; RNA Polymerase II; RNA-Binding Proteins; Transcription Factors; Triazoles; Xenograft Model Antitumor Assays

Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cytokines; Drug Discovery; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Lung Neoplasms; Mice; Molecular Targeted Therapy; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proteomics; Xenograft Model Antitumor Assays

Topics: Animals; Antineoplastic Agents; Benzimidazoles; Drug Discovery; Epigenesis, Genetic; Flavonoids; Histone Deacetylase Inhibitors; Humans; Leukemia, Myeloid, Acute; Orphan Drug Production; Piperidines; Protein Kinase Inhibitors; Pteridines; Terminology as Topic

Few published studies characterize early lymphocyte recovery after intensive chemotherapy for acute myelogenous leukemia (AML). To test the hypothesis that lymphocyte recovery mirrors ontogeny, we characterized early lymphocyte recovery in 20 consecutive patients undergoing induction timed sequential chemotherapy for newly diagnosed AML. Recovering T lymphocytes were predominantly CD4(+) and included a greatly expanded population of CD3(+)CD4(+)CD25(+)Foxp3(+) T cells. Recovering CD3(+)CD4(+)CD25(+)Foxp3(+) T cells were phenotypically activated regulatory T cells and showed suppressive activity on cytokine production in a mixed lymphocyte reaction. Despite an initial burst of thymopoiesis, most recovering regulatory T cells were peripherally derived. Furthermore, regulatory T cells showed marked oligoclonal skewing, suggesting that their peripheral expansion was antigen-driven. Overall, lymphocyte recovery after chemotherapy differs from ontogeny, specifically identifying a peripherally expanded oligoclonal population of activated regulatory T lymphocytes. These differences suggest a stereotyped immunologic recovery shared by patients with newly diagnosed AML after induction timed sequential chemotherapy. Further insight into this oligoclonal regulatory T-cell population will be fundamental toward developing effective immunomodulatory techniques to improve survival for patients with AML.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cell Separation; Cytarabine; Cytosine; Daunorubicin; Etoposide; Female; Flavonoids; Flow Cytometry; Gene Expression Profiling; Humans; Immunophenotyping; Leukemia, Myeloid, Acute; Male; Middle Aged; Mitoxantrone; Piperidines; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Vidarabine; Young Adult

Topics: Antineoplastic Combined Chemotherapy Protocols; Clinical Trials, Phase II as Topic; Combined Modality Therapy; Cytarabine; Drug Administration Schedule; Flavonoids; Follow-Up Studies; Forecasting; Hematopoietic Stem Cell Transplantation; Humans; Leukemia, Myeloid, Acute; Mitoxantrone; Piperidines; Risk; Treatment Outcome; Tumor Lysis Syndrome

Histone H1 is commonly used to assay kinase activity in vitro. As many promising targeted therapies affect kinase activity of specific enzymes involved in cancer transformation, H1 phosphorylation can serve as potential pharmacodynamic marker for drug activity within the cell. In this study we utilized a phosphoproteomic workflow to characterize histone H1 phosphorylation changes associated with two targeted therapies in the Kasumi-1 acute myeloid leukemia cell line. The phosphoproteomic workflow was first validated with standard casein phosphoproteins and then applied to the direct analysis of histone H1 from Kasumi-1 nuclear lysates. Ten H1 phosphorylation sites were identified on the H1 variants, H1.2, H1.3, H1.4, H1.5 and H1.x. LC MS profiling of intact H1s demonstrated global dephosphorylation of H1.5 associated with therapy by the cyclin-dependent kinase inhibitor, flavopiridol and the Heat Shock Protein 90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin. In contrast, independent treatments with a nucleotide analog, proteosome inhibitor and histone deacetylase inhibitor did not exhibit decreased H1.5 phosphorylation. The data presented herein demonstrate that potential of histones to assess the cellular response of reagents that have direct and indirect effects on kinase activity that alters histone phosphorylation. As such, this approach may be a highly informative marker for response to targeted therapies influencing histone phosphorylation.

Topics: Amino Acid Sequence; Benzoquinones; Biomarkers, Pharmacological; Caseins; Flavonoids; Histones; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Leukemia, Myeloid, Acute; Molecular Sequence Data; Molecular Targeted Therapy; Phosphopeptides; Piperidines; Protein Kinase Inhibitors; Tumor Cells, Cultured

Topics: Antineoplastic Agents; Azacitidine; Biomedical Research; Carbazoles; Carboplatin; Cytarabine; Daunorubicin; Decitabine; Enzyme Inhibitors; Etoposide; Flavonoids; Furans; Humans; Leukemia, Myeloid, Acute; Mitoxantrone; Piperidines; Quinolones; Staurosporine; Topotecan; Tretinoin

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

Flavopiridol is a cyclin-dependent kinase inhibitor currently undergoing human clinical trials. As clinical development is pursued, it becomes important to evaluate resistance mechanisms to flavopiridol. To elucidate the contribution of breast cancer resistance protein (BCRP) to cellular resistance to flavopiridol in acute myeloid leukemia, we studied the relationship between cellular resistance to flavopiridol and mRNA expression of BCRP or P-glycoprotein (P-gp, product of MDR1gene) in blast cells from adult patients with acute leukemia.. Twenty-one blast cell samples from 20 patients were studied. The expression of BCRP, P-gp, or beta-actin mRNA was determined by real-time reverse transcription-PCR, using fluorescent hybridization probes to evaluate codon 482, a known site of mutations in BCRP mRNA. In vitro cell viability and apoptosis were examined after 24 h exposure to flavopiridol.. BCRP mRNA expression varied over a 200-fold range. In the blast cell samples with BCRP mRNA expression > 10000 copies/pg beta-actin (n = 9), BCRP mRNA correlated proportionally with cell viability in the presence of 250 nM flavopiridol (r = 0.86, P = 0.003) and with apoptosis induced by flavopiridol (r = 0.71, P = 0.031). In contrast, MDR1mRNA expression did not correlate with either flavopiridol cytotoxicity or induction of apoptosis. Melting point analysis of the hybridization probes determined that all 21 patient samples had arginine at codon 482 of BCRP mRNA, the wild-type form.. These results suggest that unlike P-gp, BCRP may play a role in leukemia cellular resistance to flavopiridol. No mutations at codon 482 were observed in BCRP mRNA in this group of patients.

Topics: Actins; Adult; Aged; Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Bone Marrow Cells; Cell Line, Tumor; Cell Survival; Codon; DNA, Complementary; Female; Flavonoids; HL-60 Cells; Humans; In Situ Hybridization, Fluorescence; Leukemia, Myeloid, Acute; Male; Middle Aged; Mutation; Neoplasm Proteins; Piperidines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Temperature; Time Factors

The survival of adults with acute leukemias remains unsatisfactory and requires new treatment approaches. Flavopiridol modulates cell cycle progression, inhibits transcription, and induces apoptosis. We designed an in vitro model of timed sequential therapy for acute leukemia to determine whether flavopiridol can: (a). trigger apoptosis in fresh acute leukemia; and (b). recruit surviving leukemic cells to a proliferative state, thereby priming such cells for the S-phase-related cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C).. Bone marrow cells from 20 adults with relapsed and refractory acute leukemias were enriched for blasts by Ficoll Hypaque sedimentation. Blasts were cultured on day 0 in flavopiridol 250 nM for 24 h, removed from flavopiridol for 24 h, and then cultured in ara-C 1 microM for an additional 72 h (F(250)A(1)). Apoptosis and cell cycle phase distribution were estimated from cells stained with propidium iodide. Cell survival was determined after the 72 h ara-C exposure by double cytofluorescence assay with fluorescein diacetate and propidium iodide.. Flavopiridol induced a 4.3-fold increase in apoptosis in human leukemia samples within the first 24 h of culture. Subsequent removal of flavopiridol led to a 1.7-fold increase in the proportion of cells in S phase by day 2. Mean survival in F(250)A(1) cultures after 72 h exposure to ara-C was 35.6% compared with flavopiridol alone (F(250)A(0), 56.1%; P = 0.0003) and ara-C alone (F(0)A(1), 65.2%; P < 0.00001).. Flavopiridol induces apoptosis in marrow blasts from patients with refractory acute leukemias. Furthermore, flavopiridol pretreatment increases the proapoptotic and cytotoxic effects of ara-C. The advantage of sequential FP(250)A(1) over either agent alone is seen for both acute myelogenous leukemia and acute lymphoblastic leukemia. These findings support a clinical trial of timed sequential therapy where flavopiridol is given for cytoreduction and subsequent priming of remaining leukemic cells for enhanced cycle-dependent drug cytotoxicity.

Topics: Adult; Aged; Antineoplastic Agents; Apoptosis; Bone Marrow Cells; Cell Division; Cell Survival; Coloring Agents; Female; Flavonoids; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Piperidines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Propidium; S Phase; Time Factors; Tumor Cells, Cultured

The success of the phosphotyrosine kinase inhibitor imatinib mesylate (STI571, Gleevec has emphasized the significance of a growing understanding of tumor cell biology. The search has since been intensified to identify other candidate molecules in cancer cell-specific signaling transduction pathways whose disruption may result in similar therapeutic benefits. Flavonoids are potent inhibitors of cyclin-dependent kinases, but in addition also inhibit the activity of angiogenic mediators and induce apoptosis by mechanisms that are still not fully understand. In the current study by Liesveld et al., flavonoids are shown to have an antiproliferative and proapoptotic effect in leukemic cells. The implications of the results of this study on the activity of flavonoids in leukemias and their future development are being discussed.

Topics: Apoptosis; Bone Marrow Cells; Case-Control Studies; Cell Division; Cell Survival; Flavonoids; Humans; Leukemia, Myeloid, Acute; Piperidines; Quercetin

Quercetin and flavopiridol, both flavonoids which influence oxidative milieu, proliferation, and apoptosis of various cell types, were examined for their effects on acute myelogenous leukemic cells and normal progenitors. Both quercetin and flavopiridol inhibited the growth and viability of various acute myelogenous leukemia (AML) cell lines and AML blasts isolated afresh from patients with AML of various subtypes. The effects on inhibition of proliferation and decreased viability were also significant in normal CD34+ cells isolated from normal marrow donors. In certain AML cases, the effects of flavopiridol appeared to be mediated through activation of caspase 3, offering one possible mechanism for the apoptosis evident after exposure to flavopiridol as measured by annexin V expression. These flavonoid compounds might find use in various therapeutic settings in AML.

Topics: Adult; Aged; Annexin A5; Antigens, CD34; Apoptosis; Bone Marrow Cells; Case-Control Studies; Caspase 3; Caspases; Cell Division; Cell Survival; Colony-Forming Units Assay; Enzyme Activation; Female; Flavonoids; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Cell Growth Factors; Humans; Interleukin-3; Leukemia, Myeloid, Acute; Male; Middle Aged; Piperidines; Quercetin; Tumor Cells, Cultured

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