nitrophenols and Leukemia--Myeloid--Acute

Acute myeloid leukemia (AML) continues to represent an area of critical unmet need with respect to new and effective targeted therapies. The Bcl-2 family of pro- and antiapoptotic proteins stands at the crossroads of cellular survival and death, and the expression of and interactions between these proteins determine tumor cell fate. Malignant cells, which are often primed for apoptosis, are particularly vulnerable to the simultaneous disruption of cooperative survival signaling pathways. Indeed, the single agent activity of agents such as mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase kinase (MEK) inhibitors in AML has been modest. Much work in recent years has focused on strategies to enhance the therapeutic potential of the bona fide BH3-mimetic, ABT-737, which inhibits B-cell lymphoma 2 (Bcl-2) and Bcl-xL. Most of these strategies target Mcl-1, an antiapoptotic protein not inhibited by ABT-737. The phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways are central to the growth, proliferation, and survival of AML cells, and there is much interest currently in pharmacologically interrupting these pathways. Dual inhibitors of PI3K and mTOR overcome some intrinsic disadvantages of rapamycin and its derivatives, which selectively inhibit mTOR. In this review, we discuss why combining dual PI3K/mTOR blockade with inhibition of Bcl-2 and Bcl-xL, by virtue of allowing coordinate inhibition of three mutually synergistic pathways in AML cells, may be a particularly attractive therapeutic strategy in AML, the success of which may be predicted for by basal Akt activation.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; bcl-X Protein; Biphenyl Compounds; Humans; Leukemia, Myeloid, Acute; Nitrophenols; Phosphoinositide-3 Kinase Inhibitors; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases

Acute myeloid leukemia (AML) is a malignant disease of the bone marrow, comprising various subtypes. We have investigated seven different AML cell lines that showed different sensitivities toward the inducer of apoptosis ABT-737, with IC

Topics: Apoptosis; Biphenyl Compounds; Cell Differentiation; Fluorodeoxyglucose F18; Humans; Leukemia, Myeloid, Acute; Nitrophenols; Piperazines; Sulfonamides; Tretinoin

Topics: Apoptosis; Biphenyl Compounds; Cell Differentiation; Fluorodeoxyglucose F18; Humans; Leukemia, Myeloid, Acute; Nitrophenols; Piperazines; Sulfonamides

Paediatric acute myeloid leukaemia (AML) is a heterogeneous disease characterised by genetics and morphology. The introduction of intensive chemotherapy treatments together with patient stratification and supportive therapy has resulted in a moderate improvement in patient prognosis. However, overall survival rates remain unacceptably poor, with only 65% of patients surviving longer than 5 years. Recently age-specific differences in AML have been identified, highlighting the need for tailored treatments for paediatric patients. Combination therapies have the potential to improve patient prognosis, while minimising harmful side-effects. In the laboratory setting, identifying key combinations from large drug libraries can be resource-intensive, prohibiting discovery and translation into the clinic. To minimise redundancy and maximise discovery, we undertook a multiplex screen of 80 apoptotic-inducing agents in paediatric AML pre-clinical models. The screen was designed using an all-pairs testing algorithm, which ensured that all pairs of compounds could be tested, while minimising the number of wells used. We identified a combination of ABT-737, a Bcl-2 family inhibitor and Purvalanol A, a CDK inhibitor, as a potential targeted therapy for AML patients with an MLL rearrangement and an FLT3-ITD. Our approach has the potential to reduce resource-intensity and time associated with the identification of novel combination therapies.

Topics: Adolescent; Algorithms; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biphenyl Compounds; Cell Line, Tumor; Child; Child, Preschool; Databases, Genetic; Drug Screening Assays, Antitumor; Female; Humans; Leukemia, Myeloid, Acute; Male; Nitrophenols; Piperazines; Prognosis; Purines; Remission Induction; Sulfonamides

Many acute myeloid leukaemias (AMLs) express high levels of BCL-2 and MCL-1, especially after therapy. To test the impact of these anti-apoptotic proteins on AML development and treatment, we used haemopoietic reconstitution to generate MLL-AF9 AMLs expressing BCL-2 or Mcl-1 transgenes. AMLs with elevated BCL-2 or MCL-1 had a higher proportion of mature myeloid cells but, like conventional MLL-AF9 AMLs, were readily transplantable. Short-term cell lines established from multiple primary AMLs of each genotype were tested in vitro for susceptibility to chemotherapeutics currently used for treating AML (daunorubicin, etoposide, cytarabine); the proteasome inhibitor bortezomib; CDK7/9 inhibitors; and BH3 mimetics, which bind and inhibit pro-survival proteins. The BH3 mimetics tested, alone and in combination with the other drugs, were: ABT-737 which, like its clinical counterpart navitoclax, targets BCL-2, BCL-X

Topics: Animals; Antineoplastic Agents; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cell Proliferation; Daunorubicin; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Humans; Injections, Intravenous; Leukemia, Myeloid, Acute; Mice; Mice, Inbred C57BL; Myeloid Cell Leukemia Sequence 1 Protein; Myeloid-Lymphoid Leukemia Protein; Neoplasms, Experimental; Nitrophenols; Oncogene Proteins, Fusion; Piperazines; Proto-Oncogene Proteins c-bcl-2; Structure-Activity Relationship; Sulfonamides; THP-1 Cells

Cells from patients with acute myeloid leukemia (AML) that remain dormant and protected by stromal cells may escape effects of chemotherapy. We modeled dormancy in vitro and investigated the ability of Bcl-2 inhibitors ABT-199 and ABT-737 to overcome chemoprotection of dormant cells. CD34-enriched primary AML cells with aberrant leukemia-associated phenotypes (LAPs) were cultured on stromal cells. The chemosensitivity of dormant (PKH26

Topics: Animals; Antineoplastic Agents; Biphenyl Compounds; Bone Marrow; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cell Proliferation; Coculture Techniques; Disease Progression; Drug Screening Assays, Antitumor; Humans; Leukemia, Myeloid, Acute; Leukocytes, Mononuclear; Mice; Neoplasm Recurrence, Local; Nitrophenols; Piperazines; Primary Cell Culture; Proto-Oncogene Proteins c-bcl-2; Stromal Cells; Sulfonamides; Tumor Cells, Cultured

Acute myeloid leukemia (AML) is an immunophenotypically heterogenous malignant disease. The early immature CD34. Inhibition of Bcl-2 by ABT-737 effectively inhibited growth and induced apoptosis in CD34. Taken together, these findings indicate that ABT-737 may offer as a promising molecular targeting agent in CD34

Topics: Adult; Apoptosis; Biphenyl Compounds; Drug Synergism; Female; Genes, bcl-2; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Nitrophenols; Piperazines; Sulfonamides; Young Adult

Galectin 3 (LGALS3) expression is prognostic for poor survival in acute myeloid leukemia (AML) patients. GCS-100 is a novel galectin inhibitor that may prove useful for AML therapy. In this study, we found that GCS-100 induced apoptosis in AML cells. The agent reduced MCL-1 expression suggesting that GCS-100 could be more effective when combined with a BH3 mimetic. Indeed, potent synergistic cytotoxicity was achieved when GCS-100 was combined with ABT-737 or ABT-199. Furthermore, the GCS-100/ABT-199 combination was effective against primary AML blast cells from patients with FLT3 ITD mutations, which is another prognostic factor for poor outcome in AML. This activity may involve wild-type p53 as shRNA knockdown of LGALS3 or galectin 1 (LGALS1) sensitized wild-type p53 OCI-AML3 cells to GCS-100/ABT-737-induced apoptosis to a much greater extent than p53 null THP-1 cells. Suppression of LGALS3 by shRNA inhibited MCL-1 expression in OCI-AML3 cells, but not THP-1 cells, suggesting the induced sensitivity to ABT-737 may involve a MCL-1 mediated mechanism. OCI-AML3 cells with LGALS1 shRNA were also sensitized to ABT-737. However, these cells exhibited increased MCL-1 expression, so MCL-1 reduction is apparently not required in this process. A role for p53 appears important as GCS-100 induces p53 expression and shRNA knockdown of p53 protected OCI-AML3 cells from the cytotoxic effects of the GCS-100/ABT-737 treatment combination. Our results suggest that galectins regulate a survival axis in AML cells, which may be targeted via combined inhibition with drugs such as GCS-100 and ABT-199.

Topics: Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Drug Synergism; Female; Galectins; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Nitrophenols; Peptide Fragments; Piperazines; Polysaccharides; Proto-Oncogene Proteins; Sulfonamides; Tumor Suppressor Protein p53

Inhibition of the apoptosis pathway controlled by opposing members of the Bcl-2 protein family plays a central role in cancer development and resistance to therapy. To investigate how pro-apoptotic Bcl-2 homology domain 3 (BH3)-only proteins impact on acute myeloid leukemia (AML), we generated mixed lineage leukemia (MLL)-AF9 and MLL-ENL AMLs from BH3-only gene knockout mice. Disease development was not accelerated by loss of Bim, Puma, Noxa, Bmf, or combinations thereof; hence these BH3-only proteins are apparently ineffectual as tumor suppressors in this model. We tested the sensitivity of MLL-AF9 AMLs of each genotype in vitro to standard chemotherapeutic drugs and to the proteasome inhibitor bortezomib, with or without the BH3 mimetic ABT-737. Loss of Puma and/or Noxa increased resistance to cytarabine, daunorubicin and etoposide, while loss of Bim protected against cytarabine and loss of Bmf had no impact. ABT-737 increased sensitivity to the genotoxic drugs but was not dependent on any BH3-only protein tested. The AML lines were very sensitive to bortezomib and loss of Noxa conveyed significant resistance. In vivo, several MLL-AF9 AMLs responded well to daunorubicin and this response was highly dependent on Puma and Noxa but not Bim. Combination therapy with ABT-737 provided little added benefit at the daunorubicin dose trialed. Bortezomib also extended survival of AML-bearing mice, albeit less than daunorubicin. In summary, our genetic studies reveal the importance of Puma and Noxa for the action of genotoxics currently used to treat MLL-driven AML and suggest that, while addition of ABT-737-like BH3 mimetics might enhance their efficacy, new Noxa-like BH3 mimetics targeting Mcl-1 might have greater potential.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Carcinogenesis; Daunorubicin; Gene Expression Regulation, Leukemic; Gene Knockout Techniques; Leukemia, Myeloid, Acute; Mice, Inbred C57BL; Myeloid-Lymphoid Leukemia Protein; Nitrophenols; Oncogene Proteins, Fusion; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Approximately fifty percent of patients with acute myeloid leukemia can be cured with current therapeutic strategies which include, standard dose chemotherapy for patients at standard risk of relapse as assessed by cytogenetic and molecular analysis, or high-dose chemotherapy with allogeneic hematopoietic stem cell transplant for high-risk patients. Despite allogeneic hematopoietic stem cell transplant about 25% of patients still succumb to disease relapse, therefore, novel strategies are needed to improve the outcome of patients with acute myeloid leukemia.. We developed an immunotherapeutic strategy targeting the CD33 myeloid antigen, expressed in ~ 85-90% of patients with acute myeloid leukemia, using chimeric antigen receptor redirected T-cells. Considering that administration of CAR T-cells has been associated with cytokine release syndrome and other potential off-tumor effects in patients, safety measures were here investigated and reported. We genetically modified human activated T-cells from healthy donors or patients with acute myeloid leukemia with retroviral supernatant encoding the inducible Caspase9 suicide gene, a ΔCD19 selectable marker, and a humanized third generation chimeric antigen receptor recognizing human CD33. ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells had a 75±3.8% (average ± standard error of the mean) chimeric antigen receptor expression, were able to specifically lyse CD33+ targets in vitro, including freshly isolated leukemic blasts from patients, produce significant amount of tumor-necrosis-factor-alpha and interferon-gamma, express the CD107a degranulation marker, and proliferate upon antigen specific stimulation. Challenging ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells with programmed-death-ligand-1 enriched leukemia blasts resulted in significant killing like observed for the programmed-death-ligand-1 negative leukemic blasts fraction. Since the administration of 10 nanomolar of a non-therapeutic dimerizer to activate the suicide gene resulted in the elimination of only 76.4±2.0% gene modified cells in vitro, we found that co-administration of the dimerizer with either the BCL-2 inhibitor ABT-199, the pan-BCL inhibitor ABT-737, or mafosfamide, resulted in an additive effect up to complete cell elimination.. This strategy could be investigated for the safety of CAR T-cell applications, and targeting CD33 could be used as a 'bridge" therapy for patients coming to allogeneic hematopoietic stem cell transplant, as anti-leukemia activity from infusing CAR.CD33 T-cells has been demonstrated in an ongoing clinical trial. Albeit never performed in the clinical setting, our future plan is to investigate the utility of iC9-CAR.CD33 T-cells as part of the conditioning therapy for an allogeneic hematopoietic stem cell transplant for acute myeloid leukemia, together with other myelosuppressive agents, whilst the activation of the inducible Caspase9 suicide gene would grant elimination of the infused gene modified T-cells prior to stem cell infusion to reduce the risk of engraftment failure as the CD33 is also expressed on a proportion of the donor stem cell graft.

Topics: B7-H1 Antigen; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Caspase 9; Cell Engineering; Cell Line, Tumor; Cell Proliferation; Cellular Reprogramming; Clinical Trials as Topic; Cyclophosphamide; Cytotoxicity, Immunologic; Genetic Vectors; Humans; Interferon-gamma; Leukemia, Myeloid, Acute; Lysosomal-Associated Membrane Protein 1; Myeloid Cells; Nitrophenols; Piperazines; Primary Cell Culture; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; Sialic Acid Binding Ig-like Lectin 3; Sulfonamides; T-Lymphocytes; Tumor Necrosis Factor-alpha

Standard in vitro drug testing employs 2-D tissue culture plate systems to test anti-leukemic drugs against cell adhesion-mediated drug-resistant leukemic cells that harbor in 3-D bone marrow microenvironments. This drawback necessitates the fabrication of 3-D scaffolds that have cell adhesion-mediated drug-resistant properties similar to in vivo niches. We therefore aimed at exploiting the known property of polyurethane (PU)/poly-L-lactic acid (PLLA) in forming a micro-nanofibrous structure to fabricate unique, not presented before, as far as we are aware, 3-D micro-nanofibrous scaffold composites using a thermally induced phase separation technique. Among the different combinations of PU/PLLA composites generated, the unique PU/PLLA 60:40 composite displayed micro-nanofibrous morphology similar to decellularized bone marrow with increased protein and fibronectin adsorption. Culturing of acute myeloid leukemia (AML) KG1a cells in FN-coated PU/PLLA 60:40 shows increased cell adhesion and cell adhesion-mediated drug resistance to the drugs cytarabine and daunorubicin without changing the original CD34(+)/CD38(-)/CD33(-) phenotype for 168 hours compared to fibronectin tissue culture plate systems. Molecularly, as seen in vivo, increased chemoresistance is associated with the upregulation of anti-apoptotic Bcl2 and the cell cycle regulatory protein p27(Kip1) leading to cell growth arrest. Abrogation of Bcl2 activity by the Bcl2-specific inhibitor ABT 737 led to cell death in the presence of both cytarabine and daunorubicin, demonstrating that the cell adhesion-mediated drug resistance induced by Bcl2 and p27(Kip1) in the scaffold was similar to that seen in vivo. These results thus show the utility of a platform technology, wherein drug testing can be performed before administering to patients without the necessity for stromal cells.

Topics: Biphenyl Compounds; Cell Adhesion; Cell Proliferation; Cytarabine; Daunorubicin; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Fibronectins; Humans; Lactic Acid; Leukemia, Myeloid, Acute; Nanocomposites; Nanofibers; Nitrophenols; Piperazines; Polyesters; Polymers; Polyurethanes; Sulfonamides; Tissue Scaffolds

Overexpression of antiapoptotic Bcl-2 proteins such as Bcl-2, Bcl-xL, and Mcl-1 is widely associated with tumor initiation, progression, and chemoresistance. Furthermore, it has been demonstrated that Mcl-1 upregulation renders several types of cancers resistant to the Bcl-2/Bcl-xL inhibitors ABT-737 and ABT-263. The emerging importance of Mcl-1 in pathogenesis and drug resistance makes it a high-priority therapeutic target. In this study, we showed that inhibition of Mcl-1 with a novel pan-Bcl-2 inhibitor (-)BI97D6 potently induced apoptosis in acute myeloid leukemia (AML) cells. (-)BI97D6 induced hallmarks of mitochondrial apoptosis, disrupted Mcl-1/Bim and Bcl-2/Bax interactions, and stimulated cell death via the Bak/Bax-dependent mitochondrial apoptosis pathway, suggesting on-target mechanisms. As a single agent, this pan-Bcl-2 inhibitor effectively overcame AML cell apoptosis resistance mediated by Mcl-1 or by interactions with bone marrow mesenchymal stromal cells. (-)BI97D6 was also potent in killing refractory primary AML cells. Importantly, (-)BI97D6 killed AML leukemia stem/progenitor cells while largely sparing normal hematopoietic stem/progenitor cells. These findings demonstrate that pan-Bcl-2 inhibition by an Mcl-1-targeting inhibitor not only overcomes intrinsic drug resistance ensuing from functional redundancy of Bcl-2 proteins, but also abrogates extrinsic resistance caused by the protective tumor microenvironment.

Topics: Animals; Apoptosis; Biphenyl Compounds; Blotting, Western; Cell Proliferation; Drug Resistance, Neoplasm; Female; Flow Cytometry; Gossypol; Humans; Immunoenzyme Techniques; Immunoprecipitation; Leukemia, Myeloid, Acute; Mesenchymal Stem Cells; Mice; Mice, Inbred NOD; Mice, SCID; Myeloid Cell Leukemia Sequence 1 Protein; Naphthoquinones; Neoplastic Stem Cells; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Stem Cells; Sulfonamides; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

We assessed the antileukemic activity of 2-deoxy-d-glucose (2-DG) through the modulation of expression of receptor tyrosine kinases (RTK) commonly mutated in acute myeloid leukemia (AML). We used human leukemic cell lines cells, both in vitro and in vivo, as well as leukemic samples from AML patients to demonstrate the role of 2-DG in tumor cell growth inhibition. 2-DG, through N-linked glycosylation inhibition, affected the cell-surface expression and cellular signaling of both FTL3-ITD and mutated c-KIT and induced apoptotic cell death. Leukemic cells harboring these mutated RTKs (MV4-11, MOLM-14, Kasumi-1, and TF-1 c-KIT D816V) were the most sensitive to 2-DG treatment in vitro as compared with nonmutated cells. 2-DG activity was also demonstrated in leukemic cells harboring FLT3-TKD mutations resistant to the tyrosine kinase inhibitor (TKI) quizartinib. Moreover, the antileukemic activity of 2-DG was particularly marked in c-KIT-mutated cell lines and cell samples from core binding factor-AML patients. In these cells, 2-DG inhibited the cell-surface expression of c-KIT, abrogated STAT3 and MAPK-ERK pathways, and strongly downregulated the expression of the receptor resulting in a strong in vivo effect in NOD/SCID mice xenografted with Kasumi-1 cells. Finally, we showed that 2-DG decreases Mcl-1 protein expression in AML cells and induces sensitization to both the BH3 mimetic inhibitor of Bcl-xL, Bcl-2 and Bcl-w, ABT-737, and cytarabine. In conclusion, 2-DG displays a significant antileukemic activity in AML with FLT3-ITD or KIT mutations, opening a new therapeutic window in a subset of AML with mutated RTKs.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzothiazoles; Biphenyl Compounds; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytarabine; Deoxyglucose; Drug Resistance, Neoplasm; fms-Like Tyrosine Kinase 3; Glycolysis; Glycosylation; Humans; Leukemia, Myeloid, Acute; Mice, Inbred NOD; Mice, SCID; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Phenylurea Compounds; Piperazines; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-kit; Signal Transduction; Sulfonamides; Xenograft Model Antitumor Assays

Effects of concurrent inhibition of mTORC1/2 and Bcl-2/Bcl-xL in human acute myeloid leukemia cells were examined. Tetracycline-inducible Bcl-2/Bcl-xL dual knockdown markedly sensitized acute myeloid leukemia cells to the dual TORC1/2 inhibitor INK128 in vitro as well as in vivo. Moreover, INK128 co-administered with the Bcl-2/xL antagonist ABT-737 sharply induced cell death in multiple acute myeloid leukemia cell lines, including TKI-resistant FLT3-ITD mutants and primary acute myeloid leukemia blasts carrying various genetic aberrations e.g., FLT3, IDH2, NPM1, and Kras, while exerting minimal toxicity toward normal hematopoietic CD34(+) cells. Combined treatment was particularly active against CD34(+)/CD38(-)/CD123(+) primitive leukemic progenitor cells. The INK128/ABT-737 regimen was also effective in the presence of a protective stromal microenvironment. Notably, INK128 was more potent than the TORC1 inhibitor rapamycin in down-regulating Mcl-1, diminishing AKT and 4EBP1 phosphorylation, and potentiating ABT-737 activity. Mcl-1 ectopic expression dramatically attenuated INK128/ABT-737 lethality, indicating an important functional role for Mcl-1 down-regulation in INK128/ABT-737 actions. Immunoprecipitation analysis revealed that combined treatment markedly diminished Bax, Bak, and Bim binding to all major anti-apoptotic Bcl-2 members (Bcl-2/Bcl-xL/Mcl-1), while Bax/Bak knockdown reduced cell death. Finally, INK128/ABT-737 co-administration sharply attenuated leukemia growth and significantly prolonged survival in a systemic acute myeloid leukemia xenograft model. Analysis of subcutaneous acute myeloid leukemia-derived tumors revealed significant decrease in 4EBP1 phosphorylation and Mcl-1 protein level, consistent with results obtained in vitro. These findings demonstrate that co-administration of dual mTORC1/mTORC2 inhibitors and BH3-mimetics exhibits potent anti-leukemic activity in vitro and in vivo, arguing that this strategy warrants attention in acute myeloid leukemia.

Topics: Animals; bcl-X Protein; Benzoxazoles; Biphenyl Compounds; Down-Regulation; Enzyme Activation; Female; Humans; Leukemia, Myeloid, Acute; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Mice, Inbred NOD; Mice, SCID; Multiprotein Complexes; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Nucleophosmin; Piperazines; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Sulfonamides; TOR Serine-Threonine Kinases; U937 Cells; Xenograft Model Antitumor Assays

B-cell leukemia/lymphoma 2 (BCL-2) prevents commitment to programmed cell death at the mitochondrion. It remains a challenge to identify those tumors that are best treated by inhibition of BCL-2. Here, we demonstrate that acute myeloid leukemia (AML) cell lines, primary patient samples, and murine primary xenografts are very sensitive to treatment with the selective BCL-2 antagonist ABT-199. In primary patient cells, the median IC50 was approximately 10 nmol/L, and cell death occurred within 2 hours. Our ex vivo sensitivity results compare favorably with those observed for chronic lymphocytic leukemia, a disease for which ABT-199 has demonstrated consistent activity in clinical trials. Moreover, mitochondrial studies using BH3 profiling demonstrate activity at the mitochondrion that correlates well with cytotoxicity, supporting an on-target mitochondrial mechanism of action. Our protein and BH3 profiling studies provide promising tools that can be tested as predictive biomarkers in any clinical trial of ABT-199 in AML.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; Biomarkers, Tumor; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Death; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; Mice; Mitochondria; Neoplasms, Experimental; Nitrophenols; Peptide Fragments; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Xenograft Model Antitumor Assays

Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response.

Topics: Antimetabolites, Antineoplastic; Azacitidine; bcl-X Protein; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Humans; Leukemia, Myeloid, Acute; Myelodysplastic Syndromes; Myeloid Cell Leukemia Sequence 1 Protein; Myeloproliferative Disorders; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Sulfonamides

Acute myeloid leukemia (AML) is one of the deadliest leukemias for which there is an urgent and unmet need for the development of novel treatment strategies. Multiple drug resistance mechanisms mediate poor drug response and relapse in patients, and a selective Mcl-1 inhibitor has been speculated to be a promising agent in the treatment of AML. Here, we describe that maritoclax, a small molecule Mcl-1 inhibitor, induces Mcl-1 proteasomal degradation without transcriptional downregulation. Maritoclax killed AML cell lines and primary cells with elevated Mcl-1 levels through selective Mcl-1 downregulation, and synergized with ABT-737 to overcome Mcl-1-mediated ABT-737 resistance. Maritoclax was more effective than daunorubicin at inducing leukemic cell death when co-cultured with HS-5 bone marrow stroma cells, while being less toxic than daunorubicin against HS-5 stroma cells, primary mouse bone marrow cells, and hematopoietic progenitor cells. Moreover, maritoclax administration at 20 mg/kg/d intraperitoneally caused significant U937 tumor shrinkage, as well as 36% tumors remission rate in athymic nude mice, without apparent toxicity to healthy tissue or circulating blood cells. In summary, our studies suggest that maritoclax belongs to a novel class of Mcl-1 inhibitors that has the potential to be developed for the treatment of AML.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Bone Marrow Cells; Cell Line, Tumor; Coculture Techniques; Daunorubicin; Drug Resistance, Neoplasm; Drug Synergism; Hematopoietic Stem Cells; Heterografts; Humans; Leukemia, Myeloid, Acute; Male; Mice, Inbred C57BL; Mice, Nude; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Pyrroles; RNA Interference; Stromal Cells; Sulfonamides

The mechanisms underlying acute myeloid leukemia (AML) treatment failure are not clear. Here, we established a mouse model of AML by syngeneic transplantation of BXH-2 derived myeloid leukemic cells and developed an efficacious Ara-C-based regimen for treatment of these mice. We proved that leukemic cell load was correlated with survival. We also demonstrated that the susceptibility of leukemia cells to Ara-C could significantly affect the survival. To examine the molecular alterations in cells with different sensitivity, genome-wide expression of the leukemic cells was profiled, revealing that overall 366 and 212 genes became upregulated or downregulated, respectively, in the resistant cells. Many of these genes are involved in the regulation of cell cycle, cellular proliferation, and apoptosis. Some of them were further validated by quantitative PCR. Interestingly, the Ara-C resistant cells retained the sensitivity to ABT-737, an inhibitor of anti-apoptosis proteins, and treatment with ABT-737 prolonged the life span of mice engrafted with resistant cells. These results suggest that leukemic load and intrinsic cellular resistance can affect the outcome of AML treated with Ara-C. Incorporation of apoptosis inhibitors, such as ABT-737, into traditional cytotoxic regimens merits consideration for the treatment of AML in a subset of patients with resistance to Ara-C. This work provided direct in vivo evidence that leukemic load and intrinsic cellular resistance can affect the outcome of AML treated with Ara-C, suggesting that incorporation of apoptosis inhibitors into traditional cytotoxic regimens merits consideration for the treatment of AML in a subset of patients with resistance to Ara-C.

Topics: Animals; Antimetabolites, Antineoplastic; Biphenyl Compounds; Cell Line, Tumor; Cytarabine; Disease Models, Animal; Down-Regulation; Drug Resistance, Neoplasm; Gene Expression Profiling; Inhibitor of Apoptosis Proteins; Leukemia, Myeloid, Acute; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Nitrophenols; Piperazines; Sulfonamides; Survival Rate; Transplantation, Homologous; Up-Regulation

Myelodysplastic syndrome (MDS) transforms into an acute myelogenous leukemia (AML) with associated increased bone marrow (BM) blast infiltration. Using a transgenic mouse model, MRP8[NRASD12/hBCL-2], in which the NRAS:BCL-2 complex at the mitochondria induces MDS progressing to AML with dysplastic features, we studied the therapeutic potential of a BCL-2 homology domain 3 mimetic inhibitor, ABT-737. Treatment significantly extended lifespan, increased survival of lethally irradiated secondary recipients transplanted with cells from treated mice compared with cells from untreated mice, with a reduction of BM blasts, Lin-/Sca-1(+)/c-Kit(+), and progenitor populations by increased apoptosis of infiltrating blasts of diseased mice assessed in vivo by technicium-labeled annexin V single photon emission computed tomography and ex vivo by annexin V/7-amino actinomycin D flow cytometry, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, caspase 3 cleavage, and re-localization of the NRAS:BCL-2 complex from mitochondria to plasma membrane. Phosphoprotein analysis showed restoration of wild-type (WT) AKT or protein kinase B, extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase patterns in spleen cells after treatment, which showed reduced mitochondrial membrane potential. Exon specific gene expression profiling corroborates the reduction of leukemic cells, with an increase in expression of genes coding for stem cell development and maintenance, myeloid differentiation, and apoptosis. Myelodysplastic features persist underscoring targeting of BCL-2-mediated effects on MDS-AML transformation and survival of leukemic cells.

Topics: Animals; Antigens, Ly; Biphenyl Compounds; Cell Lineage; Cell Membrane; Cell Proliferation; Cell Transformation, Neoplastic; Cell Transplantation; Disease Models, Animal; Flow Cytometry; Gene Expression Regulation, Leukemic; Leukemia, Myeloid, Acute; MAP Kinase Signaling System; Membrane Proteins; Mice; Mice, Transgenic; Mitochondria; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-kit; ras Proteins; Stem Cells; Sulfonamides

Both phosphatidylinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling and antiapoptotic Bcl-2 family members are critical for survival of acute myeloid leukemia (AML) cells. Here, we demonstrate the antileukemic effects of simultaneous inhibition of PI3K by the selective class I PI3K inhibitor GDC-0941 and of Bcl-2 family members by the BH3 mimetic ABT-737 in the context of the bone marrow microenvironment, where hypoxia and interactions with bone marrow stromal cells promote AML cell survival and chemoresistance. The combination of GDC-0941 and ABT-737 profoundly downregulated antiapoptotic Mcl-1 expression levels, activated BAX, and induced mitochondrial apoptosis in AML cells co-cultured with bone marrow stromal cells under hypoxic conditions. Hypoxia caused degradation of Mcl-1 and rendered Mcl-1-overexpressing OCI-AML3 cells sensitive to ABT-737. Our findings suggest that pharmacologic PI3K inhibition by GDC-0941 enhances ABT-737-induced leukemia cell death even under the protective conditions afforded by the bone marrow microenvironment.. Combined blockade of PI3K and Bcl-2 pathways down-regulates anti-apoptotic Mcl-1 expression PI3K and Bcl-2 induced Mcl-1 down-regulation activates BAX PI3K and Bcl-2 blockage induces apoptosis in AML under hypoxic BM microenvironment.

Topics: Apoptosis; Biphenyl Compounds; Bone Marrow; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Drug Synergism; Gene Expression Regulation, Leukemic; Humans; Hypoxia; Indazoles; Leukemia, Myeloid, Acute; Mesenchymal Stem Cells; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Phosphoinositide-3 Kinase Inhibitors; Piperazines; RNA Interference; Sulfonamides; Tumor Microenvironment

Recently, strategies for acute myeloid leukemia (AML) therapy have been developed that target anti-apoptotic BCL2 family members using BH3-mimetic drugs such as ABT-737. Though effective against BCL2 and BCL-X(L), ABT-737 poorly inhibits MCL-1. Here we report that, unexpectedly, ABT-737 induces activation of the extracellular receptor activated kinase and induction of MCL-1 in AML cells. MEK inhibitors such as PD0325901 and CI-1040 have been used successfully to suppress MCL-1. We report that PD0325901 blocked ABT-737-induced MCL-1 expression, and when combined with ABT-737 resulted in potent synergistic killing of AML-derived cell lines, primary AML blast and CD34+38-123+ progenitor/stem cells. Finally, we tested the combination of ABT-737 and CI-1040 in a murine xenograft model using MOLM-13 human leukemia cells.Whereas control mice and CI-1040-treated mice exhibited progressive leukemia growth, ABT-737, and to a significantly greater extent, ABT-737+CI-1040 exerted major anti-leukemia activity. Collectively, results demonstrated unexpected anti-apoptotic interaction between the BCL2 family-targeted BH3-mimetic ABT-737 and mitogen-activated protein kinase signaling in AML cells: the BH3 mimetic is not only restrained in its activity by MCL-1, but also induces its expression. However, concomitant inhibition by BH3 mimetics and MEK inhibitors could abrogate this effect and may be developed into a novel and effective therapeutic strategy for patients with AML.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Benzamides; Biphenyl Compounds; Cell Line, Tumor; Diphenylamine; Extracellular Signal-Regulated MAP Kinases; Humans; Leukemia, Myeloid, Acute; Membrane Proteins; Mice; Mice, Nude; Mitogen-Activated Protein Kinase Kinases; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

DNA methylation and BLC-2 are potential therapeutic targets in acute myeloid leukemia (AML). We investigated pharmacologic interaction between the DNA methyltransferase inhibitor 5-azacytidine (5-AZA) and the BCL-2 inhibitor ABT-737. Increased BCL-2 expression determined by reverse phase protein analysis was associated with poor survival in AML patients with unfavorable cytogenetics (n = 195). We found that 5-AZA, which itself has modest apoptotic activity, acts synergistically with ABT-737 to induce apoptosis. The 5-AZA/ABT-737 combination enhanced mitochondrial outer membrane permeabilization, as evidenced by effective conformational activation of BAX and ∆ψ(m) loss. Although absence of p53 limited apoptotic activities of 5-AZA and ABT-737 as single agents, the combination synergistically induced apoptosis independent of p53 expression. 5-AZA down-regulated MCL-1, known to mediate resistance to ABT-737, in a p53-independent manner. The 5-AZA/ABT-737 combination synergistically induced apoptosis in AML cells in seven of eight patients. 5-AZA significantly reduced MCL-1 levels in two of three samples examined. Our data provide a molecular rationale for this combination strategy in AML therapy.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Azacitidine; Biphenyl Compounds; Cell Line, Tumor; DNA Modification Methylases; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Inhibitors; Humans; Leukemia, Myeloid, Acute; Mice; Mice, Inbred NOD; Mitochondria; Neoplasm Proteins; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

We have previously shown that inhibition of translation initiation, using the small molecule inhibitor silvestrol, induces apoptosis in a pre-clinical murine lymphoma model when combined with daunorubicin. Silvestrol blocks ribosome recruitment by targeting the RNA helicase, eIF4A, which is required for this process. Here we investigate the sensitivity of acute myelogenous leukemia (AML) cell lines to protein synthesis inhibition in combination with the standard cytotoxic agents daunorubicin, etoposide, and cytarabine. Silvestrol shows synergy with standard-of-care agents in AML cell lines and synergizes with ABT-737, a small molecule inhibitor of Bcl-X(L) and Bcl-2. The in vitro synergy between silvestrol and the cytotoxic drugs used in AML therapy provides a basis for in vivo evaluation of these combinations.

Topics: Antineoplastic Combined Chemotherapy Protocols; Biphenyl Compounds; Cell Line, Tumor; Cytarabine; Cytotoxins; Daunorubicin; Drug Evaluation, Preclinical; Drug Synergism; Etoposide; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Models, Biological; Nitrophenols; Peptide Initiation Factors; Piperazines; Protein Biosynthesis; Protein Synthesis Inhibitors; Sulfonamides; Triterpenes; U937 Cells

Since apoptosis is impaired in malignant cells overexpressing prosurvival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Of seven putative BH3 mimetics tested, only ABT-737 triggered Bax/Bak-mediated apoptosis. Despite its high affinity for Bcl-2, Bcl-x(L), and Bcl-w, many cell types proved refractory to ABT-737. We show that this resistance reflects ABT-737's inability to target another prosurvival relative, Mcl-1. Downregulation of Mcl-1 by several strategies conferred sensitivity to ABT-737. Furthermore, enforced Mcl-1 expression in a mouse lymphoma model conferred resistance. In contrast, cells overexpressing Bcl-2 remained highly sensitive to ABT-737. Hence, ABT-737 should prove efficacious in tumors with low Mcl-1 levels, or when combined with agents that inactivate Mcl-1, even to treat those tumors that overexpress Bcl-2.

Topics: Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Biphenyl Compounds; Cells, Cultured; Cytokines; Disease Models, Animal; Fibroblasts; Humans; Leukemia, Myeloid, Acute; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Nitrophenols; Piperazines; Protein Structure, Tertiary; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; RNA Interference; Sulfonamides

BCL-2 proteins are critical for cell survival and are overexpressed in many tumors. ABT-737 is a small-molecule BH3 mimetic that exhibits single-agent activity against lymphoma and small-cell lung cancer in preclinical studies. We here report that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induced the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway. In cells with phosphorylated BCL-2 or increased MCL-1, ABT-737 was inactive. Inhibition of BCL-2 phosphorylation and reduction of MCL-1 expression restored sensitivity to ABT-737. These data suggest that ABT-737 could be a highly effective antileukemia agent when the mechanisms of resistance identified here are considered.

Topics: Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Biphenyl Compounds; Cell Line; Dimerization; Drug Resistance, Neoplasm; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid, Acute; Mice; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Nitrophenols; Piperazines; Protein Conformation; Protein Structure, Tertiary; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; RNA, Small Interfering; Sulfonamides

Disruption of Mdm2-p53 interaction activates p53 signaling, disrupts the balance of antiapoptotic and proapoptotic Bcl-2 family proteins and induces apoptosis in acute myeloid leukemia (AML). Overexpression of Bcl-2 may inhibit this effect. Thus, functional inactivation of antiapoptotic Bcl-2 proteins may enhance apoptogenic effects of Mdm2 inhibition. We here investigate the potential therapeutic utility of combined targeting of Mdm2 by Nutlin-3a and Bcl-2 by ABT-737, recently developed inhibitors of protein-protein interactions. Nutlin-3a and ABT-737 induced Bax conformational change and mitochondrial apoptosis in AML cells in a strikingly synergistic fashion. Nutlin-3a induced p53-mediated apoptosis predominantly in S and G2/M cells, while cells in G1 were protected through induction of p21. In contrast, ABT-737 induced apoptosis predominantly in G1, the cell cycle phase with the lowest Bcl-2 protein levels and Bcl-2/Bax ratios. In addition, Bcl-2 phosphorylation on Ser70 was absent in G1 but detectable in G2/M, thus lower Bcl-2 levels and absence of Bcl-2 phosphorylation appeared to facilitate ABT-737-induced apoptosis of G1 cells. The complementary effects of Nutlin-3a and ABT-737 in different cell cycle phases could, in part, account for their synergistic activity. Our data suggest that combined targeting of Mdm2 and Bcl-2 proteins could offer considerable therapeutic promise in AML.

Topics: Animals; Annexin A5; Apoptosis; bcl-2-Associated X Protein; Biphenyl Compounds; Cell Line, Tumor; Drug Resistance, Neoplasm; G1 Phase; G2 Phase; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mice; Mitochondria; Mitosis; Mutant Proteins; Nitrophenols; Phosphorylation; Piperazines; Protein Conformation; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; S Phase; Sulfonamides; Tumor Suppressor Protein p53

Topics: Acid Phosphatase; Blood Platelets; Bone Marrow; Bone Marrow Cells; Chromatography, DEAE-Cellulose; Chromatography, Ion Exchange; Electrophoresis, Disc; Fluorides; Gaucher Disease; Histocytochemistry; Humans; Isoenzymes; Leukemia; Leukemia, Lymphoid; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Leukocytes; Lymphatic Diseases; Lymphocytes; Lymphoma, Non-Hodgkin; Molecular Weight; Monocytes; Nitrophenols; Polycythemia Vera; Staining and Labeling; Tartrates