nitrophenols and Precursor-Cell-Lymphoblastic-Leukemia-Lymphoma

ABT-737 belongs to a new class of anticancer agents named BH3 mimetics. ABT-737 competitively binds to surface hydrophobic grooves of anti-apoptotic proteins of Bcl-2 family, counteracting their protective effect. Resveratrol is a natural polyphenol that has been shown to inhibit the proliferation and/or induce apoptosis in a number of different types of cancer cells. The present study was designed to analyze the combined effects of ABT-737 and resveratrol on human acute lymphoblastic leukemia cells. The in vitro cytotoxic activity of these agents against MOLT-4 leukemia cells was determined using the Coulter electrical impedance method, comet assay, and flow cytometry, light microscopy and western blot techniques. The results are the first data showing that ABT-737 combined with resveratrol markedly decreased the cell viability, increased DNA damage, caused the cell cycle perturbation, and synergistically enhanced apoptosis in MOLT-4 cells, when compared to the data obtained after application of the single agent. Moreover, the simultaneous treatment of leukemia cells with ABT-737 and resveratrol resulted in a reduction in mitochondrial membrane potential, an increase of p53 protein level and up-regulation of the Bax/Bcl-2 ratio. The obtained data indicate that the combination of ABT-737 and resveratrol is a promising approach for acute lymphoblastic leukemia treatment that should be further explored.

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Cell Cycle; Cell Line, Tumor; DNA Damage; Drug Synergism; Humans; Membrane Potential, Mitochondrial; Nitrophenols; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-bcl-2; Resveratrol; Stilbenes; Sulfonamides; Tumor Suppressor Protein p53

Several chemo-resistance mechanisms including the Bcl-2 protein family overexpression and constitutive activation of the PI3K/Akt/mTOR signaling have been documented in acute lymphoblastic leukemia (ALL), encouraging targeted approaches to circumvent this clinical problem. Here we analyzed the activity of the BH3 mimetic ABT-737 in ALL, exploring the synergistic effects with the mTOR inhibitor CCI-779 on ABT-737 resistant cells. We showed that a low Mcl-1/Bcl-2 plus Bcl-xL protein ratio determined ABT-737 responsiveness. ABT-737 exposure further decreased Mcl-1, inducing apoptosis on sensitive models and primary samples, while not affecting resistant cells. Co-inhibition of Bcl-2 and the mTOR pathway resulted cytotoxic on ABT-737 resistant models, by downregulating mTORC1 activity and Mcl-1 in a proteasome-independent manner. Although Mcl-1 seemed to be critical, ectopic modulation did not correlate with apoptosis changes. Importantly, dual targeting proved effective on ABT-737 resistant samples, showing additive/synergistic effects. Together, our results show the efficacy of BH3 mimetics as single agent in the majority of the ALL samples and demonstrate that resistance to ABT-737 mostly correlated with Mcl-1 overexpression. Co-targeting of the Bcl-2 protein family and mTOR pathway enhanced drug-induced cytotoxicity by suppressing Mcl-1, providing a novel therapeutic approach to overcome BH3 mimetics resistance in ALL.

Topics: Adult; Antineoplastic Agents; Apoptosis; Biomimetics; Biphenyl Compounds; Blotting, Western; Cell Cycle; Cell Proliferation; Child; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Male; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Peptide Fragments; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

B-cell malignancies upregulate the B-cell lymphoma 2 (Bcl-2) family inhibitors of the intrinsic apoptosis pathway, making them therapy resistant. However, small-molecule inhibitors of Bcl-2 family members such as ABT-737 restore a functional apoptosis pathway in cancer cells, and its oral analog ABT-263 (Navitoclax) has entered clinical trials. Gene engineered chimeric antigen receptor (CAR) T cells also show promise in B-cell malignancy, and as they induce apoptosis via the extrinsic pathway, we hypothesized that small-molecule inhibitors of the Bcl-2 family may potentiate the efficacy of CAR T cells by engaging both apoptosis pathways. CAR T cells targeting CD19 were generated from healthy donors as well as from pre-B-ALL (precursor-B acute lymphoblastic leukemia) patients and tested together with ABT-737 to evaluate apoptosis induction in five B-cell tumor cell lines. The CAR T cells were effective even if the cell lines exhibited different apoptosis resistance profiles, as shown by analyzing the expression of apoptosis inhibitors by PCR and western blot. When combining T-cell and ABT-737 therapy simultaneously, or with ABT-737 as a presensitizer, tumor cell apoptosis was significantly increased. In conclusion, the apoptosis inducer ABT-737 enhanced the efficacy of CAR T cells and could be an interesting drug candidate to potentiate T-cell therapy.

Topics: Antigens, CD19; Antineoplastic Agents; Apoptosis; B7-2 Antigen; Biphenyl Compounds; Cell Line, Tumor; Coculture Techniques; Combined Modality Therapy; Cytotoxicity, Immunologic; fas Receptor; Gene Expression; HLA Antigens; Humans; Immunotherapy; Intercellular Adhesion Molecule-1; Nitrophenols; Phenotype; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-bcl-2; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; Sulfonamides; T-Lymphocytes

Current regimens for induction therapy of pediatric acute lymphoblastic leukemia (ALL), or for re-induction post relapse, use a combination of vincristine (VCR), a glucocorticoid, and L-asparaginase (ASP) with or without an anthracycline. With cure rates now approximately 80%, robust pre-clinical models are necessary to prioritize active new drugs for clinical trials in relapsed/refractory patients, and the ability of these models to predict synergy/antagonism with established therapy is an essential attribute. In this study, we report optimization of an induction-type regimen by combining VCR, dexamethasone (DEX) and ASP (VXL) against ALL xenograft models established from patient biopsies in immune-deficient mice. We demonstrate that the VXL combination was synergistic in vitro against leukemia cell lines as well as in vivo against ALL xenografts. In vivo, VXL treatment caused delays in progression of individual xenografts ranging from 22 to >146 days. The median progression delay of xenografts derived from long-term surviving patients was 2-fold greater than that of xenografts derived from patients who died of their disease. Pharmacokinetic analysis revealed that systemic DEX exposure in mice increased 2-fold when administered in combination with VCR and ASP, consistent with clinical findings, which may contribute to the observed synergy between the 3 drugs. Finally, as proof-of-principle we tested the in vivo efficacy of combining VXL with either the Bcl-2/Bcl-xL/Bcl-w inhibitor, ABT-737, or arsenic trioxide to provide evidence of a robust in vivo platform to prioritize new drugs for clinical trials in children with relapsed/refractory ALL.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Asparaginase; Biphenyl Compounds; Cell Line, Tumor; Child; Dexamethasone; Disease Models, Animal; Drug Synergism; Female; Humans; Induction Chemotherapy; Male; Mice; Mice, Inbred NOD; Mice, SCID; Nitrophenols; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Sulfonamides; Vincristine; Xenograft Model Antitumor Assays

The novel anticancer drug ABT-737 is a Bcl-2 Homology 3 (BH3)-mimetic that induces apoptosis by inhibiting pro-survival Bcl-2 proteins. ABT-737 binds with equal affinity to Bcl-2, Bcl-xL and Bcl-w in vitro and is expected to overrule apoptosis resistance mediated by these Bcl-2 proteins in equal measure. We have profiled ABT-737 specificity for all six pro-survival Bcl-2 proteins, in p53 wild-type or p53-mutant human T-leukemic cells. Bcl-B was untargeted, like Bfl-1 and Mcl-1, in accord with their low affinity for ABT-737 in vitro. However, Bcl-2 proved a better ABT-737 target than Bcl-xL and Bcl-w. This was reflected in differential apoptosis-sensitivity to ABT-737 alone, or combined with etoposide. ABT-737 was not equally effective in displacing BH3-only proteins or Bax from Bcl-2, as compared with Bcl-xL or Bcl-w, offering an explanation for the differential ABT-737 sensitivity of tumor cells overexpressing these proteins. Inducible expression demonstrated that BH3-only proteins Noxa, but not Bim, Puma or truncated Bid could overrule ABT-737 resistance conferred by Bcl-B, Bfl-1 or Mcl-1. These data identify Bcl-B, Bfl-1 and Mcl-1, but also Bcl-xL and Bcl-w as potential mediators of ABT-737 resistance and indicate that target proteins can be differentially sensitive to BH3-mimetics, depending on the pro-apoptotic Bcl-2 proteins they are complexed with.

Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; bcl-X Protein; Biphenyl Compounds; Boronic Acids; Bortezomib; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Etoposide; Humans; Minor Histocompatibility Antigens; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Sulfonamides; T-Lymphocytes; Tumor Suppressor Protein p53

ABT-737 is a BH3 mimetic small-molecule inhibitor that binds with high affinity to Bcl-2 to induce apoptosis in malignant cells and has shown promise as an effective anti-leukemic agent in pediatric preclinical tests. This study focuses on the effects of ABT-737 on leukemia cells with MLL rearrangement and identifies some of the biological correlates of its activity.. Cells were cultured in the presence of increasing concentrations of ABT-737 alone or in combination with other agents. After 4 days in culture, cell growth inhibition was measured by Alamar blue assay. The expression and activation of potential intracellular targets of ABT-737 activity were determined by Western blot analysis.. Significant Bcl-2 expression was detected in all infant leukemia cells investigated. ABT-737 induced cell death in all cell lines studied although the IC(50) values differed somewhat between cell lines. Western blot analysis identified the effects of ABT-737 on survival and apoptosis-regulatory proteins PARP, caspase-8, and cytochrome-c. Drug combination studies indicated synergy with distinct anti-neoplastic agents, including the multi-tyrosine kinase inhibitor sunitinib. This effective drug synergy appears to be mediated by the combined inhibition of Bcl-2 and intracellular signaling pathways.. We describe the in vitro studies to demonstrate the activity and drug combinability of ABT-737 against MLL rearranged leukemia cells. In addition, identification of the molecular changes that occur in the presence of ABT-737 provides information regarding effective target validation and target modulation analyses in future clinical trials.

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Blotting, Western; Caspase 8; Cell Line, Tumor; Cytochromes c; Drug Combinations; Gene Rearrangement; Histone-Lysine N-Methyltransferase; Humans; Infant; Male; Myeloid-Lymphoid Leukemia Protein; Nitrophenols; Piperazines; Poly(ADP-ribose) Polymerases; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Although recent decades have seen a significant improvement in the treatment outcome of leukemia in the pediatric population, those who are treated for relapsed disease still face significant morbidity and mortality. However, current salvage regimens are often assembled with agents that have similar mode of activity as the chemotherapeutics used in the initial treatment. Hence, novel therapeutic agents that are capable of distinct and diverse mechanisms of activity in, now resistant, leukemia cells are of great interest. We have investigated the opioid agonist methadone for its anti-leukemic activity, initially reported in studies with cell lines derived from adult patients. Our findings show that, compared to normal cells, methadone has enhanced cytotoxicity against specimens and cell lines established from refractory childhood leukemia. In addition, methadone's activity synergized with that of the anti-Bcl-2 agent ABT-737 and was characterized by the induction of distinct changes in tumor cell mitochondria. Data presented also identify biological correlates and a potential mechanism for methadone activity by its effects on Mcl-1 and other members of the apoptosis cascade. We provide mechanistic data for the therapeutic potential of a family of agents that is largely unexplored for anti-leukemic activity.

Topics: Age Factors; Age of Onset; Analgesics, Opioid; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Child; Child, Preschool; Drug Evaluation, Preclinical; Drug Synergism; Female; Humans; Infant; Male; Methadone; Nitrophenols; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Primary Cell Culture; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Treatment Outcome; Tumor Cells, Cultured; Up-Regulation

Antiapoptotic Bcl-2 proteins are overexpressed in a number of cancers, including leukemias, and are frequently associated with resistance to conventional chemotherapeutic drugs. ABT-737, a Bcl-2 homology domain 3 mimetic (for structure, see Nature 435:677-681, 2005) inhibits the prosurvival function of Bcl-2, Bcl-X(L), and Bcl-w. We show that ABT-737 was effective as a single agent against a panel of pediatric acute lymphoblastic leukemia (ALL) xenografts, previously established, from patient biopsies, in immunodeficient mice. Although in vitro resistance of leukemia cell lines correlated with expression of the prosurvival protein Mcl-1, there was no relationship between Mcl-1 expression and in vivo xenograft response to ABT-737. However, expression of the pro-apoptotic protein Bim, and the extent of its association with Bcl-2, significantly correlated with in vivo ABT-737 sensitivity. ABT-737 potentiated the antileukemic effects of L-asparaginase, topotecan, vincristine, and etoposide against drug-resistant xenografts in vitro and in vivo. Finally, we show that the combination of L-asparaginase (by specifically down-regulating Mcl-1 protein levels), topotecan (by activating p53 via DNA damage), and ABT-737 (by inhibiting antiapoptotic Bcl-2 family members) caused profound synergistic antileukemic efficacy both in vitro and in vivo. Rational targeting of specific components of the apoptotic pathway may be a useful approach to improve the treatment of refractory or relapsed pediatric ALL. Overall, this study supports the inclusion of the clinical derivative of ABT-737, ABT-263 (for structure, see Cancer Res 68:3421-3428, 2008), into clinical trials against relapsed/refractory pediatric ALL.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Drug Delivery Systems; Drug Synergism; HeLa Cells; HL-60 Cells; Humans; Jurkat Cells; K562 Cells; Mice; Mice, Inbred C57BL; Mice, SCID; Molecular Mimicry; Nitrophenols; Pharmaceutical Preparations; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Xenograft Model Antitumor Assays

Although the BCR/ABL tyrosine kinase inhibitor imatinib is highly effective for treatment of chronic myelogenous leukemia and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia, relapse with emerging imatinib resistance mutations in the BCR/ABL kinase domain poses a significant problem. Here, we show that the multikinase inhibitor sorafenib inhibits proliferation and induces apoptosis at much lower concentrations in Ton.B210 cells when driven by inducibly expressed BCR/ABL than when driven by interleukin-3. The increased sensitivity to sorafenib was also observed in cells inducibly expressing BCR/ABL with the imatinib-resistant E255K or T315I mutation. Sorafenib-induced apoptosis in these cells and Ph+ leukemic cells was synergistically enhanced by rottlerin, bortezomib, or ABT-737 and inhibited by the pan-caspase inhibitor BOC-d-fmk or the overexpression of Bcl-XL. It was further revealed that sorafenib activates Bax and caspase-3 and reduces mitochondrial membrane potential specifically in BCR/ABL-driven cells. Sorafenib also inhibited BCR/ABL-induced tyrosine phosphorylation of its cellular substrates and its autophosphorylation in Ton.B210. It was finally shown that sorafenib inhibits the kinase activity of BCR/ABL as well as its E255K and T315I mutants in in vitro kinase assays. These results indicate that sorafenib induces apoptosis of BCR/ABL-expressing cells, at least partly, by inhibiting BCR/ABL to activate the mitochondria-mediated apoptotic pathway. Thus, sorafenib may provide an effective therapeutic measure to treat Ph+ leukemias, particularly those expressing the T315I mutant, which is totally resistant to imatinib and the second generation BCR/ABL inhibitors.

Topics: Acetophenones; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; Benzenesulfonates; Benzopyrans; Biphenyl Compounds; Boronic Acids; Bortezomib; Caspases; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Activation; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Interleukin-3; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mitochondria; Mutation; Niacinamide; Nitrophenols; Phenylurea Compounds; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazines; Pyridines; Pyrimidines; Sorafenib; Sulfonamides

Cancer cells acquire disruptions in normal signal transduction pathways and homeostatic mechanisms that would trigger apoptosis in normal cells. These abnormalities include genomic instability, oncogene activation, and growth factor independent proliferation. Therefore, cancer cells likely require a block in apoptosis in order to survive. Overexpression of the antiapoptotic protein BCL-2 provides a block in apoptosis that is frequently observed in cancer cells. We have developed methods for the detection and analysis of BCL-2 dependence and here apply them to acute lymphoblastic leukemia (ALL). BH3 profiling, a mitochondrial assay that classifies blocks in the intrinsic apoptotic pathway, indicated a dependence on BCL-2 of both ALL cell lines and primary samples. This dependence predicted that BCL-2 would be complexed with select pro-death BH3 family proteins, a prediction confirmed by the isolation of BCL-2 complexes with BIM. Furthermore, the BH3 profiling and protein analysis predicted that ALL cell lines and primary cells would be sensitive to ABT-737 as a single agent. Finally, BH3 profiling and protein studies accurately predicted a relative degree of sensitivity to BCL-2 antagonism in cell lines. The ALL cells studied exhibit BCL-2 dependence, supporting clinical trials of BCL-2 antagonists in ALL as single agents or combination therapies.

Topics: Amino Acid Sequence; Annexin A5; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; BH3 Interacting Domain Death Agonist Protein; Biphenyl Compounds; Cell Line, Tumor; Electron Transport Complex IV; Humans; Membrane Proteins; Mitochondria; Nitrophenols; Peptide Fragments; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Tumor Cells, Cultured

ABT-737 is a pan-Bcl-2 inhibitor that has a wide range of single-agent activity against acute lymphoblastic leukemia (ALL) cell lines and xenografts. A relationship between expression of myeloid cell leukemia 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family of proteins, and resistance to ABT-737 has been reported for various cancers. The synthetic cytotoxic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) is known to generate reactive oxygen species (ROS), and ROS have been shown to activate c-Jun kinase (JNK), which in turn phosphorylates and inhibits Mcl-1. Thus, we investigated whether 4-HPR-mediated inactivation of Mcl-1 could act synergistically with ABT-737 to promote leukemia cell death.. Cytotoxicity was determined using the fluorescence-based DIMSCAN assay. Synergy was defined as a combination index (CIN) less than 1. The expression of Bcl-2 family messenger RNAs was measured by real-time reverse transcription-polymerase chain reaction, and caspase activity was measured enzymatically. Changes in Bcl-2 family proteins and release of mitochondrial cytochrome c were detected by immunoblotting. ROS, apoptosis, mitochondrial membrane depolarization, and phospho-JNK were measured by flow cytometry. Gene silencing was by small interfering RNA (siRNA). All statistical tests were two-sided.. ABT-737 decreased Mcl-1 protein expression in ABT-737-sensitive ALL cell lines but not in ABT-737-resistant lines. Using the antioxidant ascorbic acid and siRNA-mediated knockdown of JNK, we showed that 4-HPR decreased Mcl-1 via ROS generation (that phosphorylates JNK) in ABT-737-resistant cell lines. Combining ABT-737 with 4-HPR enhanced the mitochondrial apoptotic cascade (percentage of cells with depolarized mitochondrial membrane at 6 hours, ABT-737 vs ABT-737 plus 4-HPR: 24.5% vs 45.5%, difference = 20.1%, 95% CI = 18.9% to 13.9%; P < .001) and caused caspase-dependent, synergistic multilog cytotoxicity in all seven ALL cell lines examined (mean CIN = 0.57, 95% CI = 0.37 to 0.87), with minimal cytotoxicity for normal lymphocytes.. An increase of Mcl-1 protein in response to ABT-737 is one mechanism of ABT-737 resistance that can be overcome by 4-HPR, resulting in synergistic cytotoxicity of ABT-737 combined with 4-HPR in ALL cell lines.

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Caspases; Cell Line, Tumor; Cytochromes c; Drug Synergism; Enzyme Activation; Fenretinide; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Immunoblotting; JNK Mitogen-Activated Protein Kinases; Mitochondrial Membranes; Nitrophenols; Phosphorylation; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Sulfonamides

Defects in apoptosis signaling contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL), and overexpression of antiapoptotic Bcl-2 (Bcl-2 and Bcl-X(L)) family proteins has been observed in ALL. ABT-737 is a small-molecule BH3-mimetic that inhibits the antiapoptotic Bcl-2 family proteins. We evaluated the cytotoxicity of ABT-737 in combination with vincristine, dexamethasone, and L-asparaginase (VXL) in 7 ALL cell lines. Multilog synergistic cytotoxicity was observed in all 7 cell lines with ABT-737 plus L-asparaginase or vincristine, and in 5 of 7 cell lines with ABT-737 plus dexamethasone or VXL. In leukemia cells, but not in normal lymphocytes, ABT-737 plus L-asparaginase induced greater mitochondrial depolarization (JC-1 staining); mitochondrial cytochrome c release; activation of Bax, Bid, and caspases (immunoblotting); and eventually apoptosis (annexin V staining) than did either drug alone. In mouse xenografts derived from patients with ALL at diagnosis (ALL-7) or at relapse (ALL-19), event-free survival (EFS) was significantly enhanced with ABT-737 plus VXL relative to VXL or ABT-737 alone (P

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Apoptosis; Asparaginase; Biphenyl Compounds; Blotting, Western; Cell Line, Tumor; Child; Cytochromes c; Dexamethasone; Drug Therapy, Combination; Female; Flow Cytometry; Humans; In Vitro Techniques; Leukemia, T-Cell; Membrane Potential, Mitochondrial; Mice; Mice, Inbred NOD; Mice, SCID; Mitochondria; Nitrophenols; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Survival Rate; Vincristine