nitrophenols and Small-Cell-Lung-Carcinoma

Small cell lung cancer (SCLC) cannot be efficiently controlled using existing chemotherapy and radiotherapy approaches, indicating the need for new therapeutic strategies. Although ABT-737, a B-cell lymphoma-2 (BCL-2) inhibitor, exerts anticancer effects against BCL-2-expressing SCLC, monotherapy with ABT-737 is associated with limited clinical activity because of the development of resistance and toxicity. Here, we examined whether combination therapy with ABT-737 and heat shock protein 90 (HSP90) inhibitor NVP-AUY922 exerted synergistic anticancer effects on SCLC. We found that the combination of ABT-737 and NVP-AUY922 synergistically induced the apoptosis of BCL-2-expressing SCLC cells. NVP-AUY922 downregulated the expression of AKT and ERK, which activate MCL-1 to induce resistance against ABT-737. The synergistic effect was also partly due to blocking NF-κB activation, which induces anti-apoptosis protein expressions. However, interestingly, targeting BCL-2 and MCL-1 or BCL2 and NF-κB did not induce the cytotoxicity. In conclusion, our study showed that combination of BCL2 inhibitor with HSP90 inhibitor increased activity in in vitro and in vivo study in only BCL-2 expressing SCLC compared to either single BCL2 inhibitor or HSP inhibitor. The enhanced activity might be led by blocking several apoptotic pathways simultaneously rather than a specific pathway.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Biphenyl Compounds; Cell Line, Tumor; Drug Synergism; HSP90 Heat-Shock Proteins; Humans; Isoxazoles; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Random Allocation; Resorcinols; Signal Transduction; Small Cell Lung Carcinoma; Sulfonamides; Xenograft Model Antitumor Assays

Most small cell lung cancer (SCLC) patients are initially responsive to cytotoxic chemotherapy, but almost all undergo fatal relapse with progressive disease, highlighting an urgent need for improved therapies and better patient outcomes in this disease. The proapoptotic BH3 mimetic ABT-737 that targets BCL-2 family proteins demonstrated good single-agent efficacy in preclinical SCLC models. However, so far clinical trials of the BH3 mimetic Navitoclax have been disappointing. We previously demonstrated that inhibition of a PI3K/BMX cell survival signaling pathway sensitized colorectal cancer cells to ABT-737. Here, we show that SCLC cell lines, which express high levels of BMX, become sensitized to ABT-737 upon inhibition of PI3K in vitro, and this is dependent on inhibition of the PI3K-BMX-AKT/mTOR signaling pathway. Consistent with these cell line data, when combined with Navitoclax, PI3K inhibition suppressed tumor growth in both an established SCLC xenograft model and in a newly established circulating tumor cell-derived explant (CDX) model generated from a blood sample obtained at presentation from a chemorefractory SCLC patient. These data show for the first time that a PI3K/BMX signaling pathway plays a role in SCLC cell survival and that a BH3 mimetic plus PI3K inhibition causes prolonged tumor regression in a chemorefractory SCLC patient-derived model in vivo These data add to a body of evidence that this combination should move toward the clinic. Mol Cancer Ther; 15(6); 1248-60. ©2016 AACR.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biphenyl Compounds; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Synergism; Humans; Lung Neoplasms; Mice; Nitrophenols; Phosphatidylinositol 3-Kinases; Piperazines; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Signal Transduction; Small Cell Lung Carcinoma; Sulfonamides; Xenograft Model Antitumor Assays

Small cell lung carcinoma (SCLC) is an aggressive, recalcitrant cancer, often metastatic at diagnosis and unresponsive to chemotherapy upon recurrence, thus it is challenging to treat.. Sixty-three human SCLC lines and three NSCLC lines were screened for response to 103 US Food and Drug Administration-approved oncology agents and 423 investigational agents. The investigational agents library was a diverse set of small molecules that included multiple compounds targeting the same molecular entity. The compounds were screened in triplicate at nine concentrations with a 96-hour exposure time using an ATP Lite endpoint. Gene expression was assessed by exon array, and microRNA expression was derived by direct digital detection. Activity across the SCLC lines was associated with molecular characteristics using pair-wise Pearson correlations.. Results are presented for inhibitors of targets: BCL2, PARP1, mTOR, IGF1R, KSP/Eg5, PLK-1, AURK, and FGFR1. A relational map identified compounds with similar patterns of response. Unsupervised microRNA clustering resulted in three distinct SCLC subgroups. Associating drug response with micro-RNA expression indicated that lines most sensitive to etoposide and topotecan expressed high miR-200c-3p and low miR-140-5p and miR-9-5p. The BCL-2/BCL-XL inhibitors produced similar response patterns. Sensitivity to ABT-737 correlated with higher ASCL1 and BCL2. Several classes of compounds targeting nuclear proteins regulating mitosis produced a response pattern distinct from the etoposide response pattern.. Agents targeting nuclear kinases appear to be effective in SCLC lines. Confirmation of SCLC line findings in xenografts is needed. The drug and compound response, gene expression, and microRNA expression data are publicly available at http://sclccelllines.cancer.gov.

Topics: Antineoplastic Agents; Biphenyl Compounds; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Line, Tumor; Drug Screening Assays, Antitumor; Drugs, Investigational; Gene Expression; Humans; Kinesins; Lung Neoplasms; MicroRNAs; Nitrophenols; Piperazines; Polo-Like Kinase 1; Poly (ADP-Ribose) Polymerase-1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptor, Fibroblast Growth Factor, Type 1; Receptor, IGF Type 1; Receptors, Somatomedin; Small Cell Lung Carcinoma; Sulfonamides; TOR Serine-Threonine Kinases

The sensitivity to ABT-737, a prototype BH3 mimetic drug, varies in a broad range in small cell lung cancer (SCLC) cells. We have previously shown that the expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is the critical determinant of ABT-737 sensitivity. We show here that Noxa regulates the localization and stability of MCL-1, an anti-apoptotic member, which results in modulating ABT-737 sensitivity. Mutations in Noxa within the BH3 domain, the carboxyl terminus mitochondrial targeting domain, or of ubiquitinated lysines not only change the localization and stability of Noxa itself but also affect the mitochondrial localization and phosphorylation/ubiquitination status of MCL-1 and consequently modulate sensitivity to ABT-737. Results of studies utilizing these mutant proteins indicate that Noxa recruits MCL-1 from the cytosol to the mitochondria. Translocation of MCL-1 initiates its phosphorylation and subsequent ubiquitination, which triggers proteasome-mediated degradation. The precise regulatory mechanisms of Noxa/MCL-1 expression and stability could provide alternative targets to modulate apoptosis induced by BH3 mimetic drugs or other chemotherapeutic reagents.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Cell Survival; Dose-Response Relationship, Drug; HEK293 Cells; HeLa Cells; Humans; Lung Neoplasms; Mice, Knockout; Mitochondria; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Phosphorylation; Piperazines; Proteasome Endopeptidase Complex; Protein Stability; Protein Transport; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Small Cell Lung Carcinoma; Sulfonamides; Transfection; Ubiquitination

Overexpression of the antiapoptotic protein Bcl-2 is observed in the majority of small cell lung cancer (SCLC) cases and is associated with resistance to chemotherapy. While targeting Bcl-2 in hematologic malignancies continues to show signs of promise, translating the BH3 mimetic ABT-737 (or ABT-263; navitoclax) to the clinic for solid tumors has remained problematic, with limited single-agent activity in early-phase clinical trials. Here, we used patient-derived xenograft (PDX) models of SCLC to study ABT-737 resistance and demonstrated that responses to ABT-737 are short lived and coincide with decreases in HIF-1α-regulated transcripts. Combining the mTOR inhibitor rapamycin with ABT-737 rescued this resistance mechanism, was highly synergistic in vitro, and provided durable tumor regressions in vivo without notable hematologic suppression. In comparison, tumor regressions did not occur when ABT-737 was combined with etoposide, a gold-standard cytotoxic for SCLC therapy. Rapamycin exposure was consistently associated with an increase in the proapoptotic protein BAX, whereas ABT-737 caused dose-dependent decreases in BAX. As ABT-737 triggers programmed cell death in a BAX/BAK-dependent manner, we provide preclinical evidence that the efficacy of ABT-737 as a single agent is self-limiting in SCLC, but the addition of rapamycin can maintain or increase levels of BAX protein and markedly enhance the anticancer efficacy of ABT-737. These data have direct translational implications for SCLC clinical trials.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Biphenyl Compounds; Cell Line, Tumor; Drug Synergism; Etoposide; Female; Humans; Lung Neoplasms; Mice; Mice, SCID; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Random Allocation; Sirolimus; Small Cell Lung Carcinoma; Sulfonamides; Xenograft Model Antitumor Assays

New therapies are urgently needed for patients with small cell lung cancer (SCLC). Chemotherapy and targeted therapies, including the Bcl-2 inhibitor ABT-737, may induce tumor cell autophagy. Autophagy can promote survival of cancer cells under stress and comprise a pathway of escape from cytotoxic therapies.. We explored the combination of ABT-737 and chloroquine, an inhibitor of autophagy, in preclinical models of SCLC. These included cell culture analyses of viability and of autophagic and apoptotic pathway induction, as well as in vivo analyses of efficacy in multiple xenograft models.. Combination treatment of SCLC lines with ABT-737 and chloroquine decreased viability and increased caspase-3 activation over treatment with either single agent. ABT-737 induced several hallmarks of autophagy. However, knockdown of beclin-1, a key regulator of entry into autophagy, diminished the efficacy of ABT-737, suggesting either that the effects of chloroquine were nonspecific or that induction but not completion of autophagy is necessary for the combined effect of ABT-737 and chloroquine. ABT-737 and chloroquine in SCLC cell lines downregulated Mcl-1 and upregulated NOXA, both of which may promote apoptosis. Treatment of tumor-bearing mice demonstrated that chloroquine could enhance ABT-737-mediated tumor growth inhibition against NCI-H209 xenografts, but did not alter ABT-737 response in three primary patient-derived xenograft models.. These data suggest that although ABT-737 can induce autophagy in SCLC, autophagic inhibition by choroquine does not markedly alter in vivo response to ABT-737 in relevant preclinical models, arguing against this as a treatment strategy for SCLC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Biphenyl Compounds; Cell Proliferation; Cell Survival; Chloroquine; Drug Resistance, Neoplasm; Female; Gene Knockdown Techniques; Humans; Lung Neoplasms; Membrane Proteins; Mice; Mice, Nude; Nitrophenols; Piperazines; RNA, Small Interfering; Small Cell Lung Carcinoma; Sulfonamides; Tumor Burden; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

B cell lymphoma 2 (Bcl-2) is a central regulator of cell survival that is overexpressed in the majority of small-cell lung cancers (SCLC) and contributes to both malignant transformation and therapeutic resistance. The purpose of this work was to study the key factors that determine the sensitivity of SCLC cells to Bcl-2 homology domain-3 (BH3) mimetic S1 and the mechanism underlying the resistance of BH3 mimetics.. Western blot was used to evaluate the contribution of Bcl-2 family members to the cellular response of SCLC cell lines to S1. Acquired resistant cells were derived from initially sensitive H1688 cells. Quantitative PCR and gene silencing were performed to investigate Bcl-2 up-regulation.. A progressive increase in the relative levels of Bcl-2 and phosphorylated Bcl-2 (pBcl-2) characterized the increased de novo and acquired resistance of SCLC cell lines. Furthermore, acute treatment of S1 induced Bcl-2 expression and phosphorylation. We showed that BH3 mimetics, including S1 and ABT-737, induced endoplasmic reticulum (ER) stress and then activated MAPK/ERK pathway. The dual function of MAPK/ERK pathway in defining BH3 mimetics was illustrated; ERK1/2 activation leaded to Bcl-2 transcriptional up-regulation and sustained phosphorylation in naïve and acquired resistant SCLC cells. pBcl-2 played a key role in creating resistance of S1 and ABT-737 not only by sequestrating pro-apoptotic proteins, but also sequestrating a positive feedback to promote ERK1/2 activation.. These results provide significant novel insights into the molecular mechanisms for crosstalk between ER stress and endogenously apoptotic pathways in SCLC following BH3 mimetics treatment.

Topics: Antineoplastic Agents; Biphenyl Compounds; Cell Line, Tumor; Drug Resistance, Neoplasm; Endoplasmic Reticulum Stress; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; MAP Kinase Signaling System; Molecular Mimicry; Nitrophenols; Phosphorylation; Piperazines; Proto-Oncogene Proteins c-bcl-2; Small Cell Lung Carcinoma; Sulfonamides

Synergistic killing was achieved when Small Cell Lung Cancer (SCLC) cell lines were incubated with ABT-263 and an immunotoxin directed to the transferrin receptor. SCLC lines are variably sensitive to the BH-3 only peptide mimetic, ABT-263. To determine their sensitivity to toxin-based reagents, we incubated four representative SCLC lines with a model Pseudomonas exotoxin-based immunotoxin directed to the transferrin receptor. Remarkably in 4-of-4 lines, there was little evidence of immunotoxin-mediated cytotoxicity despite near complete inhibition of protein synthesis. However, when combinations of ABT-263 and immunotoxin were added to the ABT-263-resistant cell lines (H196 and H69AR), there was synergistic killing as evidenced by increased activation of caspase 3/7, annexin V staining, and loss of cell integrity. Synergistic killing was evident at 6 hr and correlated with loss of Mcl-1. This synergy was also noted when the closely related compound ABT-737 was combined with the same immunotoxin. To establish that the synergy seen in tissue culture could be achieved in vivo, H69AR cells were grown as tumors in nude mice and shown to be susceptible to the killing action of an immunotoxin-ABT-737 combination but not to either agent alone. When immunotoxin-ABT combinations were added to ABT-263-sensitive lines (H146 and H1417), killing was additive. Our data support combination approaches for treating ABT-263-resistant SCLC with ABT-263 and a second agent that provides synergistic killing action.

Topics: Aniline Compounds; Animals; Biphenyl Compounds; Caspase 3; Caspase 7; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Humans; Immunotoxins; Mice; Mice, Inbred BALB C; Mice, Nude; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Receptors, Transferrin; Small Cell Lung Carcinoma; Sulfonamides

ABT-737, which blocks the function of Bcl-2 and Bcl-X(L) but not Mcl-1, has shown single-agent activity in preclinical models of small cell lung cancer (SCLC). Elevated expression of Mcl-1 induces resistance to ABT-737 in SCLC. Based on the short half-life of Mcl-1 mRNA and protein, we hypothesized that the actinomycin D could reverse Mcl-1-induced resistance to ABT-737.. The dose-response of multiple SCLC cell lines to actinomycin D in the absence and presence of ABT-737 was followed by the assessment of Bcl-2 family expression and poly ADP ribose polymerase cleavage by Western blot, viability by tetrazolium dye reduction and clonogenic assay, and cell cycle kinetics by flow cytometry.. Actinomycin D decreased Mcl-1 expression and resulted in a cell line-dependent increase in Noxa expression. Clinically relevant concentrations of actinomycin D from 0.4 to 4 ng/mL showed single-agent activity across a panel of SCLC cell lines. When combined with low micromolar doses of ABT-737, near complete loss of viability was seen with synergistic combination indices of 0.5 to 0.7. Exposure to 4 ng/mL actinomycin was only required for the first 24 hours of the combined incubation, mimicking a clinically achievable area under the curve, but the presence of ABT-737 was required for an additional 48 hours to obtain maximal effect.. Clinically relevant concentrations of actinomycin D act synergistically with ABT-737 to induce SCLC apoptosis, which can be at least partially attributed to the actinomycin D-induced decrease in Mcl-1 and increase in Noxa expression. Taken together, these data suggest the feasibility of combining actinomycin D with BH3-mimetic drugs in the clinical setting.

Topics: Antibiotics, Antineoplastic; Apoptosis; Biphenyl Compounds; Blotting, Western; Cell Division; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dactinomycin; Dose-Response Relationship, Drug; Drug Synergism; Flow Cytometry; G2 Phase; Humans; Lung Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Small Cell Lung Carcinoma; Sulfonamides; Time Factors

To understand the molecular basis for variable sensitivity to the BH3 mimetic drug ABT-737, the abundance of Bcl-2 family members was assayed in a panel of small cell lung cancer cell lines whose sensitivity varied over a 2-log range. Elevated Noxa and Bcl-2 levels directly correlated with sensitivity to ABT-737, whereas Mcl-1 levels were similar in all cell lines tested regardless of sensitivity. Transgenically enforced expression of Noxa but not Bcl-2 resulted in increased sensitivity to ABT-737 in multiple cell lines. This increase was especially pronounced in the H209 cell line in which expression of Noxa resulted in a proportionate decline in Mcl-1 expression. Although overexpression of Noxa enhanced sensitivity of the H526 and H82 cell lines to ABT-737, it did not result in altered Mcl-1 levels. Similarly, small interfering RNA-mediated knockdown of Noxa expression in the H146 cell line, which increased resistance to ABT-737, did not result in altered Mcl-1 levels. Therefore, three of four cell lines studied failed to show Noxa-mediated regulation of Mcl-1 expression. However, despite failure to regulate Mcl-1 levels, Noxa blocked binding of Bim to Mcl-1 following its release from Bcl-2 by ABT-737. Finally, we observed that a 24-hour incubation of the H526 and WBA cell lines with ABT-737 resulted in increased Noxa expression, suggesting that Noxa may play a direct role in ABT-737-mediated apoptosis. These results indicate that Noxa expression is the critical determinant of ABT-737 sensitivity and loss of Noxa-mediated regulation of Mcl-1 expression may be an important feature of small cell lung cancer biology.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Biphenyl Compounds; Blotting, Western; Cell Proliferation; Drug Resistance, Neoplasm; Humans; Immunoprecipitation; Lung Neoplasms; Membrane Proteins; Molecular Mimicry; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Peptide Fragments; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Small Cell Lung Carcinoma; Sulfonamides; Tumor Cells, Cultured

This study evaluated epithelial cell death ELISAs that measure circulating cytokeratin 18 in mice bearing small-cell lung cancer xenografts treated with a proapoptotic dose of the BH-3 mimetic ABT-737.. H146 tumor-bearing and non-H146 tumor-bearing severe combined immunodeficient (SCID)/bg mice were treated with ABT-737 or vehicle control. Plasma collected before and 2 to 360 hours after treatment was analyzed by M30 (caspase-cleaved cytokeratin 18) and M65 (intact and cleaved cytokeratin 18) ELISA. In parallel, tumors were interrogated for cleaved caspase-3 and cleaved cytokeratin 18 as biomarkers of apoptosis.. ABT-737-treated tumors regressed by 48 hours (P < 0.01) compared with controls, correlating with increased cleaved cytokeratin 18 (P < 0.01; 6 and 24 hours) and increased intact cytokeratin 18 (P < 0.01; 24 hours). Cleaved cytokeratin 18 levels decreased below baseline between 72 and 360 hours for ABT-737-treated and control mice whereas intact cytokeratin 18 decreased below the level of detection at 8 and 15 days in ABT-737-treated mice only. Apoptosis in tumors reflected changes in circulating cytokeratin 18 (cleaved caspase-3, P < 0.05 at 2 hours and P < 0.001 at 6, 12, and 24 hours; caspase-cleaved cytokeratin 18, P < 0.05 at 15 days, for drug treated versus controls).. ABT-737 caused tumor regression by apoptosis in H146 xenografts that mapped to a drug-specific, early increase in circulating cleaved cytokeratin 18 that subsequently declined. Circulating, intact cytokeratin 18 levels correlated with tumor burden. Cleaved caspase-3 and caspase-cleaved cytokeratin 18 in tumor correlated with treatment (P < 0.05, 2 hours; P < 0.001, 6, 12, and 24 hours; cleaved caspase-3, P < 0.05, 15 days; caspase-cleaved cytokeratin 18), indicating that events in plasma were tumor derived. These circulating biomarker data will be translated to clinical trials wherein serial tumor biopsies are rarely obtained.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Biomimetic Materials; Biphenyl Compounds; Butylated Hydroxytoluene; Caspase 3; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Humans; Immunohistochemistry; Keratin-18; Lung Neoplasms; Mice; Nitrophenols; Piperazines; Small Cell Lung Carcinoma; Sulfonamides; Xenograft Model Antitumor Assays