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Oncotarget Jan 2023Overexpression of the dihydrolipoamide S-succinyltransferase () is associated with poor outcome in neuroblastoma patients and triple-negative breast cancer (TNBC) and...
Overexpression of the dihydrolipoamide S-succinyltransferase () is associated with poor outcome in neuroblastoma patients and triple-negative breast cancer (TNBC) and specifically with the oxidative phosphorylation (OXPHOS) pathway. Inhibitors of OXPHOS were previously suggested as a potential therapeutic strategy for a subset of patients with high-risk neuroblastoma. Here, we tested if cell lines with amplifications or high mRNA levels were associated with sensitivity to 250 drugs from the Genomics of Drug Sensitivity in Cancer (GDSC) dataset by comparing them to cell lines without these changes. -altered cell lines were more sensitive to 7 approved drugs, among these obatoclax mesylate, a BCL2 inhibitor that reduces OXPHOS in human leukemia stem cells. Moreover, several protein kinase inhibitors were identified to be efficient in cell lines with amplifications or high mRNA levels, suggesting a vulnerability of -altered cell lines for drugs targeting the ERK/MAPK pathway. Furthermore, increased expression in cell lines with driver mutations in supported this relationship. We therefore conclude that, in addition to OXPHOS, protein kinases could be potential targets of therapy in the presence of amplifications or high mRNA levels. The new drug candidates proposed here could serve in experimental testing on drug efficacy in knock-in cell lines and -activated tumors.
Topics: Humans; RNA, Messenger; Cell Line; Neuroblastoma; Cell Line, Tumor
PubMed: 36634214
DOI: 10.18632/oncotarget.28342 -
Oncotarget May 2017Drug combination therapies remain pivotal for the treatment of heterogeneous malignancies, such as glioblastomas. Here, we show a novel lethal interaction between Bcl-xL...
Drug combination therapies remain pivotal for the treatment of heterogeneous malignancies, such as glioblastomas. Here, we show a novel lethal interaction between Bcl-xL and c-myc inhibition accomplished by bromodomain protein inhibitors. Established, patient-derived xenograft and stem cell-like glioma cells were treated with the novel bromodomain protein inhibitors, JQ1 and OTX015, along with BH3-mimetics, ABT263 or Obatoclax. Synergy was assessed by calculation of CI values. Small interfering RNAs (siRNAs) were used for gene silencing and mechanistic studies. In vivo experiments were performed in a glioblastoma xenograft model. Single treatments with JQ1 and OTX015 had only moderate effects on the reduction of cellular viability. However, the combination treatment of BH3-mimetics along with JQ1 or OTX015 resulted in a highly synergistic reduction of cellular viability in a broad range of different model systems of malignant glioma. Similarly, knockdown of c-myc sensitized glioma cells for ABT263 mediated cell death. The enhanced loss of cellular viability in the combination treatment was mediated by activation of apoptosis with dissipation of mitochondrial membrane potential and caspase cleavage. The combination treatment led to a modulation of anti- and pro-apoptotic Bcl-2 family members with an increase in pro-apoptotic Noxa mediated by ATF4. Small interfering RNA mediated knockdown of Bak and Noxa protected glioma cells from ABT263/JQ1 mediated apoptosis. Finally, the combination treatment of ABT263 and OTX015 resulted in a regression of tumors and a significantly smaller tumor size as compared to single or vehicle treated tumors. Thus, these results warrant clinical testing for the drug combination of BH3-mimetics along with bromodain protein inhibitors.
Topics: Acetanilides; Aniline Compounds; Animals; Antineoplastic Agents; Apoptosis; Azepines; Bcl-2-Like Protein 11; Caspases; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Drug Synergism; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioma; Heterocyclic Compounds, 3-Ring; Humans; Membrane Potential, Mitochondrial; Mice; Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; RNA, Small Interfering; Sulfonamides; Triazoles; Xenograft Model Antitumor Assays
PubMed: 28418907
DOI: 10.18632/oncotarget.16365 -
Oncogene Mar 2019The poor outcomes in infant acute lymphoblastic leukemia (ALL) necessitate new treatments. Here we discover that EIF4E protein is elevated in most cases of infant ALL...
The poor outcomes in infant acute lymphoblastic leukemia (ALL) necessitate new treatments. Here we discover that EIF4E protein is elevated in most cases of infant ALL and test EIF4E targeting by the repurposed antiviral agent ribavirin, which has anticancer properties through EIF4E inhibition, as a potential treatment. We find that ribavirin treatment of actively dividing infant ALL cells on bone marrow stromal cells (BMSCs) at clinically achievable concentrations causes robust proliferation inhibition in proportion with EIF4E expression. Further, we find that ribavirin treatment of KMT2A-rearranged (KMT2A-R) infant ALL cells and the KMT2A-AFF1 cell line RS4:11 inhibits EIF4E, leading to decreases in oncogenic EIF4E-regulated cell growth and survival proteins. In ribavirin-sensitive KMT2A-R infant ALL cells and RS4:11 cells, EIF4E-regulated proteins with reduced levels of expression following ribavirin treatment include MYC, MCL1, NBN, BCL2 and BIRC5. Ribavirin-treated RS4:11 cells exhibit impaired EIF4E-dependent nuclear to cytoplasmic export and/or translation of the corresponding mRNAs, as well as reduced phosphorylation of the p-AKT1, p-EIF4EBP1, p-RPS6 and p-EIF4E signaling proteins. This leads to an S-phase cell cycle arrest in RS4:11 cells corresponding to the decreased proliferation. Ribavirin causes nuclear EIF4E to re-localize to the cytoplasm in KMT2A-AFF1 infant ALL and RS4:11 cells, providing further evidence for EIF4E inhibition. Ribavirin slows increases in peripheral blasts in KMT2A-R infant ALL xenograft-bearing mice. Ribavirin cooperates with chemotherapy, particularly L-asparaginase, in reducing live KMT2A-AFF1 infant ALL cells in BMSC co-cultures. This work establishes that EIF4E is broadly elevated across infant ALL and that clinically relevant ribavirin exposures have preclinical activity and effectively inhibit EIF4E in KMT2A-R cases, suggesting promise in EIF4E targeting using ribavirin as a means of treatment.
Topics: Cell Line, Tumor; Child, Preschool; Drug Resistance, Neoplasm; Eukaryotic Initiation Factor-4E; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Humans; Indoles; Infant; Microarray Analysis; Molecular Targeted Therapy; Multigene Family; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Biosynthesis; Pyrroles; Ribavirin; Signal Transduction
PubMed: 30478448
DOI: 10.1038/s41388-018-0567-7 -
Cancer Research Jul 2017Rational therapeutic approaches based on synthetic lethality may improve cancer management. On the basis of a high-throughput drug screen, we provide preclinical proof...
Rational therapeutic approaches based on synthetic lethality may improve cancer management. On the basis of a high-throughput drug screen, we provide preclinical proof of concept that targeting the mitochondrial Hsp90 chaperone network (mtHsp90) and inhibition of Bcl-2, Bcl-xL, and Mcl-1 is sufficient to elicit synthetic lethality in tumors recalcitrant to therapy. Our analyses focused on BH3 mimetics that are broad acting (ABT263 and obatoclax) or selective (ABT199, WEHI-539, and A1210477), along with the established mitochondrial matrix chaperone inhibitor gamitrinib-TPP. Drug combinations were tested in various therapy-resistant tumors and in murine model systems of melanoma, triple-negative breast cancer, and patient-derived orthotopic xenografts (PDX) of human glioblastoma. We found that combining BH3 mimetics and gamitrinib-TPP blunted cellular proliferation in a synergistic manner by massive activation of intrinsic apoptosis. In like manner, suppressing either Bcl-2, Bcl-xL, or Mcl-1 recapitulated the effects of BH3 mimetics and enhanced the effects of gamitrinib-TPP. Mechanistic investigations revealed that gamitrinib-TPP activated a PERK-dependent integrated stress response, which activated the proapoptotic BH3 protein Noxa and its downstream targets Usp9X and Mcl-1. Notably, in the PDX glioblastoma and BRAFi-resistant melanoma models, this drug combination safely and significantly extended host survival. Our results show how combining mitochondrial chaperone and Bcl-2 family inhibitors can synergize to safely degrade the growth of tumors recalcitrant to other treatments. .
Topics: Aniline Compounds; Animals; Antineoplastic Agents; Apoptosis; Biomimetic Materials; Cell Line, Tumor; Cell Proliferation; Female; HSP90 Heat-Shock Proteins; Humans; Indoles; Male; Mice; Mice, SCID; Mitochondria; Peptide Fragments; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides; Xenograft Model Antitumor Assays
PubMed: 28522750
DOI: 10.1158/0008-5472.CAN-16-3424 -
BMC Cancer Nov 2015Colorectal cancer is the third most common malignancy in humans and novel therapeutic approaches are urgently needed. Autophagy is an evolutionarily highly conserved...
BACKGROUND
Colorectal cancer is the third most common malignancy in humans and novel therapeutic approaches are urgently needed. Autophagy is an evolutionarily highly conserved cellular process by which cells collect unnecessary organelles or misfolded proteins and subsequently degrade them in vesicular structures in order to refuel cells with energy. Dysregulation of the complex autophagy signaling network has been shown to contribute to the onset and progression of cancer in various models. The Bcl-2 family of proteins comprises central regulators of apoptosis signaling and has been linked to processes involved in autophagy. The antiapoptotic members of the Bcl-2 family of proteins have been identified as promising anticancer drug targets and small molecules inhibiting those proteins are in clinical trials.
METHODS
Flow cytometry and colorimetric assays were used to assess cell growth and cell death. Long term 3D cell culture was used to assess autophagy in a tissue mimicking environment in vitro. RNA interference was applied to modulate autophagy signaling. Immunoblotting and q-RT PCR were used to investigate autophagy signaling. Immunohistochemistry and fluorescence microscopy were used to detect autophagosome formation and autophagy flux.
RESULTS
This study demonstrates that autophagy inhibition by obatoclax induces cell death in colorectal cancer (CRC) cells in an autophagy prone environment. Here, we demonstrate that pan-Bcl-2 inhibition by obatoclax causes a striking, late stage inhibition of autophagy in CRC cells. In contrast, ABT-737, a Mcl-1 sparing Bcl-2 inhibitor, failed to interfere with autophagy signaling. Accumulation of p62 as well as Light Chain 3 (LC3) was observed in cells treated with obatoclax. Autophagy inhibition caused by obatoclax is further augmented in stressful conditions such as starvation. Furthermore, our data demonstrate that inhibition of autophagy caused by obatoclax is independent of the essential pro-autophagy proteins Beclin-1, Atg7 and Atg12.
CONCLUSIONS
The objective of this study was to dissect the contribution of Bcl-2 proteins to autophagy in CRC cells and to explore the potential of Bcl-2 inhibitors for autophagy modulation. Collectively, our data argue for a Beclin-1 independent autophagy inhibition by obatoclax. Based on this study, we recommend the concept of autophagy inhibition as therapeutic strategy for CRC.
Topics: Antineoplastic Agents; Autophagy; Biphenyl Compounds; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Indoles; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Signal Transduction; Sulfonamides
PubMed: 26585594
DOI: 10.1186/s12885-015-1929-y -
Oncotarget Mar 2015Androgen deprivation therapy induces apoptosis or cell cycle arrest in prostate cancer (PCa) cells. Here we set out to analyze whether MCL1, a known mediator of...
Androgen deprivation therapy induces apoptosis or cell cycle arrest in prostate cancer (PCa) cells. Here we set out to analyze whether MCL1, a known mediator of chemotherapy resistance regulates the cellular response to androgen withdrawal. Analysis of MCL1 protein and mRNA expression in PCa tissue and primary cell culture specimens of luminal and basal origin, respectively, reveals higher expression in cancerous tissue compared to benign origin. Using PCa cellular models in vitro and in vivo we show that MCL1 expression is upregulated in androgen-deprived PCa cells. Regulation of MCL1 through the AR signaling axis is indirectly mediated via a cell cycle-dependent mechanism. Using constructs downregulating or overexpressing MCL1 we demonstrate that expression of MCL1 prevents induction of apoptosis when PCa cells are grown under steroid-deprived conditions. The BH3-mimetic Obatoclax induces apoptosis and decreases MCL1 expression in androgen-sensitive PCa cells, while castration-resistant PCa cells are less sensitive and react with an upregulation of MCL1 expression. Synergistic effects of Obatoclax with androgen receptor inactivation can be observed. Moreover, clonogenicity of primary basal PCa cells is efficiently inhibited by Obatoclax. Altogether, our results suggest that MCL1 is a key molecule deciding over the fate of PCa cells upon inactivation of androgen receptor signaling.
Topics: Animals; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Humans; Indoles; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Myeloid Cell Leukemia Sequence 1 Protein; Prostatic Neoplasms; Pyrroles; Random Allocation; Receptors, Androgen; Risk Factors; Transfection; Xenograft Model Antitumor Assays
PubMed: 25749045
DOI: 10.18632/oncotarget.3368 -
BMC Cancer Oct 2018Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive...
BACKGROUND
Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells.
METHODS
We examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer.
RESULTS
In this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells.
CONCLUSIONS
Our findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies.
Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Forkhead Box Protein O3; Gene Expression; Genes, erbB-2; Humans; Lapatinib; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; TNF-Related Apoptosis-Inducing Ligand
PubMed: 30305055
DOI: 10.1186/s12885-018-4852-1 -
Journal of Experimental & Clinical... Jul 2015The evasion of cell death is one of the hallmarks of cancer, contributing to both tumor progression and resistance to therapy. Dedifferentiated and anaplastic thyroid...
BACKGROUND
The evasion of cell death is one of the hallmarks of cancer, contributing to both tumor progression and resistance to therapy. Dedifferentiated and anaplastic thyroid carcinomas that do not take up radioiodine are resistant to conventional anticancer treatments and patients with these tumors are difficult to treat. BH3 mimetics are a new class of drugs that target anti-apoptotic proteins of the BCL-2 family and promote cell death. The purpose of this study was to analyze the molecular effects of the BH3 mimetic GX15-070 on thyroid carcinoma cell lines and to characterize cell death induced by GX15-070.
METHODS
A total of 17 cell lines derived from follicular, papillary, and anaplastic thyroid carcinomas were treated with GX15-070. Cell viability was measured with MTT assay while cell cycle phase distribution and subG1 peaks were determined after propidium iodide staining. We assessed cell death via the caspase 3/7 activity, caspase cleavage products, lactate dehydrogenase (LDH) liberation assays, and a LC3 analysis by western blot. Ultrastructural changes were analysed by electron microscopy of GX15-070-treated cells.
RESULTS
After GX15-070 treatment, the number of viable cells was decreased in all cell lines examined, with IC50 values ranging from 48nM to 3.25 μM. We observed biochemical markers of autophagic cell death and necrosis like LC3 conversion and LDH release after the GX15-070 treatment. Electron microscopy revealed several common characteristic ultrastructural changes like swelling of mitochondria, dilatation of rough endoplasmic reticulum, membrane blebbing and formation of vacuoles. GX15-070 treatment induced DNA fragmentation detected by subG1-peak induction and an arrest in G1 phase of the cell cycle. Caspase activation after GX15-070 incubation was detected but had no effect on viability of cells.
CONCLUSIONS
With these experiments we demonstrated the efficacy of the BH3 mimetic drug GX15-070 acting against dedifferentiated thyroid carcinoma cells of various histological origins by the induction of cell death. GX15-070-treated cells underwent non-classical cell death with signs of apoptosis, autophagy and necrosis in parallel. GX15-07 and related compounds thus may be a new therapeutic option for dedifferentiated thyroid carcinoma of various histological subtypes.
Topics: Apoptosis; Cell Death; Cell Line, Tumor; Cell Survival; Humans; Indoles; Molecular Targeted Therapy; Pyrroles; Thyroid Neoplasms
PubMed: 26198850
DOI: 10.1186/s13046-015-0186-x -
Scientific Reports Sep 2019Inhibition of the androgen receptor (AR) by second-generation anti-androgens is a standard treatment for metastatic castration resistant prostate cancer (mCRPC), but it...
Inhibition of the androgen receptor (AR) by second-generation anti-androgens is a standard treatment for metastatic castration resistant prostate cancer (mCRPC), but it inevitably leads to the development of resistance. Since the introduction of highly efficient AR signalling inhibitors, approximately 20% of mCRPC patients develop disease with AR independent resistance mechanisms. In this study, we generated two anti-androgen and castration resistant prostate cancer cell models that do not rely on AR activity for growth despite robust AR expression (AR indifferent). They are thus resistant against all modern AR signalling inhibitors. Both cell lines display cross-resistance against the chemotherapeutic drug docetaxel due to MCL1 upregulation but remain sensitive to the PARP inhibitor olaparib and the pan-BCL inhibitor obatoclax. RNA-seq analysis of the anti-androgen resistant cell lines identified hyper-activation of the E2F cell-cycle master regulator as driver of AR indifferent growth, which was caused by deregulation of cyclin D/E, E2F1, RB1, and increased Myc activity. Importantly, mCRPC tissue samples with low AR activity displayed the same alterations and increased E2F activity. In conclusion, we describe two cellular models that faithfully mimic the acquisition of a treatment induced AR independent phenotype that is cross-resistant against chemotherapy and driven by E2F hyper-activation.
Topics: Androgen Antagonists; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Docetaxel; Drug Resistance, Neoplasm; Humans; Male; Mice; Mice, Nude; Myeloid Cell Leukemia Sequence 1 Protein; Prostate; Prostatic Neoplasms, Castration-Resistant; Receptors, Androgen; Signal Transduction
PubMed: 31551480
DOI: 10.1038/s41598-019-50220-1 -
IScience Sep 2022Pharmacologically active compounds with known biological targets were evaluated for inhibition of SARS-CoV-2 infection in cell and tissue models to help identify potent...
Pharmacologically active compounds with known biological targets were evaluated for inhibition of SARS-CoV-2 infection in cell and tissue models to help identify potent classes of active small molecules and to better understand host-virus interactions. We evaluated 6,710 clinical and preclinical compounds targeting 2,183 host proteins by immunocytofluorescence-based screening to identify SARS-CoV-2 infection inhibitors. Computationally integrating relationships between small molecule structure, dose-response antiviral activity, host target, and cell interactome produced cellular networks important for infection. This analysis revealed 389 small molecules with micromolar to low nanomolar activities, representing >12 scaffold classes and 813 host targets. Representatives were evaluated for mechanism of action in stable and primary human cell models with SARS-CoV-2 variants and MERS-CoV. One promising candidate, obatoclax, significantly reduced SARS-CoV-2 viral lung load in mice. Ultimately, this work establishes a rigorous approach for future pharmacological and computational identification of host factor dependencies and treatments for viral diseases.
PubMed: 35992305
DOI: 10.1016/j.isci.2022.104925