High-content profiling reveals a unified model of copper ionophore dependent cell death in oesophageal adenocarcinoma

2021 ◽  
Author(s):  
Rebecca E Hughes ◽  
Richard JR Elliott ◽  
Xiaodun Li ◽  
Alison F Munro ◽  
Ashraff Makda ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cell Death ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Drug Sensitivity ◽  
Oesophageal Adenocarcinoma ◽  
Driver Mutations ◽  
Integrated Analysis ◽  
High Content Imaging ◽  
Ammonium Pyrrolidinedithiocarbamate

Background and Aims: Oesophageal adenocarcinoma (OAC) is of increasing global concern due to increasing incidence, a lack of effective treatments, and poor prognosis. Therapeutic target discovery and clinical trials have been hindered by the heterogeneity of the disease, lack of driver mutations, and the dominance of large-scale genomic rearrangements. In this work we have characterised three potent and selective hit compounds identified in an innovative high-content phenotypic screening assay. The three hits include two approved drugs; elesclomol and disulfiram, and another small molecule compound, ammonium pyrrolidinedithiocarbamate. We uncover their mechanism of action, discover a targetable vulnerability, and gain insight into drug sensitivity for biomarker-based clinical trials in OAC. Methods: Elesclomol, disulfiram, and ammonium pyrrolidinedithiocarbamate were systematically characterised across panels of oesophageal cell lines and patient-derived organoids. Drug treated oesophageal cell lines were morphologically profiled using a high-content, imaging platform. Compounds were assessed for efficacy across patient-derived organoids. Metabolomics and transcriptomics were assessed for the identification of oesophageal-cancer specific drug mechanisms and patient stratification hypotheses. Results: High-content profiling revealed that all three compounds were highly selective for OAC over tissue-matched controls. Comparison of gene expression and morphological signatures unveiled a unified mechanism of action involving the accumulation of copper selectively in cancer cells, leading to dysregulation of proteostasis and cancer cell death. Basal omic analyses revealed proteasome and metabolic markers of drug sensitivity, forming the basis for biomarker-based clinical trials in OAC. Conclusions: Integrated analysis of high-content imaging, transcriptomic and metabolomic data has revealed a new therapeutic mechanism for the treatment of OAC and represents an alternative target-agnostic drug discovery strategy.

scholarly journals P-OGC32 Copper-dependent cell death is a cancer specific vulnerability in oesophageal adenocarcinoma and provides the opportunity for future biomarker-stratified clinical trials

2021 ◽  
Vol 108 (Supplement_9) ◽  
Author(s):  
Rebecca E Hughes ◽  
Richard JR Elliott ◽  
Alison Munro ◽  
Ted Hupp ◽  
Neil O Carragher ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cell Death ◽  
Cell Lines ◽  
Small Molecule ◽  
Oesophageal Adenocarcinoma ◽  
Driver Mutations ◽  
Imaging Features ◽  
High Content Imaging ◽  
Chemical Structures ◽  
Dependent Cell

Abstract Background Oesophageal adenocarcinoma (OAC) is of increasing global concern due to increasing incidence, lack of effective treatments, and poor prognosis. Therapeutic target discovery and clinical trials have been hindered by the heterogeneity of the disease, dominance of large-scale genomic rearrangements, and lack of driver mutations. We have profiled small-molecule compounds using an innovative high content imaging assay in a panel of transformed and non-transformed oesophageal cell lines to identify OAC-specific cytotoxic compounds, new therapeutic targets, potential drug repurposing opportunities, and chemical starting points for the treatment of OAC.   Methods We have comprehensively profiled 19,555 small-molecule compounds using an innovative high content assay to quantify 1000’s of subcellular imaging features to capture important phenotypes missed by standard approaches. Prioritised molecules then underwent functional, transcriptomic, and metabolomic characterisation across panels of oesophageal cell lines and patient-derived organoids for the identification of OAC-specific drug mechanisms. Results We identified 72 lead compounds as exhibiting OAC-specific cytotoxicity and characterised three of the most potent and selective compounds in depth, each of different proposed classes and chemical structures. Using several orthogonal methods we uncovered a unified mechanism of action and a targetable vulnerability in OAC involving copper-dependent cell death.  Strikingly no phenotypic effects or changes in gene-expression were observed following treatment with these compounds in non-transformed oesophageal cell lines or normal gastric organoids providing support for this mechanism as a cancer-specific phenomenon.     Conclusions We have applied high content imaging, transcriptomic and metabolomic analyses to reveal a unique vulnerability in OAC.  We have defined a unified mechanism of OAC-specific copper-dependent cell death for the three highly potent compounds.  Finally, through the integration of transcriptomic and metabolomics analyses we gained insight into drug sensitivity and provide the basis for a future biomarker-stratified clinical trial of these drugs in OAC.

Activity of New Heat Shock Protein 90 (hsp90) Inhibitor NVP-AUY922 Against Myeloma Cells Sensitive and Resistant to Conventional Agents.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1587-1587 ◽  
Author(s):  
Douglas W. McMillin ◽  
Joseph Negri ◽  
Jake Delmore ◽  
Patrick Hayden ◽  
Melissa Ooi ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cell Death ◽  
Cell Lines ◽  
Single Agent ◽  
Hsp90 Inhibitor ◽  
Parental Cell Line ◽  
Normal Donor ◽  
Preclinical Studies ◽  
Protective Effects ◽  
Malignant Cells

Abstract Context: The molecular chaperone hsp90 is a major anti-cancer therapeutic target because it regulates the function of proteins with pivotal roles in tumor cell proliferation, survival and drug resistance, including mutated/chimeric oncoproteins or oncogenic kinases/receptors. Our preclinical studies on the ansamycin hsp90 inhibitor tanespimycin (17-AAG) provided the rationale for clinical trials, either alone or in combination with the proteasome inhibitor bortezomib, for treatment of relapsed/refractory MM. In this study, we report preclinical studies of the new, non-ansamycin, hsp90 inhibitor NVP-AUY922. Methods/Results: We tested 36 human MM cell lines and observed with MTT colorimetric survival assays potent time- and dose-dependent anti-MM activity of NVP-AUY922. IC50 values were <12.5 nM for 31 of 35 cell lines, which were also more sensitive than all non-malignant cells tested with NVP-AUY922, including bone marrow stromal cells (BMSCs), immunortalized human hepatocytes and normal donor PBMCs, indicating a differential selectivity of NVP-AUY922 against neoplastic tissues vs. non-malignant cells. Importantly, MM cell lines or primary MM tumor cells resistant to dexamethasone (Dex), melphalan, immunomodulatory thalidomide derivatives (IMIDs), bortezomib or TRAIL were sensitive to NVP-AUY922. In addition, MM-1S cells constitutively over-expressing Akt were equally sensitive to NVP-AUY922 compared to their parental cell line. These anti-MM effects were rapid, as MM cells were committed to cell death within 16 hrs of exposure to only 25 nM of NVP-AUY922. NVP-AUY922 overcame the protective effects conferred to MM cells by exogenous IL-6 and IGF-1, while BMSC co-culture attenuated NVP-AUY922 activity only at doses <20 nM, as evidenced by compartment-specific bioluminescence imaging (CS-BLI). Mechanistic studies of NVP-AUY922 treatment of MM-1S cells showed early G2/M arrest followed by increased cell death, accompanied by caspase-3 and -8 cleavage (as early as 16 hrs). These events were preceded by decreased levels of Akt, B-Raf, phospho-MEK, cIAP2, and XIAP; and compensatory upregulation of hsp27 and hsp70. Given the highly single-agent activity of NVP-AUY922 at even low nM concentrations, formal statistical documentation of synergy was not observed in NVP-AUY922 combinations with conventional (e.g. Dex, doxorubicin) or novel (e.g. bortezomib) anti-MM agents. Encouragingly, no evidence of antagonism with any of these combinations was observed, indicating that NVP-AUY922 can be combined with current anti-MM agents in clinical settings. Conclusion: The new hsp90 inhibitor NVP-AUY922 has potent in vitro activity against MM cells resistant to conventional therapeutics, with selectivity for malignant compared to normal cells. Ongoing in vivo experiments and studies to identify biomarkers of pronounced sensitivity to NVP-AUY922 will help provide a framework for potential clinical trials of NVP-AUY922 in MM and other neoplasias.

A Novel Non-Competitive Chemical Proteasome Inhibitor Displays Preclinical Activity in Myeloma and Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1711-1711
Author(s):  
Xiaoming Li ◽  
Tabitha E Wood ◽  
Remco Sprangers ◽  
Xinliang Mao ◽  
Xiaoming Wang ◽  
...  
Keyword(s):  
Cell Death ◽  
Tumor Cell ◽  
Enzymatic Activity ◽  
Mouse Models ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibitors ◽  
Leukemia Cells ◽  
Tumor Cell Lines

Abstract The proteasome is an enzymatic complex that rids cells of excess and misfolded proteins and possesses chymotrypin, trypsin, and caspase-like enzymatic activity. To date, all of the proteasome inhibitors approved for clinical use or in clinical trials inhibit the complex competitively by binding the active sites of the enzymes. Here, we report a novel chemical proteasome inhibitor that binds the alpha subunits of the 20S proteasome and inhibits the complex non-competitively through a dual copper-dependent and independent mechanism. In a screen of a focused chemical library for novel proteasome inhibitors, we identified 5-amino-8-hydroxyquinoline (5AHQ). When added to myeloma or leukemia intact cells or cell extracts, 5AHQ inhibited the enzymatic activity of the proteasome at low micromolar concentrations. In order to obtain further insight into the mechanism of action of 5AHQ, we carried out a kinetic analysis of inhibition of the enzymatic activity of purified T. Acidophilium proteasome. By Lineweaver-Burk plot analysis, 5AHQ inhibited the proteasome non-competitively. Next, we investigated the binding of 5AHQ to the proteasome. By NMR analysis, 5AHQ bound the half-proteasome complex comprised of a pair of α-rings, α7-α7, and clear spectral changes were observed that localized to residues Ile159, Val113, Val87, Val82, Leu112, Val89, Val134, Val24 and Leu136 inside the antechamber. In contrast, the competitive inhibitor MG132 that binds the proteolytic chamber did not produce any changes in spectra of α7-α7, as expected. 5AHQ bound copper in a 2:1 stoichiometry with a logβ′ value of 9.09, and the addition of copper to 5AHQ enhanced 5AHQ-mediated inhibition of the proteasome. However, binding intracellular copper was not sufficient to explain the effects of 5AHQ on the proteasome as analogues of 5AHQ that did not bind copper continued to inhibit the proteasome, copper-binding molecules not structurally related to 5AHQ did not affect the proteasome, and 5AHQ inhibited isolated proteasomes in buffers devoid of copper and other heavy metals. Given the effects of 5AHQ on the proteasome, we examined the effects of this molecule on the viability of leukemia and myeloma cell lines. Leukemia, myeloma and solid tumor cell lines were treated with increasing concentrations of 5AHQ for 72 hours and cell viability was measured by the MTS assay. 5AHQ induced cell death in 9/9 myeloma, 6/10 leukemia, and 3/10 solid tumor cell lines with an LD50 ≤5 uM. Cell death was confirmed by Annexin V staining. Consistent with its mechanism of action as a proteasome inhibitor, the ability of 5AHQ to induce cell death matched its ability to inhibit the proteasome. In addition, 5AHQ-mediated cell death was associated with inhibition of the NF-kappaB signalling pathway. As 5AHQ induced cell death in malignant cells, we evaluated the effects of oral 5AHQ in 3 mouse models of leukemia. Sublethally irradiated NOD-SCID mice were injected subcutaneously with OCI-AML2 or K562 human leukemia cells or intraperitoneally with MDAY-D2 murine leukemia cells. After tumor implantation, mice were treated with 5AHQ (50 mg/kg/day) or buffer control by oral gavage. Oral 5AHQ decreased tumor weight and volume in all 3 mouse models compared to control without causing weight loss or gross organ toxicity. In summary, we have identified a new strategy for inhibition of the proteasome and a lead for a new therapeutic agent for the treatment of hematologic malignancies.

Purified, Fermented, Extract of Triticum Aestivum Has Significant Independent Lymphomacidal Activity and Augments the Activity of Rituximab

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2857-2857
Author(s):  
Laura Newell ◽  
Joseph Tuscano ◽  
Robert o'Donnell ◽  
Yunpeng Ma
Keyword(s):  
Triticum Aestivum ◽  
T Cells ◽  
Cell Death ◽  
Cell Lines ◽  
Nude Mice ◽  
Mechanism Of Action ◽  
Wheat Germ ◽  
The United States

Abstract Abstract 2857 Background: Non-Hodgkin's lymphoma (NHL) affects over 400,000 people in the United States and its incidence increases with age. Treatment options include cytotoxic chemotherapy, which is often poorly tolerated by elderly patients, and monoclonal antibody (mAb) therapy. Nearly 70% of NHL patients eventually die of the disease. Development of effective alternate treatments with favorable toxicity profiles is necessary. Fermented wheat germ extract (FWGE) has shown anticancer potential in laboratory animals as well as in some small clinical studies; it is produced under GMP conditions in Europe and sold as Avemar™. The mechanism of action of FWGE is unclear, but is thought to involve metabolic pathways involved in tumor cell death. We examined the effects of FWGE on NHL and found significant lymphomacidal activity using in vitro and in vivo assays. We then further purified and characterized the active components of FWGE in order to develop a more potent form and to understand the mechanism of action, physiologic, and immunologic properties. Methods: FWGE was produced by fermenting purified wheat germ (Triticum aestivum) with Baker's yeast. The FWGE was further purified by removing insoluble material, precipitating proteins, freeze drying, fractionating with Sepharose and Sephadex columns, and then dialyzing to remove small molecules. The resultant fermented wheat germ proteins (FWGP) were assessed for in vitro cytotoxicity and pro-apoptotic activity using a panel of NHL cell lines. In vivo lymphomacidal activity was assessed in nude mice bearing Raji lymphoma xenografts. Mice were treated with increasing daily doses of FWGE by gastric lavage and compared to untreated controls as well as the commercially available fermented wheat germ product, Avemar. Results: In vitro killing assays with FWGE (regardless of the source) demonstrated lymphomacidal properties in three NHL cell lines (Jurkat, Raji, and Ramos). Pre-treatment of FWGE with heat or proteinase K reduced the lymphomacidal activity, suggesting that the active component was a protein. Nude mice bearing Raji lymphoma xenografts treated with FWGE confirmed the lymphomacidal properties of FGWE; there was no detectable toxicity as assessed by observation, mouse weight, or blood counts. The purified low molecular weight proteins (FWGP) also demonstrated lymphomacidal properties by cytotoxicity assays and murine NHL models, but at 1/1000th of the original dose. When FWGP was combined with rituximab, there was enhanced in vitro lymphomacidal activity, with over a 4000-fold reduction in the IC50. FWGP-induced NHL cell death was mediated by caspase-3-dependent apoptosis. FWGP augmented the host immune effector mechanisms, including ADCC and CDC, along with potent activation of NK-T cells (CD3/69/16), CD4+ T-cells and monocytes. Conclusions: FWGE can be easily produced and has cytotoxic effects in in vitro assays and in vivo. The purified FWGP are quantifiable, and are 10–1000 times more potent than FWGE. The mechanism of FWGP activity is based on direct pro-apoptotic effects as well as augmentation of host immune mediators. FWGP has activity against various subtypes of NHL. Studies are ongoing to further characterize the immune effects and anti-cancer properties of FWGP, as is planning for a human clinical trial +/− rituximab in patients with NHL. Disclosure: No relevant conflicts of interest to declare.

scholarly journals Phosphoproteomics Uncovers Synergy between DNA-PK and FLT3 Inhibitors in Acute Myeloid Leukaemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-12
Author(s):  
Heather Murray ◽  
Anoop Kumar Enjeti ◽  
Richard Kahl ◽  
Hayley Flanagan ◽  
David Skerrett-Byrne ◽  
...  
Keyword(s):  
Cell Death ◽  
Myeloid Leukaemia ◽  
Board Of Directors ◽  
Cell Lines ◽  
Enrichment Analysis ◽  
Driver Mutations ◽  
Repair Pathway ◽  
Advisory Committees ◽  
Nhej Repair ◽  
Flt3 Inhibitors

*These authors contributed equally to this work Background:Acute Myeloid Leukaemia (AML) is the most common and aggressive form of acute leukaemia, with a 5-year survival rate of just 24%. Activating mutations in the receptor tyrosine kinase FLT3 are the most common driver mutations in AML (25-30% of patients). Inhibiting the FLT3 receptor as a mono-therapeutic strategy in AML has proven difficult however, due to the development of treatment resistance and relapse. In order to identify improved therapeutic targets, the oncogenic signalling pathways downstream of mutant FLT3 require characterisation. Methods:Quantitative, label-based phosphoproteomics was performed on primary blasts from 7 AML patients (4 mutant-FLT3, 3 wildtype-FLT3). Differentially phosphorylated pathways were identified using Ingenuity Pathway Analysis, and kinase activation was assessed by kinase substrate enrichment analysis. Validation of results was performed using targeted mass spectrometry. Proliferation, apoptosis, and cell cycle assays were used to assess drug toxicity; drug synergy was evaluated using Chou-Talalay and Webb analyses. Results:Analysis of differentially expressed phosphoproteins in mutant-FLT3 compared to wildtype-FLT3 AML patient blasts revealed dysregulation of DNA repair pathways. Specifically, mutant-FLT3 samples displayed increased phosphorylation of proteins within the error-prone Non-Homologous End Joining (NHEJ) repair pathway, indicating NHEJ pathway activation. Kinase enrichment analysis predicted increased activity of the NHEJ core kinase, DNA-dependent protein kinase (DNA-PK), in mutant-FLT3 samples. Accordingly, proliferation assays revealed that mutant-FLT3 cell lines were sensitive to inhibition of DNA-PK. FLT3-inhibitor treatment reduced DNA-PK phosphorylation in mutant-FLT3 cells, suggesting that activation of DNA-PK is downstream of FLT3 activation. Inhibition of DNA-PK kinase activity combined with FLT3 inhibitors led to synergistic induction of cell death, selectively in mutant-FLT3 cell lines. DNA-PK inhibitors combined with FLT3 inhibitors also co-operatively induced cell death in mutant-FLT3 primary AML patient samplesex vivo, and significantly prolonged survival compared to either monotherapy in a human AML xenograft mouse model. Conclusions:Mutant-FLT3 AML is associated with activation of the error-prone NHEJ repair pathway, which may contribute to genomic instability. Targeting the NHEJ kinase, DNA-PK, in combination with FLT3 inhibitors has the potential to improve outcomes for this poor-prognosis AML subtype. Disclosures Enjeti: Bayer:Speakers Bureau;AbbVie:Membership on an entity's Board of Directors or advisory committees;Alexion:Speakers Bureau;Novartis:Membership on an entity's Board of Directors or advisory committees;Astellas:Membership on an entity's Board of Directors or advisory committees;Sanofi:Speakers Bureau.

scholarly journals JX06, a Novel PDK1 Inhibitor, Induces Myeloma Cell Apoptosis By Metabolic Reprogramming and Works Synergistically with Bortezomib

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1814-1814
Author(s):  
Takayuki Sasano ◽  
Saki Kushima ◽  
Matsushita Yutaka ◽  
Masao Matsuoka ◽  
Hiroyuki Hata ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Death ◽  
Oxidative Phosphorylation ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Pyruvate Dehydrogenase ◽  
Research Funding ◽  
Caspase 3 ◽  
Low Concentrations

Background: Despite the efficacy of novel agents, multiple myeloma (MM) is still an incurable disease. In order to achieve a cure, it is necessary to develop new therapeutic drugs, which target different pathways from the present anti-MM agents. PDK1 (pyruvate dehydrogenase kinase 1) is a glucose metabolism-related protein often induced by HIF-1. PDK1 inactivates PDH (pyruvate dehydrogenase) through phosphorylation, leading to enhanced glycolysis in the cytoplasm and suppression of oxidative phosphorylation in the mitochondria. PDK1 that is highly expressed in plasma cells is a downstream target of IRF4. We previously reported that PDK1 inhibition is a potent therapeutic strategy in MM (Fujiwara S et al. Br. J. Cancer; 108 (1): 170-178. 2013). However, PDK1 inhibitors, which are effective at low concentrations, are limited at present, making PDK1 inhibition difficult to apply in the clinic. In the present study, we examined the efficacy and mechanism of action of JX06, a novel PDK1 inhibitor, against MM cells. Materials and methods: MM cell lines (NCI-H929,KMS-12PE,KMS-12BM,U266, KMM1, RPMI-8226) were treated with PDK1 inhibitor, JX06, in vitro. Caspase inhibitor, Z-VAD-FMK, was used in combination with JX06 to study the mechanism of JX06 induced MM cell death. Mitochondrial pyruvate carrier (MPC) inhibitor, UK5099, was utilized to block pyruvate transportation into the mitochondria. Bortezomib was used in combination with or without JX06. Growth inhibition of MM cell lines by JX06 were examined by WST-8 assay. Cytotoxicity of primary MM cells by JX06 was examined using flow cytometry after staining with 7AAD. Caspase 3 activity and PDH phosphorylation of MM cell lines were determined by Western blot. Cell cycle analysis of MM cell lines treated with or without JX06 was performed by flow cytometry using BrdU. Detection of apoptosis in MM cell lines were examined by Annexin V and PI staining followed by flow cytometry analysis. Results: JX06 suppressed cell growth of various MM cell lines and primary myeloma cells at low concentrations (0.5-1.0 µM). MM cell death by JX06 accompanied caspase 3 activation and this cell death was suppressed under addition of Z-VAD-FMK, indicating that JX06 induced apoptosis in MM cells. Moreover, phosphorylation of PDH, known as a target of PDK1, was significantly suppressed under JX06 treatment, demonstrating that indeed JX06 exerts anti-MM effect by inhibiting PDK1-PDH pathway. Addition of UK5099 to JX06 suppressed JX06-induced MM cell death, demonstrating that the efficacy of JX06 depends on pyruvate transported into the mitochondria through MPC. There was no significant difference in cell cycle distribution between JX06 treated MM cells compared to control, suggesting that JX06 exerts cytotoxicity independent of cell cycle phase. Moreover, significant increase of cell death was observed in NCI-H929 cell line treated in combination with 0.25 µM JX06 and 2.5 nM bortezomib, although bortezomib alone at concentration of 2.5 nM didn't induce cell death. Conclusion: We demonstrated that JX06 could induce apoptosis of MM cell lines and primary MM cells by inhibiting PDK1. JX06-induced MM cell death is mediated by metabolic shift from glycolysis in the cytoplasm to oxidative phosphorylation in the mitochondria (Fig. 1). Considering its efficacy and the distinct mechanism of action from the current anti-MM agents, JX06 can be a promising anti-MM agent. Furthermore, JX06 not only works as single agent, but can also enhance the efficacy of current anti-MM drugs, suggesting this combination lead to better treatment response and less toxicity. Disclosures Matsuoka: Kyowa Kirin Co., Ltd.: Research Funding; Bristol-Myers Squibb Corp.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria.

Glutathione and Glutathione Utilizing Enzymes Are Key Determinants in the Sensitivity of Myeloma Cells to Arsenic Trioxide as Well as Its Mechanism of Action.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5143-5143
Author(s):  
Delia Gutman ◽  
Alejo Morales ◽  
Jennifer M. McCafferty ◽  
Emilio Volz ◽  
Tatiana Eguizabal ◽  
...  
Keyword(s):  
Cell Death ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
De Novo ◽  
Single Agent ◽  
Building Blocks ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Abstract Arsenic trioxide (ATO, Trisenox) is currently being tested in clinical trials as a single agent or in combination with other agents that have activity in multiple myeloma (MM). We and others have demonstrated that glutathione (GSH) levels can influence the ability of ATO to induce cell death in MM cell lines/patient samples and based on these data have initiated a trial to test the safety and efficacy of the combination of ATO and ascorbic acid for the treatment of refractory/relapsed MM. Therefore we performed expression profiling on 4 MM cell lines treated with ATO over a 48 timecourse. Affymetrix Hu133 2.0 plus arrays were hybridized and up to 7035 out of 54,675 probe sets displayed a change and up to 1546 probes sets displayed 2 fold or greater changes compared to untreated cells at 6 hrs. By only looking at genes that increased in all four cell lines we restricted our search to less than 365 probes sets at any given time point. Interestingly the cells appear to have initiated several pathways that are consistent with an attempt to enhance GSH synthesis. Upregulation of transporters of cysteine (xCT) and glycine (Glyt1) as well as enzymes that convert methionine to cysteine (cystathionase) and serine to glycine (serine hydroxymethyl transferase-1) was observed. Moreover the rate-limiting step of the glutathione salvage pathway gamma-glutamyltransferase is also upregulated. Together this suggests an increase in the building blocks for GSH that can be used for de novo synthesis. The rate limiting step for this reaction is performed by gamma-glutamate cysteine ligase which both the catalytic and modifier subunits are upregulated. GSH can also be regenerated from GSSG by glutathione reductase (GR) in an NADPH-dependent fashion. Both GR and the NADPH generating malic enzyme are also upregulated following treatment with ATO. While consistent with our previous findings the data do not provide much insight as to how the GSH is utilized. The only GSH utilizing enzyme that was observed to be upregulated were the cytosolic and mitochondrial forms of glutaredoxin. Glutathione peroxidase (GPx) activity is not altered by ATO treatment. However GPx baseline expression and activity do correlate with sensitivity of MM cell lines to ATO. We also determined GSTP1 activity in the cells and found that it was expressed in 4/5 MM cell lines tested. In contrast to GPx, GSTP1 baseline expression did not correlate with sensitivity to ATO. However this pattern of expression correlated with our previous findings regarding these cells demonstrating differences in caspase dependence of ATO-induced cell death. The one line that did not express GSTP1, RPMI 8226, also utilizes caspase-independent mechanisms of cell death. Transfection of the GSTP1A allele into these cells could render cells more resistant to ATO-induced apoptosis at concentrations of ATO that are not likely to be achieved in patients. Interestingly transfection of the GSTP1B allele could not render cells more resistant, however like GSTP1A it resulted in inhibition of the caspase-independent pathway. Taken together these data confirm that GSH is an important modulator of ATO therapy and that GPx expression may determine the sensitivity of cells to ATO while GSTP1 can affect the mechanism of action by which ATO-induces apoptosis.

The off-Patent Anti-Fungal Ciclopirox Olamine Displays Preclinical Activity in Leukemia and Myeloma.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 944-944
Author(s):  
Yanina Eberhard ◽  
Amudha Venugopal ◽  
Marcela Gronda ◽  
Rose Hurren ◽  
Alessandro Datti ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Ribonucleotide Reductase ◽  
Leukemia Cells ◽  
Intracellular Iron ◽  
Anti Cancer ◽  
Chemical Screen ◽  
Anti Fungal ◽  
Cancer Activity

Abstract Off-patent drugs with previously unrecognized anti-cancer activity could be rapidly repurposed for this new indication given their prior testing for safety and toxicity. To identify such compounds, we developed, automated and conducted a cell-based chemical screen of 4800 off-patent drugs and chemicals. From this chemical screen, we identified the off-patent antimicrobial, ciclopirox olamine (CPX) that is currently used for the topical treatment of cutaneous fungal infections, but has not been previously evaluated as a systemic agent for the treatment of malignancy. As an anti-fungal agent, the mechanism of action of CPX is not well understood, but appears related to binding intracellular iron and inhibiting iron containing enzymes. To explore its efficacy and mechanism of action as an anticancer agent, leukemia, myeloma, and solid tumor cell lines were treated with increasing concentrations of CPX. 72 hours after incubation, cell viability was measured by the MTS assay. CPX decreased cell viability in 5/9 leukemia, 3/6 myeloma, and 3/5 solid tumor cells with an LD50 < 5 uM, a concentration that is pharmacologically achievable based on prior animal studies investigating CPX as an anti-fungal. Cell death was confirmed by the presence of a subG1 peak by flow cytometry after staining cells with propidium iodide. In contrast, CPX was less toxic to MRC 5, LF1, and GMO 5757 non malignant fibroblasts with an LD50 > 20 uM. Next, we evaluated CPX in combination with cytarabine and daunorubicin, standard chemotherapeutic agents used in the treatment of AML. In AML cell lines, CPX synergistically enhanced the cytotoxicity of cytarabine as determined by the median effect isobologram analysis. Specifically, the combination indices (CI) at the EC50, 75 and 90 were 0.18, 0.19, and 0.24, respectively, where a CI < 1 denotes synergy. In contrast, the addition of CPX to daunorubicin produced only additive effects. Given the effects in leukemia cells lines, we evaluated the effects of oral CPX in 3 mouse models of leukemia. Sublethally irradiated NOD-SCID mice were injected subcutaneously with OCI-AML2 or K562 human leukemia cells or intraperitoneally with MDAY-D2 murine leukemia cells. After tumor implantation, mice were treated with CPX (25mg/kg) in water or water alone by oral gavage. Oral CPX decreased tumor weight and volume in all 3 mouse models by up to 65% compared to control without evidence of weight loss or gross organ toxicity. Mechanistically, CPX arrested cells in the G1/S phase of the cell cycle and downregulated the expression of survivin, Cyclin D1, and the transcription factors YY1 and FTII-D prior to the onset of cell death. Consistent with effects as an anti-fungal, CPX bound intracellular iron in the malignant cells and its ability to bind intracellular iron was functionally important for its cytotoxicity. In contrast to CPX, deferoxamine, a more avid extracellular iron chelator, was not significantly cytotoxic with an IC25 > 10uM. The highest demand for intracellular iron occurs during the late G1 and S phases due, in part, to the activity of the iron-requiring enzyme ribonucleotide reductase. Therefore, we examined the effects of CPX on the activity of ribonucleotide reductase. By electron paramagnetic resonance (EPR), CPX inhibited ribonucleotide reductase at concentrations associated with cell death. Cell lines resistant to CPX-mediated inhibition of ribonucleotide reductase were also resistant to CPX-induced cell death, supporting a mechanism of action linked to ribonucleotide reductase. Thus, in summary, the off-patent anti-fungal agent CPX induces cell death through its ability to bind intracellular iron. Its ability to inhibit the iron-containing enzyme ribonucleotide reductase appears functionally important for its mechanism of action. CPX displays previously unrecognized anti-cancer activity at concentrations that are pharmacologically achievable. Thus, CPX could be rapidly repurposed for the treatment of malignancies including leukemia and myeloma.

scholarly journals Signatures of cell death and proliferation in perturbation transcriptomics data—from confounding factor to effective prediction

2019 ◽  
Vol 47 (19) ◽  
pp. 10010-10026 ◽  
Author(s):  
Bence Szalai ◽  
Vigneshwari Subramanian ◽  
Christian H Holland ◽  
Róbert Alföldi ◽  
László G Puskás ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Cell Viability ◽  
Drug Sensitivity ◽  
Integrated Analysis ◽  
Expression Data ◽  
Conventional Drug ◽  
Transcriptomics Data ◽  
New Gene ◽  
Cellular Phenotypes

Abstract Transcriptional perturbation signatures are valuable data sources for functional genomics. Linking perturbation signatures to screenings opens the possibility to model cellular phenotypes from expression data and to identify efficacious drugs. We linked perturbation transcriptomics data from the LINCS-L1000 project with cell viability information upon genetic (Achilles project) and chemical (CTRP screen) perturbations yielding more than 90 000 signature–viability pairs. An integrated analysis showed that the cell viability signature is a major factor underlying perturbation signatures. The signature is linked to transcription factors regulating cell death, proliferation and division time. We used the cell viability–signature relationship to predict viability from transcriptomics signatures, and identified and validated compounds that induce cell death in tumor cell lines. We showed that cellular toxicity can lead to unexpected similarity of signatures, confounding mechanism of action discovery. Consensus compound signatures predicted cell-specific drug sensitivity, even if the signature is not measured in the same cell line, and outperformed conventional drug-specific features. Our results can help in understanding mechanisms behind cell death and removing confounding factors of transcriptomic perturbation screens. To interactively browse our results and predict cell viability in new gene expression samples, we developed CEVIChE (CEll VIability Calculator from gene Expression; https://saezlab.shinyapps.io/ceviche/).

scholarly journals OTS167 blocks FLT3 translation and synergizes with FLT3 inhibitors in FLT3 mutant acute myeloid leukemia

2021 ◽  
Vol 11 (3) ◽  
Author(s):  
Bartholomew J. Eisfelder ◽  
Caner Saygin ◽  
Joseph Wynne ◽  
Margaret W. Colton ◽  
Mariafausta Fischietti ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Mutant Cell ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Driver Mutations ◽  
Acute Myeloid

AbstractInternal tandem duplication (-ITD) mutations of Fms-like tyrosine kinase 3 (FLT3) provide growth and pro-survival signals in the context of established driver mutations in FLT3 mutant acute myeloid leukemia (AML). Maternal embryonic leucine zipper kinase (MELK) is an aberrantly expressed gene identified as a target in AML. The MELK inhibitor OTS167 induces cell death in AML including cells with FLT3 mutations, yet the role of MELK and mechanisms of OTS167 function are not understood. OTS167 alone or in combination with tyrosine kinase inhibitors (TKIs) were used to investigate the effect of OTS167 on FLT3 signaling and expression in human FLT3 mutant AML cell lines and primary cells. We describe a mechanism whereby OTS167 blocks FLT3 expression by blocking FLT3 translation and inhibiting phosphorylation of eukaryotic initiation factor 4E–binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4B (eIF4B). OTS167 in combination with TKIs results in synergistic induction of FLT3 mutant cell death in FLT3 mutant cell lines and prolonged survival in a FLT3 mutant AML xenograft mouse model. Our findings suggest signaling through MELK is necessary for the translation and expression of FLT3-ITD, and blocking MELK with OTS167 represents a viable therapeutic strategy for patients with FLT3 mutant AML.

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