Journal: Molecular cancer therapeutics

Article Title: The Existence of MTH1-independent 8-oxodGTPase Activity in Cancer Cells as a Compensatory Mechanism against On-target Effects of MTH1 Inhibitors

doi: 10.1158/1535-7163.MCT-19-0437

Figure Lengend Snippet: The ARGO probe–based assay is an accurate measure of MTH1-specific 8-oxodGTPase activity. A, Schematic showing how cleavage of the chimeric ARGO probe (with 8-oxodG and A base moieties) by MTH1 8-oxodGTPase activity generates ATP that can then be detected via a luciferase-based luminescent signal. B, ARGO probe–generated luminescence (in relative luminescence units, RLU) from lysates made from stable MTH1-ovexpressing BEAS2B cells or their empty vector (EV)-transduced counterparts. Each sample was run in triplicate and data are representative of a minimum of n = 2.Arepresentative Western blot run on 10 μg total protein lysates from these cell lines, with the MTH1 signal and GAPDH loading control, is shown to the right of the graph. Error bars, ± SD. C, The percent decreases in ARGO probe-generated luminescence in U2OS and H23 lysates by the three indicated MTH1 inhibitors. The log-transformed drug concentration range is indicated on the x-axis. Each concentration was run in triplicate, and the signal at each concentration is normalized to signal from the counterpart vehicle-treated lysates. Error bars, ± SD. D, Comparison of effects on ARGO probe–generated luminescence produced by shRNA-mediated MTH1 depletion versus the first-in-class MTH1 inhibitors. Data are representative of a minimum of n = 2, with each sample run in triplicate. Western blotting on 10 μg of total protein from A549 lysates and 20 mg total protein from H23 lysates, with the MTH1 signal and GAPDH or tubulin as loading controls, is shown to the right of the graph, to establish levels of MTH1 protein knockdown.

Article Snippet: Blots were washed in 0.1% TBST and then probed with antibodies against the following proteins: MTH1 (1:2,000, Novus Biologicals 100–109), actin (1:2,000, Sigma A2066), p53 (1:1,000, Santa Cruz Biotechnology, SC126), cleaved PARP (1:1,000, Cell Signaling Technology, 9541), catalase (1:2,000, Abcam, ab16731), SOD2 (1:1,000, Enzo Life Sciences, ADI-SOD-110), GAPDH (1:20,000, Abcam, ab9485), γ-tubulin (1:10,000, Sigma T6557).

Techniques: Activity Assay, Luciferase, Generated, Plasmid Preparation, Western Blot, Control, Transformation Assay, Concentration Assay, Comparison, Produced, shRNA, Knockdown

Journal: Molecular cancer therapeutics

Article Title: The Existence of MTH1-independent 8-oxodGTPase Activity in Cancer Cells as a Compensatory Mechanism against On-target Effects of MTH1 Inhibitors

doi: 10.1158/1535-7163.MCT-19-0437

Figure Lengend Snippet: Five different MTH1 inhibitors, regardless of reported cytotoxicity, similarly inhibit 8-oxodGTPase activity across a panel of cancer cell lines, revealing a “nondruggable” activity component. A, Levelsof total cellular 8-oxodGTPase activity in the indicated panel of cancer cell lines. Results are derived from two independent experiments (n = 2) with each sample run in triplicate. Error bars, ± SD. B, Levels of MTH1-specific 8-oxodGTPase activity in the panel of cancer cell lines from A. The MTH1-specific values were generated by subtracting the average of triplicate RLU values from each of the three inhibitors shown in A from the RLU value i nthe vehicle counterpart and averaged across the different inhibitors used. Results are derived from two independent experiments (n = 2) with each sample run in triplicate. Error bars, ± SD. C, Left, total cellular 8-oxodGTPase activity under vehicle and different MTH1 inhibitors in A549 and H358 cells. Baseline (DMSO) luminescence values are shown next to the RLU values obtained following lysate treatment with either TH287, IACS-4759, or AZ-21 inhibitors for each cell line. Results are derived from two independent experiments (n = 2) with each sample run in triplicate. Right, percentage change in ARGO assay–generated luminescence (relative to vehicle-treated control) in MTH1-depleted A549 cell lysates, following treatment with the three indicated MTH1 inhibitors. Note that AZ-21 suppresses activity similarly to the other two inhibitors and none of the three treatments produce any significant difference in 8-oxodGTPase activity relative to shMTH1 lysates. Each sample was run in triplicate. Error bars, ± SD. D, Comparison of how total 8-oxodGTPase activity in H23 shGFP versus shMTH1 cells is affected by the reportedly cytotoxic inhibitor TH287 versus the noncytotoxic inhibitor BAY-707. Each sample was run in triplicate. Error bars, ± SD. E, The percentage decreases in ARGO probe–generated luminescence in H1563 lysates by the three indicated MTH1 inhibitors. The log-transformed drug concentration range is indicated on the x-axis. Each concentration was run in triplicate, and the luminescent signal at each concentration is normalized to the signal from its counterpart vehicle-treated sample. Error bars, ± SD. F, Comparison of how total 8-oxodGTPase activity in H1563 cells is affected by shRNA-mediated MTH1 depletion versus the three indicated MTH1 inhibitors, run in triplicate. Error bars, ± SD. Western blotting on 10 μg of total protein lysates confirming MTH1 knockdown in the shMTH1 samples, with GAPDH as the loading control, is shown to the left of the 8-oxodGTPase activity graph.

Article Snippet: Blots were washed in 0.1% TBST and then probed with antibodies against the following proteins: MTH1 (1:2,000, Novus Biologicals 100–109), actin (1:2,000, Sigma A2066), p53 (1:1,000, Santa Cruz Biotechnology, SC126), cleaved PARP (1:1,000, Cell Signaling Technology, 9541), catalase (1:2,000, Abcam, ab16731), SOD2 (1:1,000, Enzo Life Sciences, ADI-SOD-110), GAPDH (1:20,000, Abcam, ab9485), γ-tubulin (1:10,000, Sigma T6557).

Techniques: Activity Assay, Derivative Assay, Generated, Control, Comparison, Transformation Assay, Concentration Assay, shRNA, Western Blot, Knockdown

Journal: Molecular cancer therapeutics

Article Title: The Existence of MTH1-independent 8-oxodGTPase Activity in Cancer Cells as a Compensatory Mechanism against On-target Effects of MTH1 Inhibitors

doi: 10.1158/1535-7163.MCT-19-0437

Figure Lengend Snippet: MTH2 can generate ARGO cleavage–induced luminescence. A, Comparison of ARGO cleavage–generated luminescence in response to increasing concentrations of human recombinant MTH1 and MTH2, up to 5 nmol/L. Each concentration was run in triplicate. Error bars, ± SD. B, Comparison of ARGO cleavage–generated luminescence in response to increasing concentrations of human recombinant MTH1 and MTH2 (up to 1 μM/L). Each concentration was run in triplicate. The boxed lines indicate the concentrations of MTH1 versus MTH2 at which equivalent ARGO cleavage–induced luminescence is observed. Error bars, ± SD.

Article Snippet: Blots were washed in 0.1% TBST and then probed with antibodies against the following proteins: MTH1 (1:2,000, Novus Biologicals 100–109), actin (1:2,000, Sigma A2066), p53 (1:1,000, Santa Cruz Biotechnology, SC126), cleaved PARP (1:1,000, Cell Signaling Technology, 9541), catalase (1:2,000, Abcam, ab16731), SOD2 (1:1,000, Enzo Life Sciences, ADI-SOD-110), GAPDH (1:20,000, Abcam, ab9485), γ-tubulin (1:10,000, Sigma T6557).

Techniques: Comparison, Generated, Recombinant, Concentration Assay

Journal: Molecular cancer therapeutics

Article Title: The Existence of MTH1-independent 8-oxodGTPase Activity in Cancer Cells as a Compensatory Mechanism against On-target Effects of MTH1 Inhibitors

doi: 10.1158/1535-7163.MCT-19-0437

Figure Lengend Snippet: Comparison of MTH1-specific 8-oxodGTPase activity in non–small cell lung cancer (NSCLC) human specimen with their respective MTH1 protein expression levels. A, Immunoblot showing MTH1 protein levels in matched human normal lung/lung cancer specimens derived from untreated stage I/stage II patients with NSCLC. Actin is shown as the loading control. B, Fold changes in tumor (T) versus normal (N) MTH1 protein expression. Pixel density from the immunoblot MTH1 signals from A and Supplementary Fig.2A, normalized to the loading control, were quantified via ImageJ. The horizontal line represents the normal tissue baseline set at 1 for each pair. Error bars, ±SEM. C, Total 8-oxodGTPase activity in each matched pair of NSCLC tumors. Results are derived from two independent experiments (n = 2) with each sample run in triplicate. D, Corresponding MTH1-specific 8-oxodGTPase activity, calculated by subtracting the luminescent ARGO signal from TH287-treated counterpart lysates. Results are derived from two independent experiments (n = 2) with each sample run in triplicate. Error bars, ± SD. E, Relative MTH1-specific 8-oxodGTPase activity changes in the tumor versus adjacent normal tissues is shown as a fold change between tumor (T)/normal (N) values from D. The horizontal line represents the normal tissue baseline set at 1 for each pair. Error bars, ± SD. F, Linear regression analysis comparing fold change (T/N) MTH1 protein expression against fold change (T/N) MTH1-specific 8-oxodGTPase activity in the matched normal versus tumor patient tissue. Robustness of fit and P values, calculated in GraphPad Prism, are shown.

Article Snippet: Blots were washed in 0.1% TBST and then probed with antibodies against the following proteins: MTH1 (1:2,000, Novus Biologicals 100–109), actin (1:2,000, Sigma A2066), p53 (1:1,000, Santa Cruz Biotechnology, SC126), cleaved PARP (1:1,000, Cell Signaling Technology, 9541), catalase (1:2,000, Abcam, ab16731), SOD2 (1:1,000, Enzo Life Sciences, ADI-SOD-110), GAPDH (1:20,000, Abcam, ab9485), γ-tubulin (1:10,000, Sigma T6557).

Techniques: Comparison, Activity Assay, Expressing, Western Blot, Derivative Assay, Control

Journal: Molecular cancer therapeutics

Article Title: The Existence of MTH1-independent 8-oxodGTPase Activity in Cancer Cells as a Compensatory Mechanism against On-target Effects of MTH1 Inhibitors

doi: 10.1158/1535-7163.MCT-19-0437

Figure Lengend Snippet: Relative effects on the viabilities of NSCLC cell lines produced by three different MTH1 inhibitors, TH588, TH287, or IACS-4759. A, Published chemical structures of TH287, TH588, and IACS-4759. BμF, Approximately 200–750 cells (optimized per cell line based on their proliferation rates) were plated per well in a 96-well plate. The next day, the cell lines were treated with vehicle (0.1% DMSO) or the indicated first-in-class MTH1 inhibitor for 72 hours. Viability in response to drug treatment was assessed via luminescence signal from CellTiter-Glo normalized to DMSO control. The x-axis represents the log-transformed drug treatment doses. The percent viability plotted for each data point are an average of two independent experiments, with each data point treated in triplicate. Error bars, ± SD. The IC50 values (in μmol/L) for TH588 and TH287 are shown as insets within each dose–response curve. IC50 values were generated using GraphPad Prism software as described in the Materials and Methods section. IC50 values were not determined (N.D.) for H1563 pBp treated with TH287 and TH588, for H1563 pBp KRASV12 treated with TH588 due to the viability remaining above 50%. G, The denoted cell lines were treated for 72 hours with the indicated doses of IACS-4759 and processed as in B–F. Error bars, ± SD. No IC50s could be determined for any of the cell lines. H, Comparison of cell growth following IACS versus TH287 treatment. A549 cells (approximately 150 cells per well in a 96-well plate) were treated with vehicle or the indicated drug doses in triplicate, and growth curves established via luminescence signal from CellTiter-Glo over the duration of the experiment. Vehicle or drug was replenished every 24 hours. Note that the y-axis plots raw RLU values over the 8-day time course rather than % viability normalized to a control group, to reflect overall changes in cell numbers over the 8-day treatments. Error bars, ± SD. I, Western blot analysis of MTH1 inhibitor–treated A549 cells to assess molecular markers of cell death. Cells were treated with 10 μmol/L of the indicated inhibitors for 72 hours before being harvested and lysed for total protein. Approximately 10 μg of total protein lysates from each sample was probed with p53-specific or cleaved PARP–specific antibodies. GAPDH is shown as the loading control.

Article Snippet: Blots were washed in 0.1% TBST and then probed with antibodies against the following proteins: MTH1 (1:2,000, Novus Biologicals 100–109), actin (1:2,000, Sigma A2066), p53 (1:1,000, Santa Cruz Biotechnology, SC126), cleaved PARP (1:1,000, Cell Signaling Technology, 9541), catalase (1:2,000, Abcam, ab16731), SOD2 (1:1,000, Enzo Life Sciences, ADI-SOD-110), GAPDH (1:20,000, Abcam, ab9485), γ-tubulin (1:10,000, Sigma T6557).

Techniques: Produced, Control, Transformation Assay, Generated, Software, Comparison, Western Blot

Journal: Molecular cancer therapeutics

Article Title: The Existence of MTH1-independent 8-oxodGTPase Activity in Cancer Cells as a Compensatory Mechanism against On-target Effects of MTH1 Inhibitors

doi: 10.1158/1535-7163.MCT-19-0437

Figure Lengend Snippet: The first-in-class MTH1 inhibitors produce ROS and decrease OXPHOS efficiency relative to the noncytotoxic MTH1 inhibitor, IACS-4759. A, ROS levels in untreated and MTH1 inhibitor–treated cells. H358 cells were plated at an equivalent density and treated with either DMSO or the indicated inhibitors for 17 hours. All samples were harvested via trypsinization and, following incubation with CM-H2DCF-DA, concurrently assessed for total ROS levels via signal detection in the FITC channel. The flow cytometric profiles shown are representative of two independent runs. B, Immunoblotting for antioxidant proteins in H358 cells treated with DMSO or 10 μmol/L MTH1 inhibitors for 24 hours. Approximately 10 μg of total protein lysates were immunoblotted and probed with the indicated antibodies. GAPDH is shown as a loading control. This blot is representative of two independently run blots. C, The first-in-class MTH1 inhibitor, TH287, shows indications of decreased OXPHOS efficiency. H358 cells were treated for 22 hours with the indicated MTH1 inhibitors (10 μmol/L) or vehicle (0.1% DMSO). To assess mitochondrial respiratory capacity, the treated cells were subjected to polarographic measurements of intact cell respiration. Subsequently, the cells were permeabilized with digitonin, followed by the subsequent injection of the indicated substrates, prior to measurement of respiration. Oxygen consumption rates (OCR) were calculated as specified in the Materials and Methods section (left). The respiratory control ratio (RCR, right) was calculated by dividing the KCN-sensitive CCCP OCR by the KCN-sensitive oligomycin OCR. Error bars, ± SD. D, Cells cultured at 5% oxygen tension are not protected from the cytotoxic effects of TH588 or TH287. H358 cells were cultured at 5% or 21% oxygen for 12 days prior to plating for the indicated drug treatments in triplicate. Cells were kept at their respective oxygen conditions during the 72-hour treatment period. Viability was assessed via the CellTiter-Glo assay and drug responses curves established as in Fig. 5. Error bars, ± SD.

Article Snippet: Blots were washed in 0.1% TBST and then probed with antibodies against the following proteins: MTH1 (1:2,000, Novus Biologicals 100–109), actin (1:2,000, Sigma A2066), p53 (1:1,000, Santa Cruz Biotechnology, SC126), cleaved PARP (1:1,000, Cell Signaling Technology, 9541), catalase (1:2,000, Abcam, ab16731), SOD2 (1:1,000, Enzo Life Sciences, ADI-SOD-110), GAPDH (1:20,000, Abcam, ab9485), γ-tubulin (1:10,000, Sigma T6557).

Techniques: Incubation, Western Blot, Control, Injection, Cell Culture, Glo Assay

Journal: Journal of Lipid Research

Article Title: Small molecule inducers of ABCA1 and apoE that act through indirect activation of the LXR pathway

doi: 10.1194/jlr.m081851

Figure Lengend Snippet: Figure 5: AZ-1 and AZ-2 exhibit P2X7 receptor antagonist activity. (A) Concentration dependent

Article Snippet: P2X7 loss was confirmed by western blotting using anti-P2X7 antibodies (Novus Biologicals, cat# NB100-109), anti-GAPDH 14C10 (Cell Signalling Technology, cat# 2118) and anti-rabbit HRP (Cell Signalling Technology, cat# 7074) antibodies.

Techniques: Activity Assay, Concentration Assay

Journal: Journal of Lipid Research

Article Title: Small molecule inducers of ABCA1 and apoE that act through indirect activation of the LXR pathway

doi: 10.1194/jlr.m081851

Figure Lengend Snippet: Figure 6: Structurally distinct P2X7 antagonists do not induce ABCA1 or apoE. CCF-STTG1 cells

Article Snippet: P2X7 loss was confirmed by western blotting using anti-P2X7 antibodies (Novus Biologicals, cat# NB100-109), anti-GAPDH 14C10 (Cell Signalling Technology, cat# 2118) and anti-rabbit HRP (Cell Signalling Technology, cat# 7074) antibodies.

Techniques:

Journal: Journal of Lipid Research

Article Title: Small molecule inducers of ABCA1 and apoE that act through indirect activation of the LXR pathway

doi: 10.1194/jlr.m081851

Figure Lengend Snippet: Figure 7: Induction of ABCA1 by AZ-1 and AZ-2 is P2X7 receptor independent. (A) Representative

Article Snippet: P2X7 loss was confirmed by western blotting using anti-P2X7 antibodies (Novus Biologicals, cat# NB100-109), anti-GAPDH 14C10 (Cell Signalling Technology, cat# 2118) and anti-rabbit HRP (Cell Signalling Technology, cat# 7074) antibodies.

Techniques:

Journal: Journal of Lipid Research

Article Title: Small molecule inducers of ABCA1 and apoE that act through indirect activation of the LXR pathway

doi: 10.1194/jlr.m081851

Figure Lengend Snippet: Figure 8: AZ-1 and AZ-2 induce ABCA1 in other CNS cells that lack detectable P2X7. (A, D)

Article Snippet: P2X7 loss was confirmed by western blotting using anti-P2X7 antibodies (Novus Biologicals, cat# NB100-109), anti-GAPDH 14C10 (Cell Signalling Technology, cat# 2118) and anti-rabbit HRP (Cell Signalling Technology, cat# 7074) antibodies.

Techniques:

Journal: Journal of Lipid Research

Article Title: Small molecule inducers of ABCA1 and apoE that act through indirect activation of the LXR pathway

doi: 10.1194/jlr.m081851

Figure Lengend Snippet: Figure 9: Minimal or no SREBP-1c induction by P2X7 antagonists in liver cells. (A) HepG2 cells and

Article Snippet: P2X7 loss was confirmed by western blotting using anti-P2X7 antibodies (Novus Biologicals, cat# NB100-109), anti-GAPDH 14C10 (Cell Signalling Technology, cat# 2118) and anti-rabbit HRP (Cell Signalling Technology, cat# 7074) antibodies.

Techniques: