Journal: bioRxiv

Article Title: A novel pan class-I glucose transporter inhibitor DRB18 exhibits synergistic effects in vitro and in vivo with paclitaxel against human non-small cell lung cancer

doi: 10.1101/2024.12.15.628558

Figure Lengend Snippet: 2 μM of DRB18 combined with or without other drugs to treat A549 cells for 48 hours. The inhibition of cell proliferation was compared to single compound treatment to identify anti-proliferation synergism. 4A. DRB18 combined with 10 µM V9302 showed more inhibition against A549 cells. 4B . DRB18 combined with 100 nM or 300 nM of CB839 showed more cytotoxicity against A549. 4C . DRB18 combined with 5 µM or 10 µM of sunitinib (Sutent) showed more inhibition against A549 cells. 4D . DRB18 combined with 10 µM V9302 showed more inhibition against Panc1 cells. 4E . DRB18 combined with 100 nM or 300 nM of CB839 showed more inhibition against Panc1 cells. 4F . DRB18 combined with 5 µM or 10 µM of sunitinib showed more inhibition against Panc1 cells. 4G . 5 µM of DRB18 combined with 0.75 µM of brigatinib showed more cytotoxicity against A549 cells. 4H . 5 µM of DRB18 combined with 15 µM of trametinib showed more inhibition against A549 cells.

Article Snippet: V9302 (HY-112683) and CB839 (HY-12248) were purchased from MedChemExpress.

Techniques: Inhibition

Journal: Nature Genetics

Article Title: Organismal metabolism regulates the expansion of oncogenic PIK3CA mutant clones in normal esophagus

doi: 10.1038/s41588-024-01891-8

Figure Lengend Snippet: a-b , Proportion of WT-RYFP cells in mixed cultures with Pik3ca H1047R/wt cells, after 15 days in minimal media normalized to baseline value. +/–Gluc indicates culture with/without glucose and 2-DG indicates culture with 5 mM 2-deoxyglucose. Each dot is a culture from a different animal, lines are mean values. n = 4 cultures per condition. c-e , In vitro cell competition assay. c , Protocol: Confluent mixed cultures of WT-RYFP and induced Pik3ca H1047R/wt cells with/without DCA 25 mM for 28 days in minimal medium. d , Representative confocal images of basal cell layer of cultures from c , WT-RYFP cells, yellow, DAPI, blue. Scale bar, 20 μm. e , Flow cytometric analysis from mixed cultures in c . Each dot is a culture from a different mouse and lines connect mean values (n = 7-15 cultures). Two-tailed paired t -test. f , Protocol: Cre-RYFP control and Cre-Pik3ca H1047R-YFP/wt mice were induced and treated with DCA. Clones with >1 basal cell sizes were analyzed at 28 days. Mice and clone numbers are shown. g , Heatmaps showing the differences between each treatment versus control in Cre-RYFP (upper panels) or Cre-Pik3ca H1047R-YFP/wt (lower panels) mice from f . Two-tailed 2D Kolmogorov-Smirnov test. h-i , Average basal clone sizes h and proportion of first suprabasal layer cells i for each strain and treatment from f . Data includes clones with at least one basal cell. Bars are SD. Two-tailed unpaired t -test. j , Wild-type and Pik3ca H1047R mutant basal cell clone size distributions from untreated or DCA-treated Cre-RYFP and Cre-Pik3ca H1047R-YFP/wt mice, respectively, 28 days post- induction. n = 311-799 clones from 5-8 animals per condition. Dots indicate mean and lines standard deviation. Two-tailed Kolmogorov-Smirnov test and Contrast ART-C Post-hoc test of differences of differences between distributions. k . Proportion of WT-RYFP cells in mixed cultures with Pik3ca H1047R/wt cells, at 15 days normalized to baseline value. Cells were culture in minimal media (CTL) or with Betulin (6 µg/ml), CB839 (10 µM), TOFA (30 µM) or TVB-2640 (0.1 µM). Each dot represents a culture from a different animal, lines correspond to mean values. n = 4 cultures from individual animals per condition. Two tailed paired t -test.

Article Snippet: Where indicated, mixed cultures were treated with 2 ng ml −1 or 5 μg ml −1 insulin (Sigma-Aldrich I5500), 10 ng ml −1 EGF (PeproTech EC Ltd 100-15), 10 μM PX478 (Cambridge Bioscience 10005189), 0.5 μM LY294002 (Selleckchem S1105), 5 mM 2-deoxyglucose (Sigma-Aldrich D8375), 500 nM rapamycin (Sigma-Aldrich R0395), 6 μg ml −1 betulin (Merck B9757), 10 μM CB839 (Tocris 7591), 30 μM 5-tetradecyloxy-2-furoic acid (TOFA) (Santa Cruz sc-200653), 0.1 μM TVB2640 (Selleckchem S9714), 2.5 mM metformin (Sigma-Aldrich 317240) or 25 mM DCA (Sigma-Aldrich 347795).

Techniques: In Vitro, Competitive Binding Assay, Two Tailed Test, Control, Clone Assay, Mutagenesis, Standard Deviation

Journal: Cancer research

Article Title: Undermining glutaminolysis bolsters chemotherapy while NRF2 promotes chemoresistance in KRAS-driven pancreatic cancers

doi: 10.1158/0008-5472.CAN-19-1363

Figure Lengend Snippet: A, Survival of PANC-1 cells pre-treated with CM or -Q for 72 hours followed by 36-hour gemcitabine (2μM) treatment as indicated. Values were normalized to cell survival in CM-treated condition as control, which were given a value of 100%. B, PANC-1 cells were plated and shifted to CM or media lacking Q. Cells were additionally treated with a combination of (5mM) DMKG and (1X) NEAA mixture for 72 hours following glutamine withdrawal where indicated. Gemcitabine (2μM, 36 hours) was added for respective samples, and the percent of non-viable cells was calculated using the trypan blue dye exclusion assay. C, SU.86.86 cells were plated and treated with no glutamine media as in A. Cells were treated with 0.2μM gemcitabine for the last 24 hours as indicated, and the percent of non-viable cells was calculated. D, One day after plating, HPNE-hTERT cells were shifted to CM or medium lacking Q for 48 hours then treated with or without (2μM) gemcitabine for an additional 24 hours in the presence of pretreatment as indicated. The percent of non-viable cells was determined. E, PANC-1 cells were plated in CM overnight, after which cells were treated with 10μM CB-839 or 10μM BPTES for 72 hours where indicated. An additional treatment of 2μM capecitabine, 2μM gemcitabine, or 10μM 5-FU was given for 36 hours as indicated. The percent of non-viable cells was determined. F, PANC-1 cells were treated with RSL3 (10ηM) and/or gemcitabine (2μM) for 24 hours. The percent of non-viable cells was determined as in B. A-F, Error bars represent the SEM for four independent experiments. G, Experimental design for H-P. H-O, Mice bearing tumors of KPC (H-I), MIA PaCa-2 (J-K), SU.86.86 (L-M) and PANC-1 cells (N-O) were administered drug schedules as indicated in the schematic representation G. Tumor volumes (H, J, L, N) and body weights of mice (I, K, M, O) were shown with error bars representing SEM for each group. Threshold for p-value was <0.05. P, Survival of PANC-1 tumor-bearing mice represented as a Kaplan-Meier plot. H-P, Statistical differences between treatment groups were determined with a critical p value of <0.05. Synergistic effect of CB-839+Gemcitabine is evident by comparing tumor volumes (H,J,L,N) with single compound treated groups with statistical significance whereas insignificant body weight changes has been documented (I,K,M,O) for either of four treatment groups in all cases.

Article Snippet: CB-839 (#B0084–462472) was purchased from BOC Sciences.

Techniques: Exclusion Assay

Journal: Cancer research

Article Title: Undermining glutaminolysis bolsters chemotherapy while NRF2 promotes chemoresistance in KRAS-driven pancreatic cancers

doi: 10.1158/0008-5472.CAN-19-1363

Figure Lengend Snippet: Model illustrates reprogrammed metabolic pathways in RAS-driven PDAC cells linked to KRAS mutation and NRF2 activation, which drive glutaminolysis. Mutant KRAS-mediated NRF2 activation leads to chemoresistance by regulating antioxidant genes. Modulation of nucleotide synthesis by NRF2 also supports PDAC cell growth. Anaplerotic glutamine utilization is a key feature of KRAS-driven PDAC cells. KRAS-regulated glutamine metabolic rewiring influenced the TCA cycle, which is critical for nucleotide and DNA synthesis to support cell growth and survival (12,29). CB-839 inhibits GLS, whereas gemcitabine blocks DNA synthesis. This model potentiates CB-839 treatment along with gemcitabine as a therapeutic strategy to combat chemoresistance in KRAS-driven pancreatic cancers.

Article Snippet: CB-839 (#B0084–462472) was purchased from BOC Sciences.

Techniques: Mutagenesis, Activation Assay, DNA Synthesis

Journal: Cells

Article Title: Glutaminase Inhibition on NSCLC Depends on Extracellular Alanine Exploitation

doi: 10.3390/cells9081766

Figure Lengend Snippet: ( A ) Dose–response curves of the NSCLC cell lines panel treated with increasing concentrations of CB-839. The response to the drug was assessed 72 h from the start of treatment with the MTS assay. The average of three independent experiments is reported. ( B ) Dose–response curves of the NSCLC LU99 and H358 LKB1 isogenic systems treated with increasing concentrations of CB-839. The response to the drug was assessed 72 h from the start of treatment with the MTS assay. The average of three independent experiments is reported. ( C ) GLS1 RNAseq gene expression data retrieved from the CCLE , in ten NSCLC cell lines. ( D ) Western Blot analysis of GLS1 protein levels in the ten NSCLC cell lines used. Ran was used as loading control. The figure is representative of at least three independent experiments. ( E ) Fold change in abundance (normalized peak area) of extracellular glucose uptake and lactate release in NSCLC CB-839 treated vs. untreated cells (500 nM CB-839, 6 h treatment). Mean ± SD of triplicate culture/conditions.

Article Snippet: CB-839 (TargetMol, Wellesley Hills, MA, USA) DMSO stock solutions (10 mM) were dissolved in medium just before use. l -cycloserine (Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in sterile water and diluted in medium just before use.

Techniques: MTS Assay, RNA sequencing, Gene Expression, Western Blot, Control

Journal: Cells

Article Title: Glutaminase Inhibition on NSCLC Depends on Extracellular Alanine Exploitation

doi: 10.3390/cells9081766

Figure Lengend Snippet: ( A ) Schematic representation of the 13-C glutamine labelling experiment. ( B ) M + 0 and M + 5 glutamine and glutamate abundance in two sensitive and two resistant H1299-derived clones with or without CB-839 (500 nM, 24 h treatment). ( C ) Isotope labelling of the TCA cycle in the presence of 13C 5 -glutamine with or without CB-839 (500 nM, 24 h treatment) in two sensitive (S1, KRASwt/LKB1wt, S2, KRASmut/LKB1wt) and two resistant (R1, KRASwt/LKB1del, R2, KRASmut/LKB1del) H1299-derived clones (normalized peak area). ( D ) Schematic representation of intracellular pyruvate metabolism. ( E ) Intracellular lactate levels (normalized peak area) with or without CB-839 (500 nM, 24 h treatment) in two sensitive and two resistant H1299-derived clones. ( F ) Intracellular alanine levels (normalized peak area) with or without CB-839 (500 nM, 24 h treatment) in two sensitive and two resistant H1299-derived clones. ( G ) Normalized fold change of extracellular alanine levels vs. unconditioned RPMI-1640 with or without CB-839 (500 nM, 24 h treatment) in two sensitive and two resistant H1299-derived clones. ( H ) Schematic representation of fatty acid biosynthesis. ( I ) Intracellular levels of fatty acids with or without CB-839 (500 nM, 24 h treatment) in two sensitive and two resistant H1299-derived clones. Mean ± SD of triplicate culture/conditions. Statistical significance * p < 0.05 untreated vs CB-839 treated cells, # p < 0.05 untreated sensitive vs untreated resistant cells, Wilcoxon Mann–Whitney test (M + 0, M + 2, M + 3, M + 4, M + 5).

Article Snippet: CB-839 (TargetMol, Wellesley Hills, MA, USA) DMSO stock solutions (10 mM) were dissolved in medium just before use. l -cycloserine (Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in sterile water and diluted in medium just before use.

Techniques: Derivative Assay, Clone Assay, MANN-WHITNEY

Journal: Cells

Article Title: Glutaminase Inhibition on NSCLC Depends on Extracellular Alanine Exploitation

doi: 10.3390/cells9081766

Figure Lengend Snippet: ( A ) Changes in extracellular alanine levels in the panel of ten NSCLC cell lines, CB-839-treated vs. untreated, cells (500 nM, 24 h treatment). ( B ) Unlabeled and 13C 3 -labelled alanine incorporation (M + 0, M + 3) in two sensitive (LU99, A549) and two resistant (H358, H520) cells, either untreated or CB-839 treated (500 nM, 24 h treatment). ( C ) The 13C 3 -alanine-derived carbon labelling (M + 2, M + 3, normalized peak area) of TCA cycle intermediates and citrate, malate, glutamate and glutathione derived metabolites in CB-839 treated sensitive and resistant cells. ( D ) GPT2 mRNA expression as RNA-seq, retrieved from the CCLE in the sensitive and resistant cells. ( E ) Western blot analyses of GPT2 expression in the NSCLC cell line panel. Ran was used as loading control. ( F ) Pearson correlation between alanine uptake from each cell line’s medium and GPT2 mRNA expression, retrieved from the CCLE. The average of three independent experiments is reported. Statistical significance # p < 0.05 refer to significant differences (one-way ANOVA and Tukey Kramer test) of M + 2 and M + 3 in the resistant vs. sensitive cells.

Article Snippet: CB-839 (TargetMol, Wellesley Hills, MA, USA) DMSO stock solutions (10 mM) were dissolved in medium just before use. l -cycloserine (Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in sterile water and diluted in medium just before use.

Techniques: Derivative Assay, Expressing, RNA Sequencing, Western Blot, Control

Journal: Cells

Article Title: Glutaminase Inhibition on NSCLC Depends on Extracellular Alanine Exploitation

doi: 10.3390/cells9081766

Figure Lengend Snippet: ( A ) Schematic representation of metabolic strategy to overcome CB-839 resistance, targeting GPT2 by l -cycloserine (cyclo). ( B ) Dose–response curves of H1975 (left panel) and H520 (right panel) cells treated with increasing concentrations of CB-839, alone (CB-839) or with l -cycloserine (CB + cyclo) at the reported concentrations. The response to the drugs was assessed with the MTS assay 72 h from the treatment start. ( C ) Schematic representation of metabolic strategy to induce CB-839 resistance in the sensitive cell lines by pyruvate supplementation, with inhibition of GLS1, alone or together with GPT2. ( D ) Dose–response curves of LU99 (left panel) and A549 (right panel) cells treated with increasing concentrations of CB-839, grown and treated in standard (CB-839) or pyruvate-enriched medium (CB-839 Pyr). The response to the drug was assessed with the MTS assay 72 h from treatment start. ( E ) Histograms of LU99 and A549 cells grown in standard or pyruvate-enriched medium (Pyr) and treated with CB-839 (CB, 12 nM for LU99 and 111 nM for A549) and l -cycloserine (cyclo, 75 µM for LU99 and 100 µM for A549), either alone or in combination. The response to the drug was assessed 72 h from the start of treatment with the MTS assay. ( F ) Histograms of H520 and H1975 cells grown in standard or pyruvate-enriched medium (Pyr) and treated with CB-839 (CB, 1000 nM) and l -cycloserine (cyclo), 250 µM for H520 and 500 µM for H1975), either alone or in combination. The response to the drug was assessed with the MTS assay 72 h from treatment start. The average of three independent experiments is reported. For the sake of clarity, the statistical significance one-way ANOVA and Bonferroni post-test for multiple comparisons (panel B , D , E , F ) are reported as , respectively).

Article Snippet: CB-839 (TargetMol, Wellesley Hills, MA, USA) DMSO stock solutions (10 mM) were dissolved in medium just before use. l -cycloserine (Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in sterile water and diluted in medium just before use.

Techniques: MTS Assay, Inhibition