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