Journal: Cancer research

Article Title: Activation of PP2A and inhibition of mTOR synergistically reduce MYC signaling and decrease tumor growth in pancreatic ductal adenocarcinoma

doi: 10.1158/0008-5472.CAN-18-0717

Figure Lengend Snippet: PP2A activation increases the cytotoxic effects of select kinase inhibitors. A. CI values at effective dose (ED) 75 for pancreatic cells lines treated with increasing concentrations of OP449 and select kinase inhibitors. Mean +/− SEM of biological replicates. B. CI values at ED75 of ASPC1, MIAPACA2, and PANC89 treated with INK128, PP242, BKM120, or GDC0068. Values for each cell line were generated from 2 (MIAPACA2- GDC0068) or 3 biological reps. Grand mean of all lines for each drug shown. C. PDA cell lines were treated with increasing doses of DT1154 (left) or INK128 (right) for 72 hours. Shown is the viability relative to Vehicle for each line. MIAPACA2 (MPC2) D. CellTox Green was added to HPAFII and PANC89 cells and then cells were treated with increasing concentrations of DT1154 and INK128. Fluorescent cells were imaged every 2 hours for 72 hours on an Incucyte Zoom. Shown is the area under the curve (AUC) generated from the green object count (1/mm2) over time relative to Vehicle control. Graph represents average of three biological replicates. ***P <0.001 by a 2-way ANOVA. E. HPAFII and CFPAC1 cells were plated in a soft agar colony assay and treated with Vehicle, 500nM DT1154, 10nM INK128, or combination. Colony number was quantified using ImageJ. Mean +/− SEM of three biological replicates. *p<0.05 by a two-tailed students t-test.

Article Snippet: Rappta Therapeutics, Inc. (established September 7 th , 2017) has licensed this intellectual property on 12/8/2017 for the clinical and commercial development of this series of small molecule PP2A activators.

Techniques: Activation Assay, Generated, CellTox Assay, Control, Colony Assay, Two Tailed Test

Journal: Cancer research

Article Title: Activation of PP2A and inhibition of mTOR synergistically reduce MYC signaling and decrease tumor growth in pancreatic ductal adenocarcinoma

doi: 10.1158/0008-5472.CAN-18-0717

Figure Lengend Snippet: PP2A activation suppresses MYC mediated resistance to mTOR inhibition A and B. PANC89, HPAFII, MIAPACA2 (MPC2), and PANC1 cells were treated with Vehicle (V), DT1154 (D; 10μM), INK128 (I; 0.5μM), or the combination (C) for 6 hours and lysates were probed by western blot. Representative blot (A) and quantification over GAPDH (B) of three biological replicates shown. Mean +/− SEM C. PANC89 and PANC1 cells were treated as in Figure 3A. Total MYC was immunoprecipitated and lysates were probed for phosphoS62 MYC (pS62). Inputs were probed for total MYC and GAPDH *denotes IgG heavy chain. Quantification of pS62 levels over GAPDH relative to Vehicle shown D. qPCR of MYC, NCL (Nucleolin), and E2F2 from PANC89 cells treated as in Figure 3A. Mean +/− SEM of three biological replicates. E. PANC89 cells transfected with non-targeting siRNA (siNT) or a B56α targeting siRNA pool (siB56α) were treated with increasing concentrations of INK128. Viability was assessed by MTS 72 hours after drug treatment. Mean +/− SEM of three biological replicates. F. Cells from panel E were treated with either Vehicle or 0.5μM INK128 for 6 hours. Lysates were analyzed by western blot for MYC, pAKT (S473), pS6 (S235/6) and totals. Quantification from three biological replicates is shown. Mean +/− SD. G. PANC89 cells transfected with AdGFP (GFP) or AdMYCT58A (T58A) were treated with increasing concentrations of INK128. Viability was assessed by MTS 72 hours after drug treatment. Mean +/− SEM of three biological replicates. H. Cells from panel G were treated with either Vehicle or 0.5μM INK128 for 6 hours. Lysates were analyzed by western blot as above. Quantification from three biological replicates is shown. Mean +/−SD. For all panels ***p<0.001 **p<0.01 *p<0.05 by two-tailed students t-test; #, & denotes pS6 or pAKT levels, respectively, are significantly different in INK128 conditions compared to Vehicle.

Article Snippet: Rappta Therapeutics, Inc. (established September 7 th , 2017) has licensed this intellectual property on 12/8/2017 for the clinical and commercial development of this series of small molecule PP2A activators.

Techniques: Activation Assay, Inhibition, Western Blot, Immunoprecipitation, Transfection, Two Tailed Test

Journal: Cancer research

Article Title: Activation of PP2A and inhibition of mTOR synergistically reduce MYC signaling and decrease tumor growth in pancreatic ductal adenocarcinoma

doi: 10.1158/0008-5472.CAN-18-0717

Figure Lengend Snippet: PP2A activation combined with mTOR inhibition decreases tumorigenic properties in vitro and in vivo A. PANC89 were grown subcutaneously and mice were treated with Vehicle, DT1154 (15mg/kg), INK128 (0.5 mg/kg), or combination. Tumors were calipered and tumor volume was plotted across time. Mean +/− SEM shown. n= 6 Vehicle, 8 DT1154, 6 INK128, and 9 combination treated tumors. Area under the curve was analyzed for each treatment arm, *p<0.05 by a 1-way ANOVA B. Quantification of end point tumor size from panel B. Mean +/− SEM shown. C. Quantification of necrotic tumor area from H/E images using ImageJ. Mean +/− SEM shown. D. PANC89 xenograft tumors were grown as in panel A and harvested after 7 days of treatment with Vehicle (Veh), DT1154 (DT, 15mg/kg), INK128 (INK, 0.5 mg/kg), or combination (Combo). Tissues sections were stained with the ApopTag Plus Peroxidase In Situ Kit and the number of TUNEL positive cells was quantified per high-powered field (HPF, 20x). ***p<0.001 *p<0.05 by a 1-way ANOVA. E. Lysates from tumors grown in panel D were analyzed by western blot for MYC, pAKT (S473), pS6 (S235/6), 4EBP1, and totals. F. Quantification of 3 tumors in each treatment group from panel E shown. Mean +/−SEM. **p<0.01 *p<0.05 by two-tailed students t-test

Article Snippet: Rappta Therapeutics, Inc. (established September 7 th , 2017) has licensed this intellectual property on 12/8/2017 for the clinical and commercial development of this series of small molecule PP2A activators.

Techniques: Activation Assay, Inhibition, In Vitro, In Vivo, Staining, In Situ, TUNEL Assay, Western Blot, Two Tailed Test

Journal: Cancer research

Article Title: Activation of PP2A and inhibition of mTOR synergistically reduce MYC signaling and decrease tumor growth in pancreatic ductal adenocarcinoma

doi: 10.1158/0008-5472.CAN-18-0717

Figure Lengend Snippet: PP2A activation combined with mTOR inhibition as a therapeutic strategy to reduce PDA survival. A. Oncogenic KRAS drives the activation of both the MYC and PI3K/AKT/mTOR pathways. Upon mTOR inhibition, PDA cells capitalize on low PP2A levels, signaling through MYC for survival, creating a MYCHigh/mTORLow cell state. B. Activation of PP2A combined with mTOR inhibition results in low MYC and mTOR signaling in a MYCLow/mTORLow cell state, decreasing PDA cell survival.

Article Snippet: Rappta Therapeutics, Inc. (established September 7 th , 2017) has licensed this intellectual property on 12/8/2017 for the clinical and commercial development of this series of small molecule PP2A activators.

Techniques: Activation Assay, Inhibition