Journal: Cancers

Article Title: mTORC1-Inhibition Potentiating Metabolic Block by Tyrosine Kinase Inhibitor Ponatinib in Multiple Myeloma

doi: 10.3390/cancers14112766

Figure Lengend Snippet: Metabolic screen of multiple myeloma cells. ( A ) Schematic for metabolic screening targeting glycolysis pathway in MM cells. ( B ) Assay for metabolic efficiency of compounds from NCI-drug library on RPMI8226 and U266 MM cells; HEK293 cells were used to determine the toxicity of the compounds. Each point represents a single compound from the library; the assay was performed in duplicate for reproducibility (Vo = vorinostat, Vi = vismodegib, Pa = palbociclib, Po = ponatinib, Bi = binimetinib). Cells were incubated with the drug compounds for 72 h for the assay. ( C ) Schematic diagram of the glycolysis pathway for the enzymatic links. ( D , E ) The top ten selected drug candidates were screened for inhibition of glycolytic genes by qPCR. The drug treatment was for 12 h on RPMI8226 cells. ( F ) The top 5 candidates were further screened for inhibition in lactate production using a lactate assay kit on RPMI8226 cells; drug treatment was for 24 h. Data are presented as mean ± SEM; t -test: *** p < 0.001. ( G ) Venn diagram shows ponatinib was the best candidate compound for glycolysis gene and lactate production inhibition. ( H ) HK2 and GPI signals were analyzed in normal and MM cells in cancer patients. The patient data were obtained from the R2 platform (hgserver1.amc.nl) (GEO ID: GSE2658). N = normal tissue; T = tumor. Data are plotted as mean with the shaded distributions; * p < 0.05, *** p < 0.001, provided by R2 platform search engine (via one-way ANOVA on ranks). ( I ) Kaplan–Meier overall survival plots of MM patients with respect to HK2 and GPI RNA message levels are shown. Significance p-values are indicated in the figure, provided by R2 platform search engine (hgserver1.amc.nl) (GEO ID: GSE2658) (via one-way ANOVA on ranks).

Article Snippet: For the blood toxicity tests, the mice were tested with intraperitoneal injection with 100 μL PBS (Gibco BRL) or ponatinib (Biosynth Carbosynth, Staad, Switzerland) (10 mg/kg) and sirolimus (Alfa Aesar, Thermo Fisher, Ward Hill, MA, USA) (5 mg/kg), three times a week and for two weeks.

Techniques: Incubation, Inhibition, Lactate Assay

Journal: Cancers

Article Title: mTORC1-Inhibition Potentiating Metabolic Block by Tyrosine Kinase Inhibitor Ponatinib in Multiple Myeloma

doi: 10.3390/cancers14112766

Figure Lengend Snippet: Ponatinib inhibits the glycolysis pathway in multiple-myeloma cells. ( A ) Glucose metabolism array was performed on RPMI8226 cells, using Bioneer gene-expression array plates. Cells were exposed to ponatinib (1 μM) for 12 h, and total mRNA was extracted. The gene expression was analyzed by using qPCR. The histogram represents the ‘% changes’ in gene expression for different glucose metabolism pathways. ( B , C ) Ponatinib and vehicle control, dimethylsulfoxide (DMSO), treatment were for 12 h. ( B ) Glycolytic gene-expression changes were quantified by using qPCR. ( C ) HK2 and GPI activity and pyruvate and lactate production were examined by using specific assay kits. Data are presented as mean ± SEM; t -test: * p < 0.05.

Article Snippet: For the blood toxicity tests, the mice were tested with intraperitoneal injection with 100 μL PBS (Gibco BRL) or ponatinib (Biosynth Carbosynth, Staad, Switzerland) (10 mg/kg) and sirolimus (Alfa Aesar, Thermo Fisher, Ward Hill, MA, USA) (5 mg/kg), three times a week and for two weeks.

Techniques: Expressing, Activity Assay

Journal: Cancers

Article Title: mTORC1-Inhibition Potentiating Metabolic Block by Tyrosine Kinase Inhibitor Ponatinib in Multiple Myeloma

doi: 10.3390/cancers14112766

Figure Lengend Snippet: Sirolimus potentiates ponatinib-mediated glycolysis inhibition and induces multiple myeloma cell death. ( A ) Metabolic assay was performed on RPMI8226 and U266 cells in the presence of variable doses of ponatinib alone or with 150 pM sirolimus. The histograms represent the inhibition percentages; treatment was for 72 h. Sirolimus at 150 pM potentiates the ponatinib inhibition on metabolic activity significantly. ( B ) Calcein AM and PI staining show a combination of ponatinib and sirolimus significantly increases the dead cell population in the cultures of MM cells. ( C ) Quantification for Calcein AM and PI-positive cell populations. Cells were counted by using ImageJ software and plotted as histograms, using GraphPad Prism 5 software. Scale bars for the large field are 50 µm; those for the magnified inset are 5 µm. ( D ) RPMI8226 and U266 cells were treated with ponatinib and/or sirolimus for 12 h. The glycolytic gene-expression changes were examined by qPCR. ( E ) RPMI8226 and U266 cells were treated with ponatinib and/or sirolimus for 24 h. The HK2 and GPI activity was measured by activity assay kits. Glucose, ATP, lactate, and pyruvate levels were quantified by specific assay kits. Doses used for ponatinib and sirolimus were 300 nM and 150 pM, respectively. Data are presented as mean ± SEM; t -test: * p < 0.05, ^ p < 0.01, and # p < 0.001.

Article Snippet: For the blood toxicity tests, the mice were tested with intraperitoneal injection with 100 μL PBS (Gibco BRL) or ponatinib (Biosynth Carbosynth, Staad, Switzerland) (10 mg/kg) and sirolimus (Alfa Aesar, Thermo Fisher, Ward Hill, MA, USA) (5 mg/kg), three times a week and for two weeks.

Techniques: Inhibition, Metabolic Assay, Activity Assay, Staining, Software, Expressing

Journal: Cancers

Article Title: mTORC1-Inhibition Potentiating Metabolic Block by Tyrosine Kinase Inhibitor Ponatinib in Multiple Myeloma

doi: 10.3390/cancers14112766

Figure Lengend Snippet: Ponatinib and sirolimus inhibit AKT/mTORC1 pathways and residual oxidative phosphorylation (OXPHOS) in multiple myeloma cells. ( A ) Ponatinib at 300 nM and/or 150 pM sirolimus was/were administrated to RPMI8226 and U266 cells for 12 or 24 h. The activity of ERK/AKT/mTORC1 pathways was quantified by specific phosphorylated antibodies with Western blot. Expression was quantified by ImageJ and represented as a histogram. HK2 and GPI expression was determined after 24 h of treatment and quantified. ( B ) RPMI8226 and U266 cells were treated with ponatinib, sirolimus, or with their combination for 12 and 24 h. Changes in LC3 A/B and P62 expression were quantified by Western blotting. Expression was quantified by using ImageJ software and plotted as histograms. ( C ) OXPHOS was measured in vitro, using a specific kit after 24 h of treatments; antimycin was used as a positive control for OXPHOS inhibition. Ponatinib and sirolimus doses were 300 nM and 150 pM, respectively. The uncropped Western blots are shown in . Data are presented as mean ± SEM; t -test: * p < 0.05, ^ p < 0.01, and # p < 0.001.

Article Snippet: For the blood toxicity tests, the mice were tested with intraperitoneal injection with 100 μL PBS (Gibco BRL) or ponatinib (Biosynth Carbosynth, Staad, Switzerland) (10 mg/kg) and sirolimus (Alfa Aesar, Thermo Fisher, Ward Hill, MA, USA) (5 mg/kg), three times a week and for two weeks.

Techniques: Activity Assay, Western Blot, Expressing, Software, In Vitro, Positive Control, Inhibition

Journal: Cancers

Article Title: mTORC1-Inhibition Potentiating Metabolic Block by Tyrosine Kinase Inhibitor Ponatinib in Multiple Myeloma

doi: 10.3390/cancers14112766

Figure Lengend Snippet: Ponatinib and sirolimus lead to accumulation of cellular ROS in multiple myeloma cells. ( A ) ROS accumulation in cells was quantified with flow cytometry after DCFDA staining. ( B , C ) Cellular ROS were measured by DCFDA fluorescence, with or without H 2 O 2 (50 μM) and/or NAC (3 mM) presence, and cell viability was assayed. Data are presented as mean ± SEM; t -test: *** p < 0.001.

Article Snippet: For the blood toxicity tests, the mice were tested with intraperitoneal injection with 100 μL PBS (Gibco BRL) or ponatinib (Biosynth Carbosynth, Staad, Switzerland) (10 mg/kg) and sirolimus (Alfa Aesar, Thermo Fisher, Ward Hill, MA, USA) (5 mg/kg), three times a week and for two weeks.

Techniques: Flow Cytometry, Staining, Fluorescence

Journal: Cancers

Article Title: mTORC1-Inhibition Potentiating Metabolic Block by Tyrosine Kinase Inhibitor Ponatinib in Multiple Myeloma

doi: 10.3390/cancers14112766

Figure Lengend Snippet: Ponatinib-and-sirolimus combination effectively reduces the multiple-myeloma tumor burden in athymic nude mouse. ( A ) RPMI8226 tumor images for different treatment groups. Scale bar corresponds to 1 cm (picture of n = 8 shown; there were 10 mice per arm). ( B ) Tumor volume was measured at different time points during treatments. The tumor volume was calculated and plotted as a curve, using GraphPad Prism 5 software. ( C ) The terminal tumor weight was measured and plotted by using GraphPad Prism 5 software. Achieved power analysis using G*Power 3.1.9.4 is shown in . ( D , E ) Different toxicity tests were performed for the ponatinib-and-sirolimus combination treatment group. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CREA) levels in the serum were measured biochemically. Terminal mouse body weight was measured. Data are presented as mean ± SEM; t -test: * p < 0.05, ** p < 0.01, and *** p < 0.001. ns: not significant. ( F ) H&E staining was performed on fixed tumor tissue samples; the scale bar is 20 μm. Individual tumor sizes and their volumes are in .

Article Snippet: For the blood toxicity tests, the mice were tested with intraperitoneal injection with 100 μL PBS (Gibco BRL) or ponatinib (Biosynth Carbosynth, Staad, Switzerland) (10 mg/kg) and sirolimus (Alfa Aesar, Thermo Fisher, Ward Hill, MA, USA) (5 mg/kg), three times a week and for two weeks.

Techniques: Software, Staining

Journal: Cancers

Article Title: mTORC1-Inhibition Potentiating Metabolic Block by Tyrosine Kinase Inhibitor Ponatinib in Multiple Myeloma

doi: 10.3390/cancers14112766

Figure Lengend Snippet: Ponatinib-and-sirolimus combination reduces the levels of Ki67 proliferation marker, mTORC1 activity, and glycolysis rate-limiting enzymes in multiple myeloma tumors. ( A ) Fixed tumor tissue samples were stained with antibodies specific to Ki67 (for proliferation), pS6K (for mTORC1 activity), HK2, and GPI protein levels (for glycolysis). The fluorescent images were quantified by using ImageJ software and plotted as a histogram. Scale bar is 50 μm. ( B ) Tumor tissue lysates were prepared, and HK2 and GPI activity, ATP, and lactate levels were quantified. The data are representative of 3 samples from each group. Data are presented as mean ± SEM; t -test: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: For the blood toxicity tests, the mice were tested with intraperitoneal injection with 100 μL PBS (Gibco BRL) or ponatinib (Biosynth Carbosynth, Staad, Switzerland) (10 mg/kg) and sirolimus (Alfa Aesar, Thermo Fisher, Ward Hill, MA, USA) (5 mg/kg), three times a week and for two weeks.

Techniques: Marker, Activity Assay, Staining, Software

Journal: Cancers

Article Title: mTORC1-Inhibition Potentiating Metabolic Block by Tyrosine Kinase Inhibitor Ponatinib in Multiple Myeloma

doi: 10.3390/cancers14112766

Figure Lengend Snippet: Ponatinib-and-sirolimus combination increases ROS production in multiple myeloma tumors. ( A ) Fixed tumor tissue samples were stained with antibodies specific to CHOP and PDI as ROS markers. The fluorescent images were quantified by using ImageJ software and plotted as a histogram. Scale bar is 50 μm. Data are presented as mean ± SEM; t -test: ** p < 0.01, *** p < 0.001. ( B ) Ponatinib-and-sirolimus combination inhibits glycolysis and OXPHOS to inhibit ATP production and increase ROS accumulation; AKT and mTOR signaling/activity was reduced by the drug combination.

Article Snippet: For the blood toxicity tests, the mice were tested with intraperitoneal injection with 100 μL PBS (Gibco BRL) or ponatinib (Biosynth Carbosynth, Staad, Switzerland) (10 mg/kg) and sirolimus (Alfa Aesar, Thermo Fisher, Ward Hill, MA, USA) (5 mg/kg), three times a week and for two weeks.

Techniques: Staining, Software, Activity Assay