Figure 7.
Combined MEK inhibitor and pan bromodomain and extraterminal domain inhibitor ameliorate MM phenotypes and prolong the survival of VQ myeloma mice. (A-I) Two MM cell lines were established from VQ-D2 recipient mice. (A) Schematic illustration of the procedure to establish 2 VQ MM cell lines. (B) Representative images of H&E-stained 4935 and 4938 cells; original magnification ×100. (C) Flow cytometric assay of B220 and CD138 expression on the 2 cell lines. (D) Genotyping of NrasQ61R and Vκ*MYC alleles in the 2 cell lines. (E) hMYC expression in the cell lines was detected using confocal immunofluorescence. Scale bar, 10 μm. Middle and right panels: 4′,6-diamidino-2-phenylindole (DAPI) stain. (F) Hemagglutinin (HA)-tagged hMYC expression in the cell lines was validated using western blot analysis. Left, rabbit anti-Myc antibody; right, rat anti-HA antibody conjugated with horseradish peroxidase. (G-H) Flow cytometric analysis of CD138, PD-L1, CD155 (G), MHC-I, and MHC-II (H) expression on the cell lines. (I) Quantification of retroviral and lentiviral infection rate in the cell lines based on flow cytometric analysis of green fluorescent protein (GFP). (J-K) VQ MM cell lines were treated with increasing concentrations of trametinib for 48 hours. (J) Cell growth was measured using Cell TiterGlo assay. (K) Quantification of surface expression of PDL1 and CD155 in the presence of 10 nM trametinib. (L-M) VQ MM cell lines were treated with increasing concentrations of GSK525762 for 48 hours. (L) Cell growth was measured using Cell TiterGlo assay. (M) Quantification of surface expression of PDL1 and CD155 in the presence of 3 μM GSK525762. (N-O) VQ recipients were treated with vehicle or drug(s) as described in “Methods.” (N) SPEP was performed to quantify the γ/A ratios in VQ recipient mice before treatment (Pre) and at day 21 of treatment or at moribund before day 21 (Post). Note: some of the recipients were found dead and unable to be analyzed. Paired, 2-tailed Student t tests were performed. (O) Kaplan-Meier survival curves were plotted against days after treatment. Log-rank test was performed. *P < .05; **P < .01. G, GSK525762; IB, immunoblot; MFI, mean fluorescence intensity; MSCV, murine stem cell virus; T, trametinib; TG, trametinib and GSK525762; Veh, vehicle.

Combined MEK inhibitor and pan bromodomain and extraterminal domain inhibitor ameliorate MM phenotypes and prolong the survival of VQ myeloma mice. (A-I) Two MM cell lines were established from VQ-D2 recipient mice. (A) Schematic illustration of the procedure to establish 2 VQ MM cell lines. (B) Representative images of H&E-stained 4935 and 4938 cells; original magnification ×100. (C) Flow cytometric assay of B220 and CD138 expression on the 2 cell lines. (D) Genotyping of NrasQ61R and Vκ*MYC alleles in the 2 cell lines. (E) hMYC expression in the cell lines was detected using confocal immunofluorescence. Scale bar, 10 μm. Middle and right panels: 4′,6-diamidino-2-phenylindole (DAPI) stain. (F) Hemagglutinin (HA)-tagged hMYC expression in the cell lines was validated using western blot analysis. Left, rabbit anti-Myc antibody; right, rat anti-HA antibody conjugated with horseradish peroxidase. (G-H) Flow cytometric analysis of CD138, PD-L1, CD155 (G), MHC-I, and MHC-II (H) expression on the cell lines. (I) Quantification of retroviral and lentiviral infection rate in the cell lines based on flow cytometric analysis of green fluorescent protein (GFP). (J-K) VQ MM cell lines were treated with increasing concentrations of trametinib for 48 hours. (J) Cell growth was measured using Cell TiterGlo assay. (K) Quantification of surface expression of PDL1 and CD155 in the presence of 10 nM trametinib. (L-M) VQ MM cell lines were treated with increasing concentrations of GSK525762 for 48 hours. (L) Cell growth was measured using Cell TiterGlo assay. (M) Quantification of surface expression of PDL1 and CD155 in the presence of 3 μM GSK525762. (N-O) VQ recipients were treated with vehicle or drug(s) as described in “Methods.” (N) SPEP was performed to quantify the γ/A ratios in VQ recipient mice before treatment (Pre) and at day 21 of treatment or at moribund before day 21 (Post). Note: some of the recipients were found dead and unable to be analyzed. Paired, 2-tailed Student t tests were performed. (O) Kaplan-Meier survival curves were plotted against days after treatment. Log-rank test was performed. *P < .05; **P < .01. G, GSK525762; IB, immunoblot; MFI, mean fluorescence intensity; MSCV, murine stem cell virus; T, trametinib; TG, trametinib and GSK525762; Veh, vehicle.

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