Figure 6.
Treatment with BET-PROTAC and BC2059 exerts synergistic lethal activity in BETi-sensitive and resistant PD-CD34+ sAML cells. (A) PD CD34+ sAML cells (n = 35) were treated with the indicated concentrations of OTX015 for 48 hours. After this, the percentage of propidium iodide–positive, nonviable cells were determined by flow cytometry. Samples were separated based on response to OTX015 as sensitive (mean LD50 dose = 2 µM) or resistant (mean LD25 dose = 2 µM) to OTX015 treatment. (B) Violin plot of the mean fluorescence intensity of TCF7L2 or JMJD6 staining (as determined by confocal microscopy) in 11 sAML blast samples that were sensitive or resistant to OTX015 treatment. Asterisks (****) indicate mean fluorescence intensity values that are significantly greater (P < .0001) in sAML blasts resistant to OTX015 compared with sensitive sAML blast samples. (C) Representative images of sAML blast samples analyzed for TCF7L2 and JMJD6 expression by confocal microscopy (original magnification ×100). (D) Scatterplot of relative c-Myc protein expression in 17 sAML blast samples sensitive or resistant to OTX015 treatment. Asterisk (*) indicates c-Myc expression levels that are significantly greater (P < .05) in OTX015-resistant sAML blasts compared with OTX015-sensitive sAML blasts. (E-F) PD, CD34+ sAML cells determined to be resistant to OTX015 were treated with the indicated concentrations of ARV-771 (n = 10) or BC2059 (n = 15) for 48 hours. Then, the percentage of propidium iodide–positive, nonviable cells were determined by flow cytometry. (G) PD, CD34+ sAML blast cells resistant to OTX015 were treated with ARV-771 (dose range, 20-250 nM) and/or BC2059 (10-100 nM) for 48 hours. The percentage of propidium iodide–positive, nonviable cells were determined by flow cytometry. The graph shows the range of confidence interval (CI) values in each sAML sample following the combination treatments. CI values <1.0 indicate a synergistic interaction of the drugs. (H) PD, sAML blast cells were transfected with negative control sgRNA, TCF7L2 sgRNA, or JMJD6 sgRNA and incubated on a monolayer of GFP-expressing HS5 cells for 5 days. Then, cells were cytospun onto glass slides and immunostained for TCF7L2, JMJD6, or c-Myc expression. Nuclei were stained with DAPI. Cells were imaged by confocal microscopy (original magnification ×100). (I) PD, sAML blast cells were transfected and cultured as in panel H. The cell viability of the sAML cells was monitored over 7 days. Asterisks (**) indicate cell viability values significantly less (P < .01) in TCF7L2 or JMJD6 sgRNA-transfected cells compared with negative control sgRNA-transfected cells. (J) PD, sAML blast cells were transfected and cultured as in panel H for 5 days. Then, cells were treated with the indicated concentrations of OTX015 for 48 hours. At the end of treatment, the percentage of propidium iodide–positive, nonviable cells was determined by flow cytometry.

Treatment with BET-PROTAC and BC2059 exerts synergistic lethal activity in BETi-sensitive and resistant PD-CD34+ sAML cells. (A) PD CD34+ sAML cells (n = 35) were treated with the indicated concentrations of OTX015 for 48 hours. After this, the percentage of propidium iodide–positive, nonviable cells were determined by flow cytometry. Samples were separated based on response to OTX015 as sensitive (mean LD50 dose = 2 µM) or resistant (mean LD25 dose = 2 µM) to OTX015 treatment. (B) Violin plot of the mean fluorescence intensity of TCF7L2 or JMJD6 staining (as determined by confocal microscopy) in 11 sAML blast samples that were sensitive or resistant to OTX015 treatment. Asterisks (****) indicate mean fluorescence intensity values that are significantly greater (P < .0001) in sAML blasts resistant to OTX015 compared with sensitive sAML blast samples. (C) Representative images of sAML blast samples analyzed for TCF7L2 and JMJD6 expression by confocal microscopy (original magnification ×100). (D) Scatterplot of relative c-Myc protein expression in 17 sAML blast samples sensitive or resistant to OTX015 treatment. Asterisk (*) indicates c-Myc expression levels that are significantly greater (P < .05) in OTX015-resistant sAML blasts compared with OTX015-sensitive sAML blasts. (E-F) PD, CD34+ sAML cells determined to be resistant to OTX015 were treated with the indicated concentrations of ARV-771 (n = 10) or BC2059 (n = 15) for 48 hours. Then, the percentage of propidium iodide–positive, nonviable cells were determined by flow cytometry. (G) PD, CD34+ sAML blast cells resistant to OTX015 were treated with ARV-771 (dose range, 20-250 nM) and/or BC2059 (10-100 nM) for 48 hours. The percentage of propidium iodide–positive, nonviable cells were determined by flow cytometry. The graph shows the range of confidence interval (CI) values in each sAML sample following the combination treatments. CI values <1.0 indicate a synergistic interaction of the drugs. (H) PD, sAML blast cells were transfected with negative control sgRNA, TCF7L2 sgRNA, or JMJD6 sgRNA and incubated on a monolayer of GFP-expressing HS5 cells for 5 days. Then, cells were cytospun onto glass slides and immunostained for TCF7L2, JMJD6, or c-Myc expression. Nuclei were stained with DAPI. Cells were imaged by confocal microscopy (original magnification ×100). (I) PD, sAML blast cells were transfected and cultured as in panel H. The cell viability of the sAML cells was monitored over 7 days. Asterisks (**) indicate cell viability values significantly less (P < .01) in TCF7L2 or JMJD6 sgRNA-transfected cells compared with negative control sgRNA-transfected cells. (J) PD, sAML blast cells were transfected and cultured as in panel H for 5 days. Then, cells were treated with the indicated concentrations of OTX015 for 48 hours. At the end of treatment, the percentage of propidium iodide–positive, nonviable cells was determined by flow cytometry.

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