Article Figures & Data
Figures
- Figure 1.
Cotreatment with JQ1 and AC220 synergistically induces apoptosis of FLT3-ITD–expressing AML cells. A, MOLM13 and MV4-11 cells were treated with the indicated concentrations of JQ1 and/or AC220 for 48 hours. Following this, the percentage of annexin V–positive, apoptotic cells was determined by flow cytometry. Columns, mean of 3 experiments; bars, standard error of the mean (SEM). *, apoptosis values significantly greater (P < 0.05) in cells treated with the combination compared with treatment with either single agent. B, MV4-11 and MOLM13 cells were treated with JQ1 and AC220 at a constant ratio for 48 hours. The percentage of annexin V–positive, apoptotic cells was determined by flow cytometry. Median dose effect and isobologram analyses were conducted utilizing Calcusyn. Combination index (CI) values less than 1.0 indicate a synergistic interaction of the 2 agents in the combination. C and D, MV4-11 cells were treated with the indicated concentrations of AC220 and/or JQ1 for 24 hours. At the end of treatment, immunoblot analyses were conducted as indicated. The numbers beneath the blots represent densitometry analysis conducted on representative blots.
- Figure 2.
Cotreatment with JQ1 and ponatinib synergistically induces apoptosis of FLT-ITD expressing AML cells. A, MV4-11 and MOLM13 cells were treated with the indicated concentrations of JQ1 and ponatinib for 48 hours. Following this, the percentage of annexin V–positive, apoptotic cells were determined by flow cytometry. Columns, mean of 3 experiments; bars, SEM. *, apoptosis values significantly greater in cells treated with JQ1 and ponatinib compared with cells treated with either single agent (P < 0.01). B, MV4-11 and MOLM13 cells were treated with JQ1 and ponatinib at a constant ratio for 48 hours. The percentage of annexin V–positive, apoptotic cells was determined by flow cytometry. Median dose effect and isobologram analyses were conducted utilizing Calcusyn, assuming mutual exclusivity. CI values less than 1.0 indicate a synergistic interaction of the 2 agents in the combination. C and D, MV4-11 cells were treated with the indicated concentrations of ponatinib and/or JQ1 for 24 hours. Immunoblot analyses were conducted as indicated. The numbers beneath the blots represent densitometry analysis conducted on representative blots. E, MOLM13 cells were transduced with lentiviruses expressing nontargeted (NT) shRNA or BRD4 shRNA for 48 hours. At the end of treatment, total RNA was harvested and reverse transcribed. Quantitative PCR was performed for BRD4 and p21 expression. The relative mRNA expression was normalized to GAPDH and compared with the nontargeted shRNA transduced cells. *, values significantly different in BRD4 shRNA transduced cells compared with NT shRNA transduced cells (P < 0.05). F, MOLM13 cells were transduced with NT shRNA or BRD4 shRNA for 24 hours and then treated with AC220 or ponatinib for 48 hours. Following this, the percentage of annexin V–positive, apoptotic cells were determined by flow cytometry. Columns, mean of 3 experiments; bars, SEM. *, apoptosis values significantly greater in cells with BRD4 knockdown compared with cells transduced with nontargeted shRNA (P < 0.05).
- Figure 3.
Cotreatment with JQ1 and FLT3-TKI exerts synergistic antileukemia activity against primary FLT3-ITD–expressing AML cells. A, primary CD34+ FLT3-ITD–expressing AML cells (n = 5) and normal CD34+ cells were treated with JQ1 and/or ponatinib for 48 hours. At the end of treatment, the percentage of PI-positive, nonviable cells was determined by flow cytometry. *, values significantly greater (P < 0.05) in the combination compared with treatment with either single agent; †, values significantly less (P < 0.05) in normal CD34+ cells compared with CD34+ AML cells treated with the same combination of JQ1 and ponatinib. B, primary FLT3-ITD AML cells were treated with JQ1 and ponatinib at a constant ratio for 48 hours and the percentage of nonviable cells was determined by flow cytometry. Median dose effect and isobologram analyses were performed utilizing Calcusyn. CI values less than 1.0 indicate a synergistic interaction of the 2 agents in the combination. C, primary FLT3-ITD AML cells were treated with JQ1 and AC220 at a constant ratio for 48 hours and the percentage of nonviable cells was determined by flow cytometry. CI values less than 1.0 indicate a synergistic interaction of the 2 agents in the combination. D and E, primary FLT3-ITD–expressing AML cells were treated with AC220 or ponatinib and/or JQ1 for 24 hours. Immunoblot analyses were conducted as indicated.
- Figure 4.
Treatment with BET protein antagonist, JQ1, depletes c-MYC and induces BIM and apoptosis of pro-B, Ba/F3 cells expressing FLT3-ITD with or without kinase inhibitor-resistant point mutations. A, Ba/F3-FLT3-ITD, Ba/F3-ITD-F691L, and Ba/F3-ITD-D835V cells were treated with the indicated concentrations of JQ1 for 48 hours. Following this, cells were washed with 1× PBS and stained with annexin V–FITC and TO-PRO-3 iodide. The percentages of annexin V–positive, apoptotic cells were determined by flow cytometry. Columns, mean of three experiments; bars, SEM. B, Ba/F3-FLT3-ITD, Ba/F3-ITD-F691L, and Ba/F3-ITD-D835V cells were treated with the indicated concentrations of JQ1 for 24 hours. At the end of treatment, immunoblot analyses were conducted as indicated.
- Figure 5.
Characterization of MOLM13-TKIR cells and in vitro response to antileukemia agents. A, FLT3 surface expression in MOLM13 and MOLM-TKIR cells measured by flow cytometry. B, MOLM13 and MOLM13-TKIR cells were plated at 50,000 cells/mL in complete media and cultured over 96 hours. Every 24 hours, 200 μL of cells were removed and counted with a coulter counter to determine the number of cells/mL. Columns, mean of three experiments; bars, SEM. C, cell-cycle status of MOLM13 and MOLM-TKIR cells was determined by flow cytometry. Columns, mean of three experiments; bars, SEM. D, the coding exons of FLT3 (1–23) were amplified by PCR from MOLM13 and MOLM13-TKIR cells. PCR products were annealed to adapter sequences and NGS was utilized to compare the FLT3 sequence from each with the human genome version hg19. Identified mutations were compared with those in the COSMIC mutation database. MOLM13-TKIR cells, in addition to the ITD, possess a missense mutation in exon 16 C2039T, which results in an amino acid substitution of A680V in FLT3. No other mutations were identified in the FLT3 sequence.
- Figure 6.
JQ1 treatment depletes c-MYC and BCL2 to a greater extent with concomitant greater induction of HEXIM1 and p21 in MOLM13-TKIR cells compared with MOLM13 cells. A and B, MOLM13 and MOLM13-TKIR cells were treated with the indicated concentrations of JQ1 for 8 hours. At the end of treatment, total RNA was isolated and reverse transcribed. The resulting cDNA was utilized for quantitative PCR of c-MYC, BCL2, HEXIM1, and p21 expression. The relative mRNA expression in both cell lines was normalized against GAPDH and compared with the relative expression in parental MOLM13 cells. C, MOLM13 and MOLM-TKIR cells were treated with the indicated concentrations of JQ1 for 24 hours. Following this, cells were harvested and cell lysates were prepared. Immunoblot analyses were conducted for the expression levels of c-MYC, BRD4, BCL2, Bcl-xL, p-AKT, AKT, BIM, p21, cleaved PARP, and β-actin in the cell lysates. The numbers beneath the blots represent densitometry analysis conducted on representative blots. D, MOLM13 and MOLM13-TKIR cells were treated with the indicated concentrations of JQ1 for 48 hours. The percentage of annexin V–positive, apoptotic cells was determined by flow cytometry. Columns, mean of three experiments; bars, SEM. *, apoptosis values significantly greater in MOLM13-TKIR than MOLM13 treated with the same concentration of JQ1 (P < 0.01).
- Figure 7.
Cotreatment with JQ1 and PS synergistically induces apoptosis of MOLM13 cells with and without resistance to FLT3-TKI. A, MOLM13 and MOLM13-TKIR cells were treated with the indicated concentrations of JQ1 for 24 hours. Immunoblot analyses were conducted as indicated. The numbers beneath the blots represent densitometry analysis conducted on representative blots. B, MOLM13-TKIR cells were treated with the indicated concentrations of JQ1 and/or PS for 48 hours. The percentage of annexin V–positive, apoptotic cells was determined by flow cytometry. Columns, mean of 2 experiments; bars, SD. *, apoptosis values significantly greater in the combination, compared with treatment with either agent alone (P < 0.05). C, MOLM13-TKIR cells were treated with JQ1 and PS, as indicated for 48 hours. Following this, the percentage of annexin V–positive, apoptotic cells was determined by flow cytometry. Median dose effect and isobologram analysis was performed utilizing Calcusyn. CI values less than 1.0 indicate a synergistic interaction of the 2 agents in the combination. D, MOLM13-TKIR cells were treated with the indicated concentrations of JQ1 and PS for 24 hours. Cell lysates were prepared and immunoblot analyses were conducted as indicated. The numbers beneath the blots represent densitometry analysis conducted on representative blots.
Additional Files
Supplemental Figure Legends, Figures 1 - 9, Table 1
Files in this Data Supplement:
- Data Supplement - Figure Legends for Supplemental Data.
- Data Supplement - Supplemental Figure 1: Chemical structures the agents utilized in the study. Supplemental Table 1: Clinical presentation and mutational status of the primary AML samples. Supplemental Figure 2: BET protein antagonist and FLT3-TKI induces apoptosis of AML cells. Supplemental Figure 3: BET protein antagonist is synergistic with FLT3-TK inhibitors. Supplemental Figure 4: I-BET151 and FLT3-TKI is synergistic in FLT3-ITD expressing AML. Supplemental Figure 5: JQ1 and ponatinib or AC220 is synergistic against primary AML cells. Supplemental Figure 6: JQ1 and FLT3-TKI does not deplete FLT3 phosphorylation. Supplemental Table 2: Comparison of MOLM13 and MOLM13-TKIR response to anti-leukemia agents. Supplemental Figure 7: Effects of JQ1 and FLT3-TKI treatment in MOLM13-TKIR cells. Supplemental Figure 8: Knockdown of BRD4 enhances sensitivity to JQ1 in MOLM13-TKIR cells. Supplemental Figure 9: JQ1 and panobinostat is synergistic against MV4-11 TKIR cells.
Volume 13, Issue 10
