Article Figures & Data
Figures
- Figure 1.
BCL-2 protein expression as a marker of ABT-199 sensitivity and 4-HPR + ABT-199 synergy in neuroblastoma cell lines. A, CIN values of all tested neuroblastoma cell lines for the combination cytotoxicity of ABT-199 and 4-HPR showing data for 15 ABT-199 relatively sensitive cell lines (ABT-199 IC50 < 5 μmol/L), black symbols and 17 ABT-199 relatively resistant cell lines (ABT-199 IC50 > 5 μmol/L), red symbols. CIN < 1 (synergy), CIN = 1 (additive effect), and CIN > 1 (antagonism; ref. 37). Fixed-ratio dose–response curves used to generate data for A are shown in Supplementary Figs. S1 and S2. B, Example of DIMSCAN assay dose–response curves for ABT-199 relatively sensitive (COG-N-452h) and ABT-199 relatively resistant (Felix-h) cell lines. Cells were treated with varying (fixed-ratio) concentrations of 4-HPR, ABT-199, and 4-HPR + ABT-199 (six replicates per drug concentration) for 96 hours then analyzed by the DIMSCAN assay. C, Dose–response curves of seven ABT-199 relatively sensitive and seven ABT-199 relatively resistant cell lines selected for determining the difference in basal expression of antiapoptotic proteins. D, Expression of antiapoptotic proteins in seven ABT-199 relatively sensitive cell lines and seven ABT-199 relatively resistant cell lines. E, Dot plots quantitating immunoblotting data shown in C. The BCL-2 and BCL-W protein levels were significantly higher in the ABT-199 relatively sensitive group versus the ABT-199 relatively resistant group. n.s., not significant. F, CIN values for the seven selected ABT-199 relatively sensitive cell lines compared with seven selected ABT-199 relatively resistant cell lines. 4-HPR + ABT-199 CIN values of the ABT-199 relatively sensitive lines was significantly lower than the CIN values of the ABT-199 relatively resistant lines.
- Figure 2.
4-HPR + ABT-199 + keto significantly enhanced EFS of mice carrying a high BCL-2–expressing PDX. A, BCL-2 protein expression in two PDXs established at time of death from progressive neuroblastoma, FELIX-PDX (low BCL-2 expression) and COG-N-452x (high BCL-2 expression). B, Tumor growth curves and EFS of the control, 4-HPR + keto, ABT-199 + keto, and 4-HPR + ABT-199 + keto groups for FELIX-PDX and COG-N-452x. 4-HPR + ABT-199 + keto significantly improved EFS versus the 4-HPR + keto and ABT-199 + keto groups for COG-N-452x PDX but not FELIX-PDX. C, Median EFS of the control, 4-HPR + keto, ABT-199 + keto, and 4-HPR + ABT-199 + keto groups for COG-N-452x and FELIX-PDX.
- Figure 3.
BCL-2 protein expression at DX and PD are consistent and may provide a biomarker for neuroblastoma tumors likely to respond to the combination of 4-HPR + ABT-199. A, BCL-2, NOXA, and MCL-1 protein expression for matched pair cell lines (established at DX and PD from 10 patients). High BCL-2–expressing cell lines established at DX and later at PD from the same patient show comparable BCL-2 protein expression. B, High BCL-2 protein expression was observed for both PDXs of the matched pair PDX models, COG-N-623x (DX) and COG-N-603x (PD). C, Tumor growth curves and EFS of the control, 4-HPR + keto, ABT-199 + keto, and 4-HPR + ABT-199 + keto groups for COG-N-603x and COG-N-623x. Both models showed significant improvement in survival for 4-HPR + ABT-199 + keto versus the 4-HPR + keto and ABT-199 + keto groups. D, EFS of the control and CYCLO + TOPO groups for COG-N-603x (DX) and COG-N-623x (PD) showing the increased resistance of COG-N-623x to CYCLO + TOPO compared with COG-N-603x.
- Figure 4.
Apoptosis and induction of NOXA by 4-HPR + ABT-199. A and B, 4-HPR + ABT-199 induced greater caspase 3 cleavage compared with both single agents and the control. COG-N-415h and COG-N-452h were treated with vehicle, 4-HPR (10 μmol/L), ABT-199 (2 μmol/L), or 4-HPR + ABT-199 (10 μmol/L + 2 μmol/L) for 12 or 24 hours before immunoblotting. C and D, 4-HPR + ABT-199 induced greater apoptosis compared with both single agents. COG-N-415h and COG-N-452h were treated with vehicle, 4-HPR (10 μmol/L), ABT-199 (2 μmol/L), or 4-HPR + ABT-199 (10 μmol/L+2 μmol/L) for 24 and 48 hours, respectively, before staining for the TUNEL assay. E, 4-HPR induced NOXA protein expression in multiple neuroblastoma cell lines. CHLA-119, COG-N-452h, COG-N-415h, and COG-N-623h were treated with 5 and 10 μmol/L of 4-HPR for 12 hours and subjected to immunoblotting for NOXA, BIM, BAD, BIK, and PUMA.
- Figure 5.
NOXA as a crucial mediator of 4-HPR + ABT-199 synergy. A and B, COG-N-415h and COG-N-623h, transduced with either empty vector or PMAIP1-Myc-DDK, were treated with different concentrations of doxycycline for 48 hours before being subjected to immunoblotting for NOXA (Myc-DDK tagged) protein. Cells were also treated with different concentrations of doxycycline (six replicates per drug concentration) for 96 hours then analyzed for cytotoxicity by DIMSCAN. Doxycycline (0.5 μg/mL) was selected to induce NOXA-Myc-DDK expression for both COG-N-415h and COG-N-623h. C and D, COG-N-415h and COG-N-623h, transduced with either empty vector or the PMAIP1-Myc-DDK doxycycline-inducible vector, were treated with doxycycline (0.5 μg/mL) for 48 hours before being treated with different concentration of ABT-199 for 96 hours and then analyzed for cytotoxicity by DIMSCAN. E and F, NOXA knockdown (KD) reduced the NOXA protein induced by 4-HPR (5 and 10 μmol/L at 12 hours) for both COG-N-415h and COG-N-623h and decreased the cytotoxicity of 4-HPR + ABT-199. Cell lines, transduced with either EGFP-KD or NOXA-KD shRNA, were treated with 4-HPR, ABT-199, or 4-HPR + ABT-199 for 96 hours before being analyzed for cytotoxicity by DIMSCAN. G, NOXA KD attenuated the enhanced apoptosis of 4-HPR + ABT-199 relative to 4-HPR and ABT-199 as single agents. COG-N-415h, transduced with either EGFP-KD or NOXA-KD plasmid, were treated with 4-HPR, ABT-199, or 4-HPR + ABT-199 for 24 hours before being stained for the TUNEL assay. H, NOXA protein binds explicitly to the MCL-1 protein. COG-N-623h, transduced with either empty vector or PMAIP1-Myc-DDK doxycycline-inducible vector, were treated with doxycycline (0.5 μg/mL) for 48 hours before being harvested and immunoprecipitated with anti-FLAG antibody (for NOXA-Myc-DDK) and then immunoblotted to detect BCL-2, MCL-1, BCL-W, BCL-XL, BAX, and BAK.
- Figure 6.
Induction of ATF-3 and ATF-4 transcription factors by 4-HPR–induced ROS mediate induction of NOXA expression in neuroblastoma. A, RNA-seq was carried out with control and 4-HPR–treated (5 μmol/L for 15 hours) CHLA-119 cells; expression of selected genes are shown in the heatmap. We used the PROMO database and PMAIP1 promoter sequence (−500 to 0 of the ATG transcription start site) to further narrow down RNA-seq data to transcription factors binding to the PMAIP1 promoter. ATF3, a transcription factor known to activate PMAIP1 promoter (43), showed the highest increase in response to 4-HPR. ATF4, a transcription factor known to activate ATF3 and PMAIP1 promoter (44, 45), is also upregulated. Heatmap scale was adjusted to emphasize differences in transcription factor expression. Relative differences in expression are better visualized in B. B, 4-HPR treatment significantly upregulated NOXA, ATF3, and ATF4 mRNA expression compared with controls. CHLA-119 was treated with 4-HPR (5 μmol/L) or vehicle control for 6 hours before being harvested for measuring changes in mRNA expression by real-time RT-PCR. C, 4-HPR treatment at 5 and 10 μmol/L for 12 hours induced both ATF3 and ATF4 protein expression along with NOXA protein expression in multiple tested neuroblastoma cell lines. D, Knockdown of ATF3 and ATF4 using siRNA reduced NOXA induction by 4-HPR treatment in multiple neuroblastoma cell lines. Cell lines were transfected with either nontargeted control (NTC) siRNA, ATF3 siRNA, or ATF-4 siRNA for 12 hours before being treated with 4-HPR (5 μmol/L) for 6 hours, harvested, and blotted for ATF-3, ATF-4, and NOXA. E, Mice bearing COG-N-623x (PDX) were treated with either vehicle or 4-HPR + keto for 4 days before the tumors being harvested and subjected to immunoblotting for NOXA, ATF3, and ATF4 protein. Figure shows representative data from tumors in 2 mice, control and 4-HPR treated. F, Vitamin C (Vit. C) reduced ATF3, ATF4, and NOXA induced by 4-HPR treatment. Cells were pretreated with vitamin C (150 μmol/L) for 1 hour before being treated with 4-HPR for 12 hours. G, Mechanism of 4-HPR + ABT-199 mechanism of synergy.
Additional Files
Supplementary Data
- Supplementary Data - Supplementary Data and Tables
Supplementary Data
- Supplementary Data - Supplementary Data and Tables
Volume 18, Issue 12
