https://openworks.mdanderson.org/sumexp21/1224/thumbnail.jp

Drawdy, Allyson V

Gandhi, Varsha

Tantawy, Shady

English

OpenWorks @ MD Anderson

IntroductionAPR-246APR-246 (APR) is a novel agent proposed to reactivate mutant p53 and consequential downstream biological effects, including induction of apoptosis.The mechanism by which APR-246 reactivates p53 and disrupts cellular redox along with sub sequential cytotoxic effects remain unknown.https://https://www.frontiersin.org/articles/10.3389/fonc.2016.00021/fullChronic Lymphocytic Leukemia (CLL) CLL is a B-cell malignancy most commonly treated with venetoclax or ibrutinib. Deletion of chromosome 17p and mutations of p53 hallmark poor prognosis in patients with CLL.5-8% of CLL patients incur dell17p, while p53 mutations exist in 10-15% of untreated CLL cases (40-50% in refractory disease). CLL patients with mutated p53 treated with frontline targeted therapies, venetoclax and ibrutinib, experience shorter progression-free survival.Schematic of known APR-246 MechanismMechanism of APR-246 and Sensitization of Cells to Targeted AgentsAllyson Drawdy, Shady Tantawy and Varsha GandhiDepartment of Experimental Therapeutics, MD Anderson Cancer Center, Houston, TX 77054.Role of p53 in APR-246 Cytotoxicity1. Determine role of p53 in the mechanism of APR-246-induced cytotoxicity2. Evaluate APR-246 alone and in combination with venetoclax and ibrutinib in isogenic cell line with different p53 background Does p53 status relate to APR-246 induced cytotoxicity?References1. Manning BD and Cantley LC, Cell, 129; 1261-1274, 2007ConclusionsDoes APR-246 treatment induce oxidative stress?WT KO R175H R248Q050100150200HG3 APR-induced ROS% DCFDA flourescence DMSOTBrTAPR 10umAPR 25umAPR 50umDMSO APR 1uMAPR 2.5 uMAPR 5 uMAPR 10 uMAPR 20 uM050100150200Mec1 (n=3) APR-induced ROS% DCFDA flourescence DMSOAPR 1uMAPR 2.5 uMAPR 5 uMAPR 10 uMAPR 20 uMControlAPR10uMAPR10uM+GSH100ug/ml APR10uM+GSH200ug/ml APR10uM+GSH300ug/ml 050100GSH Rescue of APR-treated Mec1 Cells% Cell viabilityHG3-WT HG3-KO HG3-R175H HG3-R248QTreatment VTX VTX+APR VTX VTX+APR VTX VTX+APR VTX VTX+APRIC50 3.46 3.24 3.42 1.989 3.695 3.403 3.697 3.376R2 0.9753 0.9272 0.9578 0.8392 0.9169 0.8361 0.8941 0.871Treatment IBR IBR+APR IBR IBR+APR IBR IBR+APR IBR IBR+APRIC50 *** 6764 *** 7400 *** 14027 *** 14566R2 0.8256 0.8431 0.8349 0.9032 0.8945 0.9013 0.7118 0.877Does combining APR-246 with each venetoclax (VTX) and ibrutinib (IBR), sensitize all HG3 cell lines to treatment?0-hr 1-hr 6-hr050100Intracellular [GSH] of APR-treated Mec1 CellsRelative GSH Illumination0-hr1-hr6-hrp53/GSH Protein Immunoblot APR-246 is equally efficacious in cells with proficient, deficient, or mutated p53.Mode of action of APR-246 is independent of p53 status, and glutathione depletion appears to be the primary mechanism.APR-246 sensitizes CLL malignant B-cells to treatment with targeted therapies venetoclax and ibrutinib.Why do these findings matter?1. APR-246 may be useful in treating all CLL patients rather than solely p53-mutated.2. APR-246 shows promise for treatment of other malignancies. 3. A phase III clinical trial has been designed for patients with CLL to test APR-246 with ibrutinib or venetoclax.Figure 2. HG3 cell line p53 protein expression was visualized via immunoblot. HG3WT had minimal expression of p53. HG3R175H and HG3R248Q both had increases in p53 expression when compared to WT. Figure 3. After both 24hrs and 48hrs of treatment with increasing doses of APR-246, there is no significant difference in cytotoxicity between HG3 cell lines and Mec1. Mec1 was not treated with APR75µM or APR100µM due its sensitivity to higher doses.Figure 4. HG3 cell lines were treated with TBrTand increasing doses of APR-246. APR50µMproduced the highest DCFDA fluorescence, indicating the highest levels of cellular ROS.Figure 5. Mec1 cell line was treated with increasing doses of APR-246 and analyzed for DCFDA fluorescence. Treatment with APR20µM produced the highest levels of ROS in comparison to lower doses.Figure 9. HG3 Cell lines were administered increasing doses of both ibrutinib alone and in combination with APR-246 for 72hrs and triplicated. In all four cell lines with varying p-53 status, ibrutinib combined with APR20µM induced significantly more cell death than ibrutinib as a single agent.Figure 8. HG3 Cell lines were administered increasing doses of both alone and in combination with 20µM APR-246 for 72hrs and triplicated. In all four cell lines with varying p-53 status, venetoclax combined with APR20µM induced significantly more cell death than venetoclax as a single agent. This effect was more pronounced at higher VTX doses.Figure 6. Mec1 cells were treated with 50µMof APR-246 for six hours. Relative illumination at 1 hour was 124, while illumination at 6 hours was 64, indicating that intracellular GSH concentration decreased by half. Figure 7. Mec1 cells were treated with APR10µMand increasing amounts of glutathione. After 24-hr incubation, cell viability was analyzed showing that Mec1 cells were rescued from cell death as dosage of GSH increased. Table 2. IC50 and R^2 Values for VTX, VTX+APR, IBR, and IBR+APR in HG3 cell lines*** IC50values for IBR alone surpassed 20000nM, so could not be calculatedFigure 1. Schematic of Isogenic HG3 cell lines ◼ Cell lines: CLL isogenic HG3 cell lines: wild-type (WT), knock-out (KO), two lines with hot-spot induced mutations: R175H and R248Q, and Mec1 cell line.◼Cell viability quantified using FITC-Annexin/Propidium Iodide Flow Cytometry.◼P53 and Glutathione (GSH)-associated protein expression  confirmed via Immunoblot.◼Cellular ROS detected by DCFDA fluorescence through flow cytometry.◼Intracellular GSH measured by Promega GSH-Glo Assay via luminometer. Materials and MethodsRole of APR-246 in Sensitization of CellsIs there a cytotoxicity-related decline in glutathione after treatment with APR-246?Time HG3-WT HG3-KO HG3-R175H HG3-R248Q Mec1IC5024hr 68.29 66.02 61.55 76.97 24.1848hr 39.06 32.36 29.73 30.96 19.91Table 1. IC50 values for HG3 and Mec1 cell lines after 24- and 48-hour treatment with APR-246HG3-WT HG3-KO HG3-R175H HG3-R248Q Mec105010024hr incubation (n=3)% Cell viabilityDMSOAPR10uMAPR25uMAPR50uMAPR75uMAPR100uMHG3-WT HG3-KO HG3-R175H HG3-R248Q Mec105010048hr Incubation (n=3)% Cell viabilityDMSOAPR10uMAPR25uMAPR50uMAPR75uMAPR100uMReferences1. Aitken M. J., Lee H. J., Post S. M., 2019 Emerging treatment options for patients with p53-pathway-deficient cll. Therapeutic Advances in Hematology 10: 204062071989135. 2. Bykov V. J., Zhang Q., Zhang M., Ceder S., Abrahmsen L., Wiman K. G.  2016 Targeting of mutant p53 and the Cellular Redox balance by APR-246 as a strategy for Efficient cancer therapy. Frontiers in Oncology 6. 3. Liu D. S., Duong C. P., Haupt S., Montgomery K. G., House C. M., Azar W. J., Pearson H. B., Fisher O. M., 4. Zhang Q., Bykov V. J., Wiman K. G., Zawacka-Pankau J., 2018 APR-246 reactivates mutant P53 by TARGETING CYSTEINES 124 and 277. Cell Death & Disease 9. ObjectivesHG3-WT HG3-KO HG3-R175H HG3-R248Q050100HG3 Cell Lines (IBR/IBR+APR)% Cell ViabilityDMSOAPR20uMIBR1000nMIBR5000nMIBR10000nMIBR20000nMIBR1000nM+APR20uMIBR5000nM+APR20uMIBR10000nM+APR20uMIBR20000nM+APR20uM0 5000 10000 15000 20000050100HG3-WT (n=3)Dose (nM)% Cell ViabilityIBRIBR+APR0 5000 10000 15000 20000050100HG3-KO (n=3)Dose (nM)% Cell ViabilityIBRIBR+APR0 5000 10000 15000 20000050100HG3-R175H (n=3)Dose (nM)% Cell ViabilityIBRIBR+APR0 5000 10000 15000 20000050100HG3-R248Q (n=3)Dose (nM)% Cell ViabilityIBRIBR+APRHG3-WT HG3-KO HG3-R175H HG3-R248Q050100% Cell ViabilityDMSOAPR20MVTX100nmVTX1000nMVTX5000nMVTX10000nMVTX100nM+APR20MVTX1000nM+APR20MVTX5000nM+APR20MVTX10000nM+APR20MHG3 Cell Lines (VTX/VTX+APR)0 1 2 3 4050100HG3-KO (n=3)log dose (nM)% Cell ViabilityVTXVTX+APR0 1 2 3 4050100HG3-R175H (n=3)log dose (nM)% Cell ViabilityVTXVTX+APR0 1 2 3 4050100HG3-R248Q (n=3)log dose (nM)% Cell ViabilityVTXVTX+APR0 1 2 3 4050100HG3-WT (n=3)log dose (nM)% Cell ViabilityVTXVTX+APR

Mechanism of APR-246 and Sensitization of Cells to Targeted Agents

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1224&context=sumexp21

Mechanism of APR-246 and Sensitization of Cells to Targeted Agents

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