Figure 2.
In vivo therapeutic efficacies of HMAs and/or BCL-2 inhibition depend on TP53 mutational status. (A) Experimental workflow for in vivo competition AML xenograft assay. Isogenic MOLM13-TP53mutant (RFP657+) and MOLM13-TP53WT (GFP+) AML cells were transplanted at a 1:1 ratio into sublethally irradiated NSG mice. After a 7-day (7d) engraftment period, treatment with vehicle, decitabine (Dec), azacitidine (Aza), venetoclax (Ven), Dec + Ven, or Aza + Ven commenced at the indicated concentrations and treatment periods. At the end of the treatment period, relative competitive fitness was measured via flow cytometric analysis as the percentage of GFP+ or RFP657+ cells within human CD45 leukemia burden in bone marrow. (B) Gating examples of flow cytometric analysis of the in vivo competition assay. Numbers adjacent to gates indicate percentages. (C) Percentage of chimerism of MOLM13-TP53KO RFP657+ cells relative to MOLM13-TP53WT GFP+ in bone marrow pretransplant as well as posttreatment. Symbols represent averages; error bars indicate standard error of the mean (SEM). Animals per group: vehicle, n = 5; Ven, n = 5; decitabine (Dec), n = 4; azacitidine (Aza), n = 5; Dec+Ven, n = 5; Aza+Ven, n = 5. ***P < .001, 1-way analysis of variance. (D) Percentage of chimerism of MOLM13-TP53R248Q/− RFP657+ cells relative to MOLM13-TP53WT GFP+ cells in bone marrow pretransplant as well as posttreatment. Symbols represent averages; error bars indicate SEM. Animals per group: vehicle, n = 5; Ven, n = 5; Dec, n = 4; Aza, n = 5; Dec+Ven, n = 3; Aza+Ven, n = 5. ***P < .001, 1-way analysis of variance. (E) Representative bioluminescent images of mice, treated with Dec (1 mg/kg of BW per day) or phosphate-buffered saline (PBS) control (ctrl.) for 5 consecutive days, at days 7 and 16 postinjection of MOLM13-TP53-GFP-luciferase cells of the indicated TP53 genotypes. (F) Quantification of bioluminescent signal (total flux per second) for each group of mice. Mice were treated with Dec (1 mg/kg of BW per day) or PBS (ctrl.) for 5 consecutive days. Animals per group: WT ctrl., n = 5; WT Dec, n = 5; KO ctrl., n = 8; KO Dec, n = 9; R248Q/− ctrl., n = 5; R248Q/− Dec, n = 5. Symbols represent averages; error bars indicate SEM. *P < .001, 2-tailed Student t test. (G) Survival analysis of NSG mice engrafted with MOLM13-TP53-GFP-luciferase isogenic cell lines with the indicated genotypes and treated with Dec (1 mg/kg of BW per day) or PBS daily for 5 consecutive days, as indicated. Numbers of mice per group are as in (F). *P < .05, WT Dec vs WT ctrl. (H) Representative bioluminescent images of mice, treated with Aza (3.5 mg/kg of BW per day) or PBS for 7 consecutive days, at days 7 and 16 postinjection of MOLM13-TP53-GFP-luciferase cells of the indicated TP53 genotypes. (I, J) Quantification of bioluminescent signal (total flux per second) for each group of mice. Mice were treated with Aza (3.5 mg/kg of BW per day) or PBS for 7 consecutive days. Animals per group: WT ctrl., n = 7; WT Aza, n = 7; KO ctrl., n = 7; KO Aza, n = 7; R248Q/− ctrl., n = 5; R248Q/− Aza, n = 5. Symbols represent averages; error bars indicate SEM. *P < .001; 2-tailed Student t test. (J) Survival analysis of NSG mice engrafted with MOLM13-TP53-GFP-luciferase isogenic cell lines with the indicated genotypes and treated with Aza (3.5 mg/kg of BW per day) or PBS daily for 7 consecutive days, as indicated. **P < .01, ***P < .001, WT Aza vs KO or R248Q/− Aza; KO or R248Q/− Aza vs WT, KO, or R248Q/− ctrl.). (K) Representative bioluminescent images of mice, treated with Aza (3.5 mg/kg of BW per day) for 7 consecutive days or with Aza (7 days) + venetoclax (A+V; 75 mg/kg of BW per day) for 14 consecutive days, at days 7 and 16 postinjection of MOLM13-TP53-GFP-luciferase cells of the indicated TP53 genotypes. (L) Quantification of bioluminescent signal (total flux per second) for each group of mice. Mice were treated with Aza for 7 consecutive days or a combination of Aza for 7 days and Ven for 14 days (A+V). Animals per group: WT Aza, n = 7; WT A+V, n = 7; KO Aza, n = 7; KO A+V, n = 7; R248Q/− Aza, n = 7; R248Q/− A+V, n = 7. Symbols represent averages; error bars indicate SEM. **P < .01, ***P < .001, 2-tailed Student t test. (M) Survival analysis of NSG mice engrafted with MOLM13-TP53-GFP-luciferase isogenic cell lines with the indicated genotypes and treated with Aza (3.5 mg/kg of BW per day) or Aza (3.5 mg/kg of BW per day) for 7 consecutive days in combination with Ven (75 mg/kg of BW per day) for 14 consecutive days, as indicated. ***P < .001, WT A+V vs WT Aza; WT Aza vs KO or R248Q/− A+V; KO or R248Q/− A+V vs KO or R248Q/− Aza). FACS, fluorescence-activated cell sorting; i.p., intraperitoneally; ns, not significant; p.o., by mouth; SSC, side scatter.

In vivo therapeutic efficacies of HMAs and/or BCL-2 inhibition depend on TP53 mutational status. (A) Experimental workflow for in vivo competition AML xenograft assay. Isogenic MOLM13-TP53mutant (RFP657+) and MOLM13-TP53WT (GFP+) AML cells were transplanted at a 1:1 ratio into sublethally irradiated NSG mice. After a 7-day (7d) engraftment period, treatment with vehicle, decitabine (Dec), azacitidine (Aza), venetoclax (Ven), Dec + Ven, or Aza + Ven commenced at the indicated concentrations and treatment periods. At the end of the treatment period, relative competitive fitness was measured via flow cytometric analysis as the percentage of GFP+ or RFP657+ cells within human CD45 leukemia burden in bone marrow. (B) Gating examples of flow cytometric analysis of the in vivo competition assay. Numbers adjacent to gates indicate percentages. (C) Percentage of chimerism of MOLM13-TP53KO RFP657+ cells relative to MOLM13-TP53WT GFP+ in bone marrow pretransplant as well as posttreatment. Symbols represent averages; error bars indicate standard error of the mean (SEM). Animals per group: vehicle, n = 5; Ven, n = 5; decitabine (Dec), n = 4; azacitidine (Aza), n = 5; Dec+Ven, n = 5; Aza+Ven, n = 5. ***P < .001, 1-way analysis of variance. (D) Percentage of chimerism of MOLM13-TP53R248Q/− RFP657+ cells relative to MOLM13-TP53WT GFP+ cells in bone marrow pretransplant as well as posttreatment. Symbols represent averages; error bars indicate SEM. Animals per group: vehicle, n = 5; Ven, n = 5; Dec, n = 4; Aza, n = 5; Dec+Ven, n = 3; Aza+Ven, n = 5. ***P < .001, 1-way analysis of variance. (E) Representative bioluminescent images of mice, treated with Dec (1 mg/kg of BW per day) or phosphate-buffered saline (PBS) control (ctrl.) for 5 consecutive days, at days 7 and 16 postinjection of MOLM13-TP53-GFP-luciferase cells of the indicated TP53 genotypes. (F) Quantification of bioluminescent signal (total flux per second) for each group of mice. Mice were treated with Dec (1 mg/kg of BW per day) or PBS (ctrl.) for 5 consecutive days. Animals per group: WT ctrl., n = 5; WT Dec, n = 5; KO ctrl., n = 8; KO Dec, n = 9; R248Q/− ctrl., n = 5; R248Q/− Dec, n = 5. Symbols represent averages; error bars indicate SEM. *P < .001, 2-tailed Student t test. (G) Survival analysis of NSG mice engrafted with MOLM13-TP53-GFP-luciferase isogenic cell lines with the indicated genotypes and treated with Dec (1 mg/kg of BW per day) or PBS daily for 5 consecutive days, as indicated. Numbers of mice per group are as in (F). *P < .05, WT Dec vs WT ctrl. (H) Representative bioluminescent images of mice, treated with Aza (3.5 mg/kg of BW per day) or PBS for 7 consecutive days, at days 7 and 16 postinjection of MOLM13-TP53-GFP-luciferase cells of the indicated TP53 genotypes. (I, J) Quantification of bioluminescent signal (total flux per second) for each group of mice. Mice were treated with Aza (3.5 mg/kg of BW per day) or PBS for 7 consecutive days. Animals per group: WT ctrl., n = 7; WT Aza, n = 7; KO ctrl., n = 7; KO Aza, n = 7; R248Q/− ctrl., n = 5; R248Q/− Aza, n = 5. Symbols represent averages; error bars indicate SEM. *P < .001; 2-tailed Student t test. (J) Survival analysis of NSG mice engrafted with MOLM13-TP53-GFP-luciferase isogenic cell lines with the indicated genotypes and treated with Aza (3.5 mg/kg of BW per day) or PBS daily for 7 consecutive days, as indicated. **P < .01, ***P < .001, WT Aza vs KO or R248Q/− Aza; KO or R248Q/− Aza vs WT, KO, or R248Q/− ctrl.). (K) Representative bioluminescent images of mice, treated with Aza (3.5 mg/kg of BW per day) for 7 consecutive days or with Aza (7 days) + venetoclax (A+V; 75 mg/kg of BW per day) for 14 consecutive days, at days 7 and 16 postinjection of MOLM13-TP53-GFP-luciferase cells of the indicated TP53 genotypes. (L) Quantification of bioluminescent signal (total flux per second) for each group of mice. Mice were treated with Aza for 7 consecutive days or a combination of Aza for 7 days and Ven for 14 days (A+V). Animals per group: WT Aza, n = 7; WT A+V, n = 7; KO Aza, n = 7; KO A+V, n = 7; R248Q/− Aza, n = 7; R248Q/− A+V, n = 7. Symbols represent averages; error bars indicate SEM. **P < .01, ***P < .001, 2-tailed Student t test. (M) Survival analysis of NSG mice engrafted with MOLM13-TP53-GFP-luciferase isogenic cell lines with the indicated genotypes and treated with Aza (3.5 mg/kg of BW per day) or Aza (3.5 mg/kg of BW per day) for 7 consecutive days in combination with Ven (75 mg/kg of BW per day) for 14 consecutive days, as indicated. ***P < .001, WT A+V vs WT Aza; WT Aza vs KO or R248Q/− A+V; KO or R248Q/− A+V vs KO or R248Q/− Aza). FACS, fluorescence-activated cell sorting; i.p., intraperitoneally; ns, not significant; p.o., by mouth; SSC, side scatter.

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