Gnas deficiency in mouse T-ALL cells results in dexamethasone resistance in vitro and in vivo. (A) Schematic of cAMP pathway highlighting genes identified in the shRNA screen using red. (B) Quantification of apoptotic 1390 T-ALL cells stably infected with shRNAs to cAMP pathway member and treated with vehicle or dexamethasone (10 nM) for 48 hours. (C) qPCR analysis of Gnas mRNA in mouse T-ALL 1390 cells stably transduced with 2 Gnas specific shRNAs or NS shRNA. (D) Relative viability of mouse T-ALL cells transduced with NS or Gnas-specific shRNAs and treated with dexamethasone (0-1 µM) for 48 hours. GI₅₀ values are reported in table. Values are normalized to vehicle treated cells. (E) Quantification of apoptotic 1390 cells stably infected with Gnas shRNAs and treated with vehicle or dexamethasone (10 nM) for 48 hours. (F) qPCR analysis of NR3C1 target genes in 1390 cells stably transduced with NS, Gnas-specific shRNAs or a Nr3c1 shRNA following dexamethasone (100 nM) treatment of 6hrs. Values are normalized to vehicle treated controls. (G) Quantification of apoptotic 5059 cells stably infected with NS or a Gnas shRNA treated with vehicle or dexamethasone (10 nM) for 48 hours. (H) qPCR analysis of Gnas, Nr3c1, Bcl2l11, Klf13, and Zfp3612 in 5059 cells stably transduced with NS or a Gnas-specific shRNA after vehicle or dexamethasone (100 nM) treatment of 6 hours. Values are normalized to vehicle-treated controls. (I) Experimental design to assay dexamethasone resistance in vivo. (J) Kaplan-Meier curves of mice transplanted with 1390 leukemic cells transduced with NS (N = 19 mice) or shNr3c1 (N = 7) or shGnas-specific shRNAs (N = 12 mice) treated with vehicle or dexamethasone (15 mg/kg) for 3 weeks (5 days on; 2 days off). The difference in overall survival between vehicle vs dexamethasone treated mice is shown. All results are averages of at least 3 independent experiments, and error bars represent SEM. *P < .05, **P < .01, ***P < .001.