Transcriptomic analysis of the in vivo effects of CDK8/19 inactivation in 22Rv1 xenografts growing in intact and castrated NSG mice
RNA-Seq analysis was carried out to investigate the effects of CDK8/19 inactivation in the tumors formed in intact and castrated NSG mice by different 22Rv1 derivatives, with or without SNX631 treatment. The 22Rv1 derivatives were generated as following: Parental 22Rv1 (Rv1-WT) were transduced with a lentivirus expressing luciferase, yielding the derivative Rv1-Luc. 22Rv1 cells with a double knockout of CDK8 and CDK19 (Rv1-dKO) were made via CRISPR/Cas9.  Rv1-dKO cells were further transduced with lentiviruses to generate Rv1-dKO re-expression derivatives that express wild-type CDK19 (Rv1-dKO-CDK19) and the kinase-inactive D173A mutant (Rv1-dKO-CDK19M).  SNX631 is a selective and orally bioavalable CDK8/19 inhibtor. Overall design: Rv1-WT, Rv1-Luc, Rv1-dKO-19 and Rv1-dKO-19M cells were inoculated s.c. (2x10^6 cells in 0.1 mL 50% Matrigel per animal) in the right flank of intact or castrated male NSG mice (8-10 weeks old). Castration was performed by either surgical orchiectomy (for Rv1-WT study) or Degarelix treatment (administered s.c. at 10 mg/kg monthly, for Rv1-Luc, Rv1-dKO-19, Rv1-dKO-19M studies), and tumor inoculation was done 10-14 days post castration. For SNX631 treatment studies, animals were randomly allocated to two study groups when the mean tumor size reached 100-200 mm^3. For Rv1-WT study, the treatment group received SNX631-medicated diet (500 ppm, producing 30-60 mg/kg/day dosage on average) and the control group received the control diet.  For Rv1-Luc study, the animals in the control group were dosed with vehicle (70% PEG-400/30% Propylene Glycol, 5 mL/kg, p.o., b.i.d.) and the treatment group was dosed with SNX631 (6 mg/mL solution in the vehicle, 5 mL/kg, p.o., b.i.d.) at 60 mg/kg/day.  At the end of studies, tumor xenografts were dissected from euthanized animals using sterilized surgical tools. Pieces of non-necrotic tumor tissues 50~100mm^3 were excised at the margin near midline section for RNA extraction.  At least four biological replicates (xenograft tumors from different animals) per study group were analyzed.