Effects of CDK7 inhibition with THZ1 on tumor xenograft development using TSC1-null HCV.29 cells, and model of effect of CDK7 inhibition. (A) TSC1-null HCV.29 xenograft mice were treated with vehicle (CTRL), rapamycin (RAP; 3 mg/kg i.p. three times per wk), THZ1 (10 mg/kg i.p. two times daily), or combined rapamycin and THZ1, for 30 d, when control tumors reached 100 mm³ in size. Tumor size was assessed every third day using digital calipers (n = 5 per group, two tumors per mouse). (B) Tumor xenograft cell proliferation was markedly reduced in mice treated with rapamycin, THZ1, or both, in comparison to control, as assessed by nuclear staining using Ki-67. This was quantified by counting four to six random fields per section. Scale bar, 50 µm. (C) Tumor volume of HCV.29.TSC1⁻ xenograft mice treated with rapamycin, THZ1, or the combination in the 60 d following treatment cessation (continuation of A). n = 4 tumors per group. (D) Apoptotic cell death was increased in tumors from mice treated with THZ1 or combined rapamycin-THZ1, in comparison to vehicle or rapamycin treatment labeled by TUNEL. This was quantified by counting four to six random fields per section. n = 4 tumors per group. Scale bar, 50 µm. (E) Diagram showing GSH synthetic pathway and ROS generation in TSC-deficient cells. Top, TSC-deficient cells have hyperactive mTORC1, leading to increased ROS, NRF2 induction, and an increase in transcription of GSH synthetic genes to yield more GSH to buffer the increased ROS. Bottom, THZ1 inhibits transcription by covalently binding to CDK7 and blocking RNA Pol II phosphorylation, leading to marked reduction in NRF2 and downstream gene expression, depletion of GSH stores, and apoptotic cell death. Each data point represents the mean ± SEM with **, P < 0.01; ***, P < 0.001.