Figure 4. Genetic or pharmacologic CDK7 inhibition diminish MM cell cell proliferation and potentiate the anti-myeloma effects of PIs and BH3-mimetics.

(A) OPM2 cells were infected with lentivirus encoding Cas9 and sgRNA targeting GFP or CDK7. After infection for 48 hr and selection with puromycin (0.2 μg/ml) for 5 days, cells were collected and the expression of CDK7 was monitored by immunoblotting. Cell vitality was determined by the cell titer-glo assay at day 2, 4, 6 and 8. **P<0.01. (B) Cells were seeded in a 48-well round-bottom plates (250 cells per well), and images were obtained on day 2, 4, 6 and 8 with an IX71-Olympus research inverted system microscope at 40× magnification. (C) OPM2 cells were infected with lentivirus encoding Cas9 and sgRNA targeting GFP or CDK7 for 48 hr without selection, and then treated with the indicated concentration (nM) of Btz (bortezomib), Cfz (carfilzomib), or ABT-199 (venetoclax) for 24hr, after which cell death was analyzed by flow cytometry following staining with 7-AAD. *P<0.05, **P<0.01. (D) U266 cells were incubated with THZ1 ± Btz, Cfz, or ABT-199 for 24 hr, after which cell death was analyzed by flow cytometry after staining with 7-AAD. **P<0.01. (E) U266 cells were exposed (24 hr) to varying concentrations of Btz ± THZ1 at a fixed ratio (1:10), ABT ± THZ1 at a fixed ratio (10:1) or Cfz ± THZ1 at a fixed ratio (1:10), after which apoptosis was determined by 7-AAD staining. Combination Index (CI) values less than 1.0 denote a synergistic interaction. The results are representative of three separate experiments.