CDK7 inhibition reduces myeloma burden and enhances survival in vivo mouse models of MM. (A) BMMNC from 3 patients with relapsed MM were treated with 500 nM YKL-5-124 or DMSO for 24 hours. Cell viability in the CD138⁺ and CD138⁻ cell populations was evaluated by flow cytometry analysis. (B) Primary CD138⁺ cells were cultured in the absence or presence of YKL-5-124 for 3 days, and apoptotic cell death was assessed by flow cytometric analysis. Percentages of annexin V⁺/DAPI⁻ (early apoptosis) and annexin V⁺/DAPI⁺ (late apoptosis) cells are shown in the graphs. (C) A schematic diagram for the subcutaneous SCID model. (D) In the early treatment model, mice injected with H929 cells were randomized and treated with either YKL-5-124 or vehicle at first detection of tumor (tumor volume ∼100 mm³). Mice received 3 different doses of YKL-5-124 for 5 consecutive days per week for 2 weeks. Tumor volume was measured in 2 perpendicular dimensions by caliper once every week. Baseline values were not significantly different among groups. (E) Sublethally irradiated SCID mice were injected subcutaneously with AMO1 cells expressing CDK7^WT (left) or CDK7^C312S (right). Mice were randomized to a 5 or 10 mg/kg group, for 5 consecutive days per week for 2 weeks. Tumor volume was evaluated by caliper measurement. P values indicate significant difference between groups. ∗∗∗P < .001. (F) Western blot analysis was performed in cell lysates from tumors excised from representative mice and blotted with Rb and p-Rb antibodies. (G) Western blot analysis was performed in cell lysates from tumors excised from representative mice and blotted with indicated antibodies. Images were analyzed with Image J software and signals normalized to loading control. (H-J) NSG mice were orthotopically xenografted after intravenous injection with Molp8-luc cells. Upon detection of MM lesions (∼2 weeks after tumor cell injection), mice were randomly assigned to receive YKL-5-124 (2.5 or 5 mg/kg, intraperitoneal, 5 days per week, for 4 weeks) or vehicle control. Whole-body bioluminescence images (BLI) (H) and measurements (mean ± SEM) (I) are shown. Survival was evaluated from the first day of treatment until death. Survival curves (Kaplan-Meier) were analyzed using GraphPad analysis software (log-rank test, P = .0002) (J). (K) Monoclonal, tumor-derived, immunoglobulin (M-protein) levels were evaluated in MM-bearing Vk∗MYC mice before and after YKL-5-124 treatment (5 and 10 mg/kg) and normalized to time 0. BMMNC, bone marrow mononuclear cells; DMSO, dimethyl sulfoxide; NSG, NOD/SCID-γ; SCID, severe combined immunodeficiency.