PARPi+CDK4/6i combination treatment leads to tumor growth inhibition through suppression of p-RB-E2F1 and E2F1 signaling, and suppression of CDK1, p-S70-BCL-2, and antiapoptotic protein MCL-1 in prostate cancer xenograft models compared with PARPi or CDK4/6i single-agent treatment. A and B, Tumor growth curves and terminal wet weights of C4–2b-ENZR and NCI-H660 xenograft tumors treated with PARPi (olaparib), CDK4/6i (palbociclib), or olaparib + palbociclib combination. Tumor volumes were measured every 3 days. Left, y-axis shows mean tumor volume (mm3), and x-axis shows days of treatment. Treatments were initiated when the tumor volume reached approximately 50 mm3. olaparib: 40 mg/kg/days, 5 days/week, i.p.; palbociclib: 100 mg/kg/day, 5 days/week, oral gavage. Right, tumor wet weights at termination. ANOVA analysis was used to determine the statistical significance of tumor volumes as indicated, and t tests were used to determine statistical significance of the differences of tumor wet weights as indicated: ns, not significance, *, P < 0.05; **, for P < 0.01, and ***, for P < 0.001. ˇ, indicates that the combination treatment revealed a moderate synergistic interaction between olaparib and palbociclib by Bliss independence analysis (44). C, Immunoblot analysis of treated xenograft tumors shows expression of proteins involved in E2F1 signaling and CDK1 activation, as well as antiapoptotic proteins including “prosurvival sensor” p-S70-BCL-2 and MCL-1 as indicated. Immunoblot signals (C) were quantified and normalized to vinculin, and the relative band intensities shown below each protein specific IB band image in the figure. D, Proposed mechanism by which PARPi+CDK4/6i combination treatment suppresses growth and NED markers and induces apoptosis in prostate cancer models in vitro and in vivo.