Effects of FAK/Pyk2 inhibitor 74718 treatment on MM tumor growth. (A) The inhibitory activity of VS-4718 against FAK and Pyk2 was measured in in vitro kinase assays (left). Dose-response curves and 50% inhibition/inhibitory concentration (IC₅₀) values are shown. The cellular inhibitory activity of VS-4718 against the autophosphorylation of FAK and Pyk2 was determined using p-FAK (Y397) and p-Pyk2 (Y402) ELISA assays (right). ELISA data were plotted as a function of concentration to determine half-maximal response (EC₅₀) values. (B) MM.1S, H929, and RPMI8226 cells were cultured in suspension or under adherent conditions on fibronectin and in the presence of IL-6 were treated with VS-4718 for 96 hours. Cell proliferation and viability were assessed using a CellTiter 96 Aqueous One Solution Proliferation assay. VS-4718 inhibited the proliferation of and viability of MM cells in both the presence and absence of fibronectin and IL-6. (C) MM.1S cells were treated with VS-4718 (0, 0.5, 1, 2.5, 5, an 10 µM/L) for 4 hours. Whole-cell lysates were subjected to immunoblotting using anti-p-Pyk2 and anti-Pyk2. (D-E) Five days after being IV injected with GFP⁺/Luc⁺-MM.1S cells (3 × 10⁶/ mouse), SCID/Beige mice were dosed with VS-4718 by oral gavage at 75 mg/kg twice daily for 4 weeks in total (n = 6 for vehicle; n = 7 for VS-4718). VS-4718 treatment effectively reduced MM tumor growth compared with vehicle control. (F) MM.1S and H929 cells were treated with VS-4718 (0, 2.5, 5, an 10 µM/L) for 24 hours. β-Catenin, c-Myc, and Cyclin D1 expression in these cells was inhibited by VS-4718. (G) SCID/Beige mice were injected subcutaneously into the left flank with either scramble probe-infected RPMI8226 cells (scr) or Pyk2-kd-RPMI8226 (PYK2 KD) cells. Tumor growth was measured twice weekly and volume (mg = mm³) calculated as (length [mm] × width²  [mm²])/2.