AZD4785 exerts anti-MM activity as demonstrated using different in vivo models . (A-C) CB-17 severe combined immunodeficiency (SCID) mice bearing subcutaneous xenograft MM.1S-Luc tumors were treated with either vehicle (phosphate-buffered saline [PBS]; 5× per week), ASO control (ctrl; 50 mg/kg; 5× per week), or AZD4785 (50 mg/kg; 5× per week). (A) KRAS and DUSP6 mRNA were measured in tumors collected 2 hours post–last dose after 5 days of dosing by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The expression was normalized to POLR2A and expressed relative to PBS. Data are shown as individual tumor data and treatment group geometric mean, and SE. ***P .0001. (B) KRAS protein in tumors collected 2 hours post–last dose after 5 days of dosing was detected by immunohistochemistry. Representative images and quantification of cytoplasmic KRAS (H score) are shown. Data represent individual tumor data and treatment group mean and standard deviation. Magnification ×20. *P = .037; **P = .002. (C) AZD4785 significantly inhibited MM.1S-Luc tumor growth compared with vehicle control (90.69%; P = .0015) 17 days after the initiation of treatment. Data are shown as the geometric mean of tumor volume and SE. (D) SCID/Bg mice were injected with MM.1S GFP⁺/Luc⁺ cells and treated with either ASO ctrl (25 mg/kg; 5× per week) or AZD4785 (25 mg/kg; 5× per week). Ex vivo quantification of KRAS membrane expression in harvested femur BM. PBS-treated mice were used as control. Magnification ×20. (E) SCID/Bg mice were injected with MM.1S GFP⁺/Luc⁺ cells and treated with ASO ctrl (25 mg/kg; 5× per week) or AZD4785 (25 mg/kg; 5× per week). Kaplan-Meier survival curve; P value determined by the log-rank test. (F) SCID/Bg mice were injected with MM.1S GFP⁺/Luc⁺ cells and treated with either vehicle PBS (5× per week), ASO ctrl (25 mg/kg; 5× per week), AZD4785 (25 mg/kg; 5× per week), bortezomib (0.5 mg/kg; 2× per week), or the combination of AZD4785 plus bortezomib. Detection of tumor growth was performed by measuring bioluminescence imaging (BLI) intensity at different time points post–MM cell injection (weeks 2, 3, and 4). (G) Visualization of MM cell BM colonization was evaluated within the indicated BM area of cleared skulls; green, MM.1S GFP⁺; red, immunostaining for murine CD31; white, bone stromal collagen visualized by second harmonic generation. Inhibition of MM cell colonization with femur BM as evaluated by immunostaining for human (h) CD138. Hematoxylin and eosin (HE) staining (magnification ×20 and ×40). (H) BM mononuclear cells were harvested ex vivo from femurs, subjected to RNA extraction, and evaluated for KRAS, DUSP6, and ETV4 mRNA levels by using qRT-PCR (2^−ΔΔCt), with normalization to GAPDH. *P < .05. s.q., subcutaneous.