ADAR1 inhibition enhances lenalidomide sensitivity in vivo in MM mouse models. (A) Tumor growth profiles of RPMI-8226-isogenic cells with differential ADAR1 and MDA5 expression in subcutaneous MM xenograft models. Double-KD represents concurrent ADAR1 and MDA5 KD. Once tumors reached 150 mm³, mice were administered with lenalidomide (25 mg/kg) or DMSO (vehicle control) intraperitoneally, for four consecutive days per week for two weeks (n = 6 mice per group). Significant differences were determined by two-tailed Student t test. (B) Tumor volume and weight of tumors isolated from the respective isogenic RPMI-8226 subcutaneous xenograft mice groups after two weeks of lenalidomide treatment (25 mg/kg). Significant differences were determined by two-tailed Student t test. (C) Western blot analysis of tumor lysates from RPMI-8226 subcutaneous xenografts, assessing levels of total ADAR1 (ab88574; Abcam), ADAR1 p150 (ab126745; Abcam), MDA5, PARP, and cleaved caspase-3 after two weeks of lenalidomide treatment (25 mg/kg). #1 #2 #3 refer to the tumors isolated from the different respective isogenic RPMI-8226 subcutaneous xenograft mice (shSCR, shMDA5, shADAR1, and Double KD) at two weeks after lenalidomide treatment (25 mg/kg). (D) Relative mRNA expression of ISGs and IFNs (IFN-α and IFN-β) in tumors isolated from the respective isogenic RPMI-8226 subcutaneous xenograft mice groups after two weeks posttreatment with lenalidomide (25 mg/kg), determined by qRT-PCR. Data are presented as mean ± SD of biological triplicates. Significant differences were determined by two-tailed Student t test. (E) Bioluminescence imaging of total body bioluminescence in intravenous (IV) disseminated xenografts with RPMI-8226 firefly luciferase-green fluorescent protein (Luc-GFP)–isogenic cells at pretreatment (day 0 of DMSO/lenalidomide randomization) and one week after treatment of DMSO/lenalidomide treatment (25 mg/kg). Significant differences were determined by 2-tailed Student t test. (F) Kaplan-Meier survival curves of the IV disseminated xenografts transplanted with RPMI-8226 Luc-GFP-isogenic cells treated with lenalidomide (25 mg/kg) or DMSO control for one week. OS was followed till mice were humanely euthanized at the first sign of morbidity. IV disseminated xenograft mice survival curves were analyzed using the Mantel-Cox log-rank test. Statistical significance is denoted by ∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001. (G) Proposed model of mechanism underlying the role of ADAR1 in regulating resistance to IMiDs in MM. Increased levels of ADAR1 in MM cells suppress IMiD-induced dsRNA-sensing pathways through RNA editing, which destabilizes dsRNA and limits their recognition by dsRNA sensors. In contrast, ADAR1 loss sensitizes myeloma cells to IMiD (lenalidomide) by activating the MDA5 dsRNA-sensing pathway, leading to increased dsRNA detection, inflammation, and IFN response.