High ADAR1 expression is associated with resistance to IMiDs. (A) Western blot analysis of whole-cell lysates for the effect of lenalidomide (1 or 10 μM) on CRBN, IKZF1/3, IRF4, MYC, and PARP in isogenic KMS-11 and MM1.S LenR cells after 72 hours of treatment (left). The bar graphs (right) summarize the levels of IKZF1, IKZF3, IRF4, MYC, and cleaved-PARP, normalized to GAPDH, with quantification performed using ImageJ. (B) Flow cytometric analysis of apoptosis in isogenic KMS-11 and MM1.S LenR cells treated with lenalidomide (10 μM) for 6 days. Representative apoptosis plots and quantification, based on annexin V/Propidium Iodide (PI) staining, are shown. (C) Volcano plot showing the correlation between protein expression levels and responses to lenalidomide in hematopoietic and lymphoid cancer tissues (n = 58), including MM cell lines from the MD Anderson Cancer Center cell lines project dataset. The horizontal line represents the significance threshold at P = 0.05 level. (D) Expression levels of ADAR1 and CRBN in isogenic KMS-11 and MM1.S LenR cells validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (left) and western blot (right). Blot with ADAR1 p150 and ADAR1 p110 was probed with ab88574 (Abcam) and the blot with only ADAR1 p150 was immunoblotted with ab126745 (Abcam). (E) Kaplan-Meier analysis of overall survival (OS) and progression-free survival (PFS) in patients with MM with high vs low ADAR1 expression who received either lenalidomide-based (n = 927) or pomalidomide-based (n = 239) treatments from the CoMMpass study (IA21 release). Survival curves were generated by splitting patients into top 40% and bottom 40% groups based on ADAR1 expression values. Survival analysis was performed using Cox proportional hazard method with the log2-transformed gene expression profile, where the hazard ratio (HR), confidence interval, and P values were indicated. Data are presented as mean ± standard deviation (SD) of biological triplicates. Statistical significance is denoted by ∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001 as determined by two-tailed Student t-test. DMSO, dimethyl sulfoxide; Len, lenalidomide; OS, overall survival; PFS, progression-free survival.

High ADAR1 expression is associated with resistance to IMiDs. (A) Western blot analysis of whole-cell lysates for the effect of lenalidomide (1 or 10 μM) on CRBN, IKZF1/3, IRF4, MYC, and PARP in isogenic KMS-11 and MM1.S LenR cells after 72 hours of treatment (left). The bar graphs (right) summarize the levels of IKZF1, IKZF3, IRF4, MYC, and cleaved-PARP, normalized to GAPDH, with quantification performed using ImageJ. (B) Flow cytometric analysis of apoptosis in isogenic KMS-11 and MM1.S LenR cells treated with lenalidomide (10 μM) for 6 days. Representative apoptosis plots and quantification, based on annexin V/Propidium Iodide (PI) staining, are shown. (C) Volcano plot showing the correlation between protein expression levels and responses to lenalidomide in hematopoietic and lymphoid cancer tissues (n = 58), including MM cell lines from the MD Anderson Cancer Center cell lines project dataset. The horizontal line represents the significance threshold at P = 0.05 level. (D) Expression levels of ADAR1 and CRBN in isogenic KMS-11 and MM1.S LenR cells validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (left) and western blot (right). Blot with ADAR1 p150 and ADAR1 p110 was probed with ab88574 (Abcam) and the blot with only ADAR1 p150 was immunoblotted with ab126745 (Abcam). (E) Kaplan-Meier analysis of overall survival (OS) and progression-free survival (PFS) in patients with MM with high vs low ADAR1 expression who received either lenalidomide-based (n = 927) or pomalidomide-based (n = 239) treatments from the CoMMpass study (IA21 release). Survival curves were generated by splitting patients into top 40% and bottom 40% groups based on ADAR1 expression values. Survival analysis was performed using Cox proportional hazard method with the log2-transformed gene expression profile, where the hazard ratio (HR), confidence interval, and P values were indicated. Data are presented as mean ± standard deviation (SD) of biological triplicates. Statistical significance is denoted by ∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001 as determined by two-tailed Student t-test. DMSO, dimethyl sulfoxide; Len, lenalidomide; OS, overall survival; PFS, progression-free survival.

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