ADAR1 reduces IMiD sensitivity in MM cells in an RNA editing–dependent manner. (A) Cell viability of ADAR1-KD RPMI-8226 and OCI-MY5 cells at 6 days after treatment with increasing concentration of lenalidomide, determined by CellTiter-Glo (CTG) assay. The 50% inhibitory concentration (IC50) values were computed with CompuSyn following Chou-Talalay method. (B) Colony formation assay of ADAR1-KD RPMI-8226 and OCI-MY5 cells in methylcellulose-based media, treated with lenalidomide (1 or 10 μM) over 14 days. (C) Apoptosis analysis in ADAR1-KD RPMI-8226 and OCI-MY5 cells treated with lenalidomide (1 or 10 μM) for 6 days, detected using annexin V/PI staining by flow cytometry. (D) Western blot analysis of whole-cell lysates for the effect of lenalidomide (1 or 10 μM) on the total ADAR1 (ab88574; Abcam), ADAR1 p150 (ab126745; Abcam), CRBN, IKZF1/3, IRF4, MYC, PARP, and cleaved caspase-3 from ADAR1-KD RPMI-8226 and OCI-MY5 cells after 144 hours (6 days) of treatment. (E) Western blot validation of the OE of ADAR1 p150 WT (ADAR1 p150-WT) or ADAR1 p150 catalytic DeAD mutant (ADAR1 p150-DeAD) in KMS-11 and MM1.S cells. Blot with ADAR1 p150 and ADAR1 p110 was probed with ab88574 (Abcam) and the blot with only ADAR1 p150 was immunoblotted with ab126745 (Abcam). (F) Sanger sequencing chromatograms illustrating the RNA editing levels of ADAR1 target genes (MAGT1 and SRP9) in KMS-11 and MM1.S cells in response to ADAR1 p150 OE (EV, ADAR1 p150-WT, and ADAR1 p150-DeAD). Percentages denote the editing frequencies of the corresponding editing site indicated by the arrow. (G) Cell viability of ADAR1 p150-overexpressed KMS-11 and MM1.S cells at 6 days after treatment with increasing concentration of lenalidomide, determined by CTG assay. The IC50 values were computed with CompuSyn following Chou-Talalay method. (H) Cell viability of ADAR1-KD RPMI-8226 and OCI-MY5 cells at 6 days after treatment with increasing concentration of pomalidomide, determined by CTG assay. The IC50 values were computed with CompuSyn following Chou-Talalay method. (I) Western blot analysis of whole-cell lysates for the effect of pomalidomide (0.1 or 1 μM) on the total ADAR1 (ab88574; Abcam), ADAR1 p150 (ab126745; Abcam), CRBN, IKZF1/3, MYC, PARP, and cleaved caspase-3 from ADAR1-KD RPMI-8226 and OCI-MY5 cells after 144 hours (6 days) of treatment. Data are presented as mean ± SD of biological triplicates. Significance differences were determined by two-tailed Student t test and for the line bracket by 1-way analysis of variance (ANOVA) with Tukey post hoc test. Statistical significance is denoted by ∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001. DeAD, catalytic deaminase domain mutant; EV, empty vector; Pom, pomalidomide; WT, wild-type.

ADAR1 reduces IMiD sensitivity in MM cells in an RNA editing–dependent manner. (A) Cell viability of ADAR1-KD RPMI-8226 and OCI-MY5 cells at 6 days after treatment with increasing concentration of lenalidomide, determined by CellTiter-Glo (CTG) assay. The 50% inhibitory concentration (IC50) values were computed with CompuSyn following Chou-Talalay method. (B) Colony formation assay of ADAR1-KD RPMI-8226 and OCI-MY5 cells in methylcellulose-based media, treated with lenalidomide (1 or 10 μM) over 14 days. (C) Apoptosis analysis in ADAR1-KD RPMI-8226 and OCI-MY5 cells treated with lenalidomide (1 or 10 μM) for 6 days, detected using annexin V/PI staining by flow cytometry. (D) Western blot analysis of whole-cell lysates for the effect of lenalidomide (1 or 10 μM) on the total ADAR1 (ab88574; Abcam), ADAR1 p150 (ab126745; Abcam), CRBN, IKZF1/3, IRF4, MYC, PARP, and cleaved caspase-3 from ADAR1-KD RPMI-8226 and OCI-MY5 cells after 144 hours (6 days) of treatment. (E) Western blot validation of the OE of ADAR1 p150 WT (ADAR1 p150-WT) or ADAR1 p150 catalytic DeAD mutant (ADAR1 p150-DeAD) in KMS-11 and MM1.S cells. Blot with ADAR1 p150 and ADAR1 p110 was probed with ab88574 (Abcam) and the blot with only ADAR1 p150 was immunoblotted with ab126745 (Abcam). (F) Sanger sequencing chromatograms illustrating the RNA editing levels of ADAR1 target genes (MAGT1 and SRP9) in KMS-11 and MM1.S cells in response to ADAR1 p150 OE (EV, ADAR1 p150-WT, and ADAR1 p150-DeAD). Percentages denote the editing frequencies of the corresponding editing site indicated by the arrow. (G) Cell viability of ADAR1 p150-overexpressed KMS-11 and MM1.S cells at 6 days after treatment with increasing concentration of lenalidomide, determined by CTG assay. The IC50 values were computed with CompuSyn following Chou-Talalay method. (H) Cell viability of ADAR1-KD RPMI-8226 and OCI-MY5 cells at 6 days after treatment with increasing concentration of pomalidomide, determined by CTG assay. The IC50 values were computed with CompuSyn following Chou-Talalay method. (I) Western blot analysis of whole-cell lysates for the effect of pomalidomide (0.1 or 1 μM) on the total ADAR1 (ab88574; Abcam), ADAR1 p150 (ab126745; Abcam), CRBN, IKZF1/3, MYC, PARP, and cleaved caspase-3 from ADAR1-KD RPMI-8226 and OCI-MY5 cells after 144 hours (6 days) of treatment. Data are presented as mean ± SD of biological triplicates. Significance differences were determined by two-tailed Student t test and for the line bracket by 1-way analysis of variance (ANOVA) with Tukey post hoc test. Statistical significance is denoted by ∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001. DeAD, catalytic deaminase domain mutant; EV, empty vector; Pom, pomalidomide; WT, wild-type.

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