Figure 4.

IKBKE/TBK1 modulates MYC through phosphorylation of YB-1. (A) Phosphorylated YB-1 is decreased in AML cells following either treatment with momelotinib or shRNA-mediated knockdown of IKBKE or TBK1, as determined by RPPA. MOLM14 or OCIAML5 cells were treated with momelotinib (CYT; 2.5 µM) for 2 or 6 hours before whole cell extracts were prepared for RPPA analysis. For shRNA experiments, cells were infected with lentiviral vectors containing either shRNA targeting luciferase (shLUC) as a negative control or the indicated IKBKE/TBK1 shRNA. Cells were selected under puromycin for 54 hours before whole cell extracts were collected for RPPA analysis. (B) MOLM14 cells were treated with momelotinib (CYT; 2.5 µM), MRT (2.5 µM), BX795 (2.5 µM), or ruxolitinib (10 µM) for 6 hours, after which protein was isolated for the indicated immunoblots (top left). MOLM14 cells were treated with momelotinib (1.25 µM) for the indicated times, and then immunoblots were performed (top right). MOLM14 or OCIAML5 cells were treated with the indicated dose of BX795 for 2 hours of treatment, after which immunoblots were performed. (C) MOLM14 cells were treated with momelotinib (CYT; 0.625 µM) or LY294002 (10 µM) for 2 hours, and OCIAML5 cells were treated with CYT (2.5 µM), MRT (2.5 µM), BX795 (2.5 µM), or LY294002 (10 µM) for 2 hours, after which protein was isolated for the indicated immunoblots. (D) MOLM14 cells were treated with the indicated concentration of momelotinib for 2 hours, after which protein was isolated for the indicated immunoblots. (E) AML cell lines were treated with vehicle control or momelotinib (CYT; 1.25 µM for 6 hours), and then the indicated immunoblots were performed. (F-G) MOLM14 or OCIAML5 cells were infected overnight with lentiviral vectors containing shLuc (control) or shRNAs targeting YB-1. After 30 hours of puromycin selection, RNA and protein were isolated for analysis. Results are representative of 3 replicates. (H-I) MOLM14 or OCIAML5 cells were infected overnight with lentiviral vectors containing shLuc or shRNAs targeting YB-1. Cells were then further expanded to low density. Relative viability was evaluated 4 days after lentiviral infection, and apoptosis was evaluated 3 days after infection by annexin V/PI staining (H). Apoptosis was also evaluated 3 days after lentiviral infection by PARP cleavage (I). (J) MOLM14 cells were infected with an empty lentiviral vector or a vector expressing constitutively activated YB-1 (YB-1-S102D). Cells were selected in puromycin for 48 hours and then treated with vehicle (DMSO) or momelotinib (CYT; 0.625 µM) for 6 hours, after which RNA and protein were isolated for analysis. (K) OCIAML5 cells were infected overnight with an empty lentiviral vector or a vector expressing YB-1-S102D. Cells were then treated with vehicle (DMSO) or momelotinib (CYT; 0.625 µM) for 48 hours and then analyzed by flow cytometry for apoptosis by annexin V/PI staining. (L) MOLM14 cells were treated with vehicle (DMSO) or momelotinib (CYT; 1.25 µM) for 6 hours, after which the cells were fixed, and chromatin was isolated for ChIP to evaluate YB-1 binding at the MYC promoter site. Results are means ± standard deviation of 4 replicates.