Targeting TBL1X results in cyclin D1 depletion and cell cycle arrest in MCL cells. Cyclin D1 protein and CCND1 RNA levels in MCL cell lines (n = 2) after (A-B) TBL1X shRNA KD with specific constructs (n = 2; sh425 and sh525) compared with EV control or (C-D) tegavivint treatment (IC50, 12-18 hours) compared with DMSO control. (E) Cyclin D1 protein levels in primary MCL patient samples (n = 3) treated with tegavivint (18 hours, 0-500 nM). (F) Cyclin D1 protein levels in MCL cell lines (n = 2) treated with proteasome inhibitor Mg132 or translation inhibitor cycloheximide combined with tegavivint compared with tegavivint-only treated cells. Cells were first treated with tegavivint (IC50), then MG132 (10 μM) or cycloheximide (10 μg/mL) was added for the last 1.5 or 1 hour(s), respectively, for a total incubation time of 18 hours. (G) Cell cycle phase analyses (propidium iodide staining and flow cytometry) and cyclin D1 protein levels (immunoblot) in pharmacologically synchronized (500 nM palbociclib) MCL cell lines (n = 2) treated with tegavivint (IC50, 6-12 hours). Tegavivint IC50 concentrations = 95 nM for JeKo-1 and 65 nM for SP-53. DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G1, gap 1 phase; G2/M, gap 2 phase/mitosis; hr, hour; ns, not significant; S, synthesis phase. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.
