Fig. 1

CB15 significantly impacts the survival of MEF2D-fusion BCP-ALL. a A schematic diagram illustrating the constructions of MEF2D-fusion forms and CABIN1 utilized in experiments. The fusion partners of MEF2D encompass HNRNPUL1 and BCL9. BD: Binding Domain (Middle, Left) Bar graphs represent luciferase activity as a proxy for transcription activity (means ± SD; n = 3). These data indicate that CB15 more effectively inhibits MEF2D-dependent transcription activity than either CABIN1 full-length or CABIN1 Δ315-2143. (Bottom, Left) The binding affinity between GAL4-MEF2D and VP16-CABIN1 peptides was observed using a mammalian two-hybrid system. pVP16-CABIN1 constructs with 10-amino acid truncations were transfected into HEK293T cells in conjunction with pGAL4-MEF2D under a Gal-promoter-luciferase reporter system (Promega) to evaluate binding affinity with luciferase activity. (means ± SD; n = 4). (Middle, Right, First) CB15 binds to all the MEF2D-fusion proteins. CB15 and MEF2D fusion proteins were co-transfected into HEK293T cells, and their interaction was verified by immunoprecipitation assays. The abbreviations are as follows: M, MEF2D; MH, MEF2D::HNRNPUL1; MS, MEF2D::SS18 (breakpoint involving exon 8 of MEF2D fused in frame to exon 6 of SS18); MD, MEF2D::DAZAP1 (breakpoint involving exon 6 of MEF2D fused in frame to exon 7 of DAZAP1); MC, MEF2D::CSF1R (breakpoint involving exon 7 of MEF2D fused in frame to exon 11 of CSF1R). MEF2D::BCL9 proteins are denoted as MB1 (breakpoint involving exon 6 of MEF2D fused in frame to exon 9 of BCL9), MB2, MB3 (breakpoint involving exon 5 of MEF2D fused in frame to exon 9 of BCL9), and MB4 (breakpoint involving exon 5 of MEF2D fused in frame to exon 10 of BCL9). (Middle, Right, Second) The interactions of CB15 with wild-type MEF2D and MEF2D fusion proteins were assessed via immunoprecipitation assays with HEK293T cells. The CB15 L2172A mutant exhibited reduced binding affinity to both wild-type MEF2D and MEF2D fusion proteins compared to the native CB15. (Bottom, Right) The direct binding of CB15 to MEF2D-fusion proteins influences their transcriptional activity. MEF2D-binding defective CB15 L2172A mutant exhibits weaker transcriptional inhibition than CB15 WT via a MEF2-luciferase assay. Bar graphs represent luciferase activity as a proxy for transcription activity (means ± SD; n = 3). Statistical comparisons were executed using two-tailed Student’s t-tests, with analyses conducted using GraphPad Prism 8. Significant differences were indicated as p-values: *p < 0.05, **p < 0.01, and ***p < 0.001. b The expression CB15 significantly reduced the growth rate in KAZUMI-7 cells. Cells were exposed to 200 ng/ml doxycycline for 6 days. Cell growth rate was assessed by measuring cell density every 2 days. (means ± SD; n = 3). Statistical differences were determined by the two-tailed Student’s t-test. p < 0.05 *, p < 0.01 **, p < 0.001 *** ns: not significant c The expression of CB15 significantly triggered cell death in KAZUMI-7 cells. Cells were exposed to doxycycline for 6 days. Cells were subjected to a flow cytometry analysis of apoptosis using Annexin-V conjugated Alexa-647 and DAPI dual staining. d Western blot analysis exhibits marker proteins indicating apoptosis and components of BCP-ALL core regulatory circuitry (CRC) during CB15 expression. ACTB served as a loading control. SE short exposure, LE long exposure