Fig. 7: Engineering ssrA into monobody to photo-manipulate leukemia cell growth with PhoBIT2 in vivo.
From: Engineering of photo-inducible binary interaction tools for biomedical applications
a Schematic illustrating inhibition of BCR-ABL kinase activity by ssrA2-fused HA4-7c12 fusion monobody upon photostimulation. In the dark state, the BCR-ABL oncofusion protein in K562 cells is not inhibited by ssrA2-inserted monobody HA4-7c12, maintaining a high kinase activity to support leukemia cell growth. Upon blue light stimulation, CRY2-sspB2 oligomerizes and enhances the binding of ssrA2-fused monobody with its antigen BCR-ABL, subsequently inhibiting phosphorylation of BCR-ABL in K562 cells to suppress leukemia. b The 3D structure showing a monobody (clone 7c12) complexed with the ABL-SH2 domain (PDB entry: 3T04), with the ssrA2 insertion sites indicated by spheres. c Quantification of light-induced mitochondrial intensity change (Δ7F/F0) of indicated ssrA2-engineered monobody variants. n = 15 cells from three biological replicates. Data are shown as mean ± s.e.m. d Confocal images of HeLa cells co-expressing fusion monobody HA4-7c12 with or without ssrA2 insertion (top panel, WT; bottom panel, S2 variant) and Ntom20-Venus-Abl-SH2 upon blue light stimulation. Enlarged mitochondria regions are shown in white boxed insets. Scale bar, 10 µm. e Constructs used for viral transduction in K562 cells. Part A: Fusion monobody HA4-7c12 with (A1) or without (A2) ssrA2 insertion (red bar). Part B: mCerulean-CRY2-sspB2. Created in BioRender. Lee, Y. (2025) https://BioRender.com/t9kki1m. f Western blot detecting endogenous BCR-ABL and its phosphorylation state in K562 cells expressing indicated constructs and the non-expression control (K562WT), with or without photostimulation. GAPDH is used as the loading control. g Schematic illustrating the in vivo experimental setup. 5 × 106 K562 leukemia cells expressing indicated constructs were injected (s.c.) in the flank of SCID mice. Starting from day 2 post-K562 inoculation, mice were either subjected to photostimulation for 11 days or maintained in the dark. Created in BioRender. Lee, Y. (2025) https://BioRender.com/t9kki1m. h Images of isolated tumors for the indicated groups on day 15. i, j Quantification of xenograft tumor sizes (i) and tumor weights (j). n  =  7 mice (mean ± s.e.m.). j Line at median for the scatter plot. Statistical analysis was performed using two-way ANOVA followed by Turkey’s post hoc test. (Source Data).
