Fig 2: Effects of CABIN1 deletion and mutations on the NFAT and MEF2B transcriptional activities. A, Western blot analysis of CABIN1 expression in WT SSK41 cells or in SSK41 cells transduced with lentiviral vectors expressing either a control shRNA or CABIN1-specific shRNAs. The expression of the housekeeping gene GAPDH was used as a loading control. B, Luciferase reporter assay for NFAT (top) and MEF2 (bottom) transcriptional activity in SSK41 cells transduced with lentiviral vectors expressing either a control shRNA or CABIN1-specific shRNAs. Where indicated, cells were stimulated for 4 hours with a-IgM F(ab’)2. *, P = 0.001; **, P < 0.0002. C, Western blot analysis of CABIN1 expression in WT SSK41 cells or in SSK41 cells in which CABIN1 was initially knocked down using 3'-UTR targeting shRNA followed by expression of HA-tagged CABIN1 WT or mutants. CABIN1 was detected using anti-CABIN1 and anti-HA antibodies, while expression of the housekeeping gene GAPDH was used as a loading control. D, Luciferase reporter assay for NFAT (top) and MEF2 (bottom) transcriptional activity in SSK41 CABIN1 KD cells and cells expressing indicated CABIN1 constructs as shown in C. Where indicated, cells were stimulated for 4 hours with a-IgM F(ab’)2, in comparison to stimulated HA-WT: *, P < 0.005; **, P < 0.0001; ***, P = 0.00006. E, Representative immunoprecipitation (IP) assays with anti-HA antibodies using whole-cell protein extracts from unstimulated and a-IgM F(ab’)2–stimulated SSK141 cells expressing different CABIN1 mutants, as shown in C followed by Western blotting using indicated antibodies. Also shown mean and SD of relative SIN3A densitometry adjusted to immunoprecipitated CABIN1 in each cell type versus WT cells from three independent experiments. Statistical analyses of relative densitometry in mutants versus WT cells. *, P < 0.05; **, P < 0.01.

Fig 3: Inhibition of DNA-PKcs reduces phosphorylation of CHK2 and stabilizes the calcineurin inhibitor, Cabin1.A) Western blot analysis of Jurkat lysates following activation with PMA+PHA and NU7441 treatment. Activation increased phosphorylation of DNA-PKcs and CHK2. DNA-PKcs inhibition reduced CHK2 phosphorylation and elevated Cabin1 expression. GAPDH was used as a loading control. B) Schematic depicting the signaling pathway in T cells used by DNA-PKcs to regulate IL-2 production. DNA-PKcs phosphorylates CHK2 which in turns phosphorylates Cabin1 targeting it for destruction. This alleviates calcineurin inhibition causing an increase in translocation of NFAT and IL-2 production. CaN, calcineurin.

Fig 4: CABIN1 alterations and expression. A, Oncoprint showing alterations occurring across genes in the NFAT signaling pathway. Columns correspond to patients while genes are listed in rows. B, Lollipop plot showing the somatic nonsilent mutations found in CABIN1 gene with annotations of the amino acid changes. C, Heat map of CN status for cytoband 22q11 including the CABIN1 locus (annotated). Across this cytoband, segment colors vary from blue for strong CN losses to red for strong CN gains. Each row represents a sample, and each column represents a reported CN segment. Sample mutation status is noted to the right. D, Immunofluorescence expression analysis of CABIN1 (red), CD20 (green, top), and CD3 (green, bottom) in normal lymph node. Magnification: top and bottom left, 4×; top and bottom right, 40×. E, IHC analysis of CABIN1 expression in different MALT lymphomas (magnification all 20×). Top left, OAMZL with WT CABIN1; top right, OAMZL with CABIN1 CN losses; bottom left, parotid MZL; bottom right, lung MZL. White bars, 50 μm.

Fig 5: Effects of CABIN1 alterations on gene expression and NFAT activation. A, Heat maps showing FPKM values of genes that are differentially expressed in WT versus CABIN1 KD SSK41 cells after α-IgM F(ab’)2 stimulation. These genes are significantly enriched for leukocyte activation signature, NFAT and MAF2B targets. B, IHC analysis of NFAT expression and localization in OAMZL tumors with WT CABIN 1 (top left) and CABIN1 CN losses (bottom left). For control, NFAT antibody staining showed cytosolic expression in germinal center of normal lymph nodes (top right), while no staining detectable in the uterus (bottom right). Magnification: top left and right and bottom left, 40×; bottom right, 10×. Insets, 100×. C, NFATc1 (green) immunofluorescence in OAMZL tissue sections with WT and mutated CABIN1. Image analysis of mean nuclear fluorescence signal intensities were done using ImageJ software. ****, P < 0.0001. D, Cell viability assay of SSK41 WT, shRNA control, and CABIN1 KD cells treated for 72 hours with increasing concentrations of cyclosporin A. HEK293 cells are not sensitive to cyclosporin A and were used as control.