Figure 5: HDAC3 inhibition induces interferon signaling and antigen presentation in both CREBBP wild-type and mutant cells.

A) Flow cytometry was performed for HLA-DR following exposure to a selection of HDAC inhibitors at 10μM for 72h. This shows that HDAC inhibitors with a range specificities are able to induce MHC class II, but HDAC3 selective inhibition using BRD3308 is sufficient for this effect. B) Dose titrations of histone deacetylase inhibitors from (A) with peripheral blood CD4 and CD8 T-cells from healthy donors. C) A heat map of interferon responsive and antigen presentation genes from RNA-seq data shows an increased expression in both CREBBP^WT and CREBBP^R1446C cells. Data represent duplicate experiments for each clone and are normalized to control treated cells from the same experiment. D) Gene set enrichment analysis of the genes that have reduced H3K27Ac in CREBBP^R1446C cells shows that the expression of these same genes are coordinately increased by BRD3308 treatment in CREBBP^WT cells. E) A heat map of hypergeometric enrichment analysis results of RNA-seq data shows that BRD3308 induces the induction of similar gene sets in both CREBBP^WT and CREBBP^R1446C cells. F) A density strip plot, normalized to the mean expression in control (BRD4097)-treated CREBBP^WT cells shows the relative expression of the set of genes with reduced H3K27Ac in CREBBP^R1446C cells. This shows that these genes are induced by BRD3308 in CREBBP^WT cells, resulting in expression levels greater than baseline. Further, CREBBP^R1446C cells can be observed to start below baseline, with the induction by BRD3308 resulting in expression levels similar to that observed in control treated CREBBP^WT cells. The 4 samples per condition represent duplicate experiments in each of the two clones for each genotype. G) The firefly luciferase luminescence of two unique IRF1 reporters (R1 and R2) is shown, normalized to renilla luciferase from a control vector and shown as fold change compared to untreated cells. CREBBP^WT cells show increased IRF1 activity following IFN-γ treatment (positive control; grey), but not following treatment with BRD3308 (green). In contrast, CREBBP^R1446C cells show increased IRF1 activity following BRD3308 treatment, to a level that is similar to that observed with IFN-γ treatment. (T-test vs control-treated cells, **P<0.01, ***P<0.001). H) The role of IFN-γ in inducing MHC class II expression following BRD3308 in CREBBP^R1446C cells was assessed with a blocking experiment. Blocking IFN-γ with a neutralizing antibody (αIFN-γ) significantly reduced the induction of MHC class II, as measured by flow cytometry for HLA-DR, but the induction by BRD3308 with α IFN-γ remained significantly higher than vehicle with αIFN-γ (T-test, ***P<0.001).