Fig. 7.

ACY-1215 alters chromatin accessibility of T-cells and downregulates mTOR signaling pathways. (a-c) ATAC-seq was performed in T-cells from three melanoma patient PBMCs following expansion with 500nM ACY-1215 or DMSO. (a) Scattered dot plot displaying significant peaks (red dots; q < 0.05) of open versus closed chromatin in ACY-1215-treated over DMSO control. Representative (b) open peaks in T-BET, CD45RO, IFNγ and CCR7 gene regions, and (c) closed peaks in AKT and S6K gene regions, in ACY-1215-treated over DMSO control. Significant regions are highlighted in red. All samples were adjusted for false discovery rate (FDR). Three patient samples were assessed. (d-g) After T-cell expansion with DMSO or HDACi, phosphorylation of (d) mTOR S2448, (e) S6K S424, (f) AKT S473 and (g) SGK1 Y238 in CD4+ and CD8+ T-cells was determined by flow cytometry and the geometric mean fluorescence (gMFI) graphed. At least four patient samples were assessed in two to three experiments for each phosphorylated marker evaluated. Each paired line represents an individual patient sample. (h) T-cells from five patient samples in two experiments were treated with GSK650394 100nM (SGK1 inhibitor), ACY-1215 500nM, or DMSO, activated and assessed for IL-4 secretion after 72 h. (i) CD8+ T-cells from four patient samples in two experiments were evaluated by flow cytometry for CD45RO + CD62L+ central memory percentage after expansion in A674563 50nM (AKT inhibitor) or ACY-1215 500nM. Comparisons were made against DMSO