DIPG-11. TRANSCRIPTIONAL REGULATION OF NEURAL PROGENITOR CELL FATE BY DLX HOMEOBOX GENES - RELEVANCE TO DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Abstract

DIPG is refractory to current therapies. The identification of histone H3.1/H3.3 K27M mutations in most DIPG has provided novel insights. DLX (distal-less) genes are expressed in the developing forebrain. The Dlx1/Dlx2 double knockout (DKO) mouse (dies at P0) has loss of tangential GABAergic interneuron migration to the neocortex. We have identified glutamic acid decarboxyase genes Gad1 and Gad2 as direct transcriptional targets of DLX1 and DLX2. In DIPG patients with H3.3 K27M there is decreased Dlx2 and increased expression of Myt1 (myelin transcription factor). We used bioinformatics and chromatin immunoprecipitation (ChIP) assays with embryonic ganglionic eminences to identify Olig2, Nkx2.2 and Myt1 promoter sequences as candidate DLX2 targets in vivo. DNA binding specificity was confirmed by electrophoretic mobility shift assays (EMSA). Co-expression of Dlx2 with luciferase reporter constructs of these ChIP-isolated promoter fragments of Olig2 and Nkx2.2 demonstrated repression of these gene targets in vitro. Assessment of target gene expression in the Dlx1/Dlx2 DKO forebrain by qPCR showed increased Olig2 and Nkx2.2 expression. Stable transfection of a murine HA-tagged DIPG cell line with Dlx2 increased expression of Gad1 and Gad2 and decreased expression of Olig2 and Nkx2.2. Of significance, using Western blotting we demonstrated decreased expression of H3.3 K27M and restoration of H3.3 K27 tri-methylation (me3). DLX transcription factors promote GABAergic interneuron differentiation and concomitant inhibition of oligodendroglial differentiation in neural progenitors by repression of a suite of genes including Olig2 and Nkx2.2. Restoration of H3 K27me3 expression in DIPG is a promising lead towards exploring differentiation as a therapeutic strategy for DIPG.