BACKGROUND: The identification of H3.3/H3.1K27M mutations in most diffuse midline gliomas (DMG), formerly called diffuse intrinsic pontine glioma (DIPG), has changed our understanding of this disease. H3K27M mutations usually demonstrate global loss of H3K27 trimethylation (me3) with gain of H3K27 acetylation (ac). Single cell RNAseq has identified the putative cell of origin as oligodendroglial progenitor cells (OPC). The distalless/DLX homeobox gene family is necessary for the differentiation and tangential migration of committed neural progenitors to become GABAergic interneurons. Dlx1/Dlx2 double knockout (DKO) cells from the ganglionic eminences (GE) transplanted into a wild-type environment become oligodendrocytes.
RESULTS: We identified DLX2 occupancy of early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required for OPC differentiation in vivo and confirmed direct DLX2 protein-promoter DNA binding in vitro. Co-expression of Dlx2 with target sequences reduced reporter gene expression in vitro. There was increased expression of OLIG2, NKX2.2 and PLP-1 expression in vivo, consistent
with de-repression in the absence of Dlx1/Dlx2 homeobox gene function. Transient overexpression of a Dlx2-GFP construct into murine DIPG cells from an RCAS-tva mediated genetically engineered mouse model (GEMM) that spontaneously develops DMG resulted in significant increases in expression of Gad isoforms with concomitant decreases in Olig2 and Nkx2.2. Dlx2-transfected mDMG cells also demonstrated reduced migration, invasion and colony formation in vitro. Of significance, there was global restoration of H3K27me3 with corresponding loss of H3K27ac expression in transfected cells when compared to controls.
CONCLUSIONS: DLX2 promotes GABAergic differentiation and migration while concomitantly repressing OPC differentiation in vivo. Developmental reprogramming of mDMG cells by DLX2 demonstrates the potential role for directed differentiation strategies towards improving patient outcomes for this devastating pediatric cancer.