Poster Presentation 35th Lorne Cancer Conference 2023

Linking determination of cell fate in the developing nervous system to diffuse midline glioma (#149)

Liam M Furst 1 2 3 , Enola M Roussel 1 3 , Ryan F Leung 1 3 , Ankita M George 3 , James Whittle 2 , Sarah Best 2 , Ron Firestein 4 5 , Maree C Faux 1 3 6 , David D Eisenstat 1 3 4 7
  1. Paediatrics, University of Melbourne, Parkville, Victoria, Australia
  2. Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Melbourne
  3. Murdoch Children's Research Institute, Parkville, VICTORIA, Australia
  4. Cancer for Cancer Research, Hudson Institute of Medical Research, Clayton, Victoria, Australia
  5. Molecular and Translational Sciences, Monash University, Clayton, Victoria, Australia
  6. Surgery, Royal Melbourne Hospital, Parkville, Victoria, Australia
  7. The Royal Children's Hospital Melbourne, Parkville, Victoria, Australia

Diffuse midline glioma, K27-altered (DMG) is a uniformly fatal cancer of the brainstem primarily diagnosed in children. Next-generation sequencing has revealed that 80% of patients with DMG harbour K27M mutations in histone variants H3.1 or H3.3, leading to global loss of H3K27me3 and a halted differentiation. This results in oncogenic reprogramming and transcriptional profiles that resemble oligodendrocyte precursor cells (OPCs). DLX2 is a homeobox transcription factor necessary for GABAergic interneuron differentiation during healthy neurogenesis, which directly represses transcription factors necessary for OPC differentiation. We hypothesise that altering DLX2 expression in OPC-like DMG cell lines will cause a change in cell fate, and may lead to a reduction in tumourigenicity. To evaluate DLX2 expression across a range of DMG cell lines, an RNA-seq database containing 60 DMG cell lines was assessed. An inverse correlation was observed between DLX2 and OPC marker (OLIG1/2) expression in histone mutated cell lines. Candidate cell lines with DLX2high/OLIG1/2low expression were selected and infected with a Cas9/mCherry lentivirus. mCherry+ cells were subsequently infected with DLX2 gRNAs lentivirus.  Single cell clones were grown and screened for knockdown of DLX2 by qPCR. Concurrently, DLX2low/OLIG1/2high cell lines were infected with a DLX2-mCherry or DNA binding mutant DLX2Q50E-mCherry lentivirus and populations of high, medium and low mCherry+ cells isolated by FACS. Immunoblot and/or qPCR analysis showed successful alteration of DLX2 expression. Analysis of histone H3 marks, K27me3 and K27ac as well as K27M in addition to OPC signature genes will be presented. Expression of altered OPC genes from a DLX1/2 knockout mouse as determined by RNA-seq and DLX2 altered cell lines will also be shown.  The manipulation of DLX2 expression in DMG cells will provide a model system for assessing the contribution of a homeobox transcription factor which is required for neurogenesis to cell fate regulation and effects on tumourigenicity in DMG.