DIPG-28. RADIATION DNA DAMAGE REPAIR INHIBITION BY GSK-J4 INDUCED CHROMATIN COMPACTION IN DIPG

Abstract

INTRODUCTION: Focal radiation therapy has long been and still remains the only treatment option for diffuse intrinsic pontine glioma (DIPG). However, all children who suffer from this inoperable and uniformly fatal cancer show evidence of disease progression within months of completing radiation therapy, especially those who harbor the H3.3K27M mutation. Since chemotherapy does not provide any significant outcome improvement, it is crucial to find a suitable radiosensitizer. Our research has shown that the JMJD3 demethylase inhibitor, GSK-J4, not only exerts potent anti-tumor activity on H3K27M mutant DIPG cells, but also restores methylation while decreasing genes that encode proteins involved in DNA double-strand break (DSB) repair. Our aim is to investigate the hypothesis that GSK-J4 may inhibit radiation-induced DNA repair via chromatin modification, making it a potential radiosensitizing agent. METHODS: We evaluated DNA damage repair via quantitative-PCR (q-PCR), immunocytochemistry of DSB markers γH2AX and 53BP1, and DNA repair assay. MNase digestion was used to analyze nucleosome assembly in cellular chromatin. We are currently conducting western blotting, DNA repair assay, FAIRE-seq and clonogenic survival assays to assess DIPG response to radiation therapy (RT) in combination with GSK-J4. In vivo response to radiation monotherapy and combination of RT + GSK-J4 will be measured by bioluminescence imaging and animal survival studies. RESULTS: qPCR results showed that GSK-J4 significantly reduces DNA DSB repair genes such as PARP1, PARP2, SMARCB1 and BRCA1, in irradiated DIPG cells. Immunocytochemistry results support that GSK- J4 sustains high levels of γH2AX and 53BP1 in irradiated DIPG cells, thereby inhibiting DNA DSB repair. MNase assay indicated that GSK-J4 may cause chromatin compaction which prevents DNA DSB repair. Results from the clonogenic assays, to assess long term effect of GSK-J4 on radiated cells, as well as western blotting, FAIRE-seq and in vivo studies will be reported at the meeting.