Fig. 3

De novo purine synthesis inhibition radiosensitizes K27M cells. A and B Immunoblots of (A) IMPDH1 and (B) HGPRT expression in DMG-H3K27M isogenic paired cell lines. Densitometric values were calculated using ImageJ software and expression values were normalized to H3K27M-KO cells. Expression ratios are listed below the blot images. C Publicly available RNAseq Z-score data for HPRT1 transcript expression from pediatric high-grade gliomas (pHGG) was obtained through PedCBioPortal and filtered to include only samples with known H3 mutational status (for both H3F3A and HIST1H3B) to include all known H3WT (n=59) and combined H3K27M (H3F3A-mut+HIST1H3B-mut) samples (n=44). D Schematic depicting the hypothesis that the K27M mutation induces defective guanylate purine salvage through HGPRT suppression, leaving K27M cells vulnerable to de novo guanylate synthesis using IMPDH inhibition using MPA. Schematic was created using BioRender.com. E And G Long-term neurosphere growth assays for (E) DIPGXIII and (G) BT245 H3K27M-expressing cell lines treated with increasing doses of RT (0, 2, 6Gy) with or without 1-10μM MPA (left) and corresponding enhancement ratio (Dbar_control/Dbar_Tx) for each concentration of MPA (right) administered with RT. Each long-term neurosphere assay was performed 3x per cell line. Statistical analyses were performed using two-tailed t-tests in GraphPad Prism 10.0. F and H Live cell imaging analysis of (F) DIPGXIII and (H) BT245 neurospheres treated with 0-6Gy +/- 10μM MPA. Images taken 11 days (DIPGXIII) and 9 days (BT245) after replating in 96-well plates. Images acquired using a Cytation 5 plate reader and attached BioSpa incubator (Agilent Technologies)