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Additional file 10: S10. Functional pathways analysis of differentially expressed genes after RT and Bromodomain inhibition. A Differentially expressed genes by DIPG cells after 48 h RT compared to DMSO control enrich for DNA double-strand break repair as a top canonical pathway on functional analysis (p-value 1.10 × 10− 21). B Differentially expressed genes by DIPG cells after 48 h JQ1 treatment compared to DMSO control enrich for nucleotide excision repair as the top canonical pathway (p-value 2.51 × 10− 48).

Additional file 3: Figure S1. Characterization of pediatric glioma cells and astrocytes with genetic modification of histone H3.3G34V expression. A) Relative proportion of H3.3G34V enrichment across gene elements in non-transduced KNS42 or NHA with DOX-induced H3.3G34V overexpression or H3.3 expression control. B) H3.3G34V protein level in non-transduced KNS42 or NHA with DOX-induced H3.3G34V overexpression or H3.3 expression control. H3.3G34V protein expression was observed to be 29% of that expressed in non-transduced KNS42. C) Cell viability in modified cell lines. No significant difference was observed in cell viability between KNS42 with G34V knockdown, and KNS42 with no doxycycline control, as well as in NHA overexpressed with H3.3G34V and overexpression control, NHA overexpressed with H3.3. X-axis: absolute luminescence. Y-axis: days following plating. Error bars represent standard error. D) Rate of cell proliferation relative to control. No significant difference was observed in cell proliferation in KNS42 with G34V knockdown compared to no doxycycline control. NHA overexpressed with H3.3G34V showed lower, but not significant, proliferation compared to NHA with overexpression control. X-axis: proliferation rate normalized to control. Y-axis: days following plating. D) Light microscopy images of cells at 10x magnification on days 3 and 7 after plating.

Additional file 3: Figure S2. GO and KEGG analysis of genes with the most differentially enriched A) H3.3G34V, B) H3.3WT, and C) H3K36me3 in KNS42 vs KNS42 with H3.3G34V knockdown. Y-axis represents the enriched GO biological process and KEGG pathways, X-axis represents the number of unique genes enriched. Statistical significance determined via FDR

Additional file 9: S9. Peptide modification states observed after bromodomain inhibition with in vitro. (Microsoft Excel format). Statistically significant differences in histone acetylation and methylation are observed in NHA, U87, SF8628, and DIPG007 cells after bromodomain inhibition with JQ1 (Independent-sample t test, two-tailed).

Additional file 3: Figure S3. KEGG analysis of gene co-enriched in G34V and H3.3 (top) and co-enriched in G34V and K36me3 (bottom). Number is −log(p-value).

Diffuse midline glioma (DMG) is a highly morbid pediatric brain tumor. Up to 80% of DMGs harbor mutations in histone H3-encoding genes, associated with poor prognosis. We previously showed the feasibility of detecting H3 mutations in circulating tumor DNA (ctDNA) in the liquid biome of children diagnosed with DMG. However, detection of low levels of ctDNA is highly dependent on platform sensitivity and sample type. To address this, we optimized ctDNA detection sensitivity and specificity across two commonly used digital droplet PCR (ddPCR) platforms (RainDance and BioRad), and validated methods for detecting H3F3A c.83A > T (H3.3K27M) mutations in DMG CSF, plasma, and primary tumor specimens across three different institutions. DNA was extracted from H3.3K27M mutant and H3 wildtype (H3WT) specimens, including H3.3K27M tumor tissue (n = 4), CSF (n = 6), plasma (n = 4), and human primary pediatric glioma cells (H3.3K27M, n = 2; H3WT, n = 1). ctDNA detection was enhanced via PCR pre-amplification and use of distinct custom primers and fluorescent LNA probes for c.83 A > T H3F3A mutation detection. Mutation allelic frequency (MAF) was determined and validated through parallel analysis of matched H3.3K27M tissue specimens (n = 3). We determined technical nuances between ddPCR instruments, and optimized sample preparation and sequencing protocols for H3.3K27M mutation detection and quantification. We observed 100% sensitivity and specificity for mutation detection in matched DMG tissue and CSF across assays, platforms and institutions. ctDNA is reliably and reproducibly detected in the liquid biome using ddPCR, representing a clinically feasible, reproducible, and minimally invasive approach for DMG diagnosis, molecular subtyping and therapeutic monitoring.

Background Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles. Methods We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n = 12) and cell lines (n = 7). Results We found 29.2 and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. Conclusion Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.