Day, Charles

Abstract Pediatric high-grade gliomas (pHGG) are among the most lethal of all human cancers. Histone H3.3 G34R/V mutations are an early event in these tumors and show reduced H3.3 K36 trimethylation; implicating epigenetic dysregulation in tumorigenesis. Here we present evidence that H3.3 G34R/V mutations promote tumor formation via the induction of chromosomal instability (CIN). Pericentromeric H3.3 is phosphorylated at S31 by Chk1 during mitosis. We observed that the H3.3 G34R mutation reduced Chk1 phosphorylation of H3.3 S31 by >90% in vitro. Furthermore, H3.3 G34 mutant cells have reduced pericentromeric H3.3 S31 phosphorylation in mitosis compared to WT H3.3 cell lines. H3.3 G34 mutant pHGG cells also have significantly elevated rates of CIN as compared to H3.3 WT cells. Overexpression of H3.3 G34R, G34V or non-phosphorylatable S31A in H3.3 WT, diploid cells caused a significant increase in CIN, but H3.3 K36M overexpression had no effect on chromosome segregation. These studies demonstrate that H3.3 G34R/V mutations are sufficient to induce CIN in normal, diploid cells. To determine if this process contributes to tumorigenesis, we used RCAS Nestin-TVA mice to overexpress H3.3 WT, G34R, or S31A – P2A-linked to PDGFB in glial precursor cells of newborn mice. Over 100 days, S31A and G34R mice had drastically reduced survival (averaging 77, 81, and 100 days for S31A, G34R, and WT). Furthermore, most G34R and S31A mice developed HGG, while H3.3 WT mice remained tumor-free. Our work implicates CIN as a driver of H3.3 G34 mutant pHGG formation.

Abstract Background H3K27M-mutant diffuse midline glioma (DMG) is a lethal brain tumor that usually occurs in children. Despite advances in our understanding of its underlying biology, efficacious therapies are severely lacking. Methods We screened a library of drugs either FDA-approved or in clinical trial using a library of patient-derived H3K27M-mutant DMG cell lines with cell viability as the outcome. Results were validated for clinical relevance and mechanistic importance using patient specimens from biopsy and autopsy, patient-derived cell lines, inhibition by gene knockdown and small molecule inhibitors, and patient-derived xenografts. Results Kinase inhibitors were highly toxic to H3K27M-mutant DMG cells. Within this class, STAT3 inhibitors demonstrated robust cytotoxic activity in vitro. Mechanistic analyses revealed one form of activated STAT3, phospho-tyrosine- 705 STAT3 (pSTAT3), was selectively upregulated in H3K27M-mutant cell lines and clinical specimens. STAT3 inhibition by CRISPR/Cas9 knockout, shRNA or small molecule inhibition reduced cell viability in vitro, and partially restored expression of the polycomb repressive mark H3K27me3, which is classically lost in H3K27M-mutant DMG. Putative STAT3-regulated genes were enriched in an H3K27M-knockout DMG cell line, indicating relative gain of STAT3 signaling in K27M-mutant cells. Treatment of patient-derived intracranial xenografts with WP1066, a STAT3 pathway inhibitor currently in clinical use for pediatric brain tumors, resulted in stasis of tumor growth, and increased overall survival. Finally, pSTAT3(Y705) was detected in circulating plasma extracellular vesicles of patients with H3K27M-mutant DMG. Conclusions STAT3 is a biologically relevant therapeutic target in H3K27M-mutant DMG. STAT3 inhibition should be considered in future clinical trials.

Abstract The histone H3.3 G34R/V mutations are known drivers of high-grade pediatric glioma (pHGG). However, the mechanism(s) for H3.3 G34R/V induced tumor formation are unclear. Chk1 phosphorylates H3.3 S31 at the pericentromere during early mitosis, suggesting a novel mitotic function. We observed that H3.3 G34 mutant pHGG cells have reduced mitotic H3.3 S31 phosphorylation compare to WT H3.3 cell lines. The H3.3 G34R mutation reduced Chk1 phosphorylation at S31 by >90% in an in vitro kinase assay. Overexpression of either H3.3 G34R or non-phosphorylatable S31A in H3.3 WT, diploid cells led to a significant increase in chromosome mis-segregations. Likewise, H3.3 G34 mutant pHGG cells have significantly elevated rates of mis-segregation as compare to H3.3 WT pHGG cells. During normal cell division, phospho-S31 is lost in late anaphase. However, when chromosome missegregation occurs, phospho-S31 spreads and stimulates p53 accumulation in G1 – thus suppressing aneuploid cell proliferation. Here we show that cells expressing mutant G34 fail to arrest following mis-segregation, despite having WT p53. These studies demonstrate that the H3.3 G34R/V mutations are sufficient to transform normal, diploid cells into proliferative, chromosomally instable cells. To determine if this process contributes to tumorigenesis, we used the RCAS/TVA mouse model to overexpress H3.3 WT, G34R, or S31A in the glial precursor cells of mice pups. Over 100 days, S31A and G34R mice had drastically reduced survival (averaging 77, 81, and 100 days for S31A, G34R, and WT mice). Furthermore, most G34R and S31A mice developed HGG, while H3.3 WT mice remained tumor-free and did not develop high-grade tumors. Our work strongly indicates that a major factor in H3.3 G34R pHGG formation is the induction of chromosomal instability – which occurs directly through the suppression of H3.3 S31 phosphorylation.

Abstract Pediatric glioblastoma and diffuse intrinsic pontine glioma are high-grade gliomas of children (pHGG) with a median overall survival of under 15 months and among the most lethal cancers. Mutations in histone H3.3 and H3.1 occur as an early event in pHGG. H3.3G34R/V-mutations occur in pHGG of cerebral hemispheres, and H3.3K27M mutations occur in midline pHGGs. Post-translational histone modifications (PTMs) serve to regulate gene expression by relaxing or compacting chromatin and by recruiting proteins, with subsequent silencing or activating effects. H3.3 Serine 31 (S31) shows reduced phosphorylation during mitosis in H3.3G34R/V and H3.3K27M mutant cell. Phosphorylation at S31 is restored in wildtype H3.3K27 CRISPR revertants. Serine to alanine (A) mutant H3.3 S31A are nonphosphorylatable in vitro. To study the influence of histone mutations and the role of altered PTM and including the loss of methylation and phosphorylation on tumorigenesis, we have developed an innovative model based on the RCAS/N-TVA mouse model. In this system, the expression of an oncogenic driver is linked to mutant histone expression using a self-cleaving peptide, and tumors develop following viral delivery to neural stem cells in newborn mice. This approach is necessary, as otherwise, clonal selection could prevent tumors from forming with mutations detrimental to growth. To establish the model, N-TVA mice were injected with RCAS H3.3K27M-P2A-PDGFB, RCAS H3.3G34R-P2A-PDGFB, or H3.3WT-P2A-PDGFB. The mean survival of mice injected with H3.3K27M and H3.3S31A was 81 and 68 days, respectively, and 100% of S31A mice developed HGG. In contrast, H3.3WT caused only low-grade tumors in 46% of the mice, and all mice survived until 100 days. In ongoing experiments with H3.3G34R, 23% of mice succumb to tumors by 80 days. These results provide mechanistic insights into the early establishment of pHGGs and established a new mouse model to study the role of histone mutation and PTMs in tumor development.

Abstract Diffuse midline gliomas with the H3.3 K27M mutation are lethal brain tumors in children. H3 K27M causes global loss of Lys27 triple methylation (Lys27me3), inducing epigenetic reprograming. Here we show that H3.3 K27M also causes decreased H3.3 Ser31 phosphorylation on mitotic chromosomes. We show that H3.3 K27M DIPG cells have reduced pericentromeric phospho-Ser31 and increased rates of chromosome missegregation compared to normal, diploid human cells. CRISPR-editing K27M to M27K restored phospho-Ser31 to WT levels and dramatically decreased the rate of chromosome missegregation. We confirm that Chk1 is the H3.3 Ser31 kinase: K27M mutant H3.3 protein exhibits ~60% reduced Chk1 phosphorylation of Ser31 in vitro. Chk1 knockdown completely abolishes phospho-Ser31 in cells and these have increased rates of chromosome missegregation. In normal, diploid cells, expression of K27M or an S31A non-phosphorylatable mutant increased chromosome missegregation; this is suppressed by expressing a phosphomimetic double mutant (K27M/S31E) that restores phospho-Ser31. WT cells arrest following chromosome missegregation. However, cells expressing H3.3 K27M or S31A fail to arrest - despite having WT p53. Finally, we expressed H3F3AS31A and PDGFb in an RCAS/TVA mouse model of DIPG and ~80% developed diffuse high-grade brain tumors and show significantly decreased survival. Our results suggest that loss of phospho-Ser31 alone is oncogenic because H3.3 S31A-expressing cells are WT for K27me3. Our results demonstrate that H3.3 K27M inhibits Ser31 phosphorylation both in vitro and in vivo, leading to both chromosome missegregation and loss of subsequent G1 arrest – thus creating diffuse midline gliomas with both dynamic, complex karyotypes and epigenetic reprogramming.

Abstract Diffuse midline gliomas (DMG) patients have a dire prognosis despite radiation therapy and there is an urgent need to develop more effective treatments. DMG are characterized by heterozygous mutations in select H3 genes resulting in the replacement of lysine 27 by methionine (K27M) that leads to global epigenetic reprogramming and drives tumorigenesis. We previously reported that pharmacological inhibition of aurora kinase (AKI) may represent a targeted approach for treating tumors with this mutation. Our analysis with both published dataset and patient samples showed that patients with higher aurora kinase A (AKA) expression were associated with worse survival. AKA phosphorylates H3S10 and H3S28 during mitosis. Intriguingly, phosphorylation of the H3S28 (H3S28ph) by AKA blocks PRC2 methyltransferase activity and decreases global H3K27me3 in certain stem cells. We propose that a similar mechanism occurs in H3K27M DMG tumors, where there is a reciprocal relationship between H3S28ph and H3K27me3. We found that AKI significantly decreases H3S28ph while increasing H3K27me3 specifically in H3K27M tumors. To further evaluate the link between the H3K27M mutation and H3 serine phosphorylation, we used CRISPR/Cas9-directed gene editing to silence H3S28ph by replacing serine with alanine (H3S28A) in DIPG cell lines. Ectopic expression of histone H3S28A leads to a prominent epigenetic changes in H3K27M tumors and is similar to AKA inhibition. Overall, this study highlights H3S28ph, one of the targets of AK, is a key driver of epigenetic changes in H3K27M tumors through both direct and indirect changes to H3K27me3 and H3K27ac across the genome.

Abstract Diffuse midline glioma (DMG) is a lethal brain tumor that typically occurs in children. Numerous studies have demonstrated the central role of the H3K27M mutation and secondary loss of H3K27 trimethylation (H3K27me3) in DMG tumorigenesis. Understanding how the H3K27M mutation alters the epigenetic landscape of the cell is necessary for revealing molecular targets that are critical to tumorigenesis. To investigate the epigenetic effects of H3K27M mutation in DMG, we developed revertant DMG cell lines with the mutant methionine residue reverted to wildtype (i.e., M27K). Revertant cells were analyzed for epigenetic changes and phenotypic differences in vitro and in vivo. H3M27K DMG cells grew in culture but displayed diminished proliferative capacity. H3M27K cells demonstrated total loss of H3K27M expression and restored trimethylation of H3K27 and H3K4. Furthermore, consistent with the hypothesis that the H3K27M mutation impacts H3 phosphorylation via expression of Aurora Kinase during mitosis, H3M27K cells demonstrated reduced expression of both Aurora Kinase A and phosphorylation of H3 serine residues 10 and 28. In line with the critical role of H3S10 phosphorylation in chromatin segregation, H3M27K cells also demonstrated restored chromosome segregation compared to H3K27M cells. In vivo data will be discussed. Revertance of the H3K27M mutation reduces tumorigenesis in DMG tumors. Isogenic H3M27K cells display reversal of key epigenetic changes associated with oncogenesis in DMG. The revertant H3M27K DMG model is a useful tool to investigate the downstream epigenetic reprogramming specific to H3K27M mutation in these tumors.