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Differential Expression in Isogenic Lines

Cell type-specific markers and pathways in single-cell transcriptomics

Glycine 34-to-tryptophan (G34W) substitutions in H3.3 arise in approximately 90% of giant cell tumor of bone (GCT). Here, we show H3.3 G34W is necessary for tumor formation. By profiling the epigenome, transcriptome, and secreted proteome of patient samples and tumor-derived cells CRISPR–Cas9-edited for H3.3 G34W, we show that H3.3K36me3 loss on mutant H3.3 alters the deposition of the repressive H3K27me3 mark from intergenic to genic regions, beyond areas of H3.3 deposition. This promotes redistribution of other chromatin marks and aberrant transcription, altering cell fate in mesenchymal progenitors and hindering differentiation. Single-cell transcriptomics reveals that H3.3 G34W stromal cells recapitulate a neoplastic trajectory from a SPP1+ osteoblast-like progenitor population toward an ACTA2+ myofibroblast-like population, which secretes extracellular matrix ligands predicted to recruit and activate osteoclasts. Our findings suggest that H3.3 G34W leads to GCT by sustaining a transformed state in osteoblast-like progenitors, which promotes neoplastic growth, pathologic recruitment of giant osteoclasts, and bone destruction.Significance:This study shows that H3.3 G34W drives GCT tumorigenesis through aberrant epigenetic remodeling, altering differentiation trajectories in mesenchymal progenitors. H3.3 G34W promotes in neoplastic stromal cells an osteoblast-like progenitor state that enables undue interactions with the tumor microenvironment, driving GCT pathogenesis. These epigenetic changes may be amenable to therapeutic targeting in GCT.See related commentary by Licht, p. 1794.This article is highlighted in the In This Issue feature, p. 1775

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Golgi Secretome Analysis in Isogenic Lines

Pathway Enrichment Analysis

Supplementary Information

A high percentage of pediatric gliomas and bone tumors reportedly harbor missense mutations at glycine 34 in genes encoding histone variant H3.3. We find that these H3.3 G34 mutations directly alter the enhancer chromatin landscape of mesenchymal stem cells by impeding methylation at lysine 36 on histone H3 (H3K36) by SETD2, but not by the NSD1/2 enzymes. The reduction of H3K36 methylation by G34 mutations promotes an aberrant gain of PRC2-mediated H3K27me2/3 and loss of H3K27ac at active enhancers containing SETD2 activity. This altered histone modification profile promotes a unique gene expression profile that supports enhanced tumor development in vivo. Our findings are mirrored in G34W-containing giant cell tumors of bone where patient-derived stromal cells exhibit gene expression profiles associated with early osteoblastic differentiation. Overall, we demonstrate that H3.3 G34 oncohistones selectively promote PRC2 activity by interfering with SETD2-mediated H3K36 methylation. We propose that PRC2-mediated silencing of enhancers involved in cell differentiation represents a potential mechanism by which H3.3 G34 mutations drive these tumors.

Glycine 34-to-tryptophan (G34W) substitutions in H3.3 arise in approximately 90% of giant cell tumor of bone (GCT). Here, we show H3.3 G34W is necessary for tumor formation. By profiling the epigenome, transcriptome, and secreted proteome of patient samples and tumor-derived cells CRISPR–Cas9-edited for H3.3 G34W, we show that H3.3K36me3 loss on mutant H3.3 alters the deposition of the repressive H3K27me3 mark from intergenic to genic regions, beyond areas of H3.3 deposition. This promotes redistribution of other chromatin marks and aberrant transcription, altering cell fate in mesenchymal progenitors and hindering differentiation. Single-cell transcriptomics reveals that H3.3 G34W stromal cells recapitulate a neoplastic trajectory from a SPP1+ osteoblast-like progenitor population toward an ACTA2+ myofibroblast-like population, which secretes extracellular matrix ligands predicted to recruit and activate osteoclasts. Our findings suggest that H3.3 G34W leads to GCT by sustaining a transformed state in osteoblast-like progenitors, which promotes neoplastic growth, pathologic recruitment of giant osteoclasts, and bone destruction. Significance: This study shows that H3.3 G34W drives GCT tumorigenesis through aberrant epigenetic remodeling, altering differentiation trajectories in mesenchymal progenitors. H3.3 G34W promotes in neoplastic stromal cells an osteoblast-like progenitor state that enables undue interactions with the tumor microenvironment, driving GCT pathogenesis. These epigenetic changes may be amenable to therapeutic targeting in GCT. See related commentary by Licht, p. 1794. This article is highlighted in the In This Issue feature, p. 1775

High-grade gliomas (HGG) defined by histone 3 K27M driver mutations exhibit global loss of H3K27 trimethylation and reciprocal gain of H3K27 acetylation, respectively shaping repressive and active chromatin landscapes. We generated tumor-derived isogenic models bearing this mutation and show that it leads to pervasive H3K27ac deposition across the genome. In turn, active enhancers and promoters are not created de novo and instead reflect the epigenomic landscape of the cell of origin. H3K27ac is enriched at repeat elements, resulting in their increased expression, which in turn can be further amplified by DNA demethylation and histone deacetylase inhibitors providing an exquisite therapeutic vulnerability. These agents may therefore modulate anti-tumor immune responses as a therapeutic modality for this untreatable disease.

Gain-of-function mutations in histone 3 (H3) variants are found in a substantial proportion of pediatric high-grade gliomas (pHGG), often in association with TP53 loss and platelet-derived growth factor receptor alpha (PDGFRA) amplification. Here, we describe a somatic mouse model wherein H3.3

There are several evidences, suggesting a relationship between hyperhomocysteinemia and various diseases of the visual system. Therefore in this study the effects of homocysteinylation on aggregation and fibrillation of lens crystallins were studied using spectroscopic techniques, SDS-PAGE and western blot analysis. The results of UV-Vis absorption studies suggest an induction of lens protein aggregation after homocysteinylation. Furthermore, the existence of fibril in the aggregate of lens proteins confirmed by Congo red absorption measurement and Thioflavin-T fluorescence assay. Taken together the results of SDS-PAGE and Western blotting, it is suggested that almost all detectable eye lens crystallins are prone to aggregation by homocysteinylation, while α-Crystallin comprises the main portion of lens protein aggregate. Overall this study may suggest lens protein homocysteinylation as a possible mechanism to explain the relationship between hyperhomocysteinimia and some impairments of the visual system.

Introduction: Pediatric glioblastomas (pGBM) are malignant brain tumors associated with a dismal prognosis. A subset of pGBMs carry mutations of either the Lysine 27 or Glycine 34 (G34) amino acid residues of histone 3 variant genes. The same G34 residue is also mutated in 85-95% of giant cell tumors of bone (GCTs), albeit to Tryptophan (G34W) in GCTs rather than to Arginine or Valine (G34R/V) as in pGBMs. The G34 mutation is predicted to impede access of histone methyltransferases like SetD2 to the nearby Lysine 36 residue, thereby altering the epigenome and transcriptome. Methods: To elucidate the tumorigenic effect of G34 mutations, we used the gene-editing technology CRISPR/Cas9 to correct the G34W mutation to wild-type in 2 GCT cell lines. We then investigated CRISPR-edited cell lines using functional assays, proteomic, epigenomic, and transcriptomic analyses. Results: Correction of the G34W mutation to wild-type in CRISPR-edited GCT cells results in phenotypic and functional changes suggestive of reduced tumorigenicity. By mass spectrometry, G34W-mutant GCT cell lines display decreased level of Lysine 36 trimethylation (H3K36me3) on the mutant G34-peptide, similar to G34-mutated pGBM cell lines. However, unlike pGBMs, GCTs display increased levels of Lysine 36 dimethylation (H3K36me2) on the mutant G34W-peptide. Ongoing Experiments and Analyses: We are currently comparing the level and distribution of multiple histone marks by performing chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) of CRISPR-edited wild-type clones relative to the parent G34W-mutant GCT line. We are also assessing differential gene expression by RNA-Seq and characterizing the methylation signature by 850K DNA methylation array of G34W-mutant GCTs and edited clones. Conclusion: The G34W-mutation clearly has an impact on tumorigenic potential, as evidenced by in vitro functional assays. The G34W-mutant peptide of GCT cell lines features a distinct profile of post-translational histone modifications compared to G34R- or G34V-mutant peptides of pGBM cell lines. Further investigation could elucidate the epigenetic mechanism(s) through which the G34W mutation confers its tumorigenic properties. Citation Format: Shriya Deshmukh, Sima Khazaei, Dylan Marchione, Ashot Harutyunyan, Benjamin Garcia, Nada Jabado. Characterizing the epigenetic effects of the histone 3.3 G34W mutation in giant cell tumors of bone [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A39.

Gain-of-function mutations in histone 3 (H3) variants are found in a substantial proportion of pediatric high-grade gliomas (pHGG), often in association with TP53 loss and platelet-derived growth factor receptor alpha (PDGFRA) amplification. Here, we describe a somatic mouse model wherein H3.3K27M and Trp53 loss alone are sufficient for neoplastic transformation if introduced in utero. H3.3K27M-driven lesions are clonal, H3K27me3 depleted, Olig2 positive, highly proliferative, and diffusely spreading, thus recapitulating hallmark molecular and histopathological features of pHGG. Addition of wild-type PDGFRA decreases latency and increases tumor invasion, while ATRX knockdown is associated with more circumscribed tumors. H3.3K27M-tumor cells serially engraft in recipient mice, and preliminary drug screening reveals mutation-specific vulnerabilities. Overall, we provide a faithful H3.3K27M-pHGG model which enables insights into oncohistone pathogenesis and investigation of future therapies.

Various chemical modifications can reduce chaperone activity of α-crystallin (α-Cry) and the loss of which has been implicated in the development of cataract diseases. The side chains of lysine residues are the target of both glycation and homocysteinylation, and lysine modification by the two reactions may similarly affect the structure and function of α- Cry. In this study, α-Cry was incubated with homocysteine thiolactone (HCTL), resulting in significant protein homocysteinylation, as determined with Ellman's assay. Homocysteinylation of α-Cry resulted in the reduction in surface hydrophobicity and alpha-helix to beta-sheet transition, as observed respectively with fluorescence and circular dichroism (CD) spectroscopy. The structural alteration of homocysteinylated α-Cry was accompanied by protein aggregation, including the formation of amyloid fibrils as detected by thioflavin T (ThT) fluorescence and Congo red (CR) absorption spectroscopy. The mobility shifts of homocysteinylated α-Cry on reducing and non-reducing SDS-PAGEs suggest that disulfide cross-linking in addition to lysine modification, also plays a role in aggregation of this protein. The chaperone activities of α-Cry, namely to prevent aggregation, to assist refolding and to restore activity of thermally stressed α-glucosidase (α-Gls) were reduced after homocysteinylation. Overall, this study suggests that similar to non-enzymatic glycation, homocysteinylation of α-Cry is a risk factor for the development of cataract disorders, for instance during hyperhomocysteinemia which is linked to the various ocular pathological disorders.

Lemon balm (Melissa officinalis) is a medicinal herb possessing functional compounds with unexplored anti-glycative action. The anti-glycative activity of Lemon balm extract was evaluated in the bovine serum albumin (BSA)/glucose system. The level of glycation, conformational alterations and protein binding to RAGE receptors were assessed by specific fluorescence, Congo red binding assay, circular dichroism, ligand and Western blotting. Ethanol fractions of Melissa leaf exhibited the highest inhibitory effects on the formation of advanced glycation end products (AGEs) and the late stage of glycation process. Significant alteration in the secondary structure of albumin was observed upon glycation, which was mitigated by applying the herb extract. Moreover, upon treatment with balm extract, glycated albumin adopts a secondary structure impeding its detection by RAGE receptors of microglial cells. Our results represent the anti-glycative properties of Melissa extract and its application for possible treatment of AGE-associated diseases.