Histone Mutations Research Articles

Incorporation of a histone mutant with H3K56 site substitution perturbs the replication machinery in mouse embryonic stem cells.

Sense mutations in several conserved modifiable sites of histone H3 have been found to be strongly correlated with multiple tissue-specific clinical cancers. These clinical site mutants acquire a distinctively new epigenetic role and mediate cancer evolution. In this study, we mimicked histone H3 at the 56th lysine (H3K56) mutant incorporation in mouse embryonic stem cells (mESCs) by lentivirus-mediated ectopic expression and analyzed the effects on replication and epigenetic regulation. The data show that two types of H3K56 mutants, namely H3 lysine 56-to-methionine (H3K56M) and H3 lysine 56-to-alanine (H3K56A), promote replication by recruiting more minichromosome maintenance complex component 3 and checkpoint kinase 1 onto chromatin compared with wild-type histone H3 and other site substitution mutants. Under this condition, the frequency of genomic copy number gain in H3K56M and H3K56A cells globally increases, especially in the Mycl1 region, a known molecular marker frequently occurring in multiple malignant cancers. Additionally, we found the disruption of H3K56 acetylation distribution in the copy-gain regions, which indicates a probable epigenetic mechanism of H3K56M and H3K56A. We then identified that H3K56M and H3K56A can trigger a potential adaptation to transcription; genes involved in the mitogen-activated protein kinase pathway are partially upregulated, whereas genes associated with intrinsic apoptotic function show obvious downregulation. The final outcome of ectopic H3K56M and H3K56A incorporation in mESCs is an enhanced ability to form carcinomas. This work indicates that H3K56 site conservation and proper modification play important roles in harmonizing the function of the replication machinery in mESCs.

495 5-ALA PDT and Targeting MEK/ERK Signaling Elicits Synergistic Antitumor Effects in Diffuse Midline Glioma

INTRODUCTION: Diffuse midline gliomas are highly invasive, unresectable tumors in children. To date, there is no effective treatment for DMG. Concurrent radiotherapy (RT) and systemic therapies, the standard of care, has only been successful in providing limited disease control. The major obstacle to therapy is the selectively permeable blood brain barrier (BBB) that limits systemic drug delivery. Given the unmet need for penetrant and minimally invasive DMG treatments, photodynamic therapy (PDT), with the precursor photosensitizing agent 5-aminolevulinic acid (5-ALA), is an oncologic treatment that holds promise. 5-ALA PDT of tumors occurs by targeting tumor cells that accumulate the 5-ALA metabolite, protoporphyrin IX (PPIX), with 635 nm light to create deadly reactive oxygen species (ROS). METHODS: Growth delay assays were conducted for each individual treatment modality or in combination. FDA-approved MEK/ERK inhibitors were administered to mice with aggressive heterotopic allograft DMGs through intraperitoneal injection. Ex vivo immunofluorescence staining was performed on those mice that received 5-ALA PDT treatment. RESULTS: We demonstrated that sub-micromolar levels of 5-ALA PDT and nanomolar levels of MEK inhibitor successfully decrease cell proliferation and induce apoptosis in DMG cell lines. Cell viability assays revealed that drug response differs based on the histone mutation (H3.1 or H3.3) of the line. Mechanisms of decreased cell survival involves the generation of reactive oxygen species that induces programmed cell death. Through the use of a DMG genetically engineered mouse model, we also found 5-ALA PDT to induce apoptosis in vivo. CONCLUSION: The synergistic effects of MEK inhibition and 5-ALA PDT in vitro and apoptotic effects of 5-ALA PDT in vivo, highlights the potential therapeutic efficacy of this treatment modality.

Deciphering the effects of post-translational modifications and mutations on histone tail dynamics and interactions

Histone tails harbor a vast number of post-translational modifications which are recognized by regulatory proteins to mediate epigenetic processes. Many recurrent mutations in histones have been identified which drive cancer development, and their effects are not well characterized. Here, we investigate the effects of post-translational modifications and mutations on histone tails using molecular dynamics simulations in conjunction with bioinformatics approaches. We have compiled a dataset of histone mutations from >10,000 tumor samples and predicted the potential oncogenic mutations.

The impact of histone mutations in cancer

The integrity of histone genes is a cautiously guarded attribute in most human cells, demonstrating the integral role that they play in cellular function. Not only are histones capable of allowing the compaction of DNA within the nucleus, but they are also involved in DNA replication, repair and expression. As histones are indispensable for cellular function, mutations that alter histone structure can prove detrimental to cell viability. Recent publications have demonstrated that some histone mutations may be capable of promoting the development of phenotypically distinguishable cancers.

A Genetically Encoded Approach for Breaking Chromatin Symmetry.

Nucleosomes frequently exist as asymmetric species in native chromatin contexts. Current methods for the traceless generation of these heterotypic chromatin substrates are inefficient and/or difficult to implement. Here, we report an application of the SpyCatcher/SpyTag system as a convenient route to assemble desymmetrized nucleoprotein complexes. This genetically encoded covalent tethering system serves as an internal chaperone, maintained through the assembly process, affording traceless asymmetric nucleosomes following proteolytic removal of the tethers. The strategy allows for generation of nucleosomes containing asymmetric modifications on single or multiple histones, thereby providing facile access to a range of substrates. Herein, we use such constructs to interrogate how nucleosome desymmetrization caused by the incorporation of cancer-associated histone mutations alters chromatin remodeling processes. We also establish that our system provides access to asymmetric dinucleosomes, which allowed us to query the geometric/symmetry constraints of the unmodified histone H3 tail in stimulating the activity of the histone lysine demethylase, KDM5B. By providing a streamlined approach to generate these sophisticated substrates, our method expands the chemical biology toolbox available for interrogating the consequences of asymmetry on chromatin structure and function.

Synthesis of Oriented Hexasomes and Asymmetric Nucleosomes Using a Template Editing Process.

Nucleosomes, the structural building blocks of chromatin, possess 2-fold pseudo symmetry which can be broken through differential modification or removal of one copy of a pair of sister histones. The resultant asymmetric nucleosomes and hexasomes have been implicated in gene regulation, yet the use of these noncanonical substrates in chromatin biochemistry is limited, owing to the lack of efficient methods for their preparation. Here, we report a strategy that allows the orientation of these asymmetric species to be tightly controlled relative to the underlying DNA sequence. Our approach is based on the use of truncated DNA templates to assemble oriented hexasomes followed by DNA ligation and, in the case of asymmetric nucleosomes, addition of the missing heterotypic histones. We show that this approach is compatible with multiple nucleosome positioning sequences, allowing the generation of desymmetrized mononucleosomes and oligonucleosomes with varied DNA overhangs and heterotypic histone H2A/H2B dimer compositions. Using this technology, we examine the functional consequences of asymmetry on BRG1/BRM associated factor (BAF) complex-mediated chromatin remodeling. Our results indicate that cancer-associated histone mutations can reprogram the inherent activity of BAF chromatin remodeling to induce aberrant chromatin structure.

Oncohistones: Hijacking the histone code.

Chromatin dysfunction has been implicated in a growing number of cancers especially in children and young adults. In addition to chromatin modifying and remodeling enzymes, mutations in histone genes are linked to human cancers. Since the first reports of hotspot missense mutations affecting key residues at histone H3 tail, studies have revealed how these so-called "oncohistones" dominantly (H3K27M and H3K36M) or locally (H3.3G34R/W) inhibit corresponding histone methyltransferases and misregulate epigenome and transcriptome to promote tumorigenesis. More recently, widespread mutations in all four core histones are identified in diverse cancer types. Furthermore, an "oncohistone-like" protein EZHIP has been implicated in driving childhood ependymomas through a mechanism highly reminiscent of H3K27M mutation. We will review recent progresses on understanding the biochemical, molecular and biological mechanisms underlying the canonical and novel histone mutations. Importantly, these mechanistic insights have identified therapeutic opportunities for oncohistone-driven tumors.

Central Nervous System Tumors in Children.

Central Nervous System Tumors in Children.

STMO-19 Impact of aggressive resection for glioblastoma of the thalamus with histone H3-K27M mutation

Glioblastoma of the thalamus occurs predominantly in childhood and young adulthood, and cases with histone mutations are thought to have a particularly poor prognosis. We studied tumor resection rate, age, type of adjuvant therapy, and histone gene mutations on progression-free survival (PFS) and overall survival (OS) in patients who underwent aggressive removal. Eight cases of thalamic glioblastoma were included in the study. The mean age at surgery was 36.1 years (10–74 years, 3 cases under 18 years). Tumor removal was performed from the parieto-occipital lobe to the thalamus via the lateral ventricles in all cases. In all cases, more than 90% of the contrast-enhancing lesions were removed. Postoperatively, one patient had sensory disturbance of the left upper limb, and the other had incomplete paralysis of the left upper and lower limbs, but both were able to walk with a cane. In the case of the patient with postoperative complications, the tumor was located in the vicinity of the internal capsule. All patients were treated with radiation therapy and temozolomide, and bevacizumab and Novo-TTF were used in cases after approval. All patients were able to return home and return to school or work after initial treatment. The mean progression-free survival (PFS) was 0.87 years, and overall survival (OS) was 1.95 years. Five patients had histone H3-K27M mutations, and three patients had no mutations. PFS and OS were 1.02 years and 0.62 years, respectively, and 2.53 years and 1.20 years, respectively, both of which were longer in patients with mutations (PFS; p=0.16, OS; p=0.23).Aggressive removal of glioblastoma of the thalamus may improve prognosis, especially in patients with histone H3-K27M mutations. In patients with tumors extending to the vicinity of the internal capsule, total removal may cause paralysis and sensory disturbance.

EPCO-06. AGE- AND REGION-SPECIFIC MULTI-OMIC CHARACTERIZATION OF H3-K27M MUTANT DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas driven by lysine27-to-methionine mutations in histone 3 (H3-K27M DMGs) are among the most fatal brain tumors. Molecular studies including single cell RNA-sequencing (scRNA-seq) of pediatric and predominantly pontine H3-K27M DMGs have shown that the H3-K27M oncohistone keeps glioma cells locked in a stem-like oligodendrocyte precursor cell (OPC) state that is capable of self-renewal and tumor-initiation. However, a comprehensive dissection of the cellular architecture of H3-K27M DMGs across different midline regions and age groups is required to better understand the cell-intrinsic and contextual regulation of H3-K27M DMG cell identities. In particular, the more recently described group of adult H3-K27M DMGs remains understudied. Here, we have collected and characterized 45 H3-K27M mutant patient tumors, spanning pontine (n=26), thalamic (n=17), and spinal (n=2) locations. Median age at surgery was 12 (2-68) years, encompassing 21 early childhood (0-10 years), 12 adolescent (11-20 years), and 12 adult (≥ 21 years) tumors. The majority of samples were obtained pre-treatment (n=28), as opposed to post-treatment or at autopsy (n=17). We profiled all 45 tumors by single cell/single nucleus RNA-seq and selected tumors were further characterized by the single cell assay for transposase-accessible chromatin (scATAC-seq). Our integrated analyses highlight the predominance of transcriptionally and epigenetically defined OPC-like tumor cells as the main cell population of H3-K27M DMGs across all age groups and locations. We further identify distinct age- and location-specific OPC-like cell subpopulations. Comparison of pediatric and adult tumors further demonstrates a significant increase of mesenchymal cell states in adult H3-K27M DMGs, which we link to differences in glioma-associated immune cell compartments between age groups. Together, this study sheds light on the effects of age- and region-dependent microenvironments in shaping cellular identities in H3-K27M DMGs.

PATH-04. LEPTOMENINGEAL DISEASE IN PATIENTS WITH HISTONE-MUTANT GLIOMAS

Abstract INTRODUCTION The 2016 WHO classification describes a subtype of midline gliomas harboring histone 3 (H3) K27M mutations, and the upcoming 2021 edition may include a new subtype of hemispheric diffuse gliomas with H3 G34R/V mutations. Since we began to perform molecular profiling at Memorial Sloan Kettering Cancer Center (MSKCC) we have observed an increased prevalence of leptomeningeal disease (LMD) in this population. However, the incidence and clinical behavior of LMD has not been well defined. METHODS This is a retrospective study of patients with H3-mutant gliomas at MSKCC diagnosed from 01/2012 to 02/2021, either by tumor biopsy or by cerebrospinal fluid (CSF). Histone mutations were identified through next-generation sequencing (NGS). RESULTS We found 40 patients (pts) harboring H3 mutations (K27M mutations in 31 pts, G34R/V in 8, and both in one), with 2/40 (5%) mutations identified through CSF NGS only and rest in tumor tissue. Median age was 20.5 years (4-70); 25 were male. Tumor location was midline for K27M-mutant tumors (thalamus [N=15], brainstem [N=10], spine [N=5], pineal gland [N=1]) and hemispheric for G34R/V-mutant tumors; tumor with both mutations was thalamic. LMD was diagnosed in 22/40 (55%) pts radiographically, including 19/31 (61%) of K27M-mutant pts and 3/8 (38%) of G34R/V-mutant pts (patient with both mutations did not develop LMD). At analysis, 10 patients remain alive. Median time from diagnosis to LMD was 8.8 months (0-44.4), with median OS of 6.5 months (0.3-34) after LMD diagnosis. CONCLUSION More than half of patients with histone-mutant gliomas develop LMD, including over a third of patients with G34R/V mutations. Neuroaxis imaging should be performed in conjunction with CSF studies in histone-mutant gliomas. Besides, CSF NGS represents an important tool to identify molecular profile of tumors when biopsy is not feasible or there is limited tissue for analysis.

CSIG-21. 5-ALA PDT AND TARGETING MEK/ERK SIGNALING ELICITS SYNERGISTIC ANTITUMOR EFFECTS IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas (DMGs) are highly invasive, unresectable tumors in children. To date, there is no effective treatment for DMGs. Fractionated radiotherapy (RT), currently the standard of care, has provided limited disease control. Current obstacles to treatment include the blood brain barrier (BBB) that limits systemic drug delivery, tumor therapy resistance, and brainstem infiltration. Given the unmet need for more effective DMG treatments, photodynamic therapy (PDT), with the precursor photosensitizing agent 5-aminolevulinic acid (5-ALA), is an oncologic treatment that holds promise. 5-ALA PDT of tumors occurs by targeting tumor cells that accumulate the 5-ALA metabolite, protoporphyrin IX (PPIX), with 635 nm light to create deadly reactive oxygen species (ROS). We explore the synergism of 5-ALA PDT with the MEK inhibitor, trametinib, since the RAS/MEK signaling pathway regulates tumor cell proliferation and survival and has been shown to therapeutically enhance PDT in select tumor models. We demonstrated that sub-micromolar levels of 5-ALA PDT and nanomolar levels of trametinib successfully decrease cell proliferation and induce apoptosis in multiple DMG cell lines. Cell viability assays revealed that drug response differs based on the histone mutation (H3.1 or H3.3) of the line. Mechanisms of decreased cell survival involves the generation of reactive oxygen species that induces programmed cell death. Through the use of a DMG genetically engineered mouse model, we also found 5-ALA PDT to induce apoptosis in vivo. The synergistic effects of MEK inhibition and 5-ALA PDT in vitro and apoptotic effects of 5-ALA PDT in vivo, highlights the potential therapeutic efficacy of this treatment modality.

ITVT-01. 5-ALA PDT and Targeting MEK/ERK Signaling Elicits Synergistic Antitumor Effects in Diffuse Midline Glioma

Abstract Diffuse midline gliomas are highly invasive, unresectable tumors in children. To date, there is no effective treatment for DMG. Concurrent radiotherapy (RT) and systemic therapies, the standard of care, has only been successful in providing limited disease control. The major obstacle to therapy is the selectively permeable blood brain barrier (BBB) that limits systemic drug delivery. Given the unmet need for penetrant and minimally invasive DMG treatments, photodynamic therapy (PDT), with the precursor photosensitizing agent 5-aminolevulinic acid (5-ALA), is an oncologic treatment that holds promise. 5-ALA PDT of tumors occurs by targeting tumor cells that accumulate the 5-ALA metabolite, protoporphyrin IX (PPIX), with 635 nm light to create deadly reactive oxygen species (ROS). We explore the synergism of 5-ALA PDT with the MEK inhibitor, trametinib, since the RAS/MEK signaling pathway regulates tumor cell proliferation and survival and has been shown to therapeutically enhance PDT in select carcinoma models. We demonstrated that sub-micromolar levels of 5-ALA PDT and nanomolar levels of trametinib successfully decrease cell proliferation and induce apoptosis in DMG cell lines. Cell viability assays revealed that drug response differs based on the histone mutation (H3.1 or H3.3) of the line. Mechanisms of decreased cell survival involves the generation of reactive oxygen species that induces programmed cell death. Through the use of a DMG genetically engineered mouse model, we also found 5-ALA PDT to induce apoptosis in vivo. The synergistic effects of MEK inhibition and 5-ALA PDT in vitro and apoptotic effects of 5-ALA PDT in vivo, highlights the potential therapeutic efficacy of this treatment modality.

CSIG-31. MULTI-OMICS TO EDGE INTO PRECISION MEDICINE FOR DIPG

Abstract DIPG is an incurable pediatric brain tumor with 80% of patients harboring H3F3A (H3.3) mutation that substitutes methionine for lysine at position 27 (K27M), resulting in global depletion of H3.3K27 me3 (trimethylation). These histone mutations modify the epigenome and alter oncogenic transcription, causing oncogenic insults to progenitor cells in early neurodevelopment (1). To determine the reprogramming pathways in the cell context of H3.3K27M tumors, we conducted LC-MS based proteomic and phosphoproteomic analysis on seven patient-derived DIPG cell lines. Three normal neuronal stem cell lines were included as non-tumor brain cells for comparison. Pathway analysis identified 29 pathways that are significantly altered in DIPG compared to normal brain cells at both the protein abundance and phosphosite level. Notably, AKT and MAPK associated PI3K signaling, VEGF signaling, mTOR signaling, and HIF1a signaling were differentially active in H3.3K27M tumors compared to healthy control cell lines. We saw significantly higher activity of multiple kinases involved in axon guidance and cytoskeletal remodeling in DIPG, such as PTK2B, DYRK2, TTBK2 and MARK2. This is the first time to report an increased abundance and kinase activity of PYK2 protein (coded by PTK2B), a close homologue of FAK and its associated signaling in DIPG. PYK2 has been proposed to act in concert with Src to link Gi- or Gq-coupled receptors with the mitogen-activated protein (MAP) kinase signaling pathway (2). Because of the shared signaling across kinase pathways, targeting activated PYK2 in DIPG may complement inhibitors of other dysregulated signaling networks in DIPG such as MAPK2, VEGFR, PI3K and Src. Our data also found that IL13RA2 was upregulated in DIPG. We conclude that for H3 K27M DIPG tumors, campaigns to target PYK2, MAPK2, VEGFR, PI3K, Src and IL13Ra2 using small molecules that traverse the blood brain barrier loom as promising opportunities for drug development.

EXTH-58. THERAPEUTIC HDAC INHIBITION IN HYPERMUTANT DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG) continues to carry a dismal prognosis despite a growing understanding of its epigenetic regulation. While generally reclassified as diffuse midline glioma, H3 K27M-mutant (DMG), a subgroup of DIPGs do not harbor the classic histone mutation, with a further subset exhibiting a hypermutant phenotype. To evaluate whether hypermutant DIPG shares transcriptional vulnerabilities with H3K27M-mutant DMG, we screened a biopsy-derived treatment-naive PMS2 mutant DIPG model (PBT-24FH) for sensitivity to a panel of HDAC inhibitors (HDACi). In vitro evaluation of cell viability revealed the low nanomolar IC50 of quisinostat (50nM) and romidepsin (2nM). Dose-dependent increases in H3 acetylation and c-PARP were confirmed by western blot. Despite romidepsin’s superior potency in vitro, quisinostat demonstrated greater efficacy in an in vivo PBT-24FH flank study. 42 days following drug initiation, quisinostat-treated mice displayed dramatic tumor regression (mean volume= 33mm3, n= 7) compared to mice treated with romidepsin (mean volume= 669mm3, n= 7)(p= 0.005), or vehicle (mean volume= 990mm3, n= 6)(p< 0.001). Immunohistochemistry of quisinostat-treated tumors revealed few residual tumor cells displaying a low proliferative index. To evaluate cross-resistance, romidepsin-treated mice (mean volume= 1158mm3, n= 2) were switched to quisinostat treatment and displayed swift tumor regression (mean volume after 25 days of quisinostat= 419mm3), emphasizing quisinostat’s in vivo cytotoxic effect against both large tumors and tumors previously treated by another HDACi. To evaluate quisinostat’s effect on other hypermutant tumors, we tested HCT-116, a colon cancer cell line bearing a biallelic MLH1 deletion and observed similar cytotoxicity. We also aim to repeat these studies utilizing additional pediatric hypermutant high grade glioma models. Transcriptomic and proteomic investigations are underway to identify the mechanism of action underlying quisinostat-induced cytotoxicity. Ultimately, we are the first to demonstrate in vivo efficacy of the HDACi quisinostat against hypermutant DIPG, supporting further investigation and clinical advancement.

Germinal Center B Cells Derived from TET2-Mutated Clonal Hematopoiesis Provide a Microenviromental Niche for Tumor Cells in Angioimmunoblastic T-Cell Lymphoma

Germinal Center B Cells Derived from TET2-Mutated Clonal Hematopoiesis Provide a Microenviromental Niche for Tumor Cells in Angioimmunoblastic T-Cell Lymphoma

Polycomb repressive complex 2 in the driver's seat of childhood and young adult brain tumours.

Deregulation of the epigenome underlies oncogenesis in numerous primary brain tumours in children and young adults. In this review, we describe how recurrent mutations in isocitrate dehydrogenases or histone 3 variants (oncohistones) in gliomas, expression of the oncohistone mimic enhancer of Zeste homologs inhibiting protein (EZHIP) in a subgroup of ependymoma, and epigenetic alterations in other embryonal tumours promote oncogenicity. We review the proposed mechanisms of cellular transformation, current tumorigenesis models and their link to development. We further stress the narrow developmental windows permissive to their oncogenic potential and how this may stem from converging effects deregulating polycomb repressive complex (PRC)2 function and targets. As altered chromatin states may be reversible, improved understanding of aberrant cancer epigenomes could orient the design of effective therapies.

Emerging Advances in Combinatorial Treatments of Epigenetically Altered Pediatric High-Grade H3K27M Gliomas.

Somatic mutations in histone encoding genes result in gross alterations in the epigenetic landscape. Diffuse intrinsic pontine glioma (DIPG) is a pediatric high-grade glioma (pHGG) and one of the most challenging cancers to treat, with only 1% surviving for 5 years. Due to the location in the brainstem, DIPGs are difficult to resect and rapidly turn into a fatal disease. Over 80% of DIPGs confer mutations in genes coding for histone 3 variants (H3.3 or H3.1/H3.2), with lysine to methionine substitution at position 27 (H3K27M). This results in a global decrease in H3K27 trimethylation, increased H3K27 acetylation, and widespread oncogenic changes in gene expression. Epigenetic modifying drugs emerge as promising candidates to treat DIPG, with histone deacetylase (HDAC) inhibitors taking the lead in preclinical and clinical studies. However, some data show the evolving resistance of DIPGs to the most studied HDAC inhibitor panobinostat and highlight the need to further investigate its mechanism of action. A new forceful line of research explores the simultaneous use of multiple inhibitors that could target epigenetically induced changes in DIPG chromatin and enhance the anticancer response of single agents. In this review, we summarize the therapeutic approaches against H3K27M-expressing pHGGs focused on targeting epigenetic dysregulation and highlight promising combinatorial drug treatments. We assessed the effectiveness of the epigenetic drugs that are already in clinical trials in pHGGs. The constantly expanding understanding of the epigenetic vulnerabilities of H3K27M-expressing pHGGs provides new tumor-specific targets, opens new possibilities of therapy, and gives hope to find a cure for this deadly disease.

Binding of regulatory proteins to nucleosomes is modulated by dynamic histone tails

Little is known about the roles of histone tails in modulating nucleosomal DNA accessibility and its recognition by other macromolecules. Here we generate extensive atomic level conformational ensembles of histone tails in the context of the full nucleosome, totaling 65 microseconds of molecular dynamics simulations. We observe rapid conformational transitions between tail bound and unbound states, and characterize kinetic and thermodynamic properties of histone tail-DNA interactions. Different histone types exhibit distinct binding modes to specific DNA regions. Using a comprehensive set of experimental nucleosome complexes, we find that the majority of them target mutually exclusive regions with histone tails on nucleosomal/linker DNA around the super-helical locations ± 1, ± 2, and ± 7, and histone tails H3 and H4 contribute most to this process. These findings are explained within competitive binding and tail displacement models. Finally, we demonstrate the crosstalk between different histone tail post-translational modifications and mutations; those which change charge, suppress tail-DNA interactions and enhance histone tail dynamics and DNA accessibility.

Post-Denosumab-Treated Giant Cell Tumor of Bone: A Retrospective Histomorphological and Immunohistochemical Study

Abstract Introduction Giant cell tumors of bone (GCTBs) are treated with surgery with or without local adjuvants. Denosumab is a human monoclonal antibody that has recently emerged to be effective in treating unresectable and recurrent GCTBs. Objective In this study, we analyzed the histomorphological changes in GCTB following treatment with denosumab. The expression of histone mutation H3.3G34W by immunohistochemistry (IHC) using mutant specific antibody was also determined. Materials and Methods Of the total 109 GCTBs encountered during the study period, 14 cases with neoadjuvant denosumab therapy were analyzed retrospectively. The post-treatment changes on histopathology were examined on routine hematoxylin and eosin-stained sections. IHC was done using antihistone H3.3G34 antibodies. Statistical analysis was limited to descriptive statistics. No hypothesis testing was performed. Results All these cases except three showed fibrosis with areas of hyalinization, prominent newly formed woven bone along with spindle cells in short fascicles and storiform pattern. There was complete absence and marked reduction in osteoclast-like giant cells in six and five patients, respectively. Only three patients showed a substantial amount of residual osteoclast-like giant cells. IHC with antihistone H3.3G34W antibody showed unequivocal nuclear positivity in the mononuclear cells in nine cases. The mononuclear cells rimming and entrapped within the woven bone were also positive on IHC. The spindle cells in the benign fibrous histiocytoma-like areas and septa of aneurysmal bone cyst-like areas also retained nuclear staining. Conclusion Awareness of post-denosumab-related histopathological changes are necessary to avoid misdiagnosis as fibroosseous lesion and low-grade central osteosarcoma. Expression of mutant-specific H3.3 G34W antibody suggests that the neoplastic stromal cells are largely retained after denosumab therapy. The positive staining of cells both within and those rimming the newly formed woven bone point toward osteoblastic phenotype of the neoplastic stromal cells.