The NSD family histone methyltransferases, including NSD1, NSD2 and NSD3, play crucial roles in chromatin regulation and are implicated in oncogenesis 1 , 2 . NSD enzymes exhibit an auto-inhibitory state that is relieved by nucleosome engagement, allowing for H3K36 di-methylation catalysis 3 – 7 . However, the molecular basis underlying this mechanism is largely unknown. Here, we have solved the cryo-EM structures of NSD2 and NSD3 bound to mononucleosomes at atomic resolution. We find that NSD2/3 mononucleosome engagement causes DNA near the linker region to unwrap, which facilitates insertion of their catalytic core in-between the histone octamer and the unwrapped segment of DNA. A network of DNA- and histone-specific contacts between the nucleosome and NSD2/3 precisely define the enzymes’ position on the nucleosome, explaining the methylation specificity for H3K36. Further, NSD-nucleosome intermolecular contacts are altered by several recurrent cancer-associated NSD2/3 mutations. NSDs harboring these mutations are catalytically hyperactive in vitro and in cells, and their ectopic expression promotes cancer cell proliferation and xenograft tumor growth. Together, our research provides molecular insights into the nucleosome-based recognition and modification mechanisms of NSD2 and NSD3, which should uncover strategies for therapeutic targeting of the NSD family of proteins.

Amplification of chromosomal region 8p11-12 is a frequent genetic alteration implicated in the etiology of lung squamous cell carcinoma (LUSC) 1 - 3 . FGFR1 (fibroblast growth factor receptor 1) is the main candidate driver within this region 4 . However, clinical trials evaluating FGFR1 inhibition as a targeted therapy have been unsuccessful 5 . Here we identify the H3K36 methyltransferase NSD3 ( n uclear receptor binding S ET d omain protein 3 ), an 8p11-12-localized gene, as a key regulator of LUSC tumorigenesis. In contrast to other 8p11-12 candidate LUSC drivers, increased NSD3 expression strongly correlates with its gene amplification. Ablation of NSD3 , but not FGFR1 , attenuates tumor growth and extends survival in a potent LUSC mouse model. We identify NSD3 T1232A as an LUSC-associated variant that increases H3K36 dimethylation (H3K36me2) catalytic activity in vitro and in vivo . Structural dynamic analyses reveal that the T1232A substitution elicits localized mobility changes throughout NSD3’s catalytic domain to relieve auto-inhibition and increase H3 substrate accessibility. NSD3 T1232A expression in vivo accelerates tumorigenesis and decreases overall survival in LUSC mouse models. Pathologic H3K36me2 generation by NSD3 T1232A rewires the chromatin landscape to promote oncogenic gene expression programming. Further, NSD3’s catalytic activity promotes human tracheobronchial cell transformation and xenograft growth of human 8p11-12-amplified LUSC cell lines. NSD3 depletion in patient-derived xenografts (PDXs) from primary LUSC harboring NSD3 amplification or the NSD3 T1232A variant attenuates neoplastic growth. Finally, NSD3-regulated LUSC PDXs are markedly sensitive to bromodomain inhibition (BETi). Together, our work identifies NSD3 as a principal 8p11-12 amplicon-associated oncogenic driver in LUSC and suggests that NSD3-dependency renders LUSC therapeutically vulnerable to BETi.

Modulation of the immune system can produce anti-tumor responses in various cancer types, including melanoma. Recently, immune checkpoint inhibitors (ICI), in single agent and combination regimens, have produced durable and long-lasting clinical responses in a subset of metastatic melanoma patients. These monoclonal antibodies, developed against CTLA-4 and PD-1, block immune-inhibitory receptors on activated T-cells, amplifying the immune response. However, even when using anti-CTLA-4 and anti-PD-1 in combination, approximately half of patients exhibit innate resistance and suffer from disease progression. Currently, it is impossible to predict therapeutic response. Here, we report the first proteomic and histone epigenetic analysis of patient metastatic melanoma tumors taken prior to checkpoint blockade, which revealed biological signatures that can stratify patients as responders or non-responders. Furthermore, our findings provide evidence of mesenchymal transition, a known mechanism of immune-escape, in non-responding melanoma tumors. We identified elevated histone H3 lysine (27) trimethylation (H3K27me3), decreased E-cadherin, and other protein features indicating a more mesenchymal phenotype in non-responding tumors. Our results have implications for checkpoint inhibitor therapy as patient specific responsiveness can be predicted through readily assayable proteins and histone epigenetic marks, and pathways activated in non-responders have been identified for therapeutic development to enhance responsiveness.

Purpose Despite a few recent reports of patients harboring truncating variants in NSD2, a gene considered critical for the Wolf–Hirschhorn syndrome (WHS) phenotype, the clinical spectrum associated with NSD2 pathogenic variants remains poorly understood. Methods We collected a comprehensive series of 18 unpublished patients carrying heterozygous missense, elongating, or truncating NSD2 variants; compared their clinical data to the typical WHS phenotype after pooling them with ten previously described patients; and assessed the underlying molecular mechanism by structural modeling and measuring methylation activity in vitro. Results The core NSD2-associated phenotype includes mostly mild developmental delay, prenatal-onset growth retardation, low body mass index, and characteristic facial features distinct from WHS. Patients carrying missense variants were significantly taller and had more frequent behavioral/psychological issues compared with those harboring truncating variants. Structural in silico modeling suggested interference with NSD2’s folding and function for all missense variants in known structures. In vitro testing showed reduced methylation activity and failure to reconstitute H3K36me2 in NSD2 knockout cells for most missense variants. Conclusion NSD2 loss-of-function variants lead to a distinct, rather mild phenotype partially overlapping with WHS. To avoid confusion for patients, NSD2 deficiency may be named Rauch–Steindl syndrome after the delineators of this phenotype.

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Normal cell growth is characterized by a regulated epigenetic program that drives cellular activities such as gene transcription, DNA replication, and DNA damage repair. Perturbation of this epigenetic program can lead to events such as mis-regulation of gene transcription and diseases such as cancer. To begin to understand the epigenetic program correlated to the development of melanoma, we performed a novel quantitative mass spectrometric analysis of histone post-translational modifications mis-regulated in melanoma cell culture as well as patient tumors. Aggressive melanoma cell lines as well as metastatic melanoma were found to have elevated histone H3 Lys 27 trimethylation (H3K27me3) accompanied by overexpressed methyltransferase EZH2 that adds the specific modification. The altered epigenetic program that led to elevated H3K27me3 in melanoma cell culture was found to directly silence transcription of the tumor suppressor genes RUNX3 and E-cadherin. The EZH2-mediated silencing of RUNX3 and E-cadherin transcription was also validated in advanced stage human melanoma tissues. This is the first study focusing on the detailed epigenetic mechanisms leading to EZH2-mediated silencing of RUNX3 and E-cadherin tumor suppressors in melanoma. This study underscores the utility of using high resolution mass spectrometry to identify misregulated epigenetic programs in diseases such as cancer, which could ultimately lead to the identification of biological markers for diagnostic and prognostic applications. Melanoma is a deadly variety of skin cancer, accounting for 75% of skin cancer-related deaths. In 2015, melanoma is expected to be the fifth most common cancer in men and the seventh most common cancer in women. According to the World Health Organization, it is estimated that melanoma will result in the death of around 65,000 people globally and 9940 people in the United States in 2015. The high mortality rate associated with metastatic melanoma suggests a lack of efficient diagnostic and prognostic biomarkers (1). EZH2 1 expression has often been positively correlated to the progression of different types of cancer (2, 3). Increased expression of EZH2 has been identified in melanoma tissues (4) as well as prostate, breast, bladder, and liver cancers and has been recognized as a prognostic marker for aggressive prostate and breast cancer (5, 6). EZH2 is a histone modifier that functions as the catalytic component of the Polycomb Repressive Complex 2 (PRC2) (7,8). It is a lysine methyltransferase and promotes the addition of the repressive marker histone H3K27me2/me3 to target chromatin, thereby inducing chromatin compaction and transcriptional repression. Chromatin condensation/compaction leads to transcriptional repression by restricting access to transcriptional regulators like RNA polymerase II and other transcription-associated factors. Hence, silencing of tumor suppressor genes by H3K27me3 is implicated in the initiation and advancement of different types of cancer (9 -11). H3K27me3-silenced tumor suppressor gene...

IntroductionArsenic is a widely distributed environmental toxicant that can cause multi-tissue pathologies. Proteomic assays allow for the identification of biological processes modulated by arsenic in diverse tissue types.MethodThe altered abundance of proteins from HaCaT human keratinocyte cell line exposed to arsenic was quantified using a label-free LC-MS/MS mass spectrometry workflow. Selected proteomics results were validated using western blot and RT-PCR. A functional annotation analytics strategy that included visual analytical integration of heterogeneous data sets was developed to elucidate functional categories. The annotations integrated were mainly tissue localization, biological process and gene family.ResultThe abundance of 173 proteins was altered in keratinocytes exposed to arsenic; in which 96 proteins had increased abundance while 77 proteins had decreased abundance. These proteins were also classified into 69 Gene Ontology biological process terms. The increased abundance of transferrin receptor protein (TFRC) was validated and also annotated to participate in response to hypoxia. A total of 33 proteins (11 increased abundance and 22 decreased abundance) were associated with 18 metabolic process terms. The Glutamate--cysteine ligase catalytic subunit (GCLC), the only protein annotated with the term sulfur amino acid metabolism process, had increased abundance while succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial precursor (SDHB), a tumor suppressor, had decreased abundance.ConclusionA list of 173 differentially abundant proteins in response to arsenic trioxide was grouped using three major functional annotations covering tissue localization, biological process and protein families. A possible explanation for hyperpigmentation pathologies observed in arsenic toxicity is that arsenic exposure leads to increased iron uptake in the normally hypoxic human skin. The proteins mapped to metabolic process terms and differentially abundant are candidates for evaluating metabolic pathways perturbed by arsenicals.