mRNA display reveals a class of high-affinity bromodomain-binding motifs that are not found in the human proteome.

Bromodomains (BDs) regulate gene expression by recognizing protein motifs containing acetyllysine. Although originally characterized as histone-binding proteins, it has since become clear that these domains interact with other acetylated proteins, perhaps most prominently transcription factors. The likely transient nature and low stoichiometry of such modifications, however, has made it challenging to fully define the interactome of any given BD. To begin to address this knowledge gap in an unbiased manner, we carried out mRNA display screens against a BD-the N-terminal BD of BRD3-using peptide libraries that contained either one or two acetyllysine residues. We discovered peptides with very strong consensus sequences and with affinities that are significantly higher than typical BD-peptide interactions. X-ray crystal structures also revealed modes of binding that have not been seen with natural ligands. Intriguingly, however, our sequences are not found in the human proteome, perhaps suggesting that strong binders to BDs might have been selected against during evolution.

Tumor suppressive role of the epigenetic master regulator BRD3 in colorectal cancer.

Bromodomain and extraterminal domain (BET) family proteins are epigenetic master regulators of gene expression via recognition of acetylated histones and recruitment of transcription factors and co-activators to chromatin. Hence, BET family proteins have emerged as promising therapeutic targets in cancer. In this study, we examined the functional role of bromodomain containing 3 (BRD3), a BET family protein, in colorectal cancer (CRC). In vitro and vivo analyses using BRD3-knockdown or BRD3-overexpressing CRC cells showed that BRD3 suppressed tumor growth and cell cycle G1/S transition and induced p21 expression. Clinical analysis of CRC datasets from our hospital or The Cancer Genome Atlas revealed that BET family genes, including BRD3, were overexpressed in tumor tissues. In immunohistochemical analyses, BRD3 was observed mainly in the nucleus of CRC cells. According to single-cell RNA sequencing in untreated CRC tissues, BRD3 was highly expressed in malignant epithelial cells, and cell cycle checkpoint-related pathways were enriched in the epithelial cells with high BRD3 expression. Spatial transcriptomic and single-cell RNA sequencing analyses of CRC tissues showed that BRD3 expression was positively associated with high p21 expression. Furthermore, overexpression of BRD3 combined with knockdown of, a driver gene in the BRD family, showed strong inhibition of CRC cells in vitro. In conclusion, we demonstrated a novel tumor suppressive role of BRD3 that inhibits tumor growth by cell cycle inhibition in part via induction of p21 expression. BRD3 activation might be a novel therapeutic approach for CRC.

Cyclic peptides can engage a single binding pocket through highly divergent modes.

Cyclic peptide library screening technologies show immense promise for identifying drug leads and chemical probes for challenging targets. However, the structural and functional diversity encoded within such libraries is largely undefined. We have systematically profiled the affinity, selectivity, and structural features of library-derived cyclic peptides selected to recognize three closely related targets: the acetyllysine-binding bromodomain proteins BRD2, -3, and -4. We report affinities as low as 100 pM and specificities of up to 106-fold. Crystal structures of 13 peptide-bromodomain complexes reveal remarkable diversity in both structure and binding mode, including both α-helical and ß-sheet structures as well as bivalent binding modes. The peptides can also exhibit a high degree of structural preorganization. Our data demonstrate the enormous potential within these libraries to provide diverse binding modes against a single target, which underpins their capacity to yield highly potent and selective ligands.

Insights into the Mechanism of Curaxin CBL0137 Epigenetic Activity: The Induction of DNA Demethylation and the Suppression of BET Family Proteins.

The development of malignant tumors is caused by a complex combination of genetic mutations and epigenetic alterations, the latter of which are induced by either external environmental factors or signaling disruption following genetic mutations. Some types of cancer demonstrate a significant increase in epigenetic enzymes, and targeting these epigenetic alterations represents a compelling strategy to reverse cell transcriptome to the normal state, improving chemotherapy response. Curaxin CBL0137 is a new potent anticancer drug that has been shown to activate epigenetically silenced genes. However, its detailed effects on the enzymes of the epigenetic system of transcription regulation have not been studied. Here, we report that CBL0137 inhibits the expression of DNA methyltransferase DNMT3a in HeLa TI cells, both at the level of mRNA and protein, and it decreases the level of integral DNA methylation in Ca Ski cells. For the first time, it is shown that CBL0137 decreases the level of BET family proteins, BRD2, BRD3, and BRD4, the key participants in transcription elongation, followed by the corresponding gene expression enhancement. Furthermore, we demonstrate that CBL0137 does not affect the mechanisms of histone acetylation and methylation. The ability of CBL0137 to suppress DNMT3A and BET family proteins should be taken into consideration when combined chemotherapy is applied. Our data demonstrate the potential of CBL0137 to be used in the therapy of tumors with corresponding aberrant epigenetic profiles.

Comparative structure-function analysis of bromodomain and extraterminal motif (BET) proteins in a gene-complementation system.

The widely expressed bromodomain and extraterminal motif (BET) proteins bromodomain-containing protein 2 (BRD2), BRD3, and BRD4 are multifunctional transcriptional regulators that bind acetylated chromatin via their conserved tandem bromodomains. Small molecules that target BET bromodomains are being tested for various diseases but typically do not discern between BET family members. Genomic distributions and protein partners of BET proteins have been described, but the basis for differences in BET protein function within a given lineage remains unclear. By establishing a gene knockout-rescue system in a Brd2-null erythroblast cell line, here we compared a series of mutant and chimeric BET proteins for their ability to modulate cell growth, differentiation, and gene expression. We found that the BET N-terminal halves bearing the bromodomains convey marked differences in protein stability but do not account for specificity in BET protein function. Instead, when BET proteins were expressed at comparable levels, their specificity was largely determined by the C-terminal half. Remarkably, a chimeric BET protein comprising the N-terminal half of the structurally similar short BRD4 isoform (BRD4S) and the C-terminal half of BRD2 functioned similarly to intact BRD2. We traced part of the BRD2-specific activity to a previously uncharacterized short segment predicted to harbor a coiled-coil (CC) domain. Deleting the CC segment impaired BRD2's ability to restore growth and differentiation, and the CC region functioned in conjunction with the adjacent ET domain to impart BRD2-like activity onto BRD4S. In summary, our results identify distinct BET protein domains that regulate protein turnover and biological activities.

Skin adnexal carcinoma with BRD3-NUTM2B fusion.

NUT carcinomas are genetically defined epithelial neoplasms. Most tumors harbor fusions of NUTM1 with BRD4 or BRD3. Their histopathologic features have been predominantly reported as undifferentiated or poorly differentiated squamous cell carcinoma, and clinically they tend to be aggressive cancers. However, recent studies have revealed a broader spectrum of NUTM1-rearranged neoplasms with several new fusion partners and associated variable histopathologic phenotypes and clinical behaviors, including benign and malignant cutaneous poroid tumors. We report herein a primary invasive carcinoma of skin adnexal origin with a previously undescribed fusion between BRD3 and NUTM2B. The tumor occurred on the shoulder of a 7-year-old girl and was excised with negative margins. A sentinel lymph node was positive. After follow-up of 23 months, and without systemic treatment, the child remains free of tumor. This case expands the spectrum of NUT carcinomas by including a skin adnexal variant with follicular infundibular differentiation, a novel genomic aberration, and preliminary evidence of a less aggressive clinical course.

Targetable BET proteins- and E2F1-dependent transcriptional program maintains the malignancy of glioblastoma.

Competitive BET bromodomain inhibitors (BBIs) targeting BET proteins (BRD2, BRD3, BRD4, and BRDT) show promising preclinical activities against brain cancers. However, the BET protein-dependent glioblastoma (GBM)-promoting transcriptional network remains elusive. Here, with mechanistic exploration of a next-generation chemical degrader of BET proteins (dBET6), we reveal a profound and consistent impact of BET proteins on E2F1- dependent transcriptional program in both differentiated GBM cells and brain tumor-initiating cells. dBET6 treatment drastically reduces BET protein genomic occupancy, RNA-Pol2 activity, and permissive chromatin marks. Subsequently, dBET6 represses the proliferation, self-renewal, and tumorigenic ability of GBM cells. Moreover, dBET6-induced degradation of BET proteins exerts superior antiproliferation effects compared to conventional BBIs and overcomes both intrinsic and acquired resistance to BBIs in GBM cells. Our study reveals crucial functions of BET proteins and provides the rationale and therapeutic merits of targeted degradation of BET proteins in GBM.

NUT Carcinoma: Clinicopathologic features, pathogenesis, and treatment.

NUT carcinoma (NC) is a rare, aggressive subtype of squamous cell carcinoma defined by rearrangement of the NUTM1 (aka NUT) gene. NC is driven by NUT-fusion oncoproteins resulting from chromosomal translocation, most commonly BRD4-NUT. This is a nearly uniformly lethal cancer affecting patients of all ages, but predominantly teens and young adults. The cell of origin is unknown, but NC most commonly arises within the thorax and head and neck. NC typically consists of sheets of monomorphic primitive round cells that can exhibit focal abrupt squamous differentiation. Diagnosis of NC is easy, and can be established by positive NUT nuclear immunohistochemical staining. Though characterization of the NUTM1-fusion gene is desirable by molecular analysis, it is not required for the diagnosis. The increasingly widespread availability of the NUT diagnostic test is leading to increasing diagnoses of this vastly underdiagnosed disease. The NUT midline carcinoma registry (www.NMCRegistry.org) serves as a central repository that has provided the main source of clinical and outcomes data for NC. Currently there is no effective therapy for NC, however small molecules directly targeting the BRD4 portion of BRD4-NUT, termed BET bromodomain inhibitors, have shown activity.

Novel rice mutants overexpressing the brassinosteroid catabolic gene CYP734A4.

KEY MESSAGE: Moderate overexpression of CYP734A4 improves grain number per main panicle and seed setting rate. Brassinosteroid (BR) homeostasis and signaling are crucial for plant growth and development. CYP734A genes encode cytochrome P450 monooxygenases that control the level of bioactive BRs by degrading BRs. However, fertile plants overexpressing CYP734As have not been reported in rice. Here, we isolated a novel semi-dominant mutant brd3-D, in which T-DNA was inserted approximately 4 kb upstream of the CYP734A4 gene (GenBank Accession AB488667), causing its overexpression. The mutant is characterized by dwarfism, small grains, and erect leaves and is less sensitive to brassinolide-induced lamina joint inclination and primary root elongation. However, increased grain number per main panicle and improved seed setting rate were also found in heterozygous brd3-D. To our knowledge, these traits have not been reported in other BR deficient mutants. Quantitative real-time PCR analysis indicated that phenotypic severity of the brd3-D mutant is positively correlated with the CYP734A4 transcription level. In accordance with the increased expression of CYP734A4, a lower castasterone (a rice BR) content was detected in the brd3-D mutants. Knockout of brd3-D by using the CRISPR/Cas9 system rescued the mutation. In addition, transgenic plants overexpressing CYP734A4 with the 35S enhancer mimicked the brd3-D phenotypes, confirming that moderate overexpression of the CYP734A4 gene can improve grain number per main panicle and the seed setting rate in rice. Further studies showed that overexpression of CYP734A4 influences the expressions of multiple genes involved in the BR pathway, and the expression of CYP734A4 is induced by exogenous brassinolide, confirming the negative regulatory role of CYP734A4 in the BR pathway. CYP734A4 might provide a useful gene resource for developing new high-yielding rice varieties.

Intensive treatment and survival outcomes in NUT midline carcinoma of the head and neck.

BACKGROUND: NUT midline carcinoma is a rare and aggressive genetically characterized subtype of squamous cell carcinoma frequently arising from the head and neck. The characteristics and optimal management of head and neck NUT midline carcinoma (HNNMC) are unclear. METHODS: A retrospective review of all known cases of HNNMC in the International NUT Midline Carcinoma Registry as of December 31, 2014, was performed. Forty-eight consecutive patients were treated from 1993 to 2014, and clinicopathologic variables and outcomes for 40 patients were available for analyses; they composed the largest HNNMC cohort studied to date. Overall survival (OS) and progression-free survival (PFS) according to patient characteristics and treatment were analyzed. RESULTS: This study identified a 5-fold increase in the diagnosis of HNNMC from 2011 to 2014. The median age was 21.9 years (range, 0.1-81.7 years); the male and female proportions were 40% and 60%, respectively; and 86% had bromodomain containing 4-nuclear protein in testis (BRD4-NUT) fusion. The initial treatment was initial surgery with or without adjuvant chemoradiation or adjuvant radiation (56%), initial radiation with or without chemotherapy (15%), or initial chemotherapy with or without surgery or radiation (28%). The median PFS was 6.6 months (range, 4.7-8.4 months). The median OS was 9.7 months (range, 6.6-15.6 months). The 2-year PFS rate was 26% (95% confidence interval [CI], 13%-40%). The 2-year OS rate was 30% (95% CI, 16%-46%). Initial surgery with or without postoperative chemoradiation or radiation (P = .04) and complete resection with negative margins (P = .01) were significant predictors of improved OS even after adjustments for age, tumor size, and neck lymphadenopathy. Initial radiation or chemotherapy and the NUT translocation type were not associated with outcomes. CONCLUSIONS: HNNMC portends a poor prognosis. Aggressive initial surgical resection with or without postoperative chemoradiation or radiation is associated with significantly enhanced survival. Chemotherapy or radiation alone is often inadequate. Cancer 2016;122:3632-40. © 2016 American Cancer Society.

In vitro cytotoxicity of Withania somnifera (L.) roots and fruits on oral squamous cell carcinoma cell lines: a study supported by flow cytometry, spectral, and computational investigations.

Oral cancer is a severe health problem that accounts for an alarmingly high number of fatalities worldwide. Withania somnifera (L.) Dunal has been extensively studied against various tumor cell lines from different body organs, rarely from the oral cavity. We thus investigated the cytotoxicity of W. somnifera fruits (W-F) and roots (W-R) hydromethanolic extracts and their chromatographic fractions against oral squamous cell carcinoma (OSCC) cell lines [Ca9-22 (derived from gingiva), HSC-2, HSC-3, and HSC-4 (derived from tongue)] and three normal oral mesenchymal cells [human gingival fibroblast (HGF), human periodontal ligament fibroblast (HPLF), and human pulp cells (HPC)] in comparison to standard drugs. The root polar ethyl acetate (W-R EtOAc) and butanol (W-R BuOH) fractions exhibited the strongest cytotoxicity against the Ca9-22 cell line (CC50 = 51.8 and 40.1 µg/mL, respectively), which is relatively the same effect as 5-FU at CC50 = 69.4 µM and melphalan at CC50 = 36.3 µM on the same cancer cell line. Flow cytometric analysis revealed changes in morphology as well as in the cell cycle profile of the W-R EtOAc and W-R BuOH-treated oral cancer Ca9-22 cells compared to the untreated control. The W-R EtOAc (125 µg/mL) exerted morphological changes and induced subG1 accumulation, suggesting apoptotic cell death. A UHPLC MS/MS analysis of the extract enabled the identification of 26 compounds, mainly alkaloids, withanolides, withanosides, and flavonoids. Pharmacophore-based inverse virtual screening proposed that BRD3 and CDK2 are the cancer-relevant targets for the annotated withanolides D (18) and O (12), and the flavonoid kaempferol (11). Molecular modeling studies highlighted the BRD3 and CDK2 as the most probable oncogenic targets of anticancer activity of these molecules. These findings highlight W. somnifera's potential as an affordable source of therapeutic agents for a range of oral malignancies.

Kinome-wide screening uncovers a role for Bromodomain Protein 3 in DNA double-stranded break repair.

Double-stranded breaks (DSBs) are toxic DNA damage and a serious threat to genomic integrity. Thus, all living organisms have evolved multiple mechanisms of DNA DSB repair, the two principal ones being classical-non homologous end joining (C-NHEJ), and homology dependent recombination (HDR). In mammals, C-NHEJ is the predominate DSB repair pathway, but how a cell chooses to repair a particular DSB by a certain pathway is still not mechanistically clear. To uncover novel regulators of DSB repair pathway choice, we performed a kinome-wide screen in a human cell line engineered to express a dominant-negative C-NHEJ factor. The intellectual basis for such a screen was our hypothesis that a C-NHEJ-crippled cell line might need to upregulate other DSB repair pathways, including HDR, in order to survive. This screen identified Bromodomain-containing Protein 3 (BRD3) as a protein whose expression was almost completely ablated specifically in a C-NHEJ-defective cell line. Subsequent experimentation demonstrated that BRD3 is a negative regulator of HDR as BRD3-null cell lines proved to be hyper-recombinogenic for gene conversion, sister chromatid exchanges and gene targeting. Mechanistically, BRD3 appears to be working at the level of Radiation Sensitive 51 (RAD51) recruitment. Overall, our results demonstrate that BRD3 is a novel regulator of human DSB repair pathway choice.

Bromodomain and Extra-Terminal Proteins in Brain Physiology and Pathology: BET-ing on Epigenetic Regulation.

BET proteins function as histone code readers of acetylated lysins that determine the positive regulation in transcription of genes involved in cell cycle progression, differentiation, inflammation, and many other pathways. In recent years, thanks to the development of BET inhibitors, interest in this protein family has risen for its relevance in brain development and function. For example, experimental evidence has shown that BET modulation affects neuronal activity and the expression of genes involved in learning and memory. In addition, BET inhibition strongly suppresses molecular pathways related to neuroinflammation. These observations suggest that BET modulation may play a critical role in the onset and during the development of diverse neurodegenerative and neuropsychiatric disorders, such as Alzheimer's disease, fragile X syndrome, and Rett syndrome. In this review article, we summarize the most recent evidence regarding the involvement of BET proteins in brain physiology and pathology, as well as their pharmacological potential as targets for therapeutic purposes.

Systematic analysis of the BET family in adrenocortical carcinoma: The expression, prognosis, gene regulation network, and regulation targets.

Background: Bromodomain and extracellular terminal (BET) family (including BRD2, BRD3, and BRD4) is considered to be a major driver of cancer cell growth and a new target for cancer therapy. Currently, more than 30 targeted inhibitors have shown significant inhibitory effects against various tumors in preclinical and clinical trials. However, the expression levels, gene regulatory networks, prognostic value, and target prediction of BRD2, BRD3, and BRD4 in adrenocortical carcinoma (ACC) have not been fully elucidated. Therefore, this study aimed to systematically analyze the expression, gene regulatory network, prognostic value, and target prediction of BRD2, BRD3, and BRD4 in patients with ACC, and elucidated the association between BET family expression and ACC. We also provided useful information on BRD2, BRD3, and BRD4 and potential new targets for the clinical treatment of ACC. Methods: We systematically analyzed the expression, prognosis, gene regulatory network, and regulatory targets of BRD2, BRD3, and BRD4 in ACC using multiple online databases, including cBioPortal, TRRUST, GeneMANIA, GEPIA, Metascape, UALCAN, LinkedOmics, and TIMER. Results: The expression levels of BRD3 and BRD4 were significantly upregulated in ACC patients at different cancer stages. Moreover, the expression of BRD4 was significantly correlated with the pathological stage of ACC. ACC patients with low BRD2, BRD3, and BRD4 expressions had longer survival than patients with high BRD2, BRD3, and BRD4 expressions. The expression of BRD2, BRD3, and BRD4 was altered by 5%, 5%, and 12% in 75 ACC patients, respectively. The frequency of gene alterations in the 50 most frequently altered BRD2, BRD3, and BRD4 neighboring genes in these ACC patients were ≥25.00%, ≥25.00%, and ≥44.44%, respectively. BRD2, BRD3, and BRD4 and their neighboring genes form a complex network of interactions mainly through co-expression, physical interactions, and shared protein domains. Molecular functions related to BRD2, BRD3, and BRD4 and their neighboring genes mainly include protein-macromolecule adaptor activity, cell adhesion molecule binding, and aromatase activity. Chemokine signaling pathway, thiamine metabolism, and olfactory transduction were found to be enriched as per the KEGG pathway analysis. SP1, NPM1, STAT3, and TP53 are key transcription factors for BRD2, BRD4, and their neighboring genes. MiR-142-3P, miR-484, and miR-519C were the main miRNA targets of BRD2, BRD3, BRD4, and their neighboring genes. We analyzed the mRNA sequencing data from 79 patients with ACC and found that ZSCAN12, DHX16, PRPF4B, EHMT1, CDK5RAP2, POMT1, WIZ, ZNF543, and AKAP8 were the top nine genes whose expression were positively associated with BRD2, BRD3, and BRD4 expression. The expression level of BRD2, BRD3, and BRD4 positively correlated with B cell and dendritic cell infiltration levels. BRD4-targeted drug PFI-1 and (BRD2, BRD3, and BRD4)-targeted drug I-BET-151 may have good inhibitory effects on the SW13 cell line. Conclusions: The findings of this study provide a partial basis for the role of BRD2, BRD3, and BRD4 in the occurrence and development of ACC. In addition, this study also provides new potential therapeutic targets for ACC, which can serve as a reference for future basic and clinical research.

Case report of a pan-cytokeratin negative NUT midline carcinoma of pulmonary origin, a BRD3-NUT variant: Challenges in cytomorphologic presentation.

NUT carcinoma is an aggressive malignancy defined genetically by a balanced translocation of the NUT gene on chromosome 15q14, most commonly associated with the bromodomain-containing protein 4 (BRD4) gene on 19p13.1 but less frequently with variant genes, including BRD3 and NSD-3. We present a case report of a metastatic pulmonary NUT carcinoma found to have a BRD3-NUT fusion and to have only focal pan-cytokeratin staining. Biopsy of the pulmonary mass revealed dyscohesive cells with enlarged nuclei, prominent nucleoli and high nuclear to cytoplasmic ratio without areas of squamous differentiation. Initial immunohistochemical stains were positive for NUT, p63 and retained SMARCA4, while negative for Lu-5 (pan-cytokeratin), TTF-1, p40, S100 protein, OCT-4, HMB-45, SMA, and PAX-8. Tempus ×T assay revealed a BRD3-NUTM1 fusion gene. Post-mortem analysis revealed an ill-defined mass abutting the trachea and superior vena cava, as well as a perirenal mass.

Establishment of environment-sensitive probes targeting BRD3/BRD4 for imaging and therapy of tumor.

The BET (bromo and extra-terminal) family proteins are epigenetic readers and master transcription coactivators, which have attracted great interests as cancer therapeutic targets. However, there are few developed labeling toolkits that can be applied for the dynamic studies of BET family proteins in living cells and tissue slices. In order to label and study the distribution of the BET family proteins in tumor cells and tumor tissues, a novel series of environment-sensitive fluorescent probes (6a-6c) were designed and evaluated for their labeling properties. Interestingly, 6a is capable of identifying tumor tissue slices and making a distinction between the tumor and normal tissues. Moreover, it can localize to the nuclear bodies in tumor slices just like BRD3 antibody. In addition, it also played an anti-tumor role through the induction of apoptosis. All these features render 6a may compatible for immunofluorescent studies and future cancer diagnosis, and guide for the discovery of new anticancer drugs.

Clinicopathological molecular characterizations of sinonasal NUT carcinoma: a report of two cases and a literature review.

Background: Nuclear protein in testis (NUT) carcinoma (NC) is a rare, aggressive tumor with a typical NUTM1 gene rearrangement. Methods: Herein, we report a series of 2 cases of sinonasal NC: one in a 16-year-old woman and one in a 37-year-old man. Immunohistochemistry (IHC) staining for NUT (C52B1), fluorescence in situ hybridization (FISH), and next generation sequencing (NGS) sequencing were performed to investigate the morphological and genetic features of sinonasal NC. Results: The two cases presented similar pathological features and IHC markers, and typical morphological changes, including undifferentiated cells and abrupt keratinization, were observed, with numerous mitotic figures and widespread tumor necrosis. Diffuse expression of NUT, CK, p63, and p40 was noted, while the tumors were negative for synaptophysin, chromogranin A, S-100, EBV-ISH, and PD-L1. Both tumors harbored a NUTM1 rearrangement. Subsequent sequencing revealed a rare BRD3::NUTM1 fusion and a classic BRD4::NUTM1 fusion. In addition, MCL1 copy number gain (2.1), low tumor mutation burden and stable microsatellites, were also confirmed. Case 1 received surgery and chemoradiotherapy but died 13 months after local recurrence and subsequent lung and bone metastasis. Case 2 underwent chemoradiotherapy and unfortunately died from the disease 6 months later. A review of all previously reported cases of sinonasal NCs (n=55) revealed that these tumors occur more frequently in female pediatric patients (n=11, male: female =3:8), whereas this sex difference is not observed in adult patients (n=44, male: female =23:21). The median survival times of pediatric and adult patients were 17 and 13.8 months, respectively. Conclusion: Sinonasal NC presents typical undifferentiated or poorly differentiated cells, abrupt keratinization features and heterogeneous genotypes, including BRD4::NUTM1 and BRD3::NUTM1 fusions, with low tumor mutation burden and stable microsatellites.

NUT carcinoma, an under-recognized malignancy: a clinicopathologic and molecular series of 6 cases showing a subset of patients with better prognosis and a rare ZNF532::NUTM1 fusion.

NUT carcinoma (NC) is a rare malignancy with aggressive clinical behavior, defined by rearrangements involving the NUTM1 gene locus. This entity is often under-recognized and its diagnosis may be challenging. In this study, we describe a subset of patients that, despite the molecularly proven diagnosis of NC, show improved outcomes. In addition, we describe one case with the novel ZNF532::NUTM1 fusion. All cases of NC diagnosed from 2013 to 2022 in our department were retrieved. FISH using dual color bring-together probes and next-generation sequencing assay were performed to characterize the fusions involving NUTM1. Among 6 patients identified, 5 were men with a median age of 35.6 years. Four patients had primary tumors in the head and neck region (2 ethmoid sinus, 1 parotid gland, and 1 lacrimal gland); 1 in the mediastinum, and another presented with a femoral bone tumor. In all cases, the initial diagnoses were not NC. The cases showed different morphological patterns, including monomorphic, rhabdoid, and pleomorphic appearances. One case showed a pseudopapillary pattern. By immunohistochemistry, all tumors showed squamous differentiation and ≥50% of neoplastic cells with nuclear positivity for NUT antibody. One case expressed WT1 (C-terminus) and other showed chromogranin positivity. Genetic study revealed a BRD4::NUTM1 fusion in all head and neck cases, BRD3::NUTM1 in mediastinum case, and ZNF532::NUTM1 fusion in the femur bone case. They were treated with surgical resection plus chemotherapy and radiotherapy. The median overall survival was 23.11 months (1.6-83.3 months) and the median disease-free survival was 14.86 months (0-54.4 months). The patients with longer overall survival were one with a lacrimal gland primary (83.3 months) and other with a parotid lesion (31.9 months). Both patients were primarily treated with complete surgical resection. Anatomic location may be directly related to the overall survival in NC cases. Resectability of the lesion is also an important factor related to survival. Pathologists should include NC in the differential diagnosis of any poorly differentiated and undifferentiated monomorphic malignancy, regardless of its anatomic location.

Case report: Immunovirotherapy as a novel add-on treatment in a patient with thoracic NUT carcinoma.

NUT carcinoma (NC) is a rare and extremely aggressive form of cancer, usually presenting with intrathoracic or neck manifestations in adolescents and young adults. With no established standard therapy regimen and a median overall survival of only 6.5 months, there is a huge need for innovative treatment options. As NC is genetically driven by a single aberrant fusion oncoprotein, it is generally characterized by a low tumor mutational burden, thus making it immunologically cold and insusceptible to conventional immunotherapy. Recently, we have demonstrated that oncolytic viruses (OVs) are able to specifically infect and lyse NC cells, thereby turning an immunologically cold tumor microenvironment into a hot one. Here, we report an intensive multimodal treatment approach employing for the first time an OV (talimogene laherparepvec (T-VEC); IMLYGIC®) together with the immune checkpoint inhibitor pembrolizumab as an add-on to a basic NC therapy (cytostatic chemotherapy, radiation therapy, epigenetic therapy) in a patient suffering from a large thoracic NC tumor which exhibits an aberrant, unique BRD3:NUTM1 fusion. This case demonstrates for the first time the feasibility of this innovative add-on immunovirotherapy regimen with a profound, repetitive and durable replication of T-VEC that is instrumental in achieving tumor stabilization and improvement in the patient´s quality of life. Further, a previously unknown BRD3:NUTM1 fusion gene was discovered that lacks the extraterminal domain of BRD3.

BMS-986158, a Small Molecule Inhibitor of the Bromodomain and Extraterminal Domain Proteins, in Patients with Selected Advanced Solid Tumors: Results from a Phase 1/2a Trial.

This phase 1/2a, open-label study (NCT02419417) evaluated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics of BMS-986158, a selective bromodomain and extraterminal domain (BET) inhibitor. Dose escalation was performed with 3 BMS-986158 dosing schedules: A (5 days on, 2 days off; range, 0.75-4.5 mg), B (14 days on, 7 days off; 2.0-3.0 mg), and C (7 days on, 14 days off; 2.0-4.5 mg). Eighty-three patients were enrolled and received ≥1 BMS-986158 dose. Diarrhea (43%) and thrombocytopenia (39%) were the most common treatment-related adverse events (TRAEs). A lower incidence of TRAEs was found with schedules A (72%) and C (72%) vs. B (100%). Stable disease was achieved in 12 (26.1%), 3 (37.5%), and 9 (31.0%) patients on schedules A, B, and C, respectively. Two patients on schedule A with a 4.5-mg starting dose (ovarian cancer, n = 1; nuclear protein in testis [NUT] carcinoma, n = 1) experienced a partial response. BMS-986158 demonstrated rapid-to-moderate absorption (median time to maximum observed plasma concentration, 1-4 h). As expected with an epigenetic modifier, expression changes in select BET-regulated genes occurred with BMS-986158 treatment. Schedule A dosing (5 days on, 2 days off) yielded tolerable safety, preliminary antitumor activity, and a dose-proportional PK profile.