Michael J. Cameron

Selective inhibition of BET bromodomains

Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic ‘writers’ and ‘erasers’. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein–protein interactions of epigenetic ‘readers’, and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family.

BRD–NUT oncoproteins: a family of closely related nuclear proteins that block epithelial differentiation and maintain the growth of carcinoma cells

An unusual group of carcinomas, here termed nuclear protein in testis (NUT) midline carcinomas (NMC), are characterized by translocations that involve NUT, a novel gene on chromosome 15. In about 2/3rds of cases, NUT is fused to BRD4 on chromosome 19. Using a candidate gene approach, we identified two NMCs harboring novel rearrangements that result in the fusion of NUT to BRD3 on chromosome 9. The BRD3–NUT fusion gene encodes a protein composed of two tandem chromatin-binding bromodomains, an extra-terminal domain, a bipartite nuclear localization sequence, and almost the entirety of NUT that is highly homologous to BRD4–NUT. The function of NUT is unknown, but here we show that NUT contains nuclear localization and export sequences that promote nuclear-cytoplasmic shuttling via a leptomycin-sensitive pathway. In contrast, BRD3–NUT and BRD4–NUT are strictly nuclear, implying that the BRD moiety retains NUT in the nucleus via interactions with chromatin. Consistent with this idea, FRAP studies show that BRD4, BRD4–NUT and BRD3–NUT have significantly slower rates of lateral nuclear diffusion than that of NUT. To investigate the functional role of BRD–NUT fusion proteins in NMCs, we investigated the effects of siRNA-induced BRD3–NUT and BRD4–NUT withdrawal. Silencing of these proteins in NMC cell lines resulted in squamous differentiation and cell cycle arrest. Together, these data suggest that BRD–NUT fusion proteins contribute to carcinogenesis by associating with chromatin and interfering with epithelial differentiation.

Diagnosis of NUT midline carcinoma using a NUT-specific monoclonal antibody.

NUT midline carcinoma (NMC) is a uniformly lethal malignancy that is defined by rearrangement of the nuclear protein in testis (NUT) gene on chromosome 15q14. NMCs are morphologically indistinguishable from other poorly differentiated carcinomas, and the diagnosis is usually made currently by fluorescence in situ hybridization (FISH). As normal NUT expression is confined to testis and ovary, we reasoned that an immunohistochemical (IHC) stain for NUT would be useful in diagnosing NMC. To this end, we raised a highly specific rabbit monoclonal antibody, C52, against a recombinant NUT polypeptide, and developed an IHC staining protocol. The sensitivity and specificity of C52 staining was evaluated in a panel of 1068 tissues, predominantly diverse types of carcinomas (n=906), including 30 NMCs. Split-apart FISH for NUT rearrangement was used as a “gold standard” diagnostic test for NMC. C52 immunoreactivity among carcinomas was confined to NMCs. IHC staining had a sensitivity of 87%, a specificity of 100%, a negative predictive value of 99%, and a positive predictive value of 100%. Two new cases of NMC containing BRD4-NUT fusions were detected by C52 IHC, but missed by conventional FISH. In both instances, these tumors contained cryptic BRD4-NUT rearrangements, as confirmed by FISH using a refined set of probes. Some germ cell tumors, including 64% of dysgerminomas, showed weak NUT immunoreactivity, consistent with the expression of NUT in normal germ cells. We conclude that IHC staining with the C52 monoclonal antibody is a highly sensitive and specific test that reliably distinguishes NMC from other forms of carcinoma. The NUT antibody is being prepared for commercial release and will be available in the near future.

Differentiation of NUT midline carcinoma by epigenomic reprogramming.

NUT midline carcinoma (NMC) is a lethal pediatric tumor defined by the presence of BRD-NUT fusion proteins that arrest differentiation. Here we explore the mechanisms underlying the ability of BRD4-NUT to prevent squamous differentiation. In both gain-of and loss-of-expression assays, we find that expression of BRD4-NUT is associated with globally decreased histone acetylation and transcriptional repression. Bulk chromatin acetylation can be restored by treatment of NMC cells with histone deacetylase inhibitors (HDACi), engaging a program of squamous differentiation and arrested growth in vitro that closely mimics the effects of siRNA-mediated attenuation of BRD4-NUT expression. The potential therapeutic utility of HDACi differentiation therapy was established in three different NMC xenograft models, where it produced significant growth inhibition and a survival benefit. Based on these results and translational studies performed with patient-derived primary tumor cells, a child with NMC was treated with the FDA-approved HDAC inhibitor, vorinostat. An objective response was obtained after five weeks of therapy, as determined by positron emission tomography. These findings provide preclinical support for trials of HDACi in patients with NMC.

MYC, a downstream target of BRD-NUT, is necessary and sufficient for the blockade of differentiation in NUT midline carcinoma

NUT midline carcinoma (NMC) is an aggressive type of squamous cell carcinoma that is defined by the presence of BRD-NUT fusion oncogenes, which encode chimeric proteins that block differentiation and maintain tumor growth. BRD-NUT oncoproteins contain two bromodomains whose binding to acetylated histones is required for the blockade of differentiation in NMC, but the mechanisms by which BRD-NUT act remain uncertain. Here, we provide evidence that MYC is a key downstream target of BRD4-NUT. Expression profiling of NMCs shows that the set of genes whose expression is maintained by BRD4-NUT is highly enriched for MYC upregulated genes, and MYC and BRD4-NUT protein expression is strongly correlated in primary NMCs. More directly, we find that BRD4-NUT associates with the MYC promoter and is required to maintain MYC expression in NMC cell lines. Moreover, both siRNA knockdown of MYC and a dominant-negative form of MYC, omomyc, induce differentiation of NMC cells. Conversely, differentiation of NMC cells induced by knockdown of BRD4-NUT is abrogated by enforced expression of MYC. Together, these findings suggest that MYC is a downstream target of BRD4-NUT that is required for maintenance of NMC cells in an undifferentiated, proliferative state. Our findings support a model in which dysregulation of MYC by BRD-NUT fusion proteins has a central role in the pathogenesis of NMC.

Pathologic characteristics of NUT midline carcinoma arising in the mediastinum.

NUT midline carcinomas (NMCs) comprise a group of highly aggressive tumors that have been reported primarily in the head, neck, and mediastinum of younger individuals. These tumors overexpress the nuclear protein in the testis (NUT), most commonly due to a chromosomal translocation that fuses the NUT gene on chromosome 15 with the BRD4 gene on chromosome 19. Although the earliest recognized cases were described in the thymus or mediastinum, an extensive survey for NMCs among malignant thymic or other mediastinal neoplasms has not been reported. We examined NUT expression in 114 cases of poorly differentiated carcinomas or unclassified mediastinal malignancies using a clinically validated NUT-specific monoclonal antibody. Four of 114 (3.5%) cases showed nuclear NUT expression. A NUT translocation was confirmed by fluorescence in situ hybridization in 3 of these cases. These tumors arose in 2 men and 2 women with a median age of 50 years (range, 28 to 68 y). Three of the tumors were originally diagnosed as undifferentiated epithelioid or round cell malignant neoplasms; 1 tumor contained focal squamous differentiation and was originally diagnosed as a poorly differentiated squamous carcinoma of probable thymic origin. We find that the incidence of NMC within the mediastinum, particularly among undifferentiated tumors, is similar to that reported at other anatomic sites. NMC should be considered in the differential diagnosis of any poorly differentiated epithelioid mediastinal tumor, regardless of age.

NUT midline carcinoma: a neoplasm with diagnostic challenges in cytology

NUT midline carcinoma (NMC) is a recently described, high-grade, extremely lethal carcinoma defined by the presence of rearrangement of the nuclear protein in the testis (NUT) gene (chr15orf55) at 15q14 with BRD4 on 19p13.1, or less often with BRD3, or other uncharacterized gene partners. The BRD-NUT family of oncogenes functions by blocking epithelial differentiation and promoting cell proliferation. Our case showed the less common BRD3 translocation, was associated with a possible germ cell tumour component or differentiation, was positive for the newly developed NUT antibody and is only the second report to include cytology.

Clinicopathological significance of NUT rearrangements in poorly differentiated malignant tumors of the upper respiratory tract.

Nuclear protein in testis (NUT) midline carcinoma (NMC) is a highly malignant carcinoma originating from the midline of the body. This study investigated the clinicopathological significance of NUT rearrangements in poorly differentiated malignant tumors (PDMTs) of the upper-respiratory tract (URT) in China. The clinical and pathological features of 155 PDMTs of the URT were reviewed. Epstein-Barr virus (EBV)-encoded RNA and NUT were investigated by in situ hybridization and immunohistochemistry (IHC), respectively. NUT-positive cases were examined by fluorescence in situ hybridization (FISH) and immunohistochemical staining with a set of cytokeratins (CKs) and neuroendocrine markers. One case was observed by transmission electron microscopy. Four cases of poorly differentiated squamous cell carcinomas and sinonasal undifferentiated carcinomas were diffuse positive for NUT by IHC and also stained for antibodies to CKs and P63 but were negative for neuroendocrine markers. Only 2 of these 4 cases showed rearrangements of the NUT and BRD4 genes by FISH; both these patients died within 12 months. The remaining 2 patients showed no NUT rearrangement by FISH and did not have an aggressive clinical course. NMC is a rare, poorly differentiated carcinoma, which occurs most often in midline organs, and in this first series from China, affected the sinonasal tract of older adults and was not associated with EBV infection. Determination of NUT protein expression and gene rearrangement can allow the differentiation of NMC from other URT PDMTs. The authors suggest that molecular determination of NUT gene rearrangements should therefore represent the gold standard for NMC diagnosis.

NUT Rearrangement is Uncommon in Human Thymic Epithelial Tumors

Introduction: Thymic carcinomas represent the most aggressive histotype of thymic epithelial tumors (TETs). The 2004 World Health Organization classification has assigned a subgroup of thymic carcinomas as t(15;19) carcinomas based on the presence of t(15;19), a translocation found in poorly differentiated and highly aggressive NUT midline carcinomas. These tumors are characterized byrearrangement of the NUT (nuclear protein in testis) gene on chromosome 15q14, which in most cases fuses to the bromodomain containing 4 (BRD4) gene on chromosome 19 p13.1 through reciprocal t(15;19) translocation, resulting in constitutive BRD4-NUT fusion protein expression. To our knowledge, NUT translocation has been reported only in four thymic carcinomas. Due to the rarity of TETs, the prevalence of NUT rearrangement in TETs has however never been systematically explored. Methods: Formalin-fixed paraffin-embedded samples of histologically confirmed TETs were evaluated for NUT expression and rearrangement by immunohistochemistry and fluorescence in situ hybridization, respectively. Results: A series of 148 TETs (37 carcinomas and 111 thymomas) were examined for NUT expression and rearrangement. Only one thymic carcinoma (2.7% of thymic carcinomas or 0.68% of TETs) was found positive for NUT expression and rearrangement. Conclusions: Rearrangement of NUT is infrequent in TETs. We propose that caution should be taken to distinguish t(15;19) thymic carcinoma from other mediastinal carcinomas, as NUT midline carcinomas are often associated with dreadful prognosis or overt lethality.

FDG PET/CT imaging of NUT midline carcinoma.

Abstract:NUT midline carcinoma is a very rare, poorly differentiated, highly aggressive cancer originating from midline body locations, most often in adolescents or young adults. Advanced local disease and distant hematogenous metastases are typical. NMC is genetically defined by chromosomal rearran