The focus of this project is a rare, highly aggressive, and incurable subtype of squamous cell carcinoma, genetically defined by rearrangement of the NUT gene, and termed midline carcinoma (NMC). The goals of the enclosed proposal are 1) to determine the pathogenesis and 2) effective therapy of NMC. NMC is characterized by a simple karyotype harboring a single translocation involving the NUT gene with members of the bromodomain-containing BET family, most commonly BRD3 or BRD4. The resultant BRD- NUT fusion protein is causative in this cancer, acting to block differentiation and maintain NMC cells in a perpetually proliferative state. Several pieces of evidence have led us formulate a model whereby BRD-NUT blocks differentiation by sequestering chromatin """"""""writers"""""""" necessary for transcription, such as histone acetyl- transferases (HATs), away from genes required for differentiation to limited genomic regions where the bromodomains of BRD """"""""read"""""""" and activate transcription of pro-growth, anti-differentiative genes. Indeed, drug inhibitors of the acetyl-histone binding bromodomains (BETi) or of histone deacetylases unblock the BRD-NUT induced blockade on differentiation, resulting in differentiation and arrested proliferation of NMC cells in vitro and in vivo. Both approaches have been used therapeutically in patients. In support of the above model, recent findings indicate that the transcriptional upregulation of the common cancer gene, MYC, by BRD4-NUT is required and can replace BRD4-NUT's function to block differentiation.
The first aim, to identify the molecular mechanisms by which BRD4-NUT leads to the MYC-dependent blockade of differentiation in NMC cells, will be accomplished by isolating and sequencing genes with which BRD4-NUT associates, and correlating with changes that occur in expression of those genes under conditions which promote differentiation in NMC cells. The gene targets of BRD4-NUT identified in these studies will be correlated with findings acquired from the genome-wide siRNA knockdown, which will identify specific genes whose expression is required for the blockade of differentiation in NMC cells. After a list of key target genes is identified and prioritized based on pathway analysis, they will be tested for their effects on MYC protein and RNA levels to identify which BRD4-NUT target genes are required to upregulate MYC levels. In addition, identification of BRD4-NUT-containing protein complexes by mass-spectrometry will be performed to identify proteins which collaborate with BRD4-NUT to upregulate MYC.
The second aim, an exploratory aim, to identify mutations in NMC cells that collaborate with BRD4-NUT in the blockade of differentiation and maintenance of growth of NMC cells, will be accomplished by sequencing all coding genes of approximately ten NMC tumors. The purpose of this aim is also to identify mutant proteins which aid BRD4-NUT oncogenic activity that can be targeted by small molecule inhibitors in the treatment of NMC. PHS 398/2590 (Rev. 09/04) Page Continuation Format Page

Public Health Relevance

The underlying subtext to the work in this proposal is to better understand the mechanism by which the BRD-NUT oncoproteins cause cancer, with the hypothesis that this exceptionally rare cancer model will, like many other rare cancers, prove a rule in more common cancers. It is anticipated that the outcome of these studies will be to more clearly define the molecular events that occur in this rare but novel oncogenic mechanism in carcinoma. Additionally, it is anticipated that more refined, yet broadly applicable therapies targeting pro-MYC upstream proteins will emerge from the proposed studies, and that their relevance will likely spill over to other, more common cancer types. PHS 398/2590 (Rev. 09/04) Page Continuation Format Page

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA124633-07S1
Application #
8839958
Study Section
Special Emphasis Panel (ZRG1 (01))
Program Officer
Mietz, Judy
Project Start
2007-08-30
Project End
2018-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
7
Fiscal Year
2014
Total Cost
$37,260
Indirect Cost
$16,184
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
Shiota, Hitoshi; Elya, Janine E; Alekseyenko, Artyom A et al. (2018) ""Z4"" Complex Member Fusions in NUT Carcinoma: Implications for a Novel Oncogenic Mechanism. Mol Cancer Res 16:1826-1833
Agaimy, Abbas; Fonseca, Isabel; Martins, Carmo et al. (2018) NUT Carcinoma of the Salivary Glands: Clinicopathologic and Molecular Analysis of 3 Cases and a Survey of NUT Expression in Salivary Gland Carcinomas. Am J Surg Pathol 42:877-884
Alekseyenko, Artyom A; Walsh, Erica M; Zee, Barry M et al. (2017) Ectopic protein interactions within BRD4-chromatin complexes drive oncogenic megadomain formation in NUT midline carcinoma. Proc Natl Acad Sci U S A 114:E4184-E4192
Hellquist, Henrik; French, Christopher A; Bishop, Justin A et al. (2017) NUT midline carcinoma of the larynx: an international series and review of the literature. Histopathology 70:861-868
Chau, Nicole G; Hurwitz, Shelley; Mitchell, Chelsey M et al. (2016) Intensive treatment and survival outcomes in NUT midline carcinoma of the head and neck. Cancer 122:3632-3640
Stathis, Anastasios; Zucca, Emanuele; Bekradda, Mohamed et al. (2016) Clinical Response of Carcinomas Harboring the BRD4-NUT Oncoprotein to the Targeted Bromodomain Inhibitor OTX015/MK-8628. Cancer Discov 6:492-500
Sholl, Lynette M; Nishino, Mizuki; Pokharel, Saraswati et al. (2015) Primary Pulmonary NUT Midline Carcinoma: Clinical, Radiographic, and Pathologic Characterizations. J Thorac Oncol 10:951-9
Alekseyenko, Artyom A; Walsh, Erica M; Wang, Xin et al. (2015) The oncogenic BRD4-NUT chromatin regulator drives aberrant transcription within large topological domains. Genes Dev 29:1507-23
French, Christopher A; Rahman, Shaila; Walsh, Erica M et al. (2014) NSD3-NUT fusion oncoprotein in NUT midline carcinoma: implications for a novel oncogenic mechanism. Cancer Discov 4:928-41
Puliyel, Mammen M; Mascarenhas, Leo; Zhou, Shengmei et al. (2014) Nuclear protein in testis midline carcinoma misdiagnosed as adamantinoma. J Clin Oncol 32:e57-60

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