Erica M. Walsh

NSD3-NUT Fusion Oncoprotein in NUT Midline Carcinoma: Implications for a Novel Oncogenic Mechanism

UNLABELLED NUT midline carcinoma (NMC) is an aggressive subtype of squamous cell carcinoma that typically harbors BRD4/3-NUT fusion oncoproteins that block differentiation and maintain tumor growth. In 20% of cases, NUT is fused to uncharacterized non-BRD gene(s). We established a new patient-derived NMC cell line (1221) and demonstrated that it harbors a novel NSD3-NUT fusion oncogene. We find that NSD3-NUT is both necessary and sufficient for the blockade of differentiation and maintenance of proliferation in NMC cells. NSD3-NUT binds to BRD4, and BRD bromodomain inhibitors induce differentiation and arrest proliferation of 1221 cells. We find further that NSD3 is required for the blockade of differentiation in BRD4-NUT-expressing NMCs. These findings identify NSD3 as a novel critical oncogenic component and potential therapeutic target in NMC. SIGNIFICANCE The existence of a family of fusion oncogenes in squamous cell carcinoma is unprecedented, and should lead to key insights into aberrant differentiation in NMC and possibly other squamous cell carcinomas. The involvement of the NSD3 methyltransferase as a component of the NUT fusion protein oncogenic complex identifies a new potential therapeutic target.

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.

Shaping the Future of Research: a perspective from junior scientists

The landscape of scientific research and funding is in flux as a result of tight budgets, evolving models of both publishing and evaluation, and questions about training and workforce stability. As future leaders, junior scientists are uniquely poised to shape the culture and practice of science in response to these challenges. A group of postdocs in the Boston area who are invested in improving the scientific endeavor, planned a symposium held on October 2 nd and 3 rd, 2014, as a way to join the discussion about the future of US biomedical research. Here we present a report of the proceedings of participant-driven workshops and the organizers’ synthesis of the outcomes.

Ectopic protein interactions within BRD4–chromatin complexes drive oncogenic megadomain formation in NUT midline carcinoma

Significance Chromatin factors generally act within large, multisubunit complexes; thus, identifying both their normal and aberrant interactors in cancer should provide important information regarding potential targets for therapeutic intervention. Here, we apply this principle to analysis of BRD4–NUT, a fusion oncoprotein that drives an aggressive subtype of squamous cell cancer. We identify ZNF532 as a prominent BRD4–NUT–interacting protein in an established NUT midline carcinoma patient cell line, and independently discover ZNF532 fused directly to NUT in a newly analyzed patient. Like BRD4–NUT, ZNF532–NUT forms unusually large (100-kb to 1-Mb) domains of hyperactive chromatin, including at the MYC locus, and drives self-reinforcing regulatory loops that are likely to be a powerful strategy for the growth advantage of cancer cells. To investigate the mechanism that drives dramatic mistargeting of active chromatin in NUT midline carcinoma (NMC), we have identified protein interactions unique to the BRD4–NUT fusion oncoprotein compared with wild-type BRD4. Using cross-linking, affinity purification, and mass spectrometry, we identified the EP300 acetyltransferase as uniquely associated with BRD4 through the NUT fusion in both NMC and non-NMC cell types. We also discovered ZNF532 associated with BRD4–NUT in NMC patient cells but not detectable in 293T cells. EP300 and ZNF532 are both implicated in feed-forward regulatory loops leading to propagation of the oncogenic chromatin complex in BRD4–NUT patient cells. Adding key functional significance to our biochemical findings, we independently discovered a ZNF532–NUT translocation fusion in a newly diagnosed NMC patient. ChIP sequencing of the major players NUT, ZNF532, BRD4, EP300, and H3K27ac revealed the formation of ZNF532–NUT–associated hyperacetylated megadomains, distinctly localized but otherwise analogous to those found in BRD4–NUT patient cells. Our results support a model in which NMC is dependent on ectopic NUT-mediated interactions between EP300 and components of BRD4 regulatory complexes, leading to a cascade of misregulation.

Abstract 2655: Oncogenic chromatin factors drive cell type-specific transcription within megadomains in NUT midline carcinoma

NUT midline carcinoma (NMC), a subtype of squamous cell cancer, is one of the most aggressive human solid malignancies known. NMC is driven by the creation of a translocation oncoprotein, BRD4-NUT, which blocks differentiation and drives growth of NMC cells. BRD4-NUT forms distinctive nuclear foci in patient tumors, which we find correlate with ∼100 unprecedented, hyperacetylated expanses of chromatin that reach up to 2 Mb in size. These ‘megadomains’ appear to be the result of aberrant, feed-forward loops of acetylation and binding of acetylated histones. Megadomains drive transcription of underlying DNA in NMC patient cells and in naive cells induced to express BRD4-NUT. Here we characterize the constituents of BRD4-NUT chromatin complexes using a crosslinking approach, BioTAP-XL. We find many transcriptional activating proteins known to associate with BRD4, along with novel interactors including p300/CBP and a previously uncharacterized BRD4-NUT Megadomain Associated Protein (BMAP1). BMAP1 is expressed in primary NMC tissue and a subset of more common head and neck squamous cell carcinomas (HNSQC). Concurrently, we discovered a patient-derived NMC harboring a novel BMAP1-NUT fusion. BMAP-NUT blocks differentiation, and like BRD4-NUT recruits p300 to form hyperacetylated megadomains, including at the MYC locus. Thus, our proteomic and genetic approaches have converged on a novel mechanism that involves reprogramming very large regulatory regions to drive oncogenic transcription. Citation Format: Mitzi I. Kuroda, Artyom A. Alekseyenko, Erica M. Walsh, Xin Wang, Adlai Grayson, Peter T. Hsi, Peter V. Kharchenko, Christopher A. French. Oncogenic chromatin factors drive cell type-specific transcription within megadomains in NUT midline carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2655.

Abstract 2476: A novel NUT translocation partner binds to BRD4 and is necessary for the blockade of differentiation in NUT midline carcinoma

NUT midline carcinoma (NMC) is an aggressive type of squamous cell carcinoma that typically harbors BRD4/3-NUT fusion oncogenes which encode chimeric proteins that block differentiation and maintain tumor growth. However, in 30% of cases NUT is fused to yet to be identified non-BRD gene(s). Using RNA sequencing, we identified a novel gene fused to NUT in a NMC cell line (1221), fusing a novel 5′ coding sequence to the NUT gene. Like Brd4/3, this gene encodes a protein that is also involved in epigenetic regulation, and we find that siRNA knockdown of this protein leads to differentiation of the 1221 cells as well as of three BRD4-NUT-positive NMC cell lines. We have established that the novel NUT fusion protein encoded by this translocated gene binds to BRD4. We have mapped the regions of Brd4 and of the novel fusion protein involved in complex formation and are exploring whether association of the novel protein with Brd4 is required for the blockade of differentiation in NMC. Differentiation of TC-797 cells induced by knockdown of BRD4-NUT is abrogated by enforced expression of the novel NUT-fusion gene. Together, these findings identify a novel BRD4-NUT-interacting protein whose expression is required for the maintenance of the undifferentiated state in NMC, and when fused to NUT, is oncogenic and can recapitulate the function of BRD4-NUT to block differentiation. Citation Format: Erica Walsh, Simone Kuhnle, Shaila Rahman, Madeleine Lemieux, Peter Howley, Christopher French. A novel NUT translocation partner binds to BRD4 and is necessary for the blockade of differentiation in NUT midline carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2476. doi:10.1158/1538-7445.AM2014-2476

Abstract B39: 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 show 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 displaced by acetyl histone mimetic BET inhibitor, JQ1, which prevents binding of BET bromodomains to acetylated histones. BRD4-NUT is also required to maintain MYC expression in NMC cell lines, as shown by a dramatic decrease in MYC expression upon JQ1 treatment and knockdown of BRD4-NUT with NUT specific siRNAs. 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. Citation Format: Erica M. Walsh, Adlai R. Grayson, Michael J. Cameron, Jernej Godec, Todd Ashworth, Alexandra B. Aserlind, Hongfang Wang, Gerard Evan, Michael J. Kluk, James E. Bradner, Jon C. Aster, Christopher A. French. MYC, a downstream target of BRD-NUT, is necessary and sufficient for the blockade of differentiation in NUT midline carcinoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B39.