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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

While epigenetic alterations are widely present in cancers, in the context of genomic instability it has been difficult to determine the degree to which epigenetic alterations serve as primary drivers of tumorigenesis. Cancers initiated by mutation of the SNF5 chromatin remodeling subunit, despite being highly aggressive and lethal, are diploid and genomically stable, making them an ideal model for this purpose. SNF5 is a core subunit of the SWI/SNF chromatin complex and is mutated in the large majority of malignant rhabdoid tumors (MRT), highly lethal cancers that occur in kidney, brain, and soft tissues of young children. Inactivating mutations in SNF5 have also recently been found to occur in a variety of other cancers and mutations in other SWI/SNF subunits occur frequently in ovarian, endometrial, renal, and lung cancers. Given our findings of genomic stability, we hypothesize that the consequences of SNF5 loss are fundamentally epigenetic in nature; that characterization of the epigenetic changes present in our model systems, rather than in cancers with genome instability, will identify those epigenetic alterations capable of driving oncogenic transformation; and lastly that cancers carrying mutations in the SWI/SNF complex can be effectively treated with epigenetically targeted therapies. Analysis of Drosophila mutants suggests that Polycomb and SWI/SNF complexes can serve antagonistic developmental roles. However, the relevance of this relationship to human disease is unclear. Here we have investigated functional relationships between these epigenetic regulators in oncogenic transformation. Mechanistically, we show that loss of the SNF5 tumor suppressor leads to elevated expression of the Polycomb gene EZH2 and that Polycomb targets are broadly H3K27-trimethylated and repressed in SNF5-deficient fibroblasts and cancers. Further, we show antagonism between SNF5 and EZH2 in the regulation of stem cell-associated programs and that Snf5 loss activates those programs. Finally, using conditional mouse models, we show that inactivation of Ezh2 blocks tumor formation driven by Snf5 loss. Collectively, we demonstrate that the rapid onset of tumor formation following SNF5 loss arises due to imbalanced epigenetic antagonism between the SWI/SNF complex and the Polycomb complex PRC2. Our work reveals essential roles for epigenetic modifications during tumor formation and demonstrates that inactivation of EZH2 can have therapeutic efficacy against cancer in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-237. doi:10.1158/1538-7445.AM2011-LB-237

Tumor vascularization is a critical component of cancer progression, malignancy, and metastasis and hypoxia inducible factor (HIF) is a key regulator of these processes. HIF transcriptional activity regulates numerous pro-angiogenic cytokines that act upon cancer cells and the surrounding tumor stroma to cooperatively stimulate tumor vascularization. HIF is constitutively activated in clear cell renal cell carcinoma (CCRCC), the most lethal and angiogenic type of kidney cancer, due to genetic or functional loss of the HIF-targeting VHL ubiquitin ligase protein. In the absence of VHL-dependent HIF degradation, pharmacologic targeting of HIF may be exploited as a means to suppress HIF-dependent signaling. HIF is also a client protein for Hsp90, a molecular chaperone regulating a number of substrates that support cancer progression. Hsp90 inhibitors possess anti-tumorigenic and anti-angiogenic properties, due in part to their ability to inhibit HIF function. However, clinical trials with the prototypic Hsp90 inhibitor 17-AAG have been unsuccessful in CCRCC and other solid tumors, highlighting the need to further evaluate the role of Hsp90 in CCRCC angiogenic processes and HIF signaling. Approach: We compared the molecular and functional effects of 3 Hsp90 inhibitors, using VHL replaced cells as HIF suppressed controls. We examined the prototypic Hsp90 inhibitor 17-AAG, a newer, more potent totally synthetic small molecule derivative (EC154), and LBH589, an HDAC inhibitor in clinical use that also impairs Hsp90 function. Our assays consisted of analysis of drug effects on 1) genome wide HIF ChIP and RT-PCR 2) cytokine profiles, 3) endothelial cell tubule assays, and 4) limited phospho-proteomic analysis of signaling proteins. Results: These inhibitors exhibit variable suppression of HIF target genes, illustrating the complexity associated with these pathways. Cytokine arrays indicated that while VEGF expression decreased in response to all drugs, a number of proteins unexpectedly increased. In addition, these changes did not always correlate with VHL-dependent HIF suppression. Although Hsp90 inhibitors initiate the destruction of a number of proteins and impact upon numerous signaling cascades, the protein-centric effects of Hsp90 inhibition in cancer cells remain unknown. To explore this, we utilized a commercial phosphoproteome array to examine the effects of Hsp90 inhibition upon the activation status of over 1300 key signaling proteins. Finally, to understand how alterations in these signaling profiles modulate angiogenic potential, conditioned medium from untreated cells, VHL replaced cells, or drug treated cells was evaluated in a tubule assay. Collectively, the results of these studies will further define the role of HIF in CCRCC tumor angiogenesis, add to our understanding of molecular events that contribute to clinical failure of Hsp90 inhibitors, and shed light on alternative approaches for CCRCC treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2391.