NEW YORK (GenomeWeb) – Researchers investigating the genetic underpinnings of a rare cancer that affects the body's midline have proposed that the so-called BRD4-NUT oncoprotein appears to cause the disease by taking over large swathes of chromatin inside cells and driving abnormal transcription.
Researchers led by Christopher French from Brigham and Women's Hospital described these expansive cell type-specific, hyperacetylated regions that the BRD4-NUT protein affects in Genes & Development this week. Ranging from 100 kb to 2 Mb in size, they've dubbed these regions "megadomains."
Functional studies conducted by French and colleagues suggest these megadomains usually appear at highly acetylated "seed regions" that promote their rapid growth until they fill entire chromosome neighborhoods, called topologically associated domains (TADs). The megadomains result from "aberrant, feed-forward loops of acetylation and binding of acetylated histones" that fuel transcription of genes in cells of patients with NUT midline carcinoma (NMC).
Moreover, in their investigations, French's group homed in on several oncogenes within these megadomains that blocked cell differentiation and fueled growth of cancer cells. "We believe that megadomains stabilize expression of pro-growth, anti-differentiation genes such as MYC and TP63 by filling entire TADs that contain both the genes themselves, and all of the genomic regions that regulate their expression, such as [long non-coding] RNAs, enhancers, and super-enhancers," French, associate professor of pathology at Harvard Medical School and Brigham & Women's Hospital, told GenomeWeb over e-mail.
NUT fusion oncogenes characterize NMC, an aggressive epithelial malignancy that occurs in the middle areas of the body, such as the nose, mouth, trachea, and upper airways. NMC is diagnosed by immunohistochemical testing for the NUT protein, followed by confirmation of the fusion oncogene by fluorescent in situ hybridization or RT-PCR. BRD4-NUT is the most common of the translocations associated with this cancer.
Although the test is easy to perform, with an 87 percent sensitivity and 100 percent specificity, diagnosing NMC is challenging because the symptoms are similar to those for other cancers. There are around 20 cases reported in the US each year, but according to the International NMC Registry, this is probably a gross underestimate given the limited number of labs with expertise testing for the NUT protein and lack of awareness of NMC in the medical community.
"At this point in time, very little is understood about which organ systems/tissues can give rise to NMC, and thus it is likely that this disease is vastly underdiagnosed in organs other than head and neck and mediastinum," said French, who runs the International NMC Registry with colleagues from Dana-Farber Cancer Institute, Children's Hospital Boston, Harvard Medical School, and Brigham & Women's. Although NMC usually arises in midline organs, he noted that the disease has also been seen in the kidney, pancreas, bone, and adrenal gland. But doctors rarely consider testing for NMC for cancers affecting these organs.
"The earlier NMC is diagnosed, the earlier appropriate intervention with either surgery or treatment with targeted BET inhibitors can be initiated, giving the patients a better chance to be cured," French said. The cure rate for NMC is less than 5 percent and average survival from time of diagnosis is less than a year. With the present publication, French's team hopes to spur more research about the cell of origin for NMC and improve outcomes for patients.
A compelling hypothesis raised by French's group in the Genes & Development paper is that NMC is an uncommon cancer not simply because of the rarity of NUT fusion oncogenes, but because only certain cell types can survive ravages of BRD-NUT overexpression. "Indeed, no cell type tested can survive prolonged overexpression of recombinant BRD4-NUT in culture," the study authors wrote in the paper. "Perhaps BRD4-NUT can only circumvent the pathways that guard against its progrowth, anti-differentiation effects in a specific subtype of primordial squamous cell that is most abundant during young adulthood, the peak age of patients with NMC."
NMC is a newly identified disease that was characterized in the US by researchers just more than a decade ago with the case of a 12-year old girl with a sore throat. She started to have trouble swallowing, developed a "muffled' voice, and wasn't responding to antibiotics. After some tests, doctors found an ulcerating mass in place of her epiglottis, the leaf-shaped flap at the entrance of the voice box. Experts at Children's Hospital treated this like a nasopharyngeal carcinoma, and for a short time, her tumor shrank.
But, it came back. "She died a horrible death, strangled by the tumor which closed off her airway," French wrote in a 2010 Cancer Genetics article about NMC. "The clinicians would not forget this tumor, but the abrupt end to her life marked the discovery of a new type of cancer."
After her death, doctors at Children’s Hospital sent her biopsy sample to Brigham & Women’s where experts noted abnormal karyotypes and identified a few other cases that resembled hers. There was a cell line created from a case in Japan, and French, then a post-doctoral fellow, was able to use it to map and identify the BRD4-NUT fusion oncogene.
Over the years, French has continued to focus on elucidating the genetic mechanisms underlying NMC. The push is now to understand the types of cells that give rise to NMC so doctors can better diagnose the disease. According to French, researchers will follow up on the hypothesis that NMC arises in squamous precursor cells by expressing BRD4-NUT in pluripotent stem cells and seeing if squamous lineage cells are "most receptive" to the transformations that the oncoprotein encourages.
"Additionally, evidence indicates that megadomains may identify gene regions, or TADs, that may be cell-lineage defining," he wrote over email. "Thus, the 'TAD signature' of NMC cells, in comparison to that of differentiating embryonic cells, can be used to identify what cell lineage NMC most closely corresponds with in embryonic stem cells."
Although this work focuses on NMC, the fact that inside these megadomains researchers found oncogenes, such as TP63, MYC, and MED24, which appear to fuel cancer growth and keep cells from differentiating, may be important for identifying the genomic drivers of other types of malignancies. According to French, this is the first time that TP63 and MED24 have been associated with fueling growth of NMC cells. "It speaks to the importance and utility of megadomains in identifying key pro-growth genes in cancer," he noted. "It is quite possible that megadomains could be used to identify key oncogenic drivers in other cancers through the experimental overexpression of BRD4-NUT in those cancer cell types."
As for treatment, there is none for NMC currently. Most NMC patients are resistant to chemotherapy regimens. However, the present study contributes knowledge that can help drugmakers. Although MYC has been "an elusive" oncogenic target, drugs that inhibit BRD4-NUT may be an option, French said, since the entire regulatory region of MYC is activated by BRD4-NUT. BET inhibitors block this oncoprotein and a number of pharmaceutical companies are investigating them in NMC.
In the Genes & Development study, French's group demonstrated that when researchers treated NMC patients' cells with the BET inhibitor JQ1, BRD4NUT megadomains diminished. "Evidence suggests that all known variants of NUT fusions, including BRD4-NUT, BRD3-NUT, and NSD3-NUT, have at the core of their function BRD4," French said. "Thus, all variants of NMC have thus far proven to be sensitive to BET inhibitors in culture."
GlaxoSmithKline, Merck/OncoEthix, Constellation, and Tensha are some companies that French said are studying BET inhibitors and enrolling NMC patients in trials. "The work herein has shown a compelling mechanism, the depletion of entire megadomains and the repression of associated pro-growth genes, including MYC, by BET inhibitors," French said. "That provides a powerful rationale for the use of these drugs in NMC."
The study was funded with grants from the National Institutes of Health, the Samuel Waxman Cancer Research Foundation, the Ellison Medical Foundation, and the St. Baldrick's Foundation.