In this study, we describe two epigenetic regulators, SETD2 and miR-21, as therapeutic targets for NMC, regardless of resistance to BET inhibitors. To the best of our knowledge, this is the first report showing the efficacy of targeted therapy with a WEE1 and miR-21 inhibitor in NMC.
First, we identified a novel SETD2 mutation in NMC. SETD2 has three conserved domains, AWS-SET-PostSET, WW, and Set2 Rpb1 interacting (SRI) domains. The SET domain (1550–1673), which mediates trimethylation of H3K36; the WW domain (2391–2420) is associated with protein-protein interactions, preferentially binding to the proline-rich region of proteins, and the SRI domain (2469–2548) binds to the phosphorylated C-terminal domain of RNA polymerase II, allowing SETD2 to move to transcription elongation complexes [32–34]. Taken together, SETD2 plays a key role in homologous recombination repair and genome stability by catalyzing trimethylation at H3K36 .
SETD2 is ubiquitously expressed in human tissues, and somatic mutations of the SETD2 gene have been reported in several types of cancer, in which loss of SETD2 function leads to decreased H3K36me3 levels . For instance, in clear cell renal cell carcinoma, widespread DNA hypomethylation associated with SETD2 loss-of-function mutations was observed, and the SETD2 mutation seemed to be related to genomic alterations leading to tumorigenesis [36, 37]. We detected the SETD2-p.Ser2382fs mutation in NMC. This frameshift mutation is located just before the WW domain, suggesting that the WW and SRI domains in the mutant SETD2 have no additional normal functions. It has been reported that SRI domain deficiency abolishes trimethylation of H3K36me3 [38, 39]. Indeed, H3K36me3 expression was decreased in NMC cells. On the other hand, H3K36me3 expression levels in Ty82 cells were different from those in HCC2429 cells. Our NGS analysis showed that the frequency of the frameshift mutation was 93.9% in HCC2429 cells and 89% in Ty82 cells; the residual SETD2 gene was mutant SETD2-p.Pro2381Leu (P2381L) in both cell lines (data not shown). Considering the mutation site, the SETD2-P2381L mutation may be a passenger mutation. The mutant SETD2-P2381L could compensate for SETD2 deficiency caused by the frameshift mutation in Ty82 cells, although the bona fide activity of the SETD2-P2381L enzyme remains unknown. If there is a monoallelic deficiency of SETD2 in Ty82 cells, they may retain the trimethyltransferase activity. The mechanistic details remain to be elucidated as to how SETD2-p.Ser2382fs and SETD2-p.Ser2382fs functions in NMC. In addition, there might be possible mechanisms that compensate for H3K36me3 in Ty82 cells.
Identification of the SETD2 mutation can lead to the development of targeted therapies for NMC. In H3K36me3-deficient tumors, WEE1 inhibition has a synthetic lethal interaction with H3K36me3 loss; the WEE1 inhibitor AZD1775 selectively kills SETD2-deficient cancer cells through dNTP starvation because of RRM2 depletion . In our study, the NMC cells were more sensitive to AZD1775 than the BET inhibitors, and AZD1775 induced DNA damage in NMC, which was concordant with the results of the present study.
The enzymes regulating epigenesis in histones are categorized as writers, erasers, readers, or others [40, 41]. Among these enzymes, SETD2 belongs to the writers that add post-translational modifications, whereas BRD4 is one of the readers that recognize acetyl groups on histone lysines. Therefore, our findings suggest that aberrant gene expression and tumorigenesis in NMC might occur through hyperacetylation by BRD4-NUT and hypomethylation by SETD2 loss. We found that the combination of the BET inhibitor and WEE1 inhibitor had additive effects on NMC. Moreover, a recent report showed that the combination therapy targeting BET and p300, which belongs to writers and acetylates histone lysine residues, was more effective than BET inhibitor alone . Given the epigenetic categories in histone modification, the combination of BET inhibitors and target inhibitors in another epigenetic category might be useful for the treatment of NMC.
Next, we found that miR-21 regulated the growth of NMC. Emerging studies have reported the functions of miRNAs in NMC. A study showed a set of 48 dysregulated miRNAs in NMC, in which the miRNAs targeting critical genes other than BRD4 and NUT were analyzed; however, miR-21 was not included in the 48 miRNAs . Another study screened an miRNA mimic library and identified miR-3140 that targets and suppresses BRD4 by binding to its coding sequence . Another report analyzed miRNA expression in NMC using clinical samples that identified three cases of sinonasal NMC, and two out of three NMCs showed upregulation of miR-21, miR-143, and miR-484 . miRNA expression is regulated by DNA methylation and histone modifications . Therefore, histone modification changes caused by SETD2 deficiency or BRD4-NUT might be associated with miR-21 expression. Indeed, altered promoter methylation of miR-21 has been reported in SETD2-deficient cancers . Additionally, H3K36me3 is required for DNA mismatch repair (MMR), while miR-21 downregulates MMR gene expression [47, 48]. Overall, together with SETD2 deficiency, miR-21 might be a key regulator in NMC.
We established HCC2429-JQR cells resistant to BET inhibitors. In our study, miR-21 expression was increased in the resistant NMC, and both the miR-21 inhibitor and AZD1775 were effective in the resistant cells. BET family proteins include BRD2, BRD3, BRD4, and BRDT. The BET proteins have conserved tandem bromodomains BD1 and BD2, which selectively bind to acetylated lysine residues in histones. BET inhibitors competitively bind to the individual or both bromodomains and inhibit BET activity [16, 17]. Therefore, tumors resistant to BET inhibitors are expected to have gatekeeper mutations at the bromodomains, as seen in epidermal growth factor receptor mutations in lung cancer . However, it is unlikely that the tumors acquire resistant mutations. In triple-negative breast cancer (TNBC), gatekeeper mutations, new driver gene alterations, and drug pump activation were not observed in BET-resistant TNBC cells . This was true in other malignant tumors such as ovarian cancer, prostate cancer, and leukemia, in which alternative signaling pathways other than BET itself were associated with acquired resistance [50–53]. Therefore, it is possible that NMC might not acquire gatekeeper mutations, although we did not evaluate the mutations in bromodomains in resistant NMC cells. Recent work has shown that adaptive kinome reprogramming is associated with acquired resistance to targeted therapies, and aberrant kinase activation has occurred in BET-resistant cancer cells without gatekeeper mutations [50, 54]. miR-21 potentially targets more than 400 genes, using data from miRDB (http://www.mirdb.org), which include various genes of receptor tyrosine kinases (RTKs). Therefore, an increase in miR-21 might be associated with acquired resistance to BET inhibitors by activating RTKs and downstream pathways in NMC. Therapeutic targets other than BET, NUT, and their associated proteins have not been reported. We demonstrated the efficacy of AZD1775 and a miR-21 inhibitor, which could overcome resistance.