Background:Nuclear protein in testis (NUT) midline carcinoma (NMC) is a rare and highly aggressive tumor with the bromodomain containing 4 (BRD4)-NUT (NUTM1) gene fusion. BRD4 is a member of the bromodomain and extra-terminal domain (BET) family of proteins, and BET inhibitors have been investigated in NMC clinical trials. However, few targeted therapies are available for NMC, and novel therapeutic targets remain to be determined. We determined the role of two epigenetic regulators as possible therapeutic targets for NMC. Methods:We performed next-generation sequencing (NGS) in NMC cell lines (HCC2429 and Ty82). H3K36me3 expression was studied using western blotting. The efficacy of AZD1775, a WEE1 inhibitor, was evaluated using the MTS and γH2AX assays. We established an NMC cell line that was resistant to BET inhibitors. The sensitivity of the cells resistant to AZD1775 was analyzed using the MTS assay. RNA sequencing was performed to determine miRNA expression levels. TaqMan miRNA assays were used to analyze miR-21 expression. The efficacy of the miR-21 inhibitor was evaluated using the MTS assay. We established a digital PCR (dPCR) assay to detect NUT gene rearrangements to identify patients with NMC. Using NGS, a patient with NMC was identified with the SETD2 mutation.Results:SETD2 mutation (p.Ser2382fs) was determined in NMC cells, in which H3K36me3 expression was depleted, indicating SETD2 loss. NMC cells were sensitive to the WEE1 inhibitor, AZD1775 in cancer cells with SETD2 deficiency. γH2AX expression was increased in NMC cells treated with AZD1775. The efficacy of the combination of AZD1775 and JQ-1 was additive. We established NMC cells that were resistant to BET inhibitors. The resistant cells were also sensitive to AZD1775. miRNA analysis revealed increased miR-21 expression in BET-inhibitor-resistant NMC cells. MiR-21 regulated the growth of NMCs. The miR-21 inhibitor suppressed the growth of BET-inhibitor-resistant cells. Thirty-two clinical samples were analyzed and NMC was identified using digital PCR assays. Tumors with the SETD2 mutation were analyzed using NGS.Conclusions:We report for the first time SET domain-containing protein 2 (SETD2) deficiency and miR-21 as novel therapeutic targets for NMC. SETD2 loss and miR-21 may be therapeutic targets for NMC.Trial registrationIn this study, we analyzed the gene alterations in human tumor samples. This study was registered in the UMIN clinical trial registered system (UMIN000043147, January 27, 2021).

BackgroundNuclear protein in testis (NUT) midline carcinoma (NMC) is a rare and highly aggressive tumor with the bromodomain containing 4 protein (BRD4)-NUT (NUTM1) gene fusion. Few targeted therapies are available for NMC, and novel therapeutic targets are required. The objectives of our study was to determine gene mutations and microRNA (miRNA) expression levels in NMC and to identify novel therapeutic targets for NMC. Methods Next-generation sequencing (NGS) was used to identify mutations in the NMC cell lines and the tumor sample from a NMC patient; we determined the BRD4-NUT fusion gene in the patient using a digital PCR assay. Trimethylation of lysine 36 on histone H3 expression (H3K36me3) was studied using western blotting. The efficacy of the WEE1 G2 checkpoint kinase (WEE1) inhibitor, AZD1775, was evaluated using the MTS assay. RNA sequencing was performed to determine miRNA expression levels in the NMC cell lines. TaqMan miRNA assays were used to analyze miR-21 expression. The efficacy of miR-21 inhibitor was evaluated using the MTS assay. Results A novel SETD2 mutation (p.Ser2382fs) was identified in the NMC cell lines and the patient tumor. H3K36me3 was depleted in the NMC cells, which was indicative of functional SETD2 loss. The NMC cells were sensitive to AZD1775, an inhibitor for SETD2-deficient cancer. miRNA analysis revealed that miR-21 expression was increased in the NMC cells resistant to the traditional BET-targeted therapy. The miR-21 inhibitor suppressed the proliferation of the NMC cells. ConclusionsSETD2 deficiency and miR-21 may serve as novel therapeutic targets for the treatment of NMC.Trial registrationIn this study, we analyzed the gene alterations in human tumor samples. This study was registered in the UMIN clinical trial registered system (UMIN000043147, January 27, 2021).