SETD2 Deficiency and Mir-21: Potent Therapeutic Targets in NUT Midline Carcinoma

SETD2 deficiency and miR-21: Potent therapeutic targets in NUT midline carcinoma. Journal Clinical Abstract Nuclear Protein in Testis (NUT) Midline Carcinoma (NMC) is a rare and highly aggressive tumor with the bromodomain containing 4 proteins (BRD4)-NUT (NUTM1) gene fusion. Few targeted therapies are available for NMC, and thus novel therapeutic targets are required. To the best of our knowledge, the present study was the first to report that SET domain-containing SETD2 mutation (p.Ser2382fs) in the NMC cell lines, HCC2429 and Ty82. Trimethylation of lysine 36 on histone H3 expression was depleted in the NMC cells, which was indicative of SETD2 loss. NMC cells were sensitive to the WEE1 G2 checkpoint kinase (WEE1) inhibitor, AZD1775, in the cancer cells with SETD2 deficiency. NMC cells that were resistant to Bromodomain and Extra-Terminal Motif (BET) inhibitors were next established, and these resistant cells were also sensitive to AZD1775. Subsequently, miRNA analysis revealed that miR-21 expression was increased in BET-inhibitor-resistant NMC cells. In addition, miR-21 regulated the proliferation of NMCs. The miR-21 inhibitor also suppressed the proliferation of BET-inhibitor-resistant cells. Additionally, a digital PCR assay was established to detect NUT gene rearrangements to identify patients with NMC. A total of 32 clinical samples were analyzed and one case of NMC was identified, in which the novel SETD2 mutation was detected. These data suggested that SETD2 loss and miR-21 may be therapeutic targets for treatment of NMC.


Introduction
Nuclear Protein in Testis (NUT) Midline Carcinoma (NMC), also referred to as NUT carcinoma, is a rare and highly aggressive tumor that predominantly affects midline structures, which occurs in both children and adults. NMC is genetically identified by the presence of the NUT gene, also known as the NUT midline carcinoma family member 1 (NUTM1) gene rearrangement [1,2]. The most frequent translocation in NMC is observed between the NUT gene at chromosome 15q14 and the Bromodomain Containing Protein 4 (BRD4) gene on chromosome 19q13.1 chromosome, which accounts for >70% of the rearrangements [3][4][5]; other NUT fusion partners include BRD3, Nuclear Receptor Binding SET Domain Protein 3 (NSD3) and zinc finger protein 532 (ZNF532), amongst other genes [6][7][8][9]. NMC lacks specific pathological features and can occur in any organ, such as the thorax, head and neck, or other midline organs [10]. Patients with NMC can be misdiagnosed with other malignant tumors due to the poor differentiation, and lack of awareness of the disease symptoms and diagnostic tests available. The prognosis of patients with NMC is considerably poor, with a median survival time of 6-9 months [3,10]. The development of novel treatment strategies for NMC is challenging. Targeted therapy has been performed based on the molecular mechanisms underlying aberrant signaling through the fusion proteins. BRD4 is a member of the bromodomain and Extra-Terminal Domain (BET) family of proteins and binds to acetylated lysine in histones [11]. BET inhibitors have been investigated, and the first-in-class BET inhibitor, JQ1, which competitively binds to bromodomains, has shown prodifferentiative and antiproliferative effects on NMC in preclinical studies [12]. More recently, novel BET inhibitors have been developed and studied in clinical trials [13,14]. However, the efficacy of these inhibitors are limited, and NMC may develop resistance to them [14,15].
To date, no other therapeutic targets have been identified in NMC. Accordingly, the development of novel treatment strategies is required. Recently, increased onco-microRNA (miRNA/miR) expression has been reported in NMC [16].
Studies that focus on the presence of simultaneous gene mutations and miRNAs may assist in identifying novel therapeutic targets. In the present study, the role of two epigenetic regulators, SET Domain Containing 2 (SETD2)

MTS assay
Cell proliferation assays were performed using the CellTiter 96 Aqueous One Solution assay (Promega Corporation).
Briefly, cells were seeded into 96-well plates (3-5x10 3 cells/well) overnight in triplicates. Following the incubation, the cells were treated with the indicated concentrations of drugs for 3 days, then the assays were performed. The experiments were performed independently at least three times.

Detection of the BRD4-NUT fusion genes in clinical samples
The

SETD2 loss-of-function mutation is present in NMC
To

miR-21 regulates the proliferation of NMC
miRNA expression analyses were performed next. miRNAs are key regulators of epigenetics and pathogenesis in almost every type of cancer [26]. However, the relationship between miRNAs and BRD4-NUT has not been fully

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siRNA-transfected cells (Figure 4b). In contrast, JQ-1 did not significantly alter miR-21 expression in HC2429 cells ( Figure 4b). miR-21 is an oncomiR, thus it was hypothesized that miR-21 may be associated with NMC cell proliferation [27]. The cell proliferation effects of miR-21 mimics or inhibitors on NMC were studied. miR-21 mimics increased the proliferation of HCC2429 cells; however, miR-21 inhibitors decreased their proliferation (Figure 4c).
The transfection efficiency with miR-21 mimics and inhibitors was shown in supplementary (Figure 1, 2).

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Short-term treatment with JQ-1 did not alter the expression levels of miR-21 in NMCs (Figure 4b). However, it was hypothesized that miR-21 may be related to BET inhibitor resistance, as studies have shown that tumors with acquired resistance to BET inhibitors do not have gatekeeper mutations and drug pump activation [28]. Next, miR-21 expression in HCC2429-JQR cells was determined using the TaqMan miRNA assay. miR-21 expression was increased in the HCC2429-JQR cells compared with that in the parental cells (Figure 5a). An MTS assay demonstrated that the miR-21 inhibitor suppressed the proliferation of HCC2429-JQR cells (Figure 5b). Since AZD1775 suppressed cell proliferation in the HCC2429-JQR cells as described, the efficacy of the combination with the miR-21 inhibitor and AZD1775 was assessed. Notably, this combination exhibited additive effects on HCC2429-JQR cells (Figure 5c). with malignant thoracic tumors, whose tumors were located in the midline of their bodies were investigated [29]. A total of 32 tumor samples collected via transbronchial biopsy were retrospectively analyzed. Screening for NMC is performed using Immunohistochemistry (IHC), which finds that NUT is expressed in the nuclei of NMC cells [30,31].
However, in some cases, IHC analysis cannot be performed due to the lack of tissue, especially in small biopsy samples.
In the present study, most of the biopsy samples were very small, and it was assumed that these samples included a small number of cancer cells. A highly sensitive digital (d) PCR assay was established to detect the BRD4-NUT fusion gene. In the preclinical study, the dPCR assay successfully    [16]. Another study screened an miRNA mimic library and identified miR-3140, which was shown to target and suppress BRD4 by binding to its coding sequence [40]. 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 expression [41]. miRNA expression is regulated by DNA methylation and histone modifications [42].
Therefore, SETD2 deficiency may be associated with miR-21 expression.
The present study could not find a relationship between miR-21 and SETD2 using miRDB (mirdb.org/index.html), but altered promoter methylation of miR-21 has been reported in SETD2-deficient cancers [32]. In the present study, combination chemotherapy had additive beneficial effects even on BET-inhibitor-resistant NMC. A recent report showed that the combination therapy targeting BET and p300 was more effective than the BET inhibitor alone [43].

Conclusion
In conclusion, the present study identified SETD2 deficiency and miR-21 as therapeutic targets for management of NMC. WEE1 and miR-21 inhibitors may serve as novel therapeutic options for the treatment of NMC.

Funding
This work was supported by the Japan Society for the