Cancer Res Treat.  2023 Apr;55(2):452-467. 10.4143/crt.2022.910.

Cancer-Specific Sequences in the Diagnosis and Treatment of NUT Carcinoma

• Lee MS
• An S
• Song JY
• Sung M
• Jung K
• Chang ES
• Choi J
• Oh DY
• Jeon YK
• Yang H
• Lakshmi C
• Park S
• Han J
• Lee SH
• Choi YL

• KMID: 2541232
• DOI: http://doi.org/10.4143/crt.2022.910

Abstract

Purpose
NUT carcinoma (NC) is a solid tumor caused by the rearrangement of NUTM1 that usually develops in midline structures, such as the thorax. No standard treatment has been established despite high lethality. Thus, we investigated whether targeting the junction region of NUTM1 fusion breakpoints could serve as a potential treatment option for NC.
Materials and Methods
We designed and evaluated a series of small interfering RNAs (siRNAs) targeting the junction region of BRD4-NUTM1 fusion (B4N), the most common form of NUTM1 fusion. Droplet digital polymerase chain reaction using the blood of patients was also tested to evaluate the treatment responses by the junction sequence of the B4N fusion transcripts.
Results
As expected, the majority of NC fusion types were B4N (12 of 18, 67%). B4N fusion-specific siRNA treatment on NC cells showed specific inhibitory effects on the B4N fusion transcript and fusion protein without affecting the endogenous expression of the parent genes, resulting in decreased relative cell growth and attenuation of tumor size. In addition, the fusion transcript levels in platelet-rich-plasma samples of the NC patients with systemic metastasis showed a negative correlation with therapeutic effect, suggesting its potential as a measure of treatment responsiveness.
Conclusion
This study suggests that tumor-specific sequences could be used to treat patients with fusion genes as part of precision medicine for a rare but deadly disease.

Keyword

NUT carcinoma; fusion gene; Junction sequence of the fusion gene

Figure

• 

  Fig. 1 Representative paired IHC images of NUT and MYC in NC patients and prevalence of NUTM1 fusion gene. (A) Immunohistochemical images in resected tumor tissue stained using anti-NUT and anti-MYC antibodies. Upper panel shows tumor images of patient No. 7 in Table 1, with NDS3-NUTM1 fusion gene in bronchus, which harbors squamous cells with pseudoglandular pattern. Lower panel presents tumor images of patient No. 15 with BRD4-NUTM1 fusion gene in lateral basal segment of right lower lobe. NUT fusion proteins formed large foci (speckled nuclear pattern) and colocalized with MYC in the nuclear region (400× magnification; scale bar=100 μm). (B) The prevalence of NUTM1 fusion gene according to the partner gene in 18 NC patients. The patient tumor samples were primarily analyzed using qRT-PCR with BRD4 exon 11 and NUTM1 exon 3 primer pairs. The samples that were not detected in the qRT-PCR were further subjected to NGS assay. For the most frequent BRD4-NUTM1 fusion samples, the sequence of the fusion breakpoint region was confirmed using Sanger sequencing. (C) Breakpoint sequence was confirmed in NUTM1 carcinoma cell lines. To determine the junction sequence of BRD4-NUTM1, mRNA was extracted from four cell lines (HCC2429, Ty-81, SNU2792-1, and SNU3178S) expressing NUTM1 fusion gene and verified using RT-PCR. The junction sequences in these cell lines were confirmed using Sanger sequencing. IHC, immunohistochemistry; NC, NUT carcinoma; NGS, next-generation sequencing; qRT-PCR, quantitative reverse transcription polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction.

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  Fig. 2 Silencing effect of siRNAs targeting the junction region of B4N fusion gene. (A) Left panel shows the scheme of junction sequence of BRD4-NUTM1 mRNA and the 15 siRNA candidates targeting the junction region, while the right panel shows the relative B4N mRNA level upon junction-specific siRNA treatment. HCC2429 cells were treated with B4N-specific siRNAs Nos. 3, 6, 7, 9, 12, and 15 at a concentration of 50 nM. After 72 hours, mRNA was extracted from the cells, and qRT-PCR was performed on the cDNA. Wild-type BRD4 level was analyzed using a primer pair for BRD4 C-terminal region, while BRD4-NUTM1 (B4N) fusion level was tested using a primer pair targeting the junction region of the fusion gene. GUSB was used as an internal control for the qRT-PCR reaction. The bar graphs represent the average values of triplicates, and error bars represent the standard deviation. (B) Immunoblots showing the expression of B4N, BRD4, and MYC in HCC2429 cells transfected with the siRNAs. The expression of B4N fusion and MYC proteins was reduced upon treatment with B4N siRNAs Nos. 7, 9, and 12 but not B4N siRNA No. 6, as compared to that in the negative control, scrambled siRNA control (indicated using a red arrowhead). (C) Cell growth assay upon B4N siRNA treatment. Cells (5×105) were seeded into 6-well plates and then treated with 50 nM siRNA. Twenty-four hours later, the cells were replated in triplicate into 96-well plates with 10,000 cells per well in 100 μL media. After 48 hours, cell growth was measured using WST assay (right panel), and representative images were acquired (left panel). p-value was determined using an unpaired Student’s t test (*p < 0.05 vs. the control group), compared with the negative control (NC) and B4N siRNA; *p < 0.05, **p < 0.01, ***p < 0.001. PC, positive control.

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  Fig. 3 The in vivo effect of siRNAs targeting the junction region of B4N fusion gene on tumor growth. (A) A subcutaneous xenograft mouse model was generated by subcutaneously inoculating HCC2429 cells (4×106) expressing B4N fusion gene in 50 μL of Matrigel and RPMI media mixture (1:1 dilution) into the left flank of BALB/c nude female mice. When the tumors reached an average volume of approximately 50 mm3, the mice were randomly divided into three groups and either left untreated or treated with 2 mg/kg or 6 mg/kg B4N siRNA No. 9. siRNA in 20 μL serum-free media per mouse was directly injected into the intra-tumoral region every 3 or 4 days for 20 days. (B) Three mice were treated with cyclophilin B siRNA, as a positive control for siRNA injection. The fluorescence was measured (red arrow) 24 hours after injection. (C) Tumor growth curves of HCC2429 cell-derived xenograft mice. Tumor volumes were calculated by taking length to be the longest diameter across the tumor, width to be the corresponding perpendicular diameter, and then using the following formula: (length×width2) mm3×0.5. Tumor volumes were measured once every 2 days until the mice were sacrificed and presented as mean±SD (n=5 mice per group). p-value was determined using an unpaired Student’s t test (*p < 0.05 vs. the control group), compared with the control groups and B4N siRNA No. 9 (2 mg/kg)-treated group. (D) The relative B4N mRNA levels in tumor tissues of B4N siRNA-treated group. The value presents the average of each group, as determined using qRT-PCR. Statistical significance was determined using an unpaired Student’s t test: *p < 0.05 vs. the control group. (E) Protein expression of NUT and β-actin in the tumor tissues of the B4N siRNA-treated mice was evaluated using western blot (upper). Quantification of protein expression form western blot using ImageJ software (lower). *p < 0.05. qRT-PCR, quantitative reverse transcription polymerase chain reaction.

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  Fig. 4 Clinical usage for targeting the junction sequence of B4N fusion gene, based on the ddPCR platform. (A) ddPCR method for detecting the B4N fusion gene in PRP of an NC patient. Whole blood sample was collected and passed through a two-step centrifugation process to obtain the platelet pellet. cfRNA was extracted from the PRP sample, and cDNA was synthesized, following which ddPCR assay was performed using primer pairs against the junction sequence of B4N fusion gene. (B) The cfRNA of B4N fusion gene detected in the PRP sample. A series of blood samples were collected once a month for 8 months and analyzed with ddPCR assay, following which the number of droplets was counted and illustrated. The patient received radiotherapy and combination chemotherapy following anti-PD-1/L1 therapy. After the first sampling, the mean duration of follow-up was 21±7 days. cfRNA, cell-free RNA; ddPCR, droplet digital polymerase chain reaction; DTX, docetaxel; GEN, gemcitabine; NVB, navelbine; PD-1, programmed death-1; PD-L1, programmed death-ligand 1; Pembro, pembrolizumab; PRP, platelet-rich plasma; Ram, ramucirumab.

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  Fig. 5 Clinical applications for fusion gene-derived cancer diagnosis and guidance of potential targeted therapies. For treatment guidance of patients with unknown solid tumors, we suggest that the tumor be sequenced and analyzed to identify the presence of tumor-specific alterations. On doing so, if a fusion gene that can be presumed to be an oncogenic driver gene is found, junction sequence of the fusion gene could be used for treatment and monitoring of therapeutic responsiveness. siRNAs targeting the junction region of the fusion gene can be developed for treatment of the patients, and cell-free RNA obtained from their blood can be used to monitor the therapeutic responsiveness. This allows for personalized treatment of individual patients, even in case of rare cancers that are ineffective with standard treatments.

Reference

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