ARV-825 Showed Antitumor Activity against BRD4-NUT Fusion Protein by Targeting the BRD4

Objective The bromodomain-containing 4 (BRD4) is a member of the bromodomain and extra terminal domain (BET) family, which is an important epigenetic reader. It is currently a promising oncology target. In some tumors, BET bromodomain inhibitors have demonstrated promising results. Proteolysis-targeting methods (PROTAC), which rapidly and effectively degrade BRD4, have displayed considerable potential in the treatment of tumors in recent years. The purpose of this study is to examine the potential impact of BRD4 PROTAC compounds ARV-825 on oncogene BRD4-NUT fused protein in NUT carcinoma. Methods The effectiveness of ARV-825 was evaluated at the cellular level using the cell counting kit 8 test, wound healing, cell transfection, western blotting analysis, and RNA sequencing. The effectiveness of ARV-825 was also examined in vivo using a xenograft model. Results The BRD4-NUT fusion gene was overexpressed in 3T3 cells, and the pathogenic fusion gene was simulated. The results showed that the overexpression of BRD4-NUT could promote the proliferation and migration of 3T3 cells, but the expression of BRD4 protein was degraded after the addition of the novel cereblon-based PROTAC compound ARV-825 against BRD4, resulting in inhibition of BRD4-NUT 3T3 cell proliferation and migration. Further RNA-seq analysis showed that overexpression of BRD4-NUT was accompanied by increased expression of gene (e.g., Myc, E2F, TRAFs, Wnt, Gadd45g, and Sox6) with significantly enriched pathway (e.g., small cell lung cancer, NF-kappa B signaling pathway, and breast cancer), promoted cell cycle from G 1 phase to S phase, and increased cell proliferation and migration, activated the antiapoptosisi signal, led to abnormal cell growth, and ultimately led to tumorigenesis. The addition of ARV-825 effectively rescued this process and effectively inhibited cell vitality, proliferation, and migration. In vivo studies demonstrated that treatment with ARV-825 greatly suppressed tumor growth without causing harmful side effects and downregulated the BRD4-NUT expression level. Conclusion Through the induction of BRD4 protein degradation, ARV-825 can successfully limit BRD4-NUT 3T3 cell proliferation in vitro and in vivo. These findings suggested that the BRD4 inhibitor ARV-825 would be an effective therapeutic strategy for treating NUT carcinoma that with the genetic feature of BRD4-NUT fusion event.


Introduction
NUTM1 (also known as NUT) gene rearrangement could lead to NUT carcinoma (NC), which is an aggressive subtype of squamous cell carcinoma.NC primarily acts on body midline organs.It presents as a monomorphic low diferentiated squamous cell carcinoma [1].Chromosomal translocation caused by BRD4-NUT fusion is the most common because of the genetically characterized disease, NC.Te age of onset of this cancer ranges from 3 to 78 years [1].NC is nearly fatal and is almost entirely resistant to currently known treatments, and even with aggressive chemotherapy, the typical survival period following diagnosis with NC is less than one year (9.5 months) [1].
Te primary member of the bromodomain and extra terminal domain (BET) family of proteins is the bromodomain-containing protein 4 (BRD4), which has two bromodomains (BD) and one extra terminal (ET) domain [2][3][4][5].BDs are capable of recognizing and interacting with lysine in acetylated histones [6].Te ET domain attracts transcriptional regulators, boosting the expression of several important oncogenes, such as c-myc, bcl-xl, and cyclin d1 [7].BRD4 dysfunction is associated with the tumorigenesis and progression of a variety of cancers, including acute myeloid leukemia, colon cancer, Burkitt's lymphoma, breast cancer, difused large B-cell lymphoma, multiple myeloma, and ovarian cancer [4,[8][9][10][11][12].Additionally, it is the site of a chromosomal translocation between chromosomes 15 and 19, which is the cause of aggressive NUT carcinoma that normally manifests as a single t (15; 19) translocation and gives rise to the novel fusion oncogene BRD4-NUT [7].
3T3 cells, a type of mouse embryonic fbroblast cell line, have become one of the most widely studied cell lines in biology since their isolation by George Todaro and Howard Green in the 1960s [13].Tese cells are commonly used in research for a variety of applications, including cell culture, transfection studies, and tumorigenicity assays.Morphologically, 3T3 cells are small and spindle-shaped with elongated nuclei, growing as adherent monolayers in tissue culture.Teir ability to undergo contact inhibition, which means they stop dividing when they come into contact with other cells, is a distinguishing feature [14].Despite their nontumorigenic nature, 3T3 cells are used as a model system for studying cellular transformation and cancer progression.Introducing oncogenes or chemical carcinogens can transform these cells into cancer cells.Te use of 3T3 cells has provided valuable insights into the molecular mechanisms underlying cancer development [15].In our study, we introduced the BRD4-NUT oncogene into 3T3 cells to facilitate the formation of tumors both in vitro and in vivo, thereby simulating nut carcinoma and enabling further investigation.
A number of BET inhibitors (BETis) have been developed during the past ten years and put through clinical studies to ascertain their potent antitumor efects for the treatment of human cancer [16].Te efectiveness of JQ1 was assessed in a variety of tumor types, and it could target superenhancers found in tumor-related genes and demonstrate antiproliferative and proapoptotic activity in a number of cancers [16,17], including pancreatic ductal adenocarcinoma [18], renal cell carcinoma [19], breast cancer [20], medulloblastoma [21], and gastric cancer [22,23].As an illustration, the JQ1 analog chemical OTX015, a BRD4 inhibitor, is now undergoing clinical phase I studies to treat patients with solid tumors and hematologic malignancies [17,20,22,23].
Although earlier research suggested that BETi like JQ1 and OTX015 have the potential to prevent tumor growth, they are not without drawbacks.Due to JQ1 and OTX015 inhibiting reversibility, BRD4 protein reaccumulates and MYC is only partially suppressed [24], necessitating the use of higher inhibitor doses.In contrast, inhibitors using proteolysis-targeting chimera (PROTAC) technology were developed to suppress BRD4 with better efciency [25].
Te PROTAC molecule is a bifunctional protein hydrolysate consisting of a ligand of the protein of interest (POI) and a covalently linked ligand of the E3 ubiquitin ligase (E3).Upon binding to POI, PROTAC can recruit E3, causing POI to ubiquitinate and then undergo proteasomemediated degradation.It has been demonstrated that ARV-825, which combines a BRD4 inhibitor with a cereblon ligand using PROTAC technology, showed to be more efectively breaking down BRD4, inhibiting tumor growth efectively and consistently [12].Te antitumor efect of ARV-825 was systematically studied in pancreatic cancer [26,27], melanoma resistant to vemurafenib [28], cholangiocarcinoma [29], thyroid carcinoma [30], acute myeloid leukemia [31,32], T-cell acute lymphoblastic leukemia, and neuroblastoma [33,34].ARV-825 has not been clinically used in the targeted treatment of NUT carcinoma.Here, we took the advantage of ARV-825 to target BRD4-NUT and tested the antitumor activity in vitro and in vivo by using a BRD4-NUT fusing construct in 3T3 cells.Te purpose of this study is to confrm ARV-825 anticancer efect against BET proteins in NUT carcinoma as well as potential underlying mechanisms.

Materials and Methods
2.1.Cell Line.3T3 and 293T-cell lines were preserved by the Applied Biology Laboratory of Shenyang University of Chemical Technology.Cells were sustained by providing 5% CO 2 at 37 °C, cultivated in the DMEM (Dulbecco's Modifed Eagle's Media) medium (Termo Fisher Scientifc), medium with 100 U/mL penicillin-streptomycin (Millipore Sigma), and 10% fetal bovine serum (FBS) (ExCell Bio).

Cell Transfection.
Te 3T3 cells were electroporated with pcDNA3.1-CMV-BRD4NUT (3 μg).Cells were gently resuspended in 1.5 mL of prewarmed DMEM media following transfection.All cells were cultivated at 37 °C in the incubator with 5% CO 2 .Te following day, the media was changed to complete the DMEM medium, and cells were kept as before.After 48 h, the geneticin (Sigma-Aldrich) was applied to select for stable cell lines.Incubate the cells for a period of 1-4 h at 37 °C in the presence of CO 2 .Measure the absorbance of the CCK-8 solution at 450 nm using a microplate reader.Te data can be analyzed using various statistical methods to determine the efects of the experimental treatment on cell proliferation.Additional controls, such as a blank well and a positive control well, are often included to ensure the accuracy of the assay.
2.6.Wound Healing Assay.Cells are frst plated in a cell culture dish and grown until they reach 100% confuency, forming a single layer of cells.A linear wound is then created on this monolayer using a sterile tool.Te dish is then washed to remove any loose cells with PBS and add fresh DMEM media containing the ARV-825 compounds.Te wound is monitored every 12 h using a microscope, and images are taken at regular intervals to track the movement of cells into the wound.Te speed and direction of cell migration can be quantifed using ImageJ software.Area recovery rate � (initial wound width-remaining wound width)/initial wound width × 100%.

RNA-Sequencing and Analysis. 3T3 cells and BRD4
-NUT 3T3 cells were cultured in 6 cm discs.BRD4-NUT 3T3 cells were treated with 0.003 μM and 0.03 μM ARV-825 for 48 h.After that, cells were collected and RNA molecules were extracted from cells using Trizol kits from Invitrogen.Te quality and quantity of RNA are then assessed using NanoDrop.In library construction, RNA is converted into cDNA using reverse transcription with random hexamers.Te resulting cDNA fragments are then fragmented, and adapters containing indexes are added to each fragment.
Tis step enables multiplexed sequencing, and the resulting libraries are amplifed using PCR.All purifed libraries were sequenced on DNBSEQ-T7RS of Geneplus to acquire 150 bp paired-end sequence reads.Te raw sequencing data undergo quality control preprocessing steps and removal of ribosomal RNA sequences before the processed reads are aligned to a reference genome or transcriptome using software STAR.Finally, the expression levels of genes are quantifed using feature counts, and diferential expression analysis can be performed using statistical methods edgeR.

Quantitative Real-Time PCR (RT-qPCR). 3T3 cells and BRD4
-NUT 3T3 cells were cultured in 6-well discs.Te BRD4-NUT 3T3 cells were then treated with 0.003 μM and 0.03 μM ARV-825 for 48 h.Following this, total RNA was extracted from BRD4-NUT 3T3 cells using Trizol (Vazyme) in accordance with the manufacturer protocol.Real-time quantitative PCR was employed to detect mRNA using the Quant Studio TM Design and Analysis Software.To synthesize cDNA, the Prime-Script RT reagent kit (TaKaRa Biotech) was utilized.
Real-time PCR using SYBR Premix Ex Taq (TaKaRa Biotech) was used to measure the mRNA levels of genes, including Sox6, Cnn1, Traf1, and Eid3.Tese levels were calculated as a ratio to the endogenous Gapdh mRNA level.Amplifcation followed a protocol of denaturation at 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 45 s.Each primer was designed to span an exon-exon junction, and the generation of individual amplicons was examined in melting curve assays.Lastly, the expression levels of diferentiation genes were analyzed using the 2 −ΔΔCT method.
2.9.In Vivo Xenografts.Te Institutional Animal Care and Use Committee (IACUC) of the authors' institutions gave its approval for the ethical care of the animals used in the research.We bought nude mice from Lan puda, Ltd. (Shenyang, China).Eight million 3T3-BRD4-NUT cells were subcutaneously implanted into the backs of four-week-old male nude mice (n � 6 per group), and when the engrafted tumor reached a size of around 200 mm 3 , either 10 mg/kg of ARV-825 or the vehicle control was administrated intraperitoneally each day.When the tumor size in the control group reached 1,000 mm 3 , the mice were sacrifced.Every three days, the size of the subcutaneous tumor was measured using calipers.Tumor volume was determined using the formula (width × width × length × 0.52).

Overexpression of BRD4-NUT Promotes Cell Proliferation and Migration in 3T3
Cells.Given that the BRD4-NUT fusion protein is the oncoprotein for NUT carcinoma [35], we constructed CMV-BRD4-NUT plasmid which can overexpress BRD4-NUT to simulate the pathogenic fusion gene of NUT carcinoma (Figure 1(a)).Exons 2-11 of BRD4 and 2-8 of hNUTM1 were seamlessly connected, mimicking the BRD4-NUT fusion in NC patients [36].Western blotting showed that BRD4-NUT was overexpressed in 3T3 cells (Figure 1(b)), but there was no appreciable morphology diference between 3T3-BRD4-NUT and 3T3 cells (Figure 1(c)).
After overexpression of BRD4-NUT fusion gene, migration ability of 3T3 cells was signifcantly enhanced (Figure 1(d)).Te CCK8 assay found there was an abnormal increase in cell proliferation demonstrated that BRD4-NUT overexpression could signifcantly promote 3T3-BRD4-NUT cell proliferation (Figure 1(e)), which could be attributed to the loss of cell contact inhibition.In conclusion, these results showed that we successfully constructed a stable cell line that overexpressed BRD4-NUT fusion protein and found that overexpression of BRD4-NUTcould promote the proliferation and migration of 3T3 cells.

ARV-825 Inhibits the Proliferation and Migration of 3T3-BRD4-NUT Cells.
In order to assess the toxic efect of ARV-825, wild-type 3T3 cells were cultured in varying concentrations of ARV-825 (0.001-0.03 µM), and it was found that there was almost no toxic efect on wild-type 3T3 cells (Figure 2(a)).Meanwhile, for the overexpressing BRD4-NUT cell lines, cells were treated with various doses of ARV-825 (0-0.1 µM) for 48 hours to assess the drug efect on the cells.After receiving ARV-825 treatment, 3T3-BRD4-NUT cell proliferation was decreased in a dose-dependent manner, and ARV-825 showed the inhibition efect even at the lowest concentration tested 0.001 µM (Figure 2

Comparative Analysis of Gene Transcript Abundance.
Next, we performed RNA-seq to investigate the potential mechanism of BRD4-NUT at the transcriptional level.We tested four sets of data consisting 3T3 cells, 3T3-BRD4-NUT cells, 0.003 µM, and 0.03 µM ARV-825-treated 3T3-BRD4-NUT cells.PCA analysis separated the four groups.Te low concentration of 0.003 µM ARV-825 treatment clustered with 0.03 µM ARV-825, not with BRD4-NUT, indicating the sensitive drug treatment (Figure 3(a)).
According to Figure 3(b), when the transcriptome data from two paired 3T3 cells with pre-and post-overexpressing BRD4-NUT were compared under the conditions of | log2fold change| > 1 and an adjusted p < 0.01, 103 genes were found to be upregulated, and 159 genes to be downregulated.Tose genes showed signifcantly diferent expression levels in 3T3-BRD4-NUT cells compared with 3T3 cells.Correlation analysis of the dysregulated genes was performed and visualized in a heat map (Figure 3(c)).We found that genes involved in spliceosome, ribosome, nucleocytoplasmic transport, amyotrophic lateral sclerosis, cell cycle, and Fanconi anemia pathway were signifcantly upregulated, indicating that these pathways may associate with overexpression of BRD4-NUT (Figure 3(d)).However, after the addition of ARV-825, the abnormally up-down-regulated genes caused by overexpression of BRD4 changed, showing the rescue trend at the transcriptional level (Figure 3(e)).We conducted an experiment to examine the expressions of Sox6, Traf1, cnn1, and Eid3 genes.Upon overexpression of BRD4-NUT, we observed a signifcant upregulation in the expressions of Sox6 and Traf1genes, while there was a signifcant downregulation in the expressions of Cnn1 and Eid3 genes.Subsequently, upon application of ARV-825 to BRD4-NUT 3T3 cells, we noted a downregulation in the expressions of Sox6 and Traf1 genes, along with an upregulation in the expressions of Cnn1 and Eid3 genes.Overtime, the expressions gradually returned to the levels observed in wild-type 3T3 cells, indicating a rescue trend that was in line with the data obtained from RNA-seq (Figure 3(f )).Similarly, in the GSEA enriched pathway, after the addition of ARV-825, the pathway changes also showed a rescue trend (Figure 4).

Discussion
NUT carcinoma is a rare, aggressive, and lethal arising carcinoma.As previously mentioned, t (15; 19) translocation generated BRD4-NUT fusion oncogene that plays a key pathological role in midline carcinoma.Despite aggressive chemotherapy and radiotherapy, the typical survival for NC patients is less than 1 year (9.5 months).NC still lacks efective treatments and strategies.Finding therapeutic targets to treat NUT carcinoma and comprehending the process of its incidence and progression are urgent and crucial tasks.
In NUT carcinoma, which is frequently caused by the fusion of the bromodomains of BRD3 or BRD4, the oncogenic characteristics of BET proteins were initially identifed [37].BET proteins became promising therapeutic candidates for the treatment of infammation and cancer with the invention of numerous small chemical inhibitors that were specifcally bound to BET bromo domains, such as benzodiazepine derivatives, I-BETs, and JQ1 [38][39][40][41].JQ1 replaces BET proteins from acetylated lysine on chromatin by competitively binding to the BET bromodomain [39].In numerous distinct hematological malignancies, inhibiting the BET bromodomain with JQ1 has shown potent anticancer efects both in vitro and in vivo [9-11, 39, 42].However, they also have drawbacks.Cell lines generated from solid tumors, such as those from cervical, breast, and lung malignancies, seem to be less resistant [11,43].Additionally, it has been demonstrated that BRD4 inhibitors may bind to BRD4 reversibly and inhibit it partially, signifcantly impairing anticancer efcacy [44].
Unlike BRD4 inhibitors, ARV-825 is a new chimeric molecule that Lu et al. have created to efciently degrade BRD4 using the PROTAC platform [44].ARV-825 had a more efective inhibitory efect on the BRD4 protein.BRD4 protein is intensely and persistently degraded as a result of ARV-825 directed recruitment of BRD4 into the E3ubiquitin ligase cereblon [44].ARV-825 caused more apoptosis induction and more efcient proliferation suppression, which may be due to its quick and long-lasting BRD4 degradation and inhibition of downstream targets such as c-myc [45].Lu et al. found that ARV-825 can degrade BRD4 protein, thus showing efective efect on cholangiocarcinoma cells [29].Consistent with the results, we found that treatment with ARV-825 led to decline in BRD4 levels in 3T3-BRD4-NUT cells.
Te efects of ARV-825 on 3T3-BRD4-NUT cell gene expression were demonstrated by RNA-seq and western blotting analysis.Te results showed that inhibition of BRD4 by ARV-825 resulted in changes in the mRNA of E2f, Trafs, Wnt, Gadd45g, and Sox6 in 3T3-BRD4-NUT cells.Further investigation of the NUT carcinoma RNA-seq data may identify novel therapeutic targets and precise signaling mechanisms.

6
Journal of Oncology

Journal of Oncology
ARV-825 inhibited the overexpression of BRD4-NUT tumor growth in the 3T3 cell xenograft model.In accordance to in vitro fndings, ARV-825 could reduce BRD4 protein levels in vivo.Tis further demonstrated that ARV-825 may efectively regulate the critical BRD4-NUT gene regulation network.Additionally, it indicated that there were no statistically signifcant diferences in body weight between mice getting ARV-825 treatment and the control group.Organs from mice treated with ARV-825 did not show any other noticeable negative efects.One study found that mice treated with JQ1 had decreased body weight and impaired adipogenesis, but no signifcant toxic efects were observed in ARV-825-treated organs in addition to body weight [46].Tese fndings make it quite evident that ARV-825 is both safe and efective.Our research has demonstrated that targeted degradation through ubiquitination is a promising   Journal of Oncology treatment approach for NUT carcinoma.Currently, we are the frst to target the BRD4-NUT fusion protein with ARV-825.Te fact that the use of ARV-825 for the treatment of NUT cancer is a novel therapeutic strategy makes it even more exciting.

Conclusion
In conclusion, our research showed that ARV-825 commenced its action within 12 h on BRD4-NUT 3T3 cells, resulting in inhibited cell proliferation and migration.
Moreover, the degradation of BRD4 protein was detectable after ARV-825 continuous administration for 72 h.As a result, the present study indicated that ARV-825 was effcient in inducing BRD4-NUT protein degradation and inhibits the growth of 3T3-BRD4-NUT cells in vitro and vivo.Our research on ectopic expression systems in cell lines has dramatically enhanced our understanding of the molecular alterations that the NUTM1 fusion proteins generated, presented novel perspectives on the creation of new targeting medications, provided theoretical support for personalized treatment, and pointed out promising avenues   Journal of Oncology for targeted therapies facilitate the progress in the treatment of NUT carcinoma.Tese fndings suggested that ARV-825 might be an efective therapeutic approach for treating NUT carcinoma that is fused with BRD4.

Figure 1 :
Figure 1: Overexpression of BRD4-NUT promotes cell proliferation and migration.(a) CMV-BRD4-NUT vector construction scheme.(b) 48 h after transfection of 3T3 cells with BRD4-NUT vectors, western blot analysis was conducted.(c) Bright feld of 3T3 cells, 3T3-BRD4-NUT cells.Scale bars, 200 μm.Cells were further cultured in a complete medium for indicated time periods.(d) Cell migration (wound healing assays).(e) Cell proliferation (CCK8 OD 450 nm) was tested.Data were presented as the mean ± standard deviation (SD, n � 3) (same for all Figures).Te experiments were repeated three times, with similar results obtained.

Figure 2 :
Figure 2: ARV-825 suppresses the proliferation and migration of BRD4-NUT 3T3 cells.(a) Diferent concentrations of ARV-825 had little efect on the proliferation of 3T3 cells.(b) Cells overexpressing BRD4-NUT decreased cell proliferation after being treated with ARV-825 at diferent concentrations in 3T3 cells.(c) 3T3 cell and (d) 3T3-BRD4-NUT cell migration changes after treatment with diferent concentrations of ARV-825 at specifc times.Scale bars, 200 μm.(e) Cells overexpressing BRD4-NUT decreased cell wound recovery after being treated with ARV-825 in 3T3 cells.(f ) Western blotting analysis showing the BRD4 protein level in 3T3-BRD4-NUT cells after being treated with ARV-825 at diferent concentrations for 72 h.(g) 3T3 cells and (h) BRD4-NUT 3T3 cells were treated with 0.03 μm ARV-825 for diferent times, and the changes of BRD4 protein expression were detected with western blotting.
XenograftTumor Model.Using 3T3-BRD4-NUT cells, a xenograft model of NUT carcinoma cancer was established in order to examine the anticancer efect of ARV-825 in vivo (Figures5(a)).When the volume of the subcutaneous tumor reached approximately 200 mm 3 , nude mice received daily intraperitoneal injections of ARV-825 at a dose of 10 mg/kg.In comparison to the control group, the tumor burden in the ARV-825 therapy group was signifcantly decreased (Figures5(b)-5(d)).We observed that treatment with ARV-825 caused a reduction in tumor weight compared to the PBS-treated control (Figures5(e)).As shown in Figure5(f ), NUTM1 labelling was markedly reduced in the ARV-825-treated group compared to the PBS control group, indicating the potential efcacy of ARV-825.In addition, the body weights did not signifcantly difer from the treatment group and control group (Figure5(b)).Tese fndings showed that ARV-825 might signifcantly slow the growth of NUT carcinoma tumors without causing any evident negative efects.

Figure 3 :
Figure 3: Transcriptome diference revealed dynamic changes with ARV-825 treatment.(a) PCA analysis of four sets of data.A projection of the input data set onto the frst two principal components.BN, 3T3-BRD4-NUT cells, BN + 0.003, 0.003 μM ARV-825 treated with 3T3-BRD4-NUT cells, BN + 0.03, 0.03 μM ARV-825 treated with 3T3-BRD4-NUT cells.(b) Volcano plot of RNA-seq analysis of gene expression changes in BRD4-NUT 3T3 and control cells.Genes highlighted in blue form the up-fold changes, and cyan indicates down regulated genes and black on behalf of unchanging genes.(c) Heatmap view of the diferentially expressed genes in 3T3-BRD4-NUTcells compared with 3T3 cells.Red indicates upregulation, and blue indicates downregulation.(d) KEGG pathway enrichment analysis signifcantly upregulated after BRD4-NUT overexpression compared with 3T3 cells (top 10).Te color represents signifcance, and the circle size represents the number of genes enriched.(e) Te top diferentially expressed genes that before and after ARV-825 treatment were subjected to functional enrichment analysis cells (p < 0.05, |log2FC| > 1).Each column represents a diferent sample, and each row represents a single gene.Color changes within a row indicate expression levels relative to the average of the same population.Red indicates upregulation, blue indicates downregulation, and white indicates the basic level of expression.(f ) q-PCR analysis relative gene expression levels of Sox6, Traf1, Cnn1, and Eid3 in each group.Data were presented as the mean ± SD. (n � 4).

Figure 4 :
Figure 4: GSEA plots showing the enrichment of genes in the gene set in RNA-Seq following ARV-825 treatment.

Figure 5 :
Figure 5: ARV-825 displays antitumor efcacy in the BRD4-NUT 3T3 xenograft model.(a) Diagram of xenograft fowchart, nude mice bearing xenograft tumors were treated by either 10 mg/kg ARV-825 or vehicle control intraperitoneally every day for 21 days.Data are the mean ± SEM (n � 6).(b) Mice body mass was weighed every 3 days.(c) Tumor volume was recorded every 3 days and calculated using the formula: (width × width × length × 0.52).(d) Photograph of xenograft tumors from ARV-825-or vehicle-treated mice.(e) Tumor size at the endpoint.(f ) Histological characteristics of subcutaneous tumors (H&E staining) and immunohistochemical staining of NUTM1 protein (representing brd4-NUTM1 fusion protein) in mice treated with ARV-825 and PBS control.