The Androgen Receptor: A Therapeutic Target in Desmoplastic Small Round Cell Sarcoma

Desmoplastic small round cell tumor (DSRCT) is an aggressive, usually incurable sarcoma subtype that predominantly occurs in post-pubertal young males. Recent evidence suggests that the androgen receptor (AR) can promote tumor progression in DSRCTs. However, the mechanism of AR-induced oncogenic stimulation remains undetermined. Herein, we demonstrate that enzalutamide and AR-directed antisense oligonucleotides (AR-ASO) block 5α-dihydrotestosterone (DHT)-induced DSRCT cell proliferation and reduce xenograft tumor burden. Gene expression analysis and chromatin immunoprecipitation sequencing (ChIP-seq) were performed to elucidate how AR signaling regulates cellular epigenetic programs. Remarkably, ChIP-seq revealed novel DSRCT-specific AR DNA binding sites adjacent to key oncogenic regulators, including WT1 (the C-terminal partner of the pathognomonic fusion protein) and FOXF1. Additionally, AR occupied enhancer sites that regulate the Wnt pathway, neural differentiation, and embryonic organ development, implicating AR in dysfunctional cell lineage commitment. Our findings have immediate clinical implications given the widespread availability of FDA-approved androgen-targeted agents used for prostate cancer. ONE SENTENCE SUMMARY We demonstrate that DSRCT, an aggressive pediatric cancer, is an AR-driven malignancy capable of responding to androgen deprivation therapy.


INTRODUCTION
Since the epigenetic effects of AR can be modified by cofactor binding and matrix metalloproteins, we assessed whether steroid receptor coactivators NCOA1/2/3 or MMP2/13 contribute to the development of DSRCT through AR-dependent mechanisms [32][33][34][35][36][37] . To accomplish this, we performed a Western blot of 11 primary DSRCT tumors and the JN-DSRCT and LNCaP PC cell lines. The three-NCOA biomarkers demonstrated heterogeneous expression in the DSRCT clinical samples proportional to their AR expression (Fig. S3B). However, the JN-DSRCT cells showed low expression of NCOA1/2 and equivalent expression of NCOA2 versus LNCaP PC cells (Fig. S3C). Further investigation with a larger sample set will be required to determine how the AR-dependent integrin/NCOA-dependent pathway impacts DSRCT cell migration and death.

In vitro stimulation and inhibition of DSRCT proliferation via AR
Though AR activation by testosterone and DHT leads to brisk PC cell proliferation 38 , it was uncertain whether DSRCT cells similarly relied upon AR signaling for proliferation, growth, and survival. To evaluate this, we performed in vitro cell proliferation assays following DHT-mediated AR stimulation in JN-DSRCT; AR-expressing LNCaP PC cells, AR-non-expressing PC3 PC cells, and ES TC71 cells that were used as positive or negative controls. As hypothesized, DHT stimulation increased cell proliferation of JN-DSRCT and LNCaP cells compared to PC3 and ES cells (Fig. 3A). As measured by Western blotting, we confirmed strong AR expression by LNCaP and JN-DSRCT cell lines following DHT stimulation in contrast to its absence in the TC71 ES and PC3 prostate cancer cells (Fig. 3B). Next, we performed confocal immunofluorescence staining of these cells to determine if (and how quickly) DHT-mediated stimulation would facilitate AR transmigration from the cytoplasm into the cell nucleus. Our results suggest that AR-upregulation begins within 5 hours of DHT exposure and peaks in JN-DSRCT, or decreases in LNCaP cells at 24 hours ( Fig. 3C-D).
Having shown that DHT stimulates DSRCT cells, we explored whether FDA-approved and experimental AR antagonists had an antiproliferative effect. Both enzalutamide (Fig. 3E) and the novel AR-ASO (IONIS 560131; formerly AZD5312) significantly slowed DSRCT cell proliferation at two weeks (Fig. 3F) and reduced AR expression (Fig. 3G). However, the in vitro antiproliferative effect was 4-fold more effective in the cells treated with the AR-targeted antisense blockade ( Fig. 3E & 3F). Notably, this antineoplastic effect required 72-hours of DHT pretreatment (Fig. S4). Altogether, this data indicates a vital role for DHT-stimulated AR expression in DSRCT cell proliferation and conclusively demonstrates a potent antineoplastic effect of AR antagonists.

Preclinical efficacy of AR-based targeted therapy for the treatment of DSRCT.
Since only one DSRCT cell line exists, we extended our evaluation of the AR antagonists to the in vivo setting using the JN-DSRCT xenograft and available DSRCT patient-derived tumor explants (PDXs). Immunocompromised NSG mice bearing JN-DSRCT xenograft tumors treated with enzalutamide or AR-ASO significantly reduced tumor burden and improved survival with the same efficacy, compared to placebo or control groups during the first two months of treatment ( Fig. 4A-B). At two months, tumor growth began to accelerate in the enzalutamide-treated mice, whereas growth suppression continued in the mice treated with either 25 or 50 mg/kg of the AR-ASO (p<0.0001; Fig. 4A). Compared to enzalutamide, the AR-targeted ASO (25 and 50 mg/kg) demonstrated superior antineoplastic activity ( Fig. 4A; p<0.0001 or p=0.006, respectively). The effects of AR-ASO and control ASOs were also assessed in NSG mice (5 mice/group) bearing a DSRCT PDX (Fig. 4D-F). As expected, tumor growth and Kaplan-Meier curves revealed that tumors treated with AR-ASO have significantly reduced tumor burden and improved survival compared to control ASO group (p=0.0097 & p<0.0001, respectively).
Though both agents delayed tumor growth, AR-ASOs were more effective than enzalutamide in both preclinical models. Therefore, our pharmacodynamic analysis focused primarily on the effect of AR-ASO treatment. Proteomic profiling by RPPA (Fig. 5A Figure 4. Consistent with prior literature in PC, loss of AR following AR-ASO treatment destabilized testosterone and reduced its intratumoral expression ( Fig.  S6) 39, 40 . As a negative control, the corticosterone levels were unchanged by AR-blockade. Additionally, since the antineoplastic action of enzalutamide works by preventing ligand-AR binding, reducing AR shuttling to the nucleus, and impairing AR DNA binding affinity -instead of reducing AR levels ( Fig. S7; panels A-E)enzalutamide-treatment did not significantly lower intratumoral testosterone.
To gain a preliminary understanding of the short-term pharmacodynamic effects of AR suppression, a group of JN-DSRCT xenografts and DSRCT PDXs was collected 10 days into their AR-ASO treatment (Fig. 5A AR-ASO PD) for analysis by RPPA to assess early compensatory pharmacodynamic changes. pS6, Akt, ER, PD-1L, pAKT, and other proteins ( Figure 5A and Supplemental Figure 7F-G) were upregulated. Others have reported that the PI3K-AKT pathway has pleiotropic effects in survival, proliferation, metabolism, and growth pathways of several malignancies 41 , and its blockade has long been of interest in managing PC, where a compensatory increase in AKT signaling can occur following AR inhibition 42 . Notably, the same AR-ASO (AZD5312) used in our preclinical experiments was well-tolerated when administered to PC patients (NCT03300505). Therefore, one could theoretically investigate this AR-ASO drug candidate in DSRCT-specific phase 2 trials without delay. Given the limited nature of our preclinical studies, future studies with enzalutamide is also of interest.

AR directly regulates important oncogenic regulators in DSRCT
To model the AR transcriptional program in a human JN-DSRCT cell line, we determined the genome-wide AR binding profiles using ChIP-Seq experiments in unstimulated or DHT-stimulated JN-DSRCT cells treated with control ASO or AR-ASO. As expected, DHT treatment enhanced AR binding to the chromatin as assessed by the average intensity plot on all significant peaks (p < 1e -7 ) and heat map (Fig. 6A). DHT stimulation led to ~4000 new peaks that were suppressed by treatment with AR ASO (Fig. 6B and Supplemental Table 1). These binding sites were enriched at known AR response elements (AREs) and in sites for FOXA1, a transcription factor known to open compacted DNA and cooperate with AR in prostate cancer 43 ( Fig. 6C and Supplemental Table 1). Consistent with DSRCT's pathogenesis, we also noted enrichment of WT1 binding motifs within AR binding peaks (Fig. 6C) suggesting potential interactions between AR, FOXA1, and WT1 in JN-DSRCT cells. To further characterize the genes adjacent to AR binding site peaks, we performed a pathway analysis using 700 genes that are direct targets of AR. Upregulated pathways included the TNFa pathway, Hippo signaling, and pluripotency regulators ( Fig. 6D and Supplemental Table 1), and key genes included WT1, CTNNB1, SOX2, GLI2, FOXF1 and GATA6 (Fig. 6E, Fig. S8D and Supplemental Table 1).
After evaluating the effects of androgen stimulation and withdrawal in JN-DSRCT cells, we next compared DSRCT to data from PC cells. Significant overlap existed at sites for AR binding at AREs (Fig. S8A), FOXA1 motifs ( Fig. S8B), and sites that regulate key cancer pathways, including WNT, TGFb, PI3K, MAPK, Hippo signaling, TNFa and epithelial-to-mesenchymal transformation (EMT) (Fig. S8C). To further evaluate the AR regulatory function in DSRCT tumor mouse models, we performed ChIP-seq on DSRCT-xenograft and PDX samples. Consistent with the cell line data, we observed suppressed AR binding to the chromatin by the treatment with AR ASO (Fig. S9A-B). Similarly, pathway analysis of the top 5000 lost AR binding sites targeted genes showed enrichment of MAPK pathway, Hippo signaling, Wnt signaling and pluripotency regulators ( Fig.  S9C-D). We also noted enrichment of AR and FOX family binding motifs within AR binding peaks in both DSRCTxenograft and PDX samples (Fig. S9E). Genes adjacent to AR binding site peaks also showed high overlap with DSRCT specific genes in both models (Fig. S9F). Key genes from cell line data (Fig.6E) also showed AR signal reduction after AR-ASO treatment (Fig. S9G).

AR-dependent enhancer reprogramming activates oncogenic pathways in DSRCT
Several studies have shown that AR establishes a pro-tumorigenic transcriptome by reprogramming the active enhancer landscape (assessed by H3K27ac profiles) in prostate cancer progression 44 . Therefore, we asked if AR plays similar roles in DSRCT by examining genome-wide profiles for H3K27ac marks in unstimulated or DHT stimulated JN-DSRCT cells treated with control ASO or AR-ASO. We noted that unstimulated cells treated with AR-ASO showed a higher intensity and a higher number of H3K27ac peaks compared to control ASO treated cells ( Fig. 7A-B, S10A, and Supplemental Table 2). Similarly, AR-ASO treatment in DHT-treated cells also increased the active enhancer peaks compared to control ASO treatment ( Fig. 7A-B, S10A, and Supplemental Table 2). This observation is contrary to those in prostate cancers where active enhancer peaks are positively associated with higher AR activity 44 . It has been previously shown that AR recruits the MLL complex and CBP/p300, which is responsible for active enhancer marking in prostate cancer 45 . To identify which enhancers were likely derived by AR binding and potential recruitment of enhancer-marking proteins, we overlapped the AR and H3K27ac peaks in DHT treated cells ( Fig. S10B and Supplemental Table 2). We then intersected these AR-targeted enhancer peaks with highly expressed genes in DSRCT ( Fig. 7C and Supplemental Table 2) (FC> 1.5, adjusted p-value < 0.05 in comparison to other sarcoma subtypes). There, we identified WNT signaling and cell-adhesion as major drivers that are regulated at the chromatin level by AR-dependent active enhancer programs (Fig. 7D). The genes with direct AR binding and enhancer gains included important oncogenes such as AXIN2 and CDK6 (Fig. 7E). Additionally, we investigated alterations in super-enhancer (SE) regions that harbor a high-density of TF binding motifs [46][47][48] . SEs in control ASO treated cells marked important oncogenes such as AKT3 and GRHL2, whereas SEs in AR-ASO treated cells marked tumor suppressor genes such as RUNX1 and CUX1 (Fig. S10C-D and Supplemental Table 3), that potentially regulate the AR-driven transcriptome. Overall, our results suggest that AR activation reprograms typical enhancers and SE to regulate key oncogenic signaling pathways in DSRCT.
Interestingly, in preclinical tumor samples we also observed similar enhancer reprogramming. AR-ASO treatment of the DSRCT-xenograft significantly increased the active enhancer and promoter binding sites compared to control ASO treatment, whereas PDX samples showed a moderate increase (Fig. S11A-D). We also observed AR-dependent active enhancers regulating PI3K-AKT-mTOR, WNT signaling, cell-adhesion pathways ( Fig. S11E-F), and key oncogenes (Fig. 7E), with direct AR binding and enhancer gains in both DSRCT-xenograft and PDX models (Fig. S11G).

DISCUSSION
Ever since Ladanyi and Gerald discovered the EWSR1-WT1 chromosomal translocation 8 , DSRCT has been treated with the same chemotherapy regimens used for ES. The recent exceptions include ES-specific agents like TK-216 that target c-terminus ETS genes (e.g., FLI1 or ERG), or pazopanib, which demonstrates preferential activity in DSRCT and other soft-tissue sarcomas 49 . Phase II studies testing neoantigen targeted monoclonal antibodies (Abs), for example 8H9 in DSRCT, are also directed at unique sarcoma subtypes 50 .
As three-quarters of all DSRCT patients typically succumb to their malignancy within 5-years, our RPPA study intended to define new molecular targets for DSRCT and expand our therapeutic arsenal of biologically targeted therapies that engage them ( Figure 1). Surprisingly, of 151 proteins assessed in the RPPA, SYK and AR were the most differentially expressed. The SYK protein -not previously reported in DSRCT -is a nonreceptor tyrosine kinase (also known as spleen tyrosine kinase) commonly found in hematological tissues. Its constitutive activation has been shown to induce malignant transformation of B-cells to lymphomas or leukemias. As such, the oral SYK inhibitors cerdulatinib (Portola Pharmaceutical) and entospletinib (Gilead Sciences) are under active clinical investigation for the treatment of certain lymphomas, chronic lymphocytic leukemia, and acute myeloid leukemia (NCT01994382 and NCT02457598). An orally active SYK inhibitor, fostamatinib, has already received FDA-approval as a treatment for immune thrombocytopenia and continues to be investigated as an experimental therapy for hematological malignancies (NCT00446095). Though tantalizing to consider that SYK hyperactivation plays an oncogenic role in DSRCT, we have not yet had the opportunity to evaluate these relatively new drugs within our preclinical DSRCT models.
In contrast to SYK, numerous FDA-approved and experimental AR antagonists were available for immediate preclinical evaluation, and potentially available to patients via compassionate access or early-phase clinical trials. Though our RPPA data and 9:1 male-to-female ratio hinted that DSRCT is an AR-driven malignancy, to prove this explicitly we proposed several criteria, akin to Koch's postulates: (a) tumors must adequately express AR, (b) DHT must stimulate DSRCT cell proliferation, and (c) AR antagonists should curtail the tumor-promoting effects of androgen stimulation. The inclusion of mechanistic studies, including those directly tying AR to androgen response elements (AREs), lends further credibility that DSRCT is a second AR-driven malignancy.
To date, the first criterion -requiring AR expression -has been reported by two prior teams that recognized the striking predilection of DSRCT for young males 15,51 . As discussed briefly in the Introduction, Fine et al. evaluated protein expression of AR, c-Kit, EGFR, and other proteins by Western blot and IHC, scored using a 5point scale that ranged between 0 (no staining) to 4+ (highly positive) 15 . Ten of twenty-seven (37%) DSRCT patients in their case series scored 2+ or higher, but we highlight that fifteen demonstrated no AR expression (Figure 2), which suggests prospective studies may wish to stratify for response by AR-status to determine whether AR expression correlates with therapeutic efficacy. A more recent study published in 2018 by Bulbul et al. at U.C.S.D., used IHC and next-generation sequencing on tumors from thirty-five DSRCT patients (86% who were males); 59% were AR-positive using a dichotomous cut-off that required ³1+ staining in ³10% of the cells 51 . In the present study, we report the most extensive series of DSRCT patients to have undergone protein and transcriptomic profiling. Though enriched in oncoproteins, our RPPA array ranked AR as the most differentially expressed protein compared to ES, its closest molecular sarcoma subtype (Figure 1). Our subsequent confirmation of the RPPA results by Western blot, and later semi-quantitative analysis by IHC, is in agreement with earlier reports and appears to substantiate AR as a bona fide target in DSRCT.
Meeting the second of Koch's postulates, a 72-hour cell proliferation assay demonstrated a significant increase in JN-DSRCT cell proliferation following exposure to physiological levels of DHT ( Figure 3A), though lower than LNCaP PC cells. As one would expect in androgen-sensitive cells, DHT also promoted the nuclear shuttling of AR into the nucleus where it would function as a transcriptional regulator of its target genes ( Figure  3C  Fulfilling the third requirement that defines an AR-driven malignancy, our team again bolsters the work by Fine et al., which had taken a prescient step more than a decade ago to evaluate CAB -in that case using Lupron and bicalutamide in six DSRCT patients that were AR-positive (3+ or 4+ by IHC) 18 . Interestingly, in their limited pilot trial, non-castrate level baseline testosterone levels were associated with modest responses lasting 3-4 months. Admittedly, having tested several DSRCT patients with the same drug combination between 2006-2015, well before the advent of modern-day androgen deprivation therapies (ADT) such as abiraterone and enzalutamide, our team observed limited clinical benefits lasting <3 months. Our renewed enthusiasm for AR targeting in DSRCT stemmed from the RPPA expression results, accompanied by the in vitro DHT stimulation studies and in vivo data using enzalutamide and the AR-ASO ( Figure 4).
In preparation for early-phase clinical trials now in development, our work takes the first step to advance our mechanistic understanding of AR signaling in DSRCT. As one of several steroid and nuclear hormone superfamily receptors that include the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), progesterone and estrogen receptors (PR & ER), and the vitamin D receptor (VDR), AR retains a conserved 66amino acid DNA-binding domain (DBD) able to join two (5'-AGAACA-3') hexameric half-sites arranged as an inverted palindrome spaced 3-b.p. apart (IR3). Due to differences in local steroid metabolism, ligand abundance, chromatin accessibility, and cofactor occupancy, the DNA binding pattern of AR varies significantly in PC compared to other tissues 52 . Interestingly, among the pioneer factors that govern the lineage-specific binding of AR to specific genomic loci in PC [53][54][55] , and that control AR-mediated transcriptional regulation of prostate genes (such as PSA) 56 , FOXA1 was the most enriched MOTIF in JN-DSRCT cells ( Figure 6C). Shared activation of the androgen signaling cascade in DSRCT and PC may explain the close transcriptomic clustering observed in Fig.  2E. Despite their similarities, ChIP-seq also identified notable differences in AR's epigenetic regulation at enhancer (Figure 7) and super-enhancer ( Figure S9C) binding sites.
Though the subject of future research, we suspect the heterotypic loss of WT1 or aberrant EWS-WT1 FP may recruit a specific set of chromatin modifiers at binding sites that differ from PC. Others have performed ChIP-seq in DSRCT patient specimens using WT1-specific antibodies, but the Santa Cruz antibody used in that publication 57 has been discontinued. Lacking suitable ChIP-seq validated WT1-specific antibodies ATAC-seq might be used before and after WT1 RNA silencing, though interpretation of that experiment wouldn't be as straightforward given the absence of selective antagonism of WT1 or EWS-WT1.
Interestingly, as occurs in castration-resistant PC 11,58,59 , our pharmacodynamic studies revealed an inverse relationship between AR and the Akt/PI3K/mTOR pathway. With numerous inhibitors of PI3K and mTOR already FDA-approved, an obvious next step would be to investigate whether co-targeting AR and either PI3K or mTOR results in synergistic anti-cancer activity. Though not explored in the present study, the estrogen receptor (ER) was also highly expressed following AR-ASO treatment. Given the shared binding DNA motifs that ER, AR, and other steroid hormone receptors have in common, this observation suggests that ER-targeted drugs might prove useful for patients with castrate-resistant DSRCT and, plausibly, the small minority of women that acquire this rare cancer type. Of course, further research in required to determine how AR and ER pathway switching affects tumor growth and survival, both in DSRCT and other hormonally-driven malignancies 60 .
Collectively, though morphologically and phenotypically distinct from PC, our data suggest that DSRCT is a second androgen-stimulated malignancy (third, if one considers the AR-positive molecular subset of triplenegative breast cancer). Shared dependence upon AR for tumor growth and survival provides an exciting opportunity to study AR signaling in a different cancer type and within a younger DSRCT-stricken patient population. Preclinical data using enzalutamide and AR-ASO raises the tantalizing possibility that AR-targeted drugs used for PC may also find utility to combat DSRCT.

Patients
The collection of DSRCT tumor patients was approved by the Institutional Review Board of MDACC under the LAB08-0151 and LAB04-0890 protocols and conducted in compliance with the principals of the Declaration of Helsinki. The charts and electronic medical records of patients with a confirmed diagnosis of DSRCT were included for analysis and archived at the MDACC biospecimen bank or the collaborator PIs laboratories. We identified 60 DSRCT patients treated at MDACC from 1990 to 2019 to generate a TMA. Also, we collected 16 DSRCT and 6 Ewing sarcoma (ES) fresh frozen tumors, all of them were profiled by RPPA. Specialist pathologists used clinical information, immunohistochemistry, and cytogenic analysis for the EWSR1-WT1 or EWSR1-FLI1 fusions to confirm the DSRCT or ES diagnoses. Blood samples (EDTA) of 5 ml were collected from 17 DSRCT patients and 3 Ewing sarcoma patients (ES) to remove serum and assay PSA.

RPPA and Western blot analyses
The available snap-frozen DSRCT (n=16) and ES (n=6) specimens collected during a core-needle biopsy or surgical debulking procedures using clinical protocols approved by MDACC's Institutional Review Board and specimens of normal-appearing mesenteric tissue adjacent to DSRCT obtained at the time of surgical debulking (n=8) were used for the proteomic analysis (Supplemental Table 4: Demographic information of DSRCT and ES patients). Lysates were created, protein concentrations were determined, and individual protein expression was measured using a well-validated reverse-phase protein array (RPPA) and Western blot (WB) technologies as previously described [61][62][63] . AR protein detection was performed using the CST antibody (#5153). Additional details about RPPA and WB analyses and normalized data are provided in the Supplementary Methods and Supplemental Table 5.

RNA Sequencing, gene expression analysis, and fusion detection
Total RNA from primary tumor samples was extracted and libraries made from cDNA using the NuGEN Ovation Ultralow Library System V2 (San Carlos, CA). RNA sequencing reads of the samples were mapped to the hg19 reference genome using the STAR aligner 64 . For calculation of gene expression, each gene's raw count data were first obtained using HTSeq 65 , and are normalized by scaling the library size using calcNormFactors in the edgeR package 66 . Then, Voom transformation was applied to normalized counts and a linear model fit to the data for differential expression analysis using the Limma package 67 . Pathway analyses of differentially expressed genes between two sample clusters were performed using Gene Set Enrichment Analysis (GSEA) 68 . Fusion transcripts were detected from RNA-seq data using MapSplice 69 .

TMA Preparation and Immunohistochemistry Analyses
A tissue microarray (TMA) was constructed from archival surgical pathology materials comprising 60 formalinfixed, paraffin-embedded tissues from 60 DSRCT patients. Areas of the viable tumor were selected by pathologist review of whole slide H&E-stained sections. Selected areas were punched and transferred, in duplicate, to a recipient block using an ATA-100 Advanced Tissue Arrayer (Chemicon International). All human specimens were utilized under an Institutional Review Board-approved research protocol (LAB04-0890) allowing for the retrospective sampling and analysis of existing archival materials collected in the course of standard patient care. Immunohistochemical studies were performed using an autostainer (Bond-Max; Leica Microsystems, Buffalo Grove, IL, USA) with anti-AR (1:30; clone AR441, Dako#M3562) antibody. Additional details about TMA slides preparation and IHC analyses are provided in the Supplementary Methods.

WST1 Cell Proliferation Assays
The JN-DSRCT, LNCaP, and TC71 cells tested for their proliferation capacity In vitro using a colorimetric assay in 96-Well plates with WST-1 reagent (Roche). The cells seeded at 3000 cells/well in triplicates with 10% FBS DMEM (JN-DSRCT) or RPMI (TC71 and LNCaP) complete media. Additional details about WST1 cell proliferation assays are provided in the Supplementary Methods. ). Additionally, cell lines are sent for 3rd-party mycoplasma testing using a sensitive PCR testing approach any time a collection of cells are cryopreserved. Monolayer JN-DSRCT cell culture in 8 chamber slides were fixed for 10 min at room temperature with 4% paraformaldehyde in phosphate-buffered saline (PBS). The primary JN-DSRCT xenograft and PDX tumors were harvested, fixed in 10% formalin, embedded in paraffin (formalin-fixed, paraffin-embedded: FFPE), and then sliced in 5 μm sections before processing them for antigen retrieval using 0.1M citrate buffer for 20 minutes and in a vegetable steamer. Altogether, monolayer and primary tumor slides were permeabilized and blocked with superblock buffer (Thermo Fisher Scientific, #37535) for 1 hour at room temperature. Slides were then incubated consecutively with primary antibodies to AR (Cell Signaling Technologies, #5153), (overnight at 4°C) and Alexa Fluor 488-labeled Goat-anti Rabbit (Thermo Fisher Scientific, #A11037) (for 1hr at room temperature). The nuclei were visualized using Hoechst (Thermo Fisher Scientific, #H357), and the immunofluorescence was acquired after subtracting the background intensities using the Nikon A1-Rsi confocal microscope (Nikon). Fluorescentdetection of proteins in the nuclei and cytosolic regions was quantified using the Imaris software (Bitplane) and its Cell module that use validated algorithms to define the segmentation by permitting the recognition of selected protein fluorescence in both nuclear and cytosolic regions.

Generation of DSRCT Xenograft/PDX mouse models and Drug Evaluation
All experiments were conducted per protocols and conditions approved by the University of Texas MD Anderson Cancer Center (MDACC; Houston, TX) Institutional Animal Care and Use Committee (eACUF Protocols #00000712-RN03). Male NOD (SCID)-IL-2Rg null mice (The Jackson Laboratory; Farmington, CT) were subcutaneously injected with JN-DSRCT cells (5X10 6 cells/animal) or received PDX explants (2 mm) to generate DSRCT xenograft and PDX mouse models. The histologic and genetic analyses of DSRCT patient and PDX tumors are available on Supplemental Figure 12. All mice were maintained under barrier conditions and treated using protocols approved by The University of Texas MD Anderson Cancer Center's Institutional Animal Care and Use Committee. Once their tumors reached a volume of 150 mm 3 , 5 mice per group received enzalutamide (25 mg/kg IP daily, 5 times per week), or AR ASOs (25 or 50 mg/kg subcutaneously daily, 5 times per week), or control ASOs (50 mg/kg subcutaneously daily, 5 times per week), or a placebo control (sterile vehicle buffer). Tumor volumes were measured using digital calipers at study initiation and 2-5 times per week after that for up to 85 days, or until their tumors reached 1500 mm 3 , whichever came first. A Kaplan-Meier analysis was performed to assess drug efficacy. Statistical analyses between control and treated group or between different treated groups were performed with the log-rank (Mantel-Cox) test using Graph-Pad Prism 8.0.

ChIP-Seq Assays
Chromatin immunoprecipitation was performed as described earlier 70 with optimized shearing conditions and minor modifications for JN-DSRCT cells. The antibodies used were: H3K27ac (Abcam ab4729) and AR (CST#5153). Briefly, 3 million cells per sample were cross-linked using 1% formaldehyde for 10 min at 37 °C. After quenching with 150 mM glycine for 5 min at 37 °C, cells were washed twice with ice-cold PBS and frozen at −80 °C for further processing. Later, cells were thawed on ice and lysed with ChIP harvest buffer (12 mM Tris-HCl, 0.1 × PBS, 6 mM EDTA, 0.5% sodium dodecyl sulfate [SDS]) for 30 min on ice. Lysed cells were sonicated with Bioruptor (Diagenode) to obtain chromatin fragment. Antibody-dynabead mixtures were incubated for 1 hr at 4 °C and cellular extracts were then incubated overnight with these mixtures. After overnight incubation, immune complexes were washed five times with RIPA buffer, twice with RIPA-500 (RIPA with 500 mM NaCl) and twice with LiCl wash buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA pH8.0, 250 mM LiCl, 0.5% NP-40, 0.1% deoxycholate). For reverse-crosslinking and elution, immune complexes were incubated overnight at 65 °C in elution buffer (10 mM Tris-HCl pH8.0, 5 mM EDTA, 300 mM NaCl, 0.5% SDS). Eluted DNA was then treated with proteinase K (20 mg/ml) and RNase A and DNA clean-up was done using SPRI beads (Beck-man-Coulter). ChIP libraries were amplified and barcoded with use of the NEBNext® Ultra™ II DNA library preparation kit (New England Biolabs). After library amplification, DNA fragments were size-selected (200 -500 bp) using AMPure XP beads (Beckman Coulter) and assessed using high sensitivity D1000 screen tape on the Bioanalyzer (Agilent Technologies). Libraries were multiplexed together and sequenced in HiSeq2000 (Illumina).

ChIP-seq Data Processing
ChIP-seq data were quality controlled and processed by pyflow-ChIPseq 71 , a snakemake 72 based ChIP-seq pipeline. Briefly, raw reads were mapped by bowtie1 73 to hg19. Duplicated reads were removed, and only uniquely mapped reads were retained. RPKM normalized bigwigs were generated by deep tools 74 , and tracks were visualized with IGV 75 . Peaks were called using macs1.4 76 with a p-value of 1e-9 for H3K27ac and 1e-7 for AR. Heatmaps were generated using R package EnrichedHeatmap. ChIP-seq peaks were annotated with the nearest genes using ChIPseeker 77 . Super-enhancers were identified using ROSE 78 based on H3K27ac ChIPseq data.

Differential Peaks Analysis
To identify variable AR or enhancer domains enriched in specific DSRCT samples, enhancer peaks that overlap with 2.5kb upstream and 2.5kb downstream of any known TSSs were removed. The unique and shared peaks within multiple groups were identified by Intervene 79 . The peaks were annotated with ChIPseeker R package 77 , using addFlankGeneInfo function for enhancers.

Identification of AR and Enhancer Associated Pathways
Differential AR binding sites and enhancers associated genes in each sample were imported into the ClusterProfiler 80 for pathway analysis, restricted to GO, KEGG, Hallmark, and WiKi gene sets. The Enrichplot package 81 was used to generate dot plot and bar plot for gene sets enriched with a false discovery rate (FDR) cut-off of < 0.05.

Enrichment of Motifs in AR-Specific Peaks
To identify the motifs over-represented within AR-specific peak sets, we used the HOMER motif database and the coordinates of AR-specific peak sets 82 . Fig 1. Proteomic comparison of DSRCT and ES. A) The protein lysates from DSRCT (red) and ES (blue) were subjected to RPPA analysis for 151 proteins and phosphoproteins (red, increased signal; green, decreased signal). Unsupervised double-hierarchical clustering using the Pearson correlation distance metric between proteins (rows) and Centroid linkage (a clustering method) separated the 22 samples into two groups by tumor type (columns). Of the 22 proteins, 8 had expression that differed significantly between ES and DSRCT (p≤0.05; fold-change ≥2). B) The mean expression intensity values of the 8 proteins associated with DSRCT or ES and their statistical significance after normalization for global protein expression by median centering across 151 antibodies in the RPPA panel. C) Western blotting was used to validate the proteins identified by RPPA as being differentially expressed between DSRCT and ES. D) Normalized protein expression is relative to b-actin.   Tumor-bearing mice volumes, and survival were reported after been treated with the enzalutamide (25mmg/kg, orange), the AR-ASO (25mg/kg, regular red; 50mg/kg, dotted red), control ASO (gray), and placebo treatment (black). A) The left panel shows the smoothed grouped median relative tumor volumes in these groups of mice. The P values for differences between the treated and control mice were performed with the log-rank (Mantel-Cox) test. B) The right panel shows the survival Kaplan-Meier curves of each treated group of mice. C-D) Therapeutic effect of AR antisense blockade in JN-DSRCT xenografts. C) Tumor-bearing mice volumes were reported through smoothed grouped median relative tumor volumes after been treated the mice with the AR-ASO (50mg/kg, red), control ASO (gray), and placebo treatment (black). D) Kaplan-Meier curves show mouse survival after drug treatment. The P values for differences between the treated and control mice were performed with the log-rank (Mantel-Cox) test. E-F) Therapeutic effect of AR antisense blockade in DSRCT PDX1 mice. E) The smoothed grouped median relative tumor volumes are shown after the mice been treated with the AR-ASO (50mg/kg, red), control ASO (gray), and placebo treatment (black). F) Kaplan-Meier curves indicate the survival rate after drug treatment. The P values for differences between the treated and control mice were performed with the log-rank (Mantel-Cox) test.

Fig 5. Proteomic evaluation of AR expression in JN-DSRCT and PDX tumors after AR-based antisense therapy. A)
The principal components analysis plot and reverse-phase protein lysate array (RPPA) evaluations of JN-DSRCT and PDX tumors after therapies, separated the 32 samples into four groups and identified 37 proteins statistically significantly associated with the treatment at a false discovery rate (FDR) of 0.05. B) Immunoblotting evaluation of JN-DSRCT xenograft and PDX-DSRCT tumors after AR-ASO treatment. C) AR normalization relative to GAPDH within the preclinical tumor samples. AR biomarker was significantly reduced in mice treated with AR-ASO compared to the control ASO group (p=0.01). D) Representative AR immunofluorescence confocal microscopy quantification of the preclinical JN-DSRCT and PDX tumor samples, within the single cell or, E) the averaged treated samples (placebo, control ASO, and AR-ASO). Bars represent standard deviations. F) Immunohistochemical evaluation images of preclinical JN-DSRCT and PDX1 tumor samples. IHC stains for AR in primary tumors of JN-DSRCT and PDX DSRCT mice after treatment with AR-ASO, control ASO, and placebo. 100 µm scale bars are shown. G) Representative IHC AR mean intensity quantification of the preclinical JN-DSRCT and PDX tumor samples, within the single cell or, H) the averaged treated samples (placebo, control ASO, and AR-ASO). Bars represent standard deviations. All tumors analyzed by PPPA were collected at tumor progression or the experiment's conclusion, except for the AR-ASO PD group, which was collected 10 days after initiating therapy with pharmacodynamic analysis.