Modeling CIC::DUX4 sarcoma reveals oncogene-mediated MHCI-dependent immune evasion

Modeling CIC::DUX4 sarcoma reveals oncogene-mediated MHCI-dependent immune evasion | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Modeling CIC::DUX4 sarcoma reveals oncogene-mediated MHCI-dependent immune evasion Ajay Vachanaram, Erdong Wei, Ana Mitanoska, William Bassett, Michael Kyba, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6516041/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Molecular Cancer → Version 1 posted 10 You are reading this latest preprint version Abstract CIC::DUX4 sarcoma (CDS) is a highly aggressive malignancy with limited therapeutic options. Here, we present a doxycycline-inducible CIC::DUX4 chimeric mouse model and a cancer line derived from it, imChCDS, that faithfully recapitulates the molecular, histological, and immunological features of human CDS. We demonstrate that CIC::DUX4 expression alone is sufficient to drive tumorigenesis in permissive lineages of soft connective tissues. The imChCDS cell line retains the transcriptional footprint of its mesenchymal cell of origin, develops tumors in immunocompetent hosts, and exhibits a clear dependency on the P300/CBP transcriptional co-activators. Notably, we identify CIC::DUX4-mediated suppression of MHC class I (MHCI) as a key mechanism of CDS immune evasion. Inactivation of CIC::DUX4 restores MHCI expression, triggers robust anti-tumor immune responses, and leads to tumor regression. Together, these models offer a versatile and physiologically relevant platform to investigate CDS pathogenesis, unravel immune evasion mechanisms, and evaluate emerging therapeutic strategies, including those targeting CIC::DUX4/P300/CBP oncogenic axis. CIC:DUX4 sarcoma immune evasion experimental models MHCI Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION CIC::DUX4 sarcoma (CDS) represents a particularly aggressive and rare subtype within the spectrum of small round blue cell tumors ( 1 , 2 ). CDS is genetically defined by chromosomal translocation involving 19q13.1 and either 4q35 or 10q26.3 fusion between CIC and DUX4 genes ( 3 – 6 ). The CIC::DUX4 fusion protein retains the DNA-binding domain of CIC but acquires a potent transcriptional activation domain from DUX4 ( 7 – 10 ). This alteration transforms CIC from a transcriptional repressor into an aberrant activator of key genes involved in cell cycle regulation and oncogenesis ( 9 , 11 ). Clinically, CDS presents as a rapidly progressing disease with high metastatic potential and a strikingly poor prognosis, often exhibiting resistance to standard chemotherapy regimens adapted from Ewing sarcoma protocols ( 12 – 15 ). Patient-derived CDS cell lines have been crucial for characterizing CIC::DUX4-driven transcriptional programs ( 16 – 22 ), but they lack the genetic, epigenetic, and cellular complexity of primary tumors. Extended culture often results in genetic drift and clonal selection, reducing their preclinical relevance ( 23 ). Advanced models such as patient-derived organoids (PDOs) and xenografts (PDXs) offer greater fidelity but are constrained by the rarity of CDS and limited availability of tumor material ( 24 ). In vivo , CDS is typically studied through xenotransplantation into immunocompromised mice ( 11 , 19 , 20 , 25 , 26 ), which, although informative for oncogenic pathway analysis, fails to recapitulate tumor initiation, native microenvironments, and functional immune systems, factors critical for investigating immune interactions and evaluating immunotherapies. Given these challenges, advancing CDS research requires complementary model systems that fully capture the disease’s complexity and better support translational applications ( 11 , 12 ). Recent studies have suggested that DUX4, through its C-terminal domain, promotes immune evasion in certain malignancies and myogenic cells by downregulating MHC class I (MHCI) antigen presentation and suppressing interferon-stimulated pathways ( 27 – 29 ). Given that CIC::DUX4 retains the same C-terminal domain, it is assumed that it may contribute to the development of an immunologically “cold” microenvironment in CDS, thereby impairing lymphocyte recruitment and activation ( 29 – 31 ). However, the mechanisms underlying immune escape in CDS and their role in tumor progression within immunocompetent hosts remain poorly understood, largely due to the absence of suitable in vivo models. This gap limits efforts to unravel immune mechanisms and hinders the development of targeted immunotherapies for CDS. Prior attempts to generate transgenic mouse models with tissue-specific CIC::DUX4 expression using Lox/Cre recombination confirmed that CIC::DUX4 can drive malignant transformation ( 31 ). However, these models were compromised by unanticipated Cre recombination, uncontrolled CIC::DUX4 expression, and failure to achieve germline transmission of the transgene. Moreover, constitutive CIC::DUX4 expression in derived cell lines limited their experimental utility ( 31 ). To overcome previous limitations, we developed a doxycycline (dox)-inducible chimeric mouse model that expresses CIC::DUX4 and forms soft tissue tumors closely resembling human CDS. From this model, we established a dox-tunable murine CDS cell line (imChCDS) that recapitulates the molecular profile of human CDS and is tumorigenic in both immunodeficient and immunocompetent hosts. Through syngeneic transplantation, we demonstrate that CIC::DUX4 suppresses MHCI presentation, thereby promoting immune evasion. This novel model provides a robust platform for investigating CDS tumor biology, tumor–immune interactions, and therapeutic strategies, ultimately advancing the development of innovative treatments. RESULTS CIC::DUX4 is sufficient to drive malignant transformation in chimeric mice Our primary goal was to develop tissue-specific CIC::DUX4 transgenic mouse models to serve as reliable tools for studying CDS pathogenesis and evaluating therapeutic strategies. As an initial step, we sought to determine whether CIC::DUX4 expression could be tightly regulated in vivo and if its expression alone could drive malignant transformation. To investigate this, we engineered dox-inducible CIC::DUX4 transgenic murine ES cells and used these to generate chimeric mice, where the inducible cells distribute across various cell types throughout the animal. This approach obviates the necessity for germline transmission and enables CIC::DUX4 expression within a permissive cellular and microenvironmental context, increasing the likelihood of initiating tumorigenesis. We engineered dox-inducible CIC::DUX4 mouse embryonic stem cells (iCD4 mESCs) by targeting the HPRT locus of ZX1 cells with the p2Lox-CIC::DUX4 construct, as previously described ( 32 ). In addition to the Lox/Cre cassette at the HPRT locus, ZX1 cells are engineered to constitutively express the reverse tetracycline transactivator (rtTA) under the control of the Rosa26 promoter ( 32 ). Neomycin-resistant clones were selected and expanded for further analysis. To assess CIC::DUX4 inducibility, clones were treated with 500 ng/mL dox for 24 hours. CIC::DUX4 expression was confirmed via immunofluorescence (Fig. 1 A), and the activation of known CIC::DUX4 target genes Etv1 , Etv4 , and Etv5 was evaluated by RT-qPCR (Fig. 1 B). Three clones demonstrating strong, non-leaky, and robust CIC::DUX4 induction were selected and microinjected into C57BL/6 blastocysts to generate chimeric mice (Fig. 1 C). CIC::DUX4 chimeric mice (iChCD4) at the age of 6 months (n = 10) with varying degrees of chimerism were fed a chow containing 625mg/kg dox to induce CIC::DUX4 expression. After 4 weeks of continuous induction, 6 mice (60%) developed palpable, macroscopic tumors under the skin (soft connective tissue) (Fig. 1 D). Two mice died of unknown causes, and the remaining 2 mice showed no observable changes. Histological analysis of the tumors revealed a striking resemblance to those formed via xenotransplantation of human CDS cell lines, such as Kitra-SRS (Kitra) and NCC-CDS-X1 (X1) (Fig. 1 E). The tumors in iChCD4 mice exhibited hallmark features of CIC::DUX4 sarcoma, including small to medium-sized neoplastic cells arranged in nodular patterns, separated by prominent collagenous septa ( 2 ). To further confirm the identity of the tumors, we performed immunofluorescence staining for CIC::DUX4 and established CDS-associated markers, including ETV4, WT1, and CD99 ( 2 , 6 , 33 , 34 ). CIC::DUX4 expression was observed in the majority of tumor cells, while subsets exhibited nuclear and cytoplasmic localization of WT1, focal expression of CD99, diffuse staining of ETV4 (Fig. 1 F) and minimum infiltration with immune cells (Supplementary Fig. 1). We performed transcriptional profiling to assess the molecular similarity of tumors arising in chimeric mice to CDS and other common sarcomas. Spearman correlation analysis revealed that the gene expression profiles of the chimeric tumors closely resembled those of tumors derived from Kitra and X1 cell lines, as well as CDS patient tumor samples. In contrast, they were more distinct from other sarcoma subtypes, including Ewing sarcoma (ES), leiomyosarcoma (LMS), osteosarcoma (OS), and synovial sarcoma (SS) (Fig. 1 G). Together, our findings demonstrate that CIC::DUX4 expression alone is sufficient to initiate malignant transformation in vivo , resulting in tumors that originate in subcutaneous connective tissues. These tumors recapitulate the key histological and molecular features of CDS, validating the chimeric mouse model as a relevant system for studying CIC::DUX4-driven sarcomagenesis. CIC::DUX4-driven murine cancer cell line imChCDS From the tumors that developed in chimeric animals, we aimed to establish a dox-inducible CIC::DUX4 murine cell line for use in syngeneic transplantation models, with the goal of studying CDS pathology within an immunocompetent environment. Tumors isolated from chimeric mice were enzymatically digested and subcutaneously transplanted into immunodeficient NSG mice, as previously described for the X1 cell line ( 35 ). Mice were kept on dox chow (625 mg/kg) to maintain sustained CIC::DUX4 expression. Palpable tumors emerged by day 45 post-inoculation and reached the maximum allowed size (2 cm³) within 8 days. Histological analysis revealed that the secondary tumors were morphologically indistinguishable from the original tumors in the chimeric mice and closely resembled human CDS tumors (Supplementary Fig. 2). Portions of these tumors were enzymatically dissociated, and the resulting single-cell suspensions were cultured in dox-containing medium (500 ng/µL) to establish a CIC::DUX4-inducible murine cell line (imChCDS). Initially, the cell culture appeared morphologically heterogeneous (Supplementary Fig. 3), but over several passages it progressively became more homogeneous, consisting of small polygonal-shaped cells by passage 9 (Fig. 2 A). Cell viability was entirely dependent on CIC::DUX4 expression, as removal of dox led to rapid cell cycle arrest, marked by the loss of Ki67 and the induction of cellular senescence, as indicated by β-galactosidase positivity (Fig. 2 A) and specific gene expression (Supplementary Fig. 4). The estimated doubling time of imChCDS cells under dox-supplemented conditions was 50.1 hours, while cells cultured without dox exhibited no proliferative capacity over a 12-day period (Fig. 2 B). imChCDS cells also demonstrated the ability to grow in 3D spheroid cultures when seeded in low-attachment plates (Fig. 2 C). Consistent with monolayer conditions, dox withdrawal in 3D cultures led to a loss of proliferative potential and progressive spheroid shrinkage (Fig. 2 C, D). Western blot analysis was performed to determine whether CIC::DUX4 induces similar transcriptional and epigenetic changes in this murine system as observed in human CDS ( 17 ). CIC::DUX4 expression induced the known target genes ETV4 and WT1 , and was associated with increased histone acetylation at H3K18 and H3K27 (Fig. 2 E). Transcriptomic analysis further confirmed that imChCDS cells cultured with dox expressed 116 of 157 genes of a well-established set of CIC::DUX4 human target genes ( 11 ) (Fig. 2 F). Three days after dox withdrawal, expression of CIC::DUX4 and its downstream targets was lost, accompanied by a morphological senescence-like phenotype (Fig. 2 A), upregulation of 59 out of 137 senescence-associated markers (Fig. 2 G, Supplementary Fig. 4). Finally, we investigated whether imChCDS cells retain the transcriptional footprint of their cell of origin by analyzing lineage-specific gene expression signatures. Among 23 distinct cell types representing all three embryonic germ layers, ectoderm, endoderm, and mesoderm ( 36 ), imChCDS cells selectively expressed markers associated with the mesoderm, particularly those related to mesenchymal and fibroadipogenic cell types (Fig. 2 H and Supplementary Fig. XX). Notably, during the proliferative phase, the cells displayed a transcriptional profile characteristic of cycling progenitors. Following dox withdrawal, this profile shifted toward that of differentiated mesenchymal stem cells (MSCs) and different subtypes of fibroblasts (Fig. 2 H). Collectively, these findings demonstrate the successful establishment of a murine CIC::DUX4 sarcoma cell line, imChCDS, which closely recapitulates key molecular and phenotypic features of CDS in vitro and retains the transcriptional footprint of its mesenchymal cell of origin. imChCDS cells exhibit CIC::DUX4-dependent tumorigenic characteristics To evaluate the tumorigenic potential of the imChCDS cell line in vivo , we transplanted 5 × 10⁵ cells subcutaneously into immunocompromised NSG mice (n = 12). Eight mice were fed a dox-containing diet (625 mg/kg; “All Dox ON ”), while the remaining four were kept on a dox-free diet to evaluate whether tumor formation could occur independently of CIC::DUX4 induction. Mice in the Dox ON group developed tumors within 14 days, whereas no tumors were observed in the Dox OFF group (Fig. 3 A), confirming that CIC::DUX4 expression is required for tumor initiation. To assess whether continued CIC::DUX4 expression was necessary for tumor maintenance, dox was withdrawn from half of the mice, and tumor progression was monitored for an additional 8 days (Dox ON>OFF ). While tumors in the continued Dox ON group exhibited progressive growth, those in the dox withdrawal group regressed significantly (Fig. 3 A–C). Histological analysis by H&E staining revealed that tumors from the Dox ON group retained typical features of CDS, including dense sheets of small, round cells. In contrast, tumors from the Dox ON>OFF group showed a marked loss of tumor cellularity and increased stromal matrix deposition (Fig. 3 D). Immunofluorescence staining confirmed the presence of CIC::DUX4 protein and active proliferation (Ki67 positivity) in the Dox ON tumors (Fig. 3 E). Additionally, these tumors expressed characteristic CDS markers, including ETV4, WT1, and CD99 (Fig. 3 E). Transcriptomic analysis further confirmed that CIC::DUX4 target genes were expressed exclusively in tumors from the Dox ON group (Fig. 3 F). Pathway enrichment analysis of differentially expressed genes revealed that dox withdrawal predominantly affected pathways involved in cellular metabolism and cell cycle regulation (Fig. 3 G). Together, these findings demonstrate that imChCDS cells possess CIC::DUX4-dependent tumorigenic potential in NSG mice and recapitulate key molecular and histopathological features of human CDS in vivo . imChCDS cell viability depends on the CIC::DUX4/P300/CBP transcriptional axis We previously found that CIC::DUX4 exerts its oncogenic activity through interaction with the transcriptional co-activators P300/CBP ( 17 ). Inhibition of P300/CBP disrupts CIC::DUX4 function, reduces cell viability and proliferation in vitro , and suppress tumor growth in xenograft models of CIC::DUX4 sarcoma ( 16 , 17 ). To assess whether the imChCDS cell line similarly depends on this axis, we performed a cell viability assay using a range of concentrations of our developed P300/CBP inhibitor, iP300w ( 17 , 37 , 38 ). The effects of iP300w treatment were compared to a control condition in which CIC::DUX4 expression was turned off. We observed a significant, dose- and time-dependent reduction in cell viability following iP300w treatment (Fig. 4 A). Notably, the most effective concentration was 0.3µM, consistent with the optimal dose previously identified for human CIC::DUX4 sarcoma cell lines ( 17 ). iP300w treatment induced morphological changes similar to those observed upon CIC::DUX4 inactivation (Dox ON>OFF ), with cells transitioning from a compact, polygonal shape to the flattened, enlarged morphology typical of senescent cells (Fig. 4 B). This change was accompanied by a loss of proliferation, as evidenced by a marked reduction in Ki67 staining (Fig. 4 B). Consistent with these findings, treatment with iP300w also led to a significant downregulation of CIC::DUX4 target gene expression, confirming that P300/CBP inhibition suppresses CIC::DUX4-driven transcriptional activity (Fig. 4 C). Collectively, these results demonstrate that imChCDS cell viability and proliferation are dependent on the CIC::DUX4/P300/CBP transcriptional axis. Pharmacologic inhibition of P300/CBP not only confirms their role in mediating CIC::DUX4 oncoactivity but also highlights the potenital of the imChCDS model for future drug screening and therapeutic development targeting this pathway. CIC::DUX4 suppresses MHCI expression Previous studies have suggested that CIC::DUX4, similar to DUX4, may contribute to immune evasion by suppressing MHCI antigen presentation, potentially through interference with interferon-gamma (IFN-γ)-induced immune response genes ( 27 , 28 ). To investigate whether CIC::DUX4 suppresses MHCI expression in the imChCDS model, as observed in CDS cell lines, we assessed the presentation of MHCI components by FACS 24 hours after dox withdrawal, with or without IFN-γ stimulation (Fig. 5 A). We found that CIC::DUX4 markedly suppressed the surface presentation of both H2-Db and β2M. Within 24 hours of dox withdrawal, H2-Db expression increased significantly even in the absence of IFN-γ, and IFN-γ stimulation further enhanced its expression, resulting in uniform receptor presentation across the cell population (Fig. 5 A, B). A similar pattern was observed for β2M, which was absent in the presence of CIC::DUX4, even with IFN-γ stimulation, but was robustly induced in all cells upon CIC::DUX4 inactivation (Fig. 5 A, B). These findings were further supported by RT-qPCR analysis, which revealed CIC::DUX4-dependent repression of MHCI-related genes, including H2-D1, H2-K1, and β2M (Fig. 5 C). Together, these results demonstrate that CIC::DUX4 suppresses MHCI expression at both the transcriptional and protein levels, likely contributing to immune evasion mechanisms in CIC::DUX4-driven sarcomas. CIC::DUX4 mediates CDS immune evasion The imChCDS cell line was derived from embryonic stem cells of 129P2 mice, enabling transplantation into 129-background mice for the study of CIC::DUX4-driven sarcomas in an immunocompetent, syngeneic setting. To assess tumorigenicity, we transplanted lentiviral GFP-labeled imChCDS cells (Supplementary Fig. 6) into 24 F1 hybrid mice (129P3/J × C57BL/6) using the same method as in NSG mice. All mice received dox-supplemented chow beginning on the day of transplantation. Within 30 days, 21 out of 24 mice developed palpable tumors. To investigate the relationship between CIC::DUX4 activity, MHCI expression, and immune cell infiltration, we conducted a time-course experiment. Tumor-bearing mice were randomized into four groups with staggered dox withdrawal schedules. Group 1 had dox removed for the entire 9-day duration of the experiment. Group 2 underwent dox withdrawal starting on day 3, for a total of 6 days without dox. Group 3 had dox withdrawn only 3 days before the experimental endpoint. Group 4 remained on dox throughout and served as the control. A progressive reduction in tumor volume was observed in the groups with longer periods of dox withdrawal, with significant decreases noted as early as 3 days post-withdrawal (Fig. 6 A–C). Histological analysis revealed that tumors in the dox-treated control group retained the characteristic CIC::DUX4 sarcoma morphology, which gradually changed following CIC::DUX4 inactivation (Fig. 6 D). Immunohistochemical analysis confirmed the expression of diagnostic markers, CIC::DUX4, ETV4, WT1, and CD99, in tumors from the Dox ON group (Supplementary Fig. 7). These markers were markedly reduced in tumors following 3 days of dox withdrawal and were undetectable after 9 days without dox (Supplementary Fig. 7). By day 3 post-dox withdrawal, tumors exhibited notable immune cell infiltration, as indicated by increased CD45-positive staining (Fig. 6 D). This infiltration progressively intensified by day 9. A similar pattern was observed for F4/80-positive macrophages and CD8-positive T cells, indicating a coordinated immune response (Fig. 6 D). To quantitatively assess MHCI expression and the dynamics of immune cell infiltration, we performed FACS analysis. As early as 3 days after dox withdrawal, there was a significant reduction in GFP + imChCDS tumor cells, accompanied by a marked increase in CD45 + hematopoietic cells (Fig. 7 A). With prolonged dox withdrawal, the percentage of GFP + tumor cells continued to decline, while CD45 + immune cell infiltration further increased (Fig. 7 A). Remarkably, only 3 days of dox withdrawal were sufficient to restore MHCI expression in fraction of GFP + tumor cells. This restoration became more pronounced over time (Fig. 7 A). The upregulation of MHCI was accompanied by a rapid and robust infiltration of T cells (Fig. 7 B). While T cells in the Dox ON group were predominantly CD4+, dox withdrawal induced a significant shift toward CD8 + cytotoxic T cells (Fig. 7 B). Macrophage infiltration also increased, with a phenotypic shift from tumor-promoting M2 macrophages in the Dox ON group to pro-inflammatory, anti-tumor M1 macrophages post-CIC::DUX4 inactivation (Fig. 7 C). Finaly, NK cell infiltration peaked at day 3 post-dox withdrawal but declined as MHCI expression and CD8 + T cell infiltration increased, falling below Dox ON levels by day 9 (Fig. 7 D). These findings demonstrate that CIC::DUX4 suppresses MHCI-mediated immune recognition in sarcomas. Its inactivation reduces tumor growth, restores MHCI expression, and triggers robust anti-tumor immune responses, characterized by enhanced CD8 + T cell infiltration, M1 macrophage reprogramming, and transient NK cell activity. DISCUSSION Here, we present a dox-inducible chimeric mouse model and a derived murine cell line (imChCDS) that faithfully recapitulate the molecular, histological, and immunological features of CIC::DUX4 sarcoma (CDS). These models address key limitations in existing systems by allowing investigation of CDS within an immunocompetent context and enabling controlled modulation of CIC::DUX4 expression. Together, they provide a robust platform to study sarcomagenesis, immune evasion, and potential therapeutic vulnerabilities. We demonstrate that CIC::DUX4 expression alone is sufficient to initiate malignant transformation and necessary to maintain it in permissive linages, producing tumors that closely resemble human CDS in histology, marker expression, and transcriptional profiles. Furthermore, we showed that CIC::DUX4 suppresses MHCI presentation to evade immune recognition, and that its inactivation restores MHCI expression, thereby triggering robust anti-tumor immune responses. We successfully generated an inducible chimeric mouse model of CDS, overcoming limitations of prior transgenic approaches, such as uncontrolled CIC::DUX4 expression and the inability to achieve germline transmission ( 31 ). We deliberately chose a chimeric strategy as an initial platform for modeling CDS as it offers a physiologically relevant and developmentally regulated microenvironment that supports tumorigenesis. This model provides a powerful means to investigate cancer initiation, progression, and tumor–microenvironment interactions in vivo ( 39 – 43 ). Furthermore, it enables direct evaluation of CIC::DUX4’s oncogenic potential without the need of additional genetic alterations or a defined cell of origin. The functionality of the dox inducible transgene not only validates the approach but also provides a strong rationale for future development of tissue-specific transgenic mouse models. This strategy also requires minimal breeding compared to conventional germline transgenic techniques, making it an efficient and scalable alternative for early-stage modeling. We demonstrated that CIC::DUX4 alone is sufficient to drive sarcomagenesis, which is consistent with previous literature highlighting the low mutational burden of CDS ( 44 ). Other studies have also shown that CIC::DUX4 can independently induce malignant transformation in mouse embryonic mesenchymal stem cells and osteochondrogenic progenitors, as well as promote tumor formation in both zebrafish and mouse models ( 22 , 31 , 45 ). Notably, although chimerism was present systemically, CDS tumors developed exclusively in subcutaneous soft tissues. This finding highlights the importance of the epigenetic and cellular context, suggesting that mesenchymal-derived cells are uniquely susceptible to CIC::DUX4-driven transformation ( 45 ). Furthermore, we demonstrated that CIC::DUX4 not only promotes cell cycle progression and transformation but also enables immune evasion, thereby supporting tumor persistence in the presence of an intact immune system ( 29 ). Tumors that developed in chimeric mice were histologically and molecularly indistinguishable from CDS tumors seen in patients ( 1 , 7 , 31 – 33 ), underscoring the relevance of this mouse model for studying CDS biology. From these tumors, we established the imChCDS cancer cell line, which closely resembles the molecular and phenotypic characteristics of CDS. It is noteworthy that while many cell lines engineered to overexpress oncogenes exhibit robust proliferative capacity in vitro , they often fail to retain tumorigenic potential in vivo . Even when tumor formation does occur, xenografted cells are prone to acquiring additional disease-nonspecific mutations due to clonal selection pressures ( 46 – 48 ). Therefore, it is crucial that transplanted tumors not only replicate the phenotypic features of the original disease but also preserve a transcriptomic landscape that closely resembles the primary tumor ( 49 – 51 ). A significant advantage of the imChCDS cell line is its ability to form tumors when transplanted into immunocompetent mice, providing a robust platform for investigating tumor–immune interactions in a physiologically relevant context. Additionally, both the chimeric model and the imChCDS cells provide tunable, temporally controlled expression of CIC::DUX4, enabling precise modulation of oncogene levels that closely mimics those observed in patients. Notably, imChCDS cells not only recapitulate the core oncogenic pathways characteristic of CDS but also retain molecular signatures of their mesenchymal cell of origin, aligning closely with established human CDS cell lines. Finally, we demonstrated that the tumorigenic potential of imChCDS cells is dependent on the CIC::DUX4/P300/CBP transcriptional axis, confirming P300/CBP as a pharmacological vulnerability in CDS ( 16 , 17 ). The dose-dependent response to P300/CBP inhibitors (iP300w, optimal at 0.3 µM) is consistent with prior studies, suggesting conserved therapeutic vulnerabilities across species and reinforcing the translational potential of our model ( 17 ). Together, these findings establish imChCDS as a highly relevant model system for studying CDS pathogenesis and as an invaluable platform for preclinical drug testing, both in vitro and in vivo ( 52 – 56 ). This cell line enhances the existing repertoire of models used to investigate CDS ( 19 , 26 , 35 ). A key finding of this study is that CIC::DUX4 suppresses MHCI expression, thereby contributing to immune evasion in CDS. Tumors often evade adaptive immune responses by downregulating MHCI, which reduces the efficacy of both endogenous and therapeutic anti-tumor T cell responses ( 57 – 60 ). Both in vitro and in vivo analyses revealed that CIC::DUX4 represses the expression of MHCI components, including H2-Db and β2M. There have been recent reports implicating the C-terminus of DUX4 in immune evasion through the downregulation of IFN-γ–mediated MHCI expression during early embryonic development and in various cancers ( 28 , 29 , 61 ). Suppression of MHCI likely contributes to the immunologically "cold" tumor microenvironment seen in CDS. Our immunocompetent transplantation experiments provide further compelling evidence that CIC::DUX4 inactivation can reverse this immune suppression. Dox withdrawal led to rapid tumor regression, upregulation of MHCI expression, and a robust immune response, marked by increased infiltration of CD8⁺ T cells, natural killer (NK) cells, and a shift in macrophage phenotype from tumor-promoting M2 to anti-tumor M1. Interestingly, NK cell infiltration peaked transiently at day 3 post-withdrawal and then declined as MHCI expression and CD8⁺ T cell activity increased. This suggests a dynamic interplay between innate and adaptive immunity following CIC::DUX4 inactivation. These findings align with prior studies demonstrating that restoring MHCI expression enhances tumor immunogenicity and facilitates effective cytotoxic T cell–mediated clearance ( 62 , 63 ). Thus, CIC::DUX4 inactivation, such as through inhibition of the P300/CBP, may offer a potent dual therapeutic strategy for CDS by both inducing cancer cell arrest and restoring MHCI expression, thereby enhancing immune recognition and accelerating tumor clearance. Furthermore, the P300/CBP inhibition strategy would likely sensitize CDS to checkpoint inhibitor therapy. Our studies provide a rationale for testing this combined approach in human CDS. In conclusion, our dox-inducible chimeric mouse model and imChCDS cell line provide powerful tools for studying CDS pathogenesis, immune evasion, and therapeutic responses. By revealing CIC::DUX4’s role in suppressing MHCI expression and mediating immune evasion, we uncover a critical mechanism driving CDS aggressiveness. These findings lay the groundwork for targeted therapies, such as P300/CBP inhibitors or immunotherapies designed to restore MHCI expression, and highlight the importance of immunocompetent models in advancing translational research for rare cancers like CDS. MATERIALS AND METHODS Cell Culture Mouse embryonic stem (ES) cells were cultured on irradiated mouse embryonic fibroblasts (MEFs) in DMEM supplemented with 15% fetal bovine serum (FBS), penicillin/streptomycin (P/S; Gibco), 2 mM GlutaMAX (Invitrogen), nonessential amino acids, 0.1 mM β-mercaptoethanol, and 100 U/mL leukemia inhibitory factor (LIF; PeproTech). Doxycycline (dox) inducible mouse CIC::DUX4 sarcoma (imChCDS) cells were cultured in DMEM (HyClone) supplemented with 10% FBS (Peak Serum, Ps-FB3, lot 293Q16), GlutaMAX, and P/S. Induction of the CIC::DUX4 transgene under the control of the TRE promoter was achieved using 500 ng/µL dox (Sigma Aldrich, CAS No. 564-25-0) dissolved in PBS. All cells were maintained at 37°C in a humidified incubator with 5% CO₂. Doubling time was assessed by passaging and counting cells every second day. For 3D culture, 2.5 × 10⁴ imChCDS cells were seeded into ultra-low attachment 96-well plates (96-well Clear Flat Bottom Ultra-Low Attachment Microplate; Corning, Inc., NY, USA) using DMEM supplemented with 10% FBS. Spheroid size was measured from 10× images using ImageJ software. GFP-expressing lentivirus was produced in 293T cells by co-transfecting the lentiviral pSAM-GFP vector along with packaging (psPAX2) and envelope (pMD2.G) plasmids. Viral supernatants were collected at 48 and 72 hours post-transfection, filtered, and used to transduce target cells. Two days after transduction, GFP-positive cells were sorted by FACS and expanded for downstream applications. Cell viability (ATP) assay Cell viability was determined using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer's protocol. Cells were seeded in 96-well plates at a density of 1 × 10⁵ cells per well one day prior to treatment. Luminescence was measured using the POLARstar Optima Microplate Reader (BMG Labtech). Histological analysis, Immunostaining and Western blotting Tumor samples were embedded in OCT compound (Scigen) and flash frozen in liquid nitrogen. Cryosections (10–12 µm) were mounted on glass slides and stained with hematoxylin and eosin (H&E) using standard protocols. For immunostaining, cells and tissue sections were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.3% Triton X-100 for 30 minutes, and blocked with 3% BSA for 1 hour at room temperature. Primary antibodies diluted in 3% BSA were incubated overnight at 4°C. Fluorophore-conjugated secondary antibodies were applied for 1 hour at room temperature. Nuclei were counterstained with DAPI (1:5000; Sigma). For β-galactosidase (β-Gal) immunofluorescence, fixed and permeabilized cells were incubated for 1 hour at 37°C with 50 µL of spider-β-Gal working solution (Dojindo, SG02-01). For western blotting, cells were lysed in RIPA buffer supplemented with a protease inhibitor cocktail (Complete, Roche). Protein samples were separated via 7.5–10% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked in 5% skim milk in TBST and incubated with primary antibodies overnight at 4°C or for 1 hour at room temperature. After washing, HRP-conjugated secondary antibodies were applied for 1 hour at room temperature. Protein signals were detected using Pierce ECL Western Blotting Substrate (Thermo Scientific). The antibodies utilized in this study included GAPDH-HRP (1:5000, Proteintech 60004), rabbit anti-DUX4 [E5.5] (1:500, Abcam ab124699), mouse anti ETV4 (1:250, Santa Cruz D2720), rabbit anti-WT1 (1:500 Cell Signaling 83535 ) rabbit anti-Histone H3K18Ac (1:500, Abcam ab1191), rabbit anti-Histone H3K27Ac (1:500, Abcam ab1791, lot: GR3297878-1), rabbit anti-Ki-67 (1:250, Cell Signaling 9129T), goat anti-rabbit/anti-mouse secondary Alexa Fluor 488/555 and 647 (1:1000, Invitrogen), HRP-conjugated anti-rabbit (1:5000, Jackson Immuno Research 111–035 − 003, lot: 149,393), and HRP-conjugated anti-rabbit/anti-mouse (1:2500, Novus NBP1-75,130, lot 58–173-090418). In vivo experiments and tumor formation assays All animal experiments were performed at the University of Minnesota Research Animal Resources facility under an Institutional Animal Care and Use Committee (IACUC)-approved protocol (#2209A-40422). NSG, C57BL/6J, and 129P3/J mice were obtained from Jackson Laboratory. Inducible CIC::DUX4 chimeric mice were generated at the University of Minnesota Mouse Genomics Laboratory. CIC::DUX4 expression was induced in chimeric, or imChCDS transplanted immunocompetent (C57BL/6J&SV129), and immunocompromised (NSG) mice via dox-containing chow (Envigo) at a concentration of 625 mg/kg. For transplantation, 5 × 10⁵ imChCDS cells were suspended in a 1:1 mixture with Matrigel and injected subcutaneously into the flank of NSG mice. Tumor dimensions were measured every 2 days, and tumor volume was calculated using the formula: volume = 0.5 × length × width². Mice were identified using number tags. Investigators were blinded to experimental conditions during tumor collection, weighing, and imaging. Tumor Dissociation Tumors were dissected, rinsed in cold PBS, then mechanically dissociated using sterile blades. Tissue fragments were digested in DMEM high glucose medium supplemented with 1 mg/mL collagenase type II (Gibco) and 1% penicillin/streptomycin (Invitrogen) for 45 minutes at 37°C on a shaker. The resulting cell suspension was washed twice by centrifugation at 1500 RPM using Ham’s F-10 medium. A second enzymatic digestion was performed using a solution containing 0.1 mg/mL collagenase type II and 0.5 mg/mL Dispase (Gibco) in Ham’s F-10 medium, incubated for 20 minutes at 37°C with shaking. The digested mixture was then homogenized using 18G needles, filtered through a 40 µm cell strainer, and washed twice more by centrifugation at 1500 RPM for 5 minutes. The final cell suspension was either cultured or processed for fluorescence-activated cell sorting (FACS) analysis. FACS analyses Tumor digests were treated with ACK lysis buffer (Gibco, A1049201) for 10 minutes at room temperature. Cells were then washed twice with PBS by centrifugation at 1500 RPM for 5 minutes. Following this, cells were incubated with anti-mouse CD16/32 antibody (0.1 µg/100 µL; TruStain FcX, clone S17011E) for 15 minutes on ice, followed by two additional washes. Staining with fluorescent-conjugated antibodies was performed at manufacturer-recommended concentrations in PBS supplemented with 1% FBS at 4°C for 60 minutes in the dark. Stained cells were then washed twice using washing buffer containing 10% FBS in PBS by centrifugation at 1500 RPM for 5 minutes. Finally, cells were resuspended in PBS containing 10% FBS and propidium iodide and analyzed by flow cytometry using a BD FACS Symphony S6. Quantification of imChCDS cells was based on GFP expression. H2-Db and β2m expression was assessed in the GFP + population gated from live cells. Immune cell subsets were quantified from the live cell population as follows: T cells (CD45+, TCR-β+, and CD4 + or CD8+), NK cells (CD45 + and NK1.1+), Macrophages (CD45+, CD11b+, and F4/80+), M1-like macrophages (CD45+, CD11b+, F4/80+, and CD206+). Data were analyzed using FlowJo (BD Biosciences). The antibodies used included: PE-Cy7-conjugated anti-H2-Db (clone: KH95, BD Biosciences), BV396-conjugated anti-β2m (clone: S19.8, BD Biosciences), BV605 or APC-conjugated anti-CD45 (clone: 30-F11, BioLegend), BV421-conjugated anti-TCR-β (clone: H57-597, BioLegend), PE-Cy7-conjugated anti-CD4 (clone: RM4-5, BioLegend), APC-conjugated anti-CD8a (clone: 53 − 6.7, BioLegend), PE-conjugated anti-CD11b (clone: M1/70, BioLegend), PE-Cy7-conjugated anti-F4/80 (clone: BM8, BioLegend), BV421-conjugated anti-CD206 (clone: HK1.4, BioLegend), PE-Cy7-conjugated anti-NK1.1 (clone: PK136, BioLegend). Live/dead discrimination was performed using propidium iodide (Thermo Scientific) at 1 µg/mL. RNA isolation, quantitative real-time RT-PCR (RT-qPCR), and RNA-seq Total RNA was isolated using the Zymo RNA extraction kit. cDNA was synthesized from 0.5 µg of total RNA using oligo(dT) primers and the Verso cDNA Synthesis Kit (Thermo Scientific), following the manufacturer’s instructions. Quantitative PCR (qPCR) was performed using either Premix Ex Taq (Probe qPCR, Takara) or SYBR Green chemistry. Primer and probe sequences used in this study are listed in Supplementary Table 1. Gene expression levels were normalized to Gapdh, and relative expression was calculated using the ∆CT method with 7500 System Software (Applied Biosystems). For RNA sequencing (RNA-Seq), libraries were prepared from 500 ng of total RNA. Sequencing libraries were generated and sequenced as paired-end 150 bp reads (2 × 150 bp; 20 million paired-end reads per sample) by Azenta Life Sciences, USA. RNA-seq data analyses RNA-seq data from CDS cell lines and mouse cell models were processed using the University of Minnesota CHURP pipeline. Reads were aligned to the GRCh38 (human) and GRCm39 (mouse) reference genomes using STAR (v2.7.10a), with gene annotations from Ensembl release 110. Gene-level quantification was performed using featureCounts with appropriate settings for strand specificity and read pairing. To assess transcriptomic similarity, Spearman correlation coefficients were computed using log-transformed, normalized gene expression values. Analyses included internally generated RNA-seq data from ichCD4 (Chimera) and two CDS cell lines (Kitra and X1), as well as publicly available datasets from biopsy samples of CDS (GSE60740), Ewing sarcoma (ES) (GSE270004), leiomyosarcoma (LMS) (GSE222045), osteosarcoma (OS) (GSE39058), and synovial sarcoma (SS) (GSE40021). Correlation matrices were visualized as heatmaps using the pheatmap package in R. Differential gene expression analysis was conducted separately for imChCDS cell lines and secondary tumors, comparing DOX ON versus DOX OFF conditions. Genes with a |log2 fold change| > 1 and an adjusted p-value < 0.05 were considered differentially expressed. DEGs were visualized using volcano plots generated with ggplot2. CDS-associated genes (157 mouse orthologs curated from Okimoto et al. ( 11 ) were highlighted to assess CIC::DUX4 gene activation. A custom senescence gene set (n = 137), compiled from the SenMayo ( 64 ) and SASP signatures ( 65 ) along with canonical markers, was also annotated in CT1 cells volcano plots to evaluate senescence-associated transcriptional responses. To investigate the cellular origin of CT1 cells, we curated a panel of lineage-specific marker genes from a single-cell RNA-seq dataset profiling teratoma development across germ layers and embryonic lineages ( 36 ). Mouse orthologs were identified for each human marker gene and mapped to imChCDS expression data. Gene expression matrices were derived from DOX ON (tumor-suppressed) and DOX OFF (tumor-induced) conditions. Heatmap analyses were performed to assess lineage-specific transcriptional signatures. Expression values were log-transformed as log2(expression + 1) to normalize signal range while retaining biological variability. Statistics Statistical analyses of the data were carried out using Graphpad Prism software, unless otherwise specified. The sample size was selected based on previous experience with the assays employed in order to ensure sufficient statistical power. The variance was comparable among the groups. Group differences were determined using one-way or two-way analysis of variance (ANOVA), accompanied by Tukey’s post-hoc testing. Differences were deemed significant at p-values of 0.05 or below. Declarations Acknowledgements This work was supported by grants to D.B from Department of Defense (HT9425-23-1-0456), Karen Wyckoff Rein in Sarcoma Foundation, and Children’s Cancer Research Fund. We are grateful to Usuk Jung, Erik Toso, Kenric Chen, Silvie Reitz, and Helen Atkins for their invaluable technical assistance in conducting experiments and preparing the manuscript. Funding declaration This work was in part supported by Department of Defense (HT9425-23-1-0456), Karen Wyckoff Rein in Sarcoma Foundation, and Children’s Cancer Research Fund to D.B.. The funding bodies had no role in the design of the study, data collection, analysis, interpretation, or manuscript preparation.. Ethics declarations Ethics approval and consent to participate: Mice were maintained under protocol 2209-40422A, approved by the University of Minnesota IACUC. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests. Data availability Sequencing reads and processed data have been deposited into GEO. The accession number will be available upon manuscript acceptance. Additional data are available upon request from the corresponding author. Contributions Investigation, all aspects of the project: AV, EW, AM, WB and DB. Analysis, all data: AV, MK and DB. Bioinformatics: EW. Conceptualization, design and supervision: DB. Funding acquisition: DB. Manuscript Preparation: AV and DB. All authors read and approved the final manuscript. Corresponding author Darko Bosnakovski; [email protected] Authors and Affiliations Dr. Ajay Vachanaram, Dr. Erdong Wei, MS. Ana Mitanoska, William Bassett, Professor Michael Kyba and Assistant professor Darko Bosnakovski Department of Pediatrics, Medical School, University of Minnesota, Minneapolis, USA Professor Michael Kyba and Assistant professor Darko Bosnakovski Lillehei Heart Institute, University of Minnesota, Minneapolis, USA References Comitani F, Nash JO, Cohen-Gogo S, Chang AI, Wen TT, Maheshwari A, Goyal B, Tio ES, Tabatabaei K, Mayoh C, Zhao R, Ho B, Brunga L, Lawrence JEG, Balogh P, Flanagan AM, Teichmann S, Huang A, Ramaswamy V, Hitzler J, Wasserman JD, Gladdy RA, Dickson BC, Tabori U, Cowley MJ, Behjati S, Malkin D, Villani A, Irwin MS, Shlien A. Diagnostic classification of childhood cancer using multiscale transcriptomics. Nat Med. 2023;29(3):656-66. Epub 2023/03/19. doi: 10.1038/s41591-023-02221-x. PubMed PMID: 36932241; PMCID: PMC10033451 transcriptional analysis to diagnose cancer and predict patient prognosis. The other authors declare no competing interests. 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Frontiers in Immunology. 2021;12. doi: ARTN 636568 10.3389/fimmu.2021.636568. PubMed PMID: WOS:000631574600001. Himeda CL, Jones PL. The Good, The Bad, and The Unexpected: Roles of DUX4 in Health and Disease. Dev Cell. 2019;50(5):525-6. Epub 2019/09/11. doi: 10.1016/j.devcel.2019.08.010. PubMed PMID: 31505171. Schreiber RD, Old LJ, Smyth MJ. Cancer Immunoediting: Integrating Immunity's Roles in Cancer Suppression and Promotion. Science. 2011;331(6024):1565-70. doi: 10.1126/science.1203486. PubMed PMID: WOS:000288754500045. Zitvogel L, Galluzzi L, Smyth MJ, Kroemer G. Mechanism of Action of Conventional and Targeted Anticancer Therapies: Reinstating Immunosurveillance. Immunity. 2013;39(1):74-88. doi: 10.1016/j.immuni.2013.06.014. PubMed PMID: WOS:000330948300007. Saul D, Kosinsky RL, Atkinson EJ, Doolittle ML, Zhang X, LeBrasseur NK, Pignolo RJ, Robbins PD, Niedernhofer LJ, Ikeno Y, Jurk D, Passos JF, Hickson LJ, Xue A, Monroe DG, Tchkonia T, Kirkland JL, Farr JN, Khosla S. A new gene set identifies senescent cells and predicts senescence-associated pathways across tissues. Nat Commun. 2022;13(1):4827. Epub 2022/08/17. doi: 10.1038/s41467-022-32552-1. PubMed PMID: 35974106; PMCID: PMC9381717 Patents on senolytic drugs and their uses and SASP biomarkers are held by Mayo Clinic and the University of Minnesota. This research has been reviewed by the Mayo Clinic Conflict of Interest Review Board and was conducted in compliance with Mayo Clinic Conflict of Interest policies. The remaining authors declare no competing interests. Coppe JP, Patil CK, Rodier F, Sun Y, Munoz DP, Goldstein J, Nelson PS, Desprez PY, Campisi J. Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol. 2008;6(12):2853-68. Epub 2008/12/05. doi: 10.1371/journal.pbio.0060301. PubMed PMID: 19053174; PMCID: PMC2592359. Additional Declarations No competing interests reported. Supplementary Files GA.pdf CT1SuppFig1.pdf CT1SuppFig2.pdf CT1SuppFig3.pdf CT1SuppFig4.pdf CT1SuppFig5.pdf CT1SuppFig6.pdf CT1SuppFig7.pdf CT1SuppFig8.pdf SuppTable1.docx Supplementaryfigurelegend.docx Cite Share Download PDF Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Molecular Cancer → Version 1 posted Editorial decision: Revision requested 01 Jun, 2025 Reviews received at journal 13 May, 2025 Reviews received at journal 08 May, 2025 Reviewers agreed at journal 04 May, 2025 Reviewers agreed at journal 02 May, 2025 Reviewers agreed at journal 02 May, 2025 Reviewers invited by journal 01 May, 2025 Editor assigned by journal 28 Apr, 2025 Submission checks completed at journal 28 Apr, 2025 First submitted to journal 23 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6516041","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":451823523,"identity":"7d9d4011-d91d-42a6-8f4b-2428b6ea8e0a","order_by":0,"name":"Ajay Vachanaram","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Ajay","middleName":"","lastName":"Vachanaram","suffix":""},{"id":451823524,"identity":"427888ef-6cc3-47f5-bb1e-e2608ee1cd1e","order_by":1,"name":"Erdong Wei","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Erdong","middleName":"","lastName":"Wei","suffix":""},{"id":451823525,"identity":"128ae6f8-25dd-4735-8d93-d60b0729c322","order_by":2,"name":"Ana Mitanoska","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"","lastName":"Mitanoska","suffix":""},{"id":451823526,"identity":"3f35e022-b10e-41ef-8854-ecdc543687b2","order_by":3,"name":"William Bassett","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"","lastName":"Bassett","suffix":""},{"id":451823527,"identity":"de2f4525-fb9e-45b0-a9eb-0d4cb1b9f6ee","order_by":4,"name":"Michael Kyba","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Kyba","suffix":""},{"id":451823528,"identity":"291d793d-c98e-4aba-a95a-ca2ac9b8f313","order_by":5,"name":"Darko Bosnakovski","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAoElEQVRIiWNgGAWjYDCCAwzMDAwVELYECVrOkKyFsY0ULXzHzz42+DjvcLQ5A/PB2zzEaJE8k26cOHPb4dydDWzJ1kRpMTiQxnyYF6hlwwEeM2nitJx/BtQyB6SF/xuRWm6kMSfzNoBtYSNOi+SNZ8yGM46l5244zGZsOYcYLXzn05glPtRY52443vzwxhtitCAAM2nKR8EoGAWjYBTgAwAV9DL9AOD6pAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Minnesota","correspondingAuthor":true,"prefix":"","firstName":"Darko","middleName":"","lastName":"Bosnakovski","suffix":""}],"badges":[],"createdAt":"2025-04-24 01:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6516041/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6516041/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12943-025-02485-6","type":"published","date":"2025-11-26T15:57:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82138973,"identity":"b149e89f-22b3-4cfc-ae31-cb5d15d1943b","added_by":"auto","created_at":"2025-05-07 06:24:56","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":869790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCIC::DUX4-driven CDS-like tumors arise in connective tissue of chimeric mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Representative immunofluorescence staining for CIC::DUX4 on iCD4mESCs, induced with 500ng/mL dox for 12 hours (Scale Bar 50 µm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e RT- qPCR analysis of CIC::DUX4 target genes in iCD4mESCs induced with 500ng/mL dox for 12 hours. Data are presented as mean ± SEM; ****p \u0026lt; 0.0001 by Student’s T-Test. Results are presented as expression relative to Gapdh (n = 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Picture of chimeric mice generated after iCD4mESCs were injected into blastocyst of female C57BL/6 mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Picture displaying gross morphology of multiple tumor formation in soft connective tissue under the skin (shown by arrows) in iChCD4 mice upon induction with 625mg/kg dox containing chow for 4 weeks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e Representative H\u0026amp;E staining images from tissue sections of tumors formed in iChCD4 mice in comparison with tumors formed by Kitra-SRS (Kitra) and NCC-X1-CDS (X1) CDS cell lines in NSG mice. (Scale Bar 50 µm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Representative immunofluorescence staining of CIC::DUX4, ETV4, WT1 and CD99 on tissue sections of tumors formed in iChCD4. (Scale Bar 20 µm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Spearmen correlation coefficients matrix visualized by heat map depicting correlation of 165 CDS target gene profile of tumors formed by iChCD4 mice compared to tumors formed by CDS cell lines Kitra and X1, biopsies obtained from CDS patients (CDS), Ewing’s Sarcoma (ES), Osteosarcoma (OS), Leiomyosarcoma (LMS) and Synovial Sarcoma (SS).\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6516041/v1/dc0c821c2a0288530d3ffe4b.jpg"},{"id":82138980,"identity":"4724dfb2-75ab-48d0-8612-7bc9358b60f9","added_by":"auto","created_at":"2025-05-07 06:24:56","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1207333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCIC::DUX4-inducible murine imChCDS cells closely resembles patient-derived CDS cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eRepresentative brightfield and immunofluorescence images showing CIC::DUX4, Ki67, and Spider-β-gal staining in imChCDS cells 72 hours after dox removal. Scale bar: 50 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eGrowth curve of imChCDS cells under CIC::DUX4\u003csup\u003eON\u003c/sup\u003e (500 ng/mL dox) and CIC::DUX4\u003csup\u003eOFF\u003c/sup\u003e conditions over a 12-day culture period. Data are presented as mean ± SEM; ****p \u0026lt; 0.0001 by two-way ANOVA (n = 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eBrightfield images showing imChCDS spheroid morphology 6 days after dox withdrawal. Scale bar: 50 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eQuantification of spheroid area in imChCDS cells after 6 days of dox withdrawal. Data are presented as mean ± SEM; ***p \u0026lt; 0.001 by t-test (n = 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e Western blot analysis of CIC::DUX4 and related markers (ETV4, WT1), along with acetylated histone marks (acH3K27, acH3K18), in imChCDS cells 72 hours after dox withdrawal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Volcano plot of differentially expressed CIC::DUX4 target genes (highlighted in red) (11) in imChCDS cells comparing CIC::DUX4\u003csup\u003eON\u003c/sup\u003e vs CIC::DUX4\u003csup\u003eOFF\u003c/sup\u003e conditions at 72 hours. Genes with log2 fold change \u0026gt; 1 and adjusted p-value \u0026lt; 0.05 are shown\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G) \u003c/strong\u003eVolcano plot of differentially expressed cellular senescence-associated genes (highlighted in red) from a curated list of 125 genes from the SenMayo dataset (64) and 54 SASP-related genes identified by Coppé et al. (65) in imChCDS cells after 72 hours of dox withdrawal. Genes with log2 fold change \u0026gt; 1 and adjusted p-value \u0026lt; 0.05 are shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H) \u003c/strong\u003eHeatmap showing the expression of gene signatures characteristic of cycling MSC/fibroblasts, MSC/fibroblasts, adipogenic MSC/fibroblasts, and fibroblasts in imChCDS cells cultured with dox (Dox\u003csup\u003eON\u003c/sup\u003e) and 3 days after dox withdrawal (Dox\u003csup\u003eOFF\u003c/sup\u003e), n = 2.\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6516041/v1/ba14fd4262e4145fe75795c1.jpg"},{"id":82143279,"identity":"1fc81035-ee85-47d3-97fc-5cac7d13ffa8","added_by":"auto","created_at":"2025-05-07 06:40:56","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":883351,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eimChCDS cell line develops CDS-like tumors in NSG recipient.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Graph showing tumor growth over a 22-day period. Data are presented as mean ± SEM; ***p \u0026lt; 0.001 by two-way ANOVA (n = 4). The Dox\u003csup\u003eOFF\u003c/sup\u003e (control) group never received dox. The remaining mice were treated with dox (625 mg/kg) for 16 days (All Dox group). On day 16, dox was withdrawn from half of these mice (Dox\u003csup\u003eON\u0026gt;OFF\u003c/sup\u003e group), while the other half continued treatment until the end of the experiment (Dox\u003csup\u003eON\u003c/sup\u003e group). Representative images show gross tumor morphology formed by imChCDS cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eTumor weights at day 22 in Dox\u003csup\u003eON\u003c/sup\u003e and Dox\u003csup\u003eON\u0026gt;OFF\u003c/sup\u003e groups. Data are presented as mean ± SEM; ****p \u0026lt; 0.0001 by t-test (n = 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eGross morphology of tumors formed by imChCDS cells at the experimental endpoint (day 22).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eRepresentative H\u0026amp;E-stained tumor sections from mice continuously treated with dox (Dox\u003csup\u003eON\u003c/sup\u003e) and those in which dox was withdrawn during the final 6 days (Dox\u003csup\u003eON\u0026gt;OFF\u003c/sup\u003e). Scale bar: 20 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e Immunofluorescence staining for CIC::DUX4, ETV4, WT1, and CD99 in tumors shown in panel (C). Scale bar: 20 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Volcano plot displaying differentially expressed CDS target genes (highlighted in red) in tumors formed by imChCDS cells, comparing Dox\u003csup\u003eON\u003c/sup\u003e and Dox\u003csup\u003eON\u0026gt;OFF\u003c/sup\u003e conditions 72 hours after dox withdrawal. Genes with Log2FC \u0026gt; 1 and adjusted p-value \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G) \u003c/strong\u003eKEGG pathway enrichment analysis on DEG from Dox\u003csup\u003eON\u003c/sup\u003e and Dox\u003csup\u003eON\u0026gt;OFF\u003c/sup\u003e conditions.\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6516041/v1/8668ac4be1152a1fbaa5d4fb.jpg"},{"id":82140854,"identity":"f7f7fddf-9992-4cf0-8d15-0528aaa04a08","added_by":"auto","created_at":"2025-05-07 06:32:57","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":840261,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eimChCDS cell viability is dependent on P300/CBP activity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eCell viability of imChCDS cells assessed by ATP assay after treatment with a range of iP300w concentrations for 24, 48, and 72 hours. Cells in the Dox\u003csup\u003eON\u003c/sup\u003e group were continuously cultured with dox (500 ng/mL), while dox was removed during treatment in the Dox\u003csup\u003eOFF\u003c/sup\u003e group. Data are presented as mean ± SEM; p \u0026lt; 0.01, *p \u0026lt; 0.0001 by one-way ANOVA (n = 3). Results are shown as percent viability relative to the Dox\u003csup\u003eON\u003c/sup\u003e group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Immunofluorescence staining for Phalloidin (F-actin), CIC::DUX4, and Ki67 in imChCDS cells under Dox\u003csup\u003eON\u003c/sup\u003e, Dox\u003csup\u003eOFF\u003c/sup\u003e, and Dox\u003csup\u003eON\u003c/sup\u003e+0.3 μM iP300w conditions following 48 hours of treatment or dox removal. Scale bar: 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e RT-qPCR analysis of CIC::DUX4 target gene expression in Dox\u003csup\u003eON\u003c/sup\u003e, Dox\u003csup\u003eOFF\u003c/sup\u003e, and Dox\u003csup\u003eON\u003c/sup\u003e+0.3 μM iP300w conditions at 24 hours of treatment. Data are presented as mean ± SEM; p \u0026lt; 0.05, p \u0026lt; 0.01, *p \u0026lt; 0.001 by one-way ANOVA. Expression levels are normalized to Gapdh (n = 3).\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6516041/v1/96c523fd059f5285ce8ad4ac.jpg"},{"id":82140851,"identity":"1b8645f6-d498-4297-b27c-8759067a0307","added_by":"auto","created_at":"2025-05-07 06:32:56","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":539259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCIC::DUX4 suppresses MHCI expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Representative FACS analysis of MHCI surface markers H2-Db and β2M expression in imChCDS cells. Cells in the CIC::DUX4\u003csup\u003eON\u003c/sup\u003e group were continuously cultured with dox (500 ng/mL), while dox was withdrawn for 48 hours in the CIC::DUX4\u003csup\u003eOFF\u003c/sup\u003e group. Where indicated, cells were treated with IFN-γ (200 ng/mL) for 24 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Quantification of H2-Db and β2M expression from (A). Results represent expression levels in the live cell population.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e RT-qPCR analysis of MHCI–related genes (H2-D1, H2-K1, and β2m) in CIC::DUX4\u003csup\u003eON\u003c/sup\u003e. Data are presented as mean ± SEM; ***p \u0026lt; 0.001, **p \u0026lt; 0.0001 by one-way ANOVA (n = 3). Expression levels are normalized to Gapdh.\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6516041/v1/91a8c9df799d497bee596397.jpg"},{"id":82138991,"identity":"a867e6da-3cd1-4497-834b-4020b1e686e0","added_by":"auto","created_at":"2025-05-07 06:24:57","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":966501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eimChCDS cells develop CDS-like “cold” tumors in immunocompetent mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eTumor formation by imChCDS cells in immunocompetent mice. Mice in the Dox\u003csup\u003eON\u003c/sup\u003e group were continuously maintained on dox-containing chow (625 mg/kg), while mice in the Dox\u003csup\u003eOFF\u003c/sup\u003e group had dox withdrawn for the last 3, 6, or 9 days of the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eBar graph showing differences in tumor volume between Dox\u003csup\u003eON\u003c/sup\u003e and Dox\u003csup\u003eOFF\u003c/sup\u003e groups at the experimental endpoint (day 31). Data are presented as mean ± SEM; *p \u0026lt; 0.05, ***p \u0026lt; 0.001, **p \u0026lt; 0.0001 by one-way ANOVA (n = 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eBar graph showing differences in tumor weight between Dox\u003csup\u003eON\u003c/sup\u003e and Dox\u003csup\u003eOFF\u003c/sup\u003e groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eRepresentative H\u0026amp;E staining showing tumor histology and immunofluorescence staining showing expression of CIC::DUX4 and infiltration of immune cells labeled with CD45, F4/80, and CD8 markers. Scale bar: 20 μm.\u003c/p\u003e","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6516041/v1/0148234c1adb2ab0c9e5b72d.jpg"},{"id":82138987,"identity":"2f25bcb0-36d3-4ba4-bc9c-fd9dc6a9401f","added_by":"auto","created_at":"2025-05-07 06:24:57","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1136600,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCIC::DUX4 inactivation restores MHCI expression and promotes immune cell infiltration in imChCDS tumors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e FACS analysis of MHCI surface markers H2-Db and β2M on GFP-labeled imChCDS cells isolated from tumors in mice maintained continuously on dox (Dox\u003csup\u003eON\u003c/sup\u003e) or withdrawn from dox for the last 3, 6, or 9 days of the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Tumor-infiltrating T cells (CD4+ and CD8+) following dox withdrawal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Tumor-infiltrating macrophages (CD11b+, F4/80+). CD206 was used to distinguish between M1 (anti-tumor) and M2 (pro-tumorigenic) macrophage populations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eFACS analysis shows NK cells defined as CD45+, NK1.1+ in the tumors. Data are presented as mean ± SEM; *p \u0026lt; 0.05, ***p \u0026lt; 0.001, **p \u0026lt; 0.0001 by one-way ANOVA (n = 5).\u003c/p\u003e","description":"","filename":"fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6516041/v1/8b76240ec38e4caa1edfa442.jpg"},{"id":97178386,"identity":"4e7ec49b-88c4-4c8e-81d6-bd3f50ab319d","added_by":"auto","created_at":"2025-12-01 16:09:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7570161,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6516041/v1/f749e570-59ac-429d-bd3c-e4b2c26add01.pdf"},{"id":82138983,"identity":"a21dfc6d-6dd3-485d-87ef-1261b8d137a0","added_by":"auto","created_at":"2025-05-07 06:24:57","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":255840,"visible":true,"origin":"","legend":"","description":"","filename":"GA.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6516041/v1/af3740ecbf26a5c26c677f7f.pdf"},{"id":82140853,"identity":"99574bf2-9b8e-4a2b-95b2-010918f37101","added_by":"auto","created_at":"2025-05-07 06:32:57","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":413701,"visible":true,"origin":"","legend":"","description":"","filename":"CT1SuppFig1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6516041/v1/85b1f7bf152597b0adebad73.pdf"},{"id":82143280,"identity":"020104f7-9679-447d-85e4-de875a8886e7","added_by":"auto","created_at":"2025-05-07 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06:24:57","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":16778,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigurelegend.docx","url":"https://assets-eu.researchsquare.com/files/rs-6516041/v1/a97ad876e1986c97a119e339.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Modeling CIC::DUX4 sarcoma reveals oncogene-mediated MHCI-dependent immune evasion","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCIC::DUX4 sarcoma (CDS) represents a particularly aggressive and rare subtype within the spectrum of small round blue cell tumors (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). CDS is genetically defined by chromosomal translocation involving 19q13.1 and either 4q35 or 10q26.3 fusion between \u003cem\u003eCIC\u003c/em\u003e and \u003cem\u003eDUX4\u003c/em\u003e genes (\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The CIC::DUX4 fusion protein retains the DNA-binding domain of CIC but acquires a potent transcriptional activation domain from DUX4 (\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). This alteration transforms CIC from a transcriptional repressor into an aberrant activator of key genes involved in cell cycle regulation and oncogenesis (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Clinically, CDS presents as a rapidly progressing disease with high metastatic potential and a strikingly poor prognosis, often exhibiting resistance to standard chemotherapy regimens adapted from Ewing sarcoma protocols (\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePatient-derived CDS cell lines have been crucial for characterizing CIC::DUX4-driven transcriptional programs (\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20 CR21\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), but they lack the genetic, epigenetic, and cellular complexity of primary tumors. Extended culture often results in genetic drift and clonal selection, reducing their preclinical relevance (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Advanced models such as patient-derived organoids (PDOs) and xenografts (PDXs) offer greater fidelity but are constrained by the rarity of CDS and limited availability of tumor material (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). \u003cem\u003eIn vivo\u003c/em\u003e, CDS is typically studied through xenotransplantation into immunocompromised mice (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), which, although informative for oncogenic pathway analysis, fails to recapitulate tumor initiation, native microenvironments, and functional immune systems, factors critical for investigating immune interactions and evaluating immunotherapies. Given these challenges, advancing CDS research requires complementary model systems that fully capture the disease\u0026rsquo;s complexity and better support translational applications (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecent studies have suggested that DUX4, through its C-terminal domain, promotes immune evasion in certain malignancies and myogenic cells by downregulating MHC class I (MHCI) antigen presentation and suppressing interferon-stimulated pathways (\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Given that CIC::DUX4 retains the same C-terminal domain, it is assumed that it may contribute to the development of an immunologically \u0026ldquo;cold\u0026rdquo; microenvironment in CDS, thereby impairing lymphocyte recruitment and activation (\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). However, the mechanisms underlying immune escape in CDS and their role in tumor progression within immunocompetent hosts remain poorly understood, largely due to the absence of suitable \u003cem\u003ein vivo\u003c/em\u003e models. This gap limits efforts to unravel immune mechanisms and hinders the development of targeted immunotherapies for CDS.\u003c/p\u003e \u003cp\u003ePrior attempts to generate transgenic mouse models with tissue-specific CIC::DUX4 expression using Lox/Cre recombination confirmed that CIC::DUX4 can drive malignant transformation (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). However, these models were compromised by unanticipated Cre recombination, uncontrolled CIC::DUX4 expression, and failure to achieve germline transmission of the transgene. Moreover, constitutive CIC::DUX4 expression in derived cell lines limited their experimental utility (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo overcome previous limitations, we developed a doxycycline (dox)-inducible chimeric mouse model that expresses CIC::DUX4 and forms soft tissue tumors closely resembling human CDS. From this model, we established a dox-tunable murine CDS cell line (imChCDS) that recapitulates the molecular profile of human CDS and is tumorigenic in both immunodeficient and immunocompetent hosts. Through syngeneic transplantation, we demonstrate that CIC::DUX4 suppresses MHCI presentation, thereby promoting immune evasion. This novel model provides a robust platform for investigating CDS tumor biology, tumor\u0026ndash;immune interactions, and therapeutic strategies, ultimately advancing the development of innovative treatments.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCIC::DUX4 is sufficient to drive malignant transformation in chimeric mice\u003c/h2\u003e \u003cp\u003eOur primary goal was to develop tissue-specific CIC::DUX4 transgenic mouse models to serve as reliable tools for studying CDS pathogenesis and evaluating therapeutic strategies. As an initial step, we sought to determine whether CIC::DUX4 expression could be tightly regulated \u003cem\u003ein vivo\u003c/em\u003e and if its expression alone could drive malignant transformation. To investigate this, we engineered dox-inducible CIC::DUX4 transgenic murine ES cells and used these to generate chimeric mice, where the inducible cells distribute across various cell types throughout the animal. This approach obviates the necessity for germline transmission and enables CIC::DUX4 expression within a permissive cellular and microenvironmental context, increasing the likelihood of initiating tumorigenesis.\u003c/p\u003e \u003cp\u003eWe engineered dox-inducible CIC::DUX4 mouse embryonic stem cells (iCD4 mESCs) by targeting the HPRT locus of ZX1 cells with the p2Lox-CIC::DUX4 construct, as previously described (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In addition to the Lox/Cre cassette at the HPRT locus, ZX1 cells are engineered to constitutively express the reverse tetracycline transactivator (rtTA) under the control of the Rosa26 promoter (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Neomycin-resistant clones were selected and expanded for further analysis. To assess CIC::DUX4 inducibility, clones were treated with 500 ng/mL dox for 24 hours. CIC::DUX4 expression was confirmed via immunofluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), and the activation of known CIC::DUX4 target genes \u003cem\u003eEtv1\u003c/em\u003e, \u003cem\u003eEtv4\u003c/em\u003e, and \u003cem\u003eEtv5\u003c/em\u003e was evaluated by RT-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Three clones demonstrating strong, non-leaky, and robust CIC::DUX4 induction were selected and microinjected into C57BL/6 blastocysts to generate chimeric mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCIC::DUX4 chimeric mice (iChCD4) at the age of 6 months (n\u0026thinsp;=\u0026thinsp;10) with varying degrees of chimerism were fed a chow containing 625mg/kg dox to induce CIC::DUX4 expression. After 4 weeks of continuous induction, 6 mice (60%) developed palpable, macroscopic tumors under the skin (soft connective tissue) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Two mice died of unknown causes, and the remaining 2 mice showed no observable changes. Histological analysis of the tumors revealed a striking resemblance to those formed via xenotransplantation of human CDS cell lines, such as Kitra-SRS (Kitra) and NCC-CDS-X1 (X1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The tumors in iChCD4 mice exhibited hallmark features of CIC::DUX4 sarcoma, including small to medium-sized neoplastic cells arranged in nodular patterns, separated by prominent collagenous septa (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). To further confirm the identity of the tumors, we performed immunofluorescence staining for CIC::DUX4 and established CDS-associated markers, including ETV4, WT1, and CD99 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). CIC::DUX4 expression was observed in the majority of tumor cells, while subsets exhibited nuclear and cytoplasmic localization of WT1, focal expression of CD99, diffuse staining of ETV4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) and minimum infiltration with immune cells (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eWe performed transcriptional profiling to assess the molecular similarity of tumors arising in chimeric mice to CDS and other common sarcomas. Spearman correlation analysis revealed that the gene expression profiles of the chimeric tumors closely resembled those of tumors derived from Kitra and X1 cell lines, as well as CDS patient tumor samples. In contrast, they were more distinct from other sarcoma subtypes, including Ewing sarcoma (ES), leiomyosarcoma (LMS), osteosarcoma (OS), and synovial sarcoma (SS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eTogether, our findings demonstrate that CIC::DUX4 expression alone is sufficient to initiate malignant transformation \u003cem\u003ein vivo\u003c/em\u003e, resulting in tumors that originate in subcutaneous connective tissues. These tumors recapitulate the key histological and molecular features of CDS, validating the chimeric mouse model as a relevant system for studying CIC::DUX4-driven sarcomagenesis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCIC::DUX4-driven murine cancer cell line imChCDS\u003c/h3\u003e\n\u003cp\u003eFrom the tumors that developed in chimeric animals, we aimed to establish a dox-inducible CIC::DUX4 murine cell line for use in syngeneic transplantation models, with the goal of studying CDS pathology within an immunocompetent environment. Tumors isolated from chimeric mice were enzymatically digested and subcutaneously transplanted into immunodeficient NSG mice, as previously described for the X1 cell line (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Mice were kept on dox chow (625 mg/kg) to maintain sustained CIC::DUX4 expression. Palpable tumors emerged by day 45 post-inoculation and reached the maximum allowed size (2 cm\u0026sup3;) within 8 days.\u003c/p\u003e \u003cp\u003eHistological analysis revealed that the secondary tumors were morphologically indistinguishable from the original tumors in the chimeric mice and closely resembled human CDS tumors (Supplementary Fig.\u0026nbsp;2). Portions of these tumors were enzymatically dissociated, and the resulting single-cell suspensions were cultured in dox-containing medium (500 ng/\u0026micro;L) to establish a CIC::DUX4-inducible murine cell line (imChCDS).\u003c/p\u003e \u003cp\u003eInitially, the cell culture appeared morphologically heterogeneous (Supplementary Fig.\u0026nbsp;3), but over several passages it progressively became more homogeneous, consisting of small polygonal-shaped cells by passage 9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Cell viability was entirely dependent on CIC::DUX4 expression, as removal of dox led to rapid cell cycle arrest, marked by the loss of Ki67 and the induction of cellular senescence, as indicated by β-galactosidase positivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and specific gene expression (Supplementary Fig.\u0026nbsp;4). The estimated doubling time of imChCDS cells under dox-supplemented conditions was 50.1 hours, while cells cultured without dox exhibited no proliferative capacity over a 12-day period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). imChCDS cells also demonstrated the ability to grow in 3D spheroid cultures when seeded in low-attachment plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Consistent with monolayer conditions, dox withdrawal in 3D cultures led to a loss of proliferative potential and progressive spheroid shrinkage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Western blot analysis was performed to determine whether CIC::DUX4 induces similar transcriptional and epigenetic changes in this murine system as observed in human CDS (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). CIC::DUX4 expression induced the known target genes \u003cem\u003eETV4\u003c/em\u003e and \u003cem\u003eWT1\u003c/em\u003e, and was associated with increased histone acetylation at H3K18 and H3K27 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTranscriptomic analysis further confirmed that imChCDS cells cultured with dox expressed 116 of 157 genes of a well-established set of CIC::DUX4 human target genes (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Three days after dox withdrawal, expression of CIC::DUX4 and its downstream targets was lost, accompanied by a morphological senescence-like phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), upregulation of 59 out of 137 senescence-associated markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, Supplementary Fig.\u0026nbsp;4). Finally, we investigated whether imChCDS cells retain the transcriptional footprint of their cell of origin by analyzing lineage-specific gene expression signatures. Among 23 distinct cell types representing all three embryonic germ layers, ectoderm, endoderm, and mesoderm (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), imChCDS cells selectively expressed markers associated with the mesoderm, particularly those related to mesenchymal and fibroadipogenic cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH and Supplementary Fig. XX). Notably, during the proliferative phase, the cells displayed a transcriptional profile characteristic of cycling progenitors. Following dox withdrawal, this profile shifted toward that of differentiated mesenchymal stem cells (MSCs) and different subtypes of fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eCollectively, these findings demonstrate the successful establishment of a murine CIC::DUX4 sarcoma cell line, imChCDS, which closely recapitulates key molecular and phenotypic features of CDS \u003cem\u003ein vitro\u003c/em\u003e and retains the transcriptional footprint of its mesenchymal cell of origin.\u003c/p\u003e\n\u003ch3\u003eimChCDS cells exhibit CIC::DUX4-dependent tumorigenic characteristics\u003c/h3\u003e\n\u003cp\u003eTo evaluate the tumorigenic potential of the imChCDS cell line \u003cem\u003ein vivo\u003c/em\u003e, we transplanted 5 \u0026times; 10⁵ cells subcutaneously into immunocompromised NSG mice (n\u0026thinsp;=\u0026thinsp;12). Eight mice were fed a dox-containing diet (625 mg/kg; \u0026ldquo;All Dox\u003csup\u003eON\u003c/sup\u003e\u0026rdquo;), while the remaining four were kept on a dox-free diet to evaluate whether tumor formation could occur independently of CIC::DUX4 induction. Mice in the Dox\u003csup\u003eON\u003c/sup\u003e group developed tumors within 14 days, whereas no tumors were observed in the Dox\u003csup\u003eOFF\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), confirming that CIC::DUX4 expression is required for tumor initiation. To assess whether continued CIC::DUX4 expression was necessary for tumor maintenance, dox was withdrawn from half of the mice, and tumor progression was monitored for an additional 8 days (Dox\u003csup\u003eON\u0026gt;OFF\u003c/sup\u003e). While tumors in the continued Dox\u003csup\u003eON\u003c/sup\u003e group exhibited progressive growth, those in the dox withdrawal group regressed significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;C). Histological analysis by H\u0026amp;E staining revealed that tumors from the Dox\u003csup\u003eON\u003c/sup\u003e group retained typical features of CDS, including dense sheets of small, round cells. In contrast, tumors from the Dox\u003csup\u003eON\u0026gt;OFF\u003c/sup\u003e group showed a marked loss of tumor cellularity and increased stromal matrix deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Immunofluorescence staining confirmed the presence of CIC::DUX4 protein and active proliferation (Ki67 positivity) in the Dox\u003csup\u003eON\u003c/sup\u003e tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Additionally, these tumors expressed characteristic CDS markers, including ETV4, WT1, and CD99 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Transcriptomic analysis further confirmed that CIC::DUX4 target genes were expressed exclusively in tumors from the Dox\u003csup\u003eON\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Pathway enrichment analysis of differentially expressed genes revealed that dox withdrawal predominantly affected pathways involved in cellular metabolism and cell cycle regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Together, these findings demonstrate that imChCDS cells possess CIC::DUX4-dependent tumorigenic potential in NSG mice and recapitulate key molecular and histopathological features of human CDS \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eimChCDS cell viability depends on the CIC::DUX4/P300/CBP transcriptional axis\u003c/h3\u003e\n\u003cp\u003eWe previously found that CIC::DUX4 exerts its oncogenic activity through interaction with the transcriptional co-activators P300/CBP (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Inhibition of P300/CBP disrupts CIC::DUX4 function, reduces cell viability and proliferation \u003cem\u003ein vitro\u003c/em\u003e, and suppress tumor growth in xenograft models of CIC::DUX4 sarcoma (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). To assess whether the imChCDS cell line similarly depends on this axis, we performed a cell viability assay using a range of concentrations of our developed P300/CBP inhibitor, iP300w (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). The effects of iP300w treatment were compared to a control condition in which CIC::DUX4 expression was turned off. We observed a significant, dose- and time-dependent reduction in cell viability following iP300w treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Notably, the most effective concentration was 0.3\u0026micro;M, consistent with the optimal dose previously identified for human CIC::DUX4 sarcoma cell lines (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). iP300w treatment induced morphological changes similar to those observed upon CIC::DUX4 inactivation (Dox\u003csup\u003eON\u0026gt;OFF\u003c/sup\u003e), with cells transitioning from a compact, polygonal shape to the flattened, enlarged morphology typical of senescent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This change was accompanied by a loss of proliferation, as evidenced by a marked reduction in Ki67 staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Consistent with these findings, treatment with iP300w also led to a significant downregulation of CIC::DUX4 target gene expression, confirming that P300/CBP inhibition suppresses CIC::DUX4-driven transcriptional activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Collectively, these results demonstrate that imChCDS cell viability and proliferation are dependent on the CIC::DUX4/P300/CBP transcriptional axis. Pharmacologic inhibition of P300/CBP not only confirms their role in mediating CIC::DUX4 oncoactivity but also highlights the potenital of the imChCDS model for future drug screening and therapeutic development targeting this pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCIC::DUX4 suppresses MHCI expression\u003c/h3\u003e\n\u003cp\u003ePrevious studies have suggested that CIC::DUX4, similar to DUX4, may contribute to immune evasion by suppressing MHCI antigen presentation, potentially through interference with interferon-gamma (IFN-γ)-induced immune response genes (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). To investigate whether CIC::DUX4 suppresses MHCI expression in the imChCDS model, as observed in CDS cell lines, we assessed the presentation of MHCI components by FACS 24 hours after dox withdrawal, with or without IFN-γ stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We found that CIC::DUX4 markedly suppressed the surface presentation of both H2-Db and β2M. Within 24 hours of dox withdrawal, H2-Db expression increased significantly even in the absence of IFN-γ, and IFN-γ stimulation further enhanced its expression, resulting in uniform receptor presentation across the cell population (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). A similar pattern was observed for β2M, which was absent in the presence of CIC::DUX4, even with IFN-γ stimulation, but was robustly induced in all cells upon CIC::DUX4 inactivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). These findings were further supported by RT-qPCR analysis, which revealed CIC::DUX4-dependent repression of MHCI-related genes, including H2-D1, H2-K1, and β2M (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Together, these results demonstrate that CIC::DUX4 suppresses MHCI expression at both the transcriptional and protein levels, likely contributing to immune evasion mechanisms in CIC::DUX4-driven sarcomas.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCIC::DUX4 mediates CDS immune evasion\u003c/h2\u003e \u003cp\u003eThe imChCDS cell line was derived from embryonic stem cells of 129P2 mice, enabling transplantation into 129-background mice for the study of CIC::DUX4-driven sarcomas in an immunocompetent, syngeneic setting. To assess tumorigenicity, we transplanted lentiviral GFP-labeled imChCDS cells (Supplementary Fig.\u0026nbsp;6) into 24 F1 hybrid mice (129P3/J \u0026times; C57BL/6) using the same method as in NSG mice. All mice received dox-supplemented chow beginning on the day of transplantation. Within 30 days, 21 out of 24 mice developed palpable tumors. To investigate the relationship between CIC::DUX4 activity, MHCI expression, and immune cell infiltration, we conducted a time-course experiment. Tumor-bearing mice were randomized into four groups with staggered dox withdrawal schedules. Group 1 had dox removed for the entire 9-day duration of the experiment. Group 2 underwent dox withdrawal starting on day 3, for a total of 6 days without dox. Group 3 had dox withdrawn only 3 days before the experimental endpoint. Group 4 remained on dox throughout and served as the control.\u003c/p\u003e \u003cp\u003eA progressive reduction in tumor volume was observed in the groups with longer periods of dox withdrawal, with significant decreases noted as early as 3 days post-withdrawal (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;C). Histological analysis revealed that tumors in the dox-treated control group retained the characteristic CIC::DUX4 sarcoma morphology, which gradually changed following CIC::DUX4 inactivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Immunohistochemical analysis confirmed the expression of diagnostic markers, CIC::DUX4, ETV4, WT1, and CD99, in tumors from the Dox\u003csup\u003eON\u003c/sup\u003e group (Supplementary Fig.\u0026nbsp;7). These markers were markedly reduced in tumors following 3 days of dox withdrawal and were undetectable after 9 days without dox (Supplementary Fig.\u0026nbsp;7). By day 3 post-dox withdrawal, tumors exhibited notable immune cell infiltration, as indicated by increased CD45-positive staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). This infiltration progressively intensified by day 9. A similar pattern was observed for F4/80-positive macrophages and CD8-positive T cells, indicating a coordinated immune response (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo quantitatively assess MHCI expression and the dynamics of immune cell infiltration, we performed FACS analysis. As early as 3 days after dox withdrawal, there was a significant reduction in GFP\u0026thinsp;+\u0026thinsp;imChCDS tumor cells, accompanied by a marked increase in CD45\u0026thinsp;+\u0026thinsp;hematopoietic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). With prolonged dox withdrawal, the percentage of GFP\u0026thinsp;+\u0026thinsp;tumor cells continued to decline, while CD45\u0026thinsp;+\u0026thinsp;immune cell infiltration further increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Remarkably, only 3 days of dox withdrawal were sufficient to restore MHCI expression in fraction of GFP\u0026thinsp;+\u0026thinsp;tumor cells. This restoration became more pronounced over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The upregulation of MHCI was accompanied by a rapid and robust infiltration of T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). While T cells in the Dox\u003csup\u003eON\u003c/sup\u003e group were predominantly CD4+, dox withdrawal induced a significant shift toward CD8\u0026thinsp;+\u0026thinsp;cytotoxic T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Macrophage infiltration also increased, with a phenotypic shift from tumor-promoting M2 macrophages in the Dox\u003csup\u003eON\u003c/sup\u003e group to pro-inflammatory, anti-tumor M1 macrophages post-CIC::DUX4 inactivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Finaly, NK cell infiltration peaked at day 3 post-dox withdrawal but declined as MHCI expression and CD8\u0026thinsp;+\u0026thinsp;T cell infiltration increased, falling below Dox\u003csup\u003eON\u003c/sup\u003e levels by day 9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). These findings demonstrate that CIC::DUX4 suppresses MHCI-mediated immune recognition in sarcomas. Its inactivation reduces tumor growth, restores MHCI expression, and triggers robust anti-tumor immune responses, characterized by enhanced CD8\u0026thinsp;+\u0026thinsp;T cell infiltration, M1 macrophage reprogramming, and transient NK cell activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eHere, we present a dox-inducible chimeric mouse model and a derived murine cell line (imChCDS) that faithfully recapitulate the molecular, histological, and immunological features of CIC::DUX4 sarcoma (CDS). These models address key limitations in existing systems by allowing investigation of CDS within an immunocompetent context and enabling controlled modulation of CIC::DUX4 expression. Together, they provide a robust platform to study sarcomagenesis, immune evasion, and potential therapeutic vulnerabilities. We demonstrate that CIC::DUX4 expression alone is sufficient to initiate malignant transformation and necessary to maintain it in permissive linages, producing tumors that closely resemble human CDS in histology, marker expression, and transcriptional profiles. Furthermore, we showed that CIC::DUX4 suppresses MHCI presentation to evade immune recognition, and that its inactivation restores MHCI expression, thereby triggering robust anti-tumor immune responses.\u003c/p\u003e \u003cp\u003eWe successfully generated an inducible chimeric mouse model of CDS, overcoming limitations of prior transgenic approaches, such as uncontrolled CIC::DUX4 expression and the inability to achieve germline transmission (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). We deliberately chose a chimeric strategy as an initial platform for modeling CDS as it offers a physiologically relevant and developmentally regulated microenvironment that supports tumorigenesis. This model provides a powerful means to investigate cancer initiation, progression, and tumor\u0026ndash;microenvironment interactions \u003cem\u003ein vivo\u003c/em\u003e (\u003cspan additionalcitationids=\"CR40 CR41 CR42\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Furthermore, it enables direct evaluation of CIC::DUX4\u0026rsquo;s oncogenic potential without the need of additional genetic alterations or a defined cell of origin. The functionality of the dox inducible transgene not only validates the approach but also provides a strong rationale for future development of tissue-specific transgenic mouse models. This strategy also requires minimal breeding compared to conventional germline transgenic techniques, making it an efficient and scalable alternative for early-stage modeling.\u003c/p\u003e \u003cp\u003eWe demonstrated that CIC::DUX4 alone is sufficient to drive sarcomagenesis, which is consistent with previous literature highlighting the low mutational burden of CDS (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Other studies have also shown that CIC::DUX4 can independently induce malignant transformation in mouse embryonic mesenchymal stem cells and osteochondrogenic progenitors, as well as promote tumor formation in both zebrafish and mouse models (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Notably, although chimerism was present systemically, CDS tumors developed exclusively in subcutaneous soft tissues. This finding highlights the importance of the epigenetic and cellular context, suggesting that mesenchymal-derived cells are uniquely susceptible to CIC::DUX4-driven transformation (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Furthermore, we demonstrated that CIC::DUX4 not only promotes cell cycle progression and transformation but also enables immune evasion, thereby supporting tumor persistence in the presence of an intact immune system (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTumors that developed in chimeric mice were histologically and molecularly indistinguishable from CDS tumors seen in patients (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), underscoring the relevance of this mouse model for studying CDS biology. From these tumors, we established the imChCDS cancer cell line, which closely resembles the molecular and phenotypic characteristics of CDS. It is noteworthy that while many cell lines engineered to overexpress oncogenes exhibit robust proliferative capacity \u003cem\u003ein vitro\u003c/em\u003e, they often fail to retain tumorigenic potential \u003cem\u003ein vivo\u003c/em\u003e. Even when tumor formation does occur, xenografted cells are prone to acquiring additional disease-nonspecific mutations due to clonal selection pressures (\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Therefore, it is crucial that transplanted tumors not only replicate the phenotypic features of the original disease but also preserve a transcriptomic landscape that closely resembles the primary tumor (\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). A significant advantage of the imChCDS cell line is its ability to form tumors when transplanted into immunocompetent mice, providing a robust platform for investigating tumor\u0026ndash;immune interactions in a physiologically relevant context. Additionally, both the chimeric model and the imChCDS cells provide tunable, temporally controlled expression of CIC::DUX4, enabling precise modulation of oncogene levels that closely mimics those observed in patients. Notably, imChCDS cells not only recapitulate the core oncogenic pathways characteristic of CDS but also retain molecular signatures of their mesenchymal cell of origin, aligning closely with established human CDS cell lines. Finally, we demonstrated that the tumorigenic potential of imChCDS cells is dependent on the CIC::DUX4/P300/CBP transcriptional axis, confirming P300/CBP as a pharmacological vulnerability in CDS (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The dose-dependent response to P300/CBP inhibitors (iP300w, optimal at 0.3 \u0026micro;M) is consistent with prior studies, suggesting conserved therapeutic vulnerabilities across species and reinforcing the translational potential of our model (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Together, these findings establish imChCDS as a highly relevant model system for studying CDS pathogenesis and as an invaluable platform for preclinical drug testing, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e (\u003cspan additionalcitationids=\"CR53 CR54 CR55\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). This cell line enhances the existing repertoire of models used to investigate CDS (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA key finding of this study is that CIC::DUX4 suppresses MHCI expression, thereby contributing to immune evasion in CDS. Tumors often evade adaptive immune responses by downregulating MHCI, which reduces the efficacy of both endogenous and therapeutic anti-tumor T cell responses (\u003cspan additionalcitationids=\"CR58 CR59\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). Both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e analyses revealed that CIC::DUX4 represses the expression of MHCI components, including H2-Db and β2M. There have been recent reports implicating the C-terminus of DUX4 in immune evasion through the downregulation of IFN-γ\u0026ndash;mediated MHCI expression during early embryonic development and in various cancers (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). Suppression of MHCI likely contributes to the immunologically \"cold\" tumor microenvironment seen in CDS. Our immunocompetent transplantation experiments provide further compelling evidence that CIC::DUX4 inactivation can reverse this immune suppression. Dox withdrawal led to rapid tumor regression, upregulation of MHCI expression, and a robust immune response, marked by increased infiltration of CD8⁺ T cells, natural killer (NK) cells, and a shift in macrophage phenotype from tumor-promoting M2 to anti-tumor M1. Interestingly, NK cell infiltration peaked transiently at day 3 post-withdrawal and then declined as MHCI expression and CD8⁺ T cell activity increased. This suggests a dynamic interplay between innate and adaptive immunity following CIC::DUX4 inactivation. These findings align with prior studies demonstrating that restoring MHCI expression enhances tumor immunogenicity and facilitates effective cytotoxic T cell\u0026ndash;mediated clearance (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). Thus, CIC::DUX4 inactivation, such as through inhibition of the P300/CBP, may offer a potent dual therapeutic strategy for CDS by both inducing cancer cell arrest and restoring MHCI expression, thereby enhancing immune recognition and accelerating tumor clearance. Furthermore, the P300/CBP inhibition strategy would likely sensitize CDS to checkpoint inhibitor therapy. Our studies provide a rationale for testing this combined approach in human CDS.\u003c/p\u003e \u003cp\u003eIn conclusion, our dox-inducible chimeric mouse model and imChCDS cell line provide powerful tools for studying CDS pathogenesis, immune evasion, and therapeutic responses. By revealing CIC::DUX4\u0026rsquo;s role in suppressing MHCI expression and mediating immune evasion, we uncover a critical mechanism driving CDS aggressiveness. These findings lay the groundwork for targeted therapies, such as P300/CBP inhibitors or immunotherapies designed to restore MHCI expression, and highlight the importance of immunocompetent models in advancing translational research for rare cancers like CDS.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eMouse embryonic stem (ES) cells were cultured on irradiated mouse embryonic fibroblasts (MEFs) in DMEM supplemented with 15% fetal bovine serum (FBS), penicillin/streptomycin (P/S; Gibco), 2 mM GlutaMAX (Invitrogen), nonessential amino acids, 0.1 mM β-mercaptoethanol, and 100 U/mL leukemia inhibitory factor (LIF; PeproTech). Doxycycline (dox) inducible mouse CIC::DUX4 sarcoma (imChCDS) cells were cultured in DMEM (HyClone) supplemented with 10% FBS (Peak Serum, Ps-FB3, lot 293Q16), GlutaMAX, and P/S. Induction of the CIC::DUX4 transgene under the control of the TRE promoter was achieved using 500 ng/\u0026micro;L dox (Sigma Aldrich, CAS No. 564-25-0) dissolved in PBS. All cells were maintained at 37\u0026deg;C in a humidified incubator with 5% CO₂. Doubling time was assessed by passaging and counting cells every second day. For 3D culture, 2.5 \u0026times; 10⁴ imChCDS cells were seeded into ultra-low attachment 96-well plates (96-well Clear Flat Bottom Ultra-Low Attachment Microplate; Corning, Inc., NY, USA) using DMEM supplemented with 10% FBS. Spheroid size was measured from 10\u0026times; images using ImageJ software. GFP-expressing lentivirus was produced in 293T cells by co-transfecting the lentiviral pSAM-GFP vector along with packaging (psPAX2) and envelope (pMD2.G) plasmids. Viral supernatants were collected at 48 and 72 hours post-transfection, filtered, and used to transduce target cells. Two days after transduction, GFP-positive cells were sorted by FACS and expanded for downstream applications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell viability (ATP) assay\u003c/h2\u003e \u003cp\u003eCell viability was determined using the CellTiter-Glo\u0026reg; Luminescent Cell Viability Assay (Promega) according to the manufacturer's protocol. Cells were seeded in 96-well plates at a density of 1 \u0026times; 10⁵ cells per well one day prior to treatment. Luminescence was measured using the POLARstar Optima Microplate Reader (BMG Labtech).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis, Immunostaining and Western blotting\u003c/h2\u003e \u003cp\u003eTumor samples were embedded in OCT compound (Scigen) and flash frozen in liquid nitrogen. Cryosections (10\u0026ndash;12 \u0026micro;m) were mounted on glass slides and stained with hematoxylin and eosin (H\u0026amp;E) using standard protocols. For immunostaining, cells and tissue sections were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.3% Triton X-100 for 30 minutes, and blocked with 3% BSA for 1 hour at room temperature. Primary antibodies diluted in 3% BSA were incubated overnight at 4\u0026deg;C. Fluorophore-conjugated secondary antibodies were applied for 1 hour at room temperature. Nuclei were counterstained with DAPI (1:5000; Sigma). For β-galactosidase (β-Gal) immunofluorescence, fixed and permeabilized cells were incubated for 1 hour at 37\u0026deg;C with 50 \u0026micro;L of spider-β-Gal working solution (Dojindo, SG02-01). For western blotting, cells were lysed in RIPA buffer supplemented with a protease inhibitor cocktail (Complete, Roche). Protein samples were separated via 7.5\u0026ndash;10% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked in 5% skim milk in TBST and incubated with primary antibodies overnight at 4\u0026deg;C or for 1 hour at room temperature. After washing, HRP-conjugated secondary antibodies were applied for 1 hour at room temperature. Protein signals were detected using Pierce ECL Western Blotting Substrate (Thermo Scientific). The antibodies utilized in this study included GAPDH-HRP (1:5000, Proteintech 60004), rabbit anti-DUX4 [E5.5] (1:500, Abcam ab124699), mouse anti ETV4 (1:250, Santa Cruz D2720), rabbit anti-WT1 (1:500 Cell Signaling 83535 ) rabbit anti-Histone H3K18Ac (1:500, Abcam ab1191), rabbit anti-Histone H3K27Ac (1:500, Abcam ab1791, lot: GR3297878-1), rabbit anti-Ki-67 (1:250, Cell Signaling 9129T), goat anti-rabbit/anti-mouse secondary Alexa Fluor 488/555 and 647 (1:1000, Invitrogen), HRP-conjugated anti-rabbit (1:5000, Jackson Immuno Research 111\u0026ndash;035\u0026thinsp;\u0026minus;\u0026thinsp;003, lot: 149,393), and HRP-conjugated anti-rabbit/anti-mouse (1:2500, Novus NBP1-75,130, lot 58\u0026ndash;173-090418).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo experiments and tumor formation assays\u003c/h2\u003e \u003cp\u003e All animal experiments were performed at the University of Minnesota Research Animal Resources facility under an Institutional Animal Care and Use Committee (IACUC)-approved protocol (#2209A-40422). NSG, C57BL/6J, and 129P3/J mice were obtained from Jackson Laboratory. Inducible CIC::DUX4 chimeric mice were generated at the University of Minnesota Mouse Genomics Laboratory. CIC::DUX4 expression was induced in chimeric, or imChCDS transplanted immunocompetent (C57BL/6J\u0026amp;SV129), and immunocompromised (NSG) mice via dox-containing chow (Envigo) at a concentration of 625 mg/kg. For transplantation, 5 \u0026times; 10⁵ imChCDS cells were suspended in a 1:1 mixture with Matrigel and injected subcutaneously into the flank of NSG mice. Tumor dimensions were measured every 2 days, and tumor volume was calculated using the formula: volume\u0026thinsp;=\u0026thinsp;0.5 \u0026times; length \u0026times; width\u0026sup2;. Mice were identified using number tags. Investigators were blinded to experimental conditions during tumor collection, weighing, and imaging.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTumor Dissociation\u003c/h2\u003e \u003cp\u003eTumors were dissected, rinsed in cold PBS, then mechanically dissociated using sterile blades. Tissue fragments were digested in DMEM high glucose medium supplemented with 1 mg/mL collagenase type II (Gibco) and 1% penicillin/streptomycin (Invitrogen) for 45 minutes at 37\u0026deg;C on a shaker. The resulting cell suspension was washed twice by centrifugation at 1500 RPM using Ham\u0026rsquo;s F-10 medium. A second enzymatic digestion was performed using a solution containing 0.1 mg/mL collagenase type II and 0.5 mg/mL Dispase (Gibco) in Ham\u0026rsquo;s F-10 medium, incubated for 20 minutes at 37\u0026deg;C with shaking. The digested mixture was then homogenized using 18G needles, filtered through a 40 \u0026micro;m cell strainer, and washed twice more by centrifugation at 1500 RPM for 5 minutes. The final cell suspension was either cultured or processed for fluorescence-activated cell sorting (FACS) analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFACS analyses\u003c/h2\u003e \u003cp\u003eTumor digests were treated with ACK lysis buffer (Gibco, A1049201) for 10 minutes at room temperature. Cells were then washed twice with PBS by centrifugation at 1500 RPM for 5 minutes. Following this, cells were incubated with anti-mouse CD16/32 antibody (0.1 \u0026micro;g/100 \u0026micro;L; TruStain FcX, clone S17011E) for 15 minutes on ice, followed by two additional washes. Staining with fluorescent-conjugated antibodies was performed at manufacturer-recommended concentrations in PBS supplemented with 1% FBS at 4\u0026deg;C for 60 minutes in the dark. Stained cells were then washed twice using washing buffer containing 10% FBS in PBS by centrifugation at 1500 RPM for 5 minutes. Finally, cells were resuspended in PBS containing 10% FBS and propidium iodide and analyzed by flow cytometry using a BD FACS Symphony S6. Quantification of imChCDS cells was based on GFP expression. H2-Db and β2m expression was assessed in the GFP\u0026thinsp;+\u0026thinsp;population gated from live cells. Immune cell subsets were quantified from the live cell population as follows: T cells (CD45+, TCR-β+, and CD4\u0026thinsp;+\u0026thinsp;or CD8+), NK cells (CD45\u0026thinsp;+\u0026thinsp;and NK1.1+), Macrophages (CD45+, CD11b+, and F4/80+), M1-like macrophages (CD45+, CD11b+, F4/80+, and CD206+). Data were analyzed using FlowJo (BD Biosciences). The antibodies used included: PE-Cy7-conjugated anti-H2-Db (clone: KH95, BD Biosciences), BV396-conjugated anti-β2m (clone: S19.8, BD Biosciences), BV605 or APC-conjugated anti-CD45 (clone: 30-F11, BioLegend), BV421-conjugated anti-TCR-β (clone: H57-597, BioLegend), PE-Cy7-conjugated anti-CD4 (clone: RM4-5, BioLegend), APC-conjugated anti-CD8a (clone: 53\u0026thinsp;\u0026minus;\u0026thinsp;6.7, BioLegend), PE-conjugated anti-CD11b (clone: M1/70, BioLegend), PE-Cy7-conjugated anti-F4/80 (clone: BM8, BioLegend), BV421-conjugated anti-CD206 (clone: HK1.4, BioLegend), PE-Cy7-conjugated anti-NK1.1 (clone: PK136, BioLegend). Live/dead discrimination was performed using propidium iodide (Thermo Scientific) at 1 \u0026micro;g/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation, quantitative real-time RT-PCR (RT-qPCR), and RNA-seq\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated using the Zymo RNA extraction kit. cDNA was synthesized from 0.5 \u0026micro;g of total RNA using oligo(dT) primers and the Verso cDNA Synthesis Kit (Thermo Scientific), following the manufacturer\u0026rsquo;s instructions. Quantitative PCR (qPCR) was performed using either Premix Ex Taq (Probe qPCR, Takara) or SYBR Green chemistry. Primer and probe sequences used in this study are listed in Supplementary Table\u0026nbsp;1. Gene expression levels were normalized to Gapdh, and relative expression was calculated using the ∆CT method with 7500 System Software (Applied Biosystems). For RNA sequencing (RNA-Seq), libraries were prepared from 500 ng of total RNA. Sequencing libraries were generated and sequenced as paired-end 150 bp reads (2 \u0026times; 150 bp; 20\u0026nbsp;million paired-end reads per sample) by Azenta Life Sciences, USA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq data analyses\u003c/h2\u003e \u003cp\u003eRNA-seq data from CDS cell lines and mouse cell models were processed using the University of Minnesota CHURP pipeline. Reads were aligned to the GRCh38 (human) and GRCm39 (mouse) reference genomes using STAR (v2.7.10a), with gene annotations from Ensembl release 110. Gene-level quantification was performed using featureCounts with appropriate settings for strand specificity and read pairing.\u003c/p\u003e \u003cp\u003eTo assess transcriptomic similarity, Spearman correlation coefficients were computed using log-transformed, normalized gene expression values. Analyses included internally generated RNA-seq data from ichCD4 (Chimera) and two CDS cell lines (Kitra and X1), as well as publicly available datasets from biopsy samples of CDS (GSE60740), Ewing sarcoma (ES) (GSE270004), leiomyosarcoma (LMS) (GSE222045), osteosarcoma (OS) (GSE39058), and synovial sarcoma (SS) (GSE40021). Correlation matrices were visualized as heatmaps using the pheatmap package in R.\u003c/p\u003e \u003cp\u003eDifferential gene expression analysis was conducted separately for imChCDS cell lines and secondary tumors, comparing DOX\u003csup\u003eON\u003c/sup\u003e versus DOX\u003csup\u003eOFF\u003c/sup\u003e conditions. Genes with a |log2 fold change| \u0026gt; 1 and an adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered differentially expressed. DEGs were visualized using volcano plots generated with ggplot2. CDS-associated genes (157 mouse orthologs curated from Okimoto et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) were highlighted to assess CIC::DUX4 gene activation. A custom senescence gene set (n\u0026thinsp;=\u0026thinsp;137), compiled from the SenMayo (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e) and SASP signatures (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e) along with canonical markers, was also annotated in CT1 cells volcano plots to evaluate senescence-associated transcriptional responses.\u003c/p\u003e \u003cp\u003eTo investigate the cellular origin of CT1 cells, we curated a panel of lineage-specific marker genes from a single-cell RNA-seq dataset profiling teratoma development across germ layers and embryonic lineages (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Mouse orthologs were identified for each human marker gene and mapped to imChCDS expression data. Gene expression matrices were derived from DOX\u003csup\u003eON\u003c/sup\u003e (tumor-suppressed) and DOX\u003csup\u003eOFF\u003c/sup\u003e (tumor-induced) conditions. Heatmap analyses were performed to assess lineage-specific transcriptional signatures. Expression values were log-transformed as log2(expression\u0026thinsp;+\u0026thinsp;1) to normalize signal range while retaining biological variability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistical analyses of the data were carried out using Graphpad Prism software, unless otherwise specified. The sample size was selected based on previous experience with the assays employed in order to ensure sufficient statistical power. The variance was comparable among the groups. Group differences were determined using one-way or two-way analysis of variance (ANOVA), accompanied by Tukey\u0026rsquo;s post-hoc testing. Differences were deemed significant at p-values of 0.05 or below.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants to D.B from Department of Defense (HT9425-23-1-0456), Karen Wyckoff Rein in Sarcoma Foundation, and Children’s Cancer Research Fund. We are grateful to Usuk Jung, Erik Toso, Kenric Chen, Silvie Reitz, and Helen Atkins for their invaluable technical assistance in conducting experiments and preparing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was in part supported by Department of Defense (HT9425-23-1-0456), Karen Wyckoff Rein in Sarcoma Foundation, and Children’s Cancer Research Fund to D.B.. The funding bodies had no role in the design of the study, data collection, analysis, interpretation, or manuscript preparation..\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/em\u003eMice were maintained under protocol 2209-40422A, approved by the University of Minnesota IACUC.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication:\u0026nbsp;\u003c/em\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests:\u0026nbsp;\u003c/em\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequencing reads and processed data have been deposited into GEO. The accession number will be available upon manuscript acceptance. Additional data are available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInvestigation, all aspects of the project: AV, EW, AM, WB and DB. Analysis, all data: AV, MK and DB. Bioinformatics: EW. Conceptualization, design and supervision: DB. Funding acquisition: DB. Manuscript Preparation: AV and DB. \u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDarko Bosnakovski; \u0026nbsp;[email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Ajay Vachanaram, Dr. Erdong Wei, MS. Ana Mitanoska, William Bassett, Professor Michael Kyba and Assistant professor Darko Bosnakovski\u003c/p\u003e\n\u003cp\u003eDepartment of Pediatrics, Medical School, University of Minnesota, Minneapolis, USA\u003c/p\u003e\n\u003cp\u003eProfessor Michael Kyba and Assistant professor Darko Bosnakovski\u003c/p\u003e\n\u003cp\u003eLillehei Heart Institute, University of Minnesota, Minneapolis, USA\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eComitani F, Nash JO, Cohen-Gogo S, Chang AI, Wen TT, Maheshwari A, Goyal B, Tio ES, Tabatabaei K, Mayoh C, Zhao R, Ho B, Brunga L, Lawrence JEG, Balogh P, Flanagan AM, Teichmann S, Huang A, Ramaswamy V, Hitzler J, Wasserman JD, Gladdy RA, Dickson BC, Tabori U, Cowley MJ, Behjati S, Malkin D, Villani A, Irwin MS, Shlien A. Diagnostic classification of childhood cancer using multiscale transcriptomics. Nat Med. 2023;29(3):656-66. Epub 2023/03/19. doi: 10.1038/s41591-023-02221-x. 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Immunity. 2013;39(1):74-88. doi: 10.1016/j.immuni.2013.06.014. PubMed PMID: WOS:000330948300007.\u003c/li\u003e\n\u003cli\u003eSaul D, Kosinsky RL, Atkinson EJ, Doolittle ML, Zhang X, LeBrasseur NK, Pignolo RJ, Robbins PD, Niedernhofer LJ, Ikeno Y, Jurk D, Passos JF, Hickson LJ, Xue A, Monroe DG, Tchkonia T, Kirkland JL, Farr JN, Khosla S. A new gene set identifies senescent cells and predicts senescence-associated pathways across tissues. Nat Commun. 2022;13(1):4827. Epub 2022/08/17. doi: 10.1038/s41467-022-32552-1. PubMed PMID: 35974106; PMCID: PMC9381717 Patents on senolytic drugs and their uses and SASP biomarkers are held by Mayo Clinic and the University of Minnesota. This research has been reviewed by the Mayo Clinic Conflict of Interest Review Board and was conducted in compliance with Mayo Clinic Conflict of Interest policies. The remaining authors declare no competing interests.\u003c/li\u003e\n\u003cli\u003eCoppe JP, Patil CK, Rodier F, Sun Y, Munoz DP, Goldstein J, Nelson PS, Desprez PY, Campisi J. Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol. 2008;6(12):2853-68. Epub 2008/12/05. doi: 10.1371/journal.pbio.0060301. PubMed PMID: 19053174; PMCID: PMC2592359.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"molc","sideBox":"Learn more about [Molecular Cancer](http://gsejournal.biomedcentral.com/)","snPcode":"12943","submissionUrl":"https://submission.nature.com/new-submission/12943/3","title":"Molecular Cancer","twitterHandle":"@SN_Oncology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"CIC:DUX4, sarcoma, immune evasion, experimental models, MHCI","lastPublishedDoi":"10.21203/rs.3.rs-6516041/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6516041/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCIC::DUX4 sarcoma (CDS) is a highly aggressive malignancy with limited therapeutic options. Here, we present a doxycycline-inducible CIC::DUX4 chimeric mouse model and a cancer line derived from it, imChCDS, that faithfully recapitulates the molecular, histological, and immunological features of human CDS. We demonstrate that CIC::DUX4 expression alone is sufficient to drive tumorigenesis in permissive lineages of soft connective tissues. The imChCDS cell line retains the transcriptional footprint of its mesenchymal cell of origin, develops tumors in immunocompetent hosts, and exhibits a clear dependency on the P300/CBP transcriptional co-activators. Notably, we identify CIC::DUX4-mediated suppression of MHC class I (MHCI) as a key mechanism of CDS immune evasion. Inactivation of CIC::DUX4 restores MHCI expression, triggers robust anti-tumor immune responses, and leads to tumor regression. Together, these models offer a versatile and physiologically relevant platform to investigate CDS pathogenesis, unravel immune evasion mechanisms, and evaluate emerging therapeutic strategies, including those targeting CIC::DUX4/P300/CBP oncogenic axis.\u003c/p\u003e","manuscriptTitle":"Modeling CIC::DUX4 sarcoma reveals oncogene-mediated MHCI-dependent immune evasion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 06:24:52","doi":"10.21203/rs.3.rs-6516041/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-01T14:59:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-13T10:37:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-08T09:31:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"162332719459030172125648589617766862170","date":"2025-05-04T09:10:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164083247868630668064429706766750150542","date":"2025-05-03T02:55:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"201149350541279288985954394637872720828","date":"2025-05-02T04:30:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-02T02:34:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-28T11:46:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-28T11:46:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Cancer","date":"2025-04-24T01:13:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"molc","sideBox":"Learn more about [Molecular Cancer](http://gsejournal.biomedcentral.com/)","snPcode":"12943","submissionUrl":"https://submission.nature.com/new-submission/12943/3","title":"Molecular Cancer","twitterHandle":"@SN_Oncology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2ecf21cf-723d-4def-ab18-d297c96fb0ed","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:01:34+00:00","versionOfRecord":{"articleIdentity":"rs-6516041","link":"https://doi.org/10.1186/s12943-025-02485-6","journal":{"identity":"molecular-cancer","isVorOnly":false,"title":"Molecular Cancer"},"publishedOn":"2025-11-26 15:57:42","publishedOnDateReadable":"November 26th, 2025"},"versionCreatedAt":"2025-05-07 06:24:52","video":"","vorDoi":"10.1186/s12943-025-02485-6","vorDoiUrl":"https://doi.org/10.1186/s12943-025-02485-6","workflowStages":[]},"version":"v1","identity":"rs-6516041","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6516041","identity":"rs-6516041","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}