Myxoid Liposarcoma Cell Research Articles

Exploiting WEE1 Kinase Activity as FUS::DDIT3-Dependent Therapeutic Vulnerability in Myxoid Liposarcoma.

The pathognomonic FUS::DDIT3 fusion protein drives myxoid liposarcoma (MLS) tumorigenesis via aberrant transcriptional activation of oncogenic signaling. As FUS::DDIT3 has so far not been pharmacologically tractable to selectively target MLS cells, this study investigated the functional role of the cell cycle regulator WEE1 as novel FUS::DDIT3-dependent therapeutic vulnerability in MLS. Immunohistochemical evaluation of the cell cycle regulator WEE1 was performed in a large cohort of MLS specimens. FUS::DDIT3 dependency and biological function of the G1/S cell cycle checkpoint were analyzed in a mesenchymal stem cell model and liposarcoma cell lines in vitro. WEE1 activity was modulated by RNAi-mediated knockdown and the small molecule inhibitor MK-1775 (adavosertib). An established MLS cell line-based chicken chorioallantoic membrane model was employed for in vivo confirmation. We demonstrate that enhanced WEE1 pathway activity represents a hallmark of FUS::DDIT3-expressing cell lines as well as MLS tissue specimens and that WEE1 is required for MLS cellular survival in vitro and in vivo. Pharmacologic inhibition of WEE1 activity results in DNA damage accumulation and cell cycle progression forcing cells to undergo apoptotic cell death. In addition, our results uncover FUS::DDIT3-dependent WEE1 expression as an oncogenic survival mechanism to tolerate high proliferation and resulting replication stress in MLS. Fusion protein-driven G1/S cell cycle checkpoint deregulation via overactive Cyclin E/CDK2 complexes thereby contributes to enhanced WEE1 inhibitor sensitivity in MLS. Our preclinical study identifies WEE1-mediated replication stress tolerance as molecular vulnerability in FUS::DDIT3-driven MLS tumorigenesis that could represent a novel target for therapeutic intervention.

S-p-bromobenzyl-glutathione cyclopentyl diester (BBGC) as novel therapeutic strategy to enhance trabectedin anti-tumor effect in soft tissue sarcoma preclinical models

Trabectedin, approved for the treatment of soft tissue sarcoma (STS), interferes with cell division and genetic transcription processes. Due to its strong anti-tumor activity in only certain histotypes, several studies on trabectedin combinations are currently ongoing to improve its efficacy. In this study, we aimed to investigate novel potential therapeutic strategies to enhance the anti-tumor effect of trabectedin using integrated in silico, in vitro, and in vivo approaches. For in silico analysis, we screened two public datasets, GSEA M5190 and TCGA SARC. Fibrosarcoma, leiomyosarcoma, dedifferentiated, and myxoid liposarcoma cell lines were used for in vitro studies. For in vivo experiments, fibrosarcoma orthotopic murine model was developed. In silico analysis identified Glo1 as the only druggable target upregulated after trabectedin treatment and correlated with poor prognosis. The specific Glo1 inhibitor, S-p-bromobenzylglutathione cyclopentyl diester (BBGC), increased trabectedin cytotoxicity in STS cells, and restored drug sensitivity in myxoid liposarcoma cells resistant to trabectedin. Moreover, the combined treatment with BBGC and trabectedin had a synergistic antitumor effect in vivo without any additional toxicity to mice. Based on these results, we believe that BBGC warrants further investigation to evaluate its potential clinical use in combination with trabectedin.

Abstract 5631: YAP1 is a critical factor for FUS::DDIT3/BAF/c-Jun complex assembly in myxoid liposarcoma

Abstract Introduction: FUS::DDIT3 is the dominant fusion protein in myxoid liposarcoma (MLS), resulting from a disease-defining chromosomal translocation. Acting as an aberrant transcription factor, FUS::DDIT3 disturbs gene regulatory networks interfering with adipogenic differentiation, while driving oncogenic gene expression and proliferation. Functionally, FUS::DDIT3 has been shown to cooperate with the transcriptional coregulator YAP1 and to interact with the BAF chromatin remodeling complex. However, mechanistic details on FUS::DDIT3’s function in transcriptional dysregulation leading to proliferative effects and the required interactome are incompletely understood. This study was performed to investigate transcriptional and epigenetic players recruited by FUS::DDIT3 to drive MLS proliferation and tumorigenesis. Experimental Procedures: To study protein-protein interactions between FUS::DDIT3 and its interacting partners, co-immunoprecipitation experiments of nuclear extracts and proximity ligation assays were performed using human MLS cell lines. To further investigate dependencies of nuclear complex assembly, combined RNA interference (RNAi) experiments were conducted. MLS cell viability and proliferation were assessed using MTT assays and xCELLigence real-time analysis, respectively. RT-qPCRs were performed to analyze the expression of FUS::DDIT3 target genes. The expression of FUS::DDIT3’s interacting proteins was evaluated by immunohistochemistry in primary human MLS specimens. Results: Co-immunoprecipitation and proximity ligation assays demonstrated nuclear interactions between FUS::DDIT3, YAP1, and the canonical BAF (cBAF) complex in MLS cell lines. In addition, a novel interaction of FUS::DDIT3 with the AP-1 transcription factor c-Jun was identified. Knockdown of the cBAF-specific subunits ARID1A and ARID1B resulted in reduced MLS cell viability and proliferation. Similarly, RNAi-mediated silencing of c-Jun impaired MLS cell viability and led to decreased expression of FUS::DDIT3 target genes. Strikingly, RNAi-mediated silencing of YAP1 resulted in decreased interaction of FUS::DDIT3 with cBAF components and c-Jun in MLS cells. Conclusions: Our results identify YAP1 as a key factor mediating the nuclear interaction of FUS::DDIT3 with the cBAF complex and the AP-1 transcription factor c-Jun, both shown to be essential for MLS cell proliferation. Citation Format: Ruth Berthold, Ilka Isfort, Lorena Heinst, Eva Wardelmann, Pierre Åman, Claudia Scholl, Marcel Trautmann, Stefan Fröhling, Wolfgang Hartmann. YAP1 is a critical factor for FUS::DDIT3/BAF/c-Jun complex assembly in myxoid liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5631.

Suppression of antitumor cytokine IL‑24 by PRG4 and PAI‑1 may promote myxoid liposarcoma cell survival.

Suppression of the antitumor cytokine interleukin-24 (IL-24) is critical for the survival of myxoid liposarcoma (MLS) cells. It has been previously demonstrated by the authors that an MLS-specific chimeric oncoprotein, translocated in liposarcoma-CCAAT/enhancer-binding protein homologous protein (TLS-CHOP), supresses IL24 mRNA expression via induction of proteoglycan 4 (PRG4) to sustain MLS cell proliferation. However, IL-24 has also been revealed to be suppressed by the ubiquitin-proteasome system in human ovarian and lung cancer cells. Therefore, the aim of the present study was to elucidate the mechanism of IL-24 suppression in MLS cells. The results revealed that the proteasome inhibitor, MG-132, induced cell death in MLS cells in vitro; this effect was reduced following IL-24 knockdown. This indicated that proteasomal degradation of IL-24 may be an important process for MLS cell survival. In addition, it was also previously revealed by the authors that knockdown of plasminogen activator inhibitor-1 (PAI-1), a TLS-CHOP downstream molecule, suppressed the growth of MLS cells, thus instigating the investigation of the effect of PAI-1 on IL-24 expression in MLS cells. Double knockdown of PAI-1 and IL-24 negated the growth-suppressive effect of PAI-1 single knockdown in MLS cells. Interestingly, PAI-1 single knockdown did not increase the mRNA expression of IL24, but it did increase the protein abundance of IL-24, indicating that PAI-1 suppressed IL-24 expression by promoting its proteasomal degradation. Moreover, treatment of MLS cells with a PAI-1 inhibitor, TM5275, induced IL-24 protein expression and apoptosis. Collectively, the results of the present as well as previous studies indicated that IL-24 expression may be suppressed at the transcriptional level by PRG4 and at the protein level by PAI-1 in MLS cells. Accordingly, PAI-1 may represent an effective therapeutic target for MLS treatment.

Lipoblastoma-Like Tumor and Fibrosarcoma-Like Lipomatous Neoplasm Represent the Same Entity: A Clinicopathologic and Molecular Genetic Study of 23 Cases Occurring in Both Men and Women at Diverse Locations

Lipoblastoma-like tumor (LLT) is a benign soft tissue tumor demonstrating mixed morphologic features of lipoblastoma, myxoid liposarcoma, and spindle cell lipoma but lacking genetic alterations associated with those tumors. LLT was originally thought to be specific to the vulva but has since been reported in the paratesticular region. The morphologic features of LLT overlap with those of “fibrosarcoma-like lipomatous neoplasm” (FLLN), a rare, indolent adipocytic neoplasm considered by some to form part of the spectrum of atypical spindle cell and pleomorphic lipomatous tumor. We compared the morphologic, immunohistochemical, and genetic features of 23 tumors previously classified as LLT (n = 17) and FLLN (n = 6). The 23 tumors occurred in 13 women and 10 men (mean age, 42 years; range, 17 to 80 years). Eighteen (78%) cases arose in the inguinogenital region, whereas 5 tumors (22%) involved noninguinogenital soft tissue, including the flank (n = 1), shoulder (n = 1), foot (n = 1), forearm (n = 1), and chest wall (n = 1). Microscopically, the tumors were lobulated and septated, with variably collagenized fibromyxoid stroma, prominent thin-walled vessels, scattered univacuolated or bivacuolated lipoblasts, and a minor component of mature adipose tissue. Using immunohistochemistry, 5 tumors (42%) showed complete RB1 loss, with partial loss in 7 cases (58%). RNA sequencing, chromosomal microarray, and DNA next-generation sequencing study results were negative for significant alterations. There were no clinical, morphologic, immunohistochemical, or molecular genetic differences between cases previously classified as LLT or FLLN. Clinical follow-up (11 patients [48%]; range, 2-276 months; mean, 48.2 months) showed all patients were alive without disease, and only one patient had experienced a single local recurrence. We conclude that LLT and FLLN represent the same entity, for which “LLT” seems most appropriate. LLT may occur in either sex and any superficial soft tissue location. Careful morphologic study and appropriate ancillary testing should allow for the distinction of LLT from its potential mimics.

Clinical analysis of 5-year survival and recurrence in giant retroperitoneal liposarcoma after surgery.

Clinical analysis of 5-year survival and recurrence in giant retroperitoneal liposarcoma after surgery.

Identification of Distinct and Common Subpopulations of Myxoid Liposarcoma and Ewing Sarcoma Cells Using Self-Organizing Maps

Myxoid liposarcoma and Ewing sarcoma are the two most common tumor types that are characterized by the FET (FUS, EWSR1 and TAF15) fusion oncogenes. These FET fusion oncogenes are considered to have the same pathological mechanism. However, the cellular similarities between cells from the different tumor entities remain unknown. Here, we profiled individual myxoid liposarcoma and Ewing sarcoma cells to determine common gene expression signatures. Five cell lines were analyzed, targeting 76 different genes. We employed unsupervised clustering, focusing on self-organizing maps, to identify biologically relevant subpopulations of tumor cells. In addition, we outlined the basic concepts of self-organizing maps. Principal component analysis and a t-distributed stochastic neighbor embedding plot showed gradual differences among all cells. However, we identified five distinct and robust subpopulations using self-organizing maps. Most cells were similar to other cells within the same tumor entity, but four out of five groups contained both myxoid liposarcoma and Ewing sarcoma cells. The major difference between the groups was the overall transcriptional activity, which could be linked to cell cycle regulation. We conclude that self-organizing maps are useful tools to define biologically relevant subpopulations and that myxoid liposarcoma and Ewing sarcoma exhibit cells with similar gene expression signatures.

Lipoblastoma-like tumor of the vulva: A rare benign mesenchymal neoplasm mimicking myxoid liposarcoma

Abstract Introduction/Objective Lipoblastoma-like tumor of the vulva (LLTV) is a rare, benign mesenchymal neoplasm of adipocytic differentiation that typically presents as a vulvar mass in young women, clinically mimicking a Bartholin gland abnormality. The tumor is well-circumscribed and multilobulated, composed of mature adipocytes, uniloculated lipoblasts and bland spindle cells. Morphologic features often overlap with lipoblastoma, myxoid liposarcoma, and spindle cell lipoma; however, absent alterations of PLAG1 and HMGA2 distinguish it from lipoblastoma, and lack of DDIT3 gene rearrangement distinguishes it from myxoid liposarcoma. While Rb1 usually demonstrates a “mosaic” pattern of expression, no definitive evidence of a structural abnormality in 13q has been identified. Methods/Case Report A 24-year-old female presented with mild discomfort and persistent swelling of the right vulva for one year, which was initially thought to be a Bartholin gland cyst. Physical examination revealed marked asymmetry between the right and left vulva, and ultrasound examination revealed an ovoid, hypoechoic, and solid- appearing vulvar mass (4.7 x 2.5 x 1.1 cm). She subsequently underwent excision of the vulvar mass for diagnosis and treatment. Results (if a Case Study enter NA) The excision specimen showed a relatively well-circumscribed and multi-lobulated subcutaneous mass with myxoid stroma and thin fibrous septa. The tumor was composed of a mixture of bland spindle cells, uniloculated lipoblasts and mature adipocytes. No significant nuclear atypia, necrosis or mitotic activity was identified. Immunohistochemical stains showed that the tumor cells were negative for keratin cocktail, S100 protein, SMA, and Desmin. CD34 highlighted vessels within the lesion, and Rb1 showed a "mosaic" pattern of weak and negative expression. Conclusion Lipoblastoma-like tumor of the vulva (LLTV) is a rare, benign tumor that tends to occur in young women, often clinically mimicking a Bartholin gland cyst. Recognition of this rare entity with distinction from mimics including myxoid liposarcoma is essential to avoid overly aggressive treatment. Complete conservative excision is recommended.

Abstract PR004: Fusion protein-driven IGF-IR signals deregulate hippo pathway promoting oncogenic cooperation of YAP1 and FUS-DDIT3

Abstract Introduction: Myxoid liposarcoma (MLS) is molecularly characterized by a recurrent chromosomal translocation which generates a chimeric FUS-DDIT3 fusion gene. The FUS-DDIT3 oncoprotein, acting as a transcriptional dysregulator, has been shown to be essential in MLS pathogenesis, among others through deregulation of IGF-IR/PI3K/AKT signaling, but its exact mode of function remains incompletely understood. Recently, a particular reliance on the Hippo pathway effector and transcriptional co-regulator YAP1 was found in MLS; however, the molecular mechanism of FUS-DDIT3-dependent YAP1 activation and its contribution to MLS pathogenesis remain unclear. Experimental Procedures: The expression of IGF-IR and YAP1 was analyzed in a large cohort of MLS specimens by immunohistochemistry. In vitro analyses were performed employing a human mesenchymal stem cell system stably expressing FUS-DDIT3 and human MLS cell lines. RNA interference-based approaches, experiments with small-molecule kinase inhibitors, co-immunoprecipitation, proximity ligation assays, transcriptome sequencing and adipogenic differentiation assays were performed to determine the interplay of FUS-DDIT3, IGF-IR-dependent signals, and YAP1 in MLS cells. Results: Immunohistochemically, a significant subset of MLS samples showed concurrent expression of IGF-IR and nuclear YAP1. In vitro, FUS-DDIT3-driven IGF-IR signaling was found to promote stability and nuclear accumulation of YAP1 via deregulation of the Hippo pathway. Co-immunoprecipitation and proximity ligation assays revealed nuclear co-localization of FUS-DDIT3 and YAP1 in FUS-DDIT3-expressing mesenchymal stem cells and MLS cell lines. Transcriptome sequencing of MLS cells demonstrated that FUS-DDIT3 and YAP1 co-regulate specific oncogenic gene signatures related to proliferation, cell cycle progression, apoptosis, and adipogenesis. In differentiation assays, FUS-DDIT3 and YAP1 were found to cooperate in adipogenic differentiation arrest. Conclusions: Our study provides molecular insights into a complex FUS-DDIT3-driven network involving IGF-IR signals acting on Hippo/YAP1, and uncovers cooperative effects of YAP1 and FUS-DDIT3 in the pathogenesis of MLS. Citation Format: Ruth Berthold, Ilka Isfort, Cihan Erkut, Lorena Heinst, Inga Grünewald, Eva Wardelmann, Thomas Kindler, Pierre Åman, Thomas G.P. Grünewald, Florencia Cidre-Aranaz, Marcel Trautmann, Stefan Fröhling, Claudia Scholl, Wolfgang Hartmann. Fusion protein-driven IGF-IR signals deregulate hippo pathway promoting oncogenic cooperation of YAP1 and FUS-DDIT3 [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR004.

Establishment and characterization of two novel patient-derived myxoid liposarcoma cell lines.

Myxoid liposarcoma (MLPS) is a lipogenic sarcoma, characterized by myxoid appearance histology and the presence of the FUS-DDIT3 fusion gene. MLPS shows frequent recurrence and poor prognosis after standard treatments, such as surgery. Therefore, novel therapeutic approaches for MLPS are needed. Development of novel treatments requires patient-derived cell lines to study the drug responses and their molecular backgrounds. Presently, only three cell lines of MLPS have been reported, and no line is available from public cell banks. Thus, this study aimed to establish and characterize novel MLPS cell lines. Using surgically resected tumor tissue from two patients with MLPS, two novel lines NCC-MLPS2-C1 and NCC-MLPS3-C1 were established. The presence of FUS-DDIT3 fusion, slow growth, spheroid formation, and invasive capability in these cell lines was confirmed. Growth retardation was monitored for 213 anti-cancer agents using NCC-MLPS2-C1 and NCC-MLPS3-C1 cells, and the results were integrated with the response to treatments in an MLPS cell line, NCC-MLPS1-C1, which was previously established in our laboratory. We found that romidepsin suppressed cell proliferation at considerably low concentrations in all three examined cell lines. NCC-MLPS2-C1 and NCC-MLPS3-C1 cell lines developed here represent a useful tool for basic and preclinical studies of MLPS.

Fusion protein-driven IGF-IR/PI3K/AKT signals deregulate Hippo pathway promoting oncogenic cooperation of YAP1 and FUS-DDIT3 in myxoid liposarcoma

Myxoid liposarcoma (MLS) represents a common subtype of liposarcoma molecularly characterized by a recurrent chromosomal translocation that generates a chimeric FUS-DDIT3 fusion gene. The FUS-DDIT3 oncoprotein has been shown to be crucial in MLS pathogenesis. Acting as a transcriptional dysregulator, FUS-DDIT3 stimulates proliferation and interferes with adipogenic differentiation. As the fusion protein represents a therapeutically challenging target, a profound understanding of MLS biology is elementary to uncover FUS-DDIT3-dependent molecular vulnerabilities. Recently, a specific reliance on the Hippo pathway effector and transcriptional co-regulator YAP1 was detected in MLS; however, details on the molecular mechanism of FUS-DDIT3-dependent YAP1 activation, and YAP1´s precise mode of action remain unclear. In elaborate in vitro studies, employing RNA interference-based approaches, small-molecule inhibitors, and stimulation experiments with IGF-II, we show that FUS-DDIT3-driven IGF-IR/PI3K/AKT signaling promotes stability and nuclear accumulation of YAP1 via deregulation of the Hippo pathway. Co-immunoprecipitation and proximity ligation assays revealed nuclear co-localization of FUS-DDIT3 and YAP1/TEAD in FUS-DDIT3-expressing mesenchymal stem cells and MLS cell lines. Transcriptome sequencing of MLS cells demonstrated that FUS-DDIT3 and YAP1 co-regulate oncogenic gene signatures related to proliferation, cell cycle progression, apoptosis, and adipogenesis. In adipogenic differentiation assays, we show that YAP1 critically contributes to FUS-DDIT3-mediated adipogenic differentiation arrest. Taken together, our study provides mechanistic insights into a complex FUS-DDIT3-driven network involving IGF-IR/PI3K/AKT signals acting on Hippo/YAP1, and uncovers substantial cooperative effects of YAP1 and FUS-DDIT3 in the pathogenesis of MLS.

When is a lipoma not a lipoma? Case report presenting a lipoblastoma-like tumor of the gluteal cleft in an older gentleman with literature review.

Introduction and importanceLipoblastoma-like tumors are rare tumors that can be confused with lipoblastomas and liposarcomas but have distinct characteristics. This tumor has previously been identified in the vulva of females, and recently in isolated cases of young males. Given its rarity, we present an instance of this tumor in an older man, demonstrating that this pathology is not limited to a specific age or sex, and surgeons and pathologists must be aware of it in their differential.Case presentationA 58-year-old male presented for evaluation of an enlarging mass in his right gluteal cleft. Prior to referral for surgical evaluation, the patient underwent an ultrasound-guided biopsy of the mass. Histologically, the tumor was a low-grade cellular spindle cell neoplasm in a fibrous to myxoid stroma. Immunohistochemical and molecular workup ruled out several malignant mesenchymal neoplasms, including myxoid liposarcoma, dedifferentiated liposarcoma, melanoma, low-grade fibromyxoid sarcoma, and sarcomatoid carcinoma. The patient initially declined surgery, but the mass continued to grow, and excision was chosen given the uncertain pathology. The tumor was resected with negative margins and histologically characterized as a “lipoblastoma-like lesion”, with features of a myxoid liposarcoma and spindle cell lipoma. Seven months post-resection, there were no signs of recurrence or metastasis.Clinical discussionDespite radiologic and pathologic similarities to malignant lipomatous tumors, lipoblastoma-like tumors are benign and have a good prognosis.ConclusionsClinicians should be aware of this entity despite its rarity as resection with negative margins is curative and may be needed to rule out more aggressive tumors.

Abstract P087: Targeting LSD1 protein scaffolding function in FET-rearranged sarcomas with SP-2577

Abstract The RNA-binding proteins, FUS, EWSR1, and TAF15 (FET protein family) are frequently fused to transcription factors, and the resulting oncogenic fusion proteins characterize various sarcomas. These fusion proteins act as aberrant transcription factors and promote oncogenic gene expression profiles. FET family rearranged sarcomas include Ewing Sarcoma, desmoplastic round cell tumors, myxoid liposarcoma, and clear cell sarcoma, amongst others. A growing body of work suggests that, independently of Lysine Specific Demethylase 1 (LSD1) demethylase activity, LSD1’s association with FET family oncogenic transcription factors is critical for FET-rearranged sarcoma development and progression. Extensive ongoing work aims to characterize the functional relationship of LSD1 and EWS/FLI, as well as the anti-tumor effect of disrupting that interaction. SP-2577 is an oral, first-in-class, small molecule with reversible, noncompetitive inhibition of LSD1 demethylase activity (IC50: 25–50 nM) and, importantly, also disrupts LSD1 protein-protein interactions. In vitro and in vivo data demonstrate SP-2577, or analogs, reverses EWS/ETS-mediated transcriptional regulation at both up- and downregulation target genes, leading to significant tumor growth inhibition in Ewing Sarcoma mouse xenograft studies. In this study, we set out to expand our understanding of SP-2577’s efficacy in diverse FET-rearranged malignancies including desmoplastic small round cell tumor (DSCRT: EWSR1/WT1), myxoid liposarcoma (ML: FUS/DDIT3), and clear cell sarcoma (CCS: EWS/ATF1). To examine whether these additional FET-rearranged sarcomas are sensitive to LSD1 scaffolding inhibition, we measured viability of Ewing sarcoma cells A673, TC32, TTC-446, and SK-N-MC, clear cell sarcoma cell lines SU-CCS-1, DTC-1, myxoid liposarcoma cell lines DL-221, 402-91, and 1765-92, and DSCRT patient derived xenograft organoids from SJDSRCT046151, X0069, SJDSRCT046155 after 96 hours of treatment with the clinical compound SP-2577, a first generation compound SP-2509, an inactive control, SP2513, and, a TCP-scaffold based, irreversible inhibitor of LSD1, OG-L002. While SP-2513 and OG-L002 had no activity against these cells SP-2509 and SP-2577 demonstrated potent activity across cell lines in vitro. Preliminary in vivo assessment of SP-2577 efficacy in DSCRT PDX SJDSRCT046151 resulted in a significant delay in time to event (p<0.02). Together, these data demonstrate that the unique scaffolding inhibition function of SP-2577 is essential for reducing cell viability through LSD1 targeting. Currently, we are elucidating changes to the transcriptome and proteome following SP-2577 treatment, in vitro. These data, in combination with results from the ongoing dose expansion phase 1 trial of SP-2577 in select sarcoma patients, may offer further mechanistic insight into the dependency of FET-rearranged sarcomas on LSD1 scaffolding functions and potentially lead to identifying biomarkers of sensitivity and response. Citation Format: Galen Rask, Emily R. Theisen, Aundrietta D. Duncan, Daniela Y. Santiesteban. Targeting LSD1 protein scaffolding function in FET-rearranged sarcomas with SP-2577 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P087.

Role of FUS-CHOP in Myxoid Liposarcoma via miR-486/CDK4 Axis.

This study aimed to explore the roles and relationship between FUsed in Sarcoma (FUS)-C/EBP HOmologous Protein (CHOP), microRNA (miR)-486 and cyclin dependent kinase 4 (CDK4) in myxoid liposarcoma, and determined whether FUS-CHOP can regulate proliferation and apoptosis of myxoid liposarcoma cells by regulating miR-486/CDK4 axis. The levels of miR-486, CDK4 and FUS-CHOP in myxoid liposarcoma samples/adjacent normal muscle tissues and myxoid liposarcoma/human adipose-derived stem cell line were evaluated using reverse transcription-quantitative polymerase chain reaction and western blotting. Cell proliferation and apoptosis were performed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide and flow cytometry, respectively. Furthermore, the apoptosis-related proteins were determined using Western blot assay. We found that miR-486 was down-regulated, FUS-CHOP and CDK4 were up-regulated in myxoid liposarcoma tissues and myxoid liposarcoma cell lines. Moreover, FUS-CHOP-siRNA distinctly suppressed FUS-CHOP level and increased miR-486 levels in 1955/91 cells. Our results demonstrated that knockdown of FUS-CHOP by siRNA inhibited 1955/91 growth, promoted cell apoptosis and enhanced cleaved Caspase3 protein expression. However, all these data were reversed by miR-486 inhibitor. Similarly, compared to mimic control, miR-486 mimic markedly reduced 1955/91 cells growth, induced cell apoptosis and fortified cleaved Caspase3 level, while these results were abolished by CDK4-plasmid. Collectively, our observations clearly suggested that FUS-CHOP regulated myxoid liposarcoma cell proliferation and apoptosis by the regulation of miR-486/CDK4 axis, indicating the potential use of FUS-CHOP-siRNA as a promising therapy for myxoid liposarcoma.

Abstract 2003: Oncogenic interplay of FUS-DDIT3 and YAP1 in myxoid liposarcoma

Abstract Introduction: Myxoid liposarcoma (MLS) represents the second most common subtype of liposarcoma. MLS is characterized by a chromosomal translocation t(12;16)(q13;p11) encoding a chimeric FUS-DDIT3 fusion gene. The resulting FUS-DDIT3 oncoprotein acts as a transcriptional dysregulator that was recently shown to mediate (i) IGF-IR/PI3K/AKT signaling and (ii) aberrant activation of the Hippo pathway effector YAP1 in MLS. This study was performed to analyze the functional interplay between IGF-IR/PI3K/AKT signals and aberrant YAP1 activity in MLS, aiming at a better functional understanding of MLS and the identification of specific molecular vulnerabilities. Experimental procedures: Immunohistochemical stainings of IGF-IR, IGF-II and YAP1 were performed in a cohort of MLS specimens (n=45). To study FUS-DDIT3-dependency in vitro, SCP-1 mesenchymal stem cells stably expressing FUS-DDIT3, and MLS cell lines expressing a doxycycline-inducible shRNA against FUS-DDIT3 were employed. Interactions between the IGF-IR/PI3K/AKT and Hippo/YAP1 pathways were investigated using RNAi approaches as well as the small molecule compounds BMS-754807 and BKM120; effects were analyzed by immunoblotting and TEAD luciferase reporter assays. To determine the impact of YAP1 in FUS-DDIT3-mediated oncogenic effects, qPCR analysis and adipogenic differentiation assays were performed. Results: Immunohistochemical analysis of human MLS tissue specimens demonstrated that expression of IGF-II and IGF-IR is associated with concomitant nuclear expression of YAP1 in a significant subset of MLS. Both, IGF-IR-dependent signals and YAP1 expression were shown to be functionally dependent on FUS-DDIT3. In MLS cell lines, inhibition of the IGF-IR/PI3K/AKT signaling cascade promoted downregulation of YAP1, accompanied by reduced TEAD transcriptional activity. Employing qPCR analyses, YAP1 was shown to co-regulate FUS-DDIT3 transcriptional targets and to be functionally involved in FUS-DDIT3-driven disruption of normal adipocytic differentiation. Conclusions: Our study provides evidence of a complex regulatory interplay in MLS with FUS-DDIT3-driven IGF-IR/PI3K/AKT signals acting as activators of nuclear YAP1 expression. Conversely, YAP1 contributes to shape FUS-DDIT3 effects on the MLS transcriptional landscape and functionally adds to an immature non-lipogenic phenotype. Our data contribute to the understanding of MLS biology and reveal specific molecular liabilities to be considered in therapeutic approaches of MLS. Citation Format: Ruth Berthold, Ilka Isfort, Jonas Breuer, Lorena Heinst, Thomas Kindler, Pierre Åman, Inga Grünewald, Eva Wardelmann, Thomas G. Grünewald, Florencia Cidre-Aranaz, Claudia Scholl, Stefan Fröhling, Marcel Trautmann, Wolfgang Hartmann. Oncogenic interplay of FUS-DDIT3 and YAP1 in myxoid liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2003.

Crosstalk between Macrophages and Myxoid Liposarcoma Cells Increases Spreading and Invasiveness of Tumor Cells.

Simple SummaryMyxoid liposarcoma (MLPS) is the second most common subtype of liposarcoma, occurs predominantly in the extremities, and tends either to recur locally and metastasize to unusual soft tissue locations. To date, the mechanisms of invasion and metastasis of MLPS remain unclear, and yet, there is a high need to identify new prognostic biomarkers to enable the development of novel targeted therapeutic strategies. This study firstly aimed to assess the role of immune cellular components that infiltrate MLPS tissues. Our data show that high grade, heavily vascularized MLPS tissues exhibit T lymphocyte-poor and M2-like macrophage-rich phenotypes, while low grade MLPS tissues are mainly infiltrated by T lymphocytes. In line with these findings, evidence is shown that a crosstalk occurring between MLPS cells and macrophages exists as MLPS cells drive an M2-like phenotype in monocytes which in turn, increase the invasive capability of MLPS cells.Myxoid liposarcoma (MLPS) is the second most common subtype of liposarcoma and has tendency to metastasize to soft tissues. To date, the mechanisms of invasion and metastasis of MLPS remain unclear, and new therapeutic strategies that improve patients’ outcomes are expected. In this study, we analyzed by immunohistochemistry the immune cellular components and microvessel density in tumor tissues from patients affected by MLPS. In order to evaluate the effects of primary human MLPS cells on macrophage polarization and, in turn, the ability of macrophages to influence invasiveness of MLPS cells, non-contact and 3D organotypic co-cultures were set up. High grade MLPS tissues were found heavily vascularized, exhibited a CD3, CD4, and CD8 positive T lymphocyte-poor phenotype and were massively infiltrated by CD163 positive M2-like macrophages. Conversely, low grade MLPS tissues were infiltrated by a discrete amount of CD3, CD4, and CD8 positive T lymphocytes and a scarce amount of CD163 positive macrophages. Kaplan–Meier analysis revealed a shorter Progression Free Survival in MLPS patients whose tumor tissues were highly vascularized and heavily infiltrated by CD163 positive macrophages, indicating a clear-cut link between M2-like macrophage abundance and poor prognosis in patients. Moreover, we documented that, in co-culture, soluble factors produced by primary human MLPS cells induce macrophage polarization toward an M2-like phenotype which, in turn, increases MLPS cell capability to spread into extracellular matrix and to cross endothelial monolayers. The identification of M2-like polarization factors secreted by MLPS cells may allow to develop novel targeted therapies counteracting MLPS progression.

Cellular Radiosensitivity of Soft Tissue Sarcoma.

Currently, all soft tissue sarcomas (STS) are irradiated by the same regimen, disregarding possible subtype-specific radiosensitivities. To gain further insight, cellular radiosensitivity was investigated in a panel of sarcoma cell lines. Fourteen sarcoma cell lines, derived from synovial sarcoma, leiomyosarcoma, fibrosarcoma and liposarcoma origin, were submitted to clonogenic survival assays. Cells were irradiated with single doses from 1-8 Gy and surviving fraction (SF) was calculated from the resulting response data. Alpha/beta (α/β) ratios were inferred from radiation-response curves using the linear-quadratic (LQ)-model. Cellular radiosensitivities varied largely in this panel, indicating a considerable degree of heterogeneity. Surviving fraction after 2 Gy (SF2) ranged from 0.27 to 0.76 with evidence of a particular radiosensitive phenotype in only few cell lines. D37% on the mean data was 3.4 Gy and the median SF2 was 0.52. The median α/β was 4.9 Gy and in six cell lines the α/β was below 4 Gy. A fairly homogeneous radiation response was observed in myxoid liposarcoma cell lines with SF2 between 0.64 and 0.67. Further comparing sarcomas of different origin, synovial sarcomas, as a group, showed the lowest SF2 values (mean 0.35) and was significantly more radiosensitive than myxoid liposarcomas and leiomyosarcomas (P = 0.0084 and 0.024, respectively). This study demonstrates a broad spectrum of radiosensitivities across STS cell lines and reveals subtype-specific radiation responses. The particular cellular radiosensitivity of synovial sarcoma cells supports consideration of the different sarcoma entities in clinical studies that aim to optimize sarcoma radiotherapy.

Establishment and characterization of NCC-MLPS1-C1: a novel patient-derived cell line of myxoid liposarcoma.

Myxoid liposarcoma is a rare mesenchymal malignancy, which is characterized by a FUS-DDIT3 fusion known as chromosomal translocation t(12;16)(q13;p11) and arises in the fat tissue. Although surgery with radiation has been established as a standard treatment, myxoid liposarcoma shows frequent recurrence and poor prognosis, thus requiring new therapeutic approaches. Patient-derived cell lines represent a critical tool for basic and preclinical research. However, only two such myxoid liposarcoma cell lines have been reported, and they are not available in cell banks. The aim of this study was to establish and characterize a novel myxoid liposarcoma cell line. Using surgically resected tumor tissue from a 47-year-old male patient, we established the NCC-MLPS1-C1 cell line. NCC-MLPS1-C1 cells were characterized by FUS-DDIT3 fusion, slow growth, spheroid formation, and invasive capability. We screened the effect of anti-cancer agents on the proliferation of NCC-MLPS1-C1 cells. The cells displayed a remarkable response to multitarget kinase inhibitors of RET, PDGFR-β, VEGFR, or FGFR. NCC-MLPS1-C1 cells and the tumor tissue shared common profiles of kinase activity including identified drug targets, such as RET and PDGFR-β. We believe that NCC-MLPS1-C1 cells will represent a useful tool for basic and preclinical studies of myxoid liposarcoma.

Suppression of MKL1 promotes adipocytic differentiation and reduces the proliferation of myxoid liposarcoma cells.

Myxoid liposarcoma (MLS) is thought to occur due to defective adipocytic differentiation in mesenchymal stem cells. A promising strategy for MLS treatment is the prevention of sarcomagenesis by promoting the terminal differentiation of MLS cells into adipocytes. Previous studies have reported that the suppression of megakaryoblastic leukemia 1 (MKL1) expression induces adipocytic differentiation in preadipocyte cell lines. The present study aimed to investigate the effects of MKL1 suppression on MLS cells. In the present study, MKL1 knockdown was demonstrated to promote the adipocytic differentiation of an MLS-derived cell line, designated 1955/91, under adipogenic conditions. This suggests that therapeutic targeting of the MKL1-associated molecular pathway has potential as a promising method of MLS treatment. However, the induction of adipogenesis by MKL knockdown was incomplete, and Oil Red O staining indicated that intracellular lipid droplets were only sporadically generated. Conversely, MKL1 knockdown reduced the growth of the MLS cells. As adipocytic differentiation in vitro requires cellular confluence, the decreased growth rate of the MLS cells following MKL1 knockdown could be attributed to the incomplete induction of adipogenesis. Translocated in liposarcoma-CCAAT/enhancer-binding protein homologous protein (TLS-CHOP) is an MLS-specific oncoprotein that is thought to play key roles in sarcomagenesis and the suppression of adipocytic differentiation. However, the results of western blotting analyses suggest that TLS-CHOP has limited effects on MKL1 expression in MLS cells and that MKL1 knockdown hardly affects TLS-CHOP expression. Thus, it is postulated that the inhibitory effect of TLS-CHOP on adipogenesis is not associated with MKL1 expression. However, MKL1 and the molecular pathway involving MKL1 appear to be attractive targets for the differentiation therapy of MLS.

Role of glyoxalase-1 in trabectedin-resistant myxoid liposarcoma.

e23566 Background: Glyoxalase-1 (Glo1) is involved in the detoxification of the endogenous reactive metabolite, methylglyoxal (MG), whose abnormal accumulation increases adduct levels and induce cell apoptosis. Previous studies demonstrated that increased Glo-1 expression was associated with cancer chemotherapy resistance. We performed bioinformatics analyses finding that Glo-1 mRNA over-expression was correlated with worse prognosis in patients affected by Soft Tissue Sarcoma (STS). On the bases of these evidences, we investigated the potential role of Glo1 expression as biomarker of tumor growth and drug resistance in STS. Methods: Trabectedin and MG cytoxicity was evaluated by MTT viability assay measured at spectrofluorometer. Apoptosis was analyzed by flow cytometry using Annexin V antibody and propidium iodide. Glo-1 expression analysis was performed by Western Blot using a mouse monoclonal anti-human Glo-1 antibody (NBP1-19015). Synergy analysis was calculated using Combenefit software and statistical analysis was performed by Student-t test using the program GraphPad-Prism. Results: As STS trabectedin resistant model, we used a myxoid-liposarcoma cell line (402-91 ET cells) that are not responsive to clinical doses of trabectedin contrary to the parental sensitive cell line (402-91 WT cells). Intriguingly, we found higher Glo-1 protein levels in 402-91 ET cells compared to 402-91 WT cells. The treatment of 402-91 ET cells with the specific Glo1 inhibitor S-p-bromobenzylglutathione cyclopentyl diester (BBGC), in combination with trabectedin (PharmaMar), significantly inhibited cell viability and increased apoptosis than trabectedin alone. In particular, the addition of BBGC reduced trabectedin EC50 (half-maximal effective concentration) from 20.4nM to 5.92nM in 402-91 ET cells, similar to that observed in 402-91 WT cells (4.92nM). To investigate if cell death was induced by MG accumulation following Glo1 inhibition, we evaluated 402-91 ET cell viability after treatment with different doses of MG in combination with trabectedin. The addition of MG restored sensitivity to trabectedin in 402-91 ET cells as well as BBGC. Conclusions: Our results highlight a new potential mechanism of trabectedin resistance mediated by Glo-1 over-expression. The use of the specific Glo-1 inhibitor, BBGC, restores trabectedin sensitivity in resistant cells leading to MG accumulation that, in turn, promotes cell death and apoptosis. These data provide a strong rationale to investigate Glo-1 inhibition strategy, in combination with trabectedin, in STS in vivo models.