Leiomyosarcoma Cell Line Research Articles

Immune evasion in lung metastasis of leiomyosarcoma: upregulation of EPCAM inhibits CD8+ T cell infiltration

BackgroundLeiomyosarcomas are among the most common histological types of soft tissue sarcoma (STS), with no effective treatment available for advanced patients. Lung metastasis, the most common site of distant metastasis, is the primary prognostic factor. We analysed the immune environment targeting lung metastasis of STS to explore new targets for immunotherapy.MethodsWe analysed the immune environment of primary and lung metastases in 38 patients with STS using immunohistochemistry. Next, we performed gene expression analyses on primary and lung metastatic tissues from six patients with leiomyosarcoma. Using human leiomyosarcoma cell lines, the effects of the identified genes on immune cells were assessed in vitro.ResultsImmunohistochemistry showed a significant decrease in CD8+ cells in the lung metastases of leiomyosarcoma. Among the genes upregulated in lung metastases, epithelial cellular adhesion molecule (EPCAM) showed the strongest negative correlation with the number of CD8+ cells. Transwell assay results showed that the migration of CD8+ T cells was significantly increased in the conditioned media obtained after inhibition or knock down of EPCAM.ConclusionsEPCAM was upregulated in lung metastases of leiomyosarcoma, suggesting inhibition of CD8+ T cell migration. Our findings suggest that EPCAM could serve as a potential novel therapeutic target for leiomyosarcoma.

Hyper-Dependence on NHEJ Enables Synergy between DNA-PK Inhibitors and Low-Dose Doxorubicin in Leiomyosarcoma.

Leiomyosarcoma (LMS) is an aggressive sarcoma for which standard chemotherapies achieve response rates under 30%. There are no effective targeted therapies against LMS. Most LMS are characterized by chromosomal instability (CIN), resulting in part from TP53 and RB1 co-inactivation and DNA damage repair defects. We sought to identify therapeutic targets that could exacerbate intrinsic CIN and DNA damage in LMS, inducing lethal genotoxicity. We performed clinical targeted sequencing in 287 LMS and genome-wide loss-of-function screens in 3 patient-derived LMS cell lines, to identify LMS-specific dependencies. We validated candidate targets by biochemical and cell-response assays in vitro and in seven mouse models. Clinical targeted sequencing revealed a high burden of somatic copy-number alterations (median fraction of the genome altered =0.62) and demonstrated homologous recombination deficiency signatures in 35% of LMS. Genome-wide short hairpin RNA screens demonstrated PRKDC (DNA-PKcs) and RPA2 essentiality, consistent with compensatory nonhomologous end joining (NHEJ) hyper-dependence. DNA-PK inhibitor combinations with unconventionally low-dose doxorubicin had synergistic activity in LMS in vitro models. Combination therapy with peposertib and low-dose doxorubicin (standard or liposomal formulations) inhibited growth of 5 of 7 LMS mouse models without toxicity. Combinations of DNA-PK inhibitors with unconventionally low, sensitizing, doxorubicin dosing showed synergistic effects in LMS in vitro and in vivo models, without discernable toxicity. These findings underscore the relevance of DNA damage repair alterations in LMS pathogenesis and identify dependence on NHEJ as a clinically actionable vulnerability in LMS.

FTIR Microspectroscopy as a new probe to study human uterine lesions: Characterization of tumor cell lines from uterine smooth muscle cells and evaluation of EPA and DHA in vitro treatments

During their life, women are likely to develop uterine diseases, which often compromise their fertile and perimenopausal age. Besides benign lesions like leiomyomas, several malignant neoplasms can occur, such as the uterine leiomyosarcoma, which represents the most frequent malignancy among the rarest uterine cancers. It presents several variants similar to both benign and malignant neoplasms, and sometimes it shares symptoms with the benign counterpart. In this scenario, for a correct diagnosis and a successful prognosis, it is mandatory to detect new reliable markers which strengthen histopathological outcomes and let define a more appropriate and less harmful therapy. Based on this concerning evidence, in the present study, Fourier Transform Infrared Microspectroscopy has been exploited at a cellular level on uterine leiomyoma and leiomyosarcoma cell lines to (1) identify specific spectral biomarkers able to distinguish between benign and malignant lesions, and (2) evaluate the efficacy of eicosapentaenoic and docosahexaenoic acids (respectively EPA and DHA), already successfully tested. Results evidenced reliable differences in the spectral signature of benign and malignant cells, mainly in terms of lipids and nucleic acids composition. Moreover, even if EPA and DHA seemed to exert different effects on the tested cell lines, no cytotoxic and/or anti-apoptotic actions were observed after omega-3 based treatments.

Heterogeneity in the gene regulatory landscape of leiomyosarcoma.

Characterizing inter-tumor heterogeneity is crucial for selecting suitable cancer therapy, as the presence of diverse molecular subgroups of patients can be associated with disease outcome or response to treatment. While cancer subtypes are often characterized by differences in gene expression, the mechanisms driving these differences are generally unknown. We set out to model the regulatory mechanisms driving sarcoma heterogeneity based on patient-specific, genome-wide gene regulatory networks. We developed a new computational framework, PORCUPINE, which combines knowledge on biological pathways with permutation-based network analysis to identify pathways that exhibit significant regulatory heterogeneity across a patient population. We applied PORCUPINE to patient-specific leiomyosarcoma networks modeled on data from The Cancer Genome Atlas and validated our results in an independent dataset from the German Cancer Research Center. PORCUPINE identified 37 heterogeneously regulated pathways, including pathways representing potential targets for treatment of subgroups of leiomyosarcoma patients, such as FGFR and CTLA4 inhibitory signaling. We validated the detected regulatory heterogeneity through analysis of networks and chromatin states in leiomyosarcoma cell lines. We showed that the heterogeneity identified with PORCUPINE is not associated with methylation profiles or clinical features, thereby suggesting an independent mechanism of patient heterogeneity driven by the complex landscape of gene regulatory interactions.

SELISARC: A Spanish Sarcoma Group (GEIS) phase I/II trial of selinexor plus gemcitabine in selected sarcoma subtypes—Results of the phase I part.

11520 Background: Selinexor (KPT-330) (S), a small-molecule inhibitor of the nuclear export mediator exportin-1 (XPO-1), is able to reduce mRNA and protein expression of DNA damage repair (DDR) gene products, being synergistic with DNA damage agents as gemcitabine (G) in preclinical experiments. We hypothesized that selinexor would have a synergistic effect with gemcitabine in selected sarcoma patients (pts). Methods: Adult progressing pts, ECOG 0-1, with up to 2 previous systemic therapies for advanced disease (localized unresectable/metastatic) with centrally confirmed diagnosis of undifferentiated pleomorphic sarcoma (UPS), leiomyosarcoma (LMS), alveolar soft part sarcoma (ASPS), or osteosarcoma (OS) were eligible. S (days 1, 8, 15) and G (days 1, 8) were given in four dose levels, L1: S 60 mg + G 1,000 mg/m2 30 min, L2: S 60 mg + G 1,000 mg/m2 (10 mg/m2/min), L3: S 60 mg + G 1,200 mg/m2 (10 mg/m2/min), and L4: S 80 mg + G 1,200 mg/m2 (10 mg/m2/min). A -1 level was defined with S 60 mg + G 800 mg/m2 (30 min). A classic 3+3 design was used to determine the RP2D based on DLTs observed during the first 21-day cycle. In vitro research was performed in LMS and OS cell lines to test the synergy of the combination (MTS and flow cytometry assays). Results: Between November 2020 and June 2022, 17 pts (M/F 9/8), ECOG 0/1 (14/3), median age 50 years (22-71) were recruited. The median of previous lines was 1 (1-2). Diagnosis was: 9 LMS (52.9%), 6 OS (35.3%), 1 ASPS and 1 synovial sarcoma (SS) (5.9%). Three pts were treated in each of the first 3 levels and 8 pts in L4 (2 pts were not evaluable for DLT due to improper dosing). Only one DLT was observed in L4 (G4 thrombocytopenia) and this level was selected as RP2D. G3/4 toxicity: Neutropenia (52.9%), thrombocytopenia (41.2%), febrile neutropenia (11.8%), anemia, nausea, asthenia, vomiting, alopecia, and lipase increased (5.9% each). There were 3 RECIST PR, 7 SD, and 7 PD. Median PFS for LMS was 7 months (95% CI: 3-11). No relevant clinical activity was observed in OS. In vitro cell viability studies shown that S+G was synergistic in the majority of LMS cell lines tested (combination index of 0.789 for CP0024, 0.791 for SK-UT-1, 0.604 for AA and 1.186 for IEC005). However, our results indicated antagonism of S+G in OS cell lines, with values for the combination index of 1.67 for MG63, 1.54 for SAOS2 and 1.23 for U2OS. Apoptosis assays by flow cytometry confirmed these observations in LMS and OS cells. Conclusions: At the RP2D, S 80 mg + G 1,200 mg/m2 (10 mg/m2/min) is a feasible scheme with manageable toxicity. S+G has shown promising clinical activity for LMS, which warrants further investigation in a phase II. Preclinical studies shown the synergy and the antagonism of S+G in LMS and OS, respectively. Clinical trial information: NCT04595994 .

Abstract 6021: Genetic and epigenetic features OF KMT2D, CREBBP, ATM, TSC2 and GNAS in uterine leyomiosarcomas

Abstract Background: Uterine leiomyosarcoma (LMS) comprises 60% to 70% of the uterine sarcoma (US). These tumors present high rates of recurrence and metastasis, and the patients’ survival rates are very low (20% in five years). It is known that somatic mutations and modifications in the DNA methylation levels are associated with several types of neoplasms. Previously, our group identified missense and loss of function mutations in KMT2D, CREBBP, ATM, TSC2, and GNAS, using a genetic screening method in LMS samples. Based on these results, we decided to investigate whether a genetic and epigenetic crosstalk could help to understand the biological processes involved in the LMS origin, development, and pathogenesis. The present study aimed to evaluate the KMT2D, CREBBP, ATM, TSC2 and GNAS gene expression profile in LMS cell line, and their methylation levels in formalin-fixed and paraffin-embedded (FFPE) patients´ samples. Methods: We selected 15 LMS - FFPE samples obtained via surgical procedures performed between 2012 and 2019 at the Instituto do Cancer do Estado de São Paulo (ICESP). Genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit and we evaluated methylation levels using the Illumina Infinium Methylation EPIC BeadChip system 850k, including samples treated with bisulfite-treated DNA. Leiomyoma (LM) (THESCs CRL-4003) and LMS (SK-UT-1) cell lines were cultivated for gene expression analysis. Cells were growth for 24, 48, 72, and 96 hours, and the total RNA was extracted by TRIzol. High-Capacity cDNA Reverse Transcription Kit was used for cDNA synthesis and for gene expression evaluation, real-time PCR reactions were performed using TaqMan Universal PCR Master Mix and inventoried TaqMan probes. Results: KMT2D and TSC2 showed increased expression in LMS compared to LM (cut-off ≤ -2 and ≥ 2 for down and upregulation, respectively), with higher expression in 24 hours of KMT2D [Fold Regulation (FR): 2.77] and TSC2 (FR: 2.58). In contrast, CREBBP was downregulated, with lowest expression in 72 hours (FR: -6.57). ATM was upregulated at 24, 48 with higher expression in 72 hours (FR: 2.32), and GNAS was downregulated, with lowest expression in 96 hours (FR: -4.31). Moreover, the methylation analyzes showed an expressive hypomethylation in LMS samples, compared to myometrium, for KMT2D (β value: 0.45; p= 0.004); CREBBP (β value: 0.51 p < 0.0001); ATM (β value: 0.47 p < 0.0001); TSC2 (β value: 0.53 p < 0.0001) and GNAS (β value: 0.41 p < 0.0001). In conclusion, our study showed that potentially pathogenic mutations may be associated with an aberrant hypomethylation profile, as well as increased expression of KMT2D, TSC2, ATM and loss expression of CREBBP and GNAS in LMS. The interconnection of these genetic and epigenetic events is essential for understanding the complex biology of these tumors, in addition to enabling identification of biomarkers. Citation Format: Laura G. dos Anjos, Daniela Bizinelli, Katia C. Carvalho. Genetic and epigenetic features OF KMT2D, CREBBP, ATM, TSC2 and GNAS in uterine leyomiosarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6021.

Abstract 346: Downregulating KIF4A inhibited growth of uterine leiomyosarcoma by regulating cell cycle

Abstract [Objective] Uterine leiomyosarcoma (LMS) has a poor prognosis, and no effective treatment other than surgery has been established. Kinesin family member4A (KIF4A) is a member of the kinesin4 subfamily and plays an important role in cell division. KIF4A has been revealed to play significant roles in some cancers with tumor proliferation. The aim of this study is to clarify the relationship between KIF4A and tumor growth of LMS. [Methods] One normal human uterine smooth muscle cell line and three LMS cell lines(SK-UT1, SK-LMS and SKN) were used to identify tumor-specific elevated proteins with iTRAQ(isobaric tags for relative and absolute quantitation). SiRNA and ShRNA were used to downregulate KIF4A in vitro and in in vivo. Cell variability was evaluated by MST-8 assay. Cell cycle analysis by fluorescence-activated cell sorting (FACS) and western blotting (WB) were performed to analyze the signal pathway. Immunostaining of 26 clinical specimen from patients operated at our university hospital since 1999 to 2021. [Results] A total of 2084 proteins were identified by iTRAQ. KIF4A was identified as a protein with more than twice the expression level of normal smooth muscle cells. WB confirmed high expression of KIF4A in all three LMS cell lines. And in immunostaining of clinical specimens, KIF4A was positive in 20/26(76.9%) cases. A trend toward worse prognosis was suggested in the group with high KIF4A expression (no significant difference due to the small number of cases). Downregulation with SiRNA significantly suppressed the growth of LMS cell lines in vitro (-37.2±4.73% in SK-LMS, -87.7±4.02% in SKN and -28.1±3.00% in SK-UT1, p<0.05). By FACS, G2/M phase cells was significantly increased in KIF4A suppressed cell lines and by WB, p-cdc2 was upregulated in KIF4A suppressed cells. In vivo, downregulation of KIF4A was found to significantly suppress tumor size in genograft mice. (1186±118mm³ vs 461±84mm³ in SK-LMS and 1704±441mm³ vs 514±230mm³ in SK-UT1, p<0.05, f). [Conclusion] Downregulating KIF4A inhibited LMS tumor growth and it can be a novel therapeutics target to treat LMS. Citation Format: Satoshi Nakagawa, Chihiro Mizuta-Odani, Mariya Kobayashi, Mamoru Kakuda, Kosuke Hiramatsu, Toshihiro Kimura, Eiji Kobayashi, Yutaka Ueda, Tadashi Kimura. Downregulating KIF4A inhibited growth of uterine leiomyosarcoma by regulating cell cycle [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 346.

Abstract 6174: ATX-101, a peptide drug targeting PCNA, enhances the effect of gemcitabine in liposarcoma and leiomyosarcoma

Abstract Soft tissue sarcomas are heterogeneous mesenchymal neoplasms and account for 1% of all cancers in adults. Over 50 sarcomas subtypes have been classified and many have very limited treatment options which include surgery, chemotherapy and radiation. Two of the most common sarcomas are liposarcoma and leiomyosarcoma which are often treated with chemotherapies such as doxorubicin, dacarbazine and gemcitabine with low response rates. There is now more than ever an urgent need for new therapies for sarcoma. PCNA (Proliferating Cell Nuclear Antigen) is considered to be a key regulator of DNA and cell cycle control. In addition, it has been implicated to have roles in metabolism, cellular signaling and some immunological functions. PCNA forms complexes with proteins bearing a novel PCNA-interacting Peptide Motif called APIM (AlkB homolog 2 PCNA Interacting Motif). This binding usually occurs during stress responses such as those achieved during cancer therapy or cancer development. By preventing stress proteins from repairing or defending themselves, cancer therapies can be made more effective. We examined the in vitro effects of ATX-101, a PCNA-APIM protein interaction blocking peptide, on liposarcoma cell lines LS141, DDLS, SW872, 93T449 and 94T778 and leiomyosarcoma cell lines SK-LMS, SK-UT1 and SK-UT-1B. Cell survival, as measured by cell proliferation assays, indicated IC50s of 7.5-15µM with ATX-101. Enhanced combination effects with ATX-101 and chemotherapies such as doxorubicin, irinotecan and gemcitabine were also observed by proliferation assay. Synergism, with ATX-101 and gemcitabine, was indicated with SynergyFinder analysis. Cell cycle changes exhibited increases of S and G2 phases when treated with ATX-101 and gemcitabine. This correlated with increase of cell cycle proteins, cyclin B1 and cyclin A2. Western blot observations included increase of apoptotic marker, cleaved caspase 3, and DNA damage marker, pH2A.X. In addition, immunofluorescence assay showed increasing pH2A.X with ATX-101 and gemcitabine. In vivo studies revealed ATX-101 enhanced gemcitabine decrease of tumor volume in leiomyosarcoma cell line xenograft, SK-UT1. Tumor target inhibition by western blot showed increased pH2A.X and cleaved PARP along with decreased RAD51 API protein with combination treatment. Taken these observations and results, there is strong evidence of combining gemcitabine with ATX-101 in liposarcoma and leiomyosarcoma. Citation Format: Elgilda Musi, Tahir Sheikh, Matthew Ingham, Sminu Bose, Gary K. Schwartz. ATX-101, a peptide drug targeting PCNA, enhances the effect of gemcitabine in liposarcoma and leiomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6174.

High levels of hypusinated eIF5A in leiomyoma and leiomyosarcoma pathologies: a possible novel therapeutic target

Research questionIs the hypusinated form of the eukaryotic translation initiation factor 5A (EIF5A) present in human myometrium, leiomyoma and leiomyosarcoma, and does it regulate cell proliferation and fibrosis? DesignThe hypusination status of eIF5A in myometrial and leiomyoma patient-matched tissues was evaluated by immunohistochemistry and Western blotting as well as in leiomyosarcoma tissues by immunohistochemistry. Myometrial, leiomyoma and leiomyosarcoma cell lines were treated with N1-guanyl-1,7-diaminoheptane (GC-7), responsible for the inhibition of the first step of eIF5A hypunization, and the proliferation rate was determined by MTT assay; fibronectin expression was analysed by Western blotting. Finally, expression of fibronectin in leiomyosarcoma tissues was detected by immunohistochemistry. ResultsThe hypusinated form of eIF5A was present in all tissues examined, with an increasing trend of hypusinated eIF5A levels from normal myometrium to neoplastic benign leiomyoma up to neoplastic malignant leiomyosarcoma. The higher levels in leiomyoma compared with myometrium were confirmed by Western blotting (P = 0.0046). The inhibition of eIF5A hypusination, with GC-7 treatment at 100 nM, reduced the cell proliferation in myometrium (P = 0.0429), leiomyoma (P = 0.0030) and leiomyosarcoma (P = 0.0044) cell lines and reduced the expression of fibronectin in leiomyoma (P = 0.0077) and leiomyosarcoma (P = 0.0280) cells. The immunohistochemical staining of leiomyosarcoma tissue revealed that fibronectin was highly expressed in the malignant aggressive (central) part of the leiomyosarcoma lesion, where hypusinated eIF5A was also highly represented. ConclusionsThese data support the hypothesis that eIF5A may be involved in the pathogenesis of myometrial benign and malignant pathologies.

Abstract B014: The role of HDAC-MEF2 axis in the epigenetic control of immune tumoral microenvironment

Abstract Epigenetic mechanisms have a main impact on cancer regulation and on microenvironment control. In the context of leiomyosarcoma (LMS), a highly aggressive and immunologically cold cancer, class IIa histone deacetylases (HDACs) and myocyte enhancer factor (MEF2) form a repressive complex that leads to the reorganization of epigenetic landscape. We have recently described the effect of some HDAC inhibitors (HDACi) on chromatin organization and in particular the increase of H3K27Ac genome occupancy near TSS and promoter regions, caused by NKL-54, a HDAC1/2/3 selective inhibitor, and the re-expression of some chemokines can be observed after the treatment with TMP-195, a specific class IIa HDACi. The hypothesis of this project is that the HDAC-MEF2 complex represses the transcription of some immune genes that may have a major role in antitumor response through an epigenetic control of chromatin. To verify this proposal, we knocked-down (KD) MEF2A in SK-UT-1, a high grade LMS cell line, or we treated the same cells with NKL-54 and TMP-195. We compared the impact of MEF2A KD and HDACs inhibition on chemokines and cytokines release by RNA-seq and Luminex profiling, while H3K27ac levels obtained by ChIP-seq were used to quantify enhancers and super-enhancers activation. To evaluate the impact of MEF2A on LMS cells proliferation we performed a clonogenic assay. To test the effectiveness of class IIa HDACs inhibitors in vivo, we aim to generate a syngeneic model of LMS by transforming uterine smooth muscle cells with Large T and Small T antigen, p53175H and by knocking out (KO) ATRX and PTEN. The preliminary data show that KD of MEF2A leads to an increased expression and release of some chemokines and cytokines such as CXCL5, CXCL2 and CXCL8 in SK-UT-1 cells. Class IIa HDACs inhibition further enhances this effect. Furthermore, the absence of MEF2A causes a reduction of colonies formation compared with WT cells, pointing out the fundamental role of MEF2A in LMS cell proliferation. Finally, ChIP-seq analysis of the genomic location of CXCL2 and CXCL5 in HDAC4 and HDAC9 KO shows an increased H3K27Ac signal at the level of the regulative elements of the two genes, indicating the epigenetic reprogramming acting on these loci and achieved by HDACs impairment. High grade LMS are characterized by the formation of HDAC IIa and MEF2 repressive complexes at some specific genomic loci required to sustain LMS malignancy and possibly to weaken LMS immunogenicity. The ablation or the specific inhibition of class IIa HDACs lead to the release of MEF2A from this repressive complex and the subsequent establishment of a transcriptional open chromatin environment that would promote tumor immune infiltration and clearance. These preliminary data, together with previous evidence, highlight the role of HDAC-MEF2 in the context of LMS microenvironment organization, suggesting that the targeting of these partners can boost the immune system activity, and this can be exploited for the development of LMS therapies. Citation Format: Martina Minisini, Emiliano Dalla, Vanessa Tolotto, Claudio Brancolini. The role of HDAC-MEF2 axis in the epigenetic control of immune tumoral microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B014.

Stathmin expression alters the antiproliferative effect of eribulin in leiomyosarcoma cells

Uterine leiomyosarcoma is an aggressive soft tissue tumor. Stathmin, a phosphoprotein that modulates microtubule dynamics, is highly expressed in many malignancies including leiomyosarcoma. The microtubule-depolymerizing agent eribulin has been recently approved for treating malignant soft tissue tumors. Although eribulin inhibits microtubule polymerization, little is known about the relationship between eribulin treatment and stathmin dynamics. In this study, we explored the role of stathmin expression in the action of eribulin in leiomyosarcoma cells. Eribulin induced phosphorylation of stathmin and reduced expression of subunits A and C of protein phosphatase 2A (PP2A) in a leiomyosarcoma cell line. The PP2A activator FTY720 reduced levels of phosphorylated stathmin. Eribulin decreased stathmin protein levels without affecting stathmin mRNA expression. Furthermore, stathmin knockdown attenuated the inhibitory effects of eribulin on cell viability, whereas stathmin overexpression enhanced the anti-proliferative effect of eribulin. Eribulin-resistant leiomyosarcoma cell lines had enhanced expression of the class Ⅰ β-tubulin TUBB1, multi-drug resistance 1 protein MDR1 and breast cancer-resistance protein BCRP, and decreased expression of stathmin. Taken together, these results suggest that stathmin expression modulates the pharmacological efficacy of eribulin in uterine leiomyosarcoma cells.

Abstract B014: Metabolic reprogramming in high-grade sarcomas: repurposing anti-cholesterol agents as a novel therapeutic strategy

Abstract Introduction and Objective: Undifferentiated pleomorphic sarcoma (UPS) and leiomyosarcoma (LMS) are treated with surgery, radiation, and/or chemotherapy. Current drug therapies, such as doxorubicin, have limited response rates and severe toxicities. We performed a drug screen with over 3000 compounds on four patient-derived UPS cell lines and uncovered sensitivity to simvastatin, which inhibits the rate limiting enzyme in the mevalonate pathway. Statins are well tolerated drugs used to lower cholesterol in patients with hypercholesterolemia. The mevalonate pathway can be exploited by cancer through metabolic reprogramming and phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling to enhance proliferation. Thus, the goals of our study are to define the mechanism(s) responsible for simvastatin sensitivity and determine the efficacy when combined with doxorubicin in vivo. Hypothesis: Dysregulation of the mevalonate pathway and/or PI3K/mTOR pathway will render certain high-grade sarcomas sensitive to simvastatin. Methods: We confirmed simvastatin sensitivity in UPS, as well as in LMS cell lines, with dose-response curves. We investigated the mechanisms whereby simvastatin inhibits UPS and LMS viability with immunoblotting and flow cytometry. Proliferation was quantified using a cell proliferation dye. To assess in vivo efficacy and toxicity of simvastatin we performed a murine study with an established xenograft UPS model from our lab. We are currently assessing the efficacy of this novel combination in an LMS xenograft. Results: We found decreased phosphorylated AKT (pAKT ser473) and increased rates of active caspase-3 in most sensitive UPS cell lines with simvastatin (1uM) treatment. This indicates downregulation of PI3K/mTOR signaling and an increase in apoptosis, respectively. Importantly, these effects were rescued with the addition of mevalonate (200uM), an effector immediately downstream of the rate limiting enzyme that statins inhibit, indicating an on-target effect. In four sensitive LMS cell lines treated with simvastatin (1.5uM or 2uM) a decrease in pAKT ser473 is not consistently seen, revealing that downregulation of PI3K/mTOR signalling may not be a universal mechanism. Furthermore, in some UPS and LMS cell lines there was a significant reduction in proliferation with simvastatin + doxorubicin (0.5uM) treatment (p<0.05). Our in vivo study demonstrated that simvastatin (50mg/kg) in combination with doxorubicin (1.2mg/kg) reduced the volume of UPS tumors in mice. Furthermore, mice treated with combination therapy had higher concentrations of simvastatin and doxorubicin within the tumor than those treated with either single agent. However, this study was not designed to look at pharmacokinetics (PK), so to confirm these results a PK study is underway in an LMS xenograft. Conclusion: UPS and LMS are sensitive to simvastatin, suggesting these sarcomas rely on metabolic reprogramming to sustain viability. Thus, simvastatin may be a novel therapy for certain sarcoma patients. Citation Format: Jen Dorsey, Yael Babichev, Rosemarie E. Venier, Richard Marcellus, Rima Al-awar, Linda Z. Penn, Albiruni A. Razak, Brendan C. Dickson, Eric Chen, Irene L. Andrulis, Jay Wunder, Rebecca A. Gladdy. Metabolic reprogramming in high-grade sarcomas: repurposing anti-cholesterol agents as a novel therapeutic strategy [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 B014.

Abstract 6074: Resistance to MK-0752 alters Notch activity and expression of stemness markers in uterine leiomyosarcoma cell lines

Abstract Introduction: Uterine leiomyosarcoma (uLMS) is a rare tumor with poor survival and few therapeutic options. The Notch pathway is evolutionarily conserved, is active in uterine processes, has oncogenic properties in many cancers, but has not been well studied in uLMS. Cancer stem cells (CSC) are subpopulations of cancer cells that are treatment resistant. In certain tumors, activation of canonical Notch signaling, via gamma secretase, supports CSC survival and the acquisition of chemoresistance. Our objective is to identify and characterize the subpopulation of uLMS cells that are viable after treatment with the half maximal inhibitory concentration (IC50) of MK-0752, a gamma secretase inhibitor (GSI) in phase I trials. We will determine Notch signaling activity and expression of markers associated with stem-like potential in GSI resistant uLMS cells. Methods: MTT assays were performed on SK-LMS-1 and SK-UT-1B, two uLMS cell lines, to identify the IC50 for MK-0752. Cells were exposed to MK-0752 at the IC50 or DMSO (control) for 24 hours and viable cells were collected. First, expression of stemness markers, CD133, cMYC and SOX2, in uLMS cells was compared to human uterine smooth muscle cells (hUT-SMC) at baseline. After treatment with MK-0752, the remaining subpopulations of resistant uLMS cells were evaluated for Notch signaling activity by expression of HES1. Stemness marker expression was also assessed. Gene expression was measured with qRT-PCR. Mean fold change (FC) gene expression was determined by FC experimental divided by FC control, relative to expression of 18S rRNA. P≤0.05 was considered significant. Results: In untreated uLMS cells, expression of CD133 and c-MYC was significantly higher in SK-LMS-1 (fibroblast morphology) vs hUT-SMC, while expression of CD133 and SOX2 was significantly higher in SK-UT-1B (epithelial morphology) vs hUT-SMC. After treatment with MK-0752 at IC50 (427.4 µM for SK-LMS-1 or 128.4µM for SK-UT-1B) expression of HES1 was decreased in SK-LMS-1 (0.35x, p≤0.05) and increased in SK-UT-1B (2.38x, p≤0.01) in treatment resistant, viable cells as compared to untreated cells. Expression of c-MYC was decreased (0.38x, p≤0.01) and CD133 was similar (1.64x, p>0.05) in GSI resistant SK-LMS-1 cells, while expression of CD133 and SOX2 was similar (1.43x and 1.30x, respectively, p>0.05) in GSI resistant SK-UT-1B cells compared to untreated cells. Conclusions: Cellular morphology, Notch signaling activity and expression of stemness markers differs in GSI resistant SK-LMS-1 compared to GSI resistant SK-UT-1B cells. The subpopulation of MK-0752 resistant SK-LMS-1 cells have reduced Notch activity and reduced expression of stemness marker, c-MYC, while the resistant SK-UT-1B cells have increased Notch activity. Further studies are required to identify additional factors associated with uLMS resistance to GSIs and the importance of this heterogeneity of uLMS in vivo. Citation Format: Yasmin Abedin, Emily Alpert, Erica Rego, Karla Larios, Emma Cheung, Sofia Gabrilovich, Qingshi Zhao, Mark H. Einstein, Nataki Douglas. Resistance to MK-0752 alters Notch activity and expression of stemness markers in uterine leiomyosarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6074.

LncRNA EGFR-AS1 facilitates leiomyosarcoma progression and immune escape via the EGFR-MYC-PD-L1 axis.

this study aimed to investigate the role of long non-coding RNA (lncRNA) epidermal growth factor receptor antisense RNA 1 (EGFR-AS1), an antisense transcript of EGFR, in leiomyosarcoma (LMS) and the underlying mechanisms. levels of EGFR-AS1 and programmed death ligand 1 (PD-L1) were measured in LMS tissues and cell lines using quantitative real-time PCR (qRT-PCR), as well as western blotting and/or immunohistochemical staining; flow cytometry was employed to validate the role of EGFR-AS1 in altering the activity of CD8+ T cells; interaction of EGFR-AS1 and EGFR was determined by fluorescent in situ hybridization (FISH) and RNA pull-down; regulation of MYC on the PD-L1 promoter was assessed by chromatin immunoprecipitation (ChIP); a xenograft in vivo tumor growth assay was applied to verify the EGFR-AS1/EGFR/MYC/PD-L1 axis in vivo. up-regulation of EGFR-AS1 and PD-L1 in LMS tissues was negatively correlated with CD8+ T-cell infiltration; EGFR-AS1 positively regulated PD-L1, thereby strengthening interaction of LMS cells and CD8+ T cells and triggering CD8+ T cell apoptosis via the PD-1/PD-L1 checkpoint; EGFR-AS1 co-localized and interacted with EGFR to promote MYC activity; MYC was identified as a transcriptional activator of PD-L1. lncRNA EGFR-AS1 was demonstrated to increase PD-L1 expression through the EGFR/MYC pathway in LMS cells, thereby repressing T-cell infiltration and contributing to immune escape.

U2 small nuclear RNA auxiliary factor 2, transcriptionally activated by the transcription factor Dp-1/E2F transcription factor 1 complex, enhances the growth and aerobic glycolysis of leiomyosarcoma cells

ABSTRACT The dysregulation of U2 Small Nuclear RNA Auxiliary Factor 2 (U2AF2) is associated with malignant behaviors of multiple types of tumors. In this study, we explored the association between U2AF2 dysregulation and the survival of patients with primary leiomyosarcoma, the regulatory effect of U2AF2 on cell growth/aerobic glycolysis, and the mechanisms of U2AF2 dysregulation at the transcriptional level. Gene expression and survival time of patients with primary leiomyosarcoma were extracted from TCGA-Sarcoma (SARC). Leiomyosarcoma cell lines SK-LMS-1 and SK-UT-1 were utilized to construct in vitro and in vivo models. Results showed that the higher U2AF2 expression group had significantly shorter progression-free survival (HR: 2.049, 95%CI: 1.136–3.697, p = 0.011) and disease-specific survival (4.656, 95%CI: 2.141–10.13, p < 0.001) compared to the lower U2AF2 expression group. U2AF2 knockdown suppressed leiomyosarcoma cell growth and aerobic glycolysis (decreased glucose uptake, lactate production, and extracellular acidification rate) in vitro. Tumors derived from SK-LMS-1 cells with U2AF2 knockdown grew significantly slower, with lower GLUT1, PGK1, and PGAM1 protein expression than the control groups. TFDP1 and E2F1 could interact with each other in leiomyosarcoma cells. Both TFDP1 and E2F1 could bind to the promoter of U2AF2 and exert a synergistic activating effect on U2AF2 transcription. In conclusion, this study revealed that U2AF2 upregulation is associated with poor survival of leiomyosarcoma. Its upregulation enhances proliferation and aerobic glycolysis of leiomyosarcoma cells in vitro and in vivo. TFDP1 and E2F1 can form a complex, which binds to the U2AF2 gene promoter and synergistically activates its transcription.

Preclinical Modeling of Leiomyosarcoma Identifies Susceptibility to Transcriptional CDK Inhibitors through Antagonism of E2F-Driven Oncogenic Gene Expression.

Leiomyosarcoma (LMS) is a neoplasm characterized by smooth muscle differentiation, complex copy-number alterations, tumor suppressor loss, and the absence of recurrent driver mutations. Clinical management for advanced disease relies on the use of empiric cytotoxic chemotherapy with limited activity, and novel targeted therapies supported by preclinical research on LMS biology are urgently needed. A lack of fidelity of established LMS cell lines to their mesenchymal neoplasm of origin has limited translational understanding of this disease, and few other preclinical models have been established. Here, we characterize patient-derived xenograft (PDX) models of LMS, assessing fidelity to their tumors of origin and performing preclinical evaluation of candidate therapies. We implanted 49 LMS surgical samples into immunocompromised mice. Engrafting tumors were characterized by histology, targeted next-generation sequencing, RNA sequencing, and ultra-low passage whole-genome sequencing. Candidate therapies were selected based on prior evidence of pathway activation or high-throughput dynamic BH3 profiling. We show that LMS PDX maintain the histologic appearance, copy-number alterations, and transcriptional program of their parental tumors across multiple xenograft passages. Transcriptionally, LMS PDX cocluster with paired LMS patient-derived samples and differ primarily in host-related immunologic and microenvironment signatures. We identify susceptibility of LMS PDX to transcriptional cyclin-dependent kinase (CDK) inhibition, which disrupts an E2F-driven oncogenic transcriptional program and inhibits tumor growth. Our results establish LMS PDX as valuable preclinical models and identify strategies to discover novel vulnerabilities in this disease. These data support the clinical assessment of transcriptional CDK inhibitors as a therapeutic strategy for patients with LMS.

The therapeutic potential of RNA Polymerase I transcription inhibitor, CX-5461, in uterine leiomyosarcoma

SummaryBackground. Uterine leiomyosarcoma is a rare aggressive smooth muscle cancer with poor survival rates. RNA Polymerase I (Pol I) activity is elevated in many cancers supporting tumour growth and prior studies in uterine leiomyosarcoma revealed enlarged nucleoli and upregulated Pol I activity-related genes. This study aimed to investigate the anti-tumour potential of CX-5461, a Pol I transcription inhibitor currently being evaluated in clinical trials for several cancers, against the human uterine leiomyosarcoma cell line, SK-UT-1. Methods. SK-UT-1 was characterised using genome profiling and western blotting. The anti-tumour effects of CX-5461 were investigated using cell proliferation assays, expression analysis using qRT-PCR, and BrdU/PI based cell cycle analysis. Results. Genetic analysis of SK-UT-1 revealed mutations in TP53, RB1, PTEN, APC and TSC1 & 2, all potentially associated with increased Pol I activity. Protein expression analysis showed dysregulated p53, RB1 and c-Myc. CX-5461 treatment resulted in an anti-proliferation response, G2 phase cell-cycle arrest and on-target activity demonstrated by reduced ribosomal DNA transcription. Conclusions. SK-UT-1 was confirmed as a representative model of uterine leiomyosarcoma and CX-5461 has significant potential as a novel adjuvant for this rare cancer.

Eribulin activity in soft tissue sarcoma monolayer and three-dimensional cell line models: could the combination with other drugs improve its antitumoral effect?

BackgroundEribulin has shown antitumour activity in some soft tissue sarcomas (STSs), but it has only been approved for advanced liposarcoma (LPS).MethodsIn this study, we evaluated the effect of eribulin on proliferation, migration and invasion capabilities in LPS, leiomyosarcoma (LMS) and fibrosarcoma (FS) models, using both monolayer (2D) and three-dimensional (3D) spheroid cell cultures. Additionally, we explored combinations of eribulin with other drugs commonly used in the treatment of STS with the aim of increasing its antitumour activity.ResultsEribulin showed activity inhibiting proliferation, 2D and 3D migration and invasion in most of the cell line models. Furthermore, we provide data that suggest, for the first time, a synergistic effect with ifosfamide in all models, and with pazopanib in LMS as well as in myxoid and pleomorphic LPS.ConclusionsOur results support the effect of eribulin on LPS, LMS and FS cell line models. The combination of eribulin with ifosfamide or pazopanib has shown in vitro synergy, which warrants further clinical research.

TJP1 Contributes to Tumor Progression through Supporting Cell-Cell Aggregation and Communicating with Tumor Microenvironment in Leiomyosarcoma.

Leiomyosarcoma (LMS) is a mesenchymal malignancy with a complex karyotype. Despite accumulated evidence, the factors contributing to the development of LMS are unclear. Here, we investigated the role of tight-junction protein 1 (TJP1), a membrane-associated intercellular barrier protein during the development of LMS and the tumor microenvironment. We orthotopically transplanted SK-LMS-1 cells and their derivatives in terms of TJP1 expression by intramuscular injection, such as SK-LMS-1 Sh-Control cells and SK-LMS-1 Sh-TJP1. We observed robust tumor growth in mice transplanted with LMS cell lines expressing TJP1 while no tumor mass was found in mice transplanted with SK-LMS-1 Sh-TJP1 cells with silenced TJP1 expression. Tissues from mice were stained and further analyzed to clarify the effects of TJP1 expression on tumor development and the tumor microenvironment. To identify the TJP1-dependent factors important in the development of LMS, genes with altered expression were selected in SK-LMS-1 cells such as cyclinD1, CSF1 and so on. The top 10% of highly expressed genes in LMS tissues were obtained from public databases. Further analysis revealed two clusters related to cell proliferation and the tumor microenvironment. Furthermore, integrated analyses of the gene expression networks revealed correlations among TJP1, CSF1 and CTLA4 at the mRNA level, suggesting a possible role for TJP1 in the immune environment. Taken together, these results imply that TJP1 contributes to the development of sarcoma by proliferation through modulating cell-cell aggregation and communication through cytokines in the tumor microenvironment and might be a beneficial therapeutic target.

KIF15 upregulation promotes leiomyosarcoma cell growth via promoting USP15-mediated DEK deubiquitylation

Kinesin Family Member 15 (KIF15) is a plus end-directed microtubule motor, which exerts complex regulations in cancer biology. This study aimed to explore the functional role of KIF15 in leiomyosarcoma (LMS). Bioinformatic analysis was carried out using data from The Cancer Genome Atlas (TCGA)-Sarcoma (SARC). LMS cell lines SK-UT-1 and SK-LMS-1 were used as in vitro cell models. Results showed that LMS patients with high KIF15 expression had significantly worse survival than the low KIF15 expression counterparts. KIF15 knockdown slowed, while KIF15 overexpression increased the proliferation of SK-UT-1 and SK-LMS-1 cells. Co-IP assay confirmed mutual interaction between endogenous KIF15 and DEK (encoded by DEK proto-oncogene). KIF15 knockdown facilitated DEK degradation, while KIF15 overexpression slowed DEK degradation. In ubiquitination assay, a significant increase in DEK polyubiquitylation was observed when KIF15 expression was suppressed. USP15 physically interacted with both DEK and KIF15 in the cells. USP15 knockdown decreased DEK protein stability and canceled KIF15-mediated DEK stabilization. USP15 overexpression enhanced DEK stability, the effect of which was impaired by KIF15 knockdown. USP15 overexpression reduced DEK polyubiquitination. USP15 knockdown increased DEK polyubiquitination and canceled the effect of KIF15 overexpression on reducing DEK polyubiquitination. DEK overexpression enhanced the proliferation of SK-UT-1 and SK-LMS-1 cells. DEK knockdown decreased cell proliferation and canceled the effect of KIF15 overexpression on cell proliferation. In conclusion, this study revealed a novel mechanism that KIF15 enhances LMS cell proliferation via preventing DEK protein from degradation by increasing USP15 mediated deubiquitylation.