Soft Tissue Sarcoma Cell Lines Research Articles

Identifying specific TLS-associated genes as potential biomarkers for predicting prognosis and evaluating the efficacy of immunotherapy in soft tissue sarcoma.

The tertiary lymphatic structure (TLS) is an important component of the tumor immune microenvironment and has important significance in patient prognosis and response to immune therapy. However, the underlying mechanism of TLS in soft tissue sarcoma remains unclear. A total of 256 RNAseq and 7 single-cell sequencing samples were collected from TCGA-SARC and GSE212527 cohorts. Based on published TLS-related gene sets, four TLS scores were established by GSVA algorithm. The immune cell infiltration was calculated via TIMER2.0 and "MCPcounter" algorithms. In addition, the univariate, LASSO, and multivariate-Cox analyses were used to select TLS-related and prognosis-significant hub genes. Single-cell sequencing dataset, clinical immunohistochemical, and cell experiments were utilized to validate the hub genes. In this study, four TLS-related scores were identified, and the total-gene TLS score more accurately reflected the infiltration level of TLS in STS. We further established two hub genes (DUSP9 and TNFSF14) prognosis markers and risk scores associated with soft tissue sarcoma prognosis and immune therapy response. Flow cytometry analysis showed that the amount of CD3, CD8, CD19, and CD11c positive immune cell infiltration in the tumor tissue dedifferentiated liposarcoma patients was significantly higher than that of liposarcoma patients. Cytological experiments showed that soft tissue sarcoma cell lines overexpressing TNFSF14 could inhibit the proliferation and migration of sarcoma cells. This study systematically explored the TLS and related genes from the perspectives of bioinformatics, clinical features and cytology experiments. The total-gene TLS score, risk score and TNFSF14 hub gene may be useful biomarkers for predicting the prognosis and immunotherapy efficacy of soft tissue sarcoma.

Establishment of a novel canine soft tissue sarcoma cell line and comparison of its characteristics with other soft tissue sarcoma cell lines

Soft tissue sarcoma (STS) is a relatively common tumor in dogs. However, very few canine STS cell lines are available. This study aimed to establish a new cell line, STS-YU1, derived from a recurrence of myxosarcoma in an 11-year-old mixed-breed dog. We examined STS-YU1 for in vitro cell proliferation, migration, anticancer drug sensitivity, transcriptome analysis using next-generation sequencing (RNA-seq), and in vivo tumorigenicity in mice and compared it with previously established STS cell lines, MUMA-G and A72. The cell proliferation and migration of STS-YU1 were higher than MUMA-G although MUMA-G only exhibited tumorigenicity in mice. STS-YU1 showed dose-dependent cytotoxicity to anticancer drugs, but with weak effects. RNA-seq analysis revealed the molecular phenotype of STS-YU1 was different from that of a previously reported cell line, A72. Hence, the use of STS-YU1 would help in efficient drug screening against canine STS in vitro.

The Effect of Photodynamic Therapy Using 5-Aminolevulinic Acid in Bone and Soft Tissue Sarcoma Cells.

5-Aminolevulinic acid (5-ALA) is a natural amino acid and a precursor of protoporphyrin IX (PpIX). Following light irradiation, the PpIX generates reactive oxygen species (ROS) in the presence of oxygen. Increased ROS levels can cause apoptotic cell death and necrosis of targeted cancer cells. This study examined whether photodynamic therapy using 5ALA (5-ALA PDT) could be used as a potential adjuvant therapy for bone and soft tissue sarcomas. The human osteosarcoma (143B), mouse osteosarcoma (LM8), human fibrosarcoma cell (HT1080) cell lines were used. In vitro, cultured cells were exposed to 5-ALA at various concentrations followed by strobe scope light irradiation for 10 min as 5-ALA PDT. Cell viability was then measured. In vivo, each tumor cell line was inoculated subcutaneously into the backs of mice. In the 5-ALA PDT group, 5-ALA (250 mg/kg) was administered intraperitoneally followed by light irradiation. Change in tumor volume by 5-ALA PDT were primarily evaluated. In vitro, treatment of sarcoma cells with 100 and 200 μg/ml 5-ALA PDT significantly inhibited cell proliferation at 24 and 48 h compared with the group treated with 0 and 10 μg/ml 5-ALA PDT. In vivo, in all cell lines, a significant inhibition of the tumor volume was observed in the 5-ALA-PDT group as compared to that in control, strobe scope light, and 5-ALA groups. 5-ALA PDT effectively inhibited proliferation of bone and soft tissue sarcoma cell lines. Further in vivo research using other subtypes of bone and soft tissue sarcoma is warranted to confirm the applicability in the clinical setting.

Antitumor effects of PRMT5 inhibition in sarcomas.

Patients with advanced soft-tissue sarcomas (STSs) have few therapeutic options. Protein arginine methyltransferase 5 (PRMT5), an anticancer target, has been extensively investigated in recent years in epithelial tumors. To date, no data related to the biological role of PRMT5 inhibition and its potential effect as a treatment in STS have been reported. To investigate the therapeutic potential of PRMT5 targeting in STS, we first evaluated the prognostic value of PRMT5 expression in 2 different cohorts of patients with STS. We then used the potent and selective GSK3326595 (GSK595) compound to investigate the antitumor effect of the pharmacological inhibition of PRMT5 in vitro via MTT, apoptosis, cell cycle, clonogenicity and proliferation assays. In vivo studies were performed with two animal models to evaluate the effects of GSK595 on tumor growth. The mechanisms of action were investigated by RNA sequencing, metabolic pathway analysis, Western blotting and glucose uptake/lactate production assays. High PRMT5 gene expression levels were significantly associated with worsened metastasis-free survival of STS patients. GSK595 decreased the global symmetric dimethylarginine level, the proliferation rate and clonogenicity of STS cell lines in vitro and tumor growth in vivo. Moreover, PRMT5 inhibition regulated aerobic glycolysis through downregulation of key enzymes of glycolysis as well as glucose uptake and lactate production. The present study demonstrated that PRMT5 regulates STS cell metabolism and thus represents a potential therapeutic target for STS. Additional studies in diverse sarcoma subtypes will be essential to confirm and expand upon these findings.

Activation of the Akt signalling pathway as a prognostic indicator in canine soft tissue sarcoma

Canine soft tissue sarcoma (STS) is relatively common in dogs and is the generic term for tumours that originate from mesenchymal cells. While histopathological grade and immunolabelling with Ki-67 have been used for estimating prognosis, additional indicators are needed for predicting prognosis. Aberrant cell signalling pathways may contribute to disease activity and, therefore, prognostic markers. However, their role in canine STS remains poorly understood. The aim of this study was to investigate expression of phosphorylated Akt (phospho-Akt) and phosphorylated S6 (phospho-S6) as potential prognostic indicators. Immunohistochemical labelling was conducted on clinical samples of canine STS (n = 67). We found that phospho-Akt expression was positively correlated with histopathological grade (P = 0.001) and Ki-67 index (P <0.01). There was no apparent relationship between the type of STS and the expression of phospho-Akt. The number of cases that expressed phospho-S6, which is the downstream molecule of the Akt signalling pathway, was higher in immunopositive phospho-Akt cases than in immunonegative phospho-Akt cases (P <0.0001). Furthermore, phospho-Akt expression was significantly higher in recurrent and metastatic cases. We also confirmed that phosphorylation of Akt occurred in conjunction with S6 phosphorylation in three canine STS cell lines. These results suggest that immunolabelling for phospho-Akt, phospho-S6 and Ki-67 could potentially be used as a prognostic indicator and therapeutic target in canine STS.

Alternative splicing (AS) regulation as a novel therapeutic target in soft-tissue sarcoma (STS).

11546 Background: There is an urgent clinical need to identify novel treatments for advanced STS, since all the clinical trials testing chemotherapy combinations have failed to improve survival compared to doxorubicin (dox) alone. AS deregulation has been associated with cancer initiation, progression and resistance to therapy. This deregulation can occur due to mutations or imbalanced expression or activity of splicing factors (SF). The alteration SF can alter thousands of pre-mRNAs, increasing cellular complexity and facilitating tumour progression. CDC-like kinases (CLK) that phosphorylate serine-arginine-rich SF (SRSFs), are key regulators of AS. SM09419 is an oral pan CLK/DYRK inhibitor that has been shown to inhibit SRSF phosphorylation, selectively modify spliceosome activity and decrease tumour growth by multiple mechanisms including the downregulation of Wnt/β-catenin signalling, a pathway that we reported to be important in sarcoma. Methods: A panel of 9 STS cell lines, including LMS (CP0024, IEC005, AA, SK-UT-1), LPS (93T449), synovial sarcoma (MCP037) and UPS (MCP016, MCP021, MCP025) were used to analyse the effect of SM09419 (provided by Biosplice) or PRI-724 (Wnt/β-catenin inhibitor) on cell viability (MTS assays). TOP flash β-catenin/TCF-responsive reporter assay was performed to evaluate the inhibition of the Wnt/β-catenin pathway in response to SM09419. In vivo experiments tested the combination of SM09419 (25mg/kg 5 days on and 2 off, for 21 days cycle) plus IP dox (5mg/kg once a week for 21 days cycle) in LMS (IEC005) and synovial sarcoma (FJD-SS-001) PDX models. Results: Our results showed that SM09419 had superior activity in STS cells compared to specific Wnt/β-catenin inhibitors with IC50 values starting at 29.8 nM in SK-UT-1 cell line, whereas PRI-724 IC50 values were clearly higher, starting from 5.18 µM in 93T449 cells. SM09419 treatment significantly inhibited the transcriptional activity of β-catenin by 40% in the MCP021 cells, demonstrating the regulatory activity of AS inhibitors on this pathway. I n vivo studies confirmed the higher efficacy of SM09419 in monotherapy compared to dox, in inhibiting tumour growth (p &lt; 0.05), which was potentiated when combined with dox. The combination was increasingly active in synovial sarcoma with tumour regressions in 3 out of 4 mice, including one complete response. Treatment schedule was adapted to a single IP administration of dox (5mg/kg) and 5 administrations of SM09419 (25mg/kg), to improve the tolerability of the combination. Conclusions: The effects of pan CLK/DYRK inhibition on growth and survival of STS cancer cell lines and PDX models, indicate that can STS may be therapeutically addressed with these AS-modulators. The combination of SM09419 with dox was active in STS preclinical models known for their resilience toward treatment. Based on these results, the exploration of such combinations in clinical trials is warranted.

Animal models of Soft Tissue Sarcoma for alternative anticancer therapy studies: characterization of the A-72 Canine Cell Line

Canine Soft Tissue Sarcoma (STS) cell line A-72 has been largely employed for antiviral and antiproliferative studies. However, there are few information on their characteristics. Our aim was to evaluate A-72 expression level of genes and proteins involved in the innate immune response and cell cycle, their ability to respond to infective stressors and their possible use as a cellular model for anti-cancer studies in human and animal medicine. For this purpose, we evaluated the basal expression of immune-related, cell cycle and DNA repair genes on this cell line and tumoral tissues. A-72 ability to respond to a wild-type strain of Salmonella typhimurium was assessed. S. typhimurium showed ability to penetrate A-72 causing pro-inflammatory response accompanied by a decrease of cell viability. IL10 and IL18 genes were not expressed in A-72 while CXCL8, NOS2, CXCR4 and PTEN were highly expressed in all samples and TP53 was slightly expressed, as shown in human STS. Our results outline the ability of A-72 to respond to a bacterial agent by modifying the expression of important genes involved in innate immune response and provide a useful model for in vitro evaluation of new therapeutic approaches that could be translated into the human oncology.

A New Glutathione-Cleavable Theranostic for Photodynamic Therapy Based on Bacteriochlorin e and Styrylnaphthalimide Derivatives

Herein, we report a new conjugate BChl-S-S-NI based on the second-generation photosensitizer bacteriochlorin e6 (BChl) and a 4-styrylnaphthalimide fluorophore (NI), which is cleaved into individual functional fragments in the intracellular medium. The chromophores in the conjugate were cross-linked by click chemistry via a bis(azidoethyl)disulfide bridge which is reductively cleaved by the intracellular enzyme glutathione (GSH). A photophysical investigation of the conjugate in solution by using optical spectroscopy revealed that the energy transfer process is realized with high efficiency in the conjugated system, leading to the quenching of the emission of the fluorophore fragment. It was shown that the conjugate is cleaved by GSH in solution, which eliminates the possibility of energy transfer and restores the fluorescence of 4-styrylnaphthalimide. The photoinduced activity of the conjugate and its imaging properties were investigated on the mouse soft tissue sarcoma cell line S37. Phototoxicity studies in vitro show that the BChl-S-S-NI conjugate has insignificant dark cytotoxicity in the concentration range from 15 to 20,000 nM. At the same time, upon photoexcitation, it exhibits high photoinduced activity.

Panobinostat enhances NK cell cytotoxicity in soft tissue sarcoma.

Sarcoma is a rare and heterogeneous class of mesenchymal malignancies with poor prognosis. Panobinostat (LBH589) as one of histone deacetylase (HDAC) inhibitors has demonstrated anti-tumor activity in patients with sarcoma, but its mechanisms remains unclear. Here, we found that LBH589 alone inhibited the proliferation and colony formation of soft tissue sarcoma (STS) cell lines. Transcriptome analysis showed that treatment with LBH589 augmented the NK cell-mediated cytotoxicity. Quantitative real-time PCR and flow cytometric analysis (FACS) further confirmed that LBH589 increased the expression of NKG2D ligands MICA/MICB. Mechanistically, LBH589 activated the Wnt/β-catenin pathway by upregulating the histone acetylation in β-catenin promoter. In vitro co-culture experiments and in vivo animal experiments showed that LBH589 increased the cytotoxicity of natural killer (NK) cells while Wnt/β-catenin inhibitor decreased the effects. Our findings suggest that LBH589 facilitates the anti-tumor effect of NK cells, highlights LBH589 an effective assistance drug in NK cell-based immunotherapies.

Alternative splicing of Apoptosis Stimulating Protein of TP53-2 (ASPP2) results in an oncogenic isoform promoting migration and therapy resistance in soft tissue sarcoma (STS)

BackgroundMetastatic soft tissue sarcoma (STS) are a heterogeneous group of malignancies which are not curable with chemotherapy alone. Therefore, understanding the molecular mechanisms of sarcomagenesis and therapy resistance remains a critical clinical need. ASPP2 is a tumor suppressor, that functions through both p53-dependent and p53-independent mechanisms. We recently described a dominant-negative ASPP2 isoform (ASPP2κ), that is overexpressed in human leukemias to promote therapy resistance. However, ASPP2κ has never been studied in STS. Materials and methodsExpression of ASPP2κ was quantified in human rhabdomyosarcoma tumors using immunohistochemistry and qRT-PCR from formalin-fixed paraffin-embedded (FFPE) and snap-frozen tissue. To study the functional role of ASPP2κ in rhabdomyosarcoma, isogenic cell lines were generated by lentiviral transduction with short RNA hairpins to silence ASPP2κ expression. These engineered cell lines were used to assess the consequences of ASPP2κ silencing on cellular proliferation, migration and sensitivity to damage-induced apoptosis. Statistical analyses were performed using Student’s t-test and 2-way ANOVA.ResultsWe found elevated ASPP2κ mRNA in different soft tissue sarcoma cell lines, representing five different sarcoma sub-entities. We found that ASSP2κ mRNA expression levels were induced in these cell lines by cell-stress. Importantly, we found that the median ASPP2κ expression level was higher in human rhabdomyosarcoma in comparison to a pool of tumor-free tissue. Moreover, ASPP2κ levels were elevated in patient tumor samples versus adjacent tumor-free tissue within individual patients. Using isogenic cell line models with silenced ASPP2κ expression, we found that suppression of ASPP2κ enhanced chemotherapy-induced apoptosis and attenuated cellular proliferation.ConclusionDetection of oncogenic ASPP2κ in human sarcoma provides new insights into sarcoma tumor biology. Our data supports the notion that ASPP2κ promotes sarcomagenesis and resistance to therapy. These observations provide the rationale for further evaluation of ASPP2κ as an oncogenic driver as well as a prognostic tool and potential therapeutic target in STS.

Canady Cold Helios Plasma Reduces Soft Tissue Sarcoma Viability by Inhibiting Proliferation, Disrupting Cell Cycle, and Inducing Apoptosis: A Preliminary Report.

Soft tissue sarcomas (STS) are a rare and highly heterogeneous group of solid tumors, originating from various types of connective tissue. Complete removal of STS by surgery is challenging due to the anatomical location of the tumor, which results in tumor recurrence. Additionally, current polychemotherapeutic regimens are highly toxic with no rational survival benefit. Cold atmospheric plasma (CAP) is a novel technology that has demonstrated immense cancer therapeutic potential. Canady Cold Helios Plasma (CHCP) is a device that sprays CAP along the surgical margins to eradicate residual cancer cells after tumor resection. This preliminary study was conducted in vitro prior to in vivo testing in a humanitarian compassionate use case study and an FDA-approved phase 1 clinical trial (IDE G190165). In this study, the authors evaluate the efficacy of CHCP across multiple STS cell lines. CHCP treatment reduced the viability of four different STS cell lines (i.e., fibrosarcoma, synovial sarcoma, rhabdomyosarcoma, and liposarcoma) in a dose-dependent manner by inhibiting proliferation, disrupting cell cycle, and inducing apoptosis-like cell death.

Abstract 1509: Genome-wide CRISPR/Cas9 library screening identified THRAP3 as a critical driver for resistance to ATR inhibitor in sarcomas revealing synthetic lethality with ATM inhibition

Abstract Background: ATR plays a crucial role in maintaining genomic integrity by responding to a large spectrum of DNA damage, including double strand breaks (DSBs) that interfere with replication. We have previously demo started that ATR inhibition is associated with anti-tumor activity in pre-clinical models of soft-tissue sarcomas (STS) (Laroche et al., 2020). We decided to investigate the mechanisms of resistance to ATR inhibition in sarcomas. Methods: We used a CRISPRa was to identify genes that mediate resistance to AZD6738 in STS cells. The anti-tumor activity of AZD6738 (ATR inhibitor) and AZD0156 (ATM inhibitor) was investigated in a panel for 10 STS cell lines and two patient-derived xenografts (PDX) established at Institut Bergonié (Bordeaux, France). Results: We used a CRISPR approach in a STS cell line resistant to AZD6738 and we made a negative-selection screen, to identify genes whose silencing conferred sensitivity to low doses of ATR inhibition. Among these genes, we identified THRAP3 which encodes a protein phosphorylated in response to DNA damage and primarily by ATR. THRAP3 is involved in the maturation and further export of transcripts encoding the ATM and its depletion results in deficient processing of transcripts encoding the ATM kinase. We validate the result of our CRISPR analysis by demonstrating that selective THRAP3 inhibition reduce significantly ATRi IC50. Given the role of role of THRAP3 on the regulation of ATM activity, we decided to investigate the potential synergy between ATR and ATM inhibition in vivo and in vitro. AZD6738 and AZD0156 displayed a synergistic or additive effect in above all the STS cell lines tested. We noted that ATRi treatment reduced phosphorylation of the ATR kinase target CHK1 but increased ATM phosphorylation, suggesting that ATM pathway takes over for DNA Damage Response and cells survival. In contrast, both ATR and ATM pathway were inhibited when AZD6738 and AZD0156 were used in combination, as shown by reduction of CHK1 and ATM phosphorylation. We validated our in vitro results in an in vivo model of liposarcoma xenograft in which AZD6738 and AZD0156 had synergistic anti-tumor activity. Conclusion: Our study demonstrates synthetically lethal interactions between ATM and ATR in vitro and in vivo. This combination deserves further investigaitons in sarcomas. Citation Format: Mariella Spalato, Audrey Laroche-Clary, Vanessa Chaire, Antoine Italiano. Genome-wide CRISPR/Cas9 library screening identified THRAP3 as a critical driver for resistance to ATR inhibitor in sarcomas revealing synthetic lethality with ATM inhibition [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 1509.

Abstract 2564: CDK9 inhibition via KB-0742 is a potential strategy to treat transcriptionally addicted cancers

Abstract Lineage-specific transcriptional networks drive cellular differentiation and development. Disruption of these specific cell programs can result in cancer and create a subset of tumors that are “transcriptionally addicted.” Sarcomas, for example, are characterized by an oncogenic fusion protein consisting of a FET family RNA-binding protein fused to a transcription factor (TF). The oncogenic fusions result in a restructuring of the transcriptome promoting cancer. In chordoma, TBXT (brachyury)—normally involved in notochord differentiation—is aberrantly expressed, resulting in promotion of cancer. As these oncogenic TFs are difficult to target directly, we and others have proposed targeting associated transcriptional regulators to inhibit their activity. Cyclin-dependent kinase 9 (CDK9) interacts with TFs to promote activation of target genes and promotes transcription elongation through phosphorylation of RNA polymerase II. We have developed a potent, selective, and orally bioavailable CDK9 inhibitor, KB-0742. Here we present the preclinical activity of KB-0742 in models of sarcoma and chordoma. We first evaluated KB-0742 activity in sarcoma in a 300-immortalized cell line screen containing 18 soft tissue sarcoma cell lines. The median IC50 of the cell lines in the study was 0.8705 µM. Sarcoma cell lines were enriched for sensitivity to KB-0742 with 61% (11/18) of the lines with an IC50 below the median. The 11 cell lines had a median IC50 of 0.679 µM. We then evaluated the activity of KB-0742 in 5 patient-derived cell line (PDC) models, with all 5 showing a cytotoxic response to treatment as measured by negative GRmax values. Last, a single patient-derived organoid (PDO) model of adult rhabdomyosarcoma was evaluated. KB-0742 treatment resulted in an IC50 of 2.75 µM and a max inhibition rate of 98.61%. For chordoma we examined the activity of KB-0742 in vivo using 2 patient-derived xenograft (PDX) models. In model CF466, we observed a dose-dependent response with increased TGI and greater reductions in RNA polymerase II phosphorylation in tumors treated with KB-0742 at 60 mg/kg as compared to 30 mg/kg. We then evaluated KB-0742 as a single agent and in combination with afatinib in the model CF539. KB-0742 as a single agent showed similar TGI activity as afatinib, whereas the combination showed increased response as compared to the two single-agent arms. These data show that transcriptionally addicted tumors are sensitive to CDK9 inhibition via KB-0742 treatment, and support the continued development of our compound to potentially treat sarcoma and chordoma. KB-0742 is currently being evaluated in a phase 1/2 clinical trial (NCT04718675) for relapsed or refractory solid tumors or non-Hodgkin lymphoma. Once a recommended phase 2 dose is established, expansion cohorts for patients with sarcoma, chordoma, and other transcriptionally addicted tumors may be opened. Citation Format: Melinda A. Day, Douglas C. Saffran, Tressa Hood, Nikolaus Obholzer, Akanksha Pandey, Charles Y. Lin, Pavan Kumar, Daniel M. Freed, Jorge DiMartino. CDK9 inhibition via KB-0742 is a potential strategy to treat transcriptionally addicted cancers [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 2564.

Abstract 3937: PRMT5 inhibition suppress soft tissue sarcomas tumorigenicity through the regulation of glucose metabolism

Abstract Introduction: Soft tissue sarcomas (STS) are a heterogeneous group of rare tumors representing 1% of solid tumors in adults and 15% in children. Despite adequate locoregional treatment, up to 40% of patients will develop metastatic disease. Doxorubicin represents the 1st line standard of care for patients in this setting. However, its activity is limited with a response rate of only 10%, a progression-free survival of less than 6-months. New therapeutic strategies are therefore urgently needed. PRMT5 belongs to a family of enzymes that catalyze the methylation of arginine residues on both histones and non-histone proteins. Recent evidence shows that PRMT5 is upregulated in a wide variety of cancers. Its expression and activity are associated with tumorigenesis and with a decrease in patient survival in several cancers, a rationale for developing highly potent inhibitors. Methods: We analyzed the transcriptome data from 255 translocation-related STS and 389 complex genomics STS with annotated clinical data from the French Sarcoma Group. We then investigated the anti-tumor activity of the first -in class PRMT5 specific inhibitor, GSK3226593 in a panel of 7 cell lines and in two xenograft models established at Institut Bergonié (Bordeaux, France). Results: We found that high PRMT5 gene expression level was significantly associated with worse metastases-free survival in both translocation-related and complex genomics STS (p=0.003 and p=0.009, respectively), support the relevance of PRMT5 as a potential therapeutic target in this indication. We then found that pharmacological inhibition of PRMT5 by using GSK3226593, suppressed the viability and the cell proliferation of 7 STS cell lines in vitro (IC50 range between 0.01 and 1.4 µM) as well as in vivo in two models of xenograft cell lines. This inhibition of cell growth was concomitant with an increase of p53 and p21 expression. However, anti-tumor activity of GSK3226593 was independent of p53 since the compound was still active in shp53 expressing cell lines. Moreover, we did not notice any significant effect of GSK3226593 on cell cycle phases or apoptosis. To decipher the mechanism of action of PRMT5 inhibition in STS, we did a comparative transcriptomic analysis (RNAseq) of STS cells before and after 10 days of treatment with GSK3226593. Gene Ontology and gene set enrichment analysis revealed downregulation of metabolic processes especially glycolysis and mTORC1 pathways. A specific metabolic pathway panel was used to confirm and deepen the underlying molecular mechanism. The downregulation of several genes by GSK3226593 such as PCK2 was confirmed at the protein level by Western blot. Glycolysis inhibition was confirmed by a decrease of glucose uptake. Conclusion: PRMT5 inhibition has anti-tumor activity in vitro and in vivo through the regulation of STS cell metabolism providing a clear rationale for its clinical investigation in this indication. Citation Format: Stephanie Verbeke, Laura Leroy, Aurelien Bourdon, Vanessa Chaire, Elodie Richard, Jean-Philippe Guegan, Alban Bessede, Antoine Italiano. PRMT5 inhibition suppress soft tissue sarcomas tumorigenicity through the regulation of glucose metabolism [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 3937.

An investigation of kV mini-GRID spatially fractionated radiation therapy: dosimetry and preclinical trial

Objective. To develop and characterize novel methods of extreme spatially fractionated kV radiation therapy (including mini-GRID therapy) and to evaluate efficacy in the context of a pre-clinical mouse study. Approach. Spatially fractionated GRIDs were precision-milled from 3 mm thick lead sheets compatible with mounting on a 225 kVp small animal irradiator (X-Rad). Three pencil-beam GRIDs created arrays of 1 mm diameter beams, and three ‘bar’ GRIDs created 1 × 20 mm rectangular fields. GRIDs projected 20 × 20 mm2 fields at isocenter, and beamlets were spaced at 1, 1.25, and 1.5 mm, respectively. Peak-to-valley ratios and dose distributions were evaluated with Gafchromic film. Syngeneic transplant tumors were induced by intramuscular injection of a soft tissue sarcoma cell line into the gastrocnemius muscle of C57BL/6 mice. Tumor-bearing mice were randomized to four groups: unirradiated control, conventional irradiation of entire tumor, GRID therapy, and hemi-irradiation (half-beam block, 50% tumor volume treated). All irradiated mice received a single fraction of 15 Gy. Results. High peak-to-valley ratios were achieved (bar GRIDs: 11.9 ± 0.9, 13.6 ± 0.4, 13.8 ± 0.5; pencil-beam GRIDs: 18.7 ± 0.6, 26.3 ± 1.5, 31.0 ± 3.3). Pencil-beam GRIDs could theoretically spare more intra-tumor immune cells than bar GRIDs, but they treat less tumor tissue (3%–4% versus 19%–23% area receiving 90% prescription, respectively). Bar GRID and hemi-irradiation treatments significantly delayed tumor growth (P < 0.05), but not as much as a conventional treatment (P < 0.001). No significant difference was found in tumor growth delay between GRID and hemi-irradiation. Significance. High peak-to-valley ratios were achieved with kV grids: two-to-five times higher than values reported in literature for MV grids. GRID irradiation and hemi-irradiation delayed tumor growth, but neither was as effective as conventional whole tumor uniform dose treatment. Single fraction GRID therapy could not initiate an anti-cancer immune response strong enough to match conventional RT outcomes, but follow-up studies will evaluate the combination of mini-GRID with immune checkpoint blockade.

HDAC Inhibition for Optimized Cellular Immunotherapy of NY-ESO-1-Positive Soft Tissue Sarcoma.

Adoptive cell therapy with NY-ESO-1-specific T cells is a promising option for the treatment of soft tissue sarcoma (STS) but achieves only transient tumor control in the majority of cases. A strategy to optimize this cell therapeutic approach might be the modulation of the expression of the cancer-testis antigen NY-ESO-1 using histone deacetylase inhibitors (HDACis). In this study, the ex vivo effect of combining NY-ESO-1-specific T cells with the clinically approved pan HDACis panobinostat or vorionstat was investigated. Our data demonstrated that STS cells were sensitive to HDACis. Administration of HDACi prior to NY-ESO-1-specific T cells exerted enhanced lysis against the NY-ESO-1+ STS cell line SW982. This correlated with an increase in the NY-ESO-1 and HLA-ABC expression of SW982 cells, as well as increased CD25 expression on NY-ESO-1-specific T cells. Furthermore, the immune reactivity of NY-ESO-1-specific CD8+ T cells in terms of cytokine release was enhanced by HDACis. In summary, pretreatment with HDACis represents a potential means of enhancing the cytotoxic efficacy of NY-ESO-1-specific T cells against NY-ESO-1-positive STS.

HIWI2 induces G2/M cell cycle arrest and apoptosis in human fibrosarcoma via the ROS/DNA damage/p53 axis

AimsPiwi-like RNA-mediated gene silencing 4 (PIWIL4) or HIWI2, are seen deregulated in human cancers and possibly play critical roles in tumorigenesis. It is unknown what role HIWI2 plays in the regulation of fibrosarcoma, an early metastatic lethal type of soft tissue sarcoma (STS). The present study aimed to investigate the role of HIWI2 in the tumorigenesis of fibrosarcoma. Main methodsThe expression of HIWI2 in HT1080 fibrosarcoma cells was determined by qRT-PCR and western blotting. The MTT assay, colony formation assay, cell cycle, and PE-AnnexinV/7AAD apoptosis assay using flow cytometry, DNA laddering assay, comet assay, and γH2AX accumulation assay were performed to study the effect of HIWI2 overexpression in HT1080 cells. Further, the effect of silencing of HIWI2 was determined by cell viability assay, transwell migration, and invasion assay. Key findingsHIWI2 is under-expressed in STS cell lines and tissues, which is associated with poor disease-free survival, disease-specific survival, and progression-free survival of the patients. Overexpression of HIWI2 in HT1080 cells causes DNA damage by increasing intracellular ROS by inhibiting the expression of antioxidant genes (SOD1, SOD2, GPX1, GPX4, and CAT). Furthermore, an increase in H2AX phosphorylation was observed, which activates p53 that promotes p21 expression and caspase-3 activation, leading to G2/M phase cell cycle arrest and apoptosis. HIWI2 silencing, on the contrary, promotes cell growth, migration, and invasion by activating MMP2 and MMP9. SignificanceThese results are the first to show that HIWI2 acts as a tumor suppressor in fibrosarcoma by modulating the ROS/DNA damage/p53 pathway.

Ribonucleotide reductase subunit M2 is a potential prognostic marker and therapeutic target for soft tissue sarcoma.

Soft tissue sarcomas (STSs) are highly aggressive malignant tumors that exhibit poor therapeutic outcomes. Hence, we aimed to track down a potential gene that can be used as a prognostic marker and therapeutic target for this malignancy. We integrated omics analysis of clinical data and in vitro studies and identified Ribonucleotide reductase subunit M2 (RRM2) as a potential oncogene associated with STS prognosis. We found RRM2 is highly expressed in STS cell lines and tissues. STS patients with increased RRM2 levels showed worse overall survival, disease-free survival, progression-free survival, and disease-specific survival. Further, overexpression of RRM2 in HT1080 cells induces proliferation, migration, invasion, and colony formation, whereas its silencing arrest the cell cycle at G0/G1 phase and induces apoptosis. Taken together, we established RRM2 to be positively associated with oncogenesis and prognosis of STS and therefore could be a promising prognostic marker and therapeutic target.

Abstract P233: TAS0953/HM06 is effective in preclinical models of diverse tumor types driven by RET alterations

Abstract Fusions involving RET receptor tyrosine kinase are a common driver of tumors across different tissue types, such as lung, thyroid, colorectal, soft tissue and others. TAS0953/HM06 (hereby referred to as HM06) is a novel 2ndgeneration RET-specific inhibitor that is effective against RET solvent front (G810) and gatekeeper (V804) mutations. Here, we evaluated the efficacy of HM06 in lung and thyroid carcinomas, and soft-tissue sarcoma cell lines and PDXs derived from RET inhibitor-naive tumor samples or from tumors with acquired resistance to selpercatinib. HM06 was more effective than the RET multi-kinase inhibitors cabozantinib and vandetanib, and as effective as selpercatinib and pralsetinib in inhibiting growth of patient-derived and isogenic lung, thyroid and sarcoma cell lines (IC50=0.02-0.1 µM) harboring different RET fusions (KIF5B-RET, CCDC6-RET, TRIM33-RET, SPECCL1-RET) or activating mutations (RET C634W). Growth of non-tumor cells was up to 80-fold less sensitive to HM06 (IC50= 1.6 µM). Treatment of RET fusion-positive lung cancer cells with HM06 resulted in a dose-dependent inhibition of RET phosphorylation (Y905 and Y1062) and the downstream effectors AKT, ERK1/2, p70S6K and S6. Caspase 3/7 activity and markers of apoptosis (BIM, cleaved PARP) were induced by HM06 to a similar extent as pralsetinib and selpercatinib (dose range: 0.05-1 µM). HM06 induced changes in the core mediators of cell cycle regulation (upregulation of p27, downregulation of CCND1) and suppressed expression of MYC and ETV5. In vivo, HM06 blocked tumor growth and/or induced regression of up to 65% in seven patient-derived xenograft (PDX) models with RET fusions (five NSCLC PDXs, one sarcoma PDX and one NSCLC cell-line xenograft) to a similar extent as pralsetinib and selpercatinib. However, 6 weeks after cessation of treatment of the SPECCL1-RET-driven sarcoma PDX model, growth of tumors treated with HM06 was suppressed completely, whereas 3/5 pralsetinib-treated tumors and 1/5 selpercatinib-treated tumor regrew. Combination of HM06 and the MET inhibitor capmatinib effectively blocked growth of PDX tumors in a model that was derived from a patient sample that expressed RET fusion and METamplification, and was resistant to selpercatinib. These results suggest that HM06 may be an effective therapy for RET-driven tumors in a tissue-type agnostic manner and can effectively address common on-target and off-target resistance mechanisms such as RET G810X and V804X mutations. HM06 is currently in a phase 1 and 2 clinical trial for patients with advanced solid tumors with RET gene abnormalities (margaRET, NCT 04683250). Citation Format: Igor Odintsov, Renate I. Kurth, Kota Ishizawa, Lukas Delasos, Allan J.W. Lui, Inna Khodos, Connor J. Hagen, Qing Chang, Marissa S. Mattar, Morana Vojnic, Siddharth Kunte, Annalisa Bonifacio, Claudio Giuliano, Elisa De Stanchina, Emily Cheng, Emanuela Lovati, Marc Ladanyi, Romel Somwar. TAS0953/HM06 is effective in preclinical models of diverse tumor types driven by RET alterations [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 P233.

The C-Circle Biomarker Is Secreted by Alternative-Lengthening-of-Telomeres Positive Cancer Cells inside Exosomes and Provides a Blood-Based Diagnostic for ALT Activity.

Simple SummaryA clinical test for alternative-lengthening-of-telomeres (ALT) could assist with cancer diagnosis and monitoring of disease progression. ALT-targeted anticancer treatments are being developed; however, there is no appropriate companion ALT diagnostic. The C-Circle biomarker is the only known ALT specific molecule and the C-Circle Assay the only quantitative ALT assay that is amenable to clinical use. We show here that C-Circles are secreted by ALT+ cancer cell lines inside the exosomes and are protected from nucleases. We also show that secreted C-Circles, like intracellular C-Circles, are an ALT-specific biomarker, and in high-risk neuroblastoma, the blood-based C-Circle Assay has the potential to be an accurate diagnostic for ALT cancer activity. Therefore, the secretion of C-Circles by ALT+ cancer cells in the exosomes provides a stable blood-based biomarker and, potentially, a clinical diagnostic for ALT activity, which is required for the development of ALT-targeted therapies as well as for the diagnosis and monitoring of ALT+ cancer.C-Circles, self-primed telomeric C-strand templates for rolling circle amplification, are the only known alternative-lengthening-of-telomeres (ALT)-specific molecule. However, little is known about the biology of C-Circles and if they may be clinically useful. Here we show that C-Circles are secreted by ALT+ cancer cells inside exosomes, and that a blood-based C-Circle Assay (CCA) can provide an accurate diagnostic for ALT activity. Extracellular vesicles were isolated by differential centrifugation from the growth media of lung adenocarcinoma, glioblastoma, neuroblastoma, osteosarcoma, and soft tissue sarcoma cell lines, and C-Circles were detected in the exosome fraction from all eleven ALT+ cancer cell lines and not in any extracellular fraction from the eight matching telomerase positive cancer cell lines or the normal fibroblast strain. The existence of C-Circles in ALT+ exosomes was confirmed with exosomes isolated by iodixanol gradient separation and CD81-immunoprecipitation, and C-Circles in the exosomes were protected from nucleases. On average, 0.4% of the total ALT+ intracellular C-Circles were secreted in the exosomes every 24 h. Comparing the serum-based and tumor-based CCAs in 35 high risk neuroblastoma patients divided randomly into ALT+ threshold derivation and validation groups, we found the serum-based CCA to have 100% sensitivity (6/6), 70% specificity (7/10), and 81% concordance (13/16). We conclude that the secretion of C-Circles by ALT+ cancer cells in the exosomes provides a stable blood-based biomarker and a potential clinical diagnostic for ALT activity.