Cell Liposarcoma Research Articles

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.

Effectiveness of tumor‑treating fields to reduce the proliferation and migration of liposarcoma cell lines.

Liposarcoma (LPS) is a rare type of soft tissue sarcoma that constitutes 20% of all sarcoma cases in adults. Effective therapeutic protocols for human LPS are not well-defined. Tumor-treating fields (TTFields) are a novel and upcoming field for antitumor therapy. TTFields combined with chemoradiotherapy have proven to be more effective than TTFields combined with radiotherapy or chemotherapy alone. The present study aimed to assess the effectiveness of TTFields in inhibiting cell proliferation and viability for the anticancer treatment of LPS. The present study used TTFields (frequency, 150 kHz; intensity, 1.0 V/cm) to treat two LPS cell lines (94T778 and SW872) and analyzed the antitumor effects. According to trypan blue and MTT assay results, TTFields markedly reduced the viability and proliferation of LPS cell lines along with the formation of colonies in three-dimensional culture. Based on the Transwell chamber assay, TTFields treatment also markedly reduced the migration of LPS cells. Furthermore, as shown by the higher activation of caspase-3 in the Caspase-3 activity assay and the results of the reactive oxygen species (ROS) assay, TTFields increased the formation of ROS in the cells and enhanced the proportion of apoptotic cells. The present study also investigated the inhibitory effect of TTFields in combination with doxorubicin (DOX) on the migratory capacity of tumor cells. The results demonstrated that TTFields treatment synergistically induced the ROS-induced apoptosis of LPS cancer cell lines and inhibited their migratory behavior. In conclusion, the present study demonstrated the potential of TTFields in improving the sensitivity of LPS cancer cells, which may lay the foundation for future clinical trials of this combination treatment strategy.

A phase II clinical trial of chidamide in combination with toripalimab in patients with advanced soft tissue sarcoma.

11566 Background: Chidamide is an oral subtype-selective histone deacetylase inhibitor which is effective on the patients with hematological tumors and reported to enhance the efficacy of checkpoint blockade therapies and regulate the host immune response. Here, we report preliminary results of Chidamide in combination with Toripalimab in soft tissue sarcoma (STS) patients. Methods: An open, single arm, phase II study of Chidamide with Toripalimab in patients with advanced soft tissue sarcoma after failure of standard treatment was conducted. Eligible patients could have any number of prior therapies, excluding HDAC inhibitors and immune checkpoint inhibitors treatment. All patients received Chidamide orally at 30mg twice weekly in combination with intravenous Toripalimab 240mg every 21 days until progression or unaccepted toxicity. The primary endpoint was RECIST1.1 objective response rate (ORR). The secondary endpoint included progression free survival (PFS), overall survival (OS), disease control rate(DCR) and safety. Results: Forty-six patients with advanced soft tissue sarcoma were enrolled. The median age of the patients was 47 years and the median prior lines of therapy were 3. The main subtypes included leiomyosarcoma (30.4%), well/dedifferentiated liposarcoma (30%), myxoid/round cell liposarcoma (7%), undifferentiated sarcoma(9%), and osteosarcoma (7%). Treatment was well tolerated with the most common adverse events mainly in grade Ⅰ and grade Ⅱ, including thrombocytopenia (43.5%), nausea(28.3%), neutropenia(23.9%), fatigue (17.4%) and hypothyroidism(15.2%). Among 46 efficacy-evaluable patients, the overall response rate and disease control rate were 23.9% and 80.4%, respectively. The median time to an initial response was 3 months (range, 2 to 14), and the 3-mon and 6-mon progression-free survival rate were 92.7% and 58.9%, respectively. Conclusions: Chidamide with Toripalimab every 21 days was well tolerated and showed promising efficacy in patients with advanced STS. Clinical trial information: NCT04025931 .

The SPEARHEAD-1 trial of afamitresgene autoleucel (afami-cel [formerly ADP-A2M4]): Analysis of overall survival in advanced synovial sarcoma.

11563 Background: Afami-cel is an autologous, T-cell receptor (TCR) T-cell therapy to treat human leukocyte antigen (HLA) A*02–eligible patients (pts) with advanced solid tumors positive for the cancer testis antigen melanoma-associated antigen A4 (MAGE-A4). The efficacy and safety of afami-cel in pretreated pts with advanced/metastatic synovial sarcoma or myxoid/round cell liposarcoma are being evaluated in the Phase 2, two-cohort, open-label, SPEARHEAD-1 (NCT04044768) trial. Here we report interim overall survival (OS) data in the pts with advanced synovial sarcoma in Cohort 1. Methods: Pts who were HLA-A*02 positive with advanced synovial sarcoma (with evidence of clinical/radiological progression) positive for MAGE-A4 received afami-cel after lymphodepleting chemotherapy containing fludarabine and cyclophosphamide. The primary endpoint was overall response rate per RECIST v1.1 by independent review. Secondary endpoints included OS and progression-free survival (PFS), estimated utilizing the Kaplan-Meier method. Results: As of August 29, 2022, 44 pts with synovial sarcoma received afami-cel (2.68–9.99×109 transduced T-cells) in Cohort 1. The median age was 41 years (range: 19–73 years), 50% of pts were female, 89% of pts were white, 96% of pts were typed as HLA-A*02:01P, and median tumor MAGE-A4 expression H-score was 257 (132–300). Pts were heavily pre-treated with a median of 3 prior lines of therapy (range: 1–12). The median follow-up time at the data cut-off was 20.8 months. Median PFS was 3.8 months (95% CI: 2.8, 5.8) and 4.1 months (95% CI: 2.8, 6.9) by independent and investigator review, respectively. Median OS by independent review was 15.4 months (95% CI: 10.9, not estimable) with 52% of pts censored at the data cut-off. The 12-month OS probability was 60% and 24-month OS probability was 40%. Twenty-one pts received additional therapy during long term follow up (systemic therapy, n = 20; radiation, n = 6; other, n = 4). In the 17 pts who had a RECIST response by independent review, the median OS was not reached, the 12-month OS probability was 90%, and 24-month OS probability was 60%. Conclusions: Pts with advanced synovial sarcoma treated with afami-cel in SPEARHEAD-1 had encouraging survival, especially those pts with a RECIST response. Clinical trial information: NCT04044768 .

The SPEARHEAD-1 trial of afamitresgene autoleucel (afami-cel [formerly ADP-A2M4]): Pooled analysis of cytokine release syndrome across cohorts in patients with advanced synovial sarcoma.

e14531 Background: Afami-cel is an autologous, T-cell receptor T-cell therapy engineered to target melanoma-associated antigen A4 (MAGE-A4) in human leukocyte antigen (HLA) A*02–eligible patients (pts) with advanced solid tumors. SPEARHEAD-1 (NCT04044768) is a Phase 2, two cohort, open-label trial to evaluate the efficacy and safety of afami-cel in pretreated pts with advanced/metastatic synovial sarcoma or myxoid/round cell liposarcoma. Here we report data on cytokine release syndrome (CRS) in the pts with advanced synovial sarcoma treated with afami-cel across Cohort 1 and Cohort 2. Methods: Eligible pts had advanced synovial sarcoma and were HLA-A*02 positive with MAGE-A4–positive tumors. Pts received afami-cel after lymphodepleting chemotherapy containing fludarabine and cyclophosphamide. Tocilizumab administration was permitted for Grade 1 CRS if symptoms persisted for ≥ 24 hours or if the pt had co-morbidities, and for Grade ≥2 CRS. Results: As of August 29, 2022, 72 pts with synovial sarcoma received afami-cel (n = 44 Cohort 1; n = 28 Cohort 2). Afami-cel dose range was 1.92–10.00×109 transduced T-cells. The median age was 39.5 years (range: 16–73 years), 47.2% of pts were female, 88.3% of pts were white, and tumor MAGE-A4 expression H-score was 244.5 (range: 66–300). CRS occurred in 72.2% of the pts with synovial sarcoma receiving afami-cel, including Grade 1 in 50% (36/72), Grade 2 in 20.8% (15/72), and Grade 3 in 1.4% (1/72) of pts as per American Society for Transplantation and Cellular Therapy grading. The median time to onset was 2.5 days (range: 1–5 days), and the median time to resolution was 3 days (range: 1 to 14 days). Twenty-seven of 72 (37.5%) pts received at least one dose of tocilizumab. Consistent with expectations, higher serum IL-6 levels were associated with CRS events and the indicated use of tocilizumab for management. RECIST v1.1 responses were observed in pts with and without CRS. Correlation of clinical factors and CRS with response will be presented. Conclusions: CRS was common after afami-cel administration but was mostly low grade. Administration of tocilizumab in SPEARHEAD-1 was effective in the management of CRS. Clinical trial information: NCT04044768 .

Impact of supraphysiologic MDM2 expression on chromatin networks and therapeutic responses in sarcoma

Amplification of MDM2 on supernumerary chromosomes is a common mechanism of P53 inactivation across tumors. Here, we investigated the impact of MDM2 overexpression on chromatin, gene expression, and cellular phenotypes in liposarcoma. Three independent regulatory circuits predominate in aggressive, dedifferentiated tumors. RUNX and AP-1 family transcription factors bind mesenchymal gene enhancers. P53 and MDM2 co-occupy enhancers and promoters associated with P53 signaling. When highly expressed, MDM2 also binds thousands of P53-independent growth and stress response genes, whose promoters engage in multi-way topological interactions. Overexpressed MDM2 concentrates within nuclear foci that co-localize with PML and YY1 and could also contribute to P53-independent phenotypes associated with supraphysiologic MDM2. Importantly, we observe striking cell-to-cell variability in MDM2 copy number and expression in tumors and models. Whereas liposarcoma cells are generally sensitive to MDM2 inhibitors and their combination with pro-apoptotic drugs, MDM2-high cells tolerate them and may underlie the poor clinical efficacy of these agents.

Strategies for Patients With Sarcomas & Merkel Cell Carcinoma

Strategies for Patients With Sarcomas & Merkel Cell Carcinoma

MicroRNA-215-5p promotes proliferation, invasion, and inhibits apoptosis in liposarcoma cells by targeting MDM2.

Liposarcoma (LPS) is one of the most common soft tissue malignancies in adults, and it is characterized by dysregulation of multiple signaling pathways, including MDM2 proto-oncogene (MDM2) amplification. MicroRNA (miRNA) regulates gene expression through incomplete complementary pairing with the 3' untranslated region of mRNAs involved in tumor progression. In this study, bioinformatics analysis, RT-qPCR, dual-luciferase reporter gene, MTT, flow cytometry, cell scratches, chamber migration, colony formation, FISH, WB, and CCK8 were used. RT-qPCR showed that the expression of MDM2 was increased when miR-215-5p was overexpressed compared with the control group. The dual-luciferase reporter gene showed that the Renilla ratio firefly fluorescence intensity was decreased in the overexpression group compared with the control group. Cell phenotype experiments revealed that the overexpression group had increased cell proliferation rate, increased apoptosis rate, increased colony formation rate, increased cell healing area ratio, and increased number of cell invasions. FISH revealed increased MDM2 expression in the overexpression group. WB suggested decreased Bax expression, increased PCNA, Bcl-2, and MDM2 expression, and decreased P53 and P21 expression in the overexpression group. In this study, we suggest that miR-215-5p can target and promote MDM2 expression, promote the proliferation and invasion of LPS cells SW-872, and inhibit apoptosis.Targeting miR-215-5p may be a novel therapeutic strategy for the treatment of LPS.

Abstract 892: Afamitresgene autoleucel (afami-cel; formerly ADP-A2M4) demonstrates durable clinical responses by inducing broad immune engagement with anti-tumor activity

Abstract Afami-cel is a mixed CD4+ CD8+ autologous T-cell therapy engineered to target the cancer testis antigen melanoma-associated antigen A4 in HLA-A*02-positive patients with advanced/metastatic synovial sarcoma or myxoid/round cell liposarcoma (MRCLS). Pooled data from the Phase 1 (NCT03132922) and Phase 2 (SPEARHEAD-1, NCT04044768) trials of afami-cel showed an acceptable benefit to risk profile with an overall response rate of 36.2% and a median duration of response of 52.0 weeks.1 To support the continued investigation of potential mechanisms of durable anti-tumor activity, we previously showed that afami-cel induces broad and enduring peripheral cytokine responses2 and that afami-cel tumoral infiltration is associated with increased presence of activated and proliferative cytotoxic T-cells in the tumor microenvironment.3 Here, we report the results of translational analyses exploring the cooperation between afami-cel-induced innate and adaptive immune responses pooled from the Phase 1 and 2 trials. Methods included measurement of 92 biomarkers related to apoptosis, chemotaxis, metabolism, tumor immunity promotion/suppression, and vascular/tissue remodeling in pre- and post-infusion serum samples from 38 patients. We also conducted multiplex immunofluorescence and gene set variation analysis of Reactome immune system pathway categories and microenvironment cell populations in RNA sequencing data from pre- and post-infusion biopsies from ≥15 patients. Serum analyses showed that patients with a clinical benefit as defined by RECIST v1.1 had significantly greater post-infusion levels of chemotactic markers (Kruskal-Wallis; p<0.05 for partial response [PR] compared to progressive disease, p<0.01 for PR compared to stable disease), indicating higher signaling related to immune-cell recruitment towards lesions. Tumor analyses showed increased expression of genes associated with innate and adaptive immunity, and cytokine signaling, in post-infusion biopsies, including T-cell receptor signaling-related expression, which was consistent with relatively greater spatial protein detection of pro-immune infiltrate. This profile was associated with longer progression-free survival. In conclusion, our data suggest that afami-cel induces peripheral and tumoral innate and adaptive immune responses, a hallmark of durable anti-tumor activity. Updated patient sample data will be presented. 1. D'Angelo SP, et al. J Clin Oncol. 2022;40:16_suppl:11562. 2. D’Angelo SP, et al. Poster 146 presented at: CTOS 2021; Virtual. 3. Van Tine, BA et al. Paper 61 presented at: CTOS 2022; Vancouver, BC, Canada. Citation Format: Cheryl McAlpine, Martin Isabelle, Robyn Broad, Revashnee Naidoo, Ashley Liddle, Elizabeth Duperret, Paul Noto, Ruoxi Wang, Dzmitry Batrakou, Sumit Middha, Chris Evans. Afamitresgene autoleucel (afami-cel; formerly ADP-A2M4) demonstrates durable clinical responses by inducing broad immune engagement with anti-tumor activity [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 892.

Abstract 6558: Defining differentiation States in well-differentiated and de-differentiated liposarcoma

Abstract Among the many sarcoma subtypes, high-grade tumors that have failed to differentiate or undergone dedifferentiation strongly correlate with metastatic potential and poor clinical outcomes. To examine the factors that regulate cell fate, lineage plasticity, and tumor grade, we focused on two common liposarcoma variants, dedifferentiated liposarcoma (DDLS) and well-differentiated liposarcomas (WDLS), that exist at both ends of the differentiation spectrum. Since tumor grading using histology is highly subjective due to widespread tumor heterogeneity, we hypothesized that an sc/snRNA-seq-based approach would be more accurate. To determine if candidate therapies modulate differentiation, we developed a quantitative metric of differentiation using scRNA-seq. The resulting mesenchymal tissue landscape (MTL) is akin to a Waddington landscape but optimized to distinguish DDLS from WDLS. After prospectively obtaining tumors from seven chemo-naïve patients, we performed scRNA-seq on three DDLS and five WDLS specimens, totaling 59,917 cells. Classical markers were used to identify cell types. Since no pre-existing liposarcoma training sets existed, malignant cells were identified by copy number variation (CNV) inferred from the scRNA-seq data. Overexpression of MDM2, CDK4, and HMGA2, known markers of LS, were positive in clusters classified by SingleR as fibroblasts, adipocytes, and myocyte - the mesenchymal subpopulation. We also inferred CNVs and found expected amplifications in 12q13-15 in the mesenchymal subpopulation. Next, we measured cell differentiation by CytoTRACE. A subcluster of malignant fibroblast-like cells positive for MDM2 and CDK4 was the most de-differentiated. Conversely, cells labeled as adipocytes and endothelial cells were the most differentiated cells. Mapping WDLS and DDLS onto the MTL demonstrated that WDLS had more mature cells than DDLS. Surprisingly, while the pathology-assigned diagnosis falls into just two categories, our approach identified a wide range of liposarcoma cells spanning the full spectrum of possible differentiation states. Though follow-on studies are needed, our data suggest that quantitative differentiation metrics can identify malignant cells in transit from a DDLS-to-WDLS state, or possibly the reverse, whereby cells undergo dedifferentiation. Both phenomena are suspected clinically, given the frequent co-existence of WDLS and DDLS in the same tumors, and the noted emergence of DDLS from WDLS diagnosed years earlier. Summarizing our key findings, we measured the degree of differentiation in liposarcoma cells as a precursor to ongoing studies aimed at understanding the molecular mechanisms that underpin LS plasticity and differentiation. Ultimately, we aim to identify the genes and pathways linked to cell fate and discover novel biologically targeted therapies capable of pushing DDLS toward a less aggressive, slower-growing state. Citation Format: Danh Truong, Hannah C. Beird, Chia-Chin Wu, Sandhya Krishnan, Davis Ingram, Alexander Lazar, Emily Keung, Christina Roland, Wantong Yao, Robert Benjamin, Neeta Somaiah, Barry W. Feig, Joseph A. Ludwig. Defining differentiation States in well-differentiated and de-differentiated liposarcoma [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 6558.

Abstract 3500: A noncatalytic role for mettl1 in oncogenic translation in soft tissue sarcoma

Abstract Amplification of chromosomal material derived from 12q is pathognomonic in a subset of soft tissue sarcomas (STS), including well-differentiated and dedifferentiated liposarcoma. However, it remains unclear which of the amplified genes are oncogenic drivers, obstacles to rational therapeutic development. We defined a minimal 12q amplicon in human liposarcoma, and tested the oncogenic activity of each recurrently amplified gene to accelerate the onset of AKT-induced STS in a genetically engineered zebrafish model of the disease. This revealed several genes whose overexpression accelerated the onset of AKT-induced STS, including METTL1, best-known for its ability to catalyze methylation of tRNAs. Surprisingly, catalytically defective mutants of METTL1 exhibited oncogenic activity that was at least as potent as the wild-type gene in zebrafish and human STS, indicating enzyme-independent oncogenic functions. We found that METTL1 promotes ribosome assembly and oncogenic translation independent of its enzymatic activity. In human liposarcoma cells and mouse embryonic fibroblasts, METTL1 overexpression was sufficient to stimulate polysome formation, and this effect was phenocopied by catalytically dead mutants of METTL1. METTL1 overexpression also robustly stimulated the rate of protein synthesis in cells (P < 0.0001), and in a cell-free biochemical assay for translation of a reporter mRNA (P < 0.001). The ability of METTL1 to stimulate ribosomal function triggered a therapeutically targetable dependency, because small molecule inhibitors of ribosome biogenesis had potent therapeutic activity against METTL1-amplified (P < 0.0001), but not METTL1 wild-type, sarcoma cells. Thus, METTL1 is a soft tissue sarcoma oncogene that stimulates oncogenic translation independent of its enzymatic activity, an oncogenic function that can be targeted therapeutically. Citation Format: Raja Hussain Ali, Brianna Silverman, Esteban A. Orellana, Alyssa Kennedy, Caitlin Bowers, Ashley Gutierrez, David Papke, Christopher D. Fletcher, Antonio P. Atayde, Akiko Shimamura, Richard I. Gregory, Alejandro Gutierrez. A noncatalytic role for mettl1 in oncogenic translation in soft tissue sarcoma [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 3500.

Abstract 3251: The immunostimulatory effect of 9-ING-41, a small molecule GSK-3 inhibitor, in sarcomas

Abstract Due to the rarity and genomic disparity of soft tissue and bone sarcomas, actionable therapeutic targets have been elusive. However, inhibition of GSK-3β has emerged as a potentially promising therapy that could be of great benefit to pediatric and adult sarcoma patients. Saos-2 osteosarcoma and 93T449 liposarcoma cell lines were pretreated at IC5072 with small molecule GSK-3β inhibitor 9-ING-41 (elraglusib). The cells were harvested for western blot analysis after 48 hours of treatment. Additionally, both cancer cell lines as well as TALL-104 T-cells and NK-92 NK cells were treated with 0.5 μM 9-ING-41 for 24 hours and harvested for Luminex cytokine profiling. The western blots demonstrated an increase in cPARP, an apoptotic marker, in both cancer cell lines after 9-ING-41 treatment. Additionally, an increase in PD-L1 expression was observed. The cytokine analysis revealed stimulation of immune cell activity in response to 9-ING-41. Treated T-cells had an increase in CXCL11, which is associated with T-cell recruitment, as well as an increased level of IL-18, which is shown to induce increased IFN-γ in Th1 cells. Additionally, NK-92 cells demonstrated an increase in IL-8 chemokine and an increase in soluble TRAIL (TRAIL/TNFSF10). The cancer cell lines showed a homogenous increase in growth factor TGF-ɑ, however, only the Saos-2 osteosarcoma cell line demonstrated an increase of IL-6. We are further validating the data with follow-up cytokine profiling. The increase in immunostimulatory cytokines as well as the increased expression of PD-L1 suggest a rationale for combining 9-ING-41 with immune checkpoint blockade therapy. The potential synergistic effect of these two therapies is currently under investigation with co-culture experiments of sarcoma and immune cells. The treatment cohorts for these experiments include 9-ING-41 combined with either anti-PD-L1, anti-PD-1, or anti-CTLA-4 immune checkpoint inhibitors. Our results suggest a promising combination therapeutic strategy for patients with soft tissue and bone sarcomas and future work will strive to better elucidate the mechanisms of efficacy. Citation Format: William J. MacDonald, Samuel Lew, Kelsey E. Huntington, Rishi R. Lulla, Bradley D. DeNardo, Wafik S. El-Deiry. The immunostimulatory effect of 9-ING-41, a small molecule GSK-3 inhibitor, in sarcomas [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 3251.

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.

Oncostatin M and Nivolumab Affect the Cytotoxic T-Cell Proportions and the Susceptibility to TRAIL-Induced Death in Non-Leukocyte Cell Subpopulations in Soft Tissue Sarcomas

Introduction: Soft tissue sarcomas (STSs) are malignant tumors arising from mesenchymal tissues. Patients with advanced and metastatic STSs have low overall survival rates and relatively limited treatment options. Oncostatin M (OSM) is a pleiotropic cytokine that was shown to carry both pro- and anti-tumorigenic properties in various cancer types. However, the role of OSM in STSs has not yet been elucidated. Moreover, the potential additive effects of combining OSM and anti-PD-1 therapy have not been carried out so far. Methods: The aim of this study was to determine the effects of in vitro OSM administration on liposarcoma, leiomyosarcoma, and myxofibrosarcoma immune cells isolated from peripheral blood and tumor tissues and the potential cooperative nature of OSM and nivolumab in treating these STSs. We designed a cohort study to explore novel histology-driven therapies in our target STSs. The immune cells were isolated from the peripheral blood and tumors of patients with STS, and the proportions and phenotypes of immune cells were evaluated with flow cytometry after cultivation with therapeutic monoclonal antibodies. Results: The proportion of peripheral CD45+ cells was not affected by OSM but was significantly increased by nivolumab, whereas both treatments had an effect on CD8+ T cells. In tumor tissues, CD8+ T cell and CD45‒ TRAIL+ cell cultures were boosted by nivolumab and significantly enriched by OSM. Our data suggest that OSM may play a role in the treatment of leiomyosarcoma, myxofibrosarcoma, and liposarcoma. Conclusion: In conclusion, the biological efficacy of OSM is reflected in the tumor microenvironment rather than in the peripheral blood of the patients in our cohort, and nivolumab could potentiate its mechanism of action in selected cases. Nevertheless, more histotype-tailored studies are needed to fully understand the functions of OSM in STSs.

5-Aminolevrinic Acid Exhibits Dual Effects on Stemness in Human Sarcoma Cell Lines under Dark Conditions.

5-aminolevulinic acid (ALA) is used for tumor-targeting phototherapy because it is converted to protoporphyrin IX (PPIX) upon excitation and induces phototoxicity. However, the effect of ALA on malignant cells under unexcited conditions is unclear. This information is essential when administering ALA systemically. We used sarcoma cell lines that usually arise deep in the body and are rarely exposed to light to examine the effects of ALA treatment under light (daylight lamp irradiation) and dark (dark room) conditions. ALA-treated human SW872 liposarcoma cells and human MG63 osteosarcoma cells cultured under light exhibited growth suppression and increased oxidative stress, while cells cultured in the dark showed no change. However, sphere-forming ability increased in the dark, and the expression of stem-cell-related genes was induced in dark, but not light, conditions. ALA administration increased heme oxygenase 1 (HO-1) expression in both cell types; when carbon monoxide (CO), a metabolite of HO-1, was administered to sarcoma cells via carbon-monoxide-releasing molecule 2 (CORM2), it enhanced sphere-forming ability. We also compared the concentration of biliverdin (BVD) (a co-product of HO-1 activity alongside CO) with sphere-forming ability when HO-1 activity was inhibited using ZnPPIX in the dark. Both cell types showed a peak in sphere-forming ability at 60-80 μM BVD. Furthermore, a cell death inhibitor assay revealed that the HO-1-induced suppression of sphere formation was rescued by apoptosis or ferroptosis inhibitors. These findings suggest that in the absence of excitation, ALA promotes HO-1 expression and enhances the stemness of sarcoma cells, although excessive HO-1 upregulation induces apoptosis and ferroptosis. Our data indicate that systemic ALA administration induces both enhanced stemness and cell death in malignant cells located in dark environments deep in the body and highlight the need to pay attention to drug delivery and ALA concentrations during phototherapy.

Amputation surgery associated with shortened survival in patients with localized soft tissue sarcoma

BackgroundThe objectives of this study were to clarify whether localized extremity soft tissue sarcoma (STS) patients who underwent amputation surgery experienced worsened survival and to identify those patients for whom amputation surgery worsened survival. MethodsUsing the Surveillance, Epidemiology, and End Results database, we identified 8897 patients with localized extremity STS between 1983 and 2016. Of these 6431 patients, 733 patients underwent amputation surgery (Amputation group), and 5698 underwent limb-sparing surgery (Limb-sparing group). ResultsAfter adjusting for patient background by propensity score matching, a total of 1346 patients were included. Patients in the Amputation group showed worsened survival (cancer-specific survival (CSS): hazard ratio (HR) = 1.42, 95% confidence interval (CI) 1.15–1.75, overall survival (OS): HR = 1.41, 95%CI 1.20–1.65). In subclass analysis, patients with high-grade STS, spindle cell sarcoma and liposarcoma in the Amputation group showed shortened survival (high-grade–CSS: HR = 1.44, 95%CI 1.16–1.77, OS: HR = 1.38, 95%CI 1.18–1.62; spindle cell sarcoma–CSS: HR = 4.75, 95%CI 1.56–14.4, OS: HR = 2.32, 95%CI 1.45–3.70; liposarcoma–CSS: HR = 2.91, 95%CI 1.54–5.50, OS: HR = 2.32, 95%CI 1.45–3.70). ConclusionsSurvival was shortened in localized extremity STS patients who received amputation surgery.

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.

Three dimensional models of dedifferentiated liposarcoma cell lines: scaffold-based and scaffold-free approaches.

Sarcomas are rare malignancies, the number of reports is limited, and this rarity makes further research difficult even though liposarcoma is one of major sarcomas. 2D cell culture remains an important role in establishing basic tumor biology research, but its various shortcomings and limitations are still of concern, and it is now well-accepted that the behavior of 3D-cultured cells is more reflective of in vivo cellular responses compared to 2D models. This study aimed to establish 3D cell culture of liposarcomas using two different methods: scaffold-based (Matrigel extracellular matrix [ECM] scaffold method) and scaffold-free (Ultra-low attachment [ULA] plate). Lipo246, Lipo224 and Lipo863 cell lines were cultured, and distinctive differences in structures were observed in Matrigel 3D model: Lipo224 and Lipo863 formed spheroids, whereas Lipo246 grew radially without forming spheres. In ULA plate approaches, all cell lines formed spheroids, but Lipo224 and Lipo863 spheroids showed bigger size and looser aggregation than Lipo246. Formalin fixed, paraffin embedded (FFPE) blocks were obtained from all 3D models, confirming the spheroid structures. The expression of MDM2, Ki-67 positivity and MDM2 amplification were confirmed by IHC and DNAscope™, respectively. Protein and DNA were extracted from all samples and MDM2 upregulation was confirmed by western blot and qPCR analysis. After treatment with MDM2 inhibitor SAR405838, DDLPS spheroids demonstrated different sensitivity patterns from 2D models. Taken together, we believed that 3D models would have a possibility to provide us a new predictability of efficacy and toxicity, and considered as one important process in in vitro pre-clinical phase prior to moving forward to clinical trials.

Epidemiology and survival analysis of non-GIST GI sarcomas: A two-decade analysis of SEER database.

812 Background: Primary adult non-GIST (gastrointestinal stromal tumors) gastrointestinal sarcomas are rare, accounting for 1-2% of all GI malignancies. Given the paucity of literature, we aimed to study the epidemiology and clinical outcomes of these sarcomas. Methods: National SEER-18 registry database was analyzed from 2001 to 2019 to identify non-GIST-GI sarcomas among adults aged 20 years and older. Demographics and clinical factors, including age at diagnosis, gender, race, primary cancer site, histology group, tumor grade and treatment, were examined in descriptive analysis. Log-rank tests, KM plots and Cox regression were utilized for observed survival analysis. P-value of <0.01 was considered statistically significant. Retroperitoneal sarcomas were excluded. Results: A total of 1277 non-GIST-GI sarcomas were identified, 53.6% were male. Common primary sites include small intestine (32.4%), stomach (27.0%), colon (23.0%), esophagus (8.4%), rectum (8.3%) and anus (0.9%). Most common histological subtype was leiomyosarcoma (52.8%) followed by sarcomatoid carcinoma (10.6%), spindle cell sarcoma (6.2%), carcinosarcoma (5.9%), liposarcoma (5.2%) and angiosarcoma (4.7%). Surgery only was the most common treatment received (61.4%). Stratified by primary tumor site, five-year survival rate was highest for colon (46%; 95% CI 40-52) followed by anus (HR 44%; 95% CI 15-70), stomach (HR 42%; 95% CI 36-47), rectum (HR 36%; 95% CI 27-46), small intestine (HR 36%; 95% CI 31-41) and esophagus (HR 30%; 95% CI 21-38).Similarly, for common histological subtypes 5-year survival was highest for liposarcoma (HR 56%; 95% CI 43-68) followed by leiomyosarcoma (HR 46%; 95% CI 42-50) and spindle cell sarcoma (HR 36%; 95% CI 25-47) and lowest for carcinosarcoma (HR 15%; 95% CI 8-25) followed by sarcomatoid carcinoma (HR 17%; 95% CI 11-24), and angiosarcoma (HR 17%; 95% CI 9-28).On multivariate cox regression analysis, factors associated with unfavorable prognosis were male gender (HR 1.21; 95% CI 1.06-1.39), small intestine (HR 1.38; 95% CI 1.15-1.65) sarcomatoid sarcoma (HR 1.42; 95% CI 1.06-1.92), poorly differentiated tumors(HR 2.90; 95% CI 1.99-2.43). Esophageal primary (HR 0.66; 95% CI 0.49-0.87) and leiomyosarcoma (HR 0.81; 95% CI 0.70-0.94) had a favorable prognosis. Conclusions: Non-GIST-GI sarcomas are very rare with slight male predominance and more than three quarters of cases diagnosed among 55 years and older. Leiomyosarcoma was the most common subtype and small intestine was most common primary site. Overall liposarcoma had the highest five year survival rate and carcinosarcoma had the lowest.On multivariate analysis male gender, small intestinal primary, sarcomatoid carcinoma had unfavorable prognosis.