Alveolar Rhabdomyosarcoma Cell Lines Research Articles

Abstract 1094: Utilizing CRISPR-Cas13 as a novel functional genomics tool to characterize PAX3-FOXO1 in alveolar rhabdomyosarcoma

Abstract Background: CRISPR-Cas13 (Cas13) is an RNA editing system that can knockdown gene transcripts with higher efficiency and specificity than RNA interference technologies. We are exploring the utility of Cas13 as a robust novel functional genomics tool to understand the biology of the PAX3-FOXO1(P3F1) gene fusion in alveolar rhabdomyosarcoma (ARMS). Our goal is to identify gene dependencies and novel druggable targets. [LB1] Aim: This study aims to functionally characterize P3F1 in paediatric cancer cell lines using Cas13. [LB2] Methods & Results: Transient transfection and viral transduction of Cas13 vector systems were used to demonstrate that Cas13 knockdown of target gene expression can be achieved in ARMS cell lines. We developed guides to specifically target P3F1 expression and tested their efficiency in HEK293T cells. Cas13 function was validated with a mCherry knockdown proof-of-concept expression in the ARMS cell lines Rh5 and Rh30. 90-95% knockdown efficiency was seen over three independent experiments. Ten P3F1 breakpoint targeting guides were designed and screened first in a HEK293T P3F1 overexpression model to select optimal high efficiency targeting guides. The highest efficiency targeting guide showed a GFP reduction of 70% in three independent experiments. High-efficiency Cas13 guides were taken forward into Rh5 cells to knockdown endogenous P3F1 expression which were validated by western blotting and RT-PCR. Western blots of Cas13 sorted Rh5 cells showed that guide 1150 was able to knockdown P3F1 protein expression by ~70%. Discussion/Conclusion: These findings demonstrate the high efficiency and specificity of the Cas13 system to silence P3F1, and other oncogenic fusion drivers. Further studies will utilize Cas13 to characterize the molecular and cellular consequences of P3F1 gene-silencing in patient-derived ARMS cell lines. Citation Format: Andrea Zhao, Lauren M. Brown, Mohamed Fareh, Paul G. Ekert. Utilizing CRISPR-Cas13 as a novel functional genomics tool to characterize PAX3-FOXO1 in alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1094.

Abstract 143: Molecular profiling of tumor-initiation events in rhabdomyosarcoma uncovers mitochondrial vulnerabilities

Abstract Background: Alveolar Rhabdomyosarcoma (ARMS) is a very aggressive soft tissue tumor affecting children and adolescents. It is found that pediatric patients with RMS harboring chimeric PAX fusion transcription factors (TFs) exhibit a greater incidence of tumor relapse, metastasis, and poor survival outcome, thereby underscoring the urgent need to develop effective therapies to treat this subtype of childhood cancer. Methods: We generated a novel ARMS model by stable transduction of myogenic progenitor cells with a cassette encoding the exact PAX3/7-FOXO1 fusion protein found in ARMS tumors. We analyzed genome-wide expression levels with RNA-seq and utilized Cut&Run to map the binding sites of PAX3/7-FOXO1, active histone marks, enhancers, and super-enhancers in our novel model. Hi-C and promoter capture HiC (pCHiC) were also performed to generate 3D chromatin conformation maps in cells expressing the oncogenic TFs. We compared these data with analogous datasets from orthotopic patient-derived xenografts (O-PDXs) of PAX3/7-FOXO1 fusion-positive ARMS tumors from St. Jude and established ARMS cell lines to validate whether our model system recapitulates ARMS tumors. CRISPRi was performed to silence key targets in two representative ARMS cell lines and proliferation was scored after silencing. Next, we found that ARMS tumors displayed anomalies in mitochondrial mass and numbers, metabolic parameters, and membrane potential by performing TEM, Seahorse assays, and FACS-based TMRM and mitotracker Red dye uptake in a panel of ARMS cells and PDX samples. We performed cell viability and colony forming assays in ARMS cells treated with single drugs and combinations of drugs targeting FGFR4 and mitochondrial metabolism. Results: Our data provide evidence to support muscle progenitors as potential cells-of-origin for ARMS. We showed that rewiring of chromatin architecture mediated by PAX3/7-FOXO1 TFs is evident across established cell lines, primary tumors as well as O-PDXs. By integrating PAX3/7-FOXO1 binding, RNA-seq, and pCHiC data, we identified key promoter-enhancer (P-En) interactions that are erased or established de novo at multiple loci of master TFs like MYOD, MYOG and MYCN. We also identified key regulators of proliferation, including FGFR4, and mitochondrial function that are essential for tumor growth and invasiveness. Our data indicated aberrant and enhanced mitochondrial metabolism in ARMS as well as selective sensitivity to mitochondrial inhibitors in RMS cells. Administration of these inhibitors preferentially inhibited cell viability and clonogenicity in ARMS, and prompted a pre-clinical phase II study to test these inhibitors at St. Jude Research Hospital. Conclusion: We present a novel treatment strategy leveraging the mitochondrial dependencies of fusion positive PAX-driven tumors which can be exploited for precision therapy in high-risk ARMS. Citation Format: Bhargab Kalita, Gerard Martinez-Cebrian, Justina McEvoy, Brittney Gordon, Kaley Blankenship, Asa Karlstorm, Elizabeth Stewart, Michael Dyer, Brian Dynlacht. Molecular profiling of tumor-initiation events in rhabdomyosarcoma uncovers mitochondrial vulnerabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 143.

A perfusion-based three-dimensional cell culture system to model alveolar rhabdomyosarcoma pathological features

Although a rare disease, rhabdomyosarcoma (RMS) is one of the most common cancers in children the more aggressive and metastatic subtype is the alveolar RMS (ARMS). Survival outcomes with metastatic disease remain dismal and the need for new models that recapitulate key pathological features, including cell-extracellular matrix (ECM) interactions, is warranted. Here, we report an organotypic model that captures cellular and molecular determinants of invasive ARMS. We cultured the ARMS cell line RH30 on a collagen sponge in a perfusion-based bioreactor (U-CUP), obtaining after 7 days a 3D construct with homogeneous cell distribution. Compared to static culture, perfusion flow induced higher cell proliferation rates (20% vs. 5%), enhanced secretion of active MMP-2, and upregulation of the Rho pathway, associated with cancer cell dissemination. Consistently, the ECM genes LAMA1 and LAMA2, the antiapoptotic gene HSP90, identified in patient databases as hallmarks of invasive ARMS, were higher under perfusion flow at mRNA and protein level. Our advanced ARMS organotypic model mimics (1) the interactions cells-ECM, (2) the cell growth maintenance, and (3) the expression of proteins that characterize tumor expansion and aggressiveness. In the future, the perfusion-based model could be used with primary patient-derived cell subtypes to create a personalized ARMS chemotherapy screening system.

EHMT1 promotes tumor progression and maintains stemness by regulating ALDH1A1 expression in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with poor prognosis. Cancer stem cells (CSCs) are seeds for tumor relapse and metastasis. However, pathways that maintain stemness genes are not fully understood. Here, we report that the enzyme euchromatic histone lysine methyltransferase 1 (EHMT1) is expressed in primary and relapse ARMS tumors. EHMT1 suppression impaired motility and induced differentiation in ARMS cell lines and reduced tumor progression in a mouse xenograft model in vivo. RNA sequencing of EHMT1-depleted cells revealed downregulation of ALDH1A1 that is associated with CSCs. Consistent with this, inhibition of ALDH1A1 expression and activity mimicked EHMT1 depletion phenotypes and reduced tumorsphere formation. Mechanistically, we demonstrate that EHMT1 does not bind to the ALDH1A1 promoter but activates it by stabilizing C/EBPβ, a known regulator of ALDH1A1 expression. Our findings identify a role for EHMT1 in maintenance of stemness by regulating ALDH1A1 expression and suggest that targeting ALDH+ cells is a promising strategy in ARMS. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cytotoxic Synergism between a Proprietary Commiphora mukul Gum Extract GU-MCT810, 2-deoxy-D-glucose, and Metformin in Human Alveolar Rhabdomyosarcoma and Hepatoma Cell Lines In vitro

In this investigation we have analyzed the synergism for the cytotoxic effect of a proprietary guggul gum extract (GU), 2-deoxy-D-glucose (2-DG) and metformin (Met) in SJRH30 human alveolar rhabdomyosarcoma and HepG2 hepatoma cell lines in vitro. 2-DG and Met as single agents have weak cytotoxic effects in both cell lines. However, the combination of GU+2-DG, GU+Met and 2-DG+Met showed synergism for cytotoxic effect by CompuSyn analysis. Therefore, GU can be included in the combination of drugs involving 2-DG and Met to have synergistic effect. GU also showed a dose-dependent increase in cellular glucose uptake in HepG2 cells like the antidiabetic drug 2,4-thiozolidine dione (TZ). The demonstration of synergism of anticancer effects between GU, metformin and 2-DG, suggest that their mechanisms are in general complementary, though further studies are required to delineate the mechanism of GU, 2-DG and metformin combinations.

Vincetoxicum arnottianum modulates motility features and metastatic marker expression in pediatric rhabdomyosarcoma by stabilizing the actin cytoskeleton

BackgroundPrevention of metastatic invasion is one of the main challenges in the treatment of alveolar rhabdomyosarcoma. Still the therapeutic options are limited. Therefore, an anti-tumor screening was initiated focusing on the anti-metastatic and anti-invasion properties of selected medicinal plant extracts and phytoestrogens, already known to be effective in the prevention and treatment of different cancer entities.MethodsTreatment effects were first evaluated by cell viability, migration, invasion, and colony forming assays on the alveolar rhabdomyosarcoma cell line RH-30 in comparison with healthy primary cells.ResultsInitial anti-tumor screenings of all substances analyzed in this study, identified the plant extract of Vincetoxicum arnottianum (VSM) as the most promising candidate, harboring the highest anti-metastatic potential. Those significant anti-motility properties were proven by a reduced ability for migration (60%), invasion (99%) and colony formation (61%) under 48 h exposure to 25 μg/ml VSM. The restricted motility features were due to an induction of the stabilization of the cytoskeleton – actin fibers were 2.5-fold longer and were spanning the entire cell. Decreased proliferation (PCNA, AMT, GCSH) and altered metastasis (e. g. SGPL1, CXCR4, stathmin) marker expression on transcript and protein level confirmed the significant lowered tumorigenicity under VSM treatment. Finally, significant alterations in the cell metabolism were detected for 25 metabolites, with levels of uracil, N-acetyl serine and propanoyl phosphate harboring the greatest alterations. Compared to the conventional therapy with cisplatin, VSM treated cells demonstrated a similar metabolic shutdown of the primary cell metabolism. Primary control cells were not affected by the VSM treatment.ConclusionsThis study revealed the VSM root extract as a potential, new migrastatic drug candidate for the putative treatment of pediatric alveolar rhabdomyosarcoma with actin filament stabilizing properties and accompanied by a marginal effect on the vitality of primary cells.Graphical abstract

Differential regulation of autophagy by STAU1 in alveolar rhabdomyosarcoma and non-transformed skeletal muscle cells.

Recent work has highlighted the therapeutic potential of targeting autophagy to modulate cell survival in a variety of diseases including cancer. Recently, we found that the RNA-binding protein Staufen1 (STAU1) is highly expressed in alveolar rhabdomyosarcoma (ARMS) and that this abnormal expression promotes tumorigenesis. Here, we asked whether STAU1 is involved in the regulation of autophagy in ARMS cells. We assessed the impact of STAU1 expression modulation in ARMS cell lines (RH30 and RH41), non-transformed skeletal muscle cells (C2C12) and STAU1-transgenic mice using complementary techniques. We found that STAU1 silencing reduces autophagy in the ARMS cell lines RH30 and RH41, while increasing their apoptosis. Mechanistically, this inhibitory effect was found to be caused by a direct negative impact of STAU1 depletion on the stability of Beclin-1 (BECN1) and ATG16L1 mRNAs, as well as by an indirect inhibition of JNK signaling via increased expression of Dual specificity phosphatase 8 (DUSP8). Pharmacological activation of JNK or expression silencing of DUSP8 was sufficient to restore autophagy in STAU1-depleted cells. By contrast, we found that STAU1 downregulation in non-transformed skeletal muscle cells activates autophagy in a mTOR-dependent manner, without promoting apoptosis. A similar effect was observed in skeletal muscles obtained from STAU1-overexpressing transgenic mice. Together, our data indicate an effect of STAU1 on autophagy regulation in ARMS cells and its differential role in non-transformed skeletal muscle cells. Our findings suggest a cancer-specific potential of targeting STAU1 for the treatment of ARMS.

Epigenetic regulator BMI1 promotes alveolar rhabdomyosarcoma proliferation and constitutes a novel therapeutic target.

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma. There are two main subtypes of RMS, alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma. ARMS typically encompasses fusion‐positive rhabdomyosarcoma, which expresses either PAX3‐FOXO1 or PAX7‐FOXO1 fusion proteins. There are no targeted therapies for ARMS; however, recent studies have begun to illustrate the cooperation between epigenetic proteins and the PAX3‐FOXO1 fusion, indicating that epigenetic proteins may serve as targets in ARMS. Here, we investigate the contribution of BMI1, given the established role of this epigenetic regulator in sustaining aggression in cancer. We determined that BMI1 is expressed across ARMS tumors, patient‐derived xenografts, and cell lines. We depleted BMI1 using RNAi and inhibitors (PTC‐209 and PTC‐028) and found that this leads to a decrease in cell growth/increase in apoptosis in vitro, and delays tumor growth in vivo. Our data suggest that BMI1 inhibition activates the Hippo pathway via phosphorylation of LATS1/2 and subsequent reduction in YAP levels and YAP/TAZ target genes. These results identify BMI1 as a potential therapeutic vulnerability in ARMS and warrant further investigation of BMI1 in ARMS and other sarcomas.

Abstract 5001: Barriers to adoptive immunotherapy of pediatric cancer

Abstract As the adoptive immunotherapy with chimeric antigen modified T cells (CAR-T) has not proven effective in pediatric solid tumors, we established a model to define which tumor- and stromal-derived factors must be surmounted to obtain effective treatment. The human alveolar rhabdomyosarcoma cell line RH30 (fusion gene positive) was administered both i.v. and i.m. to NSG mice, and subsequently treated with a FGFR4-specifc CAR-T cells (expressing the M410 binder). Although some response was noted in the i.v. model, the i.m. tumor was entirely resistant. We observed that in both primary tumors and liver metastases RH30 was surrounded with a rich stromal element when CAR-T or control activated T cells were administered. Trichrome and H&E histochemical analysis of i.m. tumors confirmed the presence of a rich collagen-containing stromal element in T cell treated mice. This was not present in untreated tumors. We therefore analyzed cell surface and soluble factors produced by RH30 by RNA array analysis, flow cytometry and ELISA in cultured RH30 alone, or treated with interferon-gamma (IFNG) to model the presence of activated T cells. RH30 expressed transcripts for both canonical and non-canonical chemokines: CCL24, GPI, MIF, CCL26, CCL20, CCL2; as well as the cytokines: IL-15, IL-32, LIF, TGFB1, and CSF1. ELISA confirmed high expression of MIF, TGFB1, CCL24 and LIF. Transcripts for the chemokines CCL8, 9, 10, and 11 were expressed at low or undetectable levels, but were strongly induced by IFNG. And although class I MHC was present, and upregulated in the presence of IFNG, so was PDL1. Surface marker analysis by flow cytometry demonstrated that CD80, CD86 and CXCR2 are absent while the CAR-T targets CD276, EGFR, FGFR4 as well as CXCR4 are present. Flow cytometry also demonstrated consistent expression of PDL2, GAL-9, and CTLA4, and strong induction of HLA class I and PDL1 by IFNG treatment of RH30. We are currently evaluating CRISPR knock-out lines of each soluble factor for effects on gene expression, IFNG-induced marker expression, and tumor stroma in vivo. Our results indicate that in this model of rhabdomyosarcoma, tumor surface expression of glycoproteins that give an immune inhibitory signal, PDL1, PDL2, CTLA4, GAL-9, as well as soluble modulators known to be associated with tumor escape from immune control, MIF, TGFB, and LIF, all must be considered in armoring CAR-T for effective immune destruction of tumor. Moreover, the numerous chemokines produced by tumor alone, as well as induced by IFNG, indicate a complex network for recruitment of local and bone marrow-derived stromal cells that create a physical as well as cellular barrier to adoptive immunotherapy. Citation Format: Rajesh Kumar, Nityashree Shivaprasad, Virginia Hoglund, Cosette LeCeil, Lingyan Wang, Yue Zhang, Dimiter S. Dimitrov, Javed Khan, Rimas J. Orentas. Barriers to adoptive immunotherapy of pediatric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5001.

Assessing alveolar rhabdomyosarcoma cell lines as tumor models by comparison of mRNA expression profiles.

Numerous cell lines for human alveolar rhabdomyosarcoma (ARMS) have been developed and are widely used to study biological processes of this myogenic cancer. The present study investigated the resemblance of commonly used ARMS cell lines to primary tumors in regards to gene expression. RNA-sequencing data was retrieved from published datasets for 4 commonly used ARMS cell lines and 35 ARMS primary tumors. The genes with most variable expression across primary tumors were used to calculate rank-based Spearman's correlation. The observed median correlations ranged from 0.36 to 0.61. RH-41 showed the highest median correlation while KYM-1 was the least correlated cell line. A significant number of genes dysregulated between tumors and non-tumors also exhibited similar expression patterns between tumors and cell lines, including The findings suggest that ARMS cell lines exhibit changes in gene expression compared to primary tumors and may not be completely representative of the disease process.

Establishment and characterization of a novel alveolar rhabdomyosarcoma cell line, NCC-aRMS1-C1

Alveolar rhabdomyosarcoma (aRMS) is a histological subtype of RMS, which is the most common pediatric and adolescent soft-tissue sarcoma, accounting for 3-4% of all pediatric malignancies. Patient-derived cells are essential tools for understanding the molecular mechanisms of poor prognosis and developing novel anti-cancer drugs. However, only a limited number of well-characterized cell lines for rhabdomyosarcoma from public cell banks is available. Therefore, we aimed to establish a novel cell line of aRMS from the tumor tissue of a patient with aRMS. The cell line was established from surgically resected tumor tissue from a 4-year-old male patient diagnosed with stage III, T2bN1M0 aRMS and was named as NCC-aRMS1-C1. The cells were maintained for more than 3months under tissue culture conditions and passaged more than 20 times. We confirmed the presence of identical fusion gene such as PAX7-FOXO1 in both the original tumor and NCC-aRMS1-C1. The cells exhibited spheroid formation and invasion. We found that docetaxel, vincristine, ifosfamide, dacarbazine, and romidepsin showed remarkable growth-suppressive effects on the NCC-aRMS1-C1 cells. In conclusion, the NCC-aRMS1-C1 cell line exhibited characteristics that may correspond to the lymph node metastasis in aRMS and mirror its less aggressive features. Thus, it may be useful for innovative seeds for novel therapeutic strategies.

Up-regulation of GLI1 in vincristine-resistant rhabdomyosarcoma and Ewing sarcoma

BackgroundThe clinical significance of GLI1 expression either through canonical Hedgehog signal transduction or through non-canonical mechanisms in rhabdomyosarcoma (RMS) or Ewing sarcoma (EWS) is incompletely understood. We tested a role for Hedgehog (HH) signal transduction and GL11 expression in development of vincristine (VCR) resistance in RMS and EWS.MethodsWe characterized baseline expression and activity of HH pathway components in 5 RMS (RD, Rh18, Ruch-2, Rh30, and Rh41) and 5 EWS (CHLA9, CHLA10, TC32, CHLA258, and TC71) cell lines. We then established VCR-resistant RMS and EWS cell lines by exposing cells to serially increasing concentrations of VCR and determining the IC50. We defined resistance as a ≥ 30-fold increase in IC50 compared with parental cells. We determined changes in gene expression in the VCR-resistant cells compared with parental cells using an 86-gene cancer drug resistance array that included GLI1 and tested the effect of GLI1 inhibition with GANT61 or GLI1 siRNA on VCR resistance.ResultsWe found evidence for HH pathway activity and GLI1 expression in RMS and EWS cell lines at baseline, and evidence that GLI1 contributes to survival and proliferation of these sarcoma cells. We were able to establish 4 VCR-resistant cell lines (Ruch-2VR, Rh30VR, Rh41VR, and TC71VR). GLI1 was significantly up-regulated in the Rh30VR, Rh41VR, and TC71VR cells. The only other gene in the drug resistance panel that was significantly up-regulated in each of these VCR-resistant cell lines compared with their corresponding parental cells was the GLI1 direct target and multidrug resistance gene, ATP-binding cassette sub-family B member 1 (MDR1). We established major vault protein (MVP), which was up-regulated in both vincristine-resistant alveolar RMS cell lines (Rh30VR and Rh41VR), as another direct target of GLI1 during development of drug resistance. Treatment of the VCR-resistant cell lines with the small molecule inhibitor GANT61 or GLI1 siRNA together with VCR significantly decreased cell viability at doses that did not reduce viability individually.ConclusionsThese experiments demonstrate that GLI1 up-regulation contributes to VCR resistance in RMS and EWS cell lines and suggest that targeting GLI1 may benefit patients with RMS or EWS by reducing multidrug resistance.

A method to culture human alveolar rhabdomyosarcoma cell lines as rhabdospheres demonstrates an enrichment in stemness and Notch signaling.

ABSTRACTThe development of three-dimensional cell culture techniques has allowed cancer researchers to study the stemness properties of cancer cells in in vitro culture. However, a method to grow PAX3-FOXO1 fusion-positive rhabdomyosarcoma (FP-RMS), an aggressive soft tissue sarcoma of childhood, has to date not been reported, hampering efforts to identify the dysregulated signaling pathways that underlie FP-RMS stemness. Here, we first examine the expression of canonical stem cell markers in human RMS tumors and cell lines. We then describe a method to grow FP-RMS cell lines as rhabdospheres and demonstrate that these spheres are enriched in expression of canonical stemness factors as well as Notch signaling components. Specifically, FP-RMS rhabdospheres have increased expression of SOX2, POU5F1 (OCT4), and NANOG, and several receptors and transcriptional regulators in the Notch signaling pathway. FP-RMS rhabdospheres also exhibit functional stemness characteristics including multipotency, increased tumorigenicity in vivo, and chemoresistance. This method provides a novel practical tool to support research into FP-RMS stemness and chemoresistance signaling mechanisms.

“Verteporfin exhibits anti-proliferative activity in embryonal and alveolar rhabdomyosarcoma cell lines”

Rhabdomyosarcoma (RMS) is a pediatric tumor, which arises from muscle precursor cells. Recently, it has been demonstrated that Hippo Pathway (Hpo), a pathway that regulates several physiological and biological features, is involved in RMS tumorigenesis. For instance, an upregulation of the Hpo downstream effector Yes-Associated Protein 1 (YAP) leads to the development of embryonal rhabdomyosarcoma (eRMS) in murine activated muscle satellite cells. On the other hand, the YAP paralog transcriptional co-activator with PDZ-binding motif (TAZ) is overexpressed in alveolar rhabdomyosarcoma (aRMS) patients with poor survival.YAP and TAZ exhibit both cytoplasmic and nuclear functions. In the nucleus, YAP binds TEADs (TEA domain family members) factors and together they constitute a complex that is able either to activate the transcription of several genes such as MYC, Tbx5 and PAX8 or to maintain the stability of others like p73. Due to the key role of YAP and TAZ in cancer, the identification and/or development of new compounds able to block their activity might be an effective antineoplastic strategy. Verteporfin (VP) is a molecule able to stop the formation of YAP/TEAD complex in the nucleus.The aim of this study is to evaluate the action of VP on RMS cell lines.This work shows that VP has an anti-proliferative activity on all RMS cell lines analyzed. Depending on RMS cell lines, VP affects cell cycle differently. Moreover, VP is able to decrease YAP protein levels, and to induce the activation of apoptosis mechanism through the cleavage of PARP-1. In addition, Annexin V assay showed the activation of apoptosis and necrosis after VP treatment.In summary, the ability of VP to disrupt RMS cell proliferation could be a novel and valuable strategy to improve the therapeutic approaches in treating rhabdomyosarcoma.

Mechanisms of simvastatin myotoxicity: The role of autophagy flux inhibition

Statins are some of the most widely used drugs worldwide, but one of their major side effects is myotoxicity. Using mouse myoblast (C2C12) and human alveolar rhabdomyosarcoma cell lines (RH30) in both 2-dimensional (2D) and 3-dimensional (3D) cell culture, we investigated the mechanisms of simvastatin's myotoxicity. We found that simvastatin significantly reduced cell viability in C2C12 cells compared to RH30 cells. However, simvastatin induced greater apoptosis in RH30 compared to C2C12 cells. Simvastatin-induced cell death is dependent on geranylgeranyl pyrophosphate (GGPP) in C2C12 cells, while in RH30 cells it is dependent on both farnesyl pyrophosphate (FPP) and GGPP. Simvastatin inhibited autophagy flux in both C2C12 and RH30 cells and inhibited lysosomal acidification in C2C12 cells, while autophagy inhibition with Bafilomycin-A1 increased simvastatin myotoxicity in both cell lines. Simvastatin induced greater cell death in RH30 cells compared to C2C12 in a 3D culture model with similar effects on autophagy flux as in 2D culture. Overall, our results suggest that simvastatin-induced myotoxicity involves both apoptosis and autophagy, where autophagy serves a pro-survival role in both cell lines. The sensitivity to simvastatin-induced myotoxicity differs between 2D and 3D culture, demonstrating that the cellular microenvironment is a critical factor in regulating simvastatin-induced cell death in myoblasts.

Inhibition of Chemoresistance in Primary Tumor Cells by Camellia sinensis non fermentatum Extract Noviphenone (NPE®).

Despite improvements in cancer therapy, life expectancy after tumor recurrence remains low. Relapsed cancer is characterized by drug resistance, often mediated through overexpression of multidrug resistance (MDR) genes. Camellia sinensis non fermentatum extract is known for its anticancer properties in several cancer cell lines and might improve cancer therapy outcome after tumor recurrence. Embryonal rhabdomyosarcoma cell lines, alveolar rhabdomyosarcoma cell lines and primary rhabdomyosarcoma MAST139 cells were used to test NPE® effects on cell viability in combination with chemotherapeutic agents. Cell viability was measured by the WST-1 assay and CV staining. Gene expression levels of chemotherapy-induced efflux pumps and their activity was assessed upon NPE® treatment by measuring doxorubicin retention through evaluation of the autofluorescence signal. Administration of increasing doxorubicin concentrations triggered immediate adaptation to the drug, which was surprisingly overcome by the addition of NPE®. Investigating the mechanism of immediate adaptation, MDR1 gene overexpression was observed upon doxorubicin treatment. Although NPE® did not alter pump gene expression, it was able to reduce pump activity, thus allowing the chemotherapeutic agent to stay inside the cells to exert its full anticancer activity. NPE® might improve chemotherapeutic treatment by re-sensitizing relapsed tumors to anticancer drugs. Fighting MDR represents the key to overcome tumor relapse and improve the overall survival of cancer patients.

Abstract 3838: Targeting epigenetic regulator BMI-1 in alveolar rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is an extremely aggressive soft tissue sarcoma which affects mainly children. There are two subtypes: alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). ARMS is characterized by PAX-FOXO1 fusion proteins, whereas subsets of ERMS harbor alterations within RAS and TP53 pathways. Currently, the outcomes for ARMS (especially when metastatic) remain dismal, thus underscoring the urgent need to identify novel targets for this cancer. The genomic landscape of many pediatric cancers, including ARMS, is relatively sparse. This led us to ask whether key epigenetic factors are driving tumor aggressiveness and could constitute novel approaches for treating ARMS. Notably, the epigenetic complexes PRC1 and PRC2 are overexpressed in a variety of sarcomas and are associated with worse overall survival. We took a hypothesis-based approach and focused on PRC1. We discovered that B lymphoma Mo-MLV insertion region 1 (BMI-1), a protein member of PRC1, is overexpressed in ARMS cells. BMI-1 is a known oncogene in other cancers, but its potential oncogenic role in ARMS and other pediatric malignancies has not yet been interrogated; thus we aim to study it within this context. Methods: To analyze the function of BMI-1 in ARMS, we depleted the protein in ARMS cell line models by both shRNA/siRNA knockdown and measured expression, cell proliferation and apoptosis. We utilized two small molecule inhibitors, PTC-209 and PTC-028, to obtain IC50s in these cell lines, then determined effects on cell proliferation and apoptosis. Results: We examined RNA-Seq tumor datasets and determined that BMI1 is robustly expressed in ARMS tumors. Additionally, we confirmed that BMI-1 is also overexpressed in ARMS cell lines at the levels of RNA and protein. Next, we depleted BMI-1 using multiple shRNAs and siRNAS and found that this led to striking (~70%) decreases in cell growth. We also observed increased levels of apoptosis within knockdown cells. Given these results, we asked whether small molecule inhibitors of BMI-1 mediated similar phenotypes, and so we used the inhibitors PTC-209 and PTC-028. PTC-209 is a first-generation BMI-1 inhibitor, while PTC-028 is a second-generation orally available inhibitor with higher potency. Both compounds inhibited BMI-1 function and greatly reduced cell proliferation in ARMS cell lines within the nanomolar range; however, as expected, PTC-028 showed a more pronounced effect compared to PTC-209. Conclusions: BMI1 supports proliferation and survival in cell line models of ARMS. Both chemical and pharmacologic inhibition of BMI1 led to striking decreases in cell proliferation. Currently, we are further investigating the molecular impact of BMI1 inhibition, with plans to investigate its effectiveness within an in vivo ARMS model. Targeting BMI-1 pharmacologically could provide a novel therapeutic option for patients with ARMS and may apply more broadly to other sarcomas. Citation Format: Cara E. Shields, Selma M. Cuya, Sarah K. Chappell, Komal Rathi, Shiv Patel, Sindhu Potlapalli, Robert W. Schnepp. Targeting epigenetic regulator BMI-1 in alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3838.

Abstract 3909: Potential antitumor effect of a pan-PI3K inhibitor ZSTK474 on human sarcoma cell lines

Abstract Phosphatidylinositol-3 kinase (PI3K) is a key signaling molecule for tumor growth and survival, thus the PI3K pathway has been thought to be a promising target for cancer therapy. We previously identified a novel phosphatidylinositol-3 kinase (PI3K) inhibitor, ZSTK474, by its similarity of antiproliferative profile across the JFCR39 cancer cell line panel to a known PI3K inhibitor, LY294002. Since then, ZSTK474 has been evaluated in preclinical models as well as in clinical trials in the US and Japan against a range of advanced solid tumors. The US trial revealed that three of four sarcoma patients exhibited stable disease, suggesting possible clinical benefit in this disease. Treatment of patients with advanced sarcoma remains challenging due to lack of effective medicine. Therefore the results of clinical trial prompted us to examine antitumor activity of this compound in various subtypes of sarcomas. To this end, we developed a sarcoma panel comprising of 17 human sarcoma cell lines from a variety of subtypes and investigated the effect of ZSTK474 across the panel and compare it with those of other molecularly targeted agents and clinically used chemotherapeutic agents for the treatment of sarcoma, such as doxorubicin, paclitaxel and gemcitabine. As a result, ZSTK474 exhibited a unique antiproliferative profile that was similar to other PI3K inhibitor but clearly different from conventional chemotherapeutic drugs examined, as we previously reported using JFCR39 carcinoma cell line panel. Indeed, ZSTK474 inhibited PI3K-downstream pathways, in parallel to growth inhibition, in all the sarcoma cell lines examined, showing proof-of-concept of PI3K inhibition. Of note, ZSTK474 exerted a potent antiproliferative effect on SW684, a fibrosarcoma cell line and MES-SA/Dx5, and a uterine sarcoma cell line that acquired resistance to doxorubicin via overexpression of P-glycoprotein, while both exhibited resistance to most of the chemotherapeutic agents examined. Moreover, ZSTK474 induced extensive apoptosis selectively in Ewing’s sarcoma and alveolar rhabdomyosarcoma cell lines, both of which harbor chromosomal translocations and resulting fusion genes, EWSR1-FLI1 and PAX3-FOXO1, respectively, whereas remaining sarcoma cell lines as well as carcinoma cell lines examined so far hardly underwent apoptosis. Additional cell lines from synovial sarcoma harboring SS18-SSX1/2 fusion gene also underwent apoptosis, suggesting preferential induction of apoptosis in oncogenic chromosomal translocation positive sarcoma cells. Finally, animal experiments using nude mice bearing human sarcoma xenograft confirmed the antitumor activity of ZSTK474 in vivo, with superior efficacy observed in translocation-positive cells. These results suggest that ZSTK474 could be a promising drug candidate for sarcomas, especially those harboring oncogenic chromosomal translocation. Citation Format: Shingo Dan, Naomi Tamaki, Nachi Namatame, Yuya Yoshizawa, Mutsumi Okamura, Yumiko Nishimura, Kanami Yamazaki, Shin-ichi Yaguchi. Potential antitumor effect of a pan-PI3K inhibitor ZSTK474 on human sarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3909.

NRF2 orchestrates the redox regulation induced by radiation therapy, sustaining embryonal and alveolar rhabdomyosarcoma cells radioresistance.

Tumor cells generally exhibit higher levels of reactive oxygen species (ROS), however, when stressed, tumor cells can undergo a process of 'Redox Resetting' to acquire a new redox balance with stronger antioxidant systems that enable cancer cells to become resistant to radiation therapy (RT). Here, we describe how RT affects the oxidant/antioxidant balance in human embryonal (RD) and alveolar (RH30) rhabdomyosarcoma (RMS) cell lines, investigating on the molecular mechanisms involved. Radiations were delivered using an x-6 MV photon linear accelerator and their effects were assessed by vitality and clonogenic assays. The expression of specific antioxidant-enzymes, such as Superoxide Dismutases (SODs), Catalase (CAT) and Glutathione Peroxidases 4 (GPx4), miRNAs (miR-22, -126, -210, -375, -146a, -34a) and the transcription factor NRF2 was analyzed by quantitative polymerase chain reaction (q-PCR) and western blotting. RNA interference experiments were performed to evaluate the role of NRF2. Doses of RT higher than 2Gy significantly affected RMS clonogenic ability by increasing ROS production. RMS rapidly and efficiently brought back ROS levels by up-regulating the gene expression of antioxidant enzymes, miRNAs as well as of NRF2. Silencing of NRF2 restrained the RMS ability to counteract RT-induced ROS accumulation, antioxidant enzyme and miRNA expression and was able to increase the abundance of γ-H2AX, a biomarker of DNA damage, in RT-treated cells. Taken together, our data suggest the strategic role of oxidant/antioxidant balance in restraining the therapeutic efficiency of RT in RMS treatment and identify NRF2 as a new potential molecular target whose inhibition might represent a novel radiosensitizing therapeutic strategy for RMS clinical management.

PARP inhibitors affect growth, survival and radiation susceptibility of human alveolar and embryonal rhabdomyosarcoma cell lines

PurposePARP inhibitors (PARPi) are used in a wide range of human solid tumours but a limited evidence is reported in rhabdomyosarcoma (RMS), the most frequent childhood soft-tissue sarcoma. The cellular and molecular effects of Olaparib, a specific PARP1/2 inhibitor, and AZD2461, a newly synthesized PARP1/2/3 inhibitor, were assessed in alveolar and embryonal RMS cells both as single-agent and in combination with ionizing radiation (IR).MethodsCell viability was monitored by trypan blue exclusion dye assays. Cell cycle progression and apoptosis were measured by flow cytometry, and alterations of specific molecular markers were investigated by, Real Time PCR, Western blotting and immunofluorescence experiments. Irradiations were carried out at a dose rate of 2 Gy (190 UM/min) or 4 Gy (380 UM/min). Radiosensitivity was assessed by using clonogenic assays.ResultsOlaparib and AZD2461 dose-dependently reduced growth of both RH30 and RD cells by arresting growth at G2/M phase and by modulating the expression, activation and subcellular localization of specific cell cycle regulators. Downregulation of phospho-AKT levels and accumulation of γH2AX, a specific marker of DNA damage, were significantly and persistently induced by Olaparib and AZD2461 exposure, this leading to apoptosis-related cell death. Both PARPi significantly enhanced the effects of IR by accumulating DNA damage, increasing G2 arrest and drastically reducing the clonogenic capacity of RMS-cotreated cells.ConclusionsThis study suggests that the combined exposure to PARPi and IR might display a role in the treatment of RMS tumours compared with single-agent exposure, since stronger cytotoxic effects are induced, and compensatory survival mechanisms are prevented.