Alveolar Rhabdomyosarcoma Cell Research Articles

CircAFF1 Is a Circular RNA with a Role in Alveolar Rhabdomyosarcoma Cell Migration.

Circular RNAs (circRNAs), covalently closed RNAs that originate from back-splicing events, participate in the control of several processes, including those that occur in the development of pathological conditions such as cancer. Hereby, we describe circAFF1, a circular RNA overexpressed in alveolar rhabdomyosarcoma. Using RH4 and RH30 cell lines, a classical cell line models for alveolar rhabdomyosarcoma, we demonstrated that circAFF1 is a cytoplasmatic circRNA and its depletion impacts cell homeostasis favouring cell migration through the downregulation of genes involved in cell adhesion pathways. The presented data underline the importance of this circular RNA as a new partial suppressor of the alveolar rhabdomyosarcoma tumour progression and as a putative future therapeutic target.

Transcription regulators and ultra-rare and other rare translocation-related sarcomas treated with trabectedin: A proof of principle from a post-hoc analysis.

Among sarcomas, which are rare cancers with an incidence of <6 per 100.000/year cases, ultra-rare sarcomas have an incidence of approximately ≤1/1,000,000/year cases and altogether account for ~20% of all soft tissue sarcomas (STS) and bone sarcomas. The Italian Sarcoma Group has recently performed a non-interventional, retrospective TrObs study with data from 512 anthracycline-pretreated patients with advanced multiple STS histologies and treated with trabectedin (Palmerini, Cancers 2021; ClinicalTrials.gov Identifier: NCT02793050). A post-hoc analysis of case series to evaluate the efficacy and safety of trabectedin on patients with ultra-rare and other rare translocation-related sarcomas included in TrObs study was performed. Main outcomes comprised investigator-assessed overall response rate (ORR), disease control rate (DCR), progression-free survival (PFS) and safety. Thirty-six patients (18 women) with ultra-rare and other rare sarcoma and a median age of 53.0 years (range: 22-81) were included. Most patients had solitary fibrous tumor (SFT; n=11) followed by epithelioid sarcoma (n=5), malignant peripheral nerve sheath tumor (MPNST; n=4), extraskeletal myxoid chondrosarcoma (EMC; n=3), desmoplastic small round cell tumor (DSRCT; n=3), and alveolar soft part sarcoma (ASPS), rhabdomyosarcoma and clear cell sarcoma (n=2 each). Thirty-five patients had metastatic disease and 23 patients received trabectedin as a second-line treatment. Among 35 patients evaluable for response, two patients with SFT and ASPS had a partial response and one patient with DSRCT obtained a complete response, reaching an ORR of 8.6% (95% CI: 2.8-23.4%). Among patients with an ORR, 6-months PFS was 100% in patients with ASPS, 45.7% in patients with SFT and 33.3% in those with DSRCT. Two patients with epithelioid sarcoma and myoepithelioma had disease stabilization lasting >24 months. Nine patients had at least one grade 3/4 adverse event, mostly being bone marrow toxicity (n=6). Trabectedin has some anti-tumor activity in some ultra-rare and other rare sarcomas, particularly translocation-related sarcomas, with the well-known manageable safety profile.

MRNA Capture Sequencing and RT-qPCR for the Detection of Pathognomonic, Novel, and Secondary Fusion Transcripts in FFPE Tissue: A Sarcoma Showcase.

We assess the performance of mRNA capture sequencing to identify fusion transcripts in FFPE tissue of different sarcoma types, followed by RT-qPCR confirmation. To validate our workflow, six positive control tumors with a specific chromosomal rearrangement were analyzed using the TruSight RNA Pan-Cancer Panel. Fusion transcript calling by FusionCatcher confirmed these aberrations and enabled the identification of both fusion gene partners and breakpoints. Next, whole-transcriptome TruSeq RNA Exome sequencing was applied to 17 fusion gene-negative alveolar rhabdomyosarcoma (ARMS) or undifferentiated round cell sarcoma (URCS) tumors, for whom fluorescence in situ hybridization (FISH) did not identify the classical pathognomonic rearrangements. For six patients, a pathognomonic fusion transcript was readily detected, i.e., PAX3-FOXO1 in two ARMS patients, and EWSR1-FLI1, EWSR1-ERG, or EWSR1-NFATC2 in four URCS patients. For the 11 remaining patients, 11 newly identified fusion transcripts were confirmed by RT-qPCR, including COPS3-TOM1L2, NCOA1-DTNB, WWTR1-LINC01986, PLAA-MOB3B, AP1B1-CHEK2, and BRD4-LEUTX fusion transcripts in ARMS patients. Additionally, recurrently detected secondary fusion transcripts in patients diagnosed with EWSR1-NFATC2-positive sarcoma were confirmed (COPS4-TBC1D9, PICALM-SYTL2, SMG6-VPS53, and UBE2F-ALS2). In conclusion, this study shows that mRNA capture sequencing enhances the detection rate of pathognomonic fusions and enables the identification of novel and secondary fusion transcripts in sarcomas.

The RNA helicase DDX5 cooperates with EHMT2 to sustain alveolar rhabdomyosarcoma growth.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood characterized by the inability to exit the proliferative myoblast-like stage. The alveolar fusion positive subtype (FP-RMS) is the most aggressive and is mainly caused by the expression of PAX3/7-FOXO1 oncoproteins, which are challenging pharmacological targets. Here, we show that the DEAD box RNA helicase 5 (DDX5) is overexpressed in alveolar RMS cells and that its depletion and pharmacological inhibition decrease FP-RMS viability and slow tumor growth in xenograft models. Mechanistically, we provide evidence that DDX5 functions upstream of the EHMT2/AKT survival signaling pathway, by directly interacting with EHMT2 mRNA, modulating its stability and consequent protein expression. We show that EHMT2 in turns regulates PAX3-FOXO1 activity in a methylation-dependent manner, thus sustaining FP-RMS myoblastic state. Together, our findings identify another survival-promoting loop in FP-RMS and highlight DDX5 as a potential therapeutic target to arrest RMS growth.

Plexin-B2 and Semaphorins Do Not Drive Rhabdomyosarcoma Proliferation or Migration.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma for which subsets of patients have longstanding unmet clinical needs. For example, children with alveolar rhabdomyosarcoma and metastases at diagnosis will experience only 8% disease-free 5-year survival for nonlocalized unresectable recurrent disease. Hence, development of novel therapeutic strategies is urgently needed to improve outcomes. The Plexin-Semaphorin pathway is largely unexplored for sarcoma research. However, emerging interest in the Plexin-Semaphorin signaling axis in pediatric sarcomas has led to phase I cooperative group dose-finding clinical trials, now completed (NCT03320330). In this study, we specifically investigated the protein expression of transmembrane receptor Plexin-B2 and its cognate SEMA4C ligands in clinical RMS tumors and cell models. By RNA interferences, we assessed the role of Plexin-B2 in cell growth and cell migration ability in selected alveolar and embryonal RMS cell model systems. Our results affirmed expression of Plexin-B2 across human samples, while also dissecting expression of the different protein subunits of Plexin-B2 along with the assessment of preferred Semaphorin ligands of Plexin-B2. Plexin-B2 knockdown had positive or negative effects on cell growth, which varied by cell model system. Migration assayed after Plexin-B2 knockdown revealed selective cell line specific migration inhibition, which was independent of Plexin-B2 expression level. Overall, these findings are suggestive of context-specific and possibly patient-specific (stochastic) role of Plexin-B2 and SEMA4 ligands in RMS.

The Effect of Direct and Indirect EZH2 Inhibition in Rhabdomyosarcoma Cell Lines.

Simple SummaryRhabdomyosarcoma is the most common soft tissue tumor in children. Its two major subtypes show epigenetic alterations that are associated with poor prognosis. Therefore, targeting these epigenetic alterations by pharmacological intervention could be a therapeutic approach. We investigated two different types of substances that interfere with the epigenetic process of histone methylation. We performed studies in two cell lines that carry characteristics of the major rhabdomyosarcoma subtypes. The aim of this study was to find out if the substances differ in their effect on tumor-related cellular functions and to find out if the tumor subtypes differ in their response to the substances. These findings may contribute to a better assessment of the feasibility of pharmacological intervention directed against histone methylation in subtypes of rhabdomyosarcoma.Enhancer of Zeste homolog 2 (EZH2) is involved in epigenetic regulation of gene transcription by catalyzing trimethylation of histone 3 at lysine 27. In rhabdomyosarcoma (RMS), increased EZH2 protein levels are associated with poor prognosis and increased metastatic potential, suggesting EZH2 as a therapeutic target. The inhibition of EZH2 can be achieved by direct inhibition which targets only the enzyme activity or by indirect inhibition which also affects activities of other methyltransferases and reduces EZH2 protein abundance. We assessed the direct inhibition of EZH2 by EPZ005687 and the indirect inhibition by 3-deazaneplanocin (DZNep) and adenosine dialdehyde (AdOx) in the embryonal RD and the alveolar RH30 RMS cell line. EPZ005687 was more effective in reducing the cell viability and colony formation, in promoting apoptosis induction, and in arresting cells in the G1 phase of the cell cycle than the indirect inhibitors. DZNep was more effective in decreasing spheroid viability and size in both cell lines than EPZ005687 and AdOx. Both types of inhibitors reduced cell migration of RH30 cells but not of RD cells. The results show that direct and indirect inhibition of EZH2 affect cellular functions differently. The alveolar cell line RH30 is more sensitive to epigenetic intervention than the embryonal cell line RD.

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.

Mechanism of Action of the Sesquiterpene Compound Helenalin in Rhabdomyosarcoma Cells.

Rhabdomyosarcoma (RMS) is the most frequent soft tissue sarcoma in paediatric patients. Relapsed or refractory RMS shows very low 5-year survival rates, which urgently necessitates new chemotherapy agents. Herein, the sesquiterpene lactone, helenalin, was investigated as a new potential therapeutic agent against the embryonal RMS (eRMS) and alveolar RMS (aRMS) cells. We have evaluated in vitro antiproliferative efficacy of helenalin on RMS cells by the MTT and wound healing assay, and estimated several cell death pathways by flow cytometry, confocal microscopy and immunoblotting. It was shown that helenalin was able to increase reactive oxygen species levels, decrease mitochondrial membrane potential, trigger endoplasmic reticulum stress and deactivate the NF-κB pathway. Confirmation was obtained through the use of antagonistic compounds which alleviated the effects of helenalin in the corresponding pathways. Our findings demonstrate that oxidative stress is the pivotal mechanism of action of helenalin in promoting RMS cell death in vitro.

Small-molecule inhibitor - tyrphostin AG1296 regulates proliferation, survival and migration of rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is the most commonly occurring malignant soft tissue tumor in children. Despite improving its treatment methods, the current outcome in the advanced stages of this tumor is not satisfactory. RMS cells are characterized by abnormal cellular signaling due to the changes in the activity of the tyrosine kinases. Thus, substances blocking the mitogenic signal transmitted by receptors with tyrosine kinase activity raise hopes for inhibition of the uncontrolled cell growth. In this study, we examined the anticancer activity of tyrphostin AG1296, a tyrosine kinase inhibitor that binds to the intracellular domain of the PDGF (platelet-derived growth factor) receptor in human RMS alveolar and embryonal cell lines. We have discovered that tyrphostin AG1296 completely inhibited cell proliferation and effectively inhibited cell viability. Tyrphostin AG1296 induced apoptosis of the RMS cells and significantly inhibited their migration. Additionally, investigated inhibitor slightly inhibited expression of AKT and phosphorylation of ERK in alveolar RMS cells. Importantly, the inhibitor exerted also potent effects on the nanomechanical properties and cytoskeleton organization of RMS cells. To conclude, tyrphostin AG1296 is a promising compound in the treatment of alveolar RMS. Undoubtedly, a better knowledge of receptor pathomechanism of tyrosine kinases may contribute to developing new, more effective ways of RMS treatment.

Abstract 629: Flavonoids are NR4A1 antagonists and target PAX3-FOXO1 and G9a in alveolar rhabdomyosarcoma cells

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma that is primarily observed in children and adolescent, and accounts for 50% of soft tissue sarcomas in children. There are two major forms of RMS, namely embryonal RMS (ERMS) and alveolar RMS (ARMS) and these tumors are readily distinguished by their histological and genomic characteristics. ERMS patients have a better prognosis than ARMS patients and both groups undergo radiation, surgical and chemotherapeutic regiments. Many ARMS patients express different oncogenes including the histone methyltransferase G9a and the PAX3-FOXO1 fusion gene that contribute to ARMS cell growth, survival and migration/invasion. Studies in this laboratory have demonstrated that both G9a and PAX3-FOXO1 are regulated by the orphan nuclear receptor 4A1 (NR4A1) and can be targeted by bis-indole derived (CDIMs) NR4A1 antagonists. A recent study showed that the flavonoid kaempferol decreased G9a expression in gastric cancer cells and we hypothesized that kaempferol may be an NR4A1 ligand. Kamepferol and quercetin were used as models and both compounds bound NR4A1 at low μM concentrations and decreased NR4A1-dependent transactivation in ARMS cells. Functional studies showed that like CDIM/NR4A1 ligands, kaempferol and quercetin decrease ARMS cell growth, survival and migration/invasion, consistent with their activity as antagonists and inhibitors of NR4A1 regulated pro-oncogenic functions. In addition, kaempferol and quercetin also decreased expression of G9a, PAX3-FOXO1 and downstream genes regulated by them in ARMS cells further demonstrating their activity as NR4A1 antagonists. These results suggest that NR4A1-active flavonoids could be readily incorporated into chemotherapies used for treatment of ARMS patients to improve their efficacy and decrease the toxic side-effects of currently used cytotoxic drugs. Citation Format: Rupesh Shrestha, Kumaravel Mohankumar, Gregory Martin, Stephen Safe. Flavonoids are NR4A1 antagonists and target PAX3-FOXO1 and G9a in alveolar rhabdomyosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 629.

Establishment of a new valid animal model for the evaluation of hyperthermic intraperitoneal chemotherapy (HIPEC) in pediatric rhabdomyosarcoma.

Cytoreductive surgery in combination with hyperthermic intraperitoneal chemotherapy has been established as a novel treatment approach for peritoneal sarcomatosis. Despite promising clinical reports, there is still a lack of knowledge regarding optimal drug usage and local effects. Therefore, we intended to establish a murine animal model for further evaluation. Alveolar rhabdomyosarcoma cells were xenotransplanted into NOD/LtSz-scid IL2Rγnullmice (n=100). The mice received a continuous intraperitoneal lavage with isotonic saline solution as control or with cisplatin (30 or 60mg/m2 ) as treatment group for 60minutes at 37°Cor 42°C (6subgroups, each n=16). Tumor spread was documented by an adapted peritoneal cancer index and MRI (n=4). Tumor and tissue samples, harvested at the end of the perfusion, were evaluated regarding morphology, proliferation, and apoptosis (H&E-, Ki-67-, cleaved caspase 3-staining, TUNEL assay). Extensive peritoneal sarcomatosis in over 91% of the cases was observed. HIPEC was feasible without acute side effects. Ki-67 staining revealed concentration- or temperature-dependent effects of cisplatin-based HIPEC on the tumors. Although cleaved caspase-3 showed only sporadic apoptotic effects. TUNEL assay detected concentration- or temperature-dependent apoptotic effects at the outer tumor surface. MRI scans confirmed the observed tumor dissemination. This is the first animal model for evaluation of HIPEC in pediatric RMS in mice. Cisplatin-based HIPEC had early effects on the proliferation whereas circumscribed apoptotic effects could be detected at the tumor surface. This model allows further insights on the possible efficiency of HIPEC in RMS. Further studies using other drug combinations and treatment will follow.

CircVAMP3: A circRNA with a Role in Alveolar Rhabdomyosarcoma Cell Cycle Progression

Circular RNAs (circRNAs), a class of covalently closed RNAs formed by a back-splicing reaction, have been involved in the regulation of diverse oncogenic processes. In this article we describe circVAMP3, a novel circular RNA overexpressed in RH4, a representative cell line of alveolar rhabdomyosarcoma. We demonstrated that circVAMP3 has a differential m6A pattern opposed to its linear counterpart, suggesting that the two isoforms can be differently regulated by such RNA modification. Moreover, we show how circVAMP3 depletion in alveolar rhabdomyosarcoma cells can impair cell cycle progression, through the alteration of the AKT-related pathways, pointing to this non-coding RNA as a novel regulator of the alveolar rhabdomyosarcoma progression and as a putative future therapeutic target.

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

The Histone Methyltransferase Gene G9A Is Regulated by Nuclear Receptor 4A1 in Alveolar Rhabdomyosarcoma Cells.

The histone methyltransferase G9A (EHMT2) gene catalyzes methylation of histone 3 lysine 9 (H3K9), and this gene silencing activity contributes to the tumor promoter-like activity of G9A in several tumor types including alveolar rhabdomyosarcoma (ARMS). Previous studies show the orphan nuclear receptor 4A1 (NR4A1, Nur77) is overexpressed in rhabdomyosarcoma and exhibits pro-oncogenic activity. In this study, we show that knockdown of NR4A1 in ARMS cells decreased expression of G9A mRNA and protein. Moreover, treatment of ARMS cells with several bis-indole-derived NR4A1 ligands (antagonists) including 1,1-bis(3'-indolyl)-1-(4-hydroxyphenyl)methane (CDIM8), 3,5-dimethyl (3,5-(CH3)2), and 3-bromo-5-methoxy (3-Br-5-OCH3) analogs also decreased G9A expression. Furthermore, NR4A1 antagonists also decreased G9A expression in breast, lung, liver, and endometrial cancer cells confirming that G9A is an NR4A1-regulated gene in ARMS and other cancer cell lines. Mechanistic studies showed that the NR4A1/Sp1 complex interacted with the GC-rich 511 region of the G9A promoter to regulate G9A gene expression. Moreover, knockdown of NR4A1 or treatment with NR4A1 receptor antagonists decreased overall H3K9me2, H3K9me2 associated with the PTEN promoter, and PTEN-regulated phospho-Akt. In vivo studies showed that the NR4A1 antagonist (3-Br-5-OCH3) inhibited tumor growth in athymic nude mice bearing Rh30 ARMS cells and confirmed that G9A was an NR4A1-regulated gene that can be targeted by NR4A1 receptor antagonists.

Infant malignant ectomesenchymoma masquerading as inguinal hernia in two patients

Infant malignant ectomesenchymoma masquerading as inguinal hernia in two patients

The Rna Helicase DDX5 Cooperates with EHMT2 to Sustain Alveolar Rhabdomyosarcoma Growth

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood characterized by the inability to exit the proliferative myoblast-like stage. The alveolar fusion positive subtype (FP-ARMS) is the most aggressive and is mainly caused by the expression of PAX3/7-FOXO1 oncoproteins, which are challenging pharmacological targets. Here, we show that the DEAD box RNA helicase 5 (DDX5) is overexpressed in alveolar RMS cells and that its depletion and pharmacological inhibition decrease FP-ARMS viability and slow tumor growth in xenograft models. Mechanistically, we provide evidence that DDX5 functions upstream the EHMT2/AKT survival signalling pathway, by directly interacting with EHMT2mRNA and modulating its protein expression. We found that EHMT2 in turns regulates PAX3-FOXO1 activity in a methylation-dependent manner, thus sustaining FP-ARMS myoblastic state. Together, our findings identify a novel survival-promoting loop in FP-ARMS and highlight DDX5 as potential therapeutic target to arrest rhabdomyosarcoma growth.

Clinically relevant radioresistant rhabdomyosarcoma cell lines: functional, molecular and immune-related characterization

BackgroundThe probability of local tumor control after radiotherapy (RT) remains still miserably poor in pediatric rhabdomyosarcoma (RMS). Thus, understanding the molecular mechanisms responsible of tumor relapse is essential to identify personalized RT-based strategies. Contrary to what has been done so far, a correct characterization of cellular radioresistance should be performed comparing radioresistant and radiosensitive cells with the same isogenic background.MethodsClinically relevant radioresistant (RR) embryonal (RD) and alveolar (RH30) RMS cell lines have been developed by irradiating them with clinical-like hypo-fractionated schedule. RMS-RR cells were compared to parental isogenic counterpart (RMS-PR) and studied following the radiobiological concept of the “6Rs”, which stand for repair, redistribution, repopulation, reoxygenation, intrinsic radioresistance and radio-immuno-biology.ResultsRMS-RR cell lines, characterized by a more aggressive and in vitro pro-metastatic phenotype, showed a higher ability to i) detoxify from reactive oxygen species; ii) repair DNA damage by differently activating non-homologous end joining and homologous recombination pathways; iii) counteract RT-induced G2/M cell cycle arrest by re-starting growth and repopulating after irradiation; iv) express cancer stem-like profile. Bioinformatic analyses, performed to assess the role of 41 cytokines after RT exposure and their network interactions, suggested TGF-β, MIF, CCL2, CXCL5, CXCL8 and CXCL12 as master regulators of cancer immune escape in RMS tumors.ConclusionsThese results suggest that RMS could sustain intrinsic and acquire radioresistance by different mechanisms and indicate potential targets for future combined radiosensitizing strategies.

Abstract 3888: Study of tumor recurrence following loss of PAX3-FOXO1 oncoprotein expression in rhabdomyosarcoma cells

Abstract We previously developed a human alveolar rhabdomyosarcoma (RMS) model using immortalized human myoblasts engineered with constitutive MYCN and doxycycline-inducible PAX3-FOXO1 (P3F) expression constructs. These cells formed primary tumors rapidly when injected into mice fed a doxycycline-containing diet. When doxycycline is discontinued to turn off P3F expression, tumors initially regress, and then recurrent tumors without P3F expression form after a variable delay. We propose that P3F is necessary for initial tumor development but is not needed in later stages due to the accumulation of additional molecular changes. We further hypothesize that these novel changes may result in dysregulated expression of one or more P3F target genes. We derived cell lines from the primary and recurrent tumors and confirmed the expression of P3F in primary tumor-derived (PTD) cells and its absence in recurrent tumor-derived (RTD) cells. By limiting dilution, we generated subclones from RTD cell lines to obtain homogeneous populations of cells with P3F-independent transformation. Microarray analysis revealed that the expression profile of RTD subclones was substantially different from that of PTD cells. However, using untreated parental cells as a reference, we found that RTD and PTD cells shared 92 upregulated genes including 14 P3F transcriptional targets; among these 14 genes, FGF8 showed the highest level of upregulation (&amp;gt;30-fold) in RTD cells. Using a CRISPR-Cas9 approach, FGF8 knockout in these RTD cells impaired transforming capability in focus formation assays and tumorigenicity in vivo. Conversely, FGF8 overexpression in non-transformed MYCN-expressing parental myoblasts induced transformation and tumorigenic capability. In medium transfer studies, conditioned media (CM) from RTD cell cultures or from parental cells overexpressing FGF8 induced in vitro transformation in parental cells, whereas CM from FGF8-knockout RTD cells or parental cells did not induce transformation. To further implicate FGF8, immunodepletion of FGF8 from the RTD conditioned media induced a substantial reduction in transforming activity in these medium transfer studies. To examine the role of FGF8 in PTD cells and human alveolar RMS cells (RH30), we used a similar CRISPR-Cas9 approach to knockout FGF8 expression and similarly found impaired in vitro transformation. Finally, FGF8 overexpression in a spontaneous non-transformed RH30 variant with low P3F expression levels resulted in stimulation of transformation in vitro and tumorigenicity in vivo. Together, our results indicate that FGF8, a P3F transcriptional target, is necessary for the oncogenicity of P3F-positive RMS cells and that dysregulated FGF8 expression leads to the development of P3F-independent recurrent RMS tumors. Citation Format: Salah Boudjadi, Thanh Nguyen, Bishwanath Chatterjee, Puspa Pandey, Wenyue Sun, Frederik Barr. Study of tumor recurrence following loss of PAX3-FOXO1 oncoprotein expression in rhabdomyosarcoma cells [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 3888.

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&amp;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.