Human Rhabdomyosarcoma Research Articles

NS-065/NCNP-01: An Antisense Oligonucleotide for Potential Treatment of Exon 53 Skipping in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD), the most common lethal heritable childhood disease, is caused by mutations in the DMD gene that result in the absence of functional dystrophin protein. Exon skipping mediated by antisense oligonucleotides has recently emerged as an effective approach for the restoration of dystrophin, and skipping of exon 51 of DMD has received accelerated approval. Identifying antisense sequences that can provide the highest possible skipping efficiency is crucial for future clinical applications. Herein, we systematically tested two-step antisense oligonucleotide walks along human DMD exon 53 in order to define sequence-dependent effects of antisense oligonucleotide binding sites in human rhabdomyosarcoma cell lines. The first rough whole-exon 53 walk enabled the identification of a target region, and a second walk of this region was used to determine an optimal antisense oligonucleotide sequence (NS-065/NCNP-01) for exon 53 skipping. This oligonucleotide strongly promoted exon 53 skipping in a dose-dependent manner during pre-mRNA splicing in rhabdomyosarcoma and DMD patient-derived cells, and it restored dystrophin protein levels in patient-derived cells. NS-065/NCNP-01, a phosphorodiamidate morpholino oligomer, appears to be a promising candidate for treating exon 53 skipping, and it is potentially applicable to 10.1% of patients with DMD.

Small molecules targeting coxsackievirus A16 capsid inactivate viral particles and prevent viral binding

Coxsackievirus A16 (CVA16) is an etiologic agent of hand, foot, and mouth disease (HFMD) that affects young children, and although typically self-limited, severe complications, and fatal cases have been reported. Due to the lack of specific medication and vaccines against CVA16, there is currently a need to develop effective antivirals to better control CVA16 infections in epidemic areas. In this study, we identified the tannins chebulagic acid (CHLA) and punicalagin (PUG) as small molecules that can efficiently disrupt the CVA16 infection of human rhabdomyosarcoma cells. Both compounds significantly reduced CVA16 infectivity at micromolar concentrations without apparent cytotoxicity. A mechanistic analysis revealed that the tannins particularly targeted the CVA16 entry phase by inactivating cell-free viral particles and inhibiting viral binding. Further examination by molecular docking analysis pinpointed the targets of the tannins in the fivefold axis canyon region of the CVA16 capsid near the pocket entrance that functions in cell surface receptor binding. We suggest that CHLA and PUG are efficient antagonists of CVA16 entry and could be of value as antiviral candidates or as starting points for developing molecules to treat CVA16 infections.

Targeting EGFR and uPAR on human rhabdomyosarcoma, osteosarcoma, and ovarian adenocarcinoma with a bispecific ligand-directed toxin.

PurposeHuman sarcomas are rare and difficult to treat cancerous tumors typically arising from soft tissue or bone. Conversely, carcinomas are the most common cancer subtype in humans and the primary cause of mortality across all cancer patients. While conventional therapeutic modalities can prolong disease-free intervals and survival in some cases, treatment of refractory or recurrent solid tumors is challenging, and tumor-related mortality remains unacceptably high. The identification of overexpressed cell surface receptors on sarcoma and carcinoma cells has provided a valuable tool to develop targeted toxins as an alternative anticancer strategy. Recent investigation of recombinant protein-linked toxins that specifically target these cancer receptors has led to the development of highly specific, cytotoxic, and deimmunized drugs that can kill cancer cells.MethodsThis study investigated a recombinant protein called epidermal growth factor bispecific angiotoxin (eBAT), which is designed to target the epidermal growth factor receptor (EGFR) on cancer cells and the urokinase plasminogen activator receptor (uPAR) on cancer cells and associated tumor vasculature. Both receptors are expressed by a variety of human sarcomas and carcinomas. Flow cytometry techniques were used to determine binding affinity of eBAT to cancer cells, and proliferation assays were performed to calculate tumor killing ability based on half-maximal inhibitory concentrations.ResultseBAT demonstrated cytotoxicity against a variety of sarcoma and carcinoma cells that overexpress EGFR and uPAR in vitro and showed greater cell killing ability and binding affinity to cancer cells compared with its monospecific counterparts.ConclusionThe results of our study are promising, and further studies will be necessary to confirm the applicability of eBAT as a supplementary therapy for a variety of sarcomas, carcinomas, and possibly other refractory malignancies that express EGFR and uPAR.

Effect of MAPK Inhibition on the Differentiation of a Rhabdomyosarcoma Cell Line Combined With CRISPR/Cas9 Technology: An In Vitro Model of Human Muscle Diseases.

The human rhabdomyosarcoma cell line TE671 has been used extensively to study different aspects of muscle biology. However, its ability to differentiate and form myotubes has not been explored. Here, we examined muscle differentiation when we specifically stopped proliferation of human TE671 (WT-TE671) cells by using 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), an MAPK inhibitor. Our data show that treated cells initiated fusion, and myotube formation and that expression levels of dysferlin and myogenin were increased, whereas those of pax7 were decreased. Treatment of WT-TE671 cells with vitamin D3 alone and cotreatment with U0126 also promoted dysferlin expression. In addition, we knocked out the DYSF gene, which is involved in muscle differentiation, using CRISPR/Cas9 technology in WT-TE671 cells (Dysf-KO TE671). No dysferlin expression was observed before and after U0126 treatment. Although myogenin expression was absent in vehicle-treated Dysf-KO TE671 cells, after addition of U0126, myogenin reached levels similar to WT-TE671. This widely available source of human cells appropriately treated with U0126 may represent a useful model to study human muscle physiology in vitro. This dysferlin-deficient cell line should allow the study of pathophysiological pathways involved in dysferlin-deficient muscle and constitute a tool for high-throughput screening of therapeutic compounds for patients with dysferlinopathy and other muscle diseases.

Laccase-Catalyzed Heterocoupling of Dihydroquercetin and p-Aminobenzoic Acid: Effect of the Reaction Product on Cultured Cells.

Derivatization of the natural flavonoid dihydroquercetin with p-aminobenzoic acid was carried out in an ethyl acetate/citric buffer biphasic system using laccase from the fungus Trametes hirsuta. The main reaction product yield was ~68 mol %. The product was characterized by 1H NMR, 13C NMR, and liquid chromatography-mass spectroscopy, and its structure was elucidated. The reaction product affected viability of cultured human rhabdomyosarcoma cells (RD cell line) in a dose-dependent manner and, therefore, can be of interest to pharmaceutical industry.

Abstract 4127: A tumor evolution screen in zebrafish identifies PlexinA1 as a driver of metastasis and growth in human rhabdomyosarcoma

Abstract Relapse and metastasis are major clinical problems facing patients with rhabdomyosarcoma (RMS), a devastating pediatric malignancy of the muscle. Alveolar Rhabdomyosarcoma (ARMS) is characterized by genetic translocations while Embryonal Rhabdomyosarcoma (ERMS) is characterized by Ras pathway activation. Treatment for either RMS subtype requires surgical resection, chemotherapy, and radiation with overall poor prognosis for patients with high-risk features including metastasis and relapse. Thus, there is great interest in elucidating key molecular pathways and genetic factors that are involved in continued RMS growth and tumor maintenance. In the Langenau lab, we utilize a powerful transgenic zebrafish model of kRASG12D- induced ERMS and have recently developed a p53-/- (null) zebrafish model. p53 is one of the most common tumor suppressor genes, and upwards of 1/3 of RMS patients have mutations or Loss-of-Heterozygosity (LOH) of this locus. This model accurately mimics the molecular underpinnings and histopathology of human ERMS and has been used to discover molecular pathways that drive relapse. Using our tp53-/- (null) zebrafish along with large-scale cell transplantation assays, we have assessed metastatic potential of our ERMS tumors following serial passaging and acquisition of new driver mutations. Specifically, we performed a tumor evolution screen using serial engraftment of ERMS into recipient fish followed by RNA sequencing to discover novel pathways that are associated with elevated tumor growth and metastasis. Plexin A1 was one of our top hits in this screen, being expressed at >8 fold in metastatic lesions of the zebrafish and expressed in >80% of human RMS. Plexins are a large family of receptors for transmembrane, secreted and GPI-anchored semaphorins. Knockdown of PLXNA1 decreased human ERMS cell proliferation, increased differentiation and impaired anchorage-independent growth and migration. Collectively, our data supports the hypothesis that PLXNA1 is an important moderator of tumor growth in a large fraction of human RMS and likely regulates local invasion and metastasis. This work will provide much needed data for discovery of novel therapeutically-actionable pathways for relapsed ERMS and likely inform future treatment for high-risk patients. Citation Format: Alessandra M. Welker, Madeline N. Hayes, Qin Tang, David M. Langenau. A tumor evolution screen in zebrafish identifies PlexinA1 as a driver of metastasis and growth in human rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4127.

Abstract 3171: Vangl2 regulates cancer stem cell self-renewal and growth in rhabdomyosarcoma

Abstract Growth and relapse are driven by cancer stem cells (CSCs) in a subset of tumors, yet mechanisms driving cancer cell fate choices, maintenance and self-renewal are not fully understood. Here, we show that Van Gogh-like 2 (Vangl2), a core regulator of the non-canonical Wnt/planar cell polarity pathway (Wnt/PCP), regulates CSCs self-renewal in human rhabdomyosarcoma (RMS) – a common pediatric cancer of muscle. Wnt/PCP signaling is essential during development and recent work has linked this pathway to cancer growth, invasion and metastasis. However, roles for Vangl2 in regulating tumor self-renewal have not been previously described. Here, we show that VANGL2 is expressed in a majority of human RMS, specifically within early mononuclear progenitor-like cells. VANGL2 depletion inhibited proliferation, reduced self-renewal, and induced differentiation of human RMS. VANGL2 was also required for continued tumor growth and maintenance following engraftment of human RMS using mouse xenografts. Using a zebrafish model of embryonal rhabdomyosarcoma (ERMS) and limiting dilution cell transplantation approaches, we identified that Vangl2 expression enriches for CSCs in vivo and when transgenically expressed, at high levels elevates cancer stem cell number by 9-fold. Mechanistic studies revealed a role for RhoA downstream of Vangl2 in regulating maintenance of stem cell programs in human RMS. Our studies offer novel opportunities to isolate and characterize RMS cancer stem cells in vivo, and identify potential therapeutic targets for patient treatment. Citation Format: Madeline Hayes, Karin McCarthy, Alexander Jin, Sowmya Iyer, Sara Garcia, Mariana L. Oliveira, Sivasish Sindiri, Berkley Gryder, Zainab Motala, G Petur Nielsen, Jean-Paul Borg, Matt van de Rijn, David Malkin, Javed Khan, Myron Ignatius, David M. Langenau. Vangl2 regulates cancer stem cell self-renewal and growth in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3171.

Abstract 2865: Ciclopirox olamine: A common antifungal agent that inhibits growth of esophageal tumor cells in vitro and in vivo

Abstract Esophageal carcinoma continues to carry a poor prognosis due presentation at advanced stages. Current chemotherapeutics often only prolong survival for months to a few years. Drug repositioning is an important pharmacologic strategy that involves investigation and implementation of known drugs for treatment of new diseases. This method significantly reduces the costs required for establishing the safety of a drug and is associated with higher rates of drug approval. Ciclopirox olamine (CPX) is an antifungal agent that has been on the market since the 1970s. The mechanism of action is believed to include disruptions of DNA repair and cell division signals. Recently, however, this drug has been studied for its antitumor properties demonstrated in human rhabdomyosarcoma, breast carcinoma, colon adenocarcinoma, bladder carcinoma, and hematologic malignancies. To date, there are no studies related to the effect of CPX on esophageal cancer. Here, we show that ciclopirox olamine causes growth inhibition of four esophageal cell lines: TE-10, SKGT4, FLO1 and ESO1. This growth inhibition is demonstrated by hexosaminidase assay performed after incubation with the drug for 24-72 h. The IC50 values range between 5-20µM depending on the cell line used. Cell death was also demonstrated by immunofluorescent staining with Hoschst and propidium iodide, which shows a decrease in the number of viable cells with increasing concentrations of CPX. Clonogenicity assay was also performed with each cell line and demonstrates a decrease in the ability of cells to form colonies after 24 and 48 hour treatment with CPX and subsequent incubation in standard media for 5-7 days. Cell cycle analysis demonstrates G0/G1 arrest in cells treated with CPX. The effects of CPX on the cell cycle are further supported by Western blot analysis showing a decrease in in CDK4 and CDK6, which are necessary for cell cycle progression from the G0/G1 phase. We have also found that treatment with CPX results in a decrease in β-catenin in TE10 cells, suggesting that the drug is affecting this pathway to cause growth inhibition of tumor cells. Finally, we used our ESO1 mouse esophageal squamous cell carcinoma cell line for a xenograft study in which tumor cells were injected into the flanks of mice. Mice treated with intraperitoneal injections of 300 mg/m2 of CPX had smaller tumor volumes compared to untreated controls. In summary, we have shown that CPX inhibits growth of esophageal tumor cells both in vitro and in vivo. Our data suggest that CPX induces cell cycle arrest of tumor cells. The mechanism of tumor cell inhibition may be related to downregulation of the WNT/β-catenin signaling pathway. In future studies, we plan to analyze the effects of CPX on other components in the WNT family from in vitro cells as well as tumor xenografts. Citation Format: Randi J. Ryan, Prabhu Ramamoorthy, Dharmalingam Subramaniam, Shrikant Anant, Scott Weir. Ciclopirox olamine: A common antifungal agent that inhibits growth of esophageal tumor cells in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2865.

Abstract 824: Targeting epidermal growth factor receptors and urokinase-type plasminogen activator receptors for sarcoma therapy

Abstract eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to epidermal growth factor (EGF) and the amino terminal fragment of urokinase. This drug was designed to use epidermal growth factor receptors (EGFR) and urokinase-type plasminogen receptors (uPAR) as baits to deliver the lethal toxin into the cells, rather than as a means to interrupt signaling by the receptors. We previously showed that eBAT is remarkably safe, and that it reduced tumor burden in mice with established human rhabdomyosarcoma xenografts. It also was safe and showed a strong efficacy signal when used in the adjuvant setting in a clinical trial for dogs with splenic hemangiosarcoma. Nevertheless, the therapeutic mechanism of action of eBAT remains to be elucidated. Our goal is to identify patterns of uPAR expression in sarcomas that promote sarcoma growth and that mediate sensitivity for therapeutic activity by eBAT. We generated uPAR-knockout (KO) cells from MC1A-C1 mouse fibrosarcoma cell line using the CRISPR-Cas system to target exon 2 of the uPAR gene. The resulting cell lines D10 and H6 are clonal uPAR knockouts, whereas K02 and K06 represent mixtures where approximately 80% and 30% of the cells retain uPAR expression. uPAR expression in K02 and K06 has remained stable over numerous passages, indicating that loss of uPAR does not create selective pressure to enhance or inhibit cell growth in vitro. Furthermore, the growth kinetics of D10 and H6 are comparable to parental MC1A-C1 cells. eBAT treatment significantly inhibited proliferation of parental cells, but not of uPAR KO cells. In contrast, all of the cells were resistant to treatment with EGF- and uPA-monospecific toxins. The engraftment efficiency and tumor growth of uPAR KO cells in syngeneic hosts was substantially diminished when compared to K02 and K06 uPAR+ cells, suggesting that, even while uPAR-KO cells might be more resistant to eBAT therapy, uPAR expression appears to be necessary to create the tumor niche and/or to promote tumor growth in vivo. In our ongoing work, we are investigating how uPAR expression in stromal cells and in cells of the innate immune system influence sarcoma growth and the response to eBAT. Citation Format: Ashley J. Graef, Jong-Hyuk Kim, Daniel A. Vallera, Jaime F. Modiano. Targeting epidermal growth factor receptors and urokinase-type plasminogen activator receptors for sarcoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 824.

Abstract 4126: DNA methyltransferase inhibitors target a YES1-YAP1 signaling axis in childhood rhabdomyosarcoma

Abstract We have previously shown that the SRC family kinase (SFK) member, YES1, is highly expressed and rapidly activated upon IGF-1R blockade in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children and adolescents. YAP1 (YES-associated protein) is also highly expressed in human RMS tumors where it functions as an oncoprotein when the upstream Hippo tumor suppressor pathway is silenced, driving cell growth and survival. Thus far, efforts to pharmacologically inhibit YAP1 have been unsuccessful. One activator of the Hippo pathway, RASSF1a, is epigenetically silenced in many cancer types including sarcomas. Here we show a loss of RASSF1a expression in RMS cell lines via promoter methylation. Treatment with DNA methyltransferase inhibitors (DNMTis) can re-activate RASSF1a expression, resulting in YAP phosphorylation and inactivation. DNMTi treatment also significantly inhibits RMS cell growth, which can be rescued by overexpression of a constitutively active YAPS127A. In the alveolar variant (aRMS) in particular, DNMTis increase apoptosis and decrease both YES1 expression and activation. This suggests DNMTi treatment can inhibit both YAP1 and YES1 signaling. To further investigate the connection between YES1 and YAP1 we performed proximity ligation assays and discovered that YES1 and YAP1 interact in the nucleus of RMS cell lines. Furthermore, dasatinib treatment, a known SFK inhibitor, prevents YAP1 nuclear accumulation suggesting a role for YES1 in regulating YAP1 cellular localization. YAP1 is also tyrosine phosphorylated, which may be one way YES1 regulates YAP1. Lastly, cells expressing a constitutively active YAPS127A have increased activation of SFK suggesting a positive feedback loop whereby YAP1 activity can further activate YES1 through an unknown mechanism. These data uncover a signaling axis between the YES1 and YAP1 oncoproteins in RMS and suggest an alternative way to silence YES1 and YAP1 through use of DNMTis. We will further analyze this signaling through shRNA knockdown of YES1 and evaluate combination treatment of a DNMTi and dasatinib in vitro and in vivo. Citation Format: Katherine Slemmons, Choh Yeung, Josh Baumgart, Amy McCalla-Martin, Lee Helman. DNA methyltransferase inhibitors target a YES1-YAP1 signaling axis in childhood rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4126.

Abstract 1115: A CRISPR/Cas9 kinome screen identifies GRK5 as a novel regulator of rhabdomyosarcoma (RMS) self-renewal

Abstract Rhabdomyosarcoma (RMS) is a devastating pediatric soft tissue sarcoma. Clinical outcome for patients with relapsed or metastatic disease remains poor. However, current treatment options remain relatively unchanged over the last two decades, so there remains an urgent need for more effective treatment options for disease relapse. Cancer stem cells (CSCs), also known as tumor-propagating cells (TPCs), have the capacity for self-renewal and have been proposed to be responsible for tumor relapse and metastasis. RMS has been shown to follow the CSC model of tumor progression. We have previously performed an siRNA library screen against the human kinome (714) and a secondary CRISPR/Cas9 screen of top candidates to narrow down to 11 novel kinase candidates that are potentially essential for self-renewal of human RMS cells. GRK5, a G-protein coupled receptor kinase, was among the top candidate genes. Preliminary data from in vitro cell-based assays and in vivo limiting-dilution experiments in xenografts showed that CRISPR/Cas9-mediated targeted disruption of GRK5 resulted in significant reduction in the self-renewal capacity of RMS cells. GRK5-deficient RMS cells also displayed significant reduction in growth secondary to cell cycle arrest at the G2/M checkpoint. The functional effects on tumor growth and self-renewal appeared unique to GRK5 among the GRK kinase subfamily, as targeted disruption of closely-related GRK4 and GRK6 did not significantly affect RMS tumor cell growth and self-renewal. We propose that targeted disruption of GRK5 by CRISPR/Cas9 results in elevated p53 protein stability and activity, leading to inhibition of proteins required for cell cycle progression. We are also characterizing the loss-of-function effects of GRK5 in a RMS TPC reporter cell line created using a CRISPR/Cas9 knockin approach that will allow for direct assessment of GRK5 and other candidate gene function within the TPC population. By characterizing the biologic role of TPC as well as the function of GRK5 in RMS, we hope to provide new biologic insights into cancer stem cell biology and GRK5 as a novel therapeutic target for the treatment of advanced RMS. Citation Format: Thao Q. Pham, Terra Vleeshouwer-Neumann, Eleanor Chen. A CRISPR/Cas9 kinome screen identifies GRK5 as a novel regulator of rhabdomyosarcoma (RMS) self-renewal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1115.

Safety markers for rhabdomyosarcoma cells using an in�vivo imaging system

In vivo imaging system (IVIS) is a novel and rapidly expanding technology that is widely applied in life sciences, including cell tracing. IVIS is able to quantify biological events, including tumor proliferation, through counting the number of photons emitted from a specimen. PLA802-enhanced green fluorescent protein (EGFP), PLA802-monomeric cherry fluorescent protein (mCherry), RH30-EGFP and RH30-mCherry tumor cells were injected into 18 BALB/c female nude mice subcutaneously with 5×106 cells in 100 µl to quantitatively analyze EGFP and mCherry cells traced by IVIS. Inversion fluorescence microscopy revealed no transfection efficiency difference between PLA802-EGFP (95.3±1.2%) and PLA802-mCherry (95.8±1.7%), or between RH30-EGFP (94.7±2.1%) and RH30-mCherry (95.2±1.9%). Transfection did not influence the cell morphology of PLA802 or RH30. The cell migration, invasion and proliferation assay results of lentivirus-EGFP and lentivirus-mCherry revealed no significant difference prior to or following transfection. Therefore, lentivirus-EGFP and lentivirus-mCherry may serve as safety biological markers for PLA802 and RH30 cells. In vivo experiments demonstrated that lentivirus-EGFP and lentivirus-mCherry tumor luminescence signals were observed in all mice by IVIS. Hematoxylin-eosin staining and immunohistochemistry indicated that PLA802-EGFP, PLA802-mCherry, RH30-EGFP and RH30-mCherry cell lines exhibited rhabdomyosarcoma (RMS) characteristics like the maternal cells. In summary, mCherry and green fluorescent protein in human RMS PLA802 and RH30 cancer cells may be safely and stably expressed for a long time in vitro and in vivo.

Construction of a tri-chromatic reporter cell line for the rapid and simple screening of splice-switching oligonucleotides targeting DMD exon 51 using high content screening.

Splice-switching oligonucleotides (SSOs) that can modulate RNA splicing are used for the treatment of many genetic disorders. To enhance the efficacy of modulating splicing, it is important to optimize SSOs with regard to target sites, GC content, melting temperature (Tm value), chemistries, and lengths. Thus, in vitro assay systems that allow for the rapid and simple screening of SSOs are essential for optimizing SSO design. In this study, we established a novel tri-chromatic reporter cell line for SSO screening. This reporter cell line is designed to express three different fluorescent proteins (blue, green, and red) and was employed for high content screening (HCS, also known as high content analysis; HCA) for the evaluation of SSO-induced exon skipping by analyzing the expression levels of fluorescent proteins. The blue fluorescent protein is stably expressed throughout the cell and is useful for data normalization using cell numbers. Furthermore, both the green and red fluorescent proteins were used for monitoring the splicing patterns of target genes. Indeed, we demonstrated that this novel reporter cell line involving HCS leads to a more rapid and simple approach for the evaluation of exon skipping than widely used methods, such as RT-PCR, western blotting, and quantitative RT-PCR. Additionally, a brief screening of Locked nucleic acids (LNA)-based SSOs targeting exon 51 in DMD was performed using the reporter cell line. The LNA-based SSO cocktail shows high exon 51 skipping in a dose-dependent manner. Furthermore, the LNA-based SSO cocktails display high exon 51 skipping activities on endogenous DMD mRNA in human rhabdomyosarcoma cells.

Cytotoxic effects of new synthesis heterocyclic derivatives of Amoxicillin on some cancer cell lines

A new Schiff base [I] was prepared by refluxing Amoxicillin trihydrate and 4-Hydroxy- 3,5-dimethoxybenzaldehyde in aqueous methanol solution using glacial acetic acid as a catalyst. The new 1,3-oxazepine derivative [II] was obtained by Diels- Alder reaction of Schiff base [I] with phthalic anhydride in dry benzene. The reaction of Schiff base [I] with thioglycolic acid in dry benzene led to the formation of thiazolidin-4-one derivative [III]. While the imidazolidin-4-one [IV] derivative was produced by reacting the mentioned Schiff base [I] with glycine and triethylamine in ethanol for 9 hrs. Tetrazole derivative [V] was synthesized by refluxing Schiff base [I] with sodium azide in dimethylformamid DMF. The structure of synthesized compounds[I-V] was characterized by their melting points, elemental analysis CHN-S and by their spectral data; FTIR and 1H NMR spectroscopy. Two cancer cell lines include: (RD) human pelvic rhabdomyosarcoma and (L20B) the mice intestines carcinoma cell line (which expresses the genes for human cellular receptor for Polio viruses) were used in this study. The cytotoxic effect of different concentrations of all the synthesized compounds for 48 hrs was examined. All compounds except [IV] and [V] showed less than 50% inhibition for (L20B), while these compounds exhibit inhibition more than 50% inhibition for (RD).

Vangl2/RhoA Signaling Pathway Regulates Stem Cell Self-Renewal Programs and Growth in Rhabdomyosarcoma.

Tumor growth and relapse are driven by tumor propagating cells (TPCs). However, mechanisms regulating TPC fate choices, maintenance, and self-renewal are not fully understood. Here, we show that Van Gogh-like 2 (Vangl2), a core regulator of the non-canonical Wnt/planar cell polarity (Wnt/PCP) pathway, affects TPC self-renewal in rhabdomyosarcoma (RMS)-a pediatric cancer of muscle. VANGL2 is expressed in a majority of human RMSand within early mononuclear progenitor cells.VANGL2 depletion inhibited cell proliferation, reduced TPC numbers, and induced differentiation of human RMS invitro and in mouse xenografts. Using a zebrafish model of embryonal rhabdomyosarcoma (ERMS), we determined that Vangl2 expression enriches for TPCs and promotes their self-renewal. Expression of constitutively active and dominant-negative isoforms of RHOA revealed that it acts downstream of VANGL2 to regulate proliferation and maintenance of TPCs in human RMS. Our studies offer insights into pathways that control TPCs and identify new potential therapeutic targets.

A novel potential role of pituitary gonadotropins in the pathogenesis of human colorectal cancer.

BackgroundColorectal cancer (CRC) is a leading cause of death in the western world, and its incidence increases with patient age. It is also known that with age there occur changes in the levels of certain hormones, including an increase in the secretion of pituitary gonadotropins (PtGs) as a result of the loss of gonadal hormone feedback. We recently reported that functional PtG receptors are expressed in human lung cancer cells, rhabdomyosarcoma cells, and malignant hematopoietic stem cells.FindingsHere we report for the first time that the receptors for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are expressed in primary tumor samples isolated from CRC patients as well as in the established human CRC cell lines HTC116 and HTB37. Moreover, we also report that PtGs stimulate chemotaxis, adhesion, and proliferation of these cell lines.ConclusionsOur results suggest that PtGs play an important and underappreciated role in CRC pathogenesis, and we call for further studies to better define their role in gastrointestinal malignancies and their direct effect on putative CRC cancer stem cells.

Functional screening of FGFR4-driven tumorigenesis identifies PI3K/mTOR inhibition as a therapeutic strategy in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and outcomes have stagnated, highlighting a need for novel therapies. Genomic analysis of RMS has revealed that alterations in the receptor tyrosine kinase (RTK)/RAS/PI3K axis are common and that FGFR4 is frequently mutated or overexpressed. Although FGFR4 is a potentially druggable receptor tyrosine kinase, its functions in RMS are undefined. This study tested FGFR4-activating mutations and overexpression for the ability to generate RMS in mice. Murine tumor models were subsequently used to discover potential therapeutic targets and to test a dual PI3K/mTOR inhibitor in a preclinical setting. Specifically, we provide the first mechanistic evidence of differential potency in the most common human RMS mutations, V550E or N535K, compared to FGFR4wt overexpression as murine myoblasts expressing FGFR4V550E undergo higher rates of cellular transformation, engraftment into mice, and rapidly form sarcomas that highly resemble human RMS. Murine tumor cells overexpressing FGFR4V550E were tested in an in vitro dose-response drug screen along with human RMS cell lines. Compounds were grouped by target class, and potency was determined using average percentage of area under the dose-response curve (AUC). RMS cells were highly sensitive to PI3K/mTOR inhibitors, in particular, GSK2126458 (omipalisib) was a potent inhibitor of FGFR4V550E tumor-derived cell and human RMS cell viability. FGFR4V550E-overexpressing myoblasts and tumor cells had low nanomolar GSK2126458 EC50 values. Mass cytometry using mouse and human RMS cell lines validated GSK2126458 specificity at single-cell resolution, decreasing the abundance of phosphorylated Akt as well as decreasing phosphorylation of the downstream mTOR effectors 4ebp1, Eif4e, and S6. Moreover, PI3K/mTOR inhibition also robustly decreased the growth of RMS tumors in vivo. Thus, by developing a preclinical platform for testing novel therapies, we identified PI3K/mTOR inhibition as a promising new therapy for this devastating pediatric cancer.

Complete genome sequence of a multi-recombinant echovirus 6 strain isolated from CSF in Ahvaz, Southwestern Iran

BackgroundEchovirus 6 (E6), is one of the main enteroviral serotypes, was initially isolated from patients with aseptic meningitis (AM) and is a major cause of hospitalization among children and adults worldwide. MethodsA cerebrospinal fluid (CSF) sample was collected from patient with clinically suspected aseptic meningitis (AM) in August 2011. Following detection of a virus and subsequent virus serotyping, the whole genome sequence was determined. The sequence of the VP1 region of the isolated strain E6 RA/E6/Ahvaz/Iran/2011 showed 79% (>75%) nucleotide and 94% (>85%) amino acid homology with prototype strain D'Amori. The isolated strain was identified as an E6 serotype. A specimen was cultured in a human rhabdomyosarcoma (RD) cell line. Following propagation, the virus was further analyzed using the plaque assay technique, reverse transcription PCR (RT-PCR), rapid amplification of CDNA ends (RACE), TA cloning, sequencing, phylogenetic analysis, Simplot and boot scanning analyses (ver. 3.5) were applied to find evidence of recombination in the isolated strain. ResultsThe isolated Echo6 strain RA/E6/Ahvaz/Iran/2011 has been recorded in GenBank with a partial and complete genome accession numbers (KX619440) (KX198605), respectively. The complete genomic sequence was 7435 nt, with a 742 bp 5′ UTR, 117 bp 3′ UTR, and an open reading frame (ORF) encoding a polypeptide of 2191 amino acids. The nucleotide analysis of the VP1 and structural genomic regions of the isolated strain showed high similarity with strain E6-10887-99 isolated from patient with facial nerve paresis in Russia in 1999. The recombinations evidence were observed in the isolated strain E6 RA/E6/Ahvaz/Iran/2011 and found to have a high levels of inter-serotypic exchanges in 2C and 3A-3C genomic regions with Echovirus13 and Echovirus14, respectively. ConclusionFull genome sequence analysis of enteroviral is required to understand the epidemiological pattern and to evaluate the new enterovirus circulating in community.

Clinical and mutational spectrum of highly differentiated, paired box 3:forkhead box protein o1 fusion-negative rhabdomyosarcoma: A report from the Children's Oncology Group.

Pediatric paired box 3:forkhead box protein O1 fusion-negative (PF-) rhabdomyosarcoma (RMS) represents a diverse spectrum of tumors with marked differences in histology, myogenic differentiation, and clinical behavior. This study sought to evaluate the clinical and mutational spectrum of 24 pediatric PF- human RMS tumors with high levels of myogenic differentiation. Tumors were sequenced with OncoPanel v.2, a panel consisting of the coding regions of 504 genes previously linked to human cancer. Most of the tumors (19 of 24) arose at head/neck or genitourinary sites, and the overall survival rate was 100% with a median follow-up time of 4.6 years (range, 1.4-8.6 years). RAS pathway gene mutations were the most common mutations in PF-, highly differentiated RMS tumors. In addition, Hedgehog (Hh) and mechanistic target of rapamycin (mTOR) gene mutations with evidence for functional relevance (high-impact) were identified in subsets of tumors. The presence of Hh and mTOR pathway gene mutations was mutually exclusive and was associated with high-impact RAS pathway gene mutations in 3 of 4 Hh-mutated tumors and in 1 of 6 mTOR-mutated tumors. Interestingly, Hh and mTOR gene mutations were previously associated with rhabdomyomas, which are also known to preferentially arise at head/neck and genitourinary sites. Findings from this study further support the idea that PF-, highly differentiated RMS tumors and rhabdomyomas may represent a continuous spectrum of tumors. Cancer 2018;124:1973-81. © 2018 American Cancer Society.

Calcium electroporation for treatment of sarcoma in preclinical studies.

Calcium electroporation (CaEP) describes the use of electric pulses (electroporation) to transiently permeabilize cells to allow supraphysiological doses of calcium to enter the cytosol. Calcium electroporation has successfully been investigated for treatment of cutaneous metastases in a clinical study. This preclinical study explores the possible use of calcium electroporation for treatment of sarcoma.A normal murine muscle cell line (C2C12), and a human rhabdomyosarcoma cell line (RD) were used in the undifferentiated and differentiated state. Electroporation was performed using 8 pulses of 100 μs at 600–1000 V/cm; with calcium (0, 0.5, 1, and 5 mM). Viability was examined by MTS assay, intracellular calcium levels were measured, and expression of plasma membrane calcium ATPase (PMCA) was investigated using western blotting. Calcium/sodium exchanger (NCX1), ryanodine receptor (RyR1) expression and cytoskeleton structure (zyxin/actin) were assessed by immunofluorescence. CaEP efficiency on RD tumors was tested in vivo in immuno-deficient mice.CaEP was significantly more efficient in RD than in normal cells. Intracellular Ca2+ levels after CaEP increased significantly in RD, whereas a lower increase was seen in normal cells. CaEP caused decreased expression of PMCA and NCX1 in malignant cells and RyR1 in both cell lines whereas normal cells exhibited increased expression of NCX1 after CaEP. Calcium electroporation also affected cytoskeleton structure in malignant cells.This study showed that calcium electroporation is tolerated significantly better in normal muscle cells than sarcoma cells and as an inexpensive and simple cancer treatment this could potentially be used in connection with sarcoma surgery for local treatment.