Soft Tissue Sarcoma In Children Research Articles

The anti-tumor growth effect of a novel agent DMAMCL in rhabdomyosarcoma in vitro and in vivo

BackgroundRhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children with poor survival. New treatment approaches are urgently needed to improve treatment efficacy in RMS patients. DMAMCL is a novel agent from Asteraceae family that has been tested in phase I clinical trials in adult glioma in Australia.MethodsFive RMS cell lines (RD, RH18, RH28, RH30 and RH41) were used. The in vitro anti-tumor effect of DMAMCL, alone or in combination with VCR or Epirubicin, was studied using MTS assay or IncuCyte-Zoom cell confluency assay, and further validated by xenograft-mouse model in vivo. Changes in caspase-3/7 activity, cell-cycle progression and generation of ROS after DMAMCL treatment were investigated. Bim mRNA expression was measured by RT-qPCR, and protein expressions of Bim and phosphorylated-NF-κB(p65) by Western blotting. Small interfering RNAs (siRNA) of Bim were used to study the role of Bim in DMAMCL-induced cell death.ResultsIn vitro, DMAMCL treatment induced a dose-dependent increase in cell death that could be blocked by pan-caspase-inhibitor-Z-VAD-fmk in five RMS cell lines. The percent of cells in SubG1 phase and activities of caspase-3/7 increased after DMAMCL treatment; The combination of DMAMCL with VCR or Epirubicin significantly increased cell death compared to each reagent alone. In vivo, DMAMCL(75 mg/kg or 100 mg/kg) inhibited tumor growth and prolonged survival of mice bearing xenograft RMS tumors (RD, RH18, RH30, RH41). Compared to treatment with DMAMCL or VCR, a combination of two reagents caused significant inhibition of tumor growth (RD, RH41), even after treatment termination. The expression of Bim increased at protein level after DMAMCL treatment both in vitro and in vivo. The expression of p-NF-κB(p65) had a transient increase and the generation of ROS increased after DMAMCL treatment in vitro. Transfection of Bim siRNA into RMS cells blocked the DMAMCL-induced increase of Bim and partially attenuated the DMAMCL-induced cell death.ConclusionDMAMCL had an anti-tumor growth effect in vitro and in vivo that potentially mediated by Bim, NF-κB pathway and ROS. A combination of DMAMCL with chemotherapeutic drugs significantly increased the treatment efficacy. Our study supports further clinical evaluation of DMAMCL in combination with conventional chemotherapy.

SENTIERI: Epidemiological Study of Residents in National Priority Contaminated Sites. Fifth Report

SENTIERI: Epidemiological Study of Residents in National Priority Contaminated Sites. Fifth Report

The palladacycle complex AJ-5 induces apoptotic cell death while reducing autophagic flux in rhabdomyosarcoma cells

Rhabdomyosarcoma (RMS) forms in skeletal muscle and is the most common soft tissue sarcoma in children and adolescents. Current treatment is associated with debilitating side effects and treatment outcomes for patients with metastatic disease are dismal. Recently, a novel binuclear palladacycle, AJ-5, was shown to exert potent cytotoxicity in melanoma and breast cancer and to present with negligible adverse effects in mice. This study investigates the anti-cancer activity of AJ-5 in alveolar and embryonal RMS. IC50 values of ≤ 0.2 µM were determined for AJ-5 and it displayed a favourable selectivity index of >2. Clonogenic and migration assays showed that AJ-5 inhibited the ability of RMS cells to survive and migrate, respectively. Western blotting revealed that AJ-5 induced levels of key DNA damage response proteins (γH2AX, p-ATM and p-Chk2) and the p38/MAPK stress pathway. This correlated with an upregulation of p21 and a G1 cell cycle arrest. Annexin V-FITC/propidium iodide staining revealed that AJ-5 induced apoptosis and necrosis. Apoptosis was confirmed by the detection of cleaved PARP and increased levels and activity of cleaved caspases-3, -7, -8 and -9. Furthermore, AJ-5 reduced autophagic flux as shown by reduced LC3II accumulation in the presence of bafilomycin A1 and a significant reduction in autophagosome flux J. Finally, pharmacokinetic studies in mice show that AJ-5 has a promising half-life and that its volume of distribution is high, its clearance low and its intraperitoneal absorption is good. Together these findings suggest that AJ-5 may be an effective chemotherapeutic with a desirable mechanism of action for treating drug-resistant and advanced sarcomas.

Gluteal region spindle cell variant embryonal rhabdomyosarcoma in infant treated with buttockectomy

IntroductionRhabdomyosarcoma (RMS) is a soft tissue tumor developed from immature mesenchymal cells and accounts for about one-half of soft tissue sarcomas in children. However, it is very rare in infants and so, little is known about. Spindle cell variant of embryonal RMS is a rare and a better differentiated variant of embryonal RMS, having a better prognosis compared to other types of rhabdomyosarcomas. So, it needs to be distinguished from classical forms of the neoplasm. Its morphological resemblance to spindle cell neoplasms like leiomyosarcomas and fibrosarcomas may pose diagnostic difficulties for the pathologist. Gluteal region is a rare site for RMS. We report an infant with embryonal spindle cell variant RMS in the gluteal region. Case reportA one-year-old infant appeared with a history of a two-month lump on the right buttock. Her mother started to notice asymmetry on her child's buttock when she was bathing him. She noticed a lump with the size of a ping-pong ball. The patient could still crawl as usual and walk with aid. Two months later, the lump grew to the size of a tennis ball. In spite of this condition, there was no complaint of fever, weight loss or signs of pain. On the MRI, the mass appeared hypo-intense at muscle on T1-weighted images and hyper-intense on T2-weighted images. Following the administration of dimeglumine-gadopentetate contrast, T1-weighted fat-saturated scans showed intense enhancement with interspersed non-enhancing areas. We decided to do a buttockectomy followed by an adjuvant chemotherapy. Results and discussionInitially, the incision was performed from the posterior iliac crest curving distally following the gluteus maximus muscle until the 2 cm distal of greater trochanter. The incision was curved slightly posterior back to the medial aspect of the thigh to the gluteal fold to form a large posterior skin flap. Sciatic nerve had been identified and preserved. The entire gluteus maximus muscle with the mass has been removed. After 6 months of follow-up treatments, the patient is able to walk in normal gait and accordance with his mileage. ConclusionThe spindle cell embryonal rhabdomyosarcomas is a rare variant of embryonal subtype of RMS, occurring more commonly in children and adolescents. Its less aggressive sarcoma and better prognosis in children or adolescents, establish the correct diagnosis crucial using combination of clinical finding, histopathology and Immunohistochemistry.

Diagnosis and Management of Genitourinary Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Genitourinary RMS accounts for 15 to 25% of all RMSs and is a heterogeneous group of soft tissue tumors that vary in presentation, distribution, and prognosis. This article reviews the pathophysiology and tumor biology of RMS. It will also describe the initial approach to its diagnosis and current tumor surveillance protocols. Furthermore, this article presents the evidence behind a number of different staging and risk stratification systems currently used to guide treatment. Lastly, this article reviews future developments of investigational studies and risk stratification under investigation by a number of large international collaborative study groups. This review contains 17 figures, 7 tables, and 68 references. Keywords: Rhabdomyosarcoma, genitourinary, staging, diagnosis, paratesticular, bladder, prostate, RMS

Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and represents a high-grade neoplasm of skeletal myoblast-like cells. Decades of clinical and basic research have gradually improved our understanding of the pathophysiology of RMS and helped to optimize clinical care. The two major subtypes of RMS, originally characterized on the basis of light microscopic features, are driven by fundamentally different molecular mechanisms and pose distinct clinical challenges. Curative therapy depends on control of the primary tumour, which can arise at many distinct anatomical sites, as well as controlling disseminated disease that is known or assumed to be present in every case. Sophisticated risk stratification for children with RMS incorporates various clinical, pathological and molecular features, and that information is used to guide the application of multifaceted therapy. Such therapy has historically included cytotoxic chemotherapy as well as surgery, ionizing radiation or both. This Primer describes our current understanding of RMS epidemiology, disease susceptibility factors, disease mechanisms and elements of clinical care, including diagnostics, risk-based care of newly diagnosed and relapsed disease and the prevention and management of late effects in survivors. We also outline potential opportunities to further translate new biological insights into improved clinical outcomes.

Analysis of mutations in primary and metastatic synovial sarcoma.

Synovial sarcoma is the most common pediatric non-rhabdomyosarcoma soft tissue sarcoma and accounts for about 8–10% of all soft tissue sarcoma in childhood and adolescence. The presence of a chromosomal translocation-associated SS18-SSX-fusion gene is causally linked to development of primary synovial sarcoma. Metastases occur in approximately 50–70% of synovial sarcoma cases with yet unknown mechanisms, which led to about 70–80% mortality rate in five years. To explore the possibilities to investigate metastatic mechanisms of synovial sarcoma, we carried out the first genome-wide search for potential genetic biomarkers and drivers associated with metastasis by comparative mutational profiling of 18 synovial sarcoma samples isolated from four patients carrying the primary tumors and another four patients carrying the metastatic tumors through whole exome sequencing. Selected from the candidates yielded from this effort, we examined the effect of the multiple missense mutations of ADAM17, which were identified solely in metastatic synovial sarcoma. The mutant alleles as well as the wild-type control were expressed in the mammalian cells harboring the SS18-SSX1 fusion gene. The ADAM17-P729H mutation was shown to enhance cell migration, a phenotype associated with metastasis. Therefore, like ADAM17-P729H, other mutations we identified solely in metastatic synovial sarcoma may also have the potential to serve as an entry point for unraveling the metastatic mechanisms of synovial sarcoma.

The HDAC3-SMARCA4-miR-27a axis promotes expression of the PAX3:FOXO1 fusion oncogene in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood with an unmet clinical need for decades. A single oncogenic fusion gene is associated with treatment resistance and a 40 to 45% decrease in overall survival. We previously showed that expression of this PAX3:FOXO1 fusion oncogene in alveolar RMS (aRMS) mediates tolerance to chemotherapy and radiotherapy and that the class I-specific histone deacetylase (HDAC) inhibitor entinostat reduces PAX3:FOXO1 protein abundance. Here, we established the antitumor efficacy of entinostat with chemotherapy in various preclinical cell and mouse models and found that HDAC3 inhibition was the primary mechanism of entinostat-induced suppression of PAX3:FOXO1 abundance. HDAC3 inhibition by entinostat decreased the activity of the chromatin remodeling enzyme SMARCA4, which, in turn, derepressed the microRNA miR-27a. This reexpression of miR-27a led to PAX3:FOXO1 mRNA destabilization and chemotherapy sensitization in aRMS cells in culture and in vivo. Furthermore, a phase 1 clinical trial (ADVL1513) has shown that entinostat is tolerable in children with relapsed or refractory solid tumors and is planned for phase 1B cohort expansion or phase 2 clinical trials. Together, these results implicate an HDAC3-SMARCA4-miR-27a-PAX3:FOXO1 circuit as a driver of chemoresistant aRMS and suggest that targeting this pathway with entinostat may be therapeutically effective in patients.

Cervical Rhabdomyosarcoma with Prolapse in a Young Girl

Rhabdomyosarcomas are malignant skeletal muscle tumours. They are rare in adults, but account for about 50% of soft tissue sarcomas in children. Most frequent sites include head and neck (35%), followed by the genitourinary tract (25%), extremities and trunk, and cervix is one of the least common sites in the genitourinary tract. We report the case of a 13-year-old girl who was evaluated for excessive discharge per vaginum and was symptomatically treated and later diagnosed as a case of cervical RMS. She developed excess bleeding per vaginum associated with prolapse of the mass for which an emergency excision had to be done. Then, she completed her chemotherapy and is disease free till date. We report this case due to its unusual presentation. She was initially treated as a case of vaginitis and diagnosis was missed. Once she was diagnosed as having cervical RMS and on chemotherapy, she developed prolapse of the mass with bleeding, which is also an unusual presentation. So cases of excessive discharge per vaginum not responding to treatment should have a detailed pelvic examination to rule out any mass lesions.

Pannexin 1 inhibits rhabdomyosarcoma progression through a mechanism independent of its canonical channel function

Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma of childhood thought to arise from impaired differentiation of skeletal muscle progenitors. We have recently identified Pannexin 1 (PANX1) channels as a novel regulator of skeletal myogenesis. In the present study, we determined that PANX1 transcript and protein levels are down-regulated in embryonal (eRMS) and alveolar RMS (aRMS) patient-derived cell lines and primary tumor specimens as compared to differentiated skeletal muscle myoblasts and tissue, respectively. While not sufficient to overcome the inability of RMS to reach terminal differentiation, ectopic expression of PANX1 in eRMS (Rh18) and aRMS (Rh30) cells significantly decreased their proliferative and migratory potential. Furthermore, ectopic PANX1 abolished 3D spheroid formation in eRMS and aRMS cells and induced regression of established spheroids through induction of apoptosis. Notably, PANX1 expression also significantly reduced the growth of human eRMS and aRMS tumor xenografts in vivo. Interestingly, PANX1 does not form active channels when expressed in eRMS (Rh18) and aRMS (Rh30) cells and the addition of PANX1 channel inhibitors did not alter or reverse the PANX1-mediated reduction of cell proliferation and migration. Moreover, expression of channel-defective PANX1 mutants not only disrupted eRMS and aRMS 3D spheroids, but also inhibited in vivo RMS tumor growth. Altogether our findings suggest that PANX1 alleviates RMS malignant properties in vitro and in vivo through a process that is independent of its canonical channel function.

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.

Abstract PR12: Hedgehog pathway drives fusion-negative rhabdomyosarcoma initiated from nonmyogenic endothelial progenitors

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite aggressive chemotherapy, radiotherapy, and surgery, clinical outcomes for RMS have not improved for three decades, emphasizing the need to uncover the molecular underpinnings of the disease. RMS has been presumed to originate from derailed muscle progenitors based on the histologic appearance and gene expression pattern of the tumors. However, an origin restricted to skeletal muscle does not explain RMS occurring in tissues devoid of skeletal muscle, such as the prostate, bladder, salivary gland, biliary tree, and the omentum. Previously, we described that activation of Sonic Hedgehog signaling through expression of a conditional, constitutively active Smoothened allele, SmoM2, under control of an adipocyte-restricted adipose protein 2 (aP2)-Cre recombinase transgene in mice gives rise to aggressive skeletal muscle tumors that display the histologic and molecular characteristics of human embryonal RMS (ERMS). With the short latency and anatomic restricted tumor location in the neck, we sought to leverage this model to explore the cell of origin of ERMS. Lineage tracing experiments identified aP2-Cre labeled cells are distinctly nonmyogenic and were identified as endothelial cells found in the interstitium between muscle fibers. We illustrate that aP2-Cre is not expressed in the quiescent or activated muscle stem cells or satellite cells. Expression of oncogenic SmoM2 with aP2-Cre results in proliferation and expansion of the aP2-Cre labeled muscle interstitial cells and myogenic transdifferentiation resulting in ERMS. Activation of the Hedgehog pathway in aP2-Cre labeled endothelial progenitors results in Tbx1 expression, which is a skeletal muscle specification factor in the head and neck. We illustrate that endothelium and skeletal muscle within the head and neck arise from Kdr (Vegfr2) expressing progenitors. Hedgehog pathway activation in committed KDR+ endothelial progenitors results in Tbx1 expression and subsequent Myod1 expression driving a partially myogenic program characteristic of ERMS. Our work identifies reprogramming cell fate as a mechanism of transformation in pediatric sarcoma. Citation Format: Catherine J. Drummond, Jason A. Hanna, Matthew R. Garcia, Daniel J. Devine, Alana J. Heyrana, David Finkelstein, Jerold E. Rehg, Mark E. Hatley. Hedgehog pathway drives fusion-negative rhabdomyosarcoma initiated from nonmyogenic endothelial progenitors [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR12.

Abstract B01: Development of an in vitro drug-profiling platform for functional guidance of treatment decisions and identification of vulnerabilities in chemoresistant relapsed rhabdomyosarcoma tumors

Abstract Over the last decades, it has become increasingly clear that tumors are characterized by inter-individual heterogeneity, which can account as one of the prominent reasons for treatment failure. To address this problem, personalized precision medicine approaches need to be applied such as comprehensive genomic profiling to identify actionable driver mutations in individual tumors. Unfortunately, genomics alone is not sufficient in tumors that are driven by mutated but otherwise undruggable targets and a typically low mutational burden. This is a characteristic of many pediatric malignancies including rhabdomyosarcoma (RMS), the most common childhood soft-tissue sarcoma. Hence, we aim to develop an in vitro drug-profiling platform to identify and prioritize treatment strategies for RMS patients. To this end, we generated a panel of patient-derived xenografts (PDXs) including some diagnostic and relapse samples. We then screened 18 different culture conditions to identify suitable parameters to establish in vitro primary cultures of PDX tumors. Interestingly, addition of fetal calf serum to cell culture media has a detrimental effect on viability of most primary RMS cell cultures (PRCCs). In contrast, defined serum-free conditions allow to grow primary cultures for several passages that closely preserve the clonal composition and phenotypic characteristics of the parental tumor, as assessed by genomic and copy number analysis. Pharmacologic profiles of PRCCs using a targeted drug library of more than 200 compounds revealed patient-specific vulnerabilities, among them an unexpected sensitivity to AKT inhibitors in some fusion-positive RMS. Interestingly, hierarchical clustering of drug sensitivities clustered PAX3-FOXO1 fusion-positive tumors together and separated them from fusion-negative RMS. Moreover, a screen to establish effective drug combinations in a highly resistant high-risk relapse sample using standard chemotherapeutics (doxorubicin, etoposide, vincristine) together with our targeted compound library revealed the BH3-only mimetic ABT-263 as the top-scoring drug capable of resensitizing recurrent PRCCs to first-line treatment. Resensitizing with ABT-263 was not a patient-specific vulnerability as it was observed in several additional PRCCs. Mechanistically, genetic loss-of-function validation experiments revealed that this occurs via blockade of the BCL-XL-MCL-1 axis and is dependent on upregulation of the BH3-only protein NOXA. Taken together, our study provides an in vitro tool kit to prioritize actionable drug targets or combinatorial options for RMS patients for whom conventional therapies are failing. Citation Format: Beat W. Schaefer, Gabriele Manzella, Michaela Roemmele, Luduo Zhang, Joëlle Tchinda, Felix Niggli, Marco Wachtel. Development of an in vitro drug-profiling platform for functional guidance of treatment decisions and identification of vulnerabilities in chemoresistant relapsed rhabdomyosarcoma tumors [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B01.

Abstract 4139: SNAI2 inhibition promotes myogenic differentiation and prevents tumorigenic features of embryonal rhabdomyosarcoma

Abstract Pediatric Rhabdomyosarcoma (RMS) is a soft tissue sarcoma of childhood that in its high risk form has a dismal prognosis. RMS includes two histological subtypes, embryonal and alveolar, which differ clinically and molecularly. Both are characterized by the expression of Myogenic Regulatory Factors (MRFs) such as MYOD and MYOG but tumor cells are unable to differentiate in skeletal muscle cells and proliferate indefinitely. For this reason, differentiation therapies have anticancer potential in this tumor. Interestingly, one of the cause of the inability of MRFs in inducing myogenic-like differentiation is related to the loss of transcriptional activity of these factors partly due to the lack of availability of DNA binding sites and/or of capability to form transcriptional complexes. We demonstrate here that the repressor SNAI2 is upregulated in embryonal RMS (ERMS) patients and cells lines compared to their normal counterparts. Silencing of SNAI2 in 2 ERMS patient-derived cell lines using a puromycin pLKO lentiviral vector expressing a SNAI2 shRNA results in a slowdown of cell proliferation associated to p21Cip1 and Myogenin upregulation starting 3 days post-selection, as compared to control vector. These molecular changes are followed by the induction of Myosin-Heavy Chain and the formation of myogenic-like multinucleated structures reminiscent of myogenic fibers. Moreover, colony formation and anchorage independent growth are both strongly impaired. Finally, SNAI2 depleted cells form smaller spheres when cultured in cancer stem cells conditions. Therefore, SNAI2 down-regulation could have therapeutic value in ERMS. Studies are ongoing to verify the restoring of MYOD transcriptional activity and to identify genes regulated by SNAI2 in our system. The work is supported by Associazione Italiana Ricerca sul Cancro project IG15312 to RR. Citation Format: Silvia Pomella, Elena Carcarino, Cristina Cossetti, Franco Locatelli, Rossella Rota. SNAI2 inhibition promotes myogenic differentiation and prevents tumorigenic features of embryonal 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 4139.

Abstract 4630: Characterizing oncogene addiction in alveolar rhabdomyosarcoma reveals novel strategies for combination therapy

Abstract Rhabdomyosarcoma is the most common soft tissue sarcoma in children. The aggressive alveolar subtype (aRMS) is characterized by chromosomal translocations, most often a t(2;13) resulting in the expression of the oncogenic fusion protein PAX3-FOXO1 which is critical for tumorigenesis and cell survival. Our aim here was to identify a pharmacological combination therapy approach interfering with PAX3-FOXO1 biology at different levels. Since loss of the fusion protein results in cell death, we first aimed to pharmacologically enhance this effect. To this end, we screened aRMS tumor cells with a library of 208 drugs while simultaneously silencing PAX3-FOXO1 by shRNA. This identified the BH3-mimetic ABT-263 to sensitize aRMS cells to cell death after PAX3-FOXO1 depletion. To further characterize the cell death mechanisms we used combined shRNA and CRISPR approaches to perform a BH3 protein profiling. In accordance with identification of ABT-263 we demonstrate that aRMS cells undergo intrinsic apoptosis in a NOXA-dependent manner upon depletion of PAX3-FOXO1. In a parallel approach, and to identify drugs altering PAX3-FOXO1 protein stability, we screened the same drug library directly measuring fusion protein levels as read-out. This revealed that inhibition of Aurora kinase A negatively affects PAX3-FOXO1 protein levels. Finally, using both aRMS cell lines and patient-derived xenografts we demonstrate that combination treatment of Aurora kinase A inhibitors together with ABT-263 synergistically induces cell death and greatly slows tumor growth in vitro and in vivo. Taken together, these data show a novel functional interaction of Aurora kinase A and PAX3-FOXO1 and suggest new opportunities for targeted combination treatment of aRMS. Citation Format: Johannes Ommer, Marco Wachtel, Beat W. Schaefer. Characterizing oncogene addiction in alveolar rhabdomyosarcoma reveals novel strategies for combination 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 4630.

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 5867: Identification of novel inhibitors of the PAX3-FOXO1 oncogenic driver in rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, representing 8% of all childhood tumors. The most aggressive subtype of RMS is associated with chromosomal translocations creating a fusion transcription factor, most commonly involving PAX3 and FOXO1 (fusion-positive (FP)-RMS). Our group has recently comprehensively characterized the genetic and epigenetic landscape of FP-RMS. We found that FP-RMS has a strikingly low somatic mutational burden and that the PAX3-FOXO1 fusion gene is the primary oncogenic driver. Specifically, we found that PAX3-FOXO1 reprograms the cis-regulatory epigenetic landscape by inducing de novo super enhancers in collaboration with the bromodomain and extra-terminal domain (BET) protein family member BRD4, and the master transcription factors MYOG, MYOD1, and MYCN. We therefore hypothesized that FP-RMS would be vulnerable to disruption of the PAX3-FOXO1 super enhancer oncogenic circuitry. Method and Results: To test this, we developed a novel readout assay to simultaneously monitor PAX3-FOXO1 super enhancer and general transcriptional activity as well as cell viability, and screened against a 62,643 small-molecule library at a concentration of 10 µM. We found a total of 573 compounds with selective ALK super enhancer inhibition, which were selected for dose response assay with four 10-fold dilutions ranging from 20 μM to 2 nM. Further prioritization using a systematic computational model reduced this number to 64. Among the compounds with known activities, the multi-kinase PKC-412/midostaurin was previously identified in screens for PAX3-FOXO1 inhibitors. Other known compounds included topoisomerase I and topoisomerase II inhibitors, where both enzymes have recently been reported to play important roles in BRD4-facilitated DNA dynamics involving super enhancers and the transcriptional machinery. Among this list we identified 33 novel compounds of unknown mechanism of action. We therefore performed RNA-seq analysis to determine the transcriptional impact of the effect of these compounds. Pathway, gene set enrichment and Connectivity Map analyses of RNA-seq expression signatures implicate several of the novel compounds as inhibitors of epigenetic regulators, including inhibitors of BRD4, histone deacetylases, histone demethylases, histone methyltransferases, and chromodomain proteins. Conclusions: Our systematic approach identified several small molecules that specifically disrupt the activity of the PAX3-FOXO1 fusion oncogene in RMS. Further characterization of the specificity and selectivity of the lead compounds is ongoing, and exact mechanism of action is currently being elucidated. Our ultimate goal is to develop a clinically effective precision therapeutic targeting the previously “undruggable” PAX3-FOXO1 oncogenic driver of aggressive FP-RMS. Citation Format: Robert G. Hawley, Girma M. Woldemichael, Berkley E. Gryder, Young Song, Sivasish Sindiri, Vineela Gangalapudi, Jun S. Wei, James B. McMahon, Jack F. Shern, Javed Khan. Identification of novel inhibitors of the PAX3-FOXO1 oncogenic driver 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 5867.

Abstract 2966: A genome-wide scan identifies a new locus associated with pediatric rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and has one of the poorest survival rates among all pediatric cancers. The two major histologic subtypes of RMS are embryonal (eRMS) and alveolar (aRMS), which display differences in terms of age-incidence patterns and somatic mutations. Approximately 10% of RMS cases are associated with germline mutations in known cancer predisposition genes (e.g., TP53, NF1), but very little is known about the genetic susceptibility to the ~90% of RMS cases that are sporadic. We conducted the first multi-institutional genome-wide association study (GWAS) of RMS in 727 cases and 3,384 controls. Methods: Phase 1 of the GWAS included 421 RMS cases from Children's Oncology Group clinical trials, Texas Children's Hospital, and the Universidad de Navarra. Controls (n=2,763) were cancer-free individuals included in previous studies at the National Cancer Institute (NCI). Phase 2 included 306 cases from the Childhood Cancer Survivor Study and 621 independent controls from NCI. Genotypes were generated using the Illumina OmniExpress or the HumanOmni5Exome array and imputed based on the 1000 Genomes Project. Analyses were restricted to those of European (EUR) ancestry, and controls were matched to the cases based on principal components and genotype platform. Assuming an additive genetic model in SNPTEST, we used multivariable logistic regression models to estimate the odds ratio (OR), 95% confidence interval (CI), and P value for each variant on RMS overall and by two RMS subtypes: eRMS and aRMS. Results: After quality control filtering and assessment of population substructure, there were 555 combined EUR RMS cases and 1,561 controls, which included: 1) 278 cases and 1,112 controls in phase 1; and 2) 277 cases and 449 controls in phase 2. In the combined set, we identified a new locus at chromosome 11p15.2 that was strongly associated with an increased risk of aRMS and significant at the genome-wide level (OR=2.3, P=2.2x10-8). Results were consistent across studies: phase 1 OR=2.3, 95% CI 1.7-3.2; and phase 2 OR=2.3, 95% CI 1.2-4.5. The top variant, rs12785926, mapped to an intron in the PSMA1 (proteasome subunit alpha 1) gene. Based on data from GTEx, rs12785926 is significantly associated with RRAS2 expression across multiple tissues. RRAS2 is involved in cell proliferation and is somatically mutated in several tumors. When evaluating eRMS and RMS overall in the combined set, there were no variants significant at the genome-wide level. Conclusion: In the first GWAS of pediatric RMS, we identified a susceptibility locus associated with the more aggressive aRMS subtype that has a poorer prognosis. Additional replication analyses are underway using DNA obtained from archived newborn blood spots linked to population-based cancer registries, as well as other institutional cohorts. Further investigation will advance understanding of RMS etiology and biology. Citation Format: Philip J. Lupo, Libby Morimoto, Eric Karlins, Xiaorong Shao, Lindsay M. Morton, Michael E. Scheurer, Smita Bhatia, Leslie L. Robison, Gregory T. Armstrong, Simone Hettmer, Javed Khan, Stephen J. Chanock, Neal D. Freedman, Kathleen Wyatt, Belynda D. Hicks, Meredith Yeager, Casey L. Dagnall, Shengchao A. Li, Stephen X. Skapek, Douglas S. Hawkins, Catherine Metayer, Lisa Mirabello. A genome-wide scan identifies a new locus associated with pediatric 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 2966.

Abstract 4132: ASO screen uncovers splicing as a therapeutic vulnerability in theinsulin-like growth factor (IGF) signaling pathway

Abstract Alternative splicing is a process contributing to structural transcript variation and proteome diversity but is often disrupted in cancer. Like most other transcripts, the insulin receptor (IN-R) undergoes alternative splicing to produce two isoforms: the full-length IN-RB and exon 11 skipped IN-RA isoform. The expression of these isoforms is tightly regulated during development, however there is an aberrant increase in IN-RA expression in childhood cancers like Rhabdomyo- & Osteo-sarcoma. This increased IN-RA expression is deleterious to the cells because it encodes for a receptor which has high affinity for both insulin & IGF2 growth hormones and it exploits the IGF pathway to accelerate the onset of tumor-cell hallmarks like proliferation & angiogenesis. Rhabdosarcoma is the 3rd most frequent solid tumor and the most common soft-tissue sarcoma in children. Despite improvement in the treatment for this disease, there is substantial inadequacy of less toxic alternative therapies. We found that there is a significantly increased expression of IN-RA levels in a cohort of 40 Rhabdosarcoma (RMS) patients (embryonal & alveolar) as compared to the control samples. We confirmed this increased expression in multiple RMS cell lines. Furthermore, we went on to show that cellular stress such as hypoxia increases alternative splicing to produce more IN-RA isoform. In order to get a mechanistic insight into this phenomenon, we utilized this hypoxia-inducible splicing system and performed an antisense-oligonucleotide (ASO) screen to characterize sequence elements and splicing factors involved in the regulation of IN-R splicing. We found that sequence elements flanking exon 11 are critical to the increased alternative splicing we see under hypoxic conditions. We then performed a refined ASO walk to target the regions important for exon inclusion or exclusion and identified a region known to be a binding site for the splicing-factor CUGBP1. RMS derived cell lines almost exclusively express IN-RA but when treated with our lead ASO compound that targets this region, they show a dramatic decrease in the IN-RA expression. More importantly, the ASO treated cells exhibit a significant reduction in cell proliferation and migration. Application of ASO compound attenuates the IGF signaling pathway measured by decreased pAKT staining. Additionally, when tested in RMS xenograft models, we see decreased extravasation of blood vessels in the grafts cells transfected with the ASO that blocks IN-RA expression compared to controls. Our current data shows promising insight into how we can impede the IGF2 pathway by reducing IN-RA expression and consequently mitigate tumor hallmarks like cell-proliferation, migration and angiogenesis. The goal is to use these ASO compounds as therapeutic interventions in conjunction with already established anti-IGF1 receptor therapies to treat pediatric Rhabdomyosarcoma. Citation Format: Safiya Khurshid, Matias Montes, Brianne Sanford, Chelsea Brown, Daniel Comiskey, Frank Rigo, Peter Houghton, Dawn Chandler. ASO screen uncovers splicing as a therapeutic vulnerability in theinsulin-like growth factor (IGF) signaling pathway [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 4132.

PD-L1 assessment in pediatric rhabdomyosarcoma: a pilot study

BackgroundRhabdomyosarcomas (RMSs) are the most frequent soft tissue sarcoma in children and adolescents, defined by skeletal muscle differentiation and the status of FOXO1 fusions. In pediatric malignancies, in particular RMS, scant and controversial observations are reported about PD-L1 expression as a putative biomarker and few immune checkpoint clinical trials are conducted.MethodsPD-L1 assessment was evaluated by immunohistochemistry (IHC) utilizing two anti-PDL1 antibodies, in a pilot cohort of 25 RMS. Results were confirmed in primary and commercial RMS cell lines by cytofluorimetric analysis and IHC.ResultsPD-L1 expression was detectable, by both anti-PD-L1 antibodies, in the immune contexture of immune cells infiltrating and/or surrounding the tumor, in 15/25 (60%) RMS, while absent expression was observed in neoplastic cells. Flow cytometry analysis and PD-L1 IHC of commercial and primary RMS cell lines confirmed a very small percentage of PD-L1 positive-tumor cells, under the detection limits of conventional IHC. Interestingly, increased PD-L1 expression was observed in the immune contexture of 4 RMS cases post chemotherapy compared to their matched pre-treatment samples.ConclusionHere we identify a peculiar pattern of PD-L1 expression in our RMS series with scanty positive-tumor cells detected by flow cytometry, and recurrent expression in the immune cells surrounding or infiltrating the tumor burden.