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NF1-associated malignant peripheral nerve sheath tumors (MPNST) are the major cause of mortality in neurofibromatosis. MPNST arise from benign peripheral nerve plexiform neurofibromas (PN) which originate in the embryonic neural crest cell lineage. Here we employ reporter transgenes that label early neural crest lineage cells in multiple NF1 MPNST mouse models to discover and characterize a rare MPNST cell population with stem cell-like properties including quiescence that is essential for tumor initiation and relapse. Following isolation of these cells we derive a cancer stem cell specific gene expression signature that includes consensus embryonic neural crest genes and also identify Nestin as a novel marker for the MPNST cell of origin. Combined targeting of cancer stem cells together with antimitotic chemotherapy yields effective tumor inhibition and prolongs survival. Finally, application of the mouse cancer stem cell signature to single cell sequencing data from a rare NF1-associated patient MPNST precursor tumor, identifies preformed unsupervised gene expression cell clusters. Enrichment of the cancer stem cell signature in cognate human tumors supports the generality and relevance of cancer stem cells to MPNST therapy development.

Alveolar and embryonal rhabdomyosarcoma (RMS) are soft-tissue cancers that affect children, adolescents, and young adults. Sometimes referred to as muscle cancer, RMS is a cancer of muscle and non-muscle origin that phenocopies incompletely differentiated myoblasts or activated satellite (muscle stem) cells. Interestingly, embryonal RMS (ERMS) has been observed to undergo terminal myogenic differentiation in response to stress induced by chemotherapy and radiation therapy4, 9, 24. Given the propensity of rhabdomyosarcoma to differentiation, in this report we explore the use of differentiation therapy combining MEK inhibitor (MEKi) cobimetinib and chemotherapy as a strategy to halt RMS growth. We evaluated a representative panel of RMS cell lines with cobimetinib and chemotherapy in two dosing schedules that mimic clinical use followed by cell growth evaluation and high content analysis (differentiation) assays. We uncovered that cobimetinib does not have significant additive or synergistic effects on cell differentiation or cell growth with chemotherapy in RMS and can have unanticipated antagonistic effects; specifically, pre-exposure of cobimetinib to cells can decrease the effectiveness of chemotherapy-mediated cell growth inhibition in vitro. Although differentiation-therapy is still a potential viable strategy in RMS, our data do not support MEKi/chemotherapy co-treatment in this context.

Reciprocal chromosomal translocations resulting in the fusion of the DNA binding domain of the transcription factor PAX3 and the transactivational domain of FOXO1 underlie most cases of alveolar rhabdomyosarcoma (aRMS). Testing of compound MTPF63 against a panel of sarcoma cell lines showed that it had an inhibitory effect on proliferation of PAX3-FOXO1 translocation-positive cells. Further evaluation of its activity in Rh4 cells transduced with a PAX3-FOXO1 luciferase reporter showed that it attenuated reporter activity. Additional confirmation for the compound's inhibitory impact on PAX3-FOXO1 activity was obtained by detecting significant attenuation in the levels of Alk1 and MyoD1 proteins whose expression is driven by PAX3-FOXO1 in aRMS cells. Cell viability assays performed under 2D and 3D culturing conditions demonstrated that MTPF63 inhibits cell growth more effectively in translocation-positive alveolar RMS than in translocation-negative embryonal RMS. A hypothesis for MTPF63's mode of action was generated using the complete pool of barcoded essential heterozygous (~1150 strains) and homozygous (~4800 strains) diploid gene deletion mutants of Saccharomyces cerevisiae to identify gene deletions that confer sensitivity to the compound. Gene ontology enrichment analysis of 255 sensitive mutants whose growth rates were significantly inhibited in the presence of the compound compared to control, not only revealed calmodulin as main target of the compound but also illustrated the compound's interference with calmodulin-dependent nuclear protein import pathway, suggesting that it might impact PAX3-FOXO1 nuclear localization. Immunostaining of nuclear-localized PAX3-FOXO1 oncoprotein in two aRMS cell lines revealed cytoplasmic localization. Immunoblot analysis of fractionated cytosolic and nuclear protein lysates for PAX3-FOXO1 in MTPF63 treated cells also showed increased amounts in the cytosolic fractions. Transient overexpression of calmodulin was shown to partially rescue the inhibitory effect of the compound on PAX3-FOXO1-driven luciferase expression in RH4 reporter cells. Taken together, the data demonstrate the involvement of calmodulin in the nuclear import of PAX3-FOXO1 and introduce calmodulin antagonists as potential therapeutics against translocation-positive childhood alveolar rhabdomyosarcoma. Citation Format: Emad Darvishi, Cheryl Lynn Thomas, Berkley Eric Gryder, John Frederick Shern, Javed Khan, Girma M Woldemichael. Calmodulin governs nuclear entry of fusion PAX3/FOXO1 oncoprotein, a target in alveolar 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 890.

10515Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. Despite aggressive therapy, the 5-year survival rate for patients with metastatic or recurrent disease remains poor. Genomic studies by our group and others have identified 39 genes as known or potential somatic driver mutations in RMS. We therefore performed a large-scale validation study through an international consortium to more accurately determine the incidence of driver mutations and their association with clinical outcome. Methods: Formalin fixed paraffin embedded material was collected from patients enrolled on Children’s Oncology Group trials and UK patients enrolled on MMT trials. Pathology was reviewed centrally and extracted DNA subjected to targeted capture sequencing. Mutations, indels, deletions, gene amplifications and genome-wide copy number variation was called using analysis pipelines developed at the NCI. Results: DNA from six hundred and thirty-one patients was suitable for analyses. A median...

Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed an siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of ten NB cell lines, those with MYCN-amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of the known target, histone H3, and upregulation of TP53 in MYCN-amplified, TP53 wild-type cells. However, both wild-type and TP53 mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials.

Disruption of muscle regulatory factors (MRFs) in muscle progenitor cells results in a failure to withdraw from cell cycle and terminal differentiation and is proposed to contribute to rhabdomyosarcoma (RMS) tumorigenesis. Zinc finger transcription factor CASZ1 is a tumor suppressor gene and regulates normal nervous system and heart development. CASZ1 is known to regulate a subset of genes that are involved in skeletal muscle development although the function of CASZ1 in normal myogenesis and RMS tumorigenesis is unknown. CASZ1 levels increase over 20-fold when C2C12 myoblasts differentiate into myotubes. To probe its role in normal myogenesis we performed genetic knockdown or overexpression experiments in C2C12 myoblasts. When CASZ1 expression is silenced using RNAi, there is a significant reduction in myotubes upon induction of differentiation. Realtime PCR shows that knockdown of CASZ1 increases Myf5, a MRF that determines early commitment of muscle precursor cells, but decreases Myogenin (2-fold, p 2-fold, p Citation Format: Zhihui Liu, Norris Lam, Arnulfo Mendoza, Jun S. Wei, John F. Shern, Marielle Yohe, Javed Khan, Carol J. Thiele. Novel myogenic differentiation transcription factor CASZ1 suppresses rhabdomyosarcoma tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5522. doi:10.1158/1538-7445.AM2017-5522

Background: Despite intensive treatment regimens, Ewing sarcoma (EWS) patients with metastatic or relapsed disease still face a 5-year overall survival of less than 20%, indicating a clear need for novel targeted therapies. Utilizing ribosomal depleted RNA sequencing of 122 EWS samples (49 cell lines and 73 tumor samples) and 96 normal samples of variable tissues, we previously reported PAPP-A as one of the top 5 genes overexpressed in EWS (> than 4-fold) compared to normal tissue. Notably, PAPP-A showed substantial expression in tumors (median log2FPKM = 3.933, n=122) and minimal expression in normal tissue (median log2FPKM = -1.564, n=96). Pregnancy Associated Plasma Protein A (PAPP-A) is a secreted zinc metalloproteinase, that anchors to cell surface of heparan sulfate proteoglycans and enhances local IGF signaling. Through the release of IGFs from IGFBP-4, IGFBP-2 and IGFBP-5, free, bioactive IGF is being increased in close proximity to its receptors. In pregnancy PAPP-A is highly expressed by placental trophoblasts and represents a key regulator of fetal growth. The impact of IGF signaling in EWS is illustrated by the modest activity of IGF-1R inhibition as a single agent treatment in clinical trials, an approach that is limited by the rapid development of resistance. Methods: In an attempt to investigate the role of PAPP-A in Ewing sarcoma, we used CRISPR/Cas9 technology to target the PAPP-A locus in EWS cell lines and generated PAPP-A knockout clones (EW8, TC32) for further investigation in vitro and in vivo. Additionally, we tested inhibition of PAPP-A proteolytic function in a xenograft NSG mouse model, as a single agent treatment as well as in conjunction with IGF1R inhibition. Results: Knockout of PAPP-A in EWS cell lines utilizing CRISPR/Cas9 technology completely abrogated PAPP-A secretion and metalloproteinase activity in single cell clones, resulting in significantly increased complexed IGFBP-4 and diminished bioactive IGF-1, together with decreased cell growth in vitro. This phenotype could be rescued by recombinant, soluble PAPP-A. In vivo experiments showed that treatment with a monoclonal PAPP-A neutralizing antibody that inhibits the IGFBP-4 proteolytic activity of PAPP-A delays tumor growth of xenografted EWS tumor cells (EW8) in NSG mice. Furthermore, in conjunction with IGF1R inhibition (mAB h7C10), PAPP-A neutralization significantly decreased tumor growth and resulted in prolonged survival of mice (p=0.021).Conclusions: PAPP-A is a novel biologically relevant and highly tumor specific cell surface target in Ewing sarcoma. Our data suggest that neutralization of PAPP-A is a new therapeutic option that merits further evaluation for the treatment of Ewing's Sarcoma. Citation Format: Sabine Heitzeneder, John F. Shern, Lee J. Helman, Javed Khan, Crystal L. Mackall. Inhibition of the novel therapeutic target pregnancy associated plasma protein A (PAPP-A) in Ewing sarcoma enhances efficacy of IGF1R targeting in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1597. doi:10.1158/1538-7445.AM2017-1597

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood and comprises two major subtypes: fusion-positive (FP, most commonly PAX3-FOXO1 or PAX7-FOXO1) and fusion-negative (FN). Our previous study demonstrated that FP and FN RMS tumors exhibit distinct DNA methylation profiles. The current study will explore these issues in a larger, separate cohort of RMS tumors and compare DNA methylation in RMS cell lines and primary tumors. METHODS: DNA methylation was examined in 48 RMS tumors (21 FP and 27 FN) as well as 10 RMS cell lines (5 FP and 5 FN) on the Illumina HumanMethylation450 BeadChip platform. RESULTS: Unsupervised clustering analysis using the most variable probes (top 1%) in the 48 RMS tumors revealed that patterns of DNA methylation segregated these tumors into two distinct subgroups; one subgroup contains all 21 FP cases along with 2 FN cases and a second subgroup contains 25 of the 27 FN cases. A principal component analysis confirmed this close association of methylation pattern and fusion status, and showed that the two “discordant” FN cases map in a region between the FP and FN clusters. The FP tumors showed substantially lower overall levels of methylation compared to FN tumors. Application of an 11-gene methylation signature developed in our earlier study classified these 48 cases into FP and FN categories with >95% accuracy. In contrast to our previous findings, there was a significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation. Though unsupervised clustering analysis indicated that FP tumors and cell lines cluster as do the FN tumors and cell lines, a principal component analysis clarified these relationships by showing that the two groups of cell lines are located at a considerable distance from the two tumor subtypes. Analysis of the most varied probes in the tumors indicated that the vast majority of these probes are hypermethylated in all RMS cell lines. In a complementary analysis with the most variable probes (top 1%) in the cell lines, most of these probes are hypomethylated in all RMS tumors, though two smaller groups of probes show differential methylation between both groups of FP and FN samples. CONCLUSIONS: This study provides an independent validation that FP and FN RMS tumors possess distinct and characteristic methylation profiles. The enrichment of PAX3-FOXO1 binding sites in genes that are differentially methylated between these FP and FN tumors suggests that the PAX3-FOXO1 fusion protein may contribute to this methylation pattern. These analyses also indicate that RMS cell lines do not faithfully recapitulate the DNA methylation patterns that characterize primary tumors. Citation Format: Wenyue Sun, Bishwanath Chatterjee, John F. Shern, Yonghong Wang, Holly S. Stevenson, Daniel C. Edelman, Paul S. Meltzer, Javed Khan, Frederic G. Barr. DNA methylation characterization of fusion-positive and fusion-negative rhabdomyosarcoma primary tumors and cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2434.

Background: Ewing sarcoma (EWS) is the second most common bone malignancy in the pediatric population. Despite intensive treatment regimens, 5-year overall survival for patients with metastatic or relapsed disease is less than 20%, indicating a clear need for novel targeted therapies. The importance of IGF signaling in EWS is illustrated by activity of antagonistic targeting of IGF-1R in clinical trials, although this approach is plagued by rapid development of resistance. Pregnancy Associated Plasma Protein A (PAPP-A) anchors to cell surface proteoglycans and mediates metalloproteinase activity, that cleaves IGFs from IGFBP-4, IGFBP-2 and IGFBP-5, thus increasing local bioavailability of bioactive, free IGF. PAPP-A is expressed at high levels by placental trophoblasts, where it induces high levels of IGFs, which are required for fetal growth. Methods: In an attempt to identify novel targetable cell surface antigens in EWS, we performed ribosomal depleted RNA sequencing in 122 EWS samples (49 cell lines and 73 tumor samples) and 96 normal samples of variable tissues. Results: PAPP-A was identified as one of the top 5 membrane associated proteins overexpressed in EWS (> than 4-fold) compared to normal tissue (p = 3.89E-30). PAPP-A showed substantial expression in tumors (median log2FPKM = 3.933, n = 122) and minimal expression in normal tissue (median log2FPKM = -1.564, n = 96). We confirmed cell surface expression of PAPP-A by flow cytometry on 10 EWS cell lines tested, and identified PAPP-A in the supernatant of 12 EWS cell lines, measured by ELISA. Targeting the PAPP-A locus in the EWS cell line EW8 utilizing CRISPR/Cas9 completely abrogated PAPP-A secretion in single cell clones and significantly diminished bioactive IGF-1 levels in EWS cultures, while increasing IGF-1 bound to IGFBP-4, results which confirm absent PAPP-A metalloproteinase activity. Gene knockout of PAPP-A in EW8 resulted in decreased cell growth in vitro. Conclusions: We have identified PAPP-A as a novel potential cell surface target in Ewing sarcoma, characterized by high level tumor expression, limited normal tissue expression and biological significance in this tumor. Ongoing work is focused on studying growth of PAPP-A KO cell lines, and assessing whether PAPP-A neutralizing antibodies may mediate antitumor effects in EWS. Citation Format: Sabine Heitzeneder, John F. Shern, Javed Khan, Crystal L. Mackall. Pregnancy associated plasma protein A (PAPP-A) is a potential novel therapeutic target in Ewing sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 571.

Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed a siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of NB cell lines, those with MYCN amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of its known target histone H3, and upregulation of p53 pathway in MYCN-amplified NB cells with wild-type TP53. Both wild-type and p53-mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials. Citation Format: Dominik Bogen, Jun S. Wei, David O. Azorsa, Pinar Ormanoglu, Eugen Buehler, Rajarshi Guha, Jonathan M. Keller, Lesley A. Mathews Griner, Marc Ferrer, Young K. Song, Hongling Liao, Arnulfo Mendoza, Berkley E. Gryder, Sivasish Sindri, Jianbin He, Xinyu Wen, Xinyu Wen, Shile Zhang, John F. Shern, Marielle E. Yohe, Sabine Taschner-Mandl, Jason Shohet, Craig J. Thomas, Scott E. Martin, Peter F. Ambros, Javed Khan. Combined siRNA and small molecule screening identifies Aurora B kinase as an effective target in MYCN-driven neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B31.

Ewing sarcoma (EWS) is the second most common bone malignancy in the pediatric population and despite aggressive treatment regimens, 5-year overall survival for patients with relapsed or metastatic disease is less than 20%. Cell surface expression of CD99 (MIC2), a heavily O-glycosylated type I transmembrane protein is a diagnostic hallmark of EWS. Nearly 100% of EWS tumors and EWS cell lines exhibit high-level, diffuse CD99 membrane staining, raising the prospect that CD99 could provide a target for monoclonal antibody derived immunotherapeutics targeting cell surface receptors. However, CD99 expression has also been reported on numerous vital tissues including non-hematopoietic cells, and T cells, B cells and monocytes. In this study, we analyzed CD99 isoforms in EWS and normal tissues, in an effort to identify CD99 isoforms with expression on EWS and diminished or absent expression in vital tissues. The Ensembl database predicted 5 protein coding CD99 isoforms (designated v001-v005). Of these, two (CD99v003 and CD99v005) are predicted to generate novel protein sequences in the extracellular domain, which could provide immunotherapeutic targets. We utilized reverse transcriptase PCR to assess CD99v001-005 isoform expression in three EWS cell lines (EW8, TC71, 5838) and in normal CD4 and CD8 T cells. EWS cell lines expressed most CD99 variants, whereas both CD99v003 and CD99v005 were absent from T cells. We next evaluated expression of CD99v001-v005 using ribosomal depleted RNAseq in 122 EWS samples (49 cell lines and 73 tumor samples) and 96 normal tissues. Expression of CD99v001(NP_002405) was significantly increased in tumors vs. normal tissues, (p Citation Format: Sabine Heitzeneder, John F. Shern, Javed Khan, Crystal L. Mackall. Preferential expression of CD99 isoform variant 5 (CD99v005) in Ewing sarcoma compared to normal tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1357. doi:10.1158/1538-7445.AM2015-1357

Background: Rhabdomyosarcoma (RMS) is most common soft tissue sarcoma in childhood and adolescence. There are two major subtypes of RMS - alveolar RMS (ARMS) and embryonal RMS (ERMS). With current treatment modalities the 5 year survival rate of patients with metastatic disease is only about 30%, thus necessitating development of novel targeted therapeutic strategies. Fibroblast Growth Factor Receptor 4 (FGFR4) is highly differentially expressed gene and activating mutations in FGFR4 is associated with increase in RMS metastasis. We hypothesize that FGFR4 can be exploited as a potential therapeutic target in RMS. Monoclonal antibodies (mAbs) against specific cancer cell surface antigens, have gained importance as therapeutic agents in other cancer types. Therefore, mAbs against FGFR4 may be developed either alone or conjugated with a chemotherapeutic agent for treatment of RMS. Methods: We have developed 15 mAbs that are of mouse, rabbit and human origin. The immunogen for rabbit antibodies is human FGFR4 extracellular domain (ECD), while human FGFR4-Fc chimeric protein or hFGFR4 transfected cells were used for mouse antibodies and were produced using hybridoma technology. Using recombinant DNA technology, human immunoglobulin library was selected using FGFR4 ECD to derive human anti-FGFR4 mAbs. All mAbs were purified using affinity chromatography and reactivity was confirmed by ELISA. These mAbs were tested for their ability to bind cell surface FGFR4 and internalization of mAb bound FGFR4 (upon incubation at 37°C) by flow cytometry using RMS cell lines. To further characterize these antibodies we have performed immunohistochemistry (IHC) on tissue microarrays (TMA) of normal tissue, xenografts of RMS cell lines, and primary tumor from RMS patients. Finally, to select a potential mAb candidate as therapeutic agent, we tested appropriate secondary antibody-drug conjugate (ADC) using cell based cytotoxic assay. Results: We observed that tested mAbs bind to both ARMS and ERMS cell lines. The level of FGFR4 expression is variable in both subtypes. Internalization assays demonstrated mAbs bound FGFR4 was internalized in RMS cell lines. Analysis by IHC on TMAs suggests that FGFR4 protein is expressed at a considerably higher level in RMS tumor tissue and xenografts compared to normal tissue. Based on these findings, we are currently screening the mAbs for their ability to elicit a cytotoxic response either alone or in conjunction with secondary ADC. Conclusion: These results suggest anti-FGFR4 mAbs can be used as therapeutic intervention for RMS. High expression of FGFR4 in other cancers such as prostate, melanoma, lung, breast, colorectal, and gastric cancers suggests the potential use of anti-FGFR4 mAbs and their derivatives in these cancers. Citation Format: Sivasubramanian Baskar, Nityashree Shivaprasad, Zhongyu Zhu, Dimiter Dimitrov, Mhairi Sigrist, Poul Sorensen, Marielle Yohe, John Shern, John Maris, Crystal Mackall, Javed Khan. FGFR4 as a potential therapeutic target for monoclonal antibody based intervention in rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2488. doi:10.1158/1538-7445.AM2015-2488

Despite improvement of survival using multimodal chemo- and immunotherapy, high mortality and morbidity is still substantial for pediatric patients with metastatic cancers. Recent large-scale sequencing studies of pediatric tumors including rhabdomyosarcoma (RMS) and neuroblastoma (NB) have been focusing on somatic mutations, and revealed a low somatic mutation rate and surprisingly few recurrently somatic mutated genes in these childhood tumors. Currently, only a small portion of pediatric cancer cases can be explained by somatic driver events; whereas the cause for the majority of these diseases remains unknown. Because both these two types of tumors are uncommon, here we hypothesize that infrequent germline mutations (frequency To test this hypothesis, we utilized sequencing data from two cancer patient cohorts consisting of RMS (n=133) and NB (n=222) patients, of which latter is a part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative for pediatric cancers. First, high-quality protein-coding changing single nucleotide variants (SNVs) were called in both paired germline and tumor genomic DNAs. Then we excluded common variants with frequency >5% in a normal human population using the 1000 Genomes data. Due to our interest in the enriched variants, we further required the frequencies of variants in our rhabdomyosarcoma and neuroblastoma patient cohorts are higher than those in the ESP dataset, a non-cancer control population comprising 6503 individuals. There are 63247 SNVs fulfilled these selection criteria. Among them, 1589 have been reported in these pediatric cancers or in other malignancies in the Cancer Genome Atlas (TCGA) project; and 1178 variants are present in the Human Gene Mutation Database (HGMD). Of these HGMD variants, 49 have been reported in human diseases and 34 of them are known disease-causing mutations for human cancers and genetic disorders including TP53, ALK, CHEK2, and PINK1. Interestingly, the most frequent germline mutations in these pediatric tumors were rarely found in the TCGA project which mostly consists of adult cancers. This observation suggests a very different genetic background of pediatric cancer patients from that of the adult cancers, and warrants a careful examination of germline mutations in these cancers. Furthermore, previous studies have highlighted the importance of expression of variant genes (including tumor suppressor genes) for identification of driver mutations in cancers. Therefore we will use 178 transcriptome sequencing experiments available for these tumors (RMS=84; NB=93) to identify expressed variants in tumor. Statistical and pathway analyses are currently underway to determine potential pathological or casual germline mutations associated with neuroblastoma and rhabdomyosarcoma. Citation Format: Jun S. Wei, Rajesh Patidar, John Shern, Shile Zhang, Trevor Pugh, Sharon J. Diskin, Sivasish Sindiri, Young K. Song, Hongling Liao, Xinyu Wen, Jianjun Wang, Stephen X. Skapek, James R. Anderson, Frederic G. Barr, Robert C. Seeger, John M. Maris, Douglas S. Hawkins, Javed Khan. Systematic identification of germline mutations in rhabdomyosarcoma and neuroblastoma using massively paralleled sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5081. doi:10.1158/1538-7445.AM2014-5081

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood with two major subtypes embryonal (ERMS) and alveolar (ARMS), and current treatment modalities have yielded event free 5-year survival in only 30% of the patients with high-risk disease. Therefore, there is an absolute need for novel strategies and to identify and validate clinically relevant targets for the treatment of RMS. The fibroblast growth factor receptor 4 (FGFR4) is a very attractive therapeutic target because: 1) the FGFR4 gene is over expressed in RMS, 2) it is crucial for survival, proliferation, metastasis and drug resistance, 3) activating mutations in the kinase domain lead to aggressive growth and poor survival in patients with alveolar RMS and 4) genetic or pharmacologic inhibition of FGFR4 signaling inhibited tumor growth in vitro and in vivo. Monoclonal antibodies (mAbs) against specific antigens expressed on cancer cell surface have gained importance as potential therapeutic agents that may be used either alone or in combination with chemotherapeutic drugs. We have developed several mAbs against human FGFR4 protein from rabbit and mouse (by hybridoma technology), and from human immunoglobulin libraries (by recombinant DNA technology). In the present study, we report the development and characterization of some of these mAbs. Biacore analysis of these antibodies showed low nM affinity to purified extracellular domain of FGFR4 (FGFR4-ECD). The ability of these mAbs to bind the native molecule was demonstrated by specific binding to RMS cell lines of both subtypes, and dose response curves exhibited higher binding in ARMS cells than ERMS cells. More importantly, significant binding was noticed in freshly isolated tumor cells from a breast metastatic nodule of a patient with ARMS. The anti-FGFR4 mAbs also bound to transfected cell line expressing FGFR4 on the surface, but not the vector control (FGFR4 negative) cell line indicating the specificity of the reaction. Furthermore, cell surface FGFR4 can mediate internalization of the bound antibody upon incubation at 37C for as little as 30 min and maximum internalization was observed at 2 h. Receptor mediated internalization of the bound mAb was inhibited by a chemical inhibitor, and ARMS cells showed more internalization than ERMS. Together, these observations support the contention that anti- FGFR4 mAb can be used as a vehicle to deliver a cytotoxic payload in the form of small molecule drugs and toxins. Ongoing investigations are aimed at developing anti-FGFR4 mAbs and their derivatives as potential therapeutic agents for the treatment of patients with RMS. This study is supported in part by the intramural research program of the National Cancer Institute, National Institutes of Health, grants from St. Baldrick's Foundation and Stand Up To Cancer - St. Baldrick's Pediatric Dream Team Translational Cancer Research Grant, Grant Number SU2C-AACR-DT11-13. Citation Format: Sivasubramanian Baskar, Zhongyu Zhu, Ramon Lorenzo Labitigan, Michelle Ovanesian, Rimas J. Orentas, Samuel Q. Li, Yohe E. Marielle, John Shern, Dimiter S. Dimitrov, John Maris, Crystal Mackall, Khan Javed. Development and characterization of anti-FGFR4 monoclonal antibodies as therapeutic agents for human rhabdomyosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 652. doi:10.1158/1538-7445.AM2014-652

Background: Ewing sarcoma (EWS) is the second most common type of primary bone tumor to affect children and adolescent and accounts for 2.9% of all childhood cancers. Despite advances in multidisciplinary treatment leading to improved outcomes over time, long term survival still remains less than 50% for patients older than 15 years of age and only 65% in younger patients. In an effort to identify novel prognostic and therapeutic targets, we utilized next generation sequencing techniques to evaluate EWS samples for somatic mutation. Methods: Whole genome paired-end sequencing using the Complete Genomics method was performed on the DNA from 6 EWS matched tumor-normal samples. Somatic mutations were identified and classified using standard bioinformatics techniques and mutations of interest were verified using capillary sequencing. To extend discoveries and evaluate for mutational recurrence, targeted sequencing was performed on a validation cohort of 61 EWS tumors and 22 EWS cell lines using a custom multiplex PCR design followed by high-coverage sequencing on the Ion Torrent system. Results: In the discovery cohort, we detected an average of 361 somatic mutations per tumor in non-repetitive regions and an average of 6 somatic mutations per tumor in protein coding regions (=0.15 mutations/Mb of coding sequence). The well-established FLI1-EWSR1 gene fusion was detected by the whole genome sequencing in all 6 samples. Additional previously reported genetic events were also seen including frequent chromosomal trisomy and deletion of the CDKN2A containing locus on chromosomal region 9p21 in 2 of 6 samples. In the validation cohort of tumors, known recurrent mutations such as in TP53 (3/61 = 4.9%) were seen at rates similar to that previously reported in EWS. Continued efforts are underway to verify novel recurrent mutations. Conclusion: Next-generation sequencing provides a powerful tool to characterize the genetic landscape and evaluate for recurrent mutations in cancer. We utilize this tool in Ewing sarcoma in a project that provides both a comprehensive genomic evaluation from whole genome sequencing data and as well evaluates for recurrent mutations using a targeted sequencing approach. Citation Format: Andrew S. Brohl, Young Song, Laura Hurd, Hongling Liao, Li Chen, Rajesh Patidar, John F. Shern, Jun S. Wei, Javed Khan. Next-generation sequencing for mutation discovery in Ewing sarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2020. doi:10.1158/1538-7445.AM2013-2020