Grant Recipients
• Grant Recipients
• 2011 SFA Research Grant Recipients
• 2010 SFA Research Grant Recipients
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• 2003 SFA Research Grant Recipients
Research Grants
2011 SFA Research Grant Recipients
Interrogating the effects of IGF1R blockade on childhood sarcoma fusion oncoprotein stability
• Poul H.B. Sorensen, MD, PhD, British Columbia Cancer Research Center
• Recipient of the $50,000 Alexander Burdo/ZIOPHARM Research Award
• Abstract: The overall goal of this proposal is to determine how IGF1R supports transformation by dominantly-acting oncoprotiens found in pediatric sarcomas. Aim 1 will focus specifically on ETV6-NTRK3 (EN), a dominant chimeric tyrosine kinase that we first discovered in pediatric saromas. We have recently gained valuable insights into how IGF1R supports EN transformation. First, we confirmed that EN transformation requires the IGF1R. Second, we found that EN physically co-localizes and interacts with IGF1R at the membrane in live cells. Third, we unexpectedly found that blocking IGF1R with the small molecule dual specificity IGF1R/insulin receptor (INSR) kinase inhibitor, BMS-536924, dramatically reduces EN protein stability in a proteasome-dependent manner. This causes robust re-localization of EN into cytoplasmic protein aggregates, where EN is ubiquitinated. Moreover, another IGF1R kinase inhibitor, BMS-754807, the IGF1R blocking antibody CP-751871, and siRNAs to IGF1R but NOT INSR, have similar effects as BMS-536924. In exciting very recent studies, we found that the E3 ligase KPC1 preferentially associates with EN in the presence of BMS-536924, and that over-expression of KPC1 induces EN degradation. Based on these findings, we hypothesize that EN becomes membrane-localized via IGF1R, and that this prevents EN degradation by KPC1/2. The goal of Aim 1 is to uncover the molecular mechanisms behind these findings. The goal of Aim 2 is to determine whether similar mechanisms control other fusion proteins found in pediatric sarcomas that have been shown to respond clinically to IGF1R inhibition. The rationale for studying this process is that a more rigorous understanding of how IGF1R contributes to transformation by such oncoproteins is essential to identify proteins other than IGF1R itself for therapeutic targeting. This is important as resistance to IGF1R inhibitors in clinical trials has already been observed. Our studies may therefore identify new pathway targets that can be co-inhibited to overcome IGF1R resistance.
Beta-catenin activation in synovial sarcomagenesis and progression
• Kevin B. Jones, MD, University of Utah - Huntsman Cancer Institute
• Recipient of the $50,000 Ashleigh Lau/Morgan Stanley Research Award
• Abstract: Synovial sarcoma is a deadly soft-tissue malignancy with a predilection for adolescents and young adults. The development of sorely needed targeted therapies depends on the identification of critical pathways in synovial sarcomagenesis. Expression of an SYT-SSX fusion oncogene generated by one of the characteristic X:18 translocations initiates synovial sarcomagenesis. Possible cooperation of β-catenin has been implicated by the prominent nuclear localization of β-catenin in a majority of human synovial sarcomas and the identification of activating β-catenin exon 3 mutations in a subset of these. The mixed cellular phenotype of synovial sarcoma, including both mesenchymal and epithelial features, further suggests a role for Wnt/β-catenin deregulation. Using the genetically engineered mouse model of synovial sarcoma developed in our laboratory, we will interrogate the contributions of β-catenin activation and disruption to synovial sarcoma initiation as well as maintenance and progression. The previously described model, which spontaneously generates tumors that faithfully recapitulate human synovial sarcoma at complete penetrance via the conditional expression of the human SYT-SSX2 cDNA in myoblasts, has recently been improved to permit in vivo monitoring of tumor maintenance and progression. In addition, a more stringent model for site-specific synovial sarcomagenesis using adenoviral delivery of Cre-recombinase permits the investigation of tumor initiation itself. These two models will be further manipulated by crossing them to available mice with conditional genetic alterations that constitutively activate or ablate the β-catenin gene. In addition, two β-catenin antagonists, PKF115-584 and AV6946, and one β-catenin activator, lithium chloride, will be used to test the role of transient enhancement or disruption of the pathway in synovial sarcoma initiation, maintenance, and progression. Our preliminary data suggests that β-catenin activation indeed enhances synovial sarcomagenesis. By these experiments, we will bring its role and its potential as a therapeutic target into better focus.
Histone deacetylase inhibitor reprograms hSNF5/INI1 regulated gene expression for the treatment of epithelioid sarcoma
• Quan-sheng Zhu, MD, PhD, The University of Texas MD Anderson Cancer Center
• Recipient of the $50,000 Mandell/Kropp Run for a Sarcoma Cure Research Award
• Abstract: Epithelioid Sarcomas (ESs) are characterized by a locally aggressive behavior and a propensity for lymphatic and metastatic spread. Current therapy follows general STS treatment guidelines with complete surgical resection being the desired (when possible) approach; the impact of chemotherapy and radiotherapy is not well defined, but seems minimal. Five-year survival rates of 60% to 75% have been reported. Survival continues to decrease with time and 10YDSS rates of ~40-50% have been observed, a critical factor when taking into account the commonly young age at ES presentation; proximal ES outcomes are even less favorable. Loss of INI1, the product of the hSNF5/SMARCB1/BAF47 gene first identified as a tumor suppressor in malignant rhabdoid tumors, has recently been demonstrated to occur in the majority of ES. This molecular alteration potentially drives a pro-tumorigenic intra-cellular epigenetic landscape. Our preliminary studies suggest that ES cells are markedly sensitive to histone deacetylase inhibition (HDACi) and we hypothesize that this occurs through the reversal of INI1-loss driven gene expression deregulations.
We propose the following molecularly-driven translational Aims:
Aim#1: To unravel INI1-loss dependent ES gene expression signatures and evaluate whether HDACis can reverse these pro-tumorigenic profiles and induce epigenetic reprogramming
Aim#2: To examine the effects of HDACis on ES cellular properties in vitro and on the growth, recurrence, and metastasis of ES in vivo
HDACi are currently being clinically tested as an anti-cancer therapeutic approach in a large and diverse array of malignancies. Insights derived from our proposed studies will enhance our understanding of ES biology and will hopefully lay the foundation for future HDACi-based clinical trials for ES.
A small molecule inhibitor of AXL is a novel therapeutic strategy in osteosarcoma
• Edward Greenfield, PhD, Case Western Reserve University
• Recipient of the $50,000 Mark Herzlich Research Award
• Abstract: Despite aggressive surgical and chemotherapeutic approaches, the survival rate among osteosarcoma patients who present with metastasis is ~25% and is ~70% in those who present without metastasis. These survival rates have not improved in the last few decades. A better understanding of osteosarcoma pathophysiology is therefore needed to allow development of novel therapeutic approaches. RTK activation is responsible for progression of many forms of cancer, and inhibitors of these RTKs are a majority of the most promising anticancer drugs. Our recent phospho-proteomic and functional genomic screening showed that a RTK known as AXL contributes to the in vitro phenotype of highly metastatic human 143B osteosarcoma cells. Moreover, we also found that a relatively specific inhibitor of AXL that blocks metastasis of other types of cancer also reduces motility, invasion, and colony formation by the osteosarcoma cells. AXL will therefore be the focus of this proposal and our overall hypothesis is that AXL is a novel metabolic target in osteosarcoma. This hypothesis will be tested by the following Aims:
AIM 1: Determine the role of AXL in tumorigenesis and metastasis in murine xenograft models of osteosarcoma. This aim will measure the in vivo effects of shRNA-mediated knockdown of AXL on tumorigenesis and metastasis by 143B human osteosarcoma cells. Experiments will include both the orthotopic tibial implantation model and the tail vein injection model.
AIM 2: Determine the therapeutic effect of the AXL inhibitor on tumorigenesis and metastasis in murine xenograft models of osteosarcoma. This aim will measure the in vivo effects of the AXL inhibitor on tumorigenesis and metastasis in the same murine models used in Aim 1.
Successful completion of this project would determine whether AXL is a promising metabolic target, and whether the inhibitor is a promising therapeutic approach, for osteosarcoma that should be evaluated in future studies.
Comprehensive mutational analysis of gastrointestinal stromal tumors lacking activating mutations in the KIT, PDGFRA, and BRAF genes
• Martin G. Belinsky, PhD, Fox Chase Cancer Center
• Recipient of a $50,000 Sarcoma Foundation of America Research Award
• Abstract: Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms that generally harbor activating kinase mutations in either KIT or PDGFRA. GISTs respond to front-line treatment with the specific kinase inhibitor imatinib mesylate (IM). However, approximately 15% of adult GISTs lack mutations in these kinases and respond poorly to IM. Activating mutations in the serine-threonine kinase BRAF have been identified in a minority (~7%) of KIT/PDGFRA mutation-negative GISTs, suggesting that somatic mutations in other kinases may be responsible for the onset of a subset of GIST.
In Aim 1 of this proposal we propose deep exome sequencing of paired constitutional and GIST DNA from four adult patients whose tumors lack KIT/PDGFRA/BRAF mutations in order to identify somatic mutations that may be oncogenic. Although we hypothesize that dominant-active kinase mutations in these tumors are responsible for GIST oncogenesis, exome-wide sequencing will ensure that variants in all genes will be examined. We will also examine other mechanisms for oncogenesis, for example by capturing genes with somatic changes in the tumors in addition to novel germline variants in the patients. Variant analysis will be facilitated by comparisons with the existing SNP database, with the database of variants from the 1,000 Genomes project, and with the growing database of exome sequences available within our institution.
Genes with confirmed somatic mutations in the exome-wide analysis will be further examined in a candidate gene analysis as described in Aim 2 of this proposal. The goal is to determine the frequency and spectrum of genetic changes in these genes within a larger panel of GIST tumors, using as comparison constitutional DNA from these patients where available as well as a large panel of constitutional DNA from unaffected individuals. The outcome of these analyses will be a set of novel therapeutic targets for the treatment of GIST refractory to standard therapy.
Epigenetic changes and epigenetic therapy in retroperitoneal liposarcomas
• Nita Ahuja, MD, Johns Hopkins University
• Recipient of a $50,000 Sarcoma Foundation of America Research Award
• Abstract: Retroperitoneal liposarcomas are rare tumors and the only effective treatment is surgery. However, local recurrence is common after surgery and is the major cause of death. Due to the rarity of these tumors, little is known about the underlying abnormalities.
Epigenetic changes, such as DNA methylation of promoter-associated CpG islands resulting in transcriptional silencing, are known to be frequent mechanisms of silencing tumor suppressor genes in most common human cancers. However, little is known about the epigenetic alterations in liposarcomas. DNA hypermethylation of tumor suppressor genes as p16 and Wnt pathway genes as APC has been described in small series of liposarcomas suggesting that epigenetic alterations may play an important role in pathogenesis of liposarcomas. Furthermore, a prior study suggested that epigenetic therapy may cause terminal differentiation of liposarcoma cell line to adipocytes. We hypothesize that epigenetic alterations occur frequently in liposarcomas and such alterations may be used to define prognostic biomarkers. Furthermore, we hypothesize that epigenetic therapy may have a role in treatment of liposarcomas.
Our laboratory has experience in developing prognostic epigenetic markers and is currently testing epigenetic therapy in solid tumors in clinical trials. Moreover, our center has a high-volume multidisciplinary sarcoma team which provides a strong foundation for our proposed sarcoma research. In the current proposal, we plan to 1) Identify the epigenetic changes that occur frequently in retroperitoneal liposarcomas; 2) Identify biomarkers that predict survival and response to treatment in retroperitoneal liposarcomas; and 3) Test the efficacy of combination epigenetic therapy in vivo mouse models of liposarcoma. Our longterm goals are to understand the epigenetic changes that occur in retroperitoneal liposarcomas and define prognostic biomarkers as well as determine the utility of epigenetic therapy in sarcomas.
Hedgehog palmitoylation inhibitors to block sarcoma growth
• Marilyn D. Resh, PhD, Memorial Sloan-Kettering Cancer Center
• Recipient of a $50,000 Sarcoma Foundation of America Research Award
• Abstract: Hedgehog (Hh) signaling has been implicated in the pathogenesis of several types of sarcomas. The goal of the proposed research is to develop drugs that block modification of Hh proteins with palmitate and thereby prevent Hh mediated growth of human sarcomas. In order to signal correctly, Hh must be modified by attachment of the 16-carbon fatty acid palmitate to its N-terminus. We aim to exploit Hh palmitoylation as a potential Achilles heel by targeting Hhat (Hedgehog acyltransferase), the enzyme that catalyzes attachment of palmitate to Hh. My laboratory developed an in vitro Hh palmitoylation assay that is dependent on Hhat activity. The assay was optimized for High Throughput Screening (HTS) to identify small molecule inhibitors of Hhat. We completed the HTS, identified multiple “hits”, and chose 4 lead compounds. Since palmitoylation is required for Hh function, Hhat inhibitors that block Hh palmitoylation could be developed into novel chemotherapeutics that will be efficacious in the treatment of sarcoma.
Aim 1) To determine the effect of Hhat and Hh depletion on sarcoma cell proliferation. Human sarcoma cells will be transduced with lentiviruses encoding ShRNAs directed against Shh, Dhh, Ihh or Hhat and the effects on cell proliferation, survival and migration will be determined.
Aim 2) To identify small molecule inhibitors of Hhat that inhibit proliferation of sarcoma cells in tissue culture and in animal models. We will assess the ability of selected Hhat inhibitors to inhibit proliferation, survival and migration of human sarcoma cell lines in vitro. Pharmacokinetics and pharmacodynamic parameters for the Hhat inhibitors will be determined in mice. The effects of Hhat inhibitors in a xenograft tumor model in vivo will then be assayed. The most effective inhibitors will be derivatized and assayed for further therapeutic development.
2010 SFA Research Grant Recipients
Identification of causative mutations for Ewing sarcoma
• Mizuki Azuma, Ph.D. University of Kansas Center for Research
• Recipient of a $25,000 research award, Dominick Rizzi Memorial Research Award
• Abstract: Ewing sarcoma is the second most common form of bone childhood cancer, and its genetic hallmark is an aberrant fusion gene known as EWS/FLI1. It is unclear whether the expression of EWS/FLI1 alone is sufficient, or additional mutations are required for malignant transformation. Identifying the common mutations among Ewing sarcoma cells is a significant process for patient treatment because of the following reasons: i) mutations can be utilized as biomarkers and ii) chemical compounds that target the mutations are strong candidates for treatment. Tumor cells are not suitable for identifying the causative mutations because additional mutations are already accumulated. And there is no Ewing sarcoma animal model. Therefore, establishing Ewing sarcoma animal model allows us not only to identify the causative mutations required for malignant transformation, but also to screen for the candidate drugs for patient treatment.
Specific aim for this proposal is to determine whether expression of EWS/FLI1 leads to aneuploidy and Ewing sarcoma in zebrafish. In recent studies we reported that the EWS/FLI1 fusion protein leads to mitotic defects in human cells and zebrafish. We subsequently demonstrated binding between EWS/FLI1 and EWS that leads to inhibition of EWS activity. The hypothesis of this proposal is that the EWS/FLI1-induced mitotic defects leading to aneuploidy and to Ewing sarcoma formation. To address the hypothesis, we generated a conditional transgenic zebrafish line expressing EWS/FLI1, and are in the process of characterizing the lines. We will investigate the mutations caused by EWS/FLI1 or additional mutations if any required for Ewing sarcoma formation. Zebrafish is a suitable model for this project because it contains the microenvironment for malignant formation, and high fecundity (200 eggs/female/week) ensures statistical reliability for genetics. Zebrafish will provide an in vivo whole animal model amenable to genetic screening for second hit mutations, or chemical compounds for patient treatment.
• Final Report: Click to view PDF
Crosstalk between EGFR and IGF1R mediated by polymorphisms in the EGFR promoter as a mechanism for resistance to IGF1R directed therapy in osteosarcoma
• E. Anders Kolb, M.D. Alfred I. duPont Hospital for Children
• Recipient of a $25,000 research award, Dominick Rizzi Memorial Research Award
• Abstract: Osteosarcoma is the most common primary malignant tumor of bone, with a pea incidence in the second decade of life. New therapies are needed to improve survival especially among patients with recurrent or metastatic disease. Insulin growth factor-I (IGF-I) is essential for growth in osteosarcoma. Several of the fully humanized monoclonal antibodies targeting the IGF-I receptor (IGF-IR) currently in clinical trials have clear evidence of growth inhibition in osteosarocoma tumor lines. However, some osteosarcoma lines remain resistant to IGF signal inhibition. Preliminary data presented using a fully humanized monoclonal anti-IGF-IR antibody, indicates that osteosarcoma tumors may employ salvage signaling through the epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (MAPK) pathways to permit growth in the presence of IGF-IR inhibition. In fact, preliminary data presented in this proposal demonstrate that single nucleotide polymorphism in the EGFR promoter predicts induction of EGFR in response to IGF1 R inhibiton. The hypothesis evaluated in Specific Aim 1 is that the activity of the EGFR pr6moter in osteosarcoma is influenced by the sequence of the Sp1 binding site of the EGFR promoter leading to differential activation of EGFR in response to IGF1R inhibition. Specifically, plasmid constructs representing the different -216 sequences of the EGFR promoter driving a luciferase reporter will be transiently transfected into osteosarcoma cells and human fibroblasts. Promoter activity in response to an antibody blocking IGF1R activity and IGF1R RNA interference (RNAi) will be measured.
The hypothesis evaluated in Specific Aim 2 is that the genotype at -216 of the EGFR promoter will serve a biomarker for response to IGF1 R inhibition. Peripheral blood mononuclear cells will be harvested from volunteer donors and EGFR expression evaluated
in response to an antibody blocking IGF1 R activity and IGF1 R RNAi. Results will be correlated with genotyping of buccal mucosal cells.
• Final Report: Click to view PDF
Functional analysis of Mirk/Dyrk 1B in osteosarcoma
• Zhenfeng Duan, M.D., Ph.D. Sarcoma Molecular Biology Laboratory, Center for Sarcoma and Connective Tissue Oncology, Massachusetts General Hospital
• Recipient of a $25,000 research award, Alexander Burdo/Ziopharm Research Award
• Abstract: Osteosarcoma is the most common primary malignant tumor of bone. Standard treatment is surgery and chemotherapy, which has significantly improved the survival rate from 11% with surgery alone to 60-70% when surgery is combined with chemotherapy. Unfortunately, progress has been limited over the past 20 years, and the identification of new therapies is critical to improving the survival rate of osteosarcoma patients. Protein kinases have recently been targeted by pharmaceutical agents to decrease tumor growth (i.e. Bcr-Abl, EGFR, Her2, c-kit). One of the less known kinases, Mirk/Dyrk1B, is a unique, multifunctional kinase, that has been observed to be amplified, upregulated, or constitutively expressed in several different types of cancer. Previously, we utilized a lentiviral shRNA kinase library to screen osteosarcoma cells and showed that knockdown of Mirk expression inhibits cell growth and induces apoptosis in osteosarcoma cell lines. We observed that Mirk is commonly overexpressed in osteosarcoma but not in normal osteoblasts. Mrik participates in multiple cellular pathways that enhance tumor growth and survival, and we observed that high levels of Mirk expression in osteosarcoma tissue are associated with more aggressive clinical behavior. Therefore, our proposed project has the following goals: 1) Determine the impact of overexpression of Mirk in human osteoblast cell lines on cell differentiation, proliferation and neoplastic transformation; and 2) Analyze the molecular consequences of inhibiting Mirk in osteosarcoma cell lines. Our long term goal is to elucidate the regulatory mechanisms controlling expression of Mirk and ultimately develop therapeutic strategies that can be used to improve the treatment of patients with osteosarcoma.
• Final Report: Click to view PDF
A Quantitative Imaging-based Biomarker for Assessment of Therapy Response in Soft Tissue Sarcomas by Differential Volume Estimation of Viable and Non-viable Tumor Fractions
• Anand K. Singh, M.D. Massachusetts General Hospital
• Recipient of a $25,000 research award
• Abstract: The rapid developments in imaging techniques and action of newer chemotherapeutic drugs have highlighted the limitations of response evaluation criteria in solid tumors (RECIST) for assessing treatment response in soft tissue sarcoma. Some chemotherapeutic agents may induce more tumor necrosis compared to another, causing enlargement of the total tumor size and thus leading to false positive interpretations of disease progression on RECIST of an otherwise stable or regressed disease state. Newer imaging techniques like tumor perfusion and positron emission tomography (PET) need standardization with regard to apparent diffusion coefficient values and tighter control on false positive detections respectively with added disadvantages of extra costs and scanning. Revisions in tumor response assessment criteria’s are therefore gaining importance.
In our preliminary study, excellent correlation was observed between the proposed MRI volumetry technique and histopathology for estimation of non-viable tumor fraction in treated and excised tumors. We thus hypothesize that non viable tissue fraction of soft tissue sarcomas can be accurately quantified by performing 3D segmentations on MRI datasets and such bio-estimates will serve as a better predictor of therapy response compared to existing RECIST criteria. We will obtain interval change in viable and non-viable tumor fractions by applying semi-automated 3D segmentation techniques on axial slices of contrast-enhanced pre-treatment and post-treatment T1weighted MRI datasets and compare them with the response obtained by RECIST criteria 1.0. We will also estimate one-dimensional tumor measurements on both MRI time-point datasets and assess therapy response based on RECIST criteria. Finally, we will compare statistical estimates of two methods generated by Kaplan-Meier survival curves for progression-free disease and overall (long-term) survival time.
After successful testing of our hypothesis, this cost-effective and feasible innovation may have significant positive impact in clinical decision-making for treatment of sarcomas where similar principles can be applied on wider latitude for other body tumors.
• Final Report: Click to view PDF
Targeting GLI1 in Ewing Sarcoma
• William A. May, M.D. Saban Research Institute, Children’s Hospital Los Angeles
• Recipient of a $25,000 research award, Mark Herzlich Research Award
• Abstract: The EWS/FLI1 oncogenic transcription factor is both unique to and indispensable to the development of Ewing’s Sarcoma and peripheral Primitive Neuroectodermal Tumor (collectively known as the Ewing Sarcoma Family Tumors or ESFT). It is imperative that we translate our understanding of EWS/FLI1 biology into improved therapeutics for Ewing Sarcoma.
We have recently published findings that EWS/FLI causes increased expression of Gli1 in ESFT in a Hedgehog independent fashion. We have further published data that inhibition of GLI1 expression by shRNA results in decreased tumorigenicity and that several known targets of EWS/FLI1 are diminished in their expression. This suggests that signaling through Gli1 may be a central and critical event in EWS/FLI1 downstream gene activation which is central to this disease. Our preliminary data demonstrates that two newly identified compounds termed GANT58 and GANT61, which inhibit this pathway at the level of GLI1, act to inhibit the HH-GLI1 pathway in Ewings cell lines in vitro. Preliminary data suggest that at least one of these compounds will also be effective in vivo. This proposal will utilize these novel compounds to fully test the potential of GLI1 inhibition in Ewing Sarcoma in vivo models. Both compounds will be tested at full doses in xenograft models of ESFT for their inhibition of tumor growth. We will assess experimental tumors for reduced expression of downstream targets of GLI1 in ESFT. We will also use in vivo imaging systems to assess the effect of the drugs on GLI1 transcriptional activity in vivo. Recently, several prominent tumor types have been shown to depend on GLI1 activation in a Hedgehog independent fashion. As these developments stimulate the development of pathway inhibitors effective in these circumstances, the data generated in this proposal will establish whether such compounds could benefit patients with Ewings Sarcoma in the near future.
• Final Report: Click to view PDF
Developing a MicroRNA-Based Strategy for Targeting Uterine Leiomyosarcoma
• Matthew L. Anderson M.D., Ph.D. Baylor College of Medicine
• Recipient of a $25,000 research award, Jay V. Jackson Memorial Research Award
• Abstract: MicroRNAs (miRNAs) are a novel class of small, non-coding RNA transcripts that broadly regulate patterns of gene expression. Recently, we used Next Generation Sequencing to dentify 49 individual miRNAs differentially expressed in uterine leiomyosarcoma (ULMS). We also identified potential targets for these miRNAs by using bioinformatic algorithms to screen gene products differentially expressed when genome-wide transcriptional profiling was used to examine our specimens. Based on these results, we hypothesize that miR-143 plays a central role in the pathogenesis of ULMS. To test this hypothesis, we intend to examine the function of miR-143 in uterine smooth muscle and smooth muscle tumors both n vitro and in vivo. Mimics and inhibitors for miR-143 will be transfected into an established LMS cell line (SK-LMS), primary cell strains derived from metastastic ULMS (285, 505 and 987) and telomerase-immortalized lines derived from human myometrium (HM) and eiomyoma (DD). Standard MTT and caspase assays will be used to compare rates of Droliferation and apoptosis. Flow cytometry will be used to measure the impact of altered miR-143 levels on the cell cycle. Scratch assays will be used to measure cell migration. Once biologic functions for miR-143 have been established, target genes will be validated using quantitative PCR and Western blotting. We also intend to test whether miR-143 sensitizes SK-LMS and primary ULMS cell strains to gemcitabine and docetexel. Lastly, we plan to examine whether miR-143 regulates the growth and metastasis of ULMS in vivo, using clones of SK-LMS stably transfected with miR-143 mimic, miR-143 inhibitor or scrambled control to create xenografts in nude mice.
We believe that the insight generated by the proposed work will establish a functional role for miR-143 in ULMS and set the stage for the clinical application of this insight. Future work would focus on how best to deliver miR-143 for therapeutic purposes.
• Final Report: Click to view PDF
Regulation of sarcomagenesis by the Piwi proteins and their interacting small RNAs (piRNAs)
• Igor Matushansky, M.D., Ph.D. Columbia University Medical Center, Irving Cancer Research Center
• Recipient of a $25,000 research award
• Abstract: Recently levels of HIWI (the human ortholog of Drosophila PIWI) HIWI, have been found to be expressed in sarcomas with higher HIWI levels correlating significantly with worse clinical outcomes. In lower organisms, PIWI family members silence mobile genomic elements (i.e., transposons) and thus help to maintain genomic integrity via regulating the production of a distinct class of small ‘silencing’ RNAs - aptly named Piwi-interacting RNAs or piRNAs. It is currently unclear as to why ‘higher’ levels of a gene that silences mobile genetic elements would correlate with worse clinical outcomes for sarcoma patients. Our studies have revealed that overexpression of HIWI in mesenchymal stem cells (MSCs; connective tissue progenitors) prevents their differentiation and generates sarcomas. Since Piwi works through generation of piRNAs we feel our models create the perfect environment to identify specific piRNAs that mediate the sarcomagenic process. Our working hypothesis is that overexpression of HIWI results in piRNA mediated suppression of genes that are essential to preventing tumorigenesis. The specific aims of this proposal are to (1) assay for the presence of piRNAs in Hiwi-mediated transformed MSCs and correlate their presence to piRNAs in sarcoma tissues; (2) identify target genes that are deregulated via Hiwi-mediated tumorigenicity; and (3) assess the role of HIWI in promoting sarcomagenesis in vivo and responsiveness to piRNA treatment.
Importantly, since Piwi/piRNA cycle is self-perpetuating, any tumorigenic process initiated by Piwi/piRNA may by permanently terminated via the one time (transient) introduction of the appropriate piRNA - and thus holds great promise as a curative cancer therapy.
• Final Report: Click to view PDF
Lipid Metabolism in Liposarcoma: A Novel Target for Therapeutic Intervention
• Nancy B. Kuemmerle, Ph.D. Saban Research Institute, Dartmouth Medical School
• Recipient of a $25,000 research award, Richard and Kathy Lobo Research Award
• Abstract: There is no effective medical therapy for patients with liposarcoma. We find that liposarcomas are dependent upon a constant supply of fatty acids to fuel their growth. Tumors may potentially acquire these lipids by a) de novo synthesis using fatty acid synthase (FASN), b) extracellular hydrolysis of circulating fat using lipoprotein lipase (LPL), followed by cellular uptake using CD36, or c) endocytosis of triglyceride-rich particles using Syndecan-1. We find that liposarcomas express high levels of FASN, LPL, CD36, and Syndecan-1. Moreover, Spot 14, a key nuclear driver of the genes encoding enzymes involved in lipogenesis, is also abundant in liposarcomas. We have demonstrated that the growth of liposarcoma cells is impaired by inhibition of lipid synthesis and is promoted by increased lipid uptake. Our Aims are 1) Identify proteins which interact with Spot 14 in liposarcoma but not in normal adipose tissues. 2) Investigate the functional interactions among the three pathways of lipid acquisition in liposarcoma cells, and determine whether inhibition of one pathway results in upregulation of the others. This work will provide understanding of the mechanisms underlying the exquisite susceptibility of liposarcoma to metabolic manipulation, and will guide the development of novel interventions to control liposarcoma, which is currently curable only by successful surgical resection.
• Final Report: Click to view PDF
How does a sarcoma circumvent fusion oncoprotein-mediated toxicity?
• Frederic G. Barr, M.D., Ph.D. University of Pennsylvania School of Medicine
• Recipient of a $25,000 research award, ARIAD and Merck Research Award
• Abstract: Alveolar rhabdomyosarcoma (ARMS) is an aggressive soft tissue sarcoma with a 2;13 translocation that generates a PAX3-FKHR fusion oncoprotein. Introduction of PAX3-FKHR into multiple cell types showed that this oncoprotein exerts growth suppression and/or cell death when expressed at levels comparable to those found in ARMS cells. Subclones from a myoblast population expressing MYCN and an inducible form of PAX3-FKHR showed increasing growth suppression in a plating assay as PAX3-FKHR activity was induced. However, when fibroblasts were added for a focus formation assay, numerous transformed foci formed when PAX3-FKHR was induced. Multiple ARMS cell lines have a similar phenotype of higher focus formation than plating efficiency. In ARMS cells, siRNA-mediated suppression of FGFR4, a PAX3-FKHR downstream target, increases plating efficiency without affecting focus formation. We hypothesize that FGFR4 signaling contributes to the mechanism of PAX3-FKHR-induced toxicity and that stromal cells attenuate the toxic effects of high PAX3-FKHR activity and promote PAX3-FKHR-induced oncogenesis. To investigate which stromal cells are capable of exerting this effect, we will use our cell culture system to study additional fibroblasts, myoblasts, and mesenchymal cell types associated with preferred sites of ARMS metastasis. We will study the role of FGFR4 signaling in PAX3-FKHR-induced toxicity and oncogenesis by extending our initial RNA interference studies. PCR and western blot approaches will then be used to analyze expression of FGFR4 and its ligands and the activity of downstream MAPK and AKT signaling pathways in these cells. Based on these findings, inhibitors will be selected to examine the role of specific components of the FGFR4 signaling pathway. These studies will provide a comprehensive approach to an important problem in sarcoma pathogenesis and lead to therapeutic strategies to undermine mechanisms by which sarcoma cells circumvent fusion and thereby become sensitized to this toxicity.
• Final Report: Click to view PDF
Identification of the target genes of the EWS/NR4A3 fusion protein expressed in extraskeletal myxoid chondrosarcoma
• Yves Labelle, Ph.D. Centre Hospitalier Universitaire de Quebec
• Recipient of a $25,000 research award, Mandell/Kropp Run for a Sarcoma Cure Research Award
• Abstract: Extraskeletal myxoid chondrosarcoma (EMC) are soft tissue tumors occurring mainly in the extremities, most commonly the thigh and knee. In approximately 75% of these tumors, at (9;22) chromosome translocation is present. This translocation encodes a fusion protein named EWS/NR4A3 which consists of the amino-terminal domain of the EWS protein fused to the complete amino acid sequence of the NR4A3 nuclear receptor. In my laboratory we have shown that EWS/NR4A3 is a highly potent transcriptional activator, suggesting that one way in which it may contribute to tumorigenesis is by activating specific target genes. There are no cell lines established from EMC tumors, so to generate a human cellular model to study EWS/NR4A3 we have used a human bone marrow mesenchymal stem cell line. This cell line, named hTERT, has retained the ability to differentiate into osteoblasts and chondrocytes, does not form foci in soft agar, has a normal karyotype, has retained inhibition of proliferation by cell-cell contact, and does not induce tumors in nude mice. We have chosen this cell line because an extensive immunohistochemical study of EMC tumors has shown that they most likely consist of primitive mesenchymal cells. We have transfected the cells with an EWS/NR.4A3 expression vector and isolated several cell lines stably expressing EWS/NR4A3. These cell lines display a loss of inhibition of proliferation by cell-cell contact. Since loss of contact inhibition is one characteristic of tumor cells, our hypothesis is that this loss of contact inhibition is due to the activation of specific target genes by EWS/NR4A3 that may also play a role in the development of EMC tumors. To identify these genes, we will perform microarray analyses of the cell lines to identify genes over-expressed in the presence of EWS/NR4A3. We will compare those genes to a list of genes which we have previously found to be specifically over-expressed in EMC tumors expressing EWS/NR4A3. Genes over-expressed in both will be considered potential targets of the fusion protein. Future experiments will involve immunohistochemical analyses of EMC tumors to confirm expression of the corresponding proteins in tumor cells. On the long-term, these proteins could be targeted to treat patients with EMC tumors expressing the fusion protein.
• Final Report: Click to view PDF
2009 SFA Research Grant Recipients
A preclinical mouse model for targeted therapy in uterine leiomyosarcoma
• Sandra Orsulic, Ph.D. Cedars-Sinai Medical Center
• Recipient of a $25,000 research award, Shelby L. Richter Memorial Research Award
• Abstract: Uterine leiomyosarcoma (ULMS) is a rare gynecologic malignancy that has a low survival rate. Currently, there is no effective treatment for ULMS. We have generated a mouse model of ULMS and demonstrated that the loss of BRCA1 function accelerates the progression of these tumors. Consistent with the hypothesis that BRCA1 plays a role in ULMS, we have shown that the BRCA1 protein is absent in 36% of human ULMS. Our findings provide a rationale for investigating therapies that target BRCA1 deficiency in ULMS. To this end, we will determine the mechanism of BRCA1 downregulation in human tumor samples and test the efficacy of BRCA-targeted therapy in a mouse model of ULMS. The results of these studies could provide justification for a clinical trial for ULMS patients with a novel BRCA-targeted therapy based on poly(ADP-ribose)-polymerase-1 (PARP-1) inhibitors.
• Final Report: Click to view PDF
Therapeutically targeting aberrant chromatin remodeling in sarcomas
• Charles W. M. Roberts, M.D., Ph.D. Dana-Farber Cancer Institute
• Recipient of a $25,000 research award, Michael Mandell/Run for a Sarcoma Cure Research Award
• Abstract: SNF5, a core subunit of the SWI/SNF chromatin remodeling complex, is a potent tumor suppressor that is specifically mutated in several types of sarcoma. It has been thought that oncogenesis in the absence of SNF5 occurs due to a loss of function of the SWI/SNF complex. However, based upon preliminary data we hypothesize that cancer instead arises due to oncogenic activation of the residual SWI/SNF complex. We propose to utilize our genetically engineered mouse models to demonstrate this, to further establish that targeted inhibition of residual activity can be therapeutically beneficial, and to establish a pre-clinical model.
• Final Report: Click to view PDF
Evaluation of the oncogenic potential of genes within the 1q23 amplicon
• Alexander Beeser, Ph.D., Kansas State University
• Recipient of a $25,000 research award, Blake Cadkin Memorial Research Award
• Abstract: Comparative genomic hybridization identified amplification of 1q23 to be associated with liposarcomas. Further analyses of 1q23 narrowed down the candidate “driver” genes to two; DUSP12 (encoding a dual-specificity phosphatase) and ATF6 (a transcription factor of the unfolded protein response). We propose to assess the specific consequences of DUSP12/ATF6 over-expression by creating stable inducible cell lines as models of 1q23 amplification. The consequences of specific DUSP12 or ATF6 over-expression on MAP Kinase and other cancer-relevant signaling pathways will be evaluated. Identification of these pathways may expose novel vulnerabilities that can be exploited in the treatment of sarcomas by accelerating translational therapeutics.
• Final Report: Click to view PDF
Development of novel dual Insulin-like Growth Factor-1 Receptor (IGF-1R)/ Epidermal Growth Factor Receptor (EGFR) inhibitors for treatment of Ewing’s sarcoma
• William Bornmann, Ph.D., M.D. Anderson Cancer Center
• Recipient of a $25,000 research award, Mary Beth Knox Memorial Research Award
• Abstract: Ewing’s sarcoma, a devastating disease in children and young adults, is in great need for a therapy. Insulin-like Growth Factor type 1Receptor (IGF-1R) and Epidermal Growth Factor Receptor (EGFR) proved to be interesting targets for different sarcomas. Here we proposed the development and synthesis of novel dual IGF-1R/EGFR inhibitors based on virtual screening. We hypothesize that simultaneous targeting of the two receptor tyrosine kinases will be an efficient treatment for Ewing’s sarcoma. Inhibitory activity and selectivity of the inhibitors will be determined in vitro.
• Final Report: Click to view PDF
Pharmacodynamic assessment of AKT and SRC inhibitors in primary human sarcoma xenograft models
• Damon Reed, M.D., Moffitt Cancer Center
• Recipient of a $25,000 research award, Marny S. Tobin Memorial Research Award
• Abstract: The poor outcome of patients with advanced sarcomas underscores the rationale supporting novel therapeutic strategies. Combination treatment with dasatinib and triciribine effectively blocks growth of sarcoma cell lines through inhibition of SRC and AKT pathways, respectively. We will determine the activity of these drugs against a set of xenografted tumors obtained from sarcoma patients and analyze pharmacodynamic markers of response using a quantitative multiplex assay platform in vivo. The studies that would be supported by this application are expected to provide a foundation to support rational clinical trial design for these agents to maximize their therapeutic potential in sarcoma.
• Final Report: Click to view PDF
Targeting kinases to reverse multidrug resistance in human sarcoma
• Zhenfeng Duan, M.D., Ph.D., Massachusetts General Hospital
• Recipient of a $25,000 research award, ARIAD and Merck Research Award
• Abstract: Protein kinases play an important role in the pathogenesis of cancer. Furthermore, kinases can be targeted by pharmaceutical agents to decrease tumor growth (i.e. EGFR, Her2, c-kit). This project examines the role of specific kinases as sarcoma cancer cells develop resistance to chemotherapeutic agents. We aim to: 1) determine the role of kinases in supporting chemoresistance to doxorubicin and methotrexate in sarcoma cell lines; and 2) determine the effectiveness of inhibiting kinases to reverse drug resistance in a sarcoma xenograft mouse model. We hope to identify the most suitable kinases for future development as new drugs for patients with sarcoma.
• Final Report: Click to view PDF
Identification of aberrantly methylated genes in Ewing sarcoma
• Scott Borinstein, M.D., Ph.D., Fred Hutchinson Cancer Research Center
• Recipient of a $25,000 research award, ARIAD and Merck Research Award
• Abstract: DNA methylation is part of a complex mechanism that regulates genes. The goal of this proposal is to identify genes that are “turned off” by DNA methylation and contribute to Ewing Sarcoma tumor formation. We will determine if the methylation pattern of these genes is associated with tumor location, tumor spread, and how the neoplasm responded to treatment. We hope that this study will lead us to better understanding of the cause of ES and will lead to the development of better diagnostic and therapeutic modalities for these patients.
• Final Report: Click to view PDF
Targeting the Ras-pathway for the treatment of embryonal rhabdomyosarcoma
• David Langenau, Ph.D., Massachusetts General Hospital
• Recipient of a $25,000 research award, PharmaMar Research Award
• Abstract: A transgenic zebrafish model of embryonal rhabdomyosarcoma was recently created that is molecularly similar to human disease and from which the cancer stem cell has been identified, a cell most similar to the normal muscle stem cell-the actived satellite cell. My hypothesis is that the RAS pathway is responsible for eliciting stem cell self-renewal programs in a subset of the RMS cell types, and that this in turn regulates tumor growth. Targeting downstream RAS pathways and self-renewal may provide a new way to curb sarcoma growth in patients affected with this devastating disease.
• Final Report: Click to view PDF
Targeting centrosomes in uterine leiomyosarcoma
• Matthew Anderson, M.D., Ph.D., Baylor College of Medicine
• Recipient of a $25,000 research award
• Abstract: Uterine leiomyosarcoma (ULMS) is characterized by its early metastases, frequent recurrences and poor outcome. Using genome-wide transcriptional profiling, we have found that the Aurora A and B centrosomal kinases are robustly overexpressed in ULMS.
AIMS:
1. Assess the clinical significance of Aurora kinase misexpression by correlating levels of expression and surrogates for function with outcome.
2. Determine the biologic impact of targeting Aurora kinases in ULMS using unique cell lines and a novel xenograft model in mice.
This work will determine whether a phase I/II trial of Aurora kinase inhibitors currently in clinical testing is warranted for ULMS patients.
• Final Report: Click to view PDF
Targeting the Wnt pathway in synovial sarcoma
• Josiane Eid, M.D., Vanderbilt University Medical Center
• Recipient of a $25,000 research award
• Abstract: Synovial Sarcoma is an aggressive soft tissue tumor (SS) that afflicts young adults. Its high fatality rate warrants the design of more efficient curative approaches. SS is characterized by a unique translocation that creates the SYT-SSX fusion protein. SYT-SSX plays a central role in the development of SS. Here we present active Wnt/beta-catenin signaling as a downstream cascade of SYT-SSX and as an ideal candidate pathway for targeting in SS. We will generate murine models to assess the extent of Wnt/beta-catenin signaling contribution to SS formation and measure the effect of a specific pharmacological beta-catenin inhibitor on SS tumor growth.
• Final Report: Click to view PDF
2008 SFA Research Grant Recipients
• Lawrence A. Donehower, Ph.D. Baylor College of Medicine
• Recipient of a $25,000 research award
• Abstract: Osteosarcoma, while relatively rare in humans (about 1000 cases per year in the U.S.), represents a continuing challenge to oncologists. Current chemotherapy regimens have a 60-70% five year survival rate. Thus, there is a need for more effective, less toxic therapies. We have extensively characterized a mouse osteosarcoma model to test therapies that may be transferable to human osteosarcomas. In this application, we propose a “proof of principle” experiment to show that downregulating the CSF-1 receptor (highly upregulated in mouse and human osteosarcomas) by siRNA and small molecule inhibitor approaches will result in significant inhibition of osteosarcoma growth in vivo.
• Final Report: Click to view PDF
Evaluation of flow cytometry for the detection of circulating Ewing sarcoma cells
• Steven DuBois, M.D. University of California San Francisco
• Recipient of a $25,000 Denis Fedorov Memorial Research Award
• Abstract: Approximately 25% of patients with Ewing sarcoma have circulating EWS fusion transcripts using RT-PCR. RT-PCR has several practical disadvantages. This study will evaluate a flow cytometry method we developed to detect circulating Ewing sarcoma cells. The primary aims are:
1. To establish the feasibility of this method and
2. To compare this method to RT-PCR results.
Twenty patients with Ewing sarcoma will enroll. Blood and bone marrow will be evaluated for circulating tumor cells using flow cytometry and RT-PCR. The flow cytometry and RT-PCR results will be compared to determine whether this method merits further evaluation in larger studies.
• Final Report: Click to view PDF
Predicting treatment response of soft tissue sarcomas to neoadjuvant therapy by imaging proliferation with a FLT-PET/CT scan: a pilot study
• Frederick C. Eilber, M.D. University of California Los Angeles
• Recipient of a $25,000 Shelby L. Richter Memorial Research Award
• Abstract: Although metabolic imaging with F18-fluorodeoxyglucose positron emission tomography(FDG-PET) is more sensitive than standard size-based criteria(RECIST) at monitoring response to therapy in high-grade soft tissue sarcomas(STS), its limited specificity restricts its utility as a functional bio-marker.
Hypothesis: Imaging proliferation with 18F-fluorodeoxythymidine-positron-emission-tomography(FLT-PET) will provide a more accurate assessment of response in patients with high-grade STS.
Aims:
1. Evaluate the ability of changes in FLT uptake and size to predict histopathologic response to systemic neoadjuvant therapy.
2. Compare change in FLT uptake with immunohistochemical markers of proliferation, cell-cycle-progression and apoptosis.
3. Correlate changes in FLT uptake, size and histopathology with recurrence and survival.
• Final Report: Click to view PDF
Mechanisms of tumor progression and kinome targeting in peripheral nerve sheath tumors
• Cristina Antonescu, M.D. Memorial Sloan-Kettering Cancer Center
• Recipient of a $25,000 research award
• Abstract: Tyrosine kinases (TK) are central regulators of signaling pathways that control critical activities in cells. A breakthrough in therapy for cancers associated with activating mutations in TKs is the development of imatinib mesylate for the treatment of CML and GIST tumors. Our hypothesis is that novel kinase oncogenic mutations might be implicated in the pathogenesis of tumor progression in peripheral nerve sheath tumors (PNST) and therefore potential targets to molecular inhibition. MPNSTs are highly aggressive neuroectodermal mesenchymal neoplasms, for which no effective cytotoxic or targeted chemotherapy is currently available. Our aim is to perform high throughput mutational analysis of 15 TKs in a group of 10 MPNST patients. The TKs selection will be based on their transcriptional expression level available from our previous microarray profiling. An additional 10 PNST samples will be studied on the microarray, focusing on malignant transformation in neurofibroma, to specifically address the transcriptional alterations related to tumor progression. The identified TK mutations in this pilot study will then be tested for constitutive activation and will be sought in a larger sarcoma subset.
• Final Report: Click to view PDF
CSF1 inhibitors in soft tissue tumors
• Paul W. Clarkson, MBChB British Columbia Cancer Agency
• Recipient of a $25,000 Brian J. Monaghan Memorial Research Award
• Abstract: Our recent microarray collaborative studies demonstrate that tenosynovial giant cell tumor and aggressive soft tissue leiomyosarcomas express macrophage colony stimulating factor, which appears to represent a key factor driving these neoplasms. New drugs inhibit this target, but appropriate models which include a tumor-host macrophage interaction are lacking. We recently found that primary tumor renal subcapsular xenografts can attract host macrophages and seek to establish if this model can serve as a preclinical assay for an anti-CSF1 targeted treatment strategy in these diseases.
• Final Report: Click to view PDF
Development of a high throughput screening assay for Ewing sarcoma oncoprotein
• Aykut Üren, M.D. Lombardi Comprehensive Cancer Center, Georgetown University
• Recipient of a $25,000 Marny S. Tobin Memorial Research Award
• Abstract: Ewing’s sarcoma presents a unique opportunity to develop tumor specific targeted therapy due to a pathognomonic chromosomal translocation. Proper function of EWS-FLI1, protein product of this chromosomal translocation, is required for tumor cell survival. We will develop a cell-based High Throughput Screening assay. We will generate Ewing’s cell-lines expressing two fluorescent proteins. GFP will be expressed from an EWS-FLI1 regulated promoter and RFP from an unrelated negative control promoter. In this dual color HTS assay, a small molecule library will be screened for inhibiting GFP but not RFP, which will provide lead compounds for novel Ewing’s sarcoma therapies.
• Final Report: Click to view PDF
Analysis of targeted biological therapies in a genetically engineered mouse model of sarcoma
• Sam Yoon, M.D. Massachusetts General Hospital
• Recipient of a $25,000 Brian W. Rybarczyk Memorial Research Award
• Abstract: The development of effective drugs in the treatment of sarcomas is limited by the lack of reliable animal models of sarcomas. This is especially true for drugs that target not only tumor cells but also the interaction between tumor cells and their host microenvironment. Bevacizumab and sunitinib are promising new biological agents that inhibit tumor angiogenesis (or new blood vessel formation) and tumor growth. We recently described an inducible, genetically engineered mouse model of sarcoma where intramuscular delivery of an adenovirus expressing Cre recombinase into mice with conditional mutations in K-ras and p53 results in high-grade sarcomas of the extremity in over 90% of mice. These sarcomas closely resemble human sarcomas based on genetic and histological analysis, and these sarcomas preferentially metastasize to the lung. This study proposes to use this newly developed mouse model of sarcoma (1) to analyze the effects of bevacizumab and sunitinib developed mouse model of sarcoma (2) to analyze the effects of bevacizumab and sunitinib in inhibiting sarcoma growth and metastasis and (3) to identify mechanisms of resistance.
• Final Report: Click to view PDF
2007 SFA Research Grant Recipients
Developing an accurate genetic model for rhabdomyosarcoma therapy testing
• Dawn Chandler, Ph.D. Children’s Research Institute
• Recipient of a $25,000 Jake’s Reindeer Race/Maynard Family Research Award
• Abstract: Rhabdomyosarcoma (RMS) is the third most common solid tumor in children and is often associated with high morbidity and mortality. The heterogenous nature of RMS along with the lack of genetically accurate mouse models has precluded this cancer from many of the therapy testing strategies currently being used for other malignancies. We hope to develop a mouse model that will accurately recapitulate the human disease including genetic heterogeneity and metastatic potential. These mice will allow testing for further classification of tumors that will aid in treatment decisions and be use to test novel therapies in pre-clinical trials for RMS treatment.
• Final Report: Click to view PDF
Inspecting the Stroma to Eliminate Metastatic Osteosarcoma
Su Young Kim, M.D., Ph.D. National Institutes of Health/National Cancer Institute
• Recipient of a $25,000 research award
• Abstract: Patients with metastatic osteosarcoma have a poor overall survival rate of 20%. The importance of stromal cells in the metastatic process is becoming well established. We have preliminary data to suggest that induction of genes in the stroma may contribute to metastasis in osteosarcoma. Our hypothesis is that the molecular signals that occur between osteosarcoma cells and the surrounding stromal cells lead to metastasis. We propose to identify a metastatic signature in stromal cells by performing microarray analysis on isolated stromal cells that surround matched metastatic and non-metastatic osteosarcoma cells.
• Final Report: Click to view PDF
Study of the mechanisms of sarcoma progression using mouse models and in vitro manipulation of mesenchymal stem cells
• Eva Hernando, Ph.D. New York University School of Medicine
• Recipient of a $25,000 Jake’s Reindeer Race/Maynard Family Research Award
• Abstract: The underlying genetic defects and the cell of origin of most human sarcomas remain unknown. Recently, alterations in the PI3K-Akt pathway have been identified in certain sarcoma subtypes. For instance, loss of heterozygosity of 10q (containing the PTEN genomic locus) has been frequently observed in human leiomyosarcomas (LMS). In addition, we have found evidences of PI3K/Akt constitutive signaling in human LMS. To determine the role of aberrant PI3K-Akt signaling in LMS pathogenesis, we have genetically inactivated Pten in the mouse smooth muscle (SM). Resultant animals carrying homozygous deletion of both PTEN alleles developed widespread SM hyperplasia, abdominal and retroperitoneal LMS with a very rapid onset and elevated incidence. This finding has led us to postulate that alterations in the PTEN-Akt pathway might constitute the initiation event of SM transformation. The present proposal will be focused on unraveling the subsequent molecular alterations required for sarcoma progression. Aim 1. Determine the genetic alterations required for sarcoma progression in mouse models of leiomyosarcoma. Aim 2. Study the capacity of Pten loss and subsequent molecular events to interfere with the differentiation program and/or induce neoplastic transformation of adult mesenchymal stem cells maturing into smooth muscle in vitro. The ultimate goal of this proposal is to establish the significance and the sequential order of events at the origin and progression of leiomysarcomas. Moreover, we will determine the effect of those alterations when introduced at specific moments of the SM differentiation process.
• Final Report: Click to view PDF
An in vivo study of molecular genetics of Rb-dependent osteosarcoma
• David Thomas, M.D., Ph.D. Peter MacCallum Cancer Center
• Recipient of a $25,000 research award
• Abstract: Osteosarcoma is 500 times more common in retinoblastoma patients compared to the normal population. The majority of osteosarcomas however do not occur as part of familial syndromes. The proposed research uses a novel 45Ca induced mouse model of osteosarcoma, as well as genome-wide transcriptional profiling and array-based comparative genomic hybridisation to identify genetics changes either unique or common to familial and sporadic osteosarcoma. These studies will identify pivotal genes and genetic changes, which initiate and allow progression of osteosarcoma in the presence and absence of Rb.
• Final Report: Click to view PDF
Identifying the sources of synovial sarcoma
• Malay Haldar University of Utah
• Recipient of a $25,000 John E. Goyert Memorial Research Award
• Abstract: Synovial sarcoma is a malignant soft tissue tumor marked by a unique t (X;18) translocation leading to expression of a chimeric SYT-SSX fusion protein. We have recently developed a mouse model of synovial sarcoma by expressing SYT-SSX fusion protein within myoblasts thereby identifying skeletal muscle lineage as a potential source of this disease. However, other potential sources of this disease remain an unknown possibility. The aim of this study is to use the mouse model to investigate the existence of other sources of synovial sarcoma and whether there is any correlation between clinical behavior of the tumor and its origin.
• Final Report: Click to view PDF
Proteomic Analysis of Sarcoma: Molecular Differences between Benign and Malignant Tumors of Bone and Soft Tissue
• Justin Cates, M.D., Ph.D. Vanderbilt University Medical Center
• Recipient of a $25,000 Shelby L. Richter Memorial Research Award
• Abstract: The proteomic profiles of bone and soft tissue sarcomas remain largely unexplored. The “proteome” represents all protein isoforms expressed within a target tissue, including those with post-translational modifications. We are using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography/tandem mass spectrometry (LC-MS/MS) to evaluate proteomic differences between selected sarcomas and their presumed benign counterparts and/or normal tissues. The specific aim of this proposal is to identify sarcoma-specific differences in protein expression that define the malignant phenotype. These findings may elucidate important mechanisms in sarcoma development and lead to novel diagnostic and therapeutic strategies.
• Final Report: Click to view PDF
2006 SFA Research Grant Recipients
Discovery of small molecule inhibitors of the MDM2-ubcH5 interaction
• Brent R. Stockwell, Ph.D. Columbia University
• Recipient of a $25,000 Bradley J. Breidinger Memorial Research Award
• Abstract: We will discover small molecules that disrupt the interaction between the oncoprotein MDM2 and UbcH5, its partner E2 ubiquitin conjugating enzyme. Such compounds will disrupt the E3 ligase function of MDM2 and are likely to inhibit its oncogenic functions, both those that are p53-dependent and p53-independent. The aims are: (i) to perform an in silico screen for compounds that disrupt E3-E2 interactions, and (ii) to test predicted inhibitors in high-throughput in vitro and cellular ubiquitination assays using MDM2/UbcH5. Inhibitors of the MDM2-UbcH5 interaction could ultimately be developed into novel therapeutics for sarcomas, which frequently involve amplification of the MDM2 oncogene.
• Final Report: Click to view PDF
Early identification of chemoresistant patients for improved risk-based therapy in pediatric osteosarcoma
• Tsz-Kwong Man, Ph.D. Baylor College of Medicine
• Recipient of a $25,000 Shelby L. Richter Memorial Research Award
• Abstract: Osteosarcoma is the most common malignant bone tumor in children. The survival rate for patients with resistance to standard chemotherapy is about 40%. The poor prognosis of chemoresistant patients indicates that new paradigms are needed to identify those patients up front, so that new treatment options can be offered initially to improve their outcome. Using a proteomic approach, we have identified two circulating biomarkers that are significantly correlated with chemoresistance. In this proposal, we plan to validate these two chemoresistant biomarkers and construct a bioinformatic model to identify chemoresistant osteosarcoma patients at initial diagnosis using sera collected in a national collaborative study.
• Final Report: Click to view PDF
Cancer stem cells and sarcoma
Karl Sylvester, M.D. Stanford University
• Recipient of a $25,000 Seth A. Bailey Memorial Research Award
• Abstract: Rhabdomyosarcomas are the most common childhood soft tissue sarcoma. We have developed a conditional mouse model of alveolar rhabdomyosarcomas by expressing the Pax3:Fkhr oncogene in skeletal muscle of juvenile mice. We propose to test that our mouse model mimics the secondary genomic and gene expression changes seen in human alveolar rhabdomyosarcomas. We will (1) compare chromosomal segment gains and losses in 8 mouse and 8 human alveolar rhabdomyosarcomas, and (2) correlate genomic imbalances of mouse and human tumors to gene expression changes of those same tumors. Understanding molecular events underlying tumor invasiveness and metastasis, we simultaneously identify potential therapeutic targets.
• Final Report: Click to view PDF
Blocking SYT-SSX activities using an inhibitory peptide
• Yuechueng Liu, Ph.D. University of Oklahoma Health Sciences Center
• Recipient of a $25,000 Paul M. Rumely Memorial Research Award
• Abstract: The long-term goal of this research is to understand the role of syt-ssx gene product in synovial sarcoma pathogenesis, and to develop novel and effective treatment for the disease. The cause of synovial sarcoma is unknown. However, a characteristic fusion of two genes, syt and ssx, has been identified. We have recently discovered a peptide that blocks SYT function in vitro. Based on the finding, I propose to further determine if this peptide is able to target SYT-SSX and block its activities in vivo. This may eventually lead to the discovery of novel treatment for synovial sarcoma.
• Final Report: Click to view PDF
Identifying native and heteroclitic epitopes for T-cell immunity to PAX3-FKHR alveolar rhabdomyosarcoma
Stacie Goldberg, M.D. Memorial Sloan Kettering Cancer Center
• Recipient of a $25,000 Brian J. Monaghan Memorial Research Award
• Abstract: Advanced liposarcoma has low overall response rates to chemotherapy. There is a need for targeted therapy for liposarcoma. Utilizing microarray and RT-PCR data, we found ribonucleotide reductase M2 (RRM2) to be upregulated in our liposarcoma cell lines and tumor samples. Triapine, (3-Aminopyridine-2-carboxaldehyde-thiosemicarbazone) and hydroxyurea are ribonucleotide reductase inhibitors that exhibit anti-proliferative activity in epithelial and hematological malignancies. We have an attenuated herpes simplex virus that requires host cell ribonucleotide reductase to replicate. We hypothesize that RRM2 is a novel target for both chemotherapeutic agents and biological viral agents in the treatment of liposarcoma and plan to validate in the laboratory RRM2 as a new target for the treatment of liposarcoma.
• Final Report: Click to view PDF
Ribonucleotide reductase as a novel target in liposarcoma
• Rula Geha, M.D Memorial Sloan Kettering Cancer Center
• Recipient of a $25,000 Close Family Research Award
• Abstract: Advanced liposarcoma has low overall response rates to chemotherapy. There is a need for targeted therapy for liposarcoma. Utilizing microarray and RT-PCR data, we found ribonucleotide reductase M2 (RRM2) to be upregulated in our liposarcoma cell lines and tumor samples. Triapine, (3-Aminopyridine-2-carboxaldehyde-thiosemicarbazone) and hydroxyurea are ribonucleotide reductase inhibitors that exhibit anti-proliferative activity in epithelial and hematological malignancies. We have an attenuated herpes simplex virus that requires host cell ribonucleotide reductase to replicate. We hypothesize that RRM2 is a novel target for both chemotherapeutic agents and biological viral agents in the treatment of liposarcoma and plan to validate in the laboratory RRM2 as a new target for the treatment of liposarcoma.
• Final Report: Click to view PDF
2005 SFA Research Grant Recipients
Development of a Mouse Model of Synovial Sarcoma
• Malay Haldar University of Utah
• Recipient of a $25,000 Bradley J. Breidinger Memorial Research Award
Abstract: Synovial sarcoma is marked by a unique and specific translocation between the SYT and SSX genes resulting into the expression of the chimeric ‘SYT-SSX’ fusion protein. We are developing a mouse model of Synovial Sarcoma by conditional expression of the human SYT-SSX protein in mouse using the technique of gene targeting. Conditional expression will be achieved by utilizing the Cre-loxP system. This strategy will enable us to investigate into: a) origin of this disease. b) the role of SYT-SSX fusion protein in tumor induction/progression and c) downstream events during tumorigenesis. This model will also enable development/evaluation of novel therapeutic strategies.
• Final Report: Click to view PDF
Immunologic Monitoring of Patients with Alveolar Soft Part Sarcoma Receiving a Long Translocation-Specific Peptide Vaccine with GM-CSF Adjuvant
• Stacie Goldberg, M.D. Memorial Sloan-Kettering Cancer Center
• Recipient of a $25,000 Bradley J. Breidinger Memorial Research Award
• Abstract: We are developing a long amino acid peptide vaccine to be used with GM-CSF adjuvant to target the ASPL/TFE3 breakpoint translocation of alveolar soft part sarcoma (ASPS). We will address two specific aims: 1) Can CD8+ and CD4+ T-cell responses be generated to this vaccine by ASPS patients of different HLA haplotypes? 2) Can these general immune responses help determine HLA-specific immunogenic peptides for use in future trials? By using a long peptide sequence we can target patients of different HLA types as well as measure class I and class II responses to optimize future vaccines capable of destroying ASPS.
• Final Report: Click to view PDF
Functional Genomic Characterization of a Conditional Mouse Model of Alveolar Rhabdomyosarcoma: Comparison to Human Tumors
• Charles Keller, M.D. Children’s Cancer Research Institute, University of Texas Health Science Center at San Antonio
• Recipient of a $25,000 Bradley J. Breidinger Memorial Research Award
• Abstract: Rhabdomyosarcomas are the most common childhood soft tissue sarcoma. We have developed a conditional mouse model of alveolar rhabdomyosarcomas by expressing the Pax3:Fkhr oncogene in skeletal muscle of juvenile mice. We propose to test that our mouse model mimics the secondary genomic and gene expression changes seen in human alveolar rhabdomyosarcomas. We will (1) compare chromosomal segment gains and losses in 8 mouse and 8 human alveolar rhabdomyosarcomas, and (2) correlate genomic imbalances of mouse and human tumors to gene expression changes of those same tumors. Understanding molecular events underlying tumor invasiveness and metastasis, we simultaneously identify potential therapeutic targets.
• Final Report: Click to view PDF
Tumor Angiogenesis in Different Organ Environments: Implications for Anti-VEGF Therapy for Soft Tissue Sarcomas
• Sam Yoon, M.D. Massachusetts General Hospital
• Recipient of a $25,000 Brian J. Monaghan Memorial Research Award
• Abstract: Anti-angiogenic agents targeting vascular endothelial growth factor (VEGF) and other angiogenic pathways are a major focus of clinical drug development for cancers including soft tissue sarcomas (STS). An underlying premise of these trials is that VEGF inhibition will have equal efficacy in all sites of tumor growth and against all types of endothelial cells. This proposal seeks to develop an angiogenic profile of STS growing in different organs and tissues and to determine if inhibition of VEGF signaling will have varying effects on tumor angiogenesis in different sites. In Specific Aim 1, expression of VEGF and other angiogenesis-related genes in STS will be assessed by ELISA and genechip microarrays. Specific Aim 2 will study the effect of VEGF inhibition using RNA interference (RNAi) and soluble VEGFR-1 (sVEGFR-1) on mouse models of STS growth in different sites. Taken together, the proposed studies should clarify the role that VEGF and other angiogenic factors play in tumor angiogenesis in different environments and provide insight into the future use of anti-VEGF therapies in STS.
• Final Report: Click to view PDF
2004 SFA Research Grant Recipients
Sarcoma Cell Line Resource with Molecular and Kinase Annotation
• Jonathan Fletcher, M.D. Brigham and Women’s Hospital; Boston, MA
• Recipient of a $25,000 Reid Brecher Memorial Research Award
• Abstract: The aim of this application is to create a sarcoma cell line resource that will be readily available to academic and industry laboratories. We will establish a molecular cytogenetic profile for each of the 30 sarcoma cell lines, and will validate that the lines are faithful biologically to the sarcomas from which they were originally established. Further, we will use a proteomic phosphokinase screening method to determine whether the cell lines contain potential therapeutic targets. These data will be integrated with cDNA array profiles from Stanford University, and the entire panel of cell lines will then be deposited with the American Type Culture Collection (ATCC) for public distribution. The proposed studies represent a substantial first step in providing a comprehensive panel of sarcoma cell lines to the research community.
• Final Report: Click to view PDF
Molecular Classification of Sarcoma Cell Lines
• Matt van de Rijn, M.D., Ph.D. Stanford University; Stanford, CA
• Recipient of a $25,000 Elizabeth Shriver Memorial Research Award
• Abstract: Currently, a lack of cell lines restrains sarcoma research. This proposal accompanies one by Dr. Jonathan Fletcher, who generated 30 sarcoma cell lines. We have extensive experience with gene array studies. We will use arrays to determine in these 30 lines levels of mRNA expression and degrees of gene amplification (or loss) for all human genes. As a result we will find novel diagnostic markers and new therapeutic targets, as we have previously done on tumor samples. These fully characterized cell lines will function as model systems for novel drug treatments and will be made available to the scientific community.
• Final Report: Click to view PDF
Molecular Dissection of a Subset of Gene Fusion-Associated Sarcomas that Lack the Gene Fusion
• Frederic Barr, M.D., Ph.D. University of Pennsylvania; Philadelphia, PA
• Recipient of a $25,000 Elizabeth Shriver Memorial Research Award
• Abstract: Alveolar rhabdomyosarcoma is characterized by recurrent PAX3-FKHR and PAX7-FKHR gene fusions. We identified a subset of cases in which these fusions are present in only a small fraction of tumor cells or are undetectable. In this project, we will use allelic loss and high sensitivity RT-PCR assays to investigate the molecular basis of these low- and null-fusion subsets. We will utilize array-CGH to assess differences in genetic changes between these cases and standard fusion-positive cases. By dissecting these novel subsets, these studies will thereby clarify the role of these fusions in alveolar rhabdomyosarcoma pathogenesis and their utility as therapeutic targets.
• Final Report: Click to view PDF Click to view PDF
The Role of RECQ Helicases in the Pathogenesis of Osteosarcoma
• Lisa Wang, M.D. Baylor College of Medicine; Houston, TX
• Recipient of a $25,000 Elizabeth Shriver Memorial Research Award
• Abstract: We have previously shown that patients with Rothmund-Thomson syndrome who carry constitutional mutations in the RECQL4 gene are highly predisposed to developing osteosarcoma. RECQL4 is a DNA helicase that functions to maintain genomic stability. The goal of this project is to determine whether RECQL4 mutations are present in sporadic osteosarcomas. Identification of such mutations by PCR and sequencing DNA from osteosarcoma samples will provide the basis for detailed investigation of the role of previously unexplored RECQ helicase pathways in the pathogenesis of sporadic osteosarcomas. These pathways may potentially serve as future therapeutic targets for osteosarcoma.
• Final Report: Click to view PDF
2003 SFA Research Grant Recipients
Targeting the Inactivation of the MYC Oncogene to Treat Osteogenic Sarcoma
• Dean Felsher, M.D., Ph.D. Stanford School of Medicine Stanford University Medical Center Stanford, California
• Recipient of a $25,000 research award
• Abstract: We have found that even brief inactivation of the MYC oncogene can result in sustained regression of osteogenic sarcoma (Jain et al, Science, 2002; Weinstein, Science, 2002). Now, we propose to perform several pre-clinical studies to evaluate the effectiveness of a novel anti-sense approach for the inactivation of MYC for the treatment of osteogenic sarcoma. We will evaluate the mechanisms by which brief inactivation of MYC induces tumor regression through cDNA microarrrays. From these experiments, we will obtain the preliminary results necessary to subsequently conduct a clinical trial to evaluate the effectiveness of the inactivation of MYC to treat human osteogenic sarcoma.
• Final Report: Our lab had previously shown if you transiently shut down a cancer gene, called MYC, that this causes osteogenic sarcoma tumors to lose their neoplastic properties - that is, the tumor regresses. As a result of this grant, we have developed a new way to shut off MYC using a synthetic RNA. We have shown that this agent shuts off the cancer properties of osteogenic sarcoma cells in tissue culture. We are evaluating if this approach can be used to suppress cancer cells in a living host such as the mouse. The results of my work will be useful towards the development of new treatments for osteogenic sarcoma patients.
Generating T cell Immunity to Sarcoma by Liposomal Vaccination with Sarcoma Fusion Breakpoints
• Mary Jo Turk, Ph.D. and Jose A. Guevara-Patino, M.D., Ph.D. Memorial Sloan Kettering Cancer Center; New York, NY
• Recipient of a $25,000 research award
• Abstract: We have developed a liposomal vaccine that delivers long peptides into the processing/presentation machinery of antigen presenting cells and generates outstanding cytotoxic T cell responses. We propose the use of this vaccine for generating T cell immunity to the breakpoint regions of four sarcoma-derived oncofusion proteins: EWS-ATF1, SYT-SSX, TLS-CHOP, and ASPL-TFE3. Experiments will be conducted to demonstrate that peptides within these breakpoints 1) are processed and presented by human antigen presenting cells, and 2) activate a population of cytotoxic T lymphocytes capable of killing human sarcoma cells. These studies will facilitate rapid clinical translation of four novel sarcoma vaccines.
• Final Report: This project was aimed at validating vaccines against four oncofusion proteins (expressed when an oncogene, such as EWS, integrates into or fuses to another gene, such as ATF1) produced by sarcomas. We injected these proteins and studied the immune reaction to see if this type of vaccine could elicit an immune response. Based upon our results, we can conclude that the human immune system does contain T cells that can potentially react against the breakpoint regions (which produce the oncofusion proteins) of these sarcoma-specific proteins. This is encouraging because these reactive T cells could potentially kill sarcoma cells containing the oncofusion proteins, and it was originally thought that there were very few T cells (1 in 100-500,000) that can recognize these antigens. We have also optimized a vaccine delivery system based on use of liposomes (microscopic spheres made with fat molecules), which will be used in conjunction with these sarcoma antigens to provide maximum T cell responses against sarcomas clinically. We believe that the results obtained in this study, in conjunction with future studies, will facilitate the development of vaccines directed against sarcoma breakpoints that can be used in sarcoma cancer patients.
Identification of Tumor Suppressor (LOH18CR) on Chromosome 18
• Antony E. Shrimpton, Ph.D. and Timothy A. Damron, M.D. State University of New York, Upstate Medical University; Syracuse, NY
• Recipient of a $20,000 research award
• Abstract: We have identified and studied a family with familial Paget’s disease of bone and Pagetoid osteosarcoma. A familial pattern is extremely unusual and indicates the presence of a putative germline mutation in a tumor suppressor gene involved in osteosarcoma. We have demonstrated loss of heterozygosity for markers located on the long arm of chromosome 18q which includes a 530 kb critical region defined by others, which is believed to contain an osteosarcoma tumor suppressor gene. We wish to sequence candidate genes, in close to this region or otherwise implicated, in order to identify a pathogenic mutation, and thus the tumor suppressor gene.
• Final Report: In our previous studies, we had identified a family with an inherited disease of bone and a rare type of bone cancer (osteosarcoma). For this to be inherited is extremely unusual and probably indicates the presence of a mutation in a gene involved in bone cancer in this family. Our studies led us to suspect that a small fragment of chromosome 18 probably has a mutation in a gene important in preventing this type of bone cancer from developing. Thus, we wanted to search for this osteosarcoma suppressor gene by testing for candidate genes on this chromosome (known to be abnormal). As a result of the grant, we have now tested twelve candidate genes. Unfortunately, we did not yet find a mutation, so as proposed, we will now test another two candidate genes from this chromosome and then two other genes known to have similar effects, but located in other chromosomes. While the search for a key mutated gene for osteosarcoma continues, we have narrowed the search by eliminating all of the above genes.
Programs & Resources
Patient Resources
Information on sarcoma subtypes, treatments, clinical trials, and other important resources for sarcoma patients and families.
Sarcoma Patient Registry
Information on the Sarcoma Patient Registry. If you are diagnosed with sarcoma, please consider joining the Registry.
Research Grants
Information on applying for a sarcoma research grant, current research funded by the SFA, and past research grants.
