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.

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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.

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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.

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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.

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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.

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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.

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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.

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