2009 Innovative Research Grant 24-Month Progress Reports
An Emerging Tumor Suppressor Pathway in Human Cancer
Fernando D. Camargo, Ph.D., Children’s Hospital Boston
Dr. Camargo’s laboratory is conducting research that will expand our understanding of the Hippo pathway and the role it plays in cancer development. In the last 6 months Dr. Camargo’s laboratory has:
- Discovered that when Hippo is turned off, the amount of Yap1, the main molecule responsible for Hippo’s expression, is increased. Studies were conducted which revealed that inactivation of the Hippo pathway in the skin of mice leads to the development of invasive squamous cell carcinoma. Similarly, inactivation of the Hippo pathway in the muscle of mice leads to the formation of rhabdomyosarcomas.
- Recent studies demonstrate that over-activation of this pathway can lead to the suppression of growth in the epidermis, but may not be helpful in the intestine.
- Finalized a genetic screen to identify which proteins can control Hippo signaling.
- Results suggest that alpha-catenin, a protein known to suppress tumors in the skin, and a different signaling pathway called JNK, are able to regulate the Hippo pathway.
- Future experiments will investigate how these molecules control Hippo signaling.
Modeling Ewing Tumor Initiation in Human Neural Crest Stem Cells
Elizabeth R. Lawlor, M.D., Ph.D., Department of Pediatrics at the University of Michigan
Tumors often look like tissues that have not developed normally. In fact, some tumors arise when abnormalities in the DNA of normal stem cells create changes that lead to the formation of malignant rather than normal tissues. We are studying whether changes in DNA methylation contribute to the genesis and growth of Ewing sarcoma family tumors (ET), highly aggressive tumors that primarily affect children and young adults. Precise control of DNA methylation is essential for the creation of normal adult tissues. We believe that ET arises from normal neural crest stem cells (NCSC) in which the expression of an abnormal gene, EWS-FLI1, induces changes to DNA methylation that result in cancer formation. To date, Dr. Lawlor’s laboratory has:
- Developed a way to generate and then differentiate neural crest stem cells in the laboratory.
- The cells are being studied to determine both their normal differentiation process and the changes that normally occur in their methylation over time.
- Analyzed gene expression data and identified a list of approximately 350 genes they suspect to be abnormally methylated in Ewing sarcomas.
- Performed experiments to determine whether abnormal DNA methylation in Ewing sarcomas can be corrected by treatment with decitabine, a targeted therapy that inhibits DNA methylation.
- Dr. Lawlor’s laboratory tested the efficacy of decitabine in Ewing sarcoma cells that are grown as tumors in mice and found that tumor growth was inhibited.
- Several genes were identified that were turned back after exposure to decitabine and will be studied further.
Cancer Cell Specific, Self-delivering Pro-Drugs
Matthew Levy, Ph.D., Albert Einstein College of Medicine of Yeshiva University
Dr. Levy’s research efforts are focused on the development of a new type of targeted therapy that can home to and deliver anti-cancer drugs specifically to the cancer cells. His initial research is focused on prostate cancer, but the technologies developed will be broadly applicable to most other cancers. His laboratory has:
- Synthesized a series of drug-laden oligonucleotides (small molecules) and aptamers with different types of modifications and/or different chemotherapy agents incorporated inside them.
- Performed tests to find the optimal drug/vehicle combination that will enable them to use two core aptamers as the basis for targeting: one that binds the human transferrin receptor (a receptor commonly overexpressed on different types of cancer cells) and one that binds the prostate specific membrane antigen (PSMA; a marker of prostate cancer).
- Both sets of minimized aptamers were confirmed for their ability to bind to and internalize into the appropriate receptor-bearing cells. The ability for the aptamers to enhance cell death have been modest. New drug “cargoes” are being developed in order to enhance the ability to increase cell death.
- Explored a number of different approaches for the targeted delivery of nucleoside analog drugs using nucleic acid aptamers.
- Initiated the production of liposomes that could bear up to 2000 drug molecules per liposome and are decorated with targeting aptamers on the outside.
- Experiments testing different and more potent drugs to be delivered by the liposomes are being performed.
- Begun development of a new transferrin specific aptamer that is optimized to work in conditions with higher transferrin.
- Begun collaboration with two different SU2C Dream Teams—Dr. Haber’s group and Dr. Baylin’s group.
Targeted Inhibition of BCL6 for Leukemia Stem Cell Eradication
Markus Müschen, M.D., Children’s Hospital Los Angeles
Dr. Müschen’s laboratory is investigating the hypothesis that BCL6 is critical for leukemia-initiation and relapse. They are examining the frequency and appearance of BCL6-dependent leukemia stem cells in human leukemia samples, and studying whether the BCL6 inhibitor RI-BPI is effective in targeting and eradicating leukemia stem cells. Dr. Müschen’s laboratory has:
- Conducted studies that showed that BCL6 enables Ph+ acute lymphoblastic leukemia (ALL) cells to survive after treatment with a BCR-ABL1 kinase inhibitor.
- BCR-ABL1tyrosine kinase inhibitors are widely used to treat patients with Ph+ ALL and chronic myeloid leukemia (CML).
- These findings identify a novel mechanism of drug resistance, and identify BCL6 as a central component of this drug resistance pathway.
- These findings demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukemia-initiating clones.
- This suggests that combining a tyrosine kinase inhibitor with a BCL6 inhibitor might potentially overcome drug resistance in Ph+ ALL.
- This led the laboratory to question which function BCL6 might have (if any) in the regulation of normal B cell development and in the regulation of cell cycle progression of differentiating pre-B cells, which represent the target cell for malignant transformation toward ALL.
- Studies to pursue this question showed that pre-B cell receptor signaling activates BCL6 and BLC6-mediated repression of proteins called MYC and CCND2.
- Conducted studies that showed that BCL6 is required for the maintenance of leukemia-initiating cells in chronic myeloid leukemia (CML).
- CML can be treated for many years with tyrosine kinase inhibitors. But unless patients use these drugs throughout their lives, the CML will eventually recur.
- This research showed that BCL6 plays a role in this process by repressing Arf and p53 in CML cells.
- It also showed that BCL6 is required to form leukemia.
- This suggests that a BCL6 inhibitor might be able to eradicate leukemia-initiating cells in CML and, in turn, limit how long CML patients must stay on a tyrosine kinase inhibitor.
- Recent work demonstrated that FoxO3A is critical for maintenance of leukemia stem cells in CML. Dr. Müschen’s laboratory identified the BCL6 protooncogene as a critical effector downstream of FoxO in self-renewal signaling of CML-initiating cells. These findings identify inhibition of BCL6 as a novel strategy to eradicate leukemia-initiating cells in CML.
- This suggests the potential for a dual strategy in which a tyrosine kinase inhibitor is used along with BCL6 inhibition to treat CML.
- Assessed gene expression data in 207 pediatric patients, in addition to an adult patient study, which demonstrated that high BCL6 expression associated with lower overall survival and relapse free survival.
Identifying Solid Tumor Kinase Fusions via Exon Capture and 454 Sequencing
William Pao, M.D., Ph.D., Vanderbilt-Ingram Cancer Center/Vanderbilt University
Dr. Pao’s laboratory is screening 120 tumor samples and cell lines from multiple cancer types, including lung, breast, and head and neck cancers, angioscarcomas, and acute T-cell leukemias/lymphomas for TK fusions. To date Dr. Pao’s laboratory has:
- Fully analyzed 72 of these samples. The other samples are in various phases of the of process, which involves quality control, DNA capture, sequencing, and computational analysis.
- Characterized a novel TK fusion in a patient with recurrent T-cell acute lymphoblastic leukemia and eosinophilia (a higher than normal level of eosinophilis, one of five major types of white blood cells.) Their studies suggested that this fusion was likely the ‘driver’ of the patient’s eosinophilia.
- Begun to use orthogonal methods to identify driver mutations in cancers that may not contain kinase fusions but may have other genetic alterations.
- Whole exome sequencing has been completed on18 pairs of tumor/normal samples from lung cancer patients, including five never smokers. Whole exome sequencing focuses on the part of the genome formed by exons, the portion of the gene that codes for proteins.
- Conventional sequencing analysis had not identified any known mutations in EGFR, KRAS, BRAF, PIK3CA, and ALK.
- Two tumors were found to have mutations in a growth factor called HGF, which binds to the TK MET both of which have been implicated in cancer progression. Studies are ongoing to further understand the role of HGF in lung cancer.
- Sequenced the expressed genes in 29 small-cell lung carcinoma tumors, which accounts for 15% of lung tumors, and found a high mutation rate and support for the role a biologic process in this cancer that is being investigated.
Therapeutically Targeting the Epigenome in Aggressive Pediatric Cancers
Charles M. Roberts, M.D., Ph.D., Dana-Farber Cancer Institute
Dr. Roberts’ laboratory is conducting studies designed to determine the role SNF5 loss plays in the development of cancer and to identify therapies that can block or reverse the epigenetic changes that result from the loss of SNF5. Dr. Roberts’ laboratory has:
- Generated an animal model that it used to test whether inactivation of a small RNA molecule, called miR-21, could block the growth of cancers driven by SNF5 loss.
- These studies determined that the absence of miR-21 has no effect upon the formation of lymphomas driven by SNF5 loss.
- Initiated experiments to evaluate whether SNF5-deficient cancer cells are susceptible to treatment with histone deacetylase inhibitors.
- The trials of DNA methylation inhibitors used SNF5 deficient cancer cells transplanted into recipient mice.
- The data suggests that SNF5-loss does indeed affect histone acetylation. But that it leads to increased, rather than decreased, acetylation.
- Pre-clinical development trials of a drug targeting EZH2, a protein that stops the expression of genes by adding a chemical modification to the DNA, have led to decreased tumor burden, however cell growth was not completely blocked and led to decreased bone marrow activity.
- Further studies are being conducted before deciding to pursue other pre-clinical trials.
Endogenous Small Molecules that Regulate Signaling Pathways in Cancer Cells
Rajat Rohatgi, M.D., Ph.D., Stanford University
Dr. Rohatgi’s laboratory is attempting to identify the undiscovered small molecule-protein pair that is believed to regulate the Hedgehog pathway. Studies suggest that Hedgehog plays a role in a diverse range of cancers and it has become an important drug target in oncology. Dr. Rohatgi’s laboratory has:
- Continued to conduct studies designed to isolate small molecule lipids that activate the Hedgehog pathway and a method to measure the activation of the pathway in a cell free system.
- Conducted studies designed to build upon their previous observation that molecules related to cholesterol, called oxysterols, can activate the Hedgehog pathway.
- Their studies demonstrated that radio-labeled directly interact with a cancer-driving protein called Smoothened that is the major drug target in the hedgehog pathway.
- This work, which has been submitted for publication, suggests both novel ways in which this pathway can be targeted by drugs and novel avenues for cross-talk between cancer signaling and lipid levels in cells.
- Performed screens to identify Oxysterol Binding Proteins (OSBPs) that activate and inactivate the Hedgehog Pathway.
- Identified several natural compounds that are toxic to several cancer cell lines.
Genetic Approaches for Next Generation of Breast Cancer Tailored Therapies
José M. Silva, Ph.D., Columbia University Medical Center
Dr. Silva’s laboratory has pioneered the development of RNAi based genetic tools for studies in mammalian cells. This technology represents a unique opportunity to identify genetic synthetic lethal interactions. Dr. Silva’s laboratory has:
- Completed and analyzed four genome-wide RNAi screens in vitro to identify target genes that upon inhibition can stop the growth of breast cancer cells that have one of the four major breast cancer alterations: ErbB2(HER2), c-Myc, Cyclin-D or RB.
- Selected 30-50 genes as synthetic lethal candidates for each of the four major breast cancer alterations.
- Developed a procedure to see whether the lethal interactions found in vitro are reproduced in vivo in mouse models.
- Identified several components involved in lipid processing that were shown to be highly significant in c-MYC alterations and have been targeted for further study
- Identified the Jak/Stat pathway as differentially activated in cells that are ErbB2(HER2)-positive. This pathway has previously been identified as a key pathway in other tumor types, including non-small cell lung cancer.
- Several small molecule inhibitors already exist that potentially could be used for translational clinical trials.
- Studies are now underway that will advance the research on this genetic interaction.
- Studies identified upregulation of multiple molecules and, importantly, their receptors. The most upregulated interleukin was interleukin-6 (IL-6), which is known to activate STAT-3.
- This suggests that cells that are HER2-postiive produce high levels of IL-6 and upregulate its receptor, which activates the Jak/Stat pathway.
- Has identified 37 new potential inhibitor molecules of STAT-3 from a screen of more than 20,000 compounds, which will be tested for their potential use as clinically relevant drugs.
Modulating Transcription Factor Abnormalities in Pediatric Cancer
Kimberly Stegmaier, M.D., Dana-Farber Cancer Institute
Rather than using standard approaches to chemical screening, Dr. Stegmaier’s laboratory developed a genomic approach that uses DNA microarrays (gene chips) to characterize genes that are turned on or off in the presence of or absence of the Ewing sarcoma protein.Over the past 18 months Dr. Stegmaier’s laboratory has:
- Developed a 123-gene signature that distinguishes Ewing sarcoma cells with the active versus inactive EWS-FLI protein.
- Adapted the signature to a robust assay that can be measured in a high-throughput format.
- Confirmed that the signature identifies the active versus inactive EWS-FLI protein across a number of different Ewing sarcoma cell lines.
- Performed a successful pilot screen of 1600 bioactive chemicals and FDA-approved drugs.
- Two chemical hits that induce the EWS-FLI inactive expression signature were identified.
- Completed a large-scale screen of more than 10,000 small molecules. This chemical collection consists of bioactive molecules (including many FDA-approved drugs), natural products, and novel chemicals created by a new approach called diversity oriented synthesis.
- Diversity oriented synthesis is expected to yield chemicals with greater similarity to these products in nature.
- These chemicals may also have greater biological activity than compounds produced by standard approaches to chemical synthesis.
- Prioritized 160 top-scoring compounds to retest in a secondary screen across multiple doses.
- Based on these findings, 36 of these compounds have been prioritized for additional testing.
- Attention is being focused on several molecules that are already FDA-approved or in clinical trials, as well as 15 diversity oriented synthesis molecules.
- Identified two classes of molecules to target: a compound which activates the protein retinoic acid receptor γ (RARγ) and a compound which inhibits a protein called focal adhesion kinase (FAK).
- Dr. Stegmaier has demonstrated that the RARγ agonist molecule, CD437, leads to a decrease of the ID2 protein, one of the downstream target proteins of EWS/FLI.
- It was shown that both CD437 treatment and genetic decrease of ID2 prevent growth of Ewing sarcoma cells
- Dr. Stegmaier has demonstrated that FAK is activated in most Ewing sarcoma tumors from patients and that inhibiting FAK with a genetic tool called shRNA prevents the growth of Ewing sarcoma cells cell culture and in mice and
- Evaluation of the activity of a FAK inhibitor in a mouse model of Ewing sarcoma is currently in progress.
Non Invasive Molecular Profiling of Cancer via Tumor-Derived Microparticles
Muneesh Tewari, M.D., Ph.D., Fred Hutchinson Cancer Research Center
Dr. Tewari is trying to develop a blood test that can efficiently capture and decode the data about a tumor that can be obtained from the tumor-derived microparticles that circulate in the patient’s bloodstream. Dr. Tewari’s laboratory has:
- Improved its procedures for concentrating the microparticles, making it easier to see them using electron microscopy.
- Obtained a Nanosight nanoparticle tracking system and analyzed its reproducibility and accuracy in sizing exosomes and microvesicles.
- Used the Nanosight system to compare plasma exosome abundance in ovarian cancer cases and controls, as well as pre- and post-surgery for ovarian cancer.
- No significant difference in bulk exosome abundance in patients with cancer vs. healthy controls was found, which differs from what others have reported.
- This indicates that capture of the tumor-derived subpopulation of exosomes will be important for successful development of an exosome-based tumor transcriptome profiling.
- Based on the above data, Dr. Tewari has optimized a protocol capturing exosomes using antibodies that recognize EpCAM, a protein which is found on ovarian cancer cells.
- This will be used to capture and elute ovarian cancer cell line-secreted exosomes and their associated microRNAs for analysis.
- Used the Nanosight system to quantitatively characterize the performance of our exosome isolation protocol.
- Found in a set of matched fresh and frozen plasma specimens from advanced prostate cancer patients, that freezing leads to a dramatic (10-100 fold) decrease in miRNA biomarker abundance in plasma
- These results suggest: (i) frozen specimens may not be suitable for exosome-based tumor transcriptome analysis, and (ii) the potential for this approach is enhanced if working with fresh samples is able to increase signal by 10-100 fold.
A Transformative Technology to Capture and Drug New Cancer Targets
Loren D. Walensky M.D., Ph.D., Dana-Farber Cancer Institute
Dr. Walensky is using modified natural peptides that bind to and capture target proteins to identify cancer-causing interactions and to develop therapies that interfere with these interactions. Dr. Walensky’s laboratory has:
- Chemically synthesized a pilot panel of “photoreactive stabilized alpha-helices” or pSAHs, which are the chemical tools designed to capture and characterize new cancer targets.
- Assessed and optimized these pSAHs so that they could be cross-linked to specific targets in the BCL-2 pathway, which regulate the cell’s life-death decision and has been implicated in the development of cancer and chemo-resistance.
- Developed a rapid method for localizing helix-target binding sites in order to identify protein interfaces, which will help in the discovery of potential new targeted therapies.
- Demonstrated the ability to successfully and reproducibly generate pSAHs that recapitulate the structure of distinct bioactive domains and deploy them to trap, purify, and identify their natural cellular targets with high fidelity.
- The laboratory is now putting these design principles into practice so that new pSAH reagents can be effectively deployed to discover and target cancer-causing protein interactions.
- This work was published in Cell’s Chemistry and Biology journal in December 2010.
- Developed and published a rapid method for localizing helix-target binding sites that will expedite the discovery of protein interfaces for targeted drug development and clinical translation.
- Expanded its panel of pSAHs for target discovery.
- The laboratory has successfully expanded to a library of 60 PSAHs that encompass 10 BCL-2 family subdomains.
- This library has been used to identify several new protein targets that are currently being confirmed.
Functional Oncogene Identification
David M. Weinstock, M.D., Dana-Farber Cancer Institute
Dr. Weinstock is using a novel molecular profiling approach to identify abnormalities that promote the growth of cancers of the blood. Dr. Weinstock’s laboratory has:
- Screened 12 types of human leukemia and lymphoma samples for new mutations.
- This screen and additional DNA sequencing led to the identification of multiple mutations that have never been described from tumor specimens.
- Mutated versions of four additional proteins in three cancer types, including proteins called kinases that can be targeted with available drugs.
- The laboratory is also defining the frequency of these mutations in other leukemia and lymphoma specimens.
- Multiple mutations are present in several lymphomas of the same type.
- Once further confirmation is completed, the laboratory will test leukemias and lymphomas dependent on these new mutations with a panel of targeted agents to define drugs for further testing in patients.
- The laboratory will then apply the same screening approach to identify targetable mutations in other blood cancers, including subtypes of leukemia, non-Hodgkin lymphoma and Hodgkin’s disease.
- Identified a donor-recipient bone marrow transplantation (BMT) pair who both developed follicular lymphoma nine years after transplantation (published in Cancer Discovery 2012;2:47-55). Sequencing identified 15 mutations that were shared between the two lymphomas as well as of mutations unique to each lymphoma.
Probing EBV-LMP-1’s Transmembrane Activation Domain with Synthetic Peptide
Hang (Hubert) Yin, Ph.D., University of Colorado at Boulder
Dr. Yin developed a new technique to identify transmembrane proteins that are part of the signaling pathways that drive cancer cell growth. Dr. Yin is currently using CHAMP-designed peptide antagonists to study the contribution of LMP-1 activation. This could lead to the development of new therapies that target transmembrane proteins like LMP-1. Dr. Yin’s laboratory has:
- Begun developing a second-generation CHAMP algorithm design tool.
- Completed the training database for the supervised-machine learning process.
- Preliminary testing showed an 85% success rate to predict/recognize protein flexible regions.
- The flexibility-prediction element has been integrated into CHAMP design package developing the second generation design tools.
- Established a method to synthesize transmembrane peptide sequences on Tentagel beads.
- A second approach to optimize these sequences that makes it possible to screen a larger peptide library has now been developed.
- Established a biophysical assay to evaluate peptide/membrane protein insertion/binding. This assay has been validated.
- This work led to the development of a novel reporter assay that the laboratory is now using to study the peptide/membrane protein interactions in E coli membranes.
- Based on his hypothesis that the fifth transmembrane domain of LMP-1 mediates LMP-1 signaling, Dr. Yin successfully designed anti-transmembrane domain 5 (TMD5) that has been shown to be able to disrupt the LMP-1 TMD5 oligomerization in cell membranes.
- Using a previously established assay to test for compounds from the NCI Diversity Set II library, Dr. Yin identified four potential compounds that may disrupt TMD5 oligomerization.
- One of the four compounds is being characterized and preliminary data shows that this compound specifically inhibits LMP-1 signaling in EBV-infected B cells.