Daniel G. Jay - US grants

The long term objective of this proposal is to understand the molecular mechanisms that determine axon adhesion. and guidance during neurodevelopment. This fundamental understanding will aid in the prevention of neurological birth defects and in the induction of nerve regeneration after injury or degenerative disease. Knowledge of the molecules required for the formation of correct neurocircuitry may lead to treatment of neurological and psychiatric disorders. The proposal focuses on adhesion molecules in grasshopper neurodevelopment. It has been suggested that these molecules dictate axon guidance by establishing labeled pathways that growth cones recognize and adhere to. A new technique called chromophore assisted laser inactivation (CALI) will be used to inactivate these adhesion molecules during the development of identified neurons in the grasshopper limb bud and CNS with an unprecedented spatial and temporal resolution. CALI uses chromophore-labeled antibodies to target laser energy to proteins of interest, thereby denaturing them, without otherwise affecting the embryo. Fasciclin I has been shown to play a role in axon adhesion in the limb bud pioneer neurons. This study will investigate when and where fasciclin I is required for correct axon guidance. The interaction of fasciclin I with Abelson tyrosine kinase will be investigated to test the hypothesis that adhesion molecules signal intracellular changes that direct axon guidance. Fasciclin II, neuroglian, Notch protein and PS2 antigen will also be inactivated at discrete times in specific cellular locations to determine their respective roles in axon guidance. This study is a molecular dissection of axon guidance using a unique approach that generates spatially and temporally specific lesions of protein function in the grasshopper embryo; this would not be otherwise possible. In addition to providing insight into the molecular specification of axon guidance, this project will develop techniques that will have significant utility in addressing many diverse questions in the biomedical sciences.

DESCRIPTION (Investigator's Abstract): The long term objective of this proposal is to understand the molecular mechanisms of growth cone motility in developing neurons. Axons are guided by extracellular cues that direct filopodial motility by locally affecting cytoskeletal dynamics. Understanding the molecular basis of this is required to understand how neurocircuitry is formed during embryonic development and to establish what factors can cause neurological birth defects. Moreover, these molecular mechanisms are likely to be used in nerve regeneration and an understanding of them will aid in designing treatment after nerve injury. Considerable work in neural development, cell biology, and signal transduction have identified candidate proteins that may be involved in filopodial motility and guidance but establishing their in vivo function in neuronal growth cones has been difficult. The long term objective will be addressed by applying microscale chromophore assisted laser inactivation (micro-CALI), a method developed in this laboratory, to inactivate specific intracellular proteins with an unprecedented level of spatial and temporal resolution. CALI has been rigorously tested and used to determine the in vivo roles of membrane proteins in neural development. It is timely to use this technique to address the molecular mechanisms of growth cone motility. Specificically, the in vivo roles of talin, vinculin, pp60c-src, and the myosins in filopodial motility will be determined by focally inactivating them in growth cones and observing the resulting behavior by video- enhanced microscopy and quantitative morphometry. Experiments are also proposed to ask if these proteins play a role in substrate-mediated guidance by using micro- CALI as growth cones reach borders on patterned substrates. These studies will be done using chick dorsal root ganglion neurons in culture, a well characterized and manipulatable system for which there exist antibodies against many proteins that are potentially involved in growth cone motility. The proposed experiments are focused on these proteins because our preliminary experiments coupled with in vitro biochemical data suggest a model for how they function and interact in the extension and regulation of filopodia. Micro-CALI will be applied to test this model by inactivating these proteins in combination to give specific phenotypes that will support or refute the proposed interactions.

Metastasis is one of the most devastating aspects of cancer. Thus, discovering therapies that inhibit metastasis is an important goal in cancer treatment. In order to do this, proteins involved in cancer cell invasiveness that could be targets for therapeutic drugs must be validated and this is often a rate-limiting step in drug discovery. The overall objective is to develop an enhanced high throughput screen (HTS) using chromophore assisted laser inactivation (CALI) for target validation of surface proteins that act in cancer cell invasiveness. CALI targets laser energy using a dye-labeled antibody to inactivate the function of the bound antigen. In the R21 phase, an HTS Transwell assay will be developed and used to show that CALI will increased markedly the number of phage display antibodies that disrupt invasiveness. Single chain Fv fusion phage libraries that bind specifically to surface proteins of HT-1080 human fibrosarcoma cells will be generated and used for a pilot screen (n=96 antibodies) using CALI. The achievement of these aims will establish the feasibility of an HTS using CALI for the validation of targets involved in cancer cell invasiveness. In the R33 phase, a full scale automated screen will be conducted (n= 10,000 antibodies) and protein targets validated by this screen will be identified using high resolution mass spectrometry. This proposed technology will provide a low cost and rapid means of target validation and will contribute a number of targets for anti-metastasis drug discovery. The methods developed are general; they will have an application to cell processes in cancer and other diseases. As such, they will be of great utility for pharmaceutical companies and academic labs.

Accurate formation of the retinotopic map is critical for our ability to see. During this process, retinal axons are guided to form a precise topographic map of connections in the brain that allow visual space to be perceived. The molecular basis of these guidance mechanisms is not yet known. The long term objective of this proposal is to understand how repulsive cues act to guide axons to form the retinotopic map. The recent discovery of a variety of repulsive cues act to guide axons to form the retinotopic map. The recent discovery of a variety of repulsive cues has modified our understanding of how axons re guided. It is timely to address how these cues function in vivo. Repulsive guidance molecule (RGM) and ephrin-A5 are expressed in high posterior to low anterior gradients in the tectum of lower vertebrates during the formation of the retinotectal projection. Current advances have suggested specific hypotheses of how these two cues act in vivo to shape the retinotectal map: 1) RGM guides initial retinotectal axon outgrowth. 2) Ephrin-A5 prevents retinal axons from exiting the tectum and modulates lateral branching after initial axon outgrowth is completed. The proposed experiments will test these hypotheses. The changes in retinotectal projects caused by perturbing RGM and ephrin-A5 in vivo will be observed. Chick and zebrafish embryos will be studied; they are well characterized and each offers distinct advantages to achieve our goals. Acute and local inactivation of RGM and ephrin-A5 during retinotectal map formation will be achieved by chromophore-assisted laser inactivation (CALI) during initial axon outgrowth and later during lateral branching. Infection with recombinant retrovirus will generate chronic misexpression of ephrin-A5 in the chick tectum. The changes in retinotectal projects will be assessed by high resolution axon tracing and live imaging. The combination of these two approaches will provide strong complementary information that will test the proposed hypotheses. As RGM and ephrin-A5 are likely used for axon guidance and formation of topographic order, the proposed experiments are of significant clinical relevance.

DESCRIPTION (Investigator's Abstract): The long term objective of this proposal is to understand the molecular mechanisms of growth cone motility in developing neurons. Axons are guided by extracellular cues that direct filopodial motility by locally affecting cytoskeletal dynamics. Understanding the molecular basis of this is required to understand how neurocircuitry is formed during embryonic development and to establish what factors can cause neurological birth defects. Moreover, these molecular mechanisms are likely to be used in nerve regeneration and an understanding of them will aid in designing treatment after nerve injury. Considerable work in neural development, cell biology, and signal transduction have identified candidate proteins that may be involved in filopodial motility and guidance but establishing their in vivo function in neuronal growth cones has been difficult. The long term objective will be addressed by applying microscale chromophore assisted laser inactivation (micro-CALI), a method developed in this laboratory, to inactivate specific intracellular proteins with an unprecedented level of spatial and temporal resolution. CALI has been rigorously tested and used to determine the in vivo roles of membrane proteins in neural development. It is timely to use this technique to address the molecular mechanisms of growth cone motility. Specificically, the in vivo roles of talin, vinculin, pp60c-src, and the myosins in filopodial motility will be determined by focally inactivating them in growth cones and observing the resulting behavior by video- enhanced microscopy and quantitative morphometry. Experiments are also proposed to ask if these proteins play a role in substrate-mediated guidance by using micro- CALI as growth cones reach borders on patterned substrates. These studies will be done using chick dorsal root ganglion neurons in culture, a well characterized and manipulatable system for which there exist antibodies against many proteins that are potentially involved in growth cone motility. The proposed experiments are focused on these proteins because our preliminary experiments coupled with in vitro biochemical data suggest a model for how they function and interact in the extension and regulation of filopodia. Micro-CALI will be applied to test this model by inactivating these proteins in combination to give specific phenotypes that will support or refute the proposed interactions.

DESCRIPTION (provided by applicant): To address tumor diversity and their varied responses to chemotherapy, we require the ability to custom design treatment for each tumor. The goal of pharmacogenomics is to generate gene expression signatures for tissue using microarrays that are then correlated with prognosis or response to therapeutics. However, much of the complexity and diversity of response may be due to proteomic differences. Thus far, probing for proteomic diversity and linking this to therapeutic efficacy has been difficult. Our overall goals are to develop surface proteome signatures (SPS) and perform functional proteomic target validation analysis directly on primary tumor tissue. There is a great need to assess the efficacy of different chemotherapy combinations directly on patient tissue samples. A major difficulty is this respect is our inability to assess the small amounts of primary tumor tissue available from patient-derived samples. As part of our previous IMAT-funded research, we developed a library of single chain (scFv) phage display antibodies that recognize approximately 500 components of the surface proteome. In this work we also developed high-throughput methods for immunocytochemistry (ICC) using scFvs and apoptosis assays that use small number of cells (< 500) per assay. Together, these developments allow us to generate SPS and measure apoptosis after chemotherapy treatment using small amounts of primary tumor tissue. Cells will be tested with a battery of drugs alone and in combination and analyzed for apoptosis. Thus, a differential response to therapeutics will be correlated with a SPS. We will develop and test these assays in the R21 phase using thymic lymphoma mouse cell lines derived from three mouse models: transgenic MyrAkt and two genetic deletions, PTEN-/+ or p53-/-. This is an ideal model system, as the primary tumors are genetically defined by single oncogenic mutations. We will establish SPS for cell lines derived from thymic lymphoma lines from these mice. We will test for the efficacy of different drug regimens to obtain the optimum combination for inducing apoptosis of the cells. We will then test these drug combinations in primary tumor tissue from MyrAkt mice. We will also address the causal link between SPS and drug response and will test the functional role of scFvs that are biomarker candidates. In the R33 phase we will test the predicted optimal drug regimen on thymic lymphomas in the three mouse models, examining tumor load and survival. We will also characterize ten of the scFvs as potential biomarkers or targets for drug discovery. Our studies complement pharmacogenomics and provide a novel route to pharmacoproteomics.

DESCRIPTION (provided by applicant): The vast majority of breast cancer-related deaths are due to secondary tumors after metastasis. There is currently no effective therapy to limit metastasis. Our long term objective is to develop new therapies that reduce cancer invasion, a critical first step in metastasis. This proposal is based on our observations that a novel extracellular form of the molecular chaperone hsp90a is required for cancer invasion by the activation of the matrix metalloproteinase MMP2. Hsp90 has been implicated in cancer and hsp90 inhibitors with anti-tumor activity are currently in clinical trials. These drugs may be problematic in that they interfere with the many intracellular functions of hsp90. Our findings suggest our main hypothesis that inhibiting extracellular hsp90a will decrease invasion and thus limit metastasis. This presents an opportunity for novel anti-cancer therapy by inhibiting invasion without interfering with the myriad intracellular functions of hsp90. To support this idea, we will address three specific aims. We will determine the mechanism of how hsp90a functions on the outside of cancer cells (Aim 1). We will then use this information to develop and test extracellular hsp90 inhibitors (Aim 2). We already have one impermeant hsp90 inhibitor in hand and several candidates for neutralizing single chain antibodies from our collaborators at NCI and Xerion Pharmaceuticals. Finally, we will test the best of these inhibitors of extracellular hsp90a for their ability to limit metastasis in a new model developed by our collaborators at Tufts using human breast cancer cells metastasizing to human bone explants in immunocompromised mice (Aim 3). Thus, these experiments take us from an initial discovery of hsp90a function with cell-based assays to in vivo animal models taking an interdisciplinary approach to address a key issue of human health: limiting metastasis to improve breast cancer prognosis. These studies aim to expedite the translation of our basic research into a potential therapy. If successful, the proposed work would provide data for developing future clinical studies and thus impact human health.

DESCRIPTION (provided by applicant): Genetic approaches have contributed greatly to our understanding of biology, but they are limited in cancer cells because genetic loss-of-function in diploid cells is obscured by expression from the wild type allele. To address this limitation, we propose to develop KillerRed Assisted Mutagenesis (KRAM) to generate an unbiased forward genetic screen for somatic cells to identify proteins required for cancer-relevant processes. There are several components to KRAM: first, enhanced retroviral mutagenesis (ERM) will be used to introduce a regulated promoter/guest exon fusion encoding the photosensitizer gene, KillerRed, randomly throughout the genome. The promoter segment provides overexpression of the fused gene, leading to gain of function; while the KillerRed fusion permits Chromophore-Assisted Light Inactivation (CALI), a light-mediated inactivation technology, to destroy the protein fusion, leading to loss of function. As will be discussed later, in contrast to genetic deletion, we expect photo damage by CALI to exert dominant effects regardless of wild type allele expression. Thus, KRAM will provide for the first time a low cost high throughput approach to address the consequences of loss-of-function and gain-of-function globally in diploid cells. KRAM selection would cost markedly less than RNAi and cDNA expression library screens, as it does not require synthesis of specific reagents for every gene. To develop and test KRAM, we will use it to identify genes that act in methotrexate resistance of chronic myelogenous leukemia (CML), a well-characterized process whose genes are known. We will then use KRAM to study imatinib resistance, an important clinical problem in CML, where we expect to identify new genes in this process. We have assembled a research team whose combined expertise is optimized for the success of the proposed work. The PI pioneered CALI and is the leading authority on this approach and its application to cancer. Dr. Cochran developed the cell lines that will be used to develop KRAM, Dr. Songyang invented ERM screening and Dr. Van Etten is an expert in CML and abl-oncogenes. To establish KRAM, we propose three Aims: 1) optimize KillerRed CALI using ¿-galactosidase and endogenous proteins implicated in drug resistance; 2) develop KRAM and test its ability to select for genes required for methotrexate resistance; and 3) conduct a full-scale KRAM selection to identify new genes important for imatinib resistance and validate them by overexpression and siRNA. Successful completion of these Aims will provide a proof-of-principle for selections that are generally applicable for other cancers and also for other cancer relevant processes such as proliferation, invasiveness and apoptosis. In addition, it will identify and validate new targets to develop drugs that prevent imatinib resistance in CML, which has potentially high clinical significance. As a generalized low cost approach for gain-of-function/loss-of-function selection in somatic cells, KRAM will have wide application across biomedicine and be a transformative technology.

? DESCRIPTION (provided by applicant): Finding drugs that target breast cancer invasion (the first step in metastasis) without affecting normal tissue is an important unmet goal. One protein target of high current interest is the molecular chaperone Hsp90, which functions in cancer progression and metastasis as well as normal cell function. Hsp90 inhibitors such as ganetespib are currently in clinical trials, however they may have serious side effects based on the many proteins that require Hsp90 for activation. We introduced the concept of inhibiting Hsp90 outside of cancer cells to circumvent this problem. My lab first showed that extracellular Hsp90 (eHsp90) is released by cancer cells and that it acts in cancer invasion by activation of Matrix Metalloproteinase-2. Since then, we and others have shown that eHsp90 activates many pro-proteins in the extracellular media to enhance invasion including Lysyl Oxidase-like 2 (LOXL2), which remodels the extracellular matrix and is well implicated in cancer. We also showed that eHsp90 is released from cells via exosomes and, in new data, we show that inhibiting Hsp90 reduces exosome release and uptake, which has been implicated in tumor communication during invasion. Inhibiting eHsp90 may prevent this thus reducing invasion and metastasis. Our long-term goal is to achieve a clinical trial for an eHsp90 inhibitor for breast cancer. Recently we showed that STA-12-7191 (an impermeant derivative of ganetespib) inhibits cancer cell motility but is 5-fold less toxic to normal cells. These findings suggest that specifically inhibiting eHsp90 will benefit cancer treatment by preventing activation of LOXL2 and exosome release, reducing invasion without the damaging effects of inhibition of Hsp90's intracellular functions. We will test this hypothesis in the following Aims: 1. Test if LOXL2 is a bona fide eHsp90 client thereby providing a biomarker for eHsp90 inhibition in vivo; 2. Determine how eHsp90 acts in exosome trafficking and show this is pro-invasive; 3. Test the association between serum eHsp90 levels and metastatic breast cancer using clinically annotated patient sera and a novel ELISA diagnostic; and 4. Test the role of eHsp90 in breast cancer metastasis in human breast-to-bone xenograft model. We bring together an outstanding collaborative team including the PI who pioneered eHsp90, Dr. Ying who developed ganetespib and STA-12-7191 and experts in Hsp90 (Neckers), breast cancer biomarkers (Seewaldt and Luo) and metastasis animal models (Kuperwasser). This study is significant because it would validate eHsp90 as an important drug target for treating metastatic breast cancer and implicate it in two important pro-invasive processes: exosome-based tumor communication and ECM remodeling by LOXL2. Importantly, this study could lead to a drug that would inhibit these processes with fewer side effects than the Hsp90 inhibitors currently in human trials. In addition, it would impact other cancers and other diseases given eHsp90's multiple roles in pathogenesis. The work is innovative because it tests a novel mechanism for exosome trafficking and introduces a novel compound to inhibit eHsp90 and testing its benefit in cancer.