Karen S. Anderson - US grants

Substrate channeling is a process by which two sequential enzymes interact to transfer a metabolite (or intermediate) directly from one enzyme active site to the next without allowing free diffusion of the metabolite. There are numerous examples of sequential enzyme pairs which are thought to exhibit channeling in glycolysis and in several biosynthetic pathways and channeling is thought to play an important role in metabolic regulation and cellular modulation of enzymatic activities. Tryptophan synthase, the final enzyme is tryptophan biosynthesis, is considered the best example for substrate channeling. The enzyme exists as an alpha2Beta tetramer with alpha and Beta subunits each catalyzing a distinct chemical reaction such that the physiological alpha Beta reaction leads to the overall conversion of indole 3-glycerol phosphate (IGP) and serine to tryptophan, glyceraldehyde-3-phosphate, and H20. In the absence of serine, IGP is cleaved to indole and glyceraldehyde-3phosphate at the alpha site, although at a slow rate. The Beta subunit catalyzes the pyridoxal-dependent reaction of indole and serine to form tryptophan. These data suggest that indole is an intermediate in the alpha/Beta reaction although indole has never been observed as an intermediate in the physiological alpha/Beta reaction. In fact, the failure to detect or trap indole during net conversion of IGP to tryptophan has led to the suggestion that indole is channeled from the alpha to the Beta subunit. Further support for channeling is provided by the recently solved three-dimensional x-ray structure of tryptophan synthase which revealed the presence of a 25 Angstroms long hydrophobic channel or connecting the alpha and Beta subunits. Although there is chemical and structural evidence suggestive of indole channeling, until now the kinetics of the reaction have not been investigated in sufficient detail to establish whether channeling occurs and to determine what elementary steps of the reaction pathway are responsible for channeling. Studies from the PI's lab have recently provided the first definitive kinetic evidence for the channeling of indole and have lead to the discovery of a novel triggering mechanism by which the alpha and Beta subunits communicate over the 25 Angstroms distance between their active sites. The goals of the current proposal are: (1) Explore the structural basis for substrate channeling and alpha/Beta intersubunit communication by time-resolved x-ray crystallography and solid-state NMR spectroscopy (2) Relate the structure of the enzyme to the dynamics of catalysis and intersubunit communications by a complete kinetic and thermodynamic analysis of enzymes containing mutations in key residues.

DESCRIPTION: (Adapted from Applicant's Abstract) The current proposal seeks to continue mechanistic studies to understand, at the molecular level, the enzyme activities of RT and interaction of nucleoside inhibitors and the resistance of these compounds which develop through mutations. To achieve these aims, the investigator proposes (1) studies of the process of tRNA3lys initiation of HIV RT using an RNA duplex substrate. Because the tRNA will most likely have substantial secondary structures, the functional properties of this RNA duplex interaction with HIV RT may be different than the DNA duplex or RNA/DNA heteroduplex substrates previously used. (2) The relevance of the nucleocapsid protein as it is involved in both the rRNA3lys initiation and elongation processes and its role in the development of mutant RT. (3) Mechanistic studies will be conducted on the inhibition of mitochondrial gamma polymerase by nucleoside analogues which may be important to understand cytotoxicity.

DESCRIPTION: A unique and integral component of the bacteria cell wall of gram-negative bacteria is the lipopolysaccharide (LPS) layer. The LPS layer has been shown to be important in the development of virulent strains of bacteria and thus selective interference with LPS formation would provide a selective strategy for the design of novel antibiotics. A key enzyme in the biosynthetic pathway for the formation of lipopolysaccharides is 2-keto-3-deoxy-D-manno-2-octulosonic acid-8-phosphate (KDO8P) synthase. The studies described in this proposal are directed toward providing an in-depth understanding of the catalytic mechanism of KDO8P with the ultimate goal of impacting inhibitor design. The current proposal is based upon a transient kinetic approach using rapid chemical quench and stopped-flow fluorescence methodologies. The important advantage of this approach is the ability to observe directly events occurring at the active site including binding events, transient enzyme intermediates, protein conformational changes, and catalysis. The rate constants of individual steps can be measured directly and any enzyme intermediates including different conformational species which might be formed can be observed directly. The specific aims are: (1) Provide a complete kinetic and thermodynamic description of the KDO8P reaction pathway. (2) Isolation and characterization of enzyme intermediates. (3) Probe the catalytic mechanism through the use of alternate analogs of PEP. (4) Site-directed mutagenesis to identify amino acids residues important for recognition and catalysis.

Key insights into protein structure-function studies and structure-based drug design can be provided by an understanding of how enzyme catalysis is occurring at the enzyme active site. The study of catalysis is at the very core of understanding enzyme function. An understanding of the chemistry, chemical species and the kinetic pathway that takes place within the active site of enzyme holds great promise for describing in detail how enzymes work and more importantly how small molecules might be designed to be more potent drugs i.e. transition state analogs. The major focus of this instrument and methodology we are developing will be in advancing studies of transient intermediates in biochemical reactions (both covalent and non-covalent). In addition, this instrument should find utility in monitoring chemical catalysis and reactions, aiding in protein folding structural and kinetic studies and further validating and characterizing the creation of engineered proteins with novel catalytic functions. This instrument will open up the possibility of studying chemically labile intermediates and new enzyme and protein Systems that otherwise could not be studied.
This instrument will provide a major advance in methods to study transient enzyme intermediates and will continue the evolution of applying high-resolution mass spectrometry techniques into biology. This area continues to grow in importance as the methods become more powerful, as new enzymes with novel mechanisms become identified, as incorporation of transient kinetic information into drug design increases and as new areas of rapid kinetic investigations such as protein folding emerge. We are, just now, witnessing the power and impact that mass spectrometry will have in the biological sciences. This instrument, which interfaces classical rapid kinetics studies of enzymes with high powered analytical detection, has the possibility of propelling enzymology to a new level.

DESCRIPTION: (Adapted from Applicant's Abstract). This revised proposal focuses on mechanistic and structural studies on thymidylate synthase/dihydrofolate reductase (TS-DHFR) . These two enzymes are crucial for DNA synthesis and one-carbon transfers. In many protozoan parasties, these two catalytic activities are located on a single polypeptide chain to form the bifunctional TS- DHFR. In mammals, these enzymes are separate and monofunctional. A considerable amount of mechanistic information is available for the human monofunctional TS and DHFR, since each enzyme has been successfully targeted with the anticancer drugs, 5-fluorouracil and methotrexate, respectively. Earlier work as well as preliminary transient kinetic studies from the PI's laboratory indicate substantial mechanistic differences in the bifunctional parasitic enzyme and the monofunctional human enzymes. The ultimate goal of the proposed research is to take advantage of the differences between the bifunctional parasitic enzyme and the human enzymes to develop novel drugs for the treatment of parasitic diseases. To accomplish this goal, the following specific aims are proposed: 1) Elucidate the molecular mechanism involved in enzyme catalysis and substrate channeling for the bifunctional TS-DHFR from Toxoplasma gondii, 2) Use site-directed mutagenesis as a tool for mechanistic and structural studies with the Leishmania TS-DHFR and the Tosoplasma TS-DHFR, and 3) Use combinatorial library screening in conjunction with computer modeling to identify compounds that interfere with substrate channeling.

DESCRIPTION (Applicant's Description): Human papillomavirus (HPV) represents a unique tumor antigen system to determine whether a cell-mediated immune response can directly alter the development of cancer. DNA from human papillomaviruses (HPV) can be found in most cervical carcinomas, and the HPV E6 and E7 gene products are implicated in cervical carcinogenesis through their interaction with p53 and Rb. An intact cell-mediated immune system is thought to be critical for control of HPV infection, since 1) the vast majority of infected HPV+ patients will spontaneously clear their infection, 2 ) the development of cervical neoplasia is HLA-linked, and 3)immunodeficiency, such as HIV infection, alters the progression of HPV infection. There are several ongoing clinical trials [involving] vaccination with HPV E6 and E7-derived antigens, but many questions regarding the endogenous iLnmune response to remain to be addressed. Hypothesis: Infection of patients with human papillomavirus (HPV) subtype 16 triggers a measurable endogenous CD8+ cytotoxic T lymphocyte (CTL) response to HPV-derived peptides, and the failure to develop functional anti-HPV CTL correlates with persistence of infection and progression to cervical neoplasia. To address this hypothesis, Dr. Anderson plans to accomplish the following aims: 1) Using a combination of tetramer and ELISpot assays, determine whether normal HLA-A2+ blood donors have measurable and functional HPV16 E6 and E7-specific CTL, and whether these CTL can be expanded in vitro. 2) Determine if HPV16-infected patients mount an anti-HPV CTL response that inversely correlates with progression of cervical neoplasia. This will be done by enumerating, expanding, and phenotyping CTL from HPV16+ patients who have a range of cervical abnormalities, from normal cytology to cervical carcinoma. Dr. Anderson's research will be performed in a laboratory at the Dana-Farber Cancer Institute under the sponsorship of Dr. Lee M, Nadler, a leader in the f i eld of tumor immunology and experienced mentor of many successful physician/scientists.

DESCRIPTION (adapted from applicant's abstract): Phosphoenol pyruvate (PEP) is a highly functionalized, chemically versatile molecule involved at several intersections of cellular energy metabolism and biosynthesis. While most enzymatic reactions utilizing PEP as a substrate involve cleavage of the high energy P-O, two types of reactions have been shown to involve the unusual cleavage of the C-O bond of PEP. These enzyme reactions fall in to two classes: (1) the first class is those that carry out an enolpyruvoyl transferase reaction and (2) the second class are those that carry out what is formally a net aldol condensation reaction. Representative enzymes in the enolpyruvoyl transferase class include EPSP synthase and MurZ. To date, there are only two known enzymes that carry out the net aldol condensation between PEP and the aldehyde moiety of a cosubstrate sugar: KDO8P synthase and DAHP synthase. Recently, a third enzyme, N-acetyl-neuraminic acid synthase, has been discovered that also appears to fall in to this category. Each of these bacterial enzymes is responsible for catalyzing the formation of key components such as lipopolysaccharide, aromatic amino acids, and capsular polysaccharides that are found in unique biosynthetic pathways and therefore may represent novel target enzymes for the design of new antibiotics. While the mechanistic aspects of the catalytic pathway for the enolpyruvoyl transferase have been delineated, the details of the catalytic mechanism for those enzymes in the second class that are involved in the net aldol condensation remain elusive. This proposal will investigate the catalytic mechanism for each of the three enzymes that catalyze a net aldol condensation and define the second class of enzyme that cleave the C-O bond of PEP. The two specific aims of this proposal are: 1) Elucidation of the KDO8P synthase molecular mechanism using transient kinetic methods to study: a) structure guided site-directed enzyme mutants and b) a series of alternate substrate and reaction intermediate analogs; and 2) Detection and characterization of novel enzyme intermediate from a series of three PEP-utilizing enzymes using a novel rapid mixing, pulsed-flow ESI-MS technique. These enzymes carry out a unique aldol type condensation of PEP and sugars of increasing length: C4 (DAHP synthase), C5 (KDO8P synthase), and C-6 (N-acetyl-neuraminic acid synthase). In addition as the investigator develops the quantitative aspects of the rapid mixing, pulsed-flow ESI-MS technique further, she will extend the studies of these enzymes to determine the full kinetic profile.

[unreadable] DESCRIPTION (provided by applicant): Opportunistic infections are the primary cause of suffering and death in individuals with AIDS. Many of these infections are produced by parasites which rarely affect individuals who are not immunocompromised. Unfortunately, successful combination therapies against the HIV-1 virus still leave most patients susceptible to opportunistic parasitic infections. The drugs which are currently available for the treatment of these parasitic infections suffer from a lack of selectivity resulting in host toxicity and untoward side effects. Thus there is a need for novel therapeutic strategies which may be more selective and less toxic. This proposal outlines mechanistic and structural studies on a unique bifunctional enzyme which will serve as basis for the design of novel antiparasitic drugs. Two enzymes crucial for DNA synthesis and one-carbon transfers are thymidylate synthase and dihydrofolate reductase. In many protozoan parasites, these two catalytic activities are located on a single polypeptide chain to form a bifunctional thymidylate synthase (TS)/dihydrofolate reductase (DHFR) enzyme. In mammalian species, the thymidylate synthase and dihydrofolate activities occur as separate catalytic activities on mono functional enzymes. A considerable amount of mechanistic information is available for the human monofunctional thymidylate synthase and dihydrofolate reductase since each enzyme has been successfully targeted with the anticancer drugs, 5-fluorouracil and methotrexate, respectively. Earlier work as well as preliminary transient kinetic studies from the Pl's lab indicate substantial mechanistic differences in the bifunctional parasitic and mono functional human enzymes. The three dimensional structure of the bifunctional TS-DHFR enzyme is available for computer modeling studies to identify inhibitors through docking programs. The central theme of this proposal is that an in-depth kinetic and structural evaluation of the bifunctional TS-enzyme at a molecular level will provide a crucial mechanistic understanding that can be exploited as a novel therapeutic approach. [unreadable] [unreadable] [unreadable]

DESCRIPTION (PROVIDED BY APPLICANT) An important aspect of protein structure-function studies and enzyme-targeted biorational inhibitor design is an understanding of how chemical catalysis is occurring a the enzyme active site. The detection and identification of enzyme reaction intermediates provide key insights into the enzyme catalytic reaction pathway. Rapid chemical quench methods involving off-line detection have proven very useful in identifying enzyme reaction intermediates. However, a limitation of this approach involves the inability to detect and characterize enzyme intermediates that are labile under the chemical quenching conditions. We have developed and demonstrated the utility for the real-time detection and characterization of enzyme intermediates on the millisecond time scale using a novel approach of rapid mixing/time-resolved electrospray ionization (ESi) mass spectrometry (MS). The overall objective of the studies described in this proposal is to further develop and demonstrate the broad applicability of the rapid mix/time-resolved ESIMS approach for detecting and quantitating enzyme intermediates and defining enzyme reaction kinetic pathways. The following specific aims are designed to accomplish this objective: 1. Demonstrate the ability of rapid mixing/time-resolved ESI-MS to quantitatively determine kinetic parameters. 2. Demonstrate the broader utility of the rapid mixing/time-resolved ESI-MS to identify putative chemically labile enzyme intermediates. 3. Demonstrate the ability of rapid mixing/time-resolved ESI-MS to monitor specific phosphorylation and dephosphorylation events in protein signaling. These studies will aid in pioneering a powerful and novel analytical methodology for detecting, characterizing and quantitating enzyme intermediates on a rapid time scale.

[unreadable] DESCRIPTION (provided by applicant): Receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR) are essential in the initiation of many protein signaling pathways. Dynamic control of multiple phosphorylation modifications of a single RTK thus can manifest critical control on multiple signal transduction pathways. Alterations of this sensitive dynamic in growth factor networks and aberrant activities of RTKs often have severe biological consequences and are linked to oncogenic processes in many human cancers. Indeed the success of tyrosine kinase inhibitors such as Iressa(tm) and Gleevec(tm) heralds a recent strategy of precisely targeted cancer therapy. An understanding of the molecular mechanisms of these early dynamic events may hold the key to understanding and predicting the nature of oncogenic behavior and for predicting the affects of RTK targeted therapy. An increased understanding of the early temporally regulated states will enable more precise strategies for selectively targeting downstream pathways and may offer a unique approach to cancer therapy based on dynamic rather than static intervention. An integrated platform of novel technologies and approaches are needed to provide temporal resolution of these rapid, early events at a molecular level both in vitro as well as in cell culture. This R21/R33 combined proposal will create a validated general set of innovative and established technologies including rapid reaction methodologies, a novel time-resolved electrospray ionization mass spectrometry (ESI-MS) technique, nanospray ESI-TOF, phosphopeptide mapping using ESI-MS LC/MS/MS and site-specific phosphotyrosine antibody detection that will be applicable for the analysis of a wide range of RTKs, their autophosphorylation patterns and downstream signaling events in the critical subsecond to multisecond time domain. We have chosen a prototypical RTK, epidermal growth factor receptor tyrosine kinase (EGFR) to develop this platform. The epidermal growth factor receptor (EGFR) tyrosine kinase pathway is linked to a large number of cancers and an important molecular target for targeted cancer therapeutics such as the small molecule ATP mimetic Iressa (gefinitib) that has recently been approved by the FDA and underscores the generation of specific kinase targeting as a new paradigm for cancer therapy. The ability to probe the molecular and temporal details of the earliest events of autophosphorylation will reveal signature patterns that will provide a new understanding of the differences between normal and oncogenic forms of EGFR and an expanded functional understanding of the emerging therapeutic class of targeted kinase inhibitors. We believe that a profile of the temporal dynamics of phosphorylation in signaling provides a unique molecular fingerprint or signature for distinguishing normal and cancer cells and the responsiveness to targeted inhibitors. New experimental tools and technologies are needed to distinguish cancer cells from normal cells at a molecular level and evaluate the effectiveness of new cancer therapies. This R21/R33 project describes a strategy to develop novel technologies that will allow us to understand how changes occur at a molecular level in a cancerous cell and a more detailed understanding of how new selectively targeted cancer therapies work. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The epidermal growth factor receptor (EGFR) tyrosine kinase pathway has been demonstrated to be a key molecular target for cancer therapeutics including TKIs such as Iressa (gefinitib) and Tarceva (erlotinib). These compounds are small molecule ATP mimetics, selectively binding to the intracellular kinase of EGFR, that have recently received FDA approval for treatment of non-small cell lung carcinoma (NSCLC). An ever growing body of data indicates that the presence of mutations in EGFR play an important role in response to TKI therapy and drug resistance. There are a subset of patients (~10%) who have a remarkable response to Iressa and Tarceva that have now been correlated to several specific activating EGFR mutations arising within the tumor. A second mutation has been identified in relapsed patients who were initially drug responsive and more recently this mutation has also been found in a family with a genetic predisposition for developing lung cancer. Recent studies from our lab and others suggest an emerging new paradigm for modulation of downstream signaling. These studies show that autophosphorylation of RTKs occurs in a specific sequential order, creating a temporal and dynamic phosphorylation pattern of different intermediate sets of phosphotyrosines. Accordingly, these discrete sets of phosphotyrosines can orchestrate the dynamic recruitment of specific downstream signaling partners in a temporal fashion that may well play an important role in the proper regulation of protein signaling. The underlying molecular mechanisms of the oncogenic activating and resistance mutations in EGFR and differential sensitivity to Iressa and Tarceva are not understood. Our preliminary data suggest that the underlying causes may be linked to alterations in nucleotide affinity for the receptor, kinase catalytic activity, and/or alterations in phosphorylation patterns that result in differential protein signaling. The studies outlined in this application will use a combination of in vitro biochemical studies and complementary cellular studies to identify specific molecular alterations that may distinguish wild type (WT) and oncogenic forms of EGFR harboring Iressa/Tarceva sensitive activating mutations as well as mutations associated with drug resistance. The overall objectives are to define the molecular mechanism of protein signaling in the EGFR pathway under normal and aberrant oncogenic processes and provide an understanding for the modulating effects of Iressa/Tarceva. This information will ultimately aid in the design of more potent and selective compounds for precisely targeted cancer therapy. Growth factors bind to their corresponding receptors located on the surface of cells and give signals for cell growth. In a cancer cell, the signals for cell growth have aberrant signaling and growth and proliferation have become out of control. The newest, most promising cancer therapies such as Iressa and Tarceva, targets these uncontrolled signaling pathways in cancer cells. Some patients with non- small cell lung carcinoma having particular mutations in their growth factor receptors respond very well to these drugs, however the molecular events are not understood. The studies described in this application are designed help unravel these molecular details to allow us to differentiate cancer cells from normal cells and assess the usefulness of current and new cancer therapies.

? DESCRIPTION (provided by applicant): The World Health Organization estimated that at the end of 2012 over 35 million individuals were infected with HIV-1 (Human Immunodeficiency Virus). Due to the advancements in highly active antiretroviral (HAART) therapy the number of AIDS-related deaths has been reduced and individuals are living longer. Despite successes with HAART, opportunistic infections remain one of major causes of death in AIDS patients. Among the most prevalent is the protozoal parasite pathogen, Cryptosporidium. Cryptosporidiosis is one of the AIDS- defining illnesses characterized by a severe chronic diarrhea for which there are currently no effective therapies and among the most frequent pathogens causing diarrheal diseases in developing countries. Efforts during the previous grant period have focused on a unique bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) found only in protozoal pathogens as a possible molecular target for inhibitor design. These proof of concept studies in Cryptosporidium hominis (Ch)TS-DHFR have identified unique species specific and allosteric/non-active target regions in this bifunctional enzymes and mutational analysis and mechanistic studies have validated these sites as essential for catalytic function. The PIs lab and their collaborators have developed a distinctive and successful computationally and mechanistically guided approach for the discovery of new inhibitors of ChTS-DHFR. This partnership of computational and detailed experimental methodologies is a unique strategy that builds optimal physiochemical and pharmacological parameters into the design. These molecules are then experimentally tested and the design iteratively refined through mechanistic, structural and cellular evaluation. Molecular docking and virtual screening coupled with structural analyses have discovered novel inhibitors of CH TS-DHFR including some with nanomolar potency and anti-Cryptosporidial activity in cell culture. The current grant period will focus on lead optimization and a novel nanotechnology strategy for parasite drug delivery to develop new therapies to treat Cryptosporidial infections.

DESCRIPTION (provided by applicant): The overall goal of this project is to identify autoantibody biomarkers in sera that can be readily used for the early detection of cancer. Antibodies are induced by tumor-specific alterations in protein expression, mutation, degradation, or localization, with high specificity. We have developed a novel programmable protein microarray technology (NAPPA), which uses printed cDNA encoding tumor antigens that are translated in vitro. We have been funded for the past four years as an EDRN Biomarker Development Laboratory to develop NAPPA as a clinical research tool and to employ it for the detection of autoantibody biomarkers for breast cancer, and have screened over 700 sera and identified 32 novel breast and 23 ovarian biomarkers that have undergone blinded validation studies. Here, we propose to increase the feature density of NAPPA arrays in order to accommodate the increasing size of our cDNA collection, which now approaches 10,000 unique genes (Aim 1) and to develop a method for producing glycoproteome arrays (Aim 2). Both of these methods will enhance autoantibody biomarker discovery by increasing the number and quality of the antigens screened. Aim 3 will use the results of Aims 1 and 2 to expand the top breast cancer biomarkers that have been identified to focus on particular subtypes of breast cancer that are more difficult to detect including Her2+ breast cancers, triple-negative (ER-PR-Her2-) breast cancers, and cancers that occur in the setting of high breast density. Association of antibody detection and tumor antigen expression will be explored. Aim 4 will assess the performance characteristics of the combined set of top breast cancer biomarkers in Phase II studies using sera collected in multicenter clinical studies. Aim 5 will focus on the utility of these biomarkers to detect disease prior to clinical diagnosis. At the end of these experiments, our goal is to identify autoantibody serum signatures that can be used by EDRN CEVCs in further Phase III independent validation studies for the early detection of breast cancer according to the EDRN biomarker discovery guideline.

DESCRIPTION (provided by applicant): Pancreatic cancer remains a highly lethal disease, with no established biomarkers or screening strategy for early detection. Somatic mutation in TP53, primarily in the DNA-binding core region of the protein, results in protein stabilization and autoantibody (AAb) generation. Using a novel method of protein display and detection of serum IgG, we have detected P53-AAb to wild-type (WT) protein in serous ovarian cancer with moderate sensitivity and high specificity, including a subset of cases with false-negative CA 125. In ovarian cancer, we detected rising p53-AAb levels starting 9 months prior to clinical diagnosis, and since the pancreatic cancer has similar, early p53 mutations as serous ovarian cancer, we predict these markers will be present in pancreatic cancer patient sera. Since the AAb were induced by mutated p53, AAb to forms of mutant p53 may improve the assay. We developed a protein microarray expressing the 68 most common p53 mutations that are present in solid tumors. Here, we propose to determine the frequency of WT and mutant-specific p53-AAb, in sera from patients with early and late-stage pancreatic cancers. We will correlate p53-AAb detection with primary and metastatic tumor p53 expression by IHC, and with TP53 mutation status.

DESCRIPTION (provided by applicant): The epidermal growth factor receptor (EGFR) tyrosine kinase pathway has been demonstrated to be a key molecular target for cancer therapeutics including TKIs such as Iressa (gefinitib) and Tarceva (erlotinib). These compounds are small molecule ATP mimetics, selectively binding to the intracellular kinase of EGFR, that have recently received FDA approval for treatment of non-small cell lung carcinoma (NSCLC). An ever growing body of data indicates that the presence of mutations in EGFR play an important role in response to TKI therapy and drug resistance. There are a subset of patients (~10%) who have a remarkable response to Iressa and Tarceva that have now been correlated to several specific activating EGFR mutations arising within the tumor. A second mutation has been identified in relapsed patients who were initially drug responsive and more recently this mutation has also been found in a family with a genetic predisposition for developing lung cancer. Recent studies from our lab and others suggest an emerging new paradigm for modulation of downstream signaling. These studies show that autophosphorylation of RTKs occurs in a specific sequential order, creating a temporal and dynamic phosphorylation pattern of different intermediate sets of phosphotyrosines. Accordingly, these discrete sets of phosphotyrosines can orchestrate the dynamic recruitment of specific downstream signaling partners in a temporal fashion that may well play an important role in the proper regulation of protein signaling. The underlying molecular mechanisms of the oncogenic activating and resistance mutations in EGFR and differential sensitivity to Iressa and Tarceva are not understood. Our preliminary data suggest that the underlying causes may be linked to alterations in nucleotide affinity for the receptor, kinase catalytic activity, and/or alterations in phosphorylation patterns that result in differential protein signaling. The studies outlined in this application will use a combination of in vitro biochemical studies and complementary cellular studies to identify specific molecular alterations that may distinguish wild type (WT) and oncogenic forms of EGFR harboring Iressa/Tarceva sensitive activating mutations as well as mutations associated with drug resistance. The overall objectives are to define the molecular mechanism of protein signaling in the EGFR pathway under normal and aberrant oncogenic processes and provide an understanding for the modulating effects of Iressa/Tarceva. This information will ultimately aid in the design of more potent and selective compounds for precisely targeted cancer therapy. Growth factors bind to their corresponding receptors located on the surface of cells and give signals for cell growth. In a cancer cell, the signals for cell growth have aberrant signaling and growth and proliferation have become out of control. The newest, most promising cancer therapies such as Iressa and Tarceva, targets these uncontrolled signaling pathways in cancer cells. Some patients with non- small cell lung carcinoma having particular mutations in their growth factor receptors respond very well to these drugs, however the molecular events are not understood. The studies described in this application are designed help unravel these molecular details to allow us to differentiate cancer cells from normal cells and assess the usefulness of current and new cancer therapies.

Routine high-sensitivity multiplexed detection of disease biomarkers in blood will have an impact on early diagnosis and therapy selection. A novel and highly effective approach for detection of broad categories of disease is multiplexed detection of antibodies using protein microarrays. As a greater number of antibody biomarkers are being identified and clinically validated for viral infection, cancer, cardiovascular disease, and autoimmune disease, there is a strong need to detect them with greater sensitivity and greater signal-to-noise ratio, while at the same time being able to perform automated biomarker analysis with low cost per test. In the proposed project, the investigators integrate photonic crystal enhanced fluorescence (PCEF) technology with a novel size-exclusion blood filtration technology to develop a multiplexed microspot fluorescent sandwich assay platform for the clinical laboratory environment. Our approach integrates several innovations into an inexpensive plastic-based sensor cartridge and desktop detection instrument: 1. A silicon-based nanostructured resonant optical photonic crystal chip with an integrated Fabry-Perot optical cavity will be used to deliver ~50x greater signal enhancement than current-generation PCEF devices, which already routinely provide <1 pg/ml limits of detection in complex media, using only 10 ?l sample volumes. 2. A laser-machined blood filter will separate plasma from a droplet of heparinized whole blood in 60 seconds, enabling the entire assay protocol to be performed automatically in <60 minutes without user intervention. 3. An innovative laser scanning approach is used to couple light from a small semiconductor laser directed to the PC surface in the optimal ?on-resonance? condition, resulting in a rugged, compact instrument with a cost of <$10K. 4. A statistical bioinformatics tool indicating the presence or absence of the biomarkers in the test sample. As an example application of the system, we will focus our effort on detection of a panel of 3 serum antibodies for human papillomavirus (HPV) as a means for identifying patients with greater susceptibility to oropharyngeal carcinoma (OPC), although the same technology can be extended to detection of broad classes of antibody biomarkers. The new instrument will be first tested upon biomarkers spiked into whole blood, with results compared against single-antibody ELISA in microplates and the Luminex bead-based system. Further validation and comparison will be performed upon clinical blood samples from patients with known HPV exposure. Our long-term goal is to demonstrate a prototype system that can be applied broadly for multiplexed serum antibody biomarker analysis.

? DESCRIPTION (provided by applicant): Currently, there are few methods available to analyze the evolution of tumor heterogeneity; micro-dissection of tumors only provides information on major species of malignant cells but is unable to detect rare therapy-resistant subclones that have the potential to regenerate tumors. Identification of these rare cells by single-cell isolatio and sequencing is both time-consuming and prohibitively expensive. Recent next-generation deep sequencing studies have demonstrated the clinical relevance of clonal heterogeneity within individual cancers, but currently rapid and cost-effective methods to measure and track the rates of co-occurrences of mutations in cell populations do not exist. Therefore, the development of rapid, flexible, single- cell technologies with the capacity to identify heterogeneous mutations of multiple genes in individual cells within bulk populations is critical for the development of effective targeted therapies that prevent tumor relapse. To overcome this challenge, we propose to adapt novel nanomolecular scaffolds (termed DNA origami) to transfect tumor cells and capture mRNA encoding known-tumor suppressor genes. We propose to test the specificity of these scaffolds in breast tumor cell lines and primary breast tumor with known mutations in p53, PTEN, and PIK3CA genes. This approach will allow the rapid quantitation of genomic diversity and evolutionary order in cells from solid tumors for improved targeting of rare malignant subclones.

Project Summary/Abstract Despite advances in screening and treatment, mortalities from breast and lung cancers have remained high in the US over the last 20 years. It is widely accepted that early detection is critical to improving outcomes in both diseases. Both also rely on imaging for screening, but false positive and false negative detection are associated with unnecessary biopsies, missed diagnoses, and costs. There is an urgent need for biochemical markers that improve the performance of imaging technologies. Our laboratories have been successful at identifying useful cancer biomarkers by exploiting patients? own ability to produce antibodies against tumor- associated antigens (TAA), referred to as tumor-associated autoantibodies (TAAb). With prior EDRN support, we developed high-throughput programmable protein display methods for the rapid detection and validation of autoantibody biomarker signatures in breast and lung cancers. Our breast cancer TAAb biomarkers have been licensed and integrated into Videssa? Breast that is now available as CLIA-certified test. Our triple negative breast cancer markers have been validated in blinded phase 2 multicenter validation studies. These demonstrate the great utility of TAAb in cancer early detection. However, the sensitivities of most TAAbs is moderate and there is a suggestion that greater sensitivity and specificity could be obtained by examining TAAb directed at aberrantly modified proteins in cancers. Our central hypothesis is that aberrant protein glycosylation, a hallmark of breast and lung cancers, induces glycoprotein-specific TAAb that can be measured as specific serum biomarkers of these cancers. Alterations in glycosylation are highly immunogenic, and there is strong historical evidence for significant antibody responses to cancer-altered glycoproteins. However, all current protein (or polypeptide) display tools allow limited or no post-translational modification. This historical roadblock has prevented the identification of these biomarkers because of the lack of screening methods that test immunogenic structural glycoproteins. We introduce a tool for the high-throughput display of full-length proteins decorated with cancer-specific O-glycan structures. This will revolutionize the opportunity to screen glycan-protein epitopes in their natural context. Our team comprises strong expertise in functional proteomics, biomarker development, glycoproteomics, medical oncology and biostatistics. Targeted proteins will include: the extra-cellular domains of relevant single pass membrane proteins, proteins known to be O-glycosylated and overexpressed in the two cancers, and all known mucins. They will be translated in situ using human ribosomes and chaperone proteins and then systematically decorated with Tn and STn O-GalNAc-type glycans by consecutive addition of recombinant glycosyltransferases and sugar nucleotides to mirror what occurs in the two cancers. Adhering to the principles of PRoBE design, we will screen these arrays with cancer patient and control sera. Our study will focus on cancer patients and non-cancer subjects with positive imaging findings. Study design will include Phase I discovery (arrays/ELISA) and Phase II validation using ELISA.

Project Summary. Despite the development of effective human papillomavirus (HPV) vaccines, it has been estimated that there will be over 200,000 new cases and over 100,000 deaths due to cervical cancer by 2020 in India, which has 25% of the global burden of cases. Cervical cancer screening by Pap smears and HPV DNA testing has become standard of care in the US and Europe, but has been too expensive and logistically challenging in low and middle income countries (LMICs). A simple, point-of-care (POC) biomarker panel for high-grade dysplasia and cervical cancer could provide a cost-effective means for triage of cervical disease in these countries. HPV infection induces systemic humoral immune responses with IgG antibodies to HPV- derived proteins. We have previously identified antibodies to a panel of five HPV antigens that detect up to 88% of patients with HPV+ oropharyngeal cancer, but less than 4% of healthy controls. Using an expanded panel of 16 HPV antigens, we have detected at least one HPV antibody in the sera of 45% of CIN II/III cervical dysplasia. These results support the development of a rapid, quantitative, and multiplexed assay for the detection of HPV-related cervical disease. We leverage our protein microarray technology with advances in fluorescent technologies to enable a fluorescent, programmable, multiplexed ELISA (serologic) assay for HPV- specific IgG antibodies in a compact and disposable configuration with high analytic sensitivity and rapid, quantitative output. Our preliminary work in this area has demonstrated detection of IgG antibodies to HPV antigens with lower limits of detection in the 10pg/mL range, which is a 100-fold improvement over existing colorimetric approaches. Under our proposed effort, we aim to develop and demonstrate a prototype multiplexed fluorescent programmable point-of-care assay with high analytical sensitivity for the simultaneous detection of 16 individual HPV-specific IgG serologic biomarkers from a single finger stick-sized patient blood sample which can be manufactured for a total reagent cost of less than $1/patient sample. We will leverage our expertise in immunoassay development with expertise in microfluidics, electrical engineering, optics, biomarker analytical and clinical validation to target the device and assays for the LMIC clinical setting. We will also provide a clinical platform and biorepository to rapidly evaluate emerging technologies for cervical screening. This proposal will adapt the engineering and biochemistry for the development of robust POC devices for detection of HPV serology specifically for low and middle-income countries. We will transfer and evaluate this technology, screening 13,000 patients in India, with a team of collaborators in the All India Institute of Medical Sciences (AIIMS) in New Delhi, India.

DEVELOPMENTAL THERAPEUTICS RESEARCH PROGRAM PROGRAM CODE: DT PROJECT SUMMARY/ABSTRACT The highly collaborative, transdisciplinary Developmental Therapeutics (DT) Research Program is a critical translational innovation hub for Yale Cancer Center (YCC), bridging drug discovery and experimental therapeutics to translate preclinical discoveries and treatment strategies into the clinic, while returning clinical advances to the bench for refinement. In achieving this mission, DT capitalizes on science derived from all seven YCC Research Programs. DT has assembled a critical mass of laboratory and clinical investigators who command expertise from basic research scientists, translational researchers, and clinical investigators, as well as proven ability to translate findings into development and evaluation of new therapies. DT?s most important focus area is the transition from a novel therapeutic strategy, through a drug candidate, into early-phase clinical trials that incorporate and leverage inventive correlative science to inform potential new targets, predictive biomarkers, and future clinical development. YCC promotes transdisciplinary collaborative research in DT and enhances cancer focus via inter- and intra-programmatic meetings, recruitment, space, pilot funding, grant applications, mentorship, and Shared Resource improvements. DT?s goals have evolved over the past cycle in response to breakthroughs in cancer medicine, including genome profiling, widespread testing of kinase inhibitors, innovations in chemical biology, insights into DNA repair, and the immunotherapy revolution. YCC members are intimately involved in these advances, and DT is poised to leverage its scientific breadth with strengthened capacity in early-phase clinical research and personalized medicine to address important barriers to progress in cancer therapeutics. The Early Phase Clinical Trials Network UM1 grant and its associated Phase II supplement, P50 Lung and Skin SPORE grants, Stand-up to Cancer grants in lung, melanoma, and colorectal cancers, and Lead Academic Participating Site grant from the NCI National Clinical Trials Network serve as a critical nidus to advance this goal and drive our translational efforts from lab to clinic and clinic to lab. DT has 66 members, representing 16 departments, with faculty from the Schools of Medicine, Public Health, Faculty of Arts and Sciences, and Engineering & Applied Sciences. This includes one Howard Hughes Medical Institute investigator, four National Academy of Sciences members, and one Institute of Medicine member. DT members published 902 papers between July 1, 2012 and June 1, 2017, with inter- programmatic (29%), intra-programmatic (19%), and intra- plus inter-programmatic (9%) collaborations demonstrating interactions among DT members within the program, as well as with YCC members in other programs. Funding is robust, with total cancer-related funding of $17.6M (direct costs). This includes $7.1M in peer-reviewed funding, of which $3.6M is from NCI. This is a 29% increase in NCI funding from $2.8M at the last renewal in 2012 (direct costs). !

ABSTRACT/SUMMARY Antiretroviral therapy (ART) has significantly reduced the mortality of HIV disease and brought viral loads in HIV patients to below detection limits (<50 copies/ml plasma). This has been critical in controlling HIV spread and there is substantial clinical evidence that zero HIV transmissions occur from HIV-infected people with non-detectable viral loads. However, side-effects and non-adherence to the compulsory daily treatment regimen limit the long-term use of ART in infected people. A foreseeable problem with antiretroviral non- adherence is the impact on non-detectable viral loads. We hypothesize that new drugs with improved pharmacological properties, and a long-acting parenteral formulation of antiretroviral drugs that could reduce the dosing to weekly, monthly, or even longer periods of time can significantly impact patient non-adherence. In recent work, we have developed a novel class of picomolar Non Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) with enhanced pharmacological properties, drug resistance profiles, and a wide margin of safety relative to the current FDA approved NNRTIs such as efavirenz and rilpivirin. Because of these novel properties, these new NNRTIs are particularly well-suited for coupling with sustained-release drug delivery technologies. A long-acting nanoformulation of our candidate NNRTI, a naphthyl catechol phenyl ether called Compound I, maintained sustained plasma levels and antiretroviral efficacy for ?3 weeks in HIV-1-infected humanized mice, confirming potential as a late-stage preclinical candidate. Given the favorable pharmacological properties and potent synergy of Compound I with other classes of currently approved FDA drugs, our overall objective is to develop Compound I as a component of long-acting combinatorial ART (cART). We envision long-acting formulations made from different biocompatible biodegradable polymers [PLGA, poly(PDL-co-DO)] that can be delivered as an intramuscular (i.m.) injection of microparticles or a subdermal biodegradable implant to provide sustained-release of NNRTIs in combination with other ARV classes over a period of months to a year. This would be similar to the FDA-approved Lupron Depot®, an 8 micron microparticle, (marketed by AbbVie and manufactured by Takeda) that provides multi-months of sustained release of the synthetic hormone, leuprolide acetate for treatment of prostate cancer and endometriosis. The aims of this application are to develop, optimize and test efficacy of Compound I as a component of long-acting cART for pre-exposure prophylaxis (PrEP) as well as late stage therapy in HIV- infected humanized NSG mice. These approaches and techniques will directly benefit patients for the treatment of HIV-1.

Abstract Oncogenic human papillomaviruses (HPV) are the causative agents of uterine cervical and an increasing portion of head and neck squamous cell carcinomas (HNSCC), but HNSCC is almost exclusively associated with HPV type 16. The oncogenic properties of HPV type 16 are largely attributed to two major HPV oncogenes, E6 and E7, that degrade p53 and retinoblastoma (RB) family members, respectively. Degradation of these tumor suppressors by E6 and E7 results in uncontrolled proliferation, diminished apoptosis and increased genomic instability that predisposes to malignant transformation. The crucial roles of E6/E7 in HPV-related carcinogenesis make them an attractive target for anti-cancer therapy, since methods for decreasing their expression or activities would restore p53 and RB activity in tumors driven by HPV. Our preliminary results indicate that treatment of HPV-positive (HPV+) HNSCC with the demethylating agent, 5-azacytidine (5-aza) at clinically relevant concentrations, resulted in remarkable downregulation of all HPV gene expression, including E6 and E7. 5-aza treatment restored p53 expression and activity in HPV+ head and neck cancer cells, which was partially responsible for the sensitivity of these cells to 5-aza. In addition to restoration of p53, 5-aza also was toxic to HPV+ HNSCC through creation of DNA double strand breaks (DSBs). Mechanistically, 5-aza-induced DNA DSBs in HPV+ HNSCC were dependent on transcription and replication and on overexpression of the cytidine deaminase, APOBEC3B (A3B). Experimental depletion of A3B inhibited 5-aza toxicity and diminished DSB formation, but also indicated that untreated HPV+ HNSCC depend on A3B for clonogenic growth. The observations that untreated HPV+ HNSCC dependent on A3B, but that A3B contributes to 5-aza toxicity and DSBs, suggests an A3B-dependent synthetic lethality upon treatment with 5-aza, and that A3B may serve as a biomarker of response. Treatment of mice bearing HPV+ tumors with 5-aza revealed significant tumor growth inhibition and prevented detection of circulating tumor cells. A window clinical trial in patients with HNSCC using standard dosing for 5 days achieved demethylation (LINE-1) similar to that seen in our in vitro experiments and confirmed that 5-aza treatment: 1) significantly decreased expression of HPV genes; 2) reactivated p53; 3) activated caspases, 4) and inhibited matrix metalloproteinase expression in HPV+ HNSCCs. This proposal is designed to determine molecular mechanisms of demethylation-induced downregulation of HPV oncogenes, elucidate the role of A3B in 5-aza-induced synthetic lethality and DNA DSBs formation, determine effect of demethylation on immune cell infiltration in HPV+ HNSCC, and explore the potential of 5-aza alone or in combination with chemotherapeutic agents to suppress HPV-associated HNSCC metastasis and inhibit growth using patient-derived xenografts. These studies will provide a basis for a new rational targeted therapy for HPV+ HNSCC, which is desperately needed to treat patients with recurrent or metastatic HPV+ HNSCC and to decrease the toxicity associated with current therapy.

SUMMARY Human papillomavirus (HPV)-associated neck squamous cell carcinoma (HNSCC) represents an increasing proportion of HNSCC. The incidence of HPV+ HNSCC has dramatically increased over the last 2 decades and in 2012 surpassed uterine cervical cancer as the most common HPV-related malignancy in the U.S. Despite the HPV vaccine, it is estimated that the ?epidemic? of HNSCC caused by HPV will not diminish until 2060. HPV+ HNSCCs occur in younger individuals and prognosis for patients with these tumors is better compared to patients with classical HNSCC; however, ~25% of patients recur with few effective therapeutic options. Based on observed hypermethylation of HPV+ HNSCC from TCGA, and understanding that HPV uses hypermethylation to impede the innate immune response, effects of the demethylating agent, 5-azacytidine (5- azaC), were tested on HPV+ HNSCC. We found that HPV+ HNSCC cells in culture and xenografts are sensitive to 5-azaC, and that 5-azaC caused double strand breaks (DSB) that were not observed after 5-azaC therapy in HPV-negative HNSCC, even with much higher doses. We found that following 5-azaC therapy, APOlipoprotein B mRNA-Editing enzyme Catalytic polypeptide 3B (APOBEC3B) was associated with chromatin in HPV+ HNSCC, but not HPV-negative cells. CRISPR knockdown of A3B prevented DSB and protected cells from 5-azaC-induced death. Despite being required for DSBs and cellular toxicity caused by 5- azaC, A3B was also required for clonogenic survival of untreated HPV+ HNSCC. These data showing that A3B is required for survival of HPV+ HNSCC cells, but that following demethylation A3B mediates toxicity and DSB. In addition, 5-azaC therapy increased type I interferon signaling as measured by increased expression of interferon-stimulated genes. These exciting pre-clinical data led to a window trial of 5days of 5-azaC. Analysis of tumor specimens confirmed in vitro data showing that 5-azaC resulted in cellular toxicity. Immunofluorescent staining of an HPV+ patient tumors pre- and post-5-azaC showed a marked increase in tumor-associated lymphocytes, possibly driven through activation of type I interferon combined with increased expression of neoantigens. In this YHN-SPORE project, we hypothesize 5-azaC therapy will enhance response to nivolumab (Nivo) through its ability to cause cell death, increase neoantigen expression, increase A3B-driven mutational load, and enhance T cell infiltration through increased type I interferon signaling. These hypotheses will be tested using established and novel in vitro assays, as well as through examination of pre- and post-therapy tumor specimens from a 3-armed clinical trial. In Aim 1, tumor specimens from the SPORE window trial will be analyzed to determine effects of 5-azaC, Nivo, or the combination on cell death, cell proliferation, immune infiltration and immune activation. Aim 2 will employ standard and novel assays to explore the role of A3B in cellular toxicity exposed by 5-azaC therapy. In Aim 3, we will determine effects of 5-azaC on activators of immune recognition and response in the presence or absence of Nivo.