Mark R. Kelley - US grants

Reactive oxygen species produced by ionizing radiation introduce a variety of lesions in DNA including strand breaks, abasic (AP) sites, and fragmented deoxyribose forms including 3-phosphoglycolate esters and 3'- phosphomonoesters. These modified 3'-termini block DNA synthesis if not identified by a class or direct acting enzymes called AP endonucleases that are capable of removing blocked 3'-termini, as well as initiating the repair of unmodified AP sites that arise spontaneously or as the product of DNA glycosylases. Since AP sites are potentially mutagenic, AP endonucleases are important enzymes to restore genetic integrity. The aims outlined in this proposal are intended to broaden our knowledge or abasic DNA repair in the genetically well-established eukaryotic organism Drosophila melanogaster. Three AP endonucleases in Drosophila will be studied, namely AP3, AP endonuclease I, and AP endonuclease fl. The cDNA encoding AP3 has already been cloned. AP3 is bound to both nuclear matrices as well as ribosomes. Its human homologue is ribosomal phosphoprotein PO, which is elevated in patients suffering from the autoimmune disease lupus. PO is also elevated in certain DNA-repair deficient human tumor cell lines, thus drawing a link between certain autoimmune diseases and DNA repair. The spectra of DNA damage recognized by this AP endonuclease will be evaluated using a number of different modified DNA substrates including lesions produced by ionizing radiation. The other two AP endonucleases will be cloned using either an antibody that specifically cross-reacts with these proteins, or oligonucleotides generated to specific regions of the purified proteins. Once cloned, products will be tested for biochemical activity similar to tests used for defining the lesions identified by AP3. All three cDNA's will be used to identify the genomic organization and upstream regulatory regions associated with these enzymes and allow us to examine how these genes respond to various environmental stimuli. Genetically, we will establish the chromosomal location of these genes through in situ hybridization, and from the knowledge gained from these studies target our search for mutants deficient for each of the AP endonucleases in Drosophila. Mutants identified will be subjected to various environmental agents to determine which of these proteins dominates for a particular repair pathway. Likewise, the rescue of E.coli mutants deficient for each of the AP endonucleases will provide important information on the major role each of these AP endonucleases play in the repair of DNA damaged by agents generating reactive oxygen species along with alkylation damage.

The Cellular and Molecular Biology Core (CMBC) will provide services to the CCEMH members using histology, immunohistochemistry (IHC) and mRNA in situ hybridization in their studies of transgenic models, as well as in the lines are being produced in the analysis of hematopoietic genes and diseases, as well as in the studies of gene therapy protocols, the ability to characterize the expression in situ, both for mRNA and protein, is becoming and more a necessity to correctly ascertain the function in these types of experimental paradigms. Furthermore, the number of these types of analyses is projected to increase rapidly, outstripping the ability of independent investigators to perform these analyses in a cost-effective or timely manner. A cohesive, efficient, and coordinated CMBC, will greatly decrease the cost and increase the reliability for these analyses to be performed for members of the CCEMH. Furthermore, as the efficiency of gene transfer improves in the future, a critical component in the evaluation of gene transfer protocols will be quantitation of transgene expression and assessment of biochemical endpoints. The Cell and Molecular Biology Core will not only assist a large number of investigators of the CCEMH working with transgenic and knock-out mice, but will also provide essential services to pre-clinical and clinical gene transfer research. The core will provide: 1) standardized processing, embedding and staining of tissues for histological analysis, 2) sectioning, embedding (paraffin or frozen) and IHC processing. Also, antibody characterization (Western blotting) and titer analysis will be performed for specificity and quality control of the antibodies used, 3) mRNA in situ analysis will be performed on frozen sections. Sense and anti-sense probes will be produced, characterized and quality controlled for each gene probe generated. Either 35S or non-radioactive probes will be used as per the investigators request, and 4) apoptosis assays on cell cytospins or tissues will be performed using the TUNEL assay on frozen or paraffin sections. Double labeling experiments can be performed using an antibody for specific gene product along with the TUNEL assay if required. Altogether, the core will provide accurate, efficient and cost-effective analysis of gene expression in vivo, with substantial savings compared to having each individuals investigator performing these analyses independently.

The recent observations that both DNA adducts and oxidative base damage are increased in the brains from Alzheimer s and Parkinson s patients and in spinal cord tissue of patients with amyotrophic lateral sclerosis (ALS) support the idea that oxidative DNA damage may contribute to the observed loss of neurons in these neurological disorders (8,9). Therefore, understanding how oxidative DNA damage is repaired (or not) is critical for understanding the pathology underlying these diseases. The overall hypothesis of this proposal is that oxidative stress induced DNA damage in neuronal cell lines leading to cellular malfunction and/or death and DNA repair enzymes are critical for the protection of neurons against oxidative stress. The following specific Aims will address this hypothesis. Specific Aim 1: Functional and biochemical characterization of the repair enzymes Ogg1, NTH, MTH, and MYH in the neuroblastoma cell lines SHSY5Y and Neuro-2A and in primary cultures of hippocampal neurons. We will measure the steady- state level of mRNA of the four repair enzymes, cytotoxocity, apoptosis, frequency of mutations, and the amount of 8-oxoG/FapyG in the absence and presence of the following agents: L-Dopa, dopamine, menadione, and H2O2. Experiments will also be performed in post-mitotic SHSY5Y cells. Specific Aim 2: Determine the effects of over-expressing these oxidative DNA repair genes on extotoxocity, apoptosis, frequency of mutations, and the amount of 8-oxoG/FapyG following exposure to L-Dopa, dopamine, menadione, or H2O2. Both mitotic and post-mitotic SHSY5Y cells and the Neuro-2A cells will be analyzed. Specific Aim 3: Determine the effects of antisence gene expression specific for the four oxidative DNA repair genes on cytotoxocity, apoptosis, frequency of mutations, and the amount of 8-oxoG/FapyG in the absence and presence of the following agents: L-Dopa, dopamine, menadione, and H2O2 in SHSY5Y mitotic and post-mitotic cells. If increasing the levels of DNA repair enzymes protects cells against oxidative stress, then this could provide a novel therapeutic basis for the treatment of neurological disorders such as ALS, aging, Alzheimer s and Parkinson s diseases.

DESCRIPTION (provided by applicant): Therapy for disseminated germ cell tumors (GCT) has been successful with 70-80% of patients being cured with front line chemotherapy. However, for those 20-30% of patients with extra-gonadal primaries or refractory disease, the response to therapy is poor with only 3-30% surviving disease-free after second line agents. One approach to treating resistant disease is the development of strategies to augment the chemotherapeutic agents that have been so successful in the majority of GCT patients. Little is known about the role of DNA repair systems in GCT's except that efficient repair appears to make tumor cells resistant to therapy. We have observed that GCT's express high levels of Ape1/ref-1 compared to normal tissues. Ape1/ref-1 is a multifunctional protein with DNA base excision repair (BER) activity and redox activity required for activation of specific transcription factors including Fos, Jun, NFkappaB, HIF-1alpha (hypoxia inducible factor), p53, and PAX5. This novel combination of functions links Ape1/ref-1 with resistance to many of the therapeutic agents (bleomycin, cisplatin, radiation, and VP-16) used to treat GCT's by acting as direct substrates for BER or indirectly by altering signaling through transcription factors regulated by Ape1/ref-l. Based on this information, we hypothesize: High level expression of Ape1/ref-1 in GCT's is a functional marker of disease which 1) is predictive of high risk disease and 2) can be manipulated to gain a therapeutic advantage. The major thrust of this proposal is to characterize the molecular biology of Ape1/ref-1 in GCT's as it relates to the clinical course of patients and the response of GCT cells to therapeutic agents. To approach this goal, we have developed four Specific Aims: Specific Aim1: Determine the relative expression of, Ape1/ref-1in good prognosis and high-risk GCT's. This takes advantage of the wealth of clinical GCT material available at Indiana University. Specific Aim 2: What is the role of Ape1/ref-1 in GCT progression and development, including cell growth, apoptosis, cell cycle, and differentiation? That is how does the high level expression of Ape1/ref-1, including over-expression of repair, redox, and nuclear localization domain mutants independently affect the ability of GCT cells to grow as cancer cells. Specific Aim 3: How do changes in the redox status of Ape1/ref-1 affect repair function? Using site-specific mutants, determine which cysteine residues specifically control repair function of Ape1/ref-l? Specific Aim 4: How do alterations in the repair and redox functioning of Ape1/ref-1 affect the response of GCT cells to therapeutic agents? Using what we learn in Aims 2 & 3, how can we alter the resistance of GCT cells to therapeutic agents. Through these analyses, we hope to determine the underlying mechanisms by which Ape1/ref-1 function is linked to the progression of testicular cancer. If any mutants are shown to sensitize the GCT cell lines to chemo-/IR agents, as expected, this will set the stage for future gene therapy approaches to sensitize GCT's to therapy.

DESCRIPTION (provided by applicant): The overall significance of this project relates to the ability to imbalance the DNA base excision repair (BER) pathway in ovarian tumor ceils, increasing their sensitivity to chemotherapeutic and ionizing radiation (IR) agents. We will attempt to accomplish this goal using mutants of the human apurinic/apyrimidinic endonuclease (APE1) enzyme, overexpression of N-methylpurine DNA glycosylase (MPG), both targeted to the nucleus and mitochondria, as well as small (short) interfering RNA (siRNA) for APE1. We will also utilize folic acid-derivatized liposomes and adenoviral targeting along with tumor specific promoter expression using the human telomerase reverste transcriptase (hTERT) promoter in both cell lines and an NOD/SCID animal model to develop the usefulness of this approach. Hypothesis: Overexpression of MPG in the nucleus and/or mitochondrial compartments, altered human APE1 proteins (dominant-negative), or siRNA for APE1 either independently, or in various combinations will enhance ovarian cancer cells to standard or decreased levels of commonly used chemotherapeutic agents (e.g. alkylators) and/or IR. The Specific Aims are: Specific Aim 1: This first aim includes determining the effectiveness of overexpressing MPG or dominant-negative APE1 in multiple ovarian cancer lines and evaluating tumor cell response to chemotherapeutic and IR treatment. This includes both nuclear and mitochondrial targeting of the MPG enzyme and overexpression and the knockdown of APE1 with siRNA. Specific Aim 2: Determine the effects of co-overexpression of nucMPG, mitoMPG and nuclMPG+mitoMPG, nucMPG+APE1 mutant, mitoMPG+APE1 mutant and nucMPG or mitoMPG and APEI-siRNA. We will monitor whether combined expression enhances the tumor cell killing effect of chemotherapeutic agents or IR. Specific Aim 3: Constructs using the hTERT promoter will be used in ovarian cancer cell lines in both plasmid (folic acid-derivatized liposome) and adenoviral based delivery systems for tumor specific expression studies using best candidate APE1 mutants, nuc- or mitoMPG, or siRNA as determined by the results in Aims 1-2. Specific Aim 4: Determine in vivo chemo- and radiosensitivity due to the expression of the various constructs of APE1 mutants, or nuc-/mitoMPG as well as APEI-siRNA in NOD/SCID mice. Adenoviral constructs with the hTERT promoter as well as folic acid-derivatized liposomes containing selected genes from the first three aims will be used with xenograft NOD/SCID mice. If successful, we feel these studies will create very effective reagents in a therapeutic gene transfer/therapy setting in the clinic, as well as shed light on the role of both nuclear and mitochondrial BER in cancer cells.

DESCRIPTION (provided by applicant): Numerous neurotoxic side effects of cancer treatments include cognitive dysfunction, commonly called chemobrain and peripheral neuropathy. The mechanisms for these side-effects and ways to protect neurons remain to be elucidated. The DNA base excision repair (BER) pathway including the abasic- endonuclease1/redox factor (Ape1/Ref-1 or Ape1) has been shown to be the major DNA repair pathway for oxidative and alkylating agent damage that occurs with chemotherapy and ionizing radiation (IR). Additionally, Ape1 interacts with a number of transcription factors, especially NF:B, AP1 and p53 to regulate their function through redox signaling. In neurons, these transcription factors mediate the expression of a number of proteins involved in neuronal survival and altered excitability in response to injury and inflammation. Thus, Ape1 could play a critical role in maintaining homeostasis in neuronal tissue through its DNA repair and/or its redox function. The overall hypothesis of the proposed work is that Ape1 acts to enhance neuronal survival and function after injury by chemotherapy or IR and helps to maintain normal neuronal function by minimizing alkylation and oxidative damage to DNA as well as by regulating the activity of AP1, NFkB and p53. We will determine if Ape1 is involved in the neurotoxicity and neuronal function associated with chemotherapy and IR using primary rat central nervous system (hippocampal) and sensory neuronal cells (dorsal root ganglia or DRG). We will reduce or augment Ape1 expression in these neurons under normal and following the addition of cancer chemotherapeutic agents and IR and ascertain the effects on various aspects of neuronal function and survival. We also will use Ape1 mutant proteins that only have either the redox or repair functions or drugs that inhibit only the repair or redox activity to ascertain which functions of Ape1 are critical for neuroprotection/function. Finally, we will determine whether the redox activity of Ape1 alters the activity of downstream stress response factors such as AP1, NFkB and p53 in neuronal cultures following chemotherapy and IR.Experiments in this application will form the basis for mechanistic studies into neurocognitive ("chemobrain") and peripheral neuropathy experienced by patients following chemotherapy and IR. Understanding the mechanism for neuroprotection during cancer therapy will be critical in providing patients with neuroprotection that can help alleviate these serious side effects.

DESCRIPTION (provided by applicant): The vast majority of people diagnosed with pancreatic cancer will undergo treatment that will ultimately fail or at best only extend their livs by ~6 - 10 weeks. Current standard-of-care consists of Gemcitabine with erlotinib, or surgery and radiation. Pancreatic tumors are especially resistant to therapy which is due at least in part to their hypoxic nature and fibrotic phenotype. Several molecular targets have been identified, however, investigation of the signaling pathways and molecular mechanisms that are major contributors to pancreatic tumor progression and its resistance to traditional therapies is lacking Thus, there is a critical need to identify novel targets in pancreatic cancer that offer the most promise for clinical utility against this dreaded disease. The long-term goal of this work is to understand the critical pathways for survival and metastasis of pancreatic ductal adenocarcinoma (PDAC) and develop a therapy that improves patient outcome by therapeutically modulating these critical pathways. The novel target, redox factor-1 (Ref-1), is a reduction-oxidation (redox) factor involved in the transcriptional regulation of gene expression. Transcriptional factors, HIF-1¿, NF?B, and AP-1 are regulated by Ref-1 and are implicated in pancreatic tumor growth and the response to hypoxia. The objective of this work is to determine the outcome of inhibiting the function of Ref-1 in PDAC as well as in the tumor microenvironment. Our data indicates that inhibition of Ref-1 in an orthotopic model reduces the number of metastatic lesions and growth of patient-derived ectopic xenografts. Furthermore, blocking the redox activity of Ref-1, using a selective inhibitor or a redox-dead Ref-1 mutant, inhibits the proliferation, migration, & adhesion of PDAC cell lines, decreases the transcriptional activation of HIF-1, NF?B, and AP-1, and interferes with stromal-induced tumor proliferation. Based on these results, the central hypothesis is that the redox function of Ref-1 is a critical regulator of pancreatic tumor growth and metastasis; therefore, inhibiting its function will interfere with hypoxia signaling pathways and markedly block pancreatic cancer progression. To address this hypothesis, three aims are proposed. Aim 1:determine the biological effects of modulating Ref-1's redox activity in tumor cells and in stromal fibroblasts; Aims 2 & 3: utilize an orthotopic mouse model of pancreatic cancer (Aim 2) and a genetic mouse model of pancreatic cancer (Aim 3) to determine the efficacy of blocking the redox function of Ref-1 alone and in combination with Gemcitabine. Upon successful completion of this project, we will establish the effects of Ref-1 inhibition on the pancreatic tumor growth and metastasis as well as how Ref-1 modulation affects hypoxia signaling pathways and pancreatic cancer progression. This new knowledge is expected to result in a fresh strategy to knock out multiple survival mechanisms within the heterogeneous milieu of pancreatic tumors. Moreover, evaluation of the effects of Ref-1 inhibition on tumor growth and proliferation, apoptosis, metastasis, and response to hypoxia will comprise a thorough assessment of the potential for Ref-1 inhibition to yield new approaches to treat PDAC.

? DESCRIPTION (provided by applicant): Although inflammation-induced peripheral sensitization (i.e. increased sensitivity of sensory neurons) can resolve as an injury heals, under pathological conditions this sensitization is maintained and contributes to chronic inflammatory pain. Studies of the cellular mechanisms mediating this maintenance of peripheral sensitization have focused on transcriptional changes that alter protein expression or post-translational modulation of various proteins, especially ion channels. To date, however, these studies have not resulted in new therapeutic approaches for treating chronic inflammatory pain. For this R21 application, we propose a novel mechanism for maintaining sensitization of sensory neurons, i.e. inflammation-induced DNA damage. This damage could result in an alteration in the phenotype of neurons from normal to the sensitized state. Recent studies performed in our laboratory provide support for examining this mechanism, since we have shown that augmenting DNA repair mechanism reverses toxicity in sensory neurons induced by cancer therapies. Furthermore, our preliminary data suggest that inflammation and the inflammatory mediators LPS, MCP-1, and, PGE2, can produce DNA damage in sensory neurons. Thus, we hypothesize that inflammation and inflammatory mediators produce oxidative DNA damage in sensory neurons that contributes to hypersensitivity and that augmenting the base excision repair pathway protects neurons from this damage and thus attenuates the enhanced excitability. To test this hypothesis we propose two specific aims. In studies for the first aim, w will determine whether CFA-induced inflammation or long-term exposure to inflammatory mediators (LPS, MCP-1 or PGE2) in isolated sensory neurons produces reactive oxygen species (ROS) and DNA damage in sensory neurons. We also will determine whether antioxidants or increasing APE1 repair activity (by overexpressing it in sensory neurons) prevents or reverses the DNA damage. In aim 2, we will determine whether augmenting APE1 activity with overexpression in sensory neurons prevents or reverses peripheral sensitization induced by CFA injection into the rat hindpaw or by long-term exposure to inflammatory mediators (LPS, MCP-1 or PGE2) in isolated sensory neurons. If we demonstrate that DNA repair reverses peripheral sensitization that occurs during inflammation, our findings have important implications for elucidating a novel therapeutic target for treating chronic pain.

PROJECT SUMMARY / ABSTRACT As cancer treatments continue to become more effective with increases in patient survival, we are recognizing clinical consequences of therapy that negatively impact the course of therapy and the quality of life of patients and survivors. Of major clinical significance is chemotherapy-induced peripheral neuropathy (CIPN), which can be severe enough to necessitate reducing or stopping treatment and thus can compromise therapy. Furthermore, CIPN can continue long after therapy is stopped and is irreversible in a significant number of patients. Compounding this problem is a lack of effective treatments available to prevent or reverse CIPN. The lack of effective prevention or treatment for CIPN is a direct consequence of not understanding the mechanisms that cause the neurotoxicity. As such, examining the provocative question of ?What are the molecular and/or cellular mechanisms that underlie the development of cancer therapy-induced severe adverse sequelae?? will be addressed in our studies using animal models and an array of endpoints measuring changes in sensory neuronal function which parallel clinical symptoms of CIPN. Most CIPN develops over time with few if any acute symptoms after initial therapy, but increases in severity with continued therapy. The delay in onset of neuropathy suggests that there is an aggregate effect of drugs over time that results in a long-term alteration in neuronal function. Consequently, it is important to examine the mechanisms by which cumulative exposure to chemotherapeutics might result in neurotoxicity. Previously, we demonstrated that reducing the activity of the DNA base excision repair (BER) pathway by reducing expression of the apurinic/apyrimidinic endonuclease/redox factor (APE1/Ref-1 or APE1) exacerbated neurotoxicity produced by anticancer treatment, whereas augmenting the repair activity of APE1 attenuated the neurotoxicity. These data support the notion that DNA damage is a critical mechanism by which the function of sensory neurons is altered by chemotherapeutics. Indeed, it is likely that in post-mitotic cells (e.g. neurons) DNA damage could result in abnormal protein production that is maintained unless the DNA damage is repaired, reversing the aberrant transcriptional effects of the neurotoxins. Therefore, we hypothesize that APE1 is a critical protein for protecting neurons from cancer therapies and that augmenting APE1 DNA repair activity will prevent and reverse chemotherapy-induced alterations in sensory neuronal function. Furthermore, fully understanding the DNA damage and the mechanisms by which the BER pathway reverses this damage will lead to the identification of novel targets for CIPN prevention or therapy. To address these hypotheses, we propose three aims which will determine whether augmenting APE1 repair activity in vivo prevents or reverses DNA damage in sensory neurons and the subsequent alterations in sensory neuronal function caused by anticancer drug administration as well as determining the mechanisms mediating APE1-induced neuroprotection of isolated sensory neurons.

PROJECT SUMMARY/ABSTRACT Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect of many efficacious anticancer drugs, including platinum drugs, taxanes, proteasome inhibitors, vinca alkaloids, epothilones, and immunomodulators. Their neurotoxic side effects can be so debilitating that treatment may need to be reduced or stopped. However, unlike other major side effects of chemotherapy (e.g. nausea, hair loss, bone marrow failure), no standard, effective treatments exist to prevent or reverse CIPN. This is largely because the cellular mechanisms for CIPN have not been identified and the symptoms of CIPN including numbness, decreased blood flow to extremities, loss of proprioception, loss of tendon reflexes, pain, allodynia, and/or increased sensitivity to cold vary greatly in patients. Because CIPN is debilitating and may be irreversible, identification of key targets to prevent neurotoxicity without compromising the tumor-killing effects of anticancer drugs is critical in developing a first-in- class therapeutic that can directly affect a patient's ability to receive optimal treatment. Our previous studies examining the hypothesis that DNA damage of sensory neurons contributes to CIPN laid the foundation for the proposed work, which is poised to develop a drug candidate. We demonstrated that reducing DNA base excision repair (BER) activity by reducing expression of the apurinic/apyrimidinic endonuclease/redox factor (APE1) augmented the neurotoxicity produced by anticancer treatment, whereas supplementing APE1's repair activity attenuated the neurotoxicity. It is likely that, in non-dividing cells like neurons, DNA damage could alter the function of sensory neurons in ways that manifest as the symptoms observed in CIPN. Consequently, DNA repair would be critical for proper genetic expression of the right types and amounts of proteins, a crucial element of genomic maintenance. For the proposed studies, we will examine whether augmenting APE1 repair activity in vivo will prevent chemotherapy-induced alterations in sensory neuronal function (manifested as CIPN) without jeopardizing the cancer treatment. Using tumor bearing mice, we will examine whether a small molecule (E3330) which was identified to enhance APE1's DNA repair function in neurons can prevent (aim 1) or reverse (aim 2) DNA damage and alterations in the function of sensory neurons caused by cisplatin, oxaliplatin or carboplatin. Furthermore, we will examine whether the small molecule (E3330) will compromise the anticancer efficacy of the platinum drugs by examining DNA damage and tumor survival following treatment (aim 3). Because E3330 has been found to act as a single agent and in combination with other cancer therapeutic drugs to decrease tumor cell growth, this molecule has the potential to offer a ?win-win? scenario; block tumor cell growth while protecting against neuronal dysfunction. Additionally, E3330 will enter a phase 1 clinical trial for solid tumors followed by phase 1b/phase 2 trials for various indications that include platinums in their SOC (e.g. colon, pancreatic). Therefore, it requires further preclinical study using an in vivo paradigm to demonstrate effectiveness in the context of neuronal protection and CIPN models.

Pancreatic ductal adenocarcinoma or PDAC is a lethal disease with a 5 year mortality rate of ~93% and little improvement has been made despite the emergence of several targeted, selective agents. Therefore, a new approach is needed. We will pursue transcriptomic-guided combination therapy as well as targets that are upstream of several signaling pathways, thereby affecting multiple downstream cellular processes and potential resistance mechanisms. Redox factor-1 (Ref-1) is one such protein, as Ref-1 regulates multiple transcriptional factors (TFs) that are critical to pancreatic cancer survival and drug resistance. In the previous funding period, we have advanced APX3330, a Ref-1 inhibitor and the first drug targeting Ref-1 to cancer clinical trials (IND 125360) as a novel, oral, first-in-class drug in humans. We have shown that APX3330 reduces tumor growth in several models of PDAC as a single agent and potentiates gemcitabine-mediated inhibition of cell growth. The mechanism of action of APX3330 has been extensively investigated and characterized by our team. Through inhibition of Ref-1, the activity of STAT3, AP-1, NFkB, and HIF-1? can be blocked leading to a decrease in survival protein expression and response to hypoxia. Recognizing that combination therapy will be necessary in PDAC, we propose to utilize transcriptomic data to identify FDA-approved agents that are likely to synergize with APX3330. Drug synthetic lethality is defined as combination therapy of molecular targets whose dual inhibition leads to potentiation of cell death much more dramatically than when administered as single agents. Single cell RNA-seq data identified HIF-1 signaling pathways as significantly down-regulated following Ref-1 knockdown (p=0.0008). Therefore, we tested the combination of Ref-1 inhibition and HIF-1 target, carbonic anhydrase (CA9) in our 3-Dimensional (3D) tumor co-culture model. Dramatic enhancement of Ref-1-induced cell killing is observed upon dual-targeting of Ref-1 and CA9. Our hypothesis is that in order to extend the survival of PDAC patients multi-targeted, combination therapy is essential; therefore we will use original, pathway-driven screening approaches to discover appropriate FDA approved agents to partner with our Ref-1 inhibitor. AIM 1- Evaluate the mechanism and efficacy of simultaneous inhibition of the Ref-1 and HIF-1? pathways using in vivo models of PDAC. AIM 2- Investigate the role of Ref-1 in sensitizing PDAC to chemotherapy currently used in PDAC treatment. Gemcitabine (Gem), one of the agents that single cell RNA-seq expression profiling predicted should work with APX3330, will be used in combination with APX3330 in the phase 1B trial. AIM 3- Screen for drug synthetic lethal hits following Ref-1 inhibition in a validated 3D model system utilizing computational and transcriptomics pathway analysis. Selective disruption of individual molecular effectors has clear limitations; our approach focuses on multi-targeted combination treatments. Thus, this project has the potential to extend pancreatic cancer survival by using appropriate disease-relevant models such as 3D co-culture spheroids, orthotopic, and GEM models.

ABSTRACT, Developmental Funds Developmental funds are a critical component of the budget used to invest in new innovations, valuable shared resources, and key areas of research. In the coming project period, the IUSCC will use developmental funds to address the following aims: 1: To identify the most promising and innovative pilot projects in cancer research. Cultivate and catalyze new research collaborations and teams around the strengths of our center through continued investment in our pilot funding program. Priority is given to the most innovative projects that could lead to future NCI-sponsored research that leads to reduced incidence and mortality rates or increased quality of life for cancer patients in Indiana and beyond; 2: To facilitate collaborative research projects and how they are conducted to maximize their success. All award recipients are mentored and monitored through the novel IUSCC Translational Research Acceleration Collaboration (ITRAC) Project Management Process. Special emphasis is made to optimize use of IUSCC Cores and to encourage collaborations towards multi-PI grant proposals; 3: To conduct rigorous monitoring and evaluation of all projects. Once approved for funding, all projects are monitored and evaluated using the ITRAC process of IUSCC. This process has been used for monitoring and evaluating all IUSCC Pilot Funded Projects since 2006. Written progress reports are submitted to the IUSCC leadership council (LC) and executive committee (EC) for review of progress. In addition, the EAB and IAB reviews the progress at their annual meetings; and 4: To develop the Translational Research Core (TRC). The TRC has been developed to assist investigators, both clinical and basic scientists, with developing and performing in-house drug development of Phase I clinical trials. The TRC also performs correlative biological assays needed to validate mechanism(s) of action of candidate drugs/therapies and to develop and test new hypotheses.

The neovascular eye diseases proliferative diabetic retinopathy and wet age-related macular degeneration are major causes of blindness through the lifespan. There is thus a critical need to find novel cellular components that could be targeted to block ocular neovascularization. The protein Ref-1 is one such component, responsible for activating redox-dependent transcription factors important for angiogenesis, and overexpressed in neovascularization. Inhibition of Ref-1?s redox function with novel small molecules blocks proliferation of ocular endothelial cells in vitro and in vivo in the laser-induced choroidal neovascularization (L-CNV) model. Ref-1 inhibition reduces signaling through hypoxia and inflammation pathways, and preliminary data reveals a novel connection to Wnt signaling. The long-term goal is to elucidate the role of Ref-1 in ocular neovascularization, and develop novel therapies targeting this protein or its pathway(s). The rationale for this research is that Ref-1 is a significant mediator of angiogenesis and inflammation, a target of multiple antiangiogenic small molecules, and a regulator of key angiogenesis factors including hypoxia-inducible factor 1? and NF-?B. The objectives in this application are to determine how Ref-1 functions as a regulator of angiogenesis and to develop new agents targeting this enzyme. The overall hypothesis is that Ref-1 activity is required for ocular angiogenesis and inflammation and that reducing the activity of Ref-1 will prevent ocular angiogenesis. Guided by exciting preliminary data, the hypothesis will be tested via two specific aims: Aim 1. Determine the Ref-1-modulated signaling pathway(s) that are key to angiogenesis and inflammation. The expression of Ref-1 in neovascularization will be assessed, and the expression and function of downstream targets (including newly identified Wnt pathway components) will be analyzed after knockdown and inhibition of this protein in endothelial cells with or without overexpression of functional mutants. Angiogenic activity will also be assessed. Aim 2. Optimize the preclinical profile of Ref-1 inhibitors in vitro and in vivo. Two novel, highly potent Ref-1 small molecule inhibitors will be explored for efficacy in vitro, and in cell and in multiple in vivo models of neovascularization, including synergy with anti-vascular endothelial growth factor therapy, target engagement and off-target effects, effects on Ref-1 target genes, and toxicity. This work is innovative, as it is the first in-depth mechanistic study of the role of Ref-1 in ocular angiogenesis, exploring this unique signaling node as an integrator of proangiogenic, proinflammatory, and newly identified Wnt signals. It will also reveal new signaling pathways relevant to angiogenesis and inflammation in the eye, and novel therapeutic leads for neovascular eye diseases. The work is highly significant because it will define Ref-1 as an ocular angiogenic mediator and determine its downstream effects, leading to development of new ways to prevent blindness. Additionally, outcomes from these studies will be the advancement of novel, anti-Ref-1 small molecule inhibitors for translation from the bench to the clinic and patient care.