Anil K. Sood - US grants

This study will evaluate rectal function in women prior to and six months after radical hysterectomy and may lead to more specific and effective treatment for patients with functional bowel abnormalities after radical hysterectomy.

DESCRIPTION (provided by applicant): Abstract In reference to NOT-OD-09-058 (Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications), we submit this Competitive Revision to the parent grant entitled "Tumor metastasis: Biobehavioral mechanisms (R01CA110793)." Psychosocial stress can elicit alterations of immunological, neurochemical, and endocrinological functions. To date, most of the research dealing with stress and tumor growth has focused on suppressed immunity;however, progressive growth of cancer is influenced by many other factors including tumor cell migration and invasion. There has been little investigation of the effect of stress hormones such as norepinephrine and epinephrine on these key processes that are required for metastasis. Focal adhesion kinase (FAK) is one of the key factors involved in tumor cell migration and invasion. We have compelling preliminary data that one of the stress hormones, norepinephrine, can directly activate FAK and promote tumor growth and these effects can be blocked by using inhibitors of beta-adrenergic receptors. Furthermore, our preliminary data suggest that FAK plays a key role in ovarian cancer migration and invasion. The parent grant has determined the underlying mechanisms and pathways by which stress hormones can affect ovarian cancer migration and invasion using in vitro assays and we have experimentally identified the in vivo effects of chronic stress on ovarian cancer progression using a well-characterized mouse model of ovarian carcinoma and are examining the associations between psychosocial factors and FAK in human ovarian cancers. During this work, we have made novel observations that chronic stress and associated increases in catecholamines protect tumor cells from undergoing anoikis, but the exact mechanisms are not well known. In the present supplement, we seek to determine whether chronic stress and associated neuroendocrine dynamics could protect ovarian cancer cells from anoikis, and to identify the underlying signaling pathways. The proposed supplement will advance the goals and objectives of the parent grant by allowing us to examine in vitro mechanisms of norepinephrine mediated protective effects against anoikis and determine in vivo effects of stress on protection of tumor cells in ascites against apoptosis (in vivo reflection of anoikis avoidance by tumor cells). Findings of this study could lead to identification of a completely new mechanism by which chronic stress affects tumor cell survival and growth, and therefore may lead to new behavioral and pharmacological therapeutic approaches. PUBLIC HEALTH RELEVANCE: Project narrative FAK is known to promote tumor cell survival and may play a significant role in avoidance of anoikis. We have previously demonstrated that catecholamines promote ovarian carcinoma growth via stimulation of angiogenic pathways. However, the mechanism by which invading tumor cells survive anoikis remains largely unknown. The proposed work here will advance the goals and objectives of the parent grant by allowing us to examine in vitro mechanisms of norepinephrine mediated protective effects against anoikis and determine in vivo effects of stress on FAK and Src and protection of cells in ascites against apoptosis (in vivo reflection of anoikis avoidance by tumor cells).

DESCRIPTION (provided by applicant): Ovarian cancer is the leading cause of death among women with gynecologic malignancies. Due to poor survival of women with epithelial ovarian cancer, identification of factors responsible for accelerated cancer growth may lead to development of novel therapeutic approaches. Stress can elicit alterations of immunological, neurochemical, and endocrinological functions. To date, most of the research dealing with stress and tumor growth has focused on suppressed immunity; however, progressive growth of cancer is influenced by many other factors including blood supply to growing tumors (angiogenesis). There has been little investigation of the effect of stress mediators such as norepinephrine and epinephrine on angiogenesis. Vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) are key factors that can stimulate tumor angiogenesis. We have compelling preliminary data that catecholamines can directly increase the production of pro-angiogenic cytokines such as VEGF and IL-8 by ovarian cancer cells and this effect can be blocked by using inhibitors of beta-adrenergic receptors. This project is designed to examine the effects of stress related mediators (norepinephrine, epinephrine, and cortisol) on secretion of pro-angiogenic cytokines by ovarian cancer cells and their resultant effects on cancer progression. We have designed a series of experiments that will determine the underlying mechanisms and pathways by which stress mediators can achieve these effects. We will also determine the in vivo effects of stress mediators on ovarian cancer growth and angiogenesis, and the effect of specific methods to block these deleterious effects using an in vivo orthotopic mouse model of ovarian cancer metastasis. Findings of this study could lead to identification of novel mechanisms underlying accelerated ovarian cancer growth and therefore may lead to new therapeutic approaches.

The progressive growth of primary ovarian cancer and metastasis is dependent on development of an adequate blood supply (angiogenesis). Vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis and consequent ovarian cancer growth and progression. VEGF blockade has shown promise in human studies. The overall goal of this proposal is to develop new strategies for targeting blood vessels in ovarian cancers. Our pre-clinical results demonstrate that a novel approach (high-affinity VEGF decoy receptor, VEGF-Trap) for VEGF blockade is highly effective in combination with taxane chemotherapy. Recent evidence suggests that VEGF-Trap may be more potent than many other existing modalities for targeting VEGF. Based on these encouraging preclinical results, we will conduct a Phase I clinical trial of VEGF-Trap and docetaxel chemotherapy in patients with recurrent ovarian carcinoma in Aim 1. In addition to endothelial cells, blood vessels consist of perivascular cells such as pericytes, which are mesenchymal cells that wrap around the vessel tube. Several functions of pericytes relevant to angiogenesis have been proposed including effects on endothelial survival, deposition of matrix, and maintenance of vessel integrity. Platelet-derived growth factor receptor (3 (PDGF-Rp) signaling is known to play a functionally significant role in pericyte development and recruitment by endothelial cells. Aim 2 will examine the mechanisms by which pericytes provide a survival advantage for endothelial cells and assess the efficacy of combinatorial approaches for targeting both endothelial cells (VEGF-blockers) and pericytes (PDGF-blockers). Most chemotherapeutic agents are traditionally administered at maximum tolerated doses. However, recently, metronomic chemotherapy (frequent administration of chemotherapeutic agents at substantially lower doses with no prolonged drug-free breaks) has been utilized for targeting endothelial cells of the growing vasculature of a tumor. Our preliminary data suggest that metronomic chemotherapy alone and in combination with other anti-vascular approaches is highly effective. Aim 3 will determine the efficacy of metronomic chemotherapy in combination with VEGF and PDGF blockers. Thus, all three Aims are complementary to each other and

DESCRIPTION (provided by applicant): The overall goal of this initiative is to investigate the cellular uptake, trafficking, and sub-cellular localization of different classes and subtypes of nanoparticles (NPs) with well-defined physiochemical properties for the creation of a reference table that relates the sub-cellular distribution of NPs to their intrinsic physiochemical properties across a range of cell lines. The subcellular fate of NPs is relevant both in terms of the therapeutic efficacy and biosafety of the NPs. The effective impact of size, shape, charge, and chemical composition of nanomaterials, in the presence of serum opsonins, on both cellular entry and subsequent subcellular localization will be investigated. The expected outcome of this project is to create a reference table that accelerates the transition of nanomaterials from the bench to the clinic by rapidly expanding our knowledge of the effect of a material's intrinsic characteristics on its intracellular destination. The final product, a comprehensive table of NPs and their subcellular locations, will guide the future development of NP drug delivery systems for rapid expansion of biomedical applications, including cancer therapy, cardiovascular imaging, and gene therapy. PUBLIC HEALTH RELEVANCE: What this project seeks to deliver is a multi-dimensional reference table that relates the subcellular distribution and toxicity of NPs to their intrinsic physiochemical properties across a range of diverse cells and cell lines. It is our hope that the data generated from this project will serve as a resource for future research and encourage model development and new insights into nanotechnologies for imaging and drug delivery.

PROJECT SUMMARY: Ovarian cancer remains the most deadly malignancy. Targeting angiogenesis is a particulariy attractive strategy because of the presumed genetic stability of endothelial cells. This is best illustrated by recent successes of anti-angiogenic therapy (e.g., bevacizumab) in patients with solid tumors. However, despite initial responses, most patients eventually develop tumor progression resulting in their demise. Therefore, new anti-angiogenesis therapeutic strategies are needed. The overall goal of this project is to develop novel nanoparticle-based strategies to target the tumor vasculature specifically. We propose to utilize two types of biocompatible therapeutic nanoparticles (chitosan and gold nanoshell nanoparticles) for the delivery of therapeutic payloads (e.g., siRNA) or near-infrared (NIR) laser mediated thermal ablation. These platforms are supported by Integrated approaches for selective delivery into the tumor vasculature using either rationally designed multi-stage carriers or surface ligands (thioaptamers) selected from screening libraries based on selective binding. Using genomic approaches, we have identified novel candidate target genes in ovarian cancer vasculature that will be targeted using RNAi approaches (Aim 1) because many are difficult to inhibit with small molecules or monoclonal antibodies. In our preliminary findings, we have identified thiophosphate oligonucleotide aptamers (thio-aptamers) that selectively bind to tumor, but not to normal endothelial cells based on counter selection strategies using freshly isolated endothelial cells from human ovarian cancer or normal ovaries. In Aim 1, we will develop thioaptamertargeted nanoparticles for selective delivery of therapeutic siRNA. On the basis of our preliminary findings regarding the critical role of size and shape in vascular localization of nanoparticles, we will pursue rational design of nanoparticles for targeting the tumor vasculature in Aim 2. Gold-based nanoshells offer unique opportunities for thermal ablation using NIR light. In Aim 3, we will develop and characterize novel approaches for thermal ablation of ovarian cancer vasculature using targeted gold nanoshells. All three aims are complementary to each other and findings of this study should allow the design and translation of new therapeutic approaches for women with ovarian cancer.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recent findings from The Sood laboratory indicate that platelets may play an important role in ovarian cancer growth and metastasis. We hypothesize that platelet ultra-structural changes reflect alterations in platelet function that facilitate disease progression in ovarian cancer patients. Thus, in order to gain better understanding of ultra-structural changes that may denote platelet pathobiology in ovarian cancer, we propose comparing platelets isolated from the peripheral blood of healthy donors to those from newly diagnosed ovarian cancer patients at the ultrastructural level using electron cryomicroscopy. Platelet electron microscopy is now used clinically to help determine if patients have an intrinsic platelet abnormality related to bleeding risk. The inherent advantages of electron cryomicroscopy make it preferable for the current investigation because cryo-EM would afford improved sample preservation.

PROJECT SUMMARY (See instructions): Despite refinements in surgery, radiation and chemotherapy, tlie mortality rates of women with advanced uterine carcinoma have remained largely unchanged for decades. Recognition of this therapeutic plateau has focused intense investigation into strategies targeting mechanisms of tumor growth and progression. Our preliminary studies have identified a novel therapeutic target, EphA2, which is overexpressed in a substantial proportion of uterine cancers, is associated with poor overall survival, and mechanistically regulates angiogenesis. While present in tumor and tumor-associated vasculature, it is low or absent in most normal tissues, making its differential presence and function an attractive therapeutic target. The overall goal of this proposal is to develop EphA2 targeted therapeutic strategies for uterine carcinoma and to characterize its biological functions in regulating uterine cancer growth and progression. We will first leverage the differential expression of EphA2 in tissue as a targeting beacon to deliver a novel molecular immunoconjugate, monomethylauristatin F (MMAF; MEDI-547). This dolastatin-analogue is attached to a monoclonal antibody, which selectively binds EphA2. Our preliminary data demonstrate that MEDI-547 inhibits tumor growth and metastasis in orthotopic animal models of uterine cancer. However, it is not known whether EphA2 expression on the tumor cells is required for MEDI-547 to be efficacious. This question will be addressed experimentally in Aim 1. The findings from Aim 1 will guide a Phase Ib clinical trial in Aim 2 that will examine the safety, toxicity, and efficacy of the MEDI-547 in patients with recurrent uterine carcinoma. In addition to targeting EphA2 for delivery of a cytotoxic agent, our preliminary data suggest that EphA2 silencing can directly affect tumor and endotiielial cell functions. These effects may be mediated via reduced activation of downstream non-receptor kinases such as focal adhesion kinase (FAK). However, the mechanisms by which EphA2 regulates tumor growth are not fully understood and will be further examined in Aim 3. In this Aim, we will also examine the therapeutic efficacy of EphA2 gene silencing. To achieve efficient systemic in vivo delivery of short interfering RNA (siRNA) for gene silencing, we have developed and characterized biocompatible nanoparticle-based delivery methods that will be utilized for the experiments proposed in Aim 3.

PROJECT SUK/IH/IARY (See instructions): The progressive growth of primary ovarian cancer and metastasis is dependent on development of an adequate blood supply (angiogenesis). Vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis and consequent ovarian cancer growrth and progression. VEGF blockade has shown promise in human studies. Our pre-clinical and clinical results from the prior funding period demonstrate that a novel approach (high-affinity VEGF decoy receptor, VEGF-Trap or aflibercept) for VEGF blockade was highly effective in combination with taxane chemotherapy. However, despite initial responses, most patients eventually develop tumor progression resulting in their demise, mainly due to the development of drug resistance. The DII4 (delta-like ligand 4)/Notch signaling pathway has recently been shown to play an important role in angiogenesis including vessel maturation, pericyte recruitment, branching and cell differentiation, proliferation, survival and apoptosis. Our preliminary data indicate that when DII4 inhibition using a monoclonal antibody (DII4-mAb or REGN421) was coupled with VEGF inhibition (aflibercept), this combination strikingly reduced tumor burden and ascites, suggesting that this anti-angiogenesis regimen holds promise as a novel therapeutic modality. However, the mechanisms of its potency are not fully understood. We hypothesize that the DII4/Notch signaling pathway plays an important role in reducing the efficacy of anti-VEGF monotherapy and targeting both VEGF and DII4/Notch signaling pathway will enhance anti-angiogenic therapy. The overall goal of this proposal is to develop novel and effective strategies for targeting ovarian cancer angiogenesis. In the current proposal, we will investigate the functional significance of DII4/Notch signaling pathway in ovarian cancer by examining whether DII4 expression in ovarian cancer cells activates Notch signaling in endothelial cells and whether blockade of DII4/Notch signaling with REGN421 deregulates angiogenesis in vitro in Aim 1. In Aim 2, we will assess the efficacy of combinatorial approaches for targeting VEGFA/EGFR/DII4/Notch signaling pathway with aflibercept and REGN421 using in vivo orthotopic ovarian cancer models. We will conduct a Phase I/lb clinical trial using the anti-DII4 monoclonal antibody REGN421 in patients with recurrent ovarian carcinoma in Aim 3. Thus, all three Aims are complementary to each other and findings of this study should allow the design of new therapeutic approaches for women with ovarian cancer.

The University of Texas Health Science Center at Houston (UTHSC-H), The University of Texas M.D. Anderson Cancer Center, Rice University and Albert Einstein College of Medicine have joined forces to form the Texas Center for Cancer Nanomedicine (TCCN). The TCCN brings together a multi-disciplinary, internationally recognized team of investigators to develop and translate nanotechnology-enabled innovation for improving the traditionally dismal outcome of ovarian and pancreatic cancers. The main research focus areas of the TCCN are: Multifunctional Nano-Therapeutics and Post-Therapy Monitoring Tools (Area 2 of the CCNE RFA), and Devices and Techniques for Cancer Prevention and Control (Area 3). By natural synergies of the underlying nano-platforms, the TCCN's investigations in focus areas 2 and 3 automatically provide a cadre of approaches for Area 1: Early Diagnosis Using In-Vitro Assays and Devices and In-Vivo Imaging Techniques. The TCCN has four projects and three cores. Projects 1 and 2 directly address ovarian cancer, and Projects 3 and 4 directly address pancreatic cancer. In each oncology focus area, one project involves multifunctional nanoplatforms for the delivery of bioactive agents to the tumors (Project 1- ovarian and Project 3- pancreatic), and the other, targeting approaches to the cancer-associated vascular endothelia (Project 2- ovarian and Project 4- pancreatic), for imaging and therapy. Both adenocarcinoma (Project 3) and endocrine pancreatic malignancies (Project 4) are considered in the TCCN. All Projects integrate fundamental investigations in cancer biology, nanotechnology platform development, and pharmaceutical sciences, albeit to different degrees. The cores are the Biomathematics Core, Targeting Core and Nanoengineering Core. All projects and Cores integrate with each other through the sharing of research results and nanotechnology platforms. This integration allows the TCCN to achieve clinical translation of its research breakthroughs, and aggressively manage the risks that are naturally associated with any highly innovative program at a rapid pace. To fuel translation to the clinic, several TCCN investigators have successfully developed spin-off companies based upon their research. Collectively, with a combination of synergistic projects supported by cores that provide services to each project and a track record of successful bench-to-bedside translation, the TCCN is uniquely positioned to bring forth highly effective nanotechnology platforms for prevention, therapy and monitoring of ovarian and pancreatic cancers. Public Health Relevance: The TCCN aims to utilize innovative nanotechnologies for new therapeutic strategies, methodologies for reliable monitoring of therapeutic efficacy, early detection approaches from biological fluids and advances in imaging, and cancer-prevention protocols for ovarian and pancreatic cancers. The TCCN will apply a diverse array of nano-platforms to achieve these aims. While the primary emphasis In the TCCN is on ovarian and pancreatic cancers, it is likely that the new approaches will have applications for many other malignancies.

DESCRIPTION (provided by applicant): To address the national shortage of academic gynecologic oncologists, the Department of Gynecologic Oncology at The University of M. D. Anderson Cancer Center has dedicated its T32 training program to producing outstanding academic Gynecologic Oncologists-both clinician investigators and physician-scientists. Our T32 trainees develop the research background skills necessary to be productive investigators. Moreover, we are committed to providing T32 training to groups that are underrepresented in academic gynecologic oncology, including minority individuals and women. Our program provides opportunities in a broad range of research training disciplines including, but not limited to, cancer biology, molecular therapeutics, tumor immunology, health disparities, epidemiology, and health services research. This 4-year program includes 24 months of research training followed by 24 months of clinical training. Two types of research training are offered: 1) a physician-scientist track in which trainees spend 24 months in the laboratory, and 2) a population-based research track in which trainees spend 24 months under the direction of one or two of the faculty mentors; the latter track may include one year of laboratory-based research followed by a year of population-based research or two years of population-based research. Additionally, during the research years, each fellow is required to complete a core of three courses and earn an advanced degree- either an M.S. degree or an M.P.H. degree-in the Graduate School of Biomedical Sciences or the UT School of Public Health, respectively. This versatility in academic training increases the likelihood of obtaining an academic position upon completion of training. The mentors in our program-physician-scientists and research scientists with active research programs supported by external peer-reviewed funding-are committed to a high quality research experience focused on gynecologic cancers. The University of Texas M. D. Anderson Cancer Center vigorously promulgates a core mission of excellence in research, education, and patient care. As a result, this is a highly stimulating environment in which aspiring academic gynecologic oncologists can receive research training under the aegis of the T32 program.

ABSTRACT Hyperthermia induced by magnetic nanoparticles in high frequency alternating magnetic fields (AMF), or Magnetic Fluid Hyperthermia (MFH), is based on the delivery of thermal energy at the nano-scale to tumors using iron oxide based magnetic nanoparticles (MNP) and an externally applied AMF. This phenomenon is the result of particle rotation or movement of the magnetic dipole. The fact that energy is only dissipated under high-frequency and moderate amplitude fields that can be constrained to the tumor region make MFH a highly promising form of non-invasive, externally activated cancer treatment To this date, the prevailing paradigm in the field is that delivery of nanoscale particles to tumors can be achieved by passive targeting due to the enhanced permeation and retention (EPR) effect. However, in vivo experiments tumors suggest otherwise, thus, posing potential limitations on the successful translation of such systems to the clinic. The aforementioned discrepancy reveals a need to understand the in vivo spatial and temporal behavior of nanoparticles as a result of their surface physicochemical properties. To our knowledge, the relationship between surface properties and the resulting temporal and spatial behavior has not been investigated in orthotopic mouse models of cancer. T The long-term goal of this project is to develop MNPs as a clinically feasible tool by providing a comprehensive understanding from fundamental particle design to clinical application. The main objective of this proposal is to pursue the optimization ofthe spatial and temporal behavior of Magnetic Nanoparticle Heaters (MNH) and perform an in vivo efficacy assessment of targeted or

DESCRIPTION (provided by applicant): RNA molecules are secreted in extracellular spaces (exRNAs) and act as endocrine signals altering the phenotypes of cancer cells. In this application we will focus on the class of small non-codingRNAs named microRNAs. We propose an innovative therapeutic concept: the depletion of oncogenic microRNAs from malignant cells by enhancing their secretion as exRNAs or blocking suppressor microRNAs' delivery in extracellular space by blocking secretion mechanisms. Our main goal is to develop new strategies using combined miRNA and siRNA to maximize therapeutic benefit by affecting the production of exRNAs. Our proposal centers on the identification and targeting of specific tumor-derived exRNA and exosomes leading to novel therapies and improved therapeutic outcomes. In UH2 phase of the grant, using ovarian cancer (OC) cell lines and cells derived from OC patient tumors, we will identify novel exRNA-based cancer therapeutic lead candidates by siRNA library genome-wide screening based on secretion measurements, by small RNA deep sequencing in paired samples from tumors, plasma and ascites from the same patient, and by functional studies for the lead exRNA candidates. In the UH3 phase, we will achieve pre-clinical optimization by testing the exRNA lead(s) identified in the UH2 for antitumor efficacy in well-established OC murine orthotopic models and their safety using well-established preclinical protocols. The aims in exploratory UH2 phase are designed to identify and validate the function of novel exRNA therapeutic candidates for pre- clinical studies. In UH3 phase, we will formulate the methods for the validation of the assays to determine the activity, pharmacology in tissue and plasma to establish dosage schedules in animal models for the design of rational anti-tumor approaches. Using our nanoliposomal delivery technology, we will also conduct target modulation and efficacy studies to generate clear evidence regarding the safety of exRNA-based therapeutic candidates in the proposed dose range. Our approach, by using a new category of regulatory exRNAs- miRNAs, could substantially enhance therapeutic efficacy for cancer treatment. We will also demonstrate, for the first time, the use of nanoliposome-siRNA-exRNA targeting to generate a synergistic boosting effect for treatment of cancers. While we are focusing on OC due to the high mortality associated with this malignancy, our findings have applications for any type of cancer. This proposal brings synergistic capabilities of scientists working for over a decade in the fields of miRNA (Dr. Calin), siRNA (Dr. Sood), nanoparticle delivery (Dr. Lopez-Berestein), and exosome biology (Dr. O'Halloran). The team has the expertise and synergistic drive to achieve novel exRNA therapeutic development by performing highly innovative research, and has developed RNA-based therapies in the past.

DESCRIPTION (provided by applicant): Elevated platelet counts are a common finding in many cancer patients, including patients with ovarian cancer. Patients with ovarian cancer and thrombocytosis have a worse prognosis compared to patients with similar stages of cancer and normal platelet counts. We have shown that platelets promote proliferation of cancer cells both in vitro and in the murine models of ovarian cancer; and reducing platelet counts decreased the size of orthotopic tumor induced in mice by ovarian cancer cells. To identify the mechanisms of the growth- enhancing effect of platelets on cancer cells, we used blocking reagents against platelets in vitro, and found that platelet activation and release of TGF¿1 are important for the proliferative effect of platelets on cancer cells. In this project, we will study the interaction between platelets and cancer cells in vivo, using both murine models of ovarian cancer and tissue samples obtained from patients with ovarian cancer. Our hypothesis is that there is a feedback loop between platelets and cancer cells. Cancer cells secrete ADP and activate platelets, and platelets secrete TGF¿1 that promotes proliferation in cancer cells. In the specific aim 1, we will investigate whether blocking ADP receptors on platelets would disrupt the growth promoting effect of platelets on orthotopic tumors in mice, using genetically modified mice or pharmacologic reagents. In the specific aim 2, we will target TGF¿1 secretion from platelets, TGF¿1 receptor on cancer cells, or TGF¿1 receptor signaling to evaluate the role of TGF¿1 on the platelet-cancer cell interaction. We will use platelet-specific TGF¿1 deficient mice, inhibitor RNAs, or pharmacologic reagents against TGF¿ receptor signaling to conduct these experiments. For the interaction between platelets and cancer cells to occur inside the tumors, platelets should exit circulation and enter into tumor microenvironment. We have shown the presence of platelets outside of blood vessel inside the implanted tumors in mice. In the specific aim 3, we will study the mechanisms of platelet exit from tumor microcirculation using immunofluorescence and electron microscopy on human and murine ovarian cancer tissue samples. We will identify the route of platelet extravasation, and evaluate the dependency of platelets on neutrophils for extravasation. The goal of our studies in this grant proposal is to evaluate the possibility of using anti-platelet reagents as an effective anticancer therapy.

DESCRIPTION (provided by applicant): RNA molecules are secreted in extracellular spaces (exRNAs) and act as endocrine signals altering the phenotypes of cancer cells. In this application we will focus on the class of small non-codingRNAs named microRNAs. We propose an innovative therapeutic concept: the depletion of oncogenic microRNAs from malignant cells by enhancing their secretion as exRNAs or blocking suppressor microRNAs' delivery in extracellular space by blocking secretion mechanisms. Our main goal is to develop new strategies using combined miRNA and siRNA to maximize therapeutic benefit by affecting the production of exRNAs. Our proposal centers on the identification and targeting of specific tumor-derived exRNA and exosomes leading to novel therapies and improved therapeutic outcomes. In UH2 phase of the grant, using ovarian cancer (OC) cell lines and cells derived from OC patient tumors, we will identify novel exRNA-based cancer therapeutic lead candidates by siRNA library genome-wide screening based on secretion measurements, by small RNA deep sequencing in paired samples from tumors, plasma and ascites from the same patient, and by functional studies for the lead exRNA candidates. In the UH3 phase, we will achieve pre-clinical optimization by testing the exRNA lead(s) identified in the UH2 for antitumor efficacy in well-established OC murine orthotopic models and their safety using well-established preclinical protocols. The aims in exploratory UH2 phase are designed to identify and validate the function of novel exRNA therapeutic candidates for pre- clinical studies. In UH3 phase, we will formulate the methods for the validation of the assays to determine the activity, pharmacology in tissue and plasma to establish dosage schedules in animal models for the design of rational anti-tumor approaches. Using our nanoliposomal delivery technology, we will also conduct target modulation and efficacy studies to generate clear evidence regarding the safety of exRNA-based therapeutic candidates in the proposed dose range. Our approach, by using a new category of regulatory exRNAs- miRNAs, could substantially enhance therapeutic efficacy for cancer treatment. We will also demonstrate, for the first time, the use of nanoliposome-siRNA-exRNA targeting to generate a synergistic boosting effect for treatment of cancers. While we are focusing on OC due to the high mortality associated with this malignancy, our findings have applications for any type of cancer. This proposal brings synergistic capabilities of scientists working for over a decade in the fields of miRNA (Dr. Calin), siRNA (Dr. Sood), nanoparticle delivery (Dr. Lopez-Berestein), and exosome biology (Dr. O'Halloran). The team has the expertise and synergistic drive to achieve novel exRNA therapeutic development by performing highly innovative research, and has developed RNA-based therapies in the past.

Project 3 SUMMARY Growing evidence suggests that EphA2 is an important therapeutic target in uterine cancer. Our recent integrative analysis of TCGA data further indicate that EphA2 upregulation is significantly correlated with poor survival. EphA2 is also expressed at high levels in the tumor vasculature and plays a critical role in regulating angiogenic functions. These findings, coupled with the low or absent expression of EphA2 in most normal adult tissues, make it a highly attractive therapeutic target. Despite its clinical benefit in patients with recurrent uterine cancer, dasatinib can result in substantial toxicity when combined with chemotherapy due to its ?off- target? engagement. Therefore, more specific therapeutic approaches for targeting EphA2 are needed. To achieve this goal, we have focused on systemically delivered short interfering RNA (siRNA) against EphA2 (EPHARNA) using a neutral nanoliposomal platform. Our overall hypotheses are that 1) EphA2 gene silencing using EPHARNA enhances the therapeutic response selectively in CAV1 overexpressing tumors; 2) Inhibition of MEK signaling increases the sensitivity to EphA2-targeted therapy in uterine cancer. The overall goal of this renewal proposal is to use EPHARNA for selective EphA2 targeting in uterine carcinoma and determine the underlying mechanisms of response and adaptive changes. The proposed Aims are complementary and will be pursued in close collaboration with the Cores.

Overall SUMMARY/ABSTRACT The overall goal of the University of Texas MD Anderson Cancer Center (MDACC) Ovarian Cancer SPORE is to improve outcomes for ovarian cancer patients by combining targeted agents based upon the molecular, cellular and clinical biology of their disease and understanding and targeting mechanisms of drug resistance. Over the last 6 years (FY2009-FY2015), MDACC has cared for 4,483 patients with ovarian, fallopian tube and peritoneal cancers. MDACC has given high priority to ovarian cancer research through recruitment, salary support, clinical facilities, laboratory space, and philanthropic funds. Over the last 6 years, MDACC has recruited six outstanding faculty members with an interest in ovarian cancer research (Drs. Amir Jazaeri, Larissa Meyer, Alpa Nick, Shannon Westin, Melinda Yates, and Behrouz Zand). Philanthropic support of the Women?s Cancer Breast-Ovarian Moon Shot has provided organization and infrastructure. Over the same time period, our previous SPORE funded 15 Developmental Research Projects (DRPs), and supported six Career Enhancement Program (CEP) awardees, and SPORE investigators have contributed 461 peer-reviewed papers pertaining to ovarian cancer with 53% (246) IF >5 and 20% (92) >10. Achievements included: 1) evaluation of a two stage screening strategy with a positive predictive value of >30% for detecting stage I-II disease in 9 of 12 cases detected; 2) identification of biomarkers that detect 18% of CA125 negative cases; 3) development of a point-of-service nanoassay for these biomarkers; 4) discovery that anti-TP53 autoantibodies rise 5 (median) -13 months (mean) prior to CA125, the first biomarker to provide earlier detection than CA125; 4) observation of a 54% objective response rate to anti-angiogenic therapy with aflibercept and docetaxel; 5) initiation of a trial targeting Dll4 and notch; 6 ) CSF1R inhibitors could deplete macrophages and reduce resistance to anti-VEGF Therapy 7) demonstration of significant activity of the MEK inhibitor selumetinib in low-grade ovarian cancers and initiation of an international phase III trial of another potent MEK inhibitor trametinib; 8) development of a robust biomarker panel that predicts response to PARP inhibitors (PARPi) and initiation of multiple trials combining PI3K and PARP inhibitors in high-grade ovarian cancer; and 9) use of mesenchymal stem cells to deliver interferon to ovarian cancers. I n t h i s new SPORE application, 4 projects will strive to: 1) validate predictive biomarkers and implement rational combination therapy with PARP inhibitors designed to overcome pre- existing and adaptive resistance; 2) validate predictive biomarkers and implement rational combination therapy with MEK inhibitor for low-grade ovarian serous cancers to overcome resistance; 3) target macrophages to overcome resistance to anti-VEGF therapies; and 4) evaluate a novel SIK2 inhibitor in a Phase IA/B trial and identify agents that produce synthetic lethality.

Project Abstract: Despite the promise of RNA interference (RNAi) approach for targeting undruggable targets, major challenges remain including specific delivery of siRNA into cell types of interest in vivo, poor stability and off-target effects. We have been at the forefront of addressing these issues and my laboratory has pioneered many studies using RNAi approaches for cancer treatment and has made key discoveries related to RNAi biology. We were among the first to demonstrate that RNAi processing machinery is deregulated in a high proportion of ovarian and other cancers (Merritt et al., New Engl J Med 2008). To achieve systemic delivery of RNAi therapeutics, we systematically identified safe and effective methods for siRNA delivery. After extensive testing, our first successful platform utilized the neutral DOPC nanoliposomal delivery system (Landen et al., Cancer Res 2005; Ahmed et al., Cancer Cell 2010), which has subsequently been tested in multiple tumor model systems (Liu et al., Nature 2015; Kim et al., Cell 2013). With a robust portfolio of preclinical studies and all of the requisite safety studies based on FDA guidance, a first-in-human phase I clinical trial with EPHARNA (EphA2 targeted siRNA in DOPC) is nearing completion for patients with solid tumors. We have made great strides in applying this technology for cancer therapy. However, despite the promise of synthetic delivery systems that we and others have developed, novel and biocompatible delivery strategies that are independent of reliance on vascular leakiness are highly desirable. In this project, we propose to develop a biomimetic exosomal system that will enable active delivery of cancer therapeutics to the tumor microenvironment. These naturally occurring particles represent a promising, and safe alternative approach for delivering RNAi therapeutics. We will package RNAi cargos into these particles and engineer their surface membrane to actively target distinct cell types. Finally, we will develop this approach for enhancing anti-tumor immune response in ovarian and other cancers. If successful, this biomimetic exosomal system can be rapidly applied more broadly to other ?undruggable? targets. Support through the R35 mechanism will greatly facilitate this undertaking, which would not otherwise be possible.

Developmental Research Program SUMMARY/ABSTRACT The purpose of the Developmental Research Program (DRP) is to fund promising projects by investigators whose current work may not focus exclusively on ovarian cancer, but who propose highly innovative translational studies of ovarian cancer that could become full SPORE projects or compete successfully for funding outside of the SPORE. The DRP provides a unique venue for making available significant financial support, and for demonstrating active institutional support, through a program that is rapidly responsive to new ideas or initiatives. Moreover, this program is rooted in a spirit of collaboration espoused by the SPORE investigators, who have an extensive track-record of bringing investigators from other disciplines into ovarian cancer research. The strength of the DRP rests in its ability to make available financial support needed to access all the critical expertise and resources within the entire SPORE. This will allow us to develop collaborative, multi-investigator, multi-institutional research projects with the support of innovative, investigator-initiated projects that have the potential to flourish into reliable and productive translational research projects that make a path from basic and/or population research projects into research focused on human clinical specimens/patient populations. Over the course of the previously-funded Ovarian SPORE grants since 2000, we have supported 48 DRPs. Overall, 14 former recipients of DRPs have received peer-reviewed funding in ovarian cancer as PI, Co- PI or Co-Investigator with eight as PIs (three DOD, two R01, one R21, one R03 and two CPRIT). Two recipients have been awarded OCRF funds (one Multi-Investigator Award and one Liz Tilberis Scholar). More recently, four recipients have been awarded six peer-reviewed ovarian cancer grants as PI (three R01, one R21, two CPRIT) and one Liz Tilberis Scholar award from the OCRF. Overall, recipients have published 281 peer-reviewed publications regarding ovarian cancer (38 specific to their DRP project) since receiving a DRP award. Additionally, six former awardees have participated in full SPORE projects after completing a DRP project.

Career Enhancement Program SUMMARY/ABSTRACT The central goal of the Career Enhancement Program (CEP) is to use the resources at MD Anderson to train exceptional young investigators who will reduce the morbidity and mortality from ovarian cancer through making advances in the early detection, prevention, and treatment of this disease. To achieve this goal, the SPORE CEP will provide two awards, each of $50,000 annually for two years, funded from the SPORE and matching funds from MD Anderson. The intent of each CEP award is to prepare the selected scientists to become international leaders in academic research relevant to ovarian cancer. We will achieve this by aggressive recruitment of the most promising young investigators and the institution of an individualized development plan with clinical and a laboratory mentors and a mentoring committee, formal course work, coaching in grant and paper writing, leadership training, attendance at national meetings, networking with ovarian cancer scholars and completing and publishing a translational ovarian cancer research program. During our past SPORE awards, the CEP has developed the careers of 21 young investigators. In the first year of the original grant, three postdoctoral trainees received CE awards, and of the 18 faculty members supported since 2000, all have remained in academic research and 13 are engaged predominantly in ovarian cancer research. Ten have achieved peer-reviewed funding and two additional awardees have received competitive foundation grants. The six awardees in the most recently funded cycle received one R01, one R03, two CPRIT awards, two Texas Center for Nanotechnology Grants, one Liz Tilberis Scholar Award from the Ovarian Cancer Research Fund and one Susan Poorman Blackie Ovarian Cancer grant. After entering the program, awardees have published over 500 peer-reviewed papers regarding ovarian cancer. Of the faculty awardees, five are project leaders, co-investigators, collaborators or core leaders on the current SPORE (Drs. Anil Sood, Jinsong Liu, KK Wong, Vikas Kundra, and Guang Peng). Moving forward, the CEP will be led by Dr. Anil Sood, who is an outstanding role model and most effective mentor.

PROJECT SUMMARY Despite advances in surgery and chemotherapy, ovarian cancer (OVCA) remains the most lethal gynecologic malignancy. The tumor microenvironment (TME) is a complex milieu of several types of cells, blood vessels and extracellular matrix proteins in which cancerous cells thrive. The cells that constitute most of the TME are fibroblasts, immune cells, endothelial cells and pericytes and are also collectively known as stroma. These cells become reactive and develop characteristics that support and even enhance tumor progression and metastasis due to proximity and constant interaction with cancer cells. Failure of traditional therapy is due to our limited understanding of how the TME can facilitate the rapid progression or recurrence of OVCA. Targeting reactive stromal cells is emerging as an attractive and viable therapy to regulate the channels of communication between stromal and cancer cells. To target non-autonomous mechanisms of cancer cell aberrations, the mechanistic underpinnings of reactive stroma vis a vis quiescent or normal stroma is required. Stromal cells such as cancer associated fibroblasts (CAFs), cancer associated mesothelial cells (CAMs), and cancer associated adipocytes (CAAs) in omental tissue have been shown to promote OVCA metastasis and growth. Although it has been recently shown that microenvironment can induce metabolic reprogramming in cancer cells, however, identification of stromal targets which make cancer cells vulnerable has remained challenging and elusive. We propose a previously unrecognized mechanism whereby metabolism of reactive stromal cells is reprogrammed through upregulated glutamine anabolic pathway. We first hypothesize that reactive stromal metabolism is altered from quiescent stroma, and is the driver for regulating cancer growth in its harsh microenvironment. Second, targeting this aberration could create metabolic vulnerability in cancer cells by disrupting the metabolic crosstalk between stromal and cancer cells. We will test these hypotheses in the proposed Aims. First, we will establish whether CAFs, CAAs, and CAMs promote OVCA cell proliferation by reprogramming glutamine (Gln) metabolism in cancer cells. We will validate upregulation of Gln anabolic pathway in reactive stromal cells compared to their normal counterparts through transcriptomic profiling. To elucidate metabolic reprogramming in reactive stromal cells we will use 13C-based metabolic flux analysis using stable isotope tracers to reveal metabolic vulnerabilities in stromal cells and unravel metabolic symbiosis between stromal and epithelial cells. Second, we will elucidate stroma-secreted Gln's role in maintaining OVCA cells' drug resistance. Our results will reveal an alternative modality in the treatment of recurrent OVCA. Third, we will determine the efficacy of targeting stromal Gln metabolism using orthotopic models of ovarian carcinoma and perform tracing of metabolic fates of different nutrients in tumors using in vivo tracer analysis in orthotopic models proposed for targeting stromal metabolism. In summary, our proposed study can lead to novel therapeutics targeting communication between cancer cells and their microenvironment.

Project 3 SUMMARY/ABSTRACT Angiogenesis is known to play a critical role in cancer growth and metastasis. Among the many potential targets, vascular endothelial growth factor (VEGF) has been well recognized to play an important role in angiogenesis, and drugs targeting this pathway have been used against ovarian and other cancers. Clinical use of anti-VEGF therapy, however, has yielded only modest improvement in progression-free or overall survival of patients with ovarian cancer, likely due to adaptive changes in the tumor microenvironment. There remains an unmet need to develop methods to enhance efficacy of anti-VEGF therapy and block growth- promoting adaptive changes. The mechanisms of adaptive resistance to anti-VEGF treatment are largely unknown. Understanding the adaptive resistance to anti-VEGF treatment has the potential to significantly enhance the efficacy of anti-VEGF therapy in ovarian cancer patients. Our preliminary findings suggest that tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) are substantially increased in the anti-VEGF therapy-resistant tumors and TAM depletion (with CSF1R inhibitor) can improve the effectiveness of anti-VEGF therapy; however, the mechanisms by which this occurs are not well understood. In this proposal, we will explore the mechanisms by which macrophages contribute to adaptive resistance to anti-VEGF treatment and test the efficacy of dual targeting of VEGF and TAMs/MDSCs. Our central hypothesis is that targeting TAMs in the microenvironment will reverse the immunophenotypical alterations induced by bevacizumab and improve clinical efficacy. We will conduct a novel, induction, randomized supplementation clinical study to assess the impact of adding a CSF1R inhibitor to identify and overcome these effects as measured by objective response and event-free survival. The proposed work is highly translational and has the potential to significantly enhance the efficacy of anti-VEGF therapy in ovarian cancer patients.