Melanie Cobb - US grants

DESCRIPTION (provided by applicant): The Cell and Molecular Biology (CMB) Training Program at the University of Texas Southwestern Medical Center at Dallas fosters the development of Ph.D. scientists with the skills and resources necessary to succeed as independent researchers in a rapidly changing scientific environment. In response to this changing environment, the CMB Training Program at UT Southwestern is largely driven by the trainees. The program focuses on Cellular and Molecular Biology as it applies to medical advances, reflecting the research interests of the students and the laboratories in which they train. It provides formal training in statistical analysis of biological data and emphasizes the importance of collaborative research in acquiring the breadth of knowledge and skills needed for the rapid pace of developments. The program offers unique small group settings for the trainees to acquire new knowledge, form scientific hypotheses, and critically analyze data. Students compete for positions in this training program by writing a research summary and personal statement about why they would like to participate. The application process is open to students in their second year of a Ph.D. program or in their first or second graduate school year of the MSTP program. The new trainees are chosen by the CMB Steering Committee. Most students remain in the training program for up to 3 years, usually including one year of advanced didactic training and up to 2 years of independent research. Currently, there are 14 granted slots, with 13 funded due to budget constraints of the NIH. We believe that the growth of our student population, along with the strength of our program within the biomedical research community, justifies a modest increase in the number of positions funded, although we are well aware of the limited resources available. The interdisciplinary, student-focused CMB program is structured to stimulate the trainees'critical thinking and to present novel opportunities to question and evaluate cellular and molecular research important for human health. Scientists equipped with these skills will make discoveries that increase quality and years of healthy life, one of the overall goals of the Department of Health and Human Service's Healthy People 2010 Report. Faculty members in the CMB program are involved in basic research relating to many of the Healthy People 2010 focus areas.

DESCRIPTION (provided by applicant): Diabetes mellitus is a huge health burden due to decreased quality of life and the escalating cost of treatment. In the United States alone, ~8% of the population is diabetic and nearly one-third of adults are now estimated to be at risk to develop the disease. Obesity, insulin resistance and metabolic abnormalities in liver, adipose, and muscle are important factors in disease. Yet, most of the gene loci recently found associated with type 2 diabetes encode proteins that enable insulin production from pancreatic cells. Nutrients and hormones regulate not only insulin secretion but also the capacity of cells to continue to produce insulin. In this proposal we focus on mechanisms of action of nutrients and agents that enhance insulin production in cells. The overarching goals are to elucidate mechanisms that can be manipulated to improve -cell function and overcome the physiological changes in cells that occur during prolonged hyperglycemia, contributing to inadequate insulin release. In aim 1 we will determine the functions of the taste receptor complex (T1R1/T1R3) in pancreatic cells. This dimeric G protein-coupled receptor (GPR) is involved in amino acid-sensing. This receptor, identified in gustatory neurons, binds to a variety of amino acids, but has not been studied on cells. We find that stable knockdown of T1R3 reduces insulin secretion, and insulin content, and causes events associated with autophagy. We will explore the bases for these phenotypic changes and we will also examine the underlying signaling mechanisms. In the second aim we will examine molecular mechanisms of action of small molecules that enhance beta-cell function. These molecules stimulate insulin production by cells, improve oral glucose tolerance of db/db mice, and restore insulin production by human islets in long term culture. We have identified a number of changes that take place in cells treated with these drugs, including epigenetic alterations, and changes in concentrations of key transcription factors. We will evaluate candidates that may mediate the actions of these drugs identified by drug-affinity chromatography. Finally, we will continue to investigate ERK1/2-dependent signaling events that control insulin gene transcription by focusing on mechanisms of recruitment of the coactivator p300. These studies should provide new understanding of how amino acids regulate insulin production, stability and release and will exploit a new pharmacological tool to identify mechanisms to enhance insulin production and insulin transcription in normal and failing islets. PUBLIC HEALTH RELEVANCE: Type 2 diabetes mellitus is now found not only in adults but also in children in the United States. These studies will explore new mechanisms to enhance insulin production in diabetes focusing on a nutrient sensing receptor and a novel small molecule that enhances insulin production from poorly functioning islets.

DESCRIPTION (provided by applicant): The Cell and Molecular Biology (CMB) Training Program at the University of Texas Southwestern Medical Center at Dallas fosters the development of Ph.D. scientists with the skills and resources necessary to succeed as independent researchers in a rapidly changing scientific environment. In response to this changing environment, the CMB Training Program at UT Southwestern is largely driven by the trainees. The program focuses on Cellular and Molecular Biology as it applies to medical advances, reflecting the research interests of the students and the laboratories in which they train. It provides formal training in statistical analysis of biological data and emphasizes the importance of collaborative research in acquiring the breadth of knowledge and skills needed for the rapid pace of developments. The program offers unique small group settings for the trainees to acquire new knowledge, form scientific hypotheses, and critically analyze data. Students compete for positions in this training program by writing a research summary and personal statement about why they would like to participate. The application process is open to students in their second or later year of a Ph.D. program or in their first or second graduate school year of the MSTP program. The new trainees are chosen by the CMB Steering Committee based on personal statement, motivation, and diversity of research topic. Most students remain in the training program for 3 years, usually including one year of advanced didactic training and up to 2 years of independent research. Currently, there are 14 granted slots, with 13 funded due to budget constraints of the NIH. The interdisciplinary, student-focused CMB program is structured to stimulate the trainees' critical thinking and to present novel opportunities to question and evaluate cellular and molecular research important for human health. This interdisciplinary training program provides didactic and laboratory training of students in a range of fields that encompass many aspects of cell and molecular biology, culminating in the Ph.D. degree. Multiple training experiences include a round table journal club covering diverse scientific questions and methodologies and designed to stimulate critical analysis, breadth of thinking, and dialog; a faculty seminar series that combines a discussion of career path and cutting edge research; a poster with all CMB faculty; and a retreat in which students' present their ongoing research and plan the following year's activities. Scientists equipped with skills provided by this training program will perform inspired research that crosses disciplines to make discoveries that benefit human health in the future.

The National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP) is a highly competitive, federal fellowship program. GRFP helps ensure the vitality and diversity of the scientific and engineering workforce of the United States. The program recognizes and supports outstanding graduate students who are pursuing research-based master's and doctoral degrees in science and engineering. GRFP provides three years of support for the graduate education of individuals who have demonstrated their potential for significant achievements in science and engineering. This award supports the NSF Graduate Fellows pursuing graduate education at this GRFP institution.

DESCRIPTION (provided by applicant): The Cell and Molecular Biology (CMB) Training Program at the University of Texas Southwestern Medical Center at Dallas fosters the development of Ph.D. scientists with the skills and resources necessary to succeed as independent researchers in a rapidly changing scientific environment. In response to this changing environment, the CMB Training Program at UT Southwestern is largely driven by the trainees. The program focuses on Cellular and Molecular Biology as it applies to medical advances, reflecting the research interests of the students and the laboratories in which they train. It provides formal training in statistical analysis of biological data and emphasizes the importance of collaborative research in acquiring the breadth of knowledge and skills needed for the rapid pace of developments. The program offers unique small group settings for the trainees to acquire new knowledge, form scientific hypotheses, and critically analyze data. Students compete for positions in this training program by writing a research summary and personal statement about why they would like to participate. The application process is open to students in their second or later year of a Ph.D. program or in their first or second graduate school year of the MSTP program. The new trainees are chosen by the CMB Steering Committee based on personal statement, motivation, and diversity of research topic. Most students remain in the training program for 3 years, usually including one year of advanced didactic training and up to 2 years of independent research. Currently, there are 14 granted slots, with 13 funded due to budget constraints of the NIH. The interdisciplinary, student-focused CMB program is structured to stimulate the trainees' critical thinking and to present novel opportunities to question and evaluate cellular and molecular research important for human health. This interdisciplinary training program provides didactic and laboratory training of students in a range of fields that encompass many aspects of cell and molecular biology, culminating in the Ph.D. degree. Multiple training experiences include a round table journal club covering diverse scientific questions and methodologies and designed to stimulate critical analysis, breadth of thinking, and dialog; a faculty seminar series that combines a discussion of career path and cutting edge research; a poster with all CMB faculty; and a retreat in which students' present their ongoing research and plan the following year's activities. Scientists equipped with skills provided by this training program will perform inspired research that crosses disciplines to make discoveries that benefit human health in the future.

? DESCRIPTION (provided by applicant): The University of Texas Southwestern Simmons Cancer Center is submitting this competitive renewal application for a Cancer Center Support Grant, Years 6-10. The Simmons Cancer Center is organized as a matrix center that integrates cancer research, clinical cancer care, and cancer control outreach across the University of Texas Southwestern (UTSW) Medical Center and its affiliated hospital systems, UTSW Health System, Parkland Health and Hospital System, Children's Medical Center of Dallas, and UTSW Moncrief Cancer Institute in Fort Worth. A research affiliation with the Dallas Regional Campus of the University of Texas School of Public Health, an MPH and PhD granting program located on the UTSW campus, brings public health expertise and training opportunities into the center. The overriding mission of the Simmons Cancer Center is to leverage these affiliations to create new knowledge to improve cancer outcomes in Dallas, North Texas, and the nation. The 164 members of the Simmons Cancer Center are organized into five scientific programs- Development and Cancer, Cancer Cell Networks, Chemistry and Cancer, Experimental Therapeutics of Cancer, and Population Science and Cancer Control. These programs are highly interactive, with 22 multi-investigator grants awarded and a 52% collaborative publication ratio over the past grant period. Funds are requested in the application for Senior Leaders and Program Leaders, Planning and Evaluation, Developmental Funds, Administration, Early Phase Clinical Research Support, Protocol Review and Monitoring System, Clinical Protocol and Data Management, and six shared resources including two in a research design and data analytics grouping (biostatistics, bioinformatics); and four in a translational grouping (high throughput screening, small animal imaging, live cell imaging, and tissue management). CCSG support for these components is supplemented by institutional funds to ensure that the center has the infrastructure required to achieve its mission. In addition, the Simmons Cancer Center has created dynamic educational and training opportunities designed to equip a new generation of scientists, physicians, and other health care providers to make a difference in cancer discovery, clinical care, and control. The Simmons Cancer Center integrates cancer research, clinical cancer care, and cancer control outreach across the University of Texas Southwestern Medical Center and its affiliated hospital systems. The overriding mission of the center is to leverage these affiliations and the exceptional resources available to improve cancer outcomes in the Dallas-Fort Worth (DFW) region and the nation.

Abstract The long term goal of this research is to determine the molecular mechanisms underlying the interactions of WNK1 with TGF? signaling on endothelial plasticity and homeostasis. Failed angiogenesis in endothelial-specific WNK1 null mice led to embryonic death. We have found two processes important for endothelial plasticity that are coordinately regulated by TGF? and WNK1: conversion of cells to a migratory phenotype and tight junction breakdown that occurs with complete endothelial-mesenchymal transition. These processes contribute to normal vascular physiology and to pathophysiology. We will investigate how signaling pathways regulated by TGF? and WNK1 intersect in controlling endothelial cell behavior and characteristics. WNK-selective kinase inhibitors, protein depletion, gene editing, proximity ligation, gene expression and other biochemical means will be used in cell- and tissue-based assays to discover the mechanisms through which WNK1 mediates dynamic reorganization of endothelial cell structures underlying normal and pathophysiological angiogenesis in tandem with TGF?. In the first specific aim, we will determine interactions between TGF? and WNK1 signaling pathways that regulate expression of the mesenchymal transcription factor Slug (Snai2) and subsequent induction of endothelial cell motility. We hypothesize that WNK1 activates Slug expression and migration through actions on TGF?-regulated SMADs. Slug promotes endothelial cell migration and remodeling by inducing proteins that repress cell-cell adhesion and enhance a migratory mesenchymal phenotype. In the second specific aim, we will determine how WNK1 promotes TGF?-induced tight junction disassembly. In response to TGF?, the WNK1 substrate kinase OSR1 binds to tight junction proteins along with other signaling proteins and TGF? receptors to break down tight junctions. Inhibiting WNK1 kinase activity prevents co-immunoprecipitation of OSR1 with occludin and prevents TGF?-induced tight junction disassembly in endothelial cells. We hypothesize that WNK1 is required for TGF?-induced tight junction disassembly through actions of its substrate kinase OSR1. We will analyze how WNK1/OSR1 participate in TGF?-initiated tight junction break down to discover the steps requiring their cooperation. We will identify components in OSR1-occludin complexes and the effects of interfering with OSR1 function on tight junction breakdown. Essential events will be established using rescue strategies. Our results will define the extent of cooperation between TGF? and WNK1 signaling mechanisms and uncover opportunities for therapeutic targeting of the WNK1 pathway in disease. Our findings will lead to a better understanding of normal and pathological angiogenesis.

Project Summary Insulin signaling is critical for multiple facets of animal physiology. Its dysregulation causes insulin resistance syndromes, such as type 2 diabetes. The spindle checkpoint ensures the fidelity of chromosome segregation and guards against aneuploidy. The key spindle checkpoint proteins Mad2 and BubR1 can simultaneously bind to Cdc20, converting it from an anaphase promoting complex/cyclosome (APC/C) activator to a subunit of an APC/C-inhibitory complex called the mitotic checkpoint complex (MCC). During checkpoint inactivation, a critical inhibitor of Mad2, p31comet promotes checkpoint inactivation and timely chromosome segregation. Recently, combining approaches in mouse genetics, cell biology, biochemistry, and single-cell genomics, we have discovered a critical role of the p31comet?Mad2?BubR1 module of mitotic regulators in insulin signaling through regulating insulin receptor (IR) endocytosis. In the mouse, p31comet ablation diminishes IR at the plasma membrane prior to insulin binding and causes defective insulin signaling in multiple tissues and metabolic syndrome. Mechanistically, Mad2 directly binds to IR through a canonical Mad2-interacting motif (MIM). IR-bound Mad2 facilitates BubR1-dependent recruitment of the clathrin adaptor AP2 to IR. p31comet blocks Mad2-BubR1 association and prevents spontaneous IR endocytosis. Mad2 and BubR1 are also required for insulin-stimulated IR endocytosis. This unexpected link between mitotic regulators and insulin signaling raises several outstanding questions that we wish to address in this proposal. In Aim 1, we will further elucidate the mechanism and regulation of insulin-stimulated IR endocytosis. In particular, we will determine how the newly discovered Mad2?BubR1 mechanism cooperates with previously described mechanisms to mediate proper IR endocytosis. We will establish how these mechanisms are regulated by insulin signaling. In Aim 2, we will test the intriguing hypothesis that insulin signaling reciprocally regulates the spindle checkpoint. In preliminary results, we have created a knock-in mouse (Insr4A/4A) with mutated IR alleles (4A) deficient for Mad2 binding. IR 4A cells have a weakened spindle checkpoint. We will determine the mechanisms by which IR promotes spindle checkpoint signaling through cellular and in vitro reconstitution experiments. In Aim 3, we will define the physiological functions of the mutual regulation between IR and mitotic regulators by examining the phenotypes of the Insr4A/4A mouse. We will test whether defective IR plasma membrane localization contributes to type 2 diabetes by comparing IR localization in liver biopsies from non-diabetic and diabetic patients. Collectively, the proposed research will further clarify the mechanism and function of the unexpected link between mitotic regulators and insulin signaling, and may establish the Mad2? BubR1?AP2 module as a novel therapeutic target for treating diabetes.

Despite their discovery over 20 years ago, TAO protein kinases (TAO1-3) remain understudied. In particular, we lack an understanding of the mechanisms regulating TAO activation, how they recognize substrates, and how these kinases contribute to key physiological processes. We originally identified TAOs through a large- scale effort to find membrane proximal components of MAPK cascades and showed that TAOs are MAP3Ks in the p38 pathway. Recent work now reveals that TAOs are essential for schistosome and viral infections and viral RNA processing and export from the nucleus. These findings suggest that TAOs are drug targets for a range of pathophysiological conditions and beg for a greater understanding of how their activities are controlled. Here, we propose to develop a paradigm for the biochemical regulation of TAO protein kinases through their integration with phosphoprotein phosphatase 1 (PP1). These results should advance future drug discovery efforts to provide new therapeutic entry points for treating a range of diverse pathological conditions. An obstacle in realizing their therapeutic potential is the limited knowledge of their regulation and partners. In searching for regulatory interactions, we found that TAOs directly bind PP1 and its R7 regulatory subunit. While PP1 dephosphorylates half the proteins in the cell, its activity is largely restricted within heteromeric complexes by regulatory subunits. Recent literature indicates that R7 maintains PP1 in an inactive state. We propose that TAOs modulate PP1 phosphatase activity via direct interactions and by R7 phosphorylation. The connection with PP1 offers TAOs wide opportunities to impact cellular control mechanisms. Our specific aims are to 1) determine how the TAO-PP1 complex regulates TAO activity; and 2) determine how TAO through R7 regulates PP1 activity. Biochemical and cell biological studies will take advantage of a model of a TAO2-PP1 complex based on our crystal structure of the TAO2 kinase domain that shows the PP1 binding motif on TAO. The relevance of this interaction is supported by our recent work revealing co-localization of TAO2 and PP1 in structures in the nucleus and the cytoplasm. Our extensive experience in identifying and characterizing TAOs, determining the structure of the TAO kinase domain, identifying chemically tractable inhibitors, and elucidating TAO-dependent pathways, since our discovery of these kinases, puts us in a unique position to determine biochemical processes that will provide a foundation for TAO kinases as subjects of drug development.

The Cellular Networks in Cancer (CNC) Program is a scientifically rich, collaborative, and productive research program co-led by Melanie Cobb, PhD, and Ralph DeBerardinis, MD, PhD. The vision of the CNC Program is to advance the fundamental knowledge of both intercellular and intracellular networks that contribute to cancer initiation, tumorigenesis, and metastasis, then to translate these findings into novel approaches to cancer prevention and therapy. CNC?s overarching scientific goal is to promote discoveries in how perturbation in these cellular networks contribute to altered tissue physiology and promote cancer. The CNC Program provides a highly interactive research environment that capitalizes on the UT Southwestern Medical Center?s (UTSW) longstanding tradition of basic science discoveries and fosters translation of these discoveries into the development of new biomarkers, diagnostic technologies, and therapeutic interventions in clinical oncology- especially malignancies relevant to the SCCC catchment area (e.g., cancer of the lung and kidney). The program?s structure is purposefully designed to stimulate interdisciplinary intra- and interprogrammatic collaborations with the objective of delivering transformative discoveries. To accomplish this, the Co-Leaders established four vibrant and synergistic subprograms that deliberately align with the strengths of program members and specifically relate to states of perturbed tissue homeostasis in cancer: (1) Signaling and Cell Biology, (2) Epigenetics and Gene Regulation, (3) Immunobiology, and (4) Metabolism. Each subprogram is directed by an investigator who is highly respected for his or her science, mentoring, and collaborative spirit. The subprogram structure and activities have directly contributed to exciting new collaborative projects such as the SPORE in Kidney Cancer. The CNC Program is composed of 59 basic, computational, and physician-scientists from 19 departments and centers at UT Southwestern Medical Center. Seven members of the CNC are Howard Hughes Medical Institute investigators. Annual direct peer-reviewed funding to the CNC Program was $19.3M in 2019. This represents an increase of $3.8M as compared with 2014 data, which were adjusted to conform to revised NCI guidelines. NCI funding has likewise increased from $3M in 2014 to $3.9M in 2019. Program members have authored 750 publications since 2014: 14% represent intraprogrammatic work, 34% are interprogrammatic collaborations, 32% are inter-institutional, and 37% are in journals with an impact factor ? 10. CNC Program members are heavily reliant upon all six CCSG Shared Resources?especially Live Cell Imaging and Tissue Management?in furtherance of their scientific goals. The CNC Program?s success is supported by SCCC?s strong infrastructure and grounded in the program?s solid interactions among CNC members and the subprograms, as well as with other SCCC Research Program members and external collaborators.