Brigham And Women'S Hospital

NIH Research Projects · FY 2026 · 2010-05

PROJECT SUMMARY/ABSTRACT Highly secretory intestinal epithelial cells (IEC) are exquisitely sensitive to endoplasmic reticulum (ER) stress and depend upon the unfolded protein response (UPR) to maintain homeostasis and avoid intestinal inflamma- tion and neoplasia. Although a dysregulated IEC-associated UPR can directly cause intestinal inflammation, it is also evident it can orchestrate barrier protective responses. Recently, we discovered that an IEC-associated UPR can induce the generation of T helper 17 (Th17) cells with potentially non-pathogenic properties in a host- intrinsic and microbial independent manner that involves a final common pathway derived from IEC production of xanthine. These UPR-induced Th17 cells are interestingly also marked by expression of GATA3, a canonical marker of Th2 cells. The current research proposal addresses the unanswered question of how an IEC-associ- ated UPR induces a novel population of Th17/Th2 cells and the role played by the intestinal microbiota. Our long-term goals are to unravel the precise mechanisms about the relationship between an IEC-associated UPR and Th17 cells. The objective of this research is to understand how an IEC-associated UPR causes the sterile induction of Th17/Th2 double-positive cells in intestinal tissues and how these are phenotypically and functionally modified by the commensal microbiota and other IEC-associated events. Our central hypothesis is that IEC- associated ER stress is associated with xanthine production and increased differentiation of IECs into Tuft cells which together result in the development of an unusual subset of Th17 cells that co-express GATA3 but whose origin and function in intestines is poorly understood. The rationale for our proposed research is that developing such insights into these mechanisms may allow for the rationale induction of Th17 cells with non-pathogenic and potentially protective activities in intestines. Our central hypothesis will be tested with three specific aims: 1) investigate the function of Th17 cells generated in the setting of uncontrolled ER stress in IECs and the T-cell intrinsic signals required for their induction; 2) identify the signals emanating from the epithelium under ER stress that promote Th17/Th2 development and the regulation of Th17/Th2 cells induced by ER stress in IECs by the microbiota, and; 3) investigate the effect of xanthine, released in response to ER stress by epithelial cells, on host health and its mechanism in supporting Th17 development. In Aim 1, we will determine whether IEC-asso- ciated, UPR-induced Th17 cells are non-pathogenic and if this property is dependent upon GATA3. Aim 2 will show how an ER-stress-induced, Tuft cell expansion and specific commensal microbiota influence the differen- tiation and function of Th17 cells. Aim 3 will ascertain the therapeutic potential of Th17 cell induction by xanthine in intestinal diseases. Overall, this proposal is significant as it will provide new dimensions on our understanding of Th17 cell development, deeper insights into how an IEC-associated, UPR regulates CD4+ T cell differentiation, provide the first identification of Th17/Th2 cells in intestinal tissues and determine if a UPR-derived metabolite can be therapeutically co-opted for potential therapeutic use.

NIH Research Projects · FY 2025 · 2009-09

ABSTRACT This is a renewal application to extend post-intervention observational follow-up for 5 yrs in the VITamin D and OmegA-3 TriaL (VITAL), a randomized, placebo-controlled factorial trial of daily vitamin D3 (2000 IU) and marine omega-3 fatty acids (n-3 FAs; 1-g Omacor® fish-oil capsule, with eicosapentaenoic acid [460 mg] + docosahexaenoic acid [380 mg]) in the primary prevention of cancer and cardiovascular disease (CVD) among 25,871 US men aged ≥50 and women aged ≥55 yrs, including 5,106 African Americans. Median treatment was 5.3 yrs. Funding for the ongoing 2-yr post-intervention follow-up (plus data closeout/analysis) ends on May 31, 2020. A 5-yr extension will yield a median post-intervention follow-up of 7 yrs and median cumulative (intervention + post-intervention) follow-up of 12 yrs. During the trial, although vitamin D did not significantly reduce total invasive cancer incidence in the overall cohort, there was a promising signal for a reduction in total cancer mortality, and subgroup analyses indicated cancer protection in African Americans and those with normal body mass index. Although n-3 FAs did not reduce major CVD events in the total cohort, there were significant reductions in myocardial infarction (MI) and other coronary endpoints; subgroup analyses showed reductions in major CVD events in those with low baseline fish intake and in MI in African Americans. Longer follow-up to account for latency effects and additional research to learn which individuals may be most likely to derive a net supplementation benefit is warranted. Annual surveys will update risk factors and endpoint occurrence. Reported endpoints will be confirmed by medical record review and augmented with Medicare linkage surveillance for maximal and unbiased endpoint capture. Deaths will be ascertained via the National Death Index-Plus. Archived baseline blood/DNA samples will allow examination of whether effects of the trial agents on cancer and CVD vary by (a) genetic factors (targeted variants in genes related to vitamin D metabolism, absorption, or receptor function or n-3 FA synthesis and activation; gene-based ancestry; genetic risk scores); (b) vitamin K and magnesium status; and (c) novel vitamin D and n-3 FA biomarkers. Moreover, continued infrastructure funding will enhance the value of ongoing ancillary studies evaluating the trial agents’ role in prevention of other diseases (23 funded studies to date, including diabetes, cognitive decline, bone disorders, and autoimmune conditions) and allow for future ones, including an examination of intervention effects on tumor molecular markers, DNA methylation, and gene expression. Building upon VITAL’s strengths—including a large, well-characterized, racially diverse cohort with high compliance throughout the trial; archived blood samples; dietary and lifestyle assessments; and rigorously adjudicated endpoints—our proposal offers an exceptionally innovative and cost-efficient (<$100/participant/yr in direct costs) opportunity to advance knowledge about the role of vitamin D and n-3 FAs in human health and disease. This is a high- impact study due to the potential for major clinical and public health implications of the findings.

NIH Research Projects · FY 2025 · 2008-09

ABSTRACT Stroke is the third leading cause of death in women and the leading cause of disability in the US. New proteomic techniques measure a wide profile of proteins, providing a comprehensive picture of protein functions. Since proteins perform most biological functions, they may be leveraged as biomarkers or drug therapy targets. Although results for proteomics and cardiovascular disease have been promising, only one small prospective proteomics study of ischemic stroke has been conducted. The over-arching goal of this study is to discover novel proteome-wide and multi-omic associations for incident ischemic stroke. Building on an established record of success in this R01, we will measure 3072 proteins (Olink platform) in the Nurses’ Health Studies (NHS; 460 ischemic strokes/ 460 controls) and VITamin D Omega3 TriAL (VITAL; 300 ischemic strokes/ 300 controls), as well as leverage separately measured data from the Women’s Health Initiative (WHI; 214 ischemic strokes), Framingham Heart Study Offspring Cohort (FHSOC; 103 ischemic strokes), and UK Biobank (UKB; ~450 ischemic strokes). In Aim 1, we will test candidate protein hypotheses in 1334 strokes in meta-analyses using Olink data from the NHS, VITAL, WHI, FHSOC and UKB. Ischemic stroke subtypes, and interactions by sex and race will be tested. In Aim 2, we will perform robust, proteome-wide discovery in NHS and VITAL with validation of significant proteins in meta- analyses of WHI, FHSOC, and UKB. Validated proteins will be evaluated for causal associations using mendelian randomization (MR) analyses. Using proteomic and genetic data from UKB (53,000), we will generate protein polygenic allele risk scores for the validated proteins and test these protein polygenetic risk scores for ischemic stroke in MEGASTROKE and SiGN. Ischemic stroke subtypes, and interactions by sex and race will be explored. In Aim 3, we will leverage prior measures of metabolomic profiles and extensive biomarker and risk factor data in NHS, VITAL and WHI, using novel integrative approaches, including similarity network fusion, to combine proteomic and metabolomic data, biomarker and risk factor data, in associations for ischemic stroke and stroke subtypes. Findings will be compared in NHS, VITAL and WHI. The proposed study leverages Olink proteomic data from five cohorts to identify proteins and multi-omic signatures integral to the incidence of stroke, which can be used to improve stroke prediction and prevention strategies and identify new drug targets for stroke prevention.

NIH Research Projects · FY 2025 · 2008-09

The Boston Older Americans Independence Center (Boston OAIC) has transcended the institutional boundaries to forge an interdisciplinary research network among aging researchers from Boston's leading academic centers to foster translational research in function promoting therapies (FPTs) – pharmacologic, physical, nutritional, technological and behavioral interventions that reduce the burden of disabling functional limitations. During its prior funding cycle, the Boston OAIC established 3 resource cores and supported 17 external projects, 9 pilot projects, 9 research education career (REC) awards, and 4 developmental projects. These investments contributed directly to the advancement of several highly talented early career scientists, including 3 Beeson awardees; enhanced research innovation and productivity, reflected in high quality peer- reviewed publications; provided research core support to many NIH-funded innovative projects by OAIC investigators; enabled substantial expansion of OAIC's collaborations with other Boston area aging centers and with other OAICs; and spurred new technologies, inventions, and patents. During the past cycle, the OAIC's center of gravity shifted to Harvard-affiliated medical centers due to the relocation of the PI. With the inclusion of several Harvard geriatricians in its leadership, the Boston OAIC is now well integrated with the Harvard Geriatrics and Gerontology research community and programs, including its T32 training grant, Harvard Clinical Translational Science Institute, the Roybal Center, The New England Geriatrics Research Clinical Education Center, and the Glenn Foundation Center for Biology of Aging. During the next funding cycle, the Boston OAIC will maintain its thematic focus on FPTs and its positioning across the entire spectrum of translational science from mechanism elucidation, preclinical proof-of-concept studies, biomarker validation, epidemiologic investigation to randomized trials of FPTs. The OAIC’s research program is founded on a platform of four cores: Leadership and Administrative Core (LAC), Research Education Core (REC), Pilot and Exploratory Studies Core (PESC), and Resource Cores (RC). The OAIC will integrate 19 externally-funded studies, and REC, PESC and developmental projects into a cohesive interdisciplinary program that is supported by 3 resource cores: Function Assessment Core, Preclinical Discovery Core, Biostatistics and Data Science Core. The REC will recruit the most promising stars from a vast reservoir of talent at Harvard, Tufts and BU, and train them through didactic and mentored research programs. The OAIC’s strengths include: 1) its focus on FPTs; 2) access to a large pool of talented young investigators, especially physician scientists, including 3 Beeson scholars; 3) strong collaborative leadership which has maintained high research productivity and grant funding; 4) strong institutional support; 5) success in developing intellectual property; 5) supporting several seminal randomized trials of FPTs; and 6) its success in forging synergistic collaborations with other Boston area aging research programs and with other OAICs.

NIH Research Projects · FY 2026 · 2007-08

Project Summary Abstract. NUT carcinoma (NC, formerly NUT midline carcinoma) is an aggressive squamous carcinoma, affecting all ages, that is highly (>90%) lethal. The over-arching goal of this proposal is to improve survival of these patients through mechanism-driven identification of key oncogenic targets. NC is defined by NUTM1-fusion oncogenes, most commonly (~78%) encoding the BRD4-NUT protein. BRD4-NUT drives NC growth through the blockade of differentiation and maintenance of proliferation by forming very large (100kb-2MB), acetyl-histone-rich super-enhancers called megadomains (MD) that we described. MDs arise from the recruitment of p300, a histone acetyl-transferase (HAT), by NUT to acetylated histones bound by the dual bromodomains of BRD4, a BET family protein. We have shown that BRD4-NUT drives transcription of key oncogenic targets through the additional recruitment of numerous transcriptional activators by BRD4. Our demonstration that treatment with BET bromodomain inhibitors (BETi) that competitively inhibit binding of BET bromodomains to chromatin, can inhibit growth of NC in humans led to a new field investigating the role of BRD4 in cancer. Unfortunately, the efficacy of BETi monotherapy is limited by toxicity. In this next phase, we are investigating novel oncogenic mechanisms whose targeting can synergize with BETi therapeutically. Our preliminary data, together with what is established, has led us to formulate a mechanistic two-domain model of how NC growth is epigenetically driven. The model will be tested in the aims below with the overall goal of identifying a therapeutic combination of compounds optimized for maximally selective, synergistic tumor inhibition with minimized toxicity. The model proposes that the first, transcriptionally repressive domain formed by PRC2 enables NC growth by repressing the expression of pro-differentiation, tumor suppressive genes (tested in aim 1). The second, physically separate permissive MD is comprised of BRD4-NUT-p300 bound to acetylated histones that drive transcription of oncogenic genes. In this model, MD formation requires cooperative binding of both BRD4-NUT bromodomains (BD1 and BD2) to acetylated histones (tested in aim 2); and the acetyl- histone-permissive state that enables MD formation requires mono-methylation of H3K79 by DOT1L (aim 3). Inhibition of both of these domains would 1. repress transcription of oncogenic genes, such as MYC, and 2. de- repress transcription of genes critical for differentiation, senescence, and exit from cell cycle. Indeed, we have found that co-inhibition of EZH2 and BRD4-NUT is synergistic in vitro and in vivo. Specific Aim 1. Determine the oncogenic role of EZH2 in NUT carcinoma. Specific Aim 2. Determine the oncogenic roles of BRD4 bromodomains 1 and 2 in NUT carcinoma. Specific Aim 3. What is the role of DOT1L in BRD4-NUT megadomain formation and oncogenic function?

NIH Research Projects · FY 2025 · 2007-04

Overall - Project Summary/Abstract Please see each project/core for individual Project Summary/Abstract

NIH Research Projects · FY 2025 · 2007-04

Project Summary Most of what we know about visual search comes from experiments where observers perform hundreds of searches for the same things (e.g. several hundred searches for a vowel among other letters). However, most searches in the world are not performed in blocks. People look for one thing and then another. Emergency Department (ED) radiology where a radiologist might look for pneumonia in a chest X-ray, followed by a search for a possible stroke in a head CT, and so on, represents a socially important real-world example that we will study here. Decades of research have established rules of search, based on roughly uniform blocks of searches. Do those rules continue to apply if searches are mixed? The importance of this question can be seen if we consider the problem of when to end a search. Quit too soon and targets (like that stroke) can be missed. Quit too late and time is wasted, perhaps dangerously (e.g. if a driver perseverates on a search on the dashboard control panel). Models of search termination often propose an adaptive process where responses on previous trials serve to adjust the quitting threshold on subsequent trials. How can such an adaptive process work if the preceding trials come from many different tasks? We hypothesize that multiple adaptive rules coexist, but that they may be less optimal in the mixed environment. The answer to such questions will shape how we attempt to minimize error in settings like ED radiology. There are four series of proposed experiments. In Exp 1, trials of several different tasks are run, either in single-task blocks or mixed together. In Exp 2, the target remains the same on all trials but the distractors (and, thus, the search task) change. This is intended to emulate situations like screening mammography where the same target (e.g. a mass) is presented on visually different backgrounds (fatty vs dense breast parenchyma). In Exp 3, multiple tasks are available simultaneously (as in driving or managing the control panel of a complex machine). Here, the observer can choose, to some extent, whether to perform blocked or mixed searches. Finally, in Exp 4, we compare mixed and blocked searches using radiologists as observers in a version of the ED radiology task. Significantly, this will be the first medical image perception study of the ED task. While we expect that many of the basic principles of search will be similar in mixed and blocked conditions, we hypothesize that some rules, notably those pertaining to errors in search, will differ in mixed and blocked situations. Understanding the rules of search when the specific search task changes from moment to moment will be critical to reducing the errors that plague real world searches from the inconsequential failure to find a pen to those with potential life and death consequences.

NIH Research Projects · FY 2026 · 2007-01

The Boston/Jackson/Providence HIV Clinical Trials Unit (BJP HIV CTU) will conduct clinical trials to address research priorities of three NIAID HIV/AIDS Clinical Trials Networks (CTNs): HIV therapeutics; integrated strategies for HIV prevention; and HIV vaccines. The BJP HIV CTU brings together a collaborative group of highly experienced investigators with decades of experience in HIV-related treatment, prevention and vaccine research. The BJP HIV CTU comprises five existing Clinical Research Sites (CRSs): therapeutics CRSs at Brigham and Women’s Hospital (BWH) and Massachusetts General Hospital in Boston, MA and The Miriam Hospital in Providence, RI; a vaccine CRS at BWH; and a combined prevention and vaccine CRS at Fenway Health (also in Boston). Two new CRSs are proposed—a prevention CRS at University of Mississippi Medical Center in Jackson, MS and a vaccine CRS at Beth Israel Deaconess Medical Center (BIDMC) in Boston. The BJP HIV CTU and its component CRSs will function as an integrated, collaborative consortium to coordinate and implement clinical trials designed by the Leadership Groups of the NIAID-funded HIV/AIDS CTNs. The five existing CRSs have been extremely effective in conducting studies and contributing substantially to the scientific agendas of their respective CTNs. Expansion of our existing CTU to include two new CRSs devoted to HIV prevention and vaccine research, respectively, substantially enhances our capacity to conduct clinical trials critical to developing more effective means of HIV prevention and testing candidate vaccines. The proposed UMMC Prevention CRS is located in Jackson, MS a city in the southeastern United States with a predominantly African American population that ranked 4th highest in HIV infection and 5th highest in the rate of AIDS diagnoses among US metropolitan areas. The proposed Vaccine CRS within the Center for Virology and Vaccine Research at BIDMC will engage investigators who have made major and sustained scientific contributions to the HIV vaccine field over the past 15 years. The BJP HIV CTU will be led by three highly experienced PIs, Drs. Daniel Kuritzkes, Kenneth Mayer and Lindsey Baden, and will include highly accomplished CRS leaders and collaborating investigators. The CTU will function as an integrated, highly collaborative consortium that will have centralized planning, resource allocation, decision-making and financial management through an efficient administration plan. Centralized resources include a Clinical Research Laboratory, a Research Pharmacy Coordinator, Data and Quality Management Plans and a Community Engagement Core. The CTU has diverse and accessible populations for study, representing communities most affected by HIV. Each CRS has well- grounded connections in the communities in which they are based. The BJP HIV CTU is well-poised to carry out efficient high-quality clinical trials to address major questions in HIV clinical research. Its leadership and administrative structure will facilitate the conduct of studies that cross traditional CTN boundaries and will enable rapid responses to new scientific priorities as they emerge.

NIH Research Projects · FY 2025 · 2005-09

PROJECT SUMMARY/ABSTRACT Women and men are at different risks for disorders that occur at different stages of the lifespan from development through aging and their sex differences have critical implications for therapeutic efficacy. However, many of the mechanisms that explain these sex differences or disorders specific to women are still unclear. The mission of our Harvard BIRCWH is to develop the next generation of scientist-clinicians as leaders in the field of women’s health and sex differences who will contribute to understanding sex-dependent vulnerabilities to clinical disorders and those specific to women. This competing renewal application seeks continued support of a successful integrated interdisciplinary training program that is based on a translational approach to understanding conditions with a higher incidence or different expression in women than men. The program is modeled in the context of a lifespan perspective to identify etiologic mechanisms during fetal development, puberty, adulthood, and aging, including the child-bearing years and menopause. Further, an underlying assumption of our BIRCWH program is that an understanding of the role of hormones and genes will provide the basis for understanding sex-dependent vulnerabilities to clinical disorders. The Division of Women’s Health at Brigham and Women’s Hospital (BWH) and the Innovation Center on Sex Differences in Medicine, ICON-X (MGH), are the home sites for this Harvard-wide training program. The program capitalizes on the long tradition of interdisciplinary research in women’s health and sex differences with mentors across Harvard institutions: BWH, MGH, Beth Israel-Deaconess Medical Center, Boston Children’s Hospital, Dana Farber Cancer Institute, McLean Hospital, Harvard T.H. Chan School of Public Health, Harvard Medical School and the Eli & Edythe Broad Institute. Each BIRCWH scholar is assigned a team of mentors to operationalize translational thinking. Primary Mentors are in clinical or basic research and provide the site at which the scholar works. Secondary Mentors are in basic or clinical research (as a counterpart to the Primary) and help to guide thinking, coursework and readings, depending on the scholar’s interests. Career Mentors advise scholars in the relevant departmental and academic structures for career advancement. Mentors in Health Disparities expose scholars to thinking about disparities and how health processes are influenced by socioenvironmental factors. The Harvard BIRCWH program focuses on disorders with differences in incidence and/or expression in women than men, including: Cardiovascular Disorders; Reproductive Endocrine & Neuroendocrine Disorders; Neuropsychiatric Disorders; Autoimmune Disorders; and Female Cancers. By capitalizing on the vast resources and faculty at Harvard and our 19-year successful BIRCWH history, Harvard is an ideal site for continuing to offer an integrated, interdisciplinary and truly translational program that will continue to train the next generation of leaders in women’s health and sex differences in medicine.

NIH Research Projects · FY 2025 · 2005-07

The Ras pathway is one of the most commonly deregulated pathways in human cancer, yet there are still no effective therapies for Ras-driven tumors. In this application we will be studying two genetically distinct Ras-driven malignancies: NF1-mutant nervous system tumors (MPNSTs) and KRAS-mutant pancreatic cancers. Specifically, we aim to elucidate an important mechanism by which these Ras-driven cancers are protected from MEK inhibitors and will use this insight as a basis for developing and testing rational combination therapies. Collectively, these studies will reveal novel insight into innate mechanisms that protect these tumor types from Ras pathway inhibitors and will also lead to the development of several new therapeutic strategies that can be translated into clinical trials.

NIH Research Projects · FY 2025 · 2005-07

SUMMARY Increased intestinal permeability, i.e., barrier loss, is a common characteristic of many gastrointestinal and systemic disorders and has been hypothesized to be a key contributor to disease progression. Animal models of disease provide strong support for this hypothesis, but, due to the absence of drugs that reverse disease- associated permeability increases, the therapeutic potential of intestinal barrier restoration is untested. Myosin light chain kinase (MLCK) is a signaling node that, when activated, increases permeability. In inflammatory bowel disease (IBD) patient biopsies, MLCK activity is upregulated and correlates with disease severity. Recent studies have shown that inflammatory stimuli trigger recruitment of a specific MLCK isoform, MLCK1, to the perijunctional cytoskeleton and that this MLCK1 recruitment is required for barrier loss. Further, disruption of the MLCK1 interactome that directs recruitment is therapeutically superior to anti-TNF in experimental IBD. Therefore, the long-term goal of this program is to create the detailed understanding that will enable development of agents that target the MLCK1 interactome to restore barrier function and benefit IBD patients. The overall objectives of this application are to i) discover the molecular basis of MLCK1 interactome function and ii) apply this knowledge to block MLCK1 recruitment in experimental IBD. The central hypothesis is that prevention of MLCK1 interactome-dependent recruitment can restore the intestinal barrier. The rationale for this project is that elucidating the molecular mechanisms of MLCK1 interactome function and assessing the preclinical efficacy of targeting interactome components will create the scientific foundation needed for translation to the clinic. The central hypothesis will be tested via three specific aims: i) define mechanisms that regulate interactions between MLCK1 interactome components and identify sites that can be targeted to disrupt these interactions; ii) determine how, when, and where these interactions are modified by inflammatory signals and the potential of obstructing key interactions as a means to block MLCK1 recruitment and restore barrier function in intestinal stem cell models; and iii) characterize the impact of interfering with dynamic regulation of the MLCK1 interactome in preclinical mouse models and assess interactome staus in patient biopsies. This proposal is conceptually and technically innovative because it will define function and regulation of the newly-recognized MLCK1 interactome using sophisticated in vitro and in vivo models as well as patient biopsies. These studies are significant because they are expected to create the structural and mechanistic insight required for development of molecular therapies that modify unique MLCK1 interactome functions to prevent or reverse barrier loss in IBD. Ultimately, advances that build on this critical information will create a foundation for development of safe, non-immunosuppressive means to restore the intestinal barrier and induce and maintain remission in IBD. These agents are also likely to provide benefit in other barrier loss- associated disorders, including celiac disease, food allergy, diabetes, and metabolic syndrome.

NIH Research Projects · FY 2025 · 2005-06

Project Summary/ Abstract Kidney Injury Molecule-1 (KIM-1) is the most upregulated protein in proximal tubular epithelial cells in various states characterized by epithelial cell dedifferentiation: ischemia, toxic renal injury, and renal cell carcinoma. We have cloned, generated cells and animals expressing wild-type and mutant KIM-1, and created monoclonal and polyclonal antibodies to, human, mouse, rat, pig, dog, and zebrafish KIM-1. We have reported that the KIM-1 ectodomain is cleaved into the blood and urine of subjects with acute (AKI) and chronic (CKD) kidney injury and is a sensitive and specific kidney injury biomarker to detect kidney injury and predict progression of CKD. KIM-1 has been qualified by the FDA for preclinical and clinical use in kidney safety studies. We have discovered that KIM-1 transforms kidney epithelial cells into semiprofessional phagocytes making it the first nonmyeloid phosphatidylserine receptor. We have described a novel phagocytosis pathway that links autophagy to KIM-1-mediated phagosome maturation and MHC restricted antigen presentation in epithelial cells. We have shown that KIM-1 expression in early AKI is adaptive, but chronic expression leads to CKD with severe fibrosis, secondary hypertension, and cardiac hypertrophy. A mouse lacking the extracellular mucin domain, important for phagocytosis, is protected against development of fibrosis. We have found that KIM-1 mediates uptake of palmitate-bound albumin and recently found an inhibitor of KIM-1- mediated phagocytosis by screening a small molecule library. The inhibitor reduces cell lipotoxicity and fibrosis in a novel mouse model of diabetic kidney disease. The current competing renewal application builds upon and extends our prior findings. Our goal is to further characterize KIM-1-mediated uptake of fatty acid bound albumin (FA-Albumin), and the implications of this uptake for cellular injury and maladaptive repair, including cell senescence leading to profibrotic and proinflammatory responses that ultimately lead to progressive CKD. KIM-1 may be a drug target to prevent and treat CKD. We hypothesize that persistent KIM-1-mediated endocytosis of FA-Albumin and subsequent signaling leads to toxicity. FA-Albumin uptake leads to a mitochondrial dysfunction, DNA damage response (DDR), G2/M arrest, mTOR signaling, TASCC formation, and a prosecretory fibrotic phenotype. In addition KIM-1-FA-Albumin uptake leads to chronic tissue inflammation in part due to tertiary lymphoid tissue development through LTaβ/LTβR signaling. In Specific Aim 1 we will characterize binding of KIM-1 to FA-Albumin and determine the architecture, structural dynamics and molecular basis for FA-Albumin binding to KIM-1. In Specific Aim 2 we will characterize the intracellular consequences of KIM-1 mediated FA-Albumin endocytosis, particularly on mitochondrial function, DNA damage, the DDR, cell cycle arrest and the profibrotic secretome. In Specific Aim 3 we will evaluate the role of KIM-1 mediated FA- Albumin uptake and DDR in LTaβ/LTβR signaling leading to tertiary lymphoid tissue (TLT) formation with consequent pro-inflammatory consequences in animal models of AKI to CKD transition.

NIH Research Projects · FY 2025 · 2002-09

PROJECT SUMMARY CD1 autoreactive T cells in human immune mediated skin disease D. Branch Moody, PI Human skin contains over ten billion resident T cells that participate in host defense, barrier function and autoimmune skin disease. For decades, research into T cell autoreactivity has emphasized T cell receptor (TCR) recognition of MHC-peptide complexes. This R01 program proposes basic research into a different mechanism involving TCR contact with human CD1a proteins expressed on Langerhans cells and lipid antigens. Building on prior studies that discovered lipid antigens and showed the molecular basis for their display by CD1a, we recently discovered an unexpected model whereby T cell receptors directly contact CD1a, and certain endogenous lipids bound to CD1a inhibit TCR contact. The new mechanism of negative regulation expands the scope of CD1a function on Langerhans cells and has potentially broad implications for the control of CD1a autoreactive Th22 T cells in human skin. Using collagen matrices and CD1a tetramers to isolate pure CD1a autoreactive T cells, we will determine T cell receptor patterns, effector functions and the identities of antagonist and agonist lipids in disease settings. We will determine the natural functions of lipid blockers by measuring their production in healthy and diseased skin, as well as in Langerhans cells and related cell types. We will determine the therapeutic potential of lipid blockers by synthesizing analogs to optimize their T cell inhibitory properties. Since we now know that contact allergens can function as CD1a presented antigens, we will test a new model of contact hypersensitivity that involves direct recognition of allergens by lipid specific T cells. Further, we will test the ability of CD1a blockers to prevent T cell response in vivo in CD1a transgenic mice and in human skin explants. In contrast to the ~ 5000 MHC I and II allomorphs present in humans, nearly all humans express the same kind of CD1a proteins. This simplified and relatively uniform T cell system suggests that lipid antigens, lipid antagonists and T cell receptor patterns discovered here will be applicable to all people and can therefore be used to develop therapies against autoimmune skin diseases.

NIH Research Projects · FY 2024 · 2002-09

PROJECT SUMMARY CD1 autoreactive T cells in human immune mediated skin disease D. Branch Moody, PI Human skin contains over ten billion resident T cells that participate in host defense, barrier function and autoimmune skin disease. For decades, research into T cell autoreactivity has emphasized T cell receptor (TCR) recognition of MHC-peptide complexes. This R01 program proposes basic research into a different mechanism involving TCR contact with human CD1a proteins expressed on Langerhans cells and lipid antigens. Building on prior studies that discovered lipid antigens and showed the molecular basis for their display by CD1a, we recently discovered an unexpected model whereby T cell receptors directly contact CD1a, and certain endogenous lipids bound to CD1a inhibit TCR contact. The new mechanism of negative regulation expands the scope of CD1a function on Langerhans cells and has potentially broad implications for the control of CD1a autoreactive Th22 T cells in human skin. Using collagen matrices and CD1a tetramers to isolate pure CD1a autoreactive T cells, we will determine T cell receptor patterns, effector functions and the identities of antagonist and agonist lipids in disease settings. We will determine the natural functions of lipid blockers by measuring their production in healthy and diseased skin, as well as in Langerhans cells and related cell types. We will determine the therapeutic potential of lipid blockers by synthesizing analogs to optimize their T cell inhibitory properties. Since we now know that contact allergens can function as CD1a presented antigens, we will test a new model of contact hypersensitivity that involves direct recognition of allergens by lipid specific T cells. Further, we will test the ability of CD1a blockers to prevent T cell response in vivo in CD1a transgenic mice and in human skin explants. In contrast to the ~ 5000 MHC I and II allomorphs present in humans, nearly all humans express the same kind of CD1a proteins. This simplified and relatively uniform T cell system suggests that lipid antigens, lipid antagonists and T cell receptor patterns discovered here will be applicable to all people and can therefore be used to develop therapies against autoimmune skin diseases.

NIH Research Projects · FY 2025 · 2001-09

ABSTRACT The incidence of immune mediated diseases, including inflammatory bowel disease, has grown exponentially over the last decades. This has been linked to a variety of factors, including dietary preservatives and processed food. Interest has recently turned to salt (NaCl) is a potential culprit. Abundant data show that high salt diets can activate intestinal mucosal inflammation and increase severity up to a variety of experimental disorders, including inflammatory bowel disease and multiple sclerosis, and experimental animals. Further, some epidemiologic studies have correlated salt intake with frequency of immune-media disease. We recently discovered that genetic or pharmacological inhibition of claudin-2, which forms a paracellular Na+ channel within the intestinal epithelium, is an effective intervention in experimental, immune-mediated inflammatory bowel disease. Our additional studies indicate that claudin-2 overexpression in transgenic mice promotes Th17 polarization of mucosal immune cells while claudin-2 deletion promotes Tregs development. A high salt diet also activates mucosal IL-17 production in claudin-2 transgenic, but not knockout, mice. Thus, expression of the paracellular Na+ channel protein claudin-2 synergizes with dietary salt to promote mucosal inflammation. This is relevant to wide variety of inflammatory intestinal disorders, especially inflammatory bowel disease, because claudin-2 expression is markedly upregulated in these settings. Thus, this is the potential of creating a vicious cycle where claudin-2 and dietary salt promote inflammation and, in turn, the inflammation signals back to the epithelium and causes further upregulation of claudin-2. High salt diets are also well-recognized to affect the microbiota. We found that the effect of high salt diet on the microbiome was modified by claudin-2 overexpression or deletion. This suggests that the immune changes induced by claudin-2 expression or deletion also modify the microbiome or, alternatively, microbial changes feedback to alter the immune system. Examples of both mechanisms have been reported in other settings. Finally, is possible that claudin-2 and dietary salt affect immune system and microbiota separately. Regardless of which affect his primary, these data provide strong support for the conclusion that claudin-2 inhibitors should be developed as potential therapeutic agents. Unfortunately, structural data and general understanding of how claudin-2 paracellular channels are created and regulated are limited. Thus, the second aim of this proposal seeks to define structure-function relationships in order to understand the molecular mechanisms that determine claudin-2 biology and to identify potential molecular interfaces to be targeted therapeutically.

NIH Research Projects · FY 2025 · 2001-05

Project Summary/Abstract Mutations in the Presenilin (PSEN) genes account for ~90% of all causative mutations in familial Alzheimer's disease (FAD). During the last two decades, our large numbers of genetic studies using conditional knockout and knockin mice have demonstrated that Presenilin (PS) is essential for learning and memory, synaptic function and neuronal survival, and that PSEN1 mutations cause loss of its essential function and g- secretase activity. Our genetic findings are further supported by cell culture and biochemical studies showing that ~90% of PSEN1 mutations cause loss of g-secretase activity. In this renewal application (YEARS 20-24), we propose to extend our successful PS studies to the development of novel therapies for FAD. Specifically, we will perform preclinical studies using recombinant adeno-associated virus to deliver wild-type human PS1 (hPS1) to PS mutant mouse brains and then determine whether hPS1 rescues impaired g-secretase activity, neurodegeneration, synaptic and memory deficits caused by PS mutations (Aim 1). The amyloid precursor protein (APP) was the first protein associated with sporadic AD, and the APP V717I mutation was the first FAD mutation identified. The genetic link of APP mutations to FAD and the presence of amyloid plaques in sporadic AD brains highlight the importance of APP in AD pathogenesis. Despite its importance, APP normal function in the cerebral cortex remains unclear due to genetic redundancy of its family members, APLP1 and APLP2, and perinatal lethality of APP/APLP1/APLP2 triple knockout mice. We therefore propose to investigate the normal physiological role of APP family through the generation and multidisciplinary analysis of APP/APLP1/APLP2 conditional triple knockout mice, in which all APP family members are selectively inactivated in excitatory or inhibitory neurons of the cerebral cortex (Aim 2). Despite the abundance of APP, APLP1 and APLP2 in excitatory neurons of the cerebral cortex, their inactivation does not lead to early lethality, loss of cortical neurons or increases of apoptosis. We will investigate the role of APP family in excitatory and inhibitory neurons and how APP family regulates hippocampal local circuits. The completion of the proposed study will provide preclinical proof-of-concept data on PS based therapy for FAD and shed light on how APP family regulates neuronal excitability and synaptic plasticity in the hippocampal network.

NIH Research Projects · FY 2025 · 2001-03

Project Summary Activation of  T cells is the mainstay of host defense against Mycobacterium tuberculosis (Mtb) infection and tuberculosis disease (TB). For decades,  T cells were thought to recognize solely peptide antigens bound to polymorphic antigen presenting molecules encoded in the MHC locus. However, the discovery that human CD1 proteins present mycobacterial antigens to T cells provides fundamentally new perspectives on the role of T cells in host defense. First, the foundational studies for this R01 proposal showed that CD1 proteins present mycobacterial glycolipids, phospholipids and lipopeptides to  and  T cells. Thus, lipids must now be considered a diverse class of natural antigens for the human immune system. Second, whereas the MHC locus is the most polymorphic of the human genome, the non-polymorphic nature of CD1 genes creates a situation in which T cell responses are not restricted to an individual’s genotype. Such ‘donor-unrestricted T cells’ have simplified patterns of antigen recognition and T cell receptor usage that could be exploited for therapy. Building on substantial published and unpublished data, this human-focused renewal proposal seeks to test a general model of antigen recognition in which T cell receptors (TCRs) bind onto a roof structure in CD1, or shift away from the roof to contact bacterial antigen. Using recently validated human CD1a, CD1b and CD1c tetramers, we will discover new conserved TCRs that are equivalent to NKT TCRs in the CD1d system. We propose to determine the effector functions of newly discovered innate T cell types in humans, focusing on genes that define an innateness gradient and the known effector functions needed to protect the host against Mtb. Finally, we combine newly produced reagents, including tetramers, transfectants and monoclonal antibodies, to use the guinea pig as a tractable model for study of host T cell responses in the lung and over time. These studies contribute to an innovative view that the human T cell system recognizes and responds to lipid antigens.

NIH Research Projects · FY 2025 · 2001-02

We have been at the forefront in elucidating the mechanisms and physiology of Coat Protein I (COPI) transport. We are proposing four lines of future investigation to maintain this track record of achievement. First, following up on our recent discovery that COPI generates not only vesicles but also tubules, and the small GTPase Cdc42 promotes COPI tubule formation through an intrinsic ability to bend membrane, we will elucidate how Cdc42 achieves this remarkable feat. In addition, as Cdc42 belongs to the Rho family of small GTPases, and we have found that other Rho members also affect COPI transport, we will elucidate how they could affect COPI vesicle and tubule formation. Second, we will address a current controversy regarding how COPI bends membrane. Whereas coat proteins are predicted to assemble into protein lattices with regular geometry in bending membrane, COPI has been found recently to assemble into lattices with irregular geometry. We will examine whether this apparent exception is due to the reconstitution of COPI vesicles that has thus far not accounted for all the factors needed for a physiology reconstitution of COPI vesicles. Third, we will follow up on our recent discovery that has identified a novel role for a ciliary protein, known as IFT20. We have found that IFT20 exists at the Golgi, where it promotes COPI tubular transport. Thus, we will elucidate how IFT20 exerts this novel role. Fourth, we will elucidate how a point mutation in a core component of the COPI complex, known as yl-COP, leads to immunodeficiency in affected individuals. We have already elucidated one explanation, which involves defect in COPI binding to the KDEL receptor, leading to stress in the endoplasmic reticulum (ER) to impair the function of T and B cells. However, because COPI binds to other cargo proteins, including a large family of proteins that promote exit from the ER, known as ER cargo receptors, we will identify those ER cargo receptors affected by the yl-COP mutation and then elucidate how their defective binding by COPI leads to altered cellular functions. In addition, as we have found that the y l -COP mutation also impairs COPI tubular transport, we will elucidate a mechanistic explanation for this additional effect of the mutation. We anticipate that the completion of these four aims will advance a basic understanding of how COPI acts to generate transport carriers, as well shed physiologic insights into cellular processes that requires this transport.

NIH Research Projects · FY 2025 · 1999-09

ABSTRACT Autoimmune disease is the third most common disease category after cancer and heart disease that afflicts 23.5 million Americans. This grant renewal continues to focus on the role of neutrophil FcγRs in autoimmune diseases. However, it has shifted from understanding the role and regulation of neutrophil FcγRs in neutrophil recruitment following intravascular IgG-immune complex deposition in glomerulonephritis (GN) to examining how engaging FcγRs converts neutrophils into highly immunogenic antigen presenting cells (nAPC) and their contribution to autoimmune disorders effecting the kidney. This new direction is based on our recent studies showing that engaging mouse FcγRs or human FcγRIIA or IIIB on mature neutrophils with IgG-complexed antigen (immune complexes) leads to their differentiation into highly active APCs. These cells are comparable to cDCs in their ability to activate naïve T cells and cross-present soluble antigen to CD8 T cells, properties previously assigned almost exclusively to cDCs. Engaging FcγR with an anti-FcγRIIIB-antigen conjugate recapitulates the activity of immune complexes and its administration in FcγR humanized mice generates nAPCs in vivo that elicit robust acquired immunity. Studies in lupus patient samples indicate that nAPC frequency in blood correlates with clinical disease scores, which suggests that nAPCs are pathogenic. Single cell transcriptional analyses and validation studies implicate the pioneer transcription factor PU.1 in neutrophil to nAPC conversion and suggest that transcriptionally defined neutrophil populations convert to two nAPC subsets with distinct gene signatures and functionality. Furthermore, we provide evidence that FcγR internalization and a defined epigenetic regulator play a key role in conversion. Here we propose to 1) Elucidate the functionality of nAPC subsets and the role of epigenetic regulation in neutrophil to nAPC conversion, 2) Elucidate the route of intracellular trafficking of FcγR bound to antibody-antigen complexes and identify mechanisms of FcγR induced generation of immunogenic nAPCs using CRISPR-Cas9 based genetic screens, and 3) Interrogate the role of nAPCs in Antineutrophil cytoplasmic antibody (ANCA)- associated glomerulonephritis and the effect of antigen-tolerized nAPCs in disease outcomes. Dysregulation of the immune system is the basis of many autoimmune diseases for which current treatments are effective in only a subset of patients and are often neither curative nor durable. Neutrophils with APC markers have been observed in diseases from autoimmune diseases to cancer. Results from this proposal may provide important insights into the molecular and cellular pathways governing neutrophil conversion to immunogenic APCs. This could provide insights into the pathogenesis of many immune related disorders and lay the groundwork for new treatments that potentially non-invasively generate a large pool of antigen carrying APCs designed to elicit tolerance or acquired immunity.

NIH Research Projects · FY 2026 · 1998-09

PROJECT SUMMARY/ABSTRACT CEACAM1 is a single-pass type I transmembrane protein and the primordial member of the carcinoembryonic antigen (CEA) family of immunoglobulin molecules that is expressed on the apical and basolateral surfaces of intestinal epithelial cells (IEC) and on a wide range of immune cell types. However, its functions on intestinal epithelial cells (IECs) are unknown. The current research proposal addresses the unanswered question of whether CEACAM1 acts to convert microbial or host signals from the apical and/or basolateral surface into the production of antimicrobial peptides (AMP) by IECs. The ability of the IEC to sense the presence of microbes and deliver antimicrobial peptides (AMP) into the lumen represents a major mechanism of mucosal defense. Our long-term goals are to determine whether CEACAM1-mediated stimulation of AMPs protects from commensal and pathogenic microbe invasion of the IEC and susceptibility to colitis, the vectoral direction required for this stimulation (apical and/or basolateral), whether it is specifically dependent upon CEACAM1 isoforms with a short (S) cytoplasmic tail and if high-affinity ligands for CEACAM1 can be developed to stimulate these protective activities. The objective of this research is to elucidate how CEACAM1 stimulates AMP production and whether this information together can be co-opted for therapeutic purposes. Our central hypothesis is that CEACAM1 functions as a novel microbial sensor that responds to specific microbes or host ligands with production of AMPs that are important to barrier protection. This rationale is derived from recent studies that a specific deficiency of CEACAM1 in mouse IEC in vivo leads to decreased AMP expression by IECs coupled to increased susceptibility to colitis and invasion by pathogenic microbes. Our central hypothesis will be tested with three specific aims: 1) Determine whether IEC-associated CEACAM1 regulates the production of AMP and host susceptibility to colitis and enteropathogens; 2) Determine whether specific CEACAM1 isoforms are responsible for delivering activat- ing signals to induce AMPs, and; 3) Define a therapeutic strategy to enhance human CEACAM1 ligands that interact homophilically to induce these protective responses. In Aim 1, we will determine the innate and/or adap- tive origins of the intestinal inflammation that ensues from CEACAM1-deficiency in the IEC and whether this involves increased bacterial translocation of founding populations in the lumen. Aim 2 will determine whether CEACAM1-S isoforms can stimulate IECs to produce AMPs in response to pharmacologic and microbial signals and whether such stimulation can occur in response to ligation of CEACAM1 on the apical and basolateral sur- faces. In Aim 3, we seek to generate human CEACAM1 variants with enhanced ability to stimulate IEC produc- tion of AMP in vitro and in vivo and as such provide protection from experimental colitis. Overall, this proposal is significant because it will define CEACAM1 as a microbial sensor, identify novel microbial sources as ligands for CEACAM1 and create high-affinity ligands with therapeutic potential that together have important implications for inflammatory bowel disease and mucosal defense mechanisms.

NIH Research Projects · FY 2025 · 1998-08

In its most recent research plan, the NHLBI’s National Center for Sleep Disorders Research identified the need to foster a strong, highly qualified and well-trained workforce as its central priority. The Mass General Brigham Program for Training in Sleep, Circadian and Respiratory Neurobiology is based at the Brigham and Women’s Hospital in Boston. Over the past 27 years, this program has evolved to rise to the new challenges in our field, including providing training in state-of-the-art research techniques from molecular biology to population sciences related to sleep, circadian and respiratory neurobiology. This program provides targeted, structured, and comprehensive research training to prepare outstanding individuals at multiple levels of scholarly achievement, from medical students to post-doctoral fellows, for academic positions in the broad fields of sleep, circadian and respiratory neurobiology. For each trainee, the training program consists of core required courses and activities, elective courses and activities, and an intensive research experience. Interdisciplinary and translational research is a highlight of this program, and formal mentoring and tracking components are integral features. Intensive research training experiences are available across the breadth of sleep, circadian and respiratory neurobiology areas, including basic, translational, and clinical research opportunities, with a program project that spans multiple laboratories and institutions. There are 27 Full Preceptors that span 9 institutions, including 2 medical schools, with extensive experience and demonstrated success at training pre-doctoral and post-doctoral fellows, well-funded research programs (supported by $31 million of direct costs annually), and outstanding resources that trainees utilize for research. In addition, we have 7 Associate Preceptors who are actively being trained to be our next generation of mentors. Our training record over the past decade reveals the success of our efforts to train leaders in academic sleep science. Of our pre- and post-doctoral trainees funded by this training grant over the last 15 years, 96% and 94% of our pre-doctoral and post-doctoral trainees, respectively, are currently active in academic/research-intensive careers, with 77% of our post-doctoral trainees currently employed in academic research careers. Of the 37 post-doctoral trainees who were supported by this training grant before 2014, 54% report being PI on a research grant (54%), almost all of whom have a faculty rank of Assistant Professor or higher. These data demonstrate the successful record of our participating faculty at training both pre- and post-doctoral trainees for research-intensive careers. Funds are requested to support four pre-doctoral graduate students, three pre-doctoral short-term summer medical students, and eight post-doctoral trainees. Based on our highly competitive application process, we are confident these positions will be filled by outstanding future leaders. This program has grown and been refined over the two decades since its inception and meets the nationally-recognized need to increase the number of highly-qualified investigators in sleep and circadian science and medicine.

NIH Research Projects · FY 2025 · 1998-01

Project Summary Cholera is a severe dehydrating diarrheal disease caused by Vibrio cholerae. This Gram-negative rod has the unusual capacity to colonize the small intestine and to cause explosive epidemics. Here we will address fundamental questions in V. cholerae-host interactions, leveraging many of the approaches and tools we have created in the past decades. V. cholerae O1, the cause of pandemic cholera, is divided into Ogawa and Inaba serotypes, which differ only by the presence or absence of methylation of the terminal O-antigen sugar respectively. Switching of the Ogawa and Inaba serotypes during cholera epidemics has been recognized for over a century, but the consequences of serotype conversion on pathogen fitness are not clear. We discovered that the Ogawa serotype has greater in vivo fitness than the Inaba serotype and that the two serotypes rely on distinct metabolic process for growth in vivo. Thus, there are unexpected direct or indirect phenotypic and physiological consequences of O-antigen methylation on V. cholerae growth in vivo. The consequences and mechanisms that underlie the in vivo fitness differences of the V. cholerae serotypes will be determined in Aim 1. Cholera toxin (CT) triggers the intestinal fluid secretion that largely accounts for choleric diarrhea. We found that CT also leads to the secretion of hundreds of host proteins identified in diarrheal fluid and re-models the intestinal epithelial transcriptional response to V. cholerae. Many of these proteins and transcripts are linked to innate immune responses and we found that one of these proteins, surfactant protein D (SP-D), restricts V. cholerae growth in the intestine. In Aim 2, we will analyze the protective mechanisms mediated by SP-D and investigate the roles of additional V. cholerae-induced secreted host proteins in impeding the pathogen’s colonization, to uncover host innate axes that protect against V. cholerae infection. Cholera epidemics often spread extremely rapidly, and host passaging increases V. cholerae infectivity, but the V. cholerae and host genes that govern cholera transmission are largely unknown. In Aim 3, we will leverage our experience with pathogen barcoding and a new computational framework to extend analyses developed in Aims 1 and 2, examining the roles of serotype and CT in modulating host-priming of infectivity. Additional pathogen pathways and host processes that control V. cholerae infectivity will also be elucidated, to deepen understanding of cholera transmission. Collectively, the proposed research will yield new understanding of the interconnected processes that govern V. cholerae intestinal colonization and infectivity as well as the host factors and mechanisms that limit colonization and control transmission. This work will provide new perspectives on the biology of the two V. cholerae serotypes, and on the actions of cholera toxin in stimulating innate host defense responses against the pathogen. Our findings will also have important translational implications for design of new cholera therapeutics and vaccines.

NIH Research Projects · FY 2025 · 1997-04

Abstract This is a competing renewal application of our longstanding R01 Grant CA74305 that concerns the development and application of chemical approaches to enhance the understanding of cell signaling pathways and the attachment, removal, and function of protein post-translational modifications (PTMs). In the next cycle, we plan to investigate how ubiquitination regulates two key cancer-related signaling enzymes, PTEN and Akt, how several cancer-connected E3 ubiquitin (Ub) ligases target and catalyze Ub transfer to their protein substrates, and how mTORC2 phosphorylates its major substrate Akt. PTEN is one of the most commonly mutated tumor suppressor genes and catalyzes the conversion of phospholipid phosphatidylinositol-3,4,5-triphosphate (PIP3) to PIP2 and in this way antagonizes the action of the PI3-kinase/Akt oncogenic signaling pathway. Akt is a protein Ser/Thr kinase that is activated by PIP3 and drives tumor formation. Akt is an intensively studied cancer therapeutic target. E3 Ub ligases are a large family (>600 members) of enzymes that target Akt, PTEN, and thousands of cellular substrates for Lys ubiquitination. Our research program will continue to develop and employ protein semisynthetic methods including ubiquitin hydrazide installation to generate mimics of mono-ubiquitinated proteins and covalent E3-Ub-substrate ternary complexes. Our three specific aims are: 1. Elucidate the basis of PTEN and Akt regulation by Lys ubiquitination; 2. Delineate the catalytic mechanisms and protein substrate recognition of key E3 ubiquitin ligases; 3. Determine the molecular basis of mTORC2 mediated- phosphorylation of Akt. With site-specifically modified signaling proteins in hand, we will integrate kinetic assays, structural analysis, binding measurements, and cell-based studies to clarify key regulatory and signaling features. Upon completion of this research effort, we will broaden the knowledge of how Lys ubiquitination targets critical cancer- related proteins and influences their functions. We will also deepen our understanding of how the multisubunit mTORC2 complex is able to activate Akt. Moreover, these studies should pave the way for new therapeutic strategies to combat pathway dysregulation in cancer. This research program will also enable the training of the next generation of biochemical investigators with an interest in cancer.

NIH Research Projects · FY 2025 · 1996-02

PROJECT SUMMARY/ABSTRACT Altered lipid and glucose homeostasis in response to overnutrition is central to the pathogenesis of non- alcoholic fatty liver disease (NAFLD). Because current management options remain limited, the discovery of new metabolic pathways will serve to identify novel opportunities for intervention. This research proposal addresses the unanswered question of how membrane lipids regulate nutrient homeostasis. Our long-term goal is to understand the relationships between membranes and metabolic regulation and to leverage these for therapeutic purposes. The objective of this research is to elucidate lipid-mediated regulation of acetyl- CoA metabolism in health and overnutrition. Acyl-CoA thioesterase 12 (Acot12), which is highly expressed in liver and intestine, is the enzyme responsible for the cytosolic conversion of acetyl-CoA to acetate plus CoA. Acot12 comprises tandem N-terminal enzymatic domains and a C-terminal lipid-binding domain. The central hypothesis is that Acot12 activity controls lipid and glucose homeostasis by limiting utilization of acetyl-CoA for lipogenesis and for transcription factor acetylation. The rationale is that identifying the molecular mechanisms that underlie this novel lipid-regulated pathway should reveal distinct new insights into the pathogenesis of NAFLD. Guided by extensive preliminary data, the central hypothesis will be tested in three specific aims: 1) To define mechanisms whereby Acot12 controls lipid and glucose metabolism in liver; 2) To demonstrate key roles for Acot12 in intestinal lipid absorption and barrier function; and 3) To elucidate the cellular and molecular determinants of Acot12 activity. Aim 1 will test the hypothesis that Acot12-mediated hydrolysis of acetyl-CoA limits substrate availability for lipogenesis and reduces transcriptional expression of glycerolipid biosynthetic and gluconeogenic genes through acetylation of transcriptional regulators. We will further examine whether reduced production of lysophosphatidic acid functions to preserve Hippo signaling, thereby inhibiting hepatocarcinogenesis. Aim 2 will test the hypothesis that upregulation of intestinal Acot12 in response to overnutrition promotes glycerolipid trafficking with enterocytes, as well as the assembly and assimilation of chylomicrons. We will further investigate whether Acot12 maintains the barrier function of the intestinal epithelium in the setting of overnutrition by reducing inflammation and necroptotic cell death, thereby preventing hepatic injury in the setting of diet-induced steatosis. Aim 3 will test the hypothesis that Acot12 localizes preferentially to the cytoplasm or nucleus depending on metabolic conditions. We will define the lipid ligand(s) of Acot12 and demonstrate how lipid binding regulates both cellular localization and enzymatic activity. Overall, this proposal will elucidate new mechanisms of lipid-mediated metabolic regulation, which is significant because membrane lipid composition varies in health and disease. These studies are expected to inform new therapeutic strategies for the management of NAFLD.

NIH Research Projects · FY 2025 · 1992-09

PROJECT SUMMARY/ABSTRACT CD1d-restricted, invariant natural killer T cells (iNKT) cells play a critical role in regulating the commensal micro- biota and resistance to mucosal pathogens. Conversely, iNKT cell levels are suppressed by microbiota in early (pre-weaned), but not later (post-weaned), life. If critical microbial signals are not provided during early-life, co- lonic iNKT cell levels remain elevated leading to increased susceptibility to colitis in later-life. Recently, we dis- covered that colon iNKT cells are resident cells that establish themselves in early-life in a pathway controlled locally by macrophages of fetal origin and a novel stromal cell population that is marked by Wnt4 which are in turn regulated by the commensal microbiota. The current research proposal addresses the unanswered question about how this newly identified stromal cell population regulates intestinal iNKT cells in early-life. Our long-term goals are to parse out the mechanisms that microbes and the host use to establish an iNKT cell niche that influences mucosal disease such as infection and colitis in later-life. The objective of this research is to better understand the pathways that regulate iNKT cells in early-life as a paradigm for imprinting cellular residency during a critical period of development. Our central hypothesis is that a unique early-life colonic stromal cell population is part of a local tissue niche that directly imprints iNKT cells through bone morphogenic protein (BMP) signals and determines the host’s susceptibility to inflammatory bowel disease (IBD) and enteropathogens in later-life. Specifically, it is proposed that embryonic macrophages and microbes regulate a unique colonic stromal cell population that is marked by Wnt4 and determines the tissue receptivity to early-life engraftment of thymic iNKT cell emigrants through stromal cell-derived cognate (CD1d) and non-cognate (BMP2) signals. The rationale for our proposed research is that there is very little known about early-life colonic stromal cells and their interac- tions with immune cells. Our central hypothesis will be tested with four specific aims: 1) determine whether colonic Wnt4+ stromal cells are an early-life specific population that forms a tissue niche with embryonic macro- phages to support local iNKT cells; 2) Identify the specific molecules generated from colonic Wnt4+ stromal cells that could directly support colonic iNKT proliferation during early-life and specifically the role of BMP2; 3) deter- mine whether Wnt4+ stromal cells specifically imprint colonic iNKT cells during early-life which results in durable effects that influence later-life disease, and; 4) determine how microbial signals regulate the cell abundance and transcriptome of colonic Wnt4+ stromal cells during early-life. Specifically, we expect we will show that this dis- tinctive type of stromal cell forms a tissue niche that supports iNKT cells in early-life (Aim 1) and operates through BMP2 and CD1d expression (Aim 2) causing durable changes in iNKT cell number and function that impacts later-life susceptibility to colitis (Aim 3) in pathways that are modulated by commensal microbes during the “win- dow of opportunity” in early-life (Aim 4). Overall, this proposal is significant because it will define a new population of early-life stromal cells that are potentially of fetal origin which regulate later-life susceptibility to IBD.