Case Western Reserve University

NIH Research Projects · FY 2025 · 2021-04

Preterm birth (PTB) affects 10-15% of pregnancies in the US and causes the majority of neonatal mortality and morbidity. To prevent PTB a clearer understanding is needed of the hormonal control of human parturition. In this regard, the steroid hormone progesterone (P4) acting via the nuclear P4 receptor (PR) isoforms, PR-A and PR-B, is a critical factor. For most of pregnancy P4/PR promotes uterine quiescence and blocks labor, and disruption of P4/PR signaling triggers parturition. The mechanism for these critical P4/PR actions are, however, unclear. Infection/inflammation in the uterine and gestational tissues are major drivers of term and preterm parturition, but the mechanism for these effects are also uncertain. The proposed research addresses these major knowledge gaps by exploring a mechanism linking inflammation and P4/PR signaling in myometrial cells via a phosphorylated form of PR-A that we hypothesize plays a central role in the causal pathway for inflammation-induced parturition. Our published and preliminary data suggest that P4/PR-B inhibits myometrial cell responsiveness to pro-inflammatory stimuli by interacting with and repressing the transcriptional activity of the activator protein 1 (AP-1) transcription factors at promotors of a subset of IL-1ß-responsive genes. Our data also suggest that P4/PR-A upon phosphorylation at the serine-344/345 locus (pSer344/345-PRA) interacts with AP-1 to disrupt P4/PR-B anti-inflammatory activity. We also found that generation of pSer344/345-PRA in myometrial cells is catalyzed by mitogen activated protein kinases (MAPKs) in response to pro-inflammatory stimuli. Based on those data we hypothesize that: 1) P4/PR-B exerts anti-inflammatory activity in myometrial cells by binding to AP-1 to inhibit transcription at a subset of inflammatory gene promoters; 2) pSer344/345-PRA inhibits P4/PR-B anti-inflammatory activity by disrupting the PR-B/AP-1 interaction, and 3) generation of pSer344/345-PRA in myometrial cells is catalyzed by specific MAPKs in response to pro-inflammation stimuli. This hypothesis will be tested in human myometrial cell lines and human myometrium obtained from c-section deliveries, and Rhesus macaque and mouse models of inflammation induced parturition. Two Specific Aims will be achieved: Specific Aim 1: Determine the mechanism by which P4/PR-B exerts anti-inflammatory activity in myometrial cells and how this is affected by pSer344/345-PRA; and Specific Aim 2: Determine how pSer344/345- PRA generation is controlled in myometrial cells. The proposed research is novel and groundbreaking and will advance understanding of the fundamental biology of human parturition and contribute to the development of effective P4/PR-based anti-inflammatory therapies to promote uterine quiescence and prevent preterm birth.

NIH Research Projects · FY 2025 · 2021-04

X-linked spinal and bulbar muscular atrophy (also known as SBMA or Kennedy's disease) is a rare neuromuscular disorder characterized by adult-onset proximal muscle weakness due to lower motor neuron degeneration. SBMA patients display signs of androgen insensitivity, including gynecomastia, reduced fertility, and testicular atrophy. SBMA, is caused by a CAG- polyglutamine (polyQ) repeat expansion in the androgen receptor (AR) gene and is one member of a family of nine CAG-polyQ repeat disorders that includes Huntington’s disease. For decades, research into the basis of neurological disease focused upon the contribution of neuronal dysfunction to disease pathogenesis. However, over the last ten years, there has been growing evidence in the motor neuron disease field that challenges the prevailing neurocentric theory of the etiology of many neurological diseases. . In the case of SBMA, there is increasing evidence implicating muscle dysfunction as a major component of disease pathogenesis. For therapeutic purposes, however, different groups have demonstrated successful treatments targeting either the skeletal muscles or the central nervous system using different SBMA animal models. Consequently, this lack of consensus in the literature underscores the need for studies of the SBMA AR-mutation on affected cell types - skeletal muscles and motor neurons - in a human background. We hypothesized that polyQ mutation cause disruption in AR binding to the DNA which leads to transcriptional dysregulation that can be pathogenic in motor neurons, skeletal muscles or both. Therefore, the goal of this proposal is to combine AR genome wide occupancy and gene expression data sets generated from SBMA and CRISPR engineered isogenic controls iPSC-derived skeletal muscle and motor neurons. We will then, co-culture the iPSC-derived skeletal muscles with iPSC-derived motor neurons to modulate the AR targets identified in the AR transcriptional network and investigate neuromuscular junctions (NMJ) by analyzing electrophysiological activity of the motor neurons and the skeletal muscles. This work will advance understanding on the molecular mechanisms of human mutant AR to SBMA pathogenesis and evaluate the utility of iPSC-derived skeletal muscles and motor neurons tool to develop SBMA in vitro studies.

NIH Research Projects · FY 2025 · 2021-03

ABSTRACT The metastatic spread of cancer cells and recurrence are intimately linked to therapy failure, which remains an important clinical challenge for patients with metastatic Triple Negative Breast Cancer (TNBC). Two important factors that regulate TNBC metastasis and recurrence are: (i) the differentiation status of the cancer cells, and (ii) the functionality of the immune system within the tumor microenvironment (TME). Both the cancer cells and the immune cells are highly influenced by the cytokines Oncostatin M (OSM) and Interferon-beta (IFNB). We find that the actions of OSM and IFNB oppose one another in regulating both cancer cell differentiation and immune system function within the TME. OSM and its receptor (OSMR) are frequently upregulated in aggressive, metastatic and therapy-resistant recurrent cancers, such as TNBC. Mechanistically, OSM/OSMR elicits robust activation of STAT3, which forms a transcriptional complex with SMAD3, and induces stem-like/mesenchymal programs that enhance TNBC aggressiveness and resistance to chemo- and immuno-therapy. Moreover, the cooperation of STAT3 and SMAD3 on co-dependent gene promoters produces a pro-inflammatory, tumor- promoting TME by suppressing the production of immune-activating IFNB and inducing immune-inhibitory cytokines. Restoring IFNB signaling prevents and reverses stem-like/mesenchymal programming stimulated by OSMR and STAT3/SMAD3. Thus, the cooperation between STAT3 and SMAD3 to inactivate IFNB :STAT1/2 signaling axis represents a key step in metastatic progression and tumor recurrence. Based on these and other compelling findings, we will test the hypothesis that the relative balance between OSM:STAT3/SMAD3 and IFNB :STAT1/2 signaling in both TNBC cells and immune cells dictates metastatic relapse and ultimately, patient outcomes. We’ll test this hypothesis in two Specific Aims: Aim 1 will determine how IFNB suppresses OSMR- mediated STAT3/SMAD3 co-dependent gene expression; Aim 2 will define how OSMR/ERK signaling activates STAT3/SMAD3 to drive stem-like/mesenchymal reprogramming and the suppression of IFNB/ISGs. Together, the results from our studies will provide a greater understanding of the molecular mechanisms by which OSM and IFNB antagonize one another and test novel therapeutic approaches to shift the balance towards active IFNB :STAT1/2 and away from OSMR:STAT3/SMAD3 in both TNBC and immune cells, with the goal of improving outcomes for patients with metastatic TNBC.

NIH Research Projects · FY 2025 · 2021-03

Project Summary/Abstract Regulatory T cell (Treg) immunosuppression is critical for maintaining immune tolerance to a diverse array of potential antigens in the intestinal mucosa. In patients with inflammatory bowel disease (IBD), chronic intestinal inflammation overwhelms local Treg function, allowing inflammation to persist. Our previous work and that of others have identified important roles for 17β-estradiol (estrogen, E2) signaling in promoting Treg differentiation and function. E2 signals through two nuclear receptors, alpha and beta (ERα, ERβ), to modulate gene transcription in target cells. Although they share high sequence homology, ERα and ERβ mediate distinct and often opposing functions on gene regulation. In previously published work, we showed that shifting the balance of E2 signaling towards ERα is generally pro-inflammatory, whereas shifting towards ERβ is generally protective. In recent preliminary studies using IBD patient samples, we observed significantly diminished ERβ expression in intestinal biopsy tissues and peripheral T cells from females with active Crohn’s disease (CD). We also found that ERβ-deficient T cells are resistant to ex vivo, TGFβ-dependent Treg differentiation, and that deletion of ERβ in a spontaneous ileitis model (SAMP/YitFC, “SAMP” mice) results in significant impairment of Treg transcriptional and functional responses, contributing to exacerbated inflammation. Therefore, the goal of this project is to determine the molecular and cellular mechanism(s) by which altered E2 signaling impacts Treg differentiation and function, contributing to intestinal inflammation. The mechanisms by which E2 signaling cross-talks with inflammatory signals to influence immune cell function are poorly understood. This proposal seeks to address this knowledge gap through our central hypothesis that dysregulated E2 signaling contributes to Treg transcriptional remodeling and loss-of-function, facilitating sustained intestinal inflammation in IBD. In Aim 1, we propose to delineate the molecular mechanisms by which ERα- and ERβ cross-talk with signaling downstream of TGFβ in primary T cells, influencing TGFβ-dependent Foxp3 expression and function. Aim 2 will determine the functional impact of rebalancing Treg-specific E2 signaling on intestinal inflammation in vivo, testing our hypothesis that augmenting Treg-specific ERβ signaling may prevent and/or rescue intestinal inflammation. Experiments will include adoptive transfer of ERβ-expressing Tregs to SAMP mice, as well as in vivo assays using a T cell-dependent colitis model. In Aim 3, we plan to determine the transcriptional and functional effects of rebalancing E2 signaling in CD patient Tregs using novel MaxCyte transfection technology, assessing ERα- and ERβ-specific effects on (i) TGFβ-dependent Foxp3 induction in naïve T cells and (ii) ex vivo suppressive function of Tregs. Successful completion of our proposed Aims will provide key mechanistic insight into the functional impact of E2 signaling in Tregs, an under-studied area with broad applicability to numerous diseases exhibiting dysregulation of ER expression and/or activation and subsequent reductions in Treg function.

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY Crohn’s disease (CD) represents a significant public health challenge with more than 1 million individuals affected by this condition in the US. To date, a complete cure of CD is not available. CD has a multifactorial etiology, characterized by a complex interplay between environmental and genetic factors, particularly an inappropriate inflammatory response to commensal microbes in genetically affected individuals. Fungal infection represents one of the most serious health hazards, especially for immuno-compromised patients. C andidiasis has dramatically increased worldwide, especially in neutropenic patients or patients with altered gut microbiome due to increased antibiotic use. Recent studies performed by our group have demonstrated that the abundance C. tropicalis is significantly higher in patients with CD compared to their non-diseased first-degree relatives. C. tropicalis and C. albicans have been recognized as the most common pathogenic fungi and are part of the human commensal flora. C. tropicalis and C. albicans are commonly found in the gastrointestinal tract. However, the role of Candida infections in intestinal inflammation in patients with CD has not been fully elucidated. Therefore, there is a need for mechanistic studies aimed at delineating the molecular mechanisms by which Candida infection may result in exacerbation of CD. Studies proposed herein take advantage of a unique resource, a spontaneous murine model of CD-like ileitis (SAMP1/YitFc or SAMP), to decipher the mechanistic role of candidiasis in experimental ileitis. Strong preliminary data from our group have demonstrated that C57BL/6 (B6) mice infected with C. tropicalis are significantly more susceptible to develop dextran sodium sulfate (DSS)- induced colitis compared to uninfected littermates, with increased gene expression of key Th1 response- associated genes, such as Tnf and Ifn, and decreased expression of Th2 response-associated genes, such as Il4 and Il13. 16S rRNA analysis of fecal samples from infected and uninfected mice have shown that C. tropicalis significantly altered the microbiome population of B6 mice, with increased abundance of mucin degrading bacteria, such as Akkermansia (A.) muciniphila and Ruminococcus (R.) gnavus. Based on its biological effects, we hypothesized that Candida infection may alter the gut microbiome community resulting in dysbiosis that, in turn, increases the susceptibility of the host to develop IBD, or triggers flare in CD patients. The goals of this proposal are: (1) demonstrate that Candida infections contribute to worsen experimental intestinal inflammation; and (2) identify the specific microbes and mechanisms responsible for the increased intestinal inflammation in B6 and SAMP mice. Understanding the mechanisms by which candidiasis affects the gut microbiome is essential to maintain remission in CD patients and the design of novel antimicrobial therapies with increased efficacy and decreased side-effects.

NIH Research Projects · FY 2025 · 2021-03

Project Summary The inflammatory bowel disease (IBD) subtype, Crohn’s disease (CD) is a chronic and relapsing inflammatory disorder of the gastrointestinal tract. Although the precise etiology of IBD is not known, evidence suggests that environmental factors, including diet, contribute to its pathogenesis.1-3 Specifically, dietary amino acids serve as key regulatory factors in cellular and microbial metabolic pathways, and disturbances in their metabolism,3 as well as altered presence of amino acid concentrations (blood/feces/urine), are observed in CD patients.4 Diet has been shown to have a pro-inflammatory effect in CD, but not much is known about anti-inflammatory diets (e.g., soy).5, 6 Gut microbial modulation via diet is a needed strategy for the therapeutic management of CD; however, no specific recommendations exist for CD patients. Moreover, studies show that the person-specific changes elicited by a dietary intervention on host immune/metabolic function reflect unique microbiota signatures. This proposal will focus on the microbiota-mediated effects of dietary soy and individual amino acid supplementation in patients with active CD compared to healthy controls. The central hypothesis of this proposal is that a soy diet induces anti-inflammatory microbiota in CD patients and that the ‘level of response’ for each individual can be predicted by metabolic and microbiome biomarkers. We will test this hypothesis directly in humans with active CD and mechanistically focus on the effect of dietary soy on the ‘pro-inflammatory potential’ of gut microbiota in CD patients. Experimentally, we will use our validated human gut microbiota SAMP1/YitFC; SAMP (hGM-SAMP) mouse model of CD-ileitis to quantify and mechanistically validate the functional effect of human feces on the severity of CD-ileitis after transplantation into GF SAMP. As a main objective, we will determine to what extent a soy-based diet could induce changes in fecal/blood inflammatory biomarkers in patients with active CD. The following aims are a continuation of our efforts to understand the microbiota-mediated effects of diet on intestinal inflammation. AIM 1: will characterize the effect of dietary soy in humans with active CD and quantify the inflammatory potential of their gut microbiome using a ‘rapid screening’ hGM-SAMP DSS-colitis model. By stratifying inflammatory microbiome/blood markers, we will identify biomarkers that could predict ‘responders’/‘non-responders’ to diet. We expect to generate a list of metabolic and microbiome clinical biomarkers that could be used to monitor response to diet in CD patients. AIM 2: will determine the impact of soy-associated amino acid dietary supplementation on the microbiome, metabolome and immunology of a spontaneous CD-like ileitis in hGM-SAMP and SPF SAMP, and AKR control mice. We will identify functional metabolic mechanisms associated with the severity of mouse CD-ileitis in response to diet. This proposal is based on strong preliminary data in mouse models of IBD that a soy-based diet treats and prevents the severity of intestinal inflammation. To translate this information to human CD represents a very significant area of investigation in this field of research and will ultimately inform the direction of future studies.

NIH Research Projects · FY 2025 · 2021-02

The overarching goals of this proposal are to determine if Erbb3 kinase plays a critical role in colorectal tumorigenesis and to test if colorectal cancer with oncogenic Erbb3 mutations respond better to anti-PD1 antibody. Erbb3, a member of the EGFR family of receptor protein tyrosine kinases, is mutated in a variety of human cancers including colorectal cancer (CRC). Unlike other EGFR family members, the kinase domain of Erbb3 is thought to be a pseudo-kinase without enzymatic activity. Although it is well documented that Erbb3 plays an oncogenic role in tumorigenesis, the mechanisms by which Erbb3 activation drives tumorigenesis are largely unknown. We made the paradigm-shifting discovery that the pseudo-kinase domain of Erbb3 is a serine/threonine (S/T) kinase activated by phosphorylation of a serine site in the juxatomembrane domain, and that its kinase activity is required for robust tumor growth in both xenograft and genetically engineered mouse models. Moreover, we found that a Erbb3 kinase-dead mutant impairs interferon-γ (IFN-γ)-induced PD-L1 expression in CRCs. Interestingly, knockin of an oncogenic Erbb3 mutation in mouse colon cancer cell lines renders them sensitive to anti-PD1 antibody therapy. Here we propose two aims to determine the role of Erbb3 S/T kinase in (1) colorectal tumorigenesis and (2) the response of colorectal cancers to immune checkpoint inhibitors. Successful completion of our proposed studies will lay a solid foundation for targeting Erbb3 mutant CRCs with Erbb3 kinase inhibitors and/or immune checkpoint inhibitors. Given that aberrant activation of Erbb3 causes resistance to various cancer therapies including Herceptin in breast cancer and EGFR inhibitors in lung cancer, the impact of our studies will have a broad impact on cancer therapy.

NIH Research Projects · FY 2025 · 2021-01

Abstract The main goal of this proposal is to develop biomaterial-based technologies that can modulate the functions of DCs and T-cells in the draining lymph nodes in the presence of systemically delivered metabolic inhibitors. The hypothesis of this proposal is that polymeric biomaterials-based particles generated from central-carbon metabolites (targeting DCs via phagocytosis) can restart glycolysis/TCA cycle in DCs in the presence of metabolic inhibitors and will also induce robust vaccine responses in immunocompetent mice. Notably, we have generated polymers of central-carbon metabolites from glycolysis and TCA cycle, which were able to activate DCs even in the presence of metabolic inhibitors. Moreover, these particles were able to rescue the metabolic inhibition, as observed by up-regulated extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in bone marrow derived DCs. In vivo PEGS particle formulations delivering TRP-2 peptide (without any adjuvant), were able to prevent the growth of subcutaneous B16F10 tumors in the presence of CB-839 a glutaminase inhibitor. Similarly, F16BP vaccine particles delivering TRP2 peptide antigen along with poly(I:C) as adjuvant and PFK15, a glycolytic inhibitor, were able to reverse the growth of subcutaneous YUMM1.1 tumors. The hypothesis of this proposal will be tested using the following specific aims: Aim 1: Evaluate if F16BP particles induce antigen-specific long-term memory T cell responses in immunocompetent mice in the presence of glycolytic inhibitor PFK15. Aim 2: Determine if PEGS particles can induce antigen-specific long-term T cell responses in immunocompetent mice in the presence of glutaminase inhibitor CB-839. Aim 3: Determine toxicity profile and maximum tolerable doses of vaccines. The results obtained from these experiments will shed light on the effect of metabolic reprogramming on the efficacy of vaccine therapy.

NIH Research Projects · FY 2025 · 2021-01

This proposal examines how a novel TLR glial signaling pathway drives phagocytic competence in glia, and defines its function in pruning neuronal number and connectivity across lifespan. Glia provide an extensive support system for healthy neurons by promoting their survival, connectivity, and synaptic function. Remarkably, glia can rapidly switch roles to precisely eliminate dying neurons or unwanted neurites/synapses by phagocytosis. These diametrically opposed functions necessitate fail-safe signaling mechanisms between neurons and glia; yet hese crucial regulatory mechanisms have remained largely obscure. Toll-like receptor (TLR) pathways were first identified for their roles in embryonic patterning and have since been defined as a conserved centerpiece of innate immunity. Our lab made the unexpected discovery that one of the most pronounced phenotypes associated with loss of a Drosophila TLR, a dramatic increase in the number of apoptotic neurons during development, is caused by selective loss of the TLR in glia. We demonstrated that release of the TLR ligand from dying neurons activates a novel TLR pathway in glia to drive phagocytic competence. In this proposal we build on our novel preliminary findings to establish how this pathway regulates the speed and specificity of debris clearance, and define its roles in neuron-glia interactions in synapse, neurite, and neuron removal across lifespan. Our unifying hypothesis is that non-canonical TLR signaling underlies the speed and specificity of debris clearance critical for proper CNS development and function. In the first aim, we focus on elucidating how glia are transformed into phagocytes during development by defining how information is relayed through the TLR pathway to elucidate how glia are primed to become phagocytic. In the second aim, we seek to extend our published work to investigate whether TLR signaling is a widespread early detection system to alert glia to the presence of neuronal debris. And in the third aim, we examine the function of TLR signaling in sculpting circuits in the olfactory system based on our preliminary findings that glial TLR signaling constrains synapse number in this well defined circuit. Here we propose to leverage the fly olfactory circuit as a model for defining glial phagocytic function in synapse maintenance. Together, these studies will shed critical light on the early signaling interactions between glia and their phagocytic substrates essential for brain health across lifespan.

NIH Research Projects · FY 2025 · 2020-12

The fusion protein AML 1/ETO (AE), resulting from the t(8;21) translocation, is a leukemia-initiating transcription factor that is frequently associated with acute myeloid leukemia (AML). Despite being defined as a "favorable" subtype of AML, many AE positive (AE+) patients relapse and die with largely unknown causes. It is also unclear how AE mediates a disease-predictable DNA methylation signature. The long-term goals are to elucidate further the mechanisms of AE+ AML leukemogenesis, discover new therapeutic targets and develop effective targeted therapies. The objective of this proposal is to explore the molecular basis of a disease-predictable DNA methylation signature underlying AE+ AML with a focus on the impact of hypoxia-independent HIF1alpha-DNMT3a signaling axis activation. The rationale underlying this proposal is that the hypoxia-independent HIF1alpha signaling activation is a new hallmark of cancer. In relation to this project, the hyperactive HIF1alpha signaling may be a disease-promoting factor and an epigenetic mediator in AE+ AML. HIF1alpha forms a feedforward loop with AE and transactivates DNMT3a, another prognostic marker in AE+ AML. HIF1alpha inhibition suppresses AML cell growth. However, the detailed mechanistic and biochemical links between HIF1alpha signaling and AE AML pathogenesis and disease recurrence are poorly defined. The central hypothesis is that HIF1alpha promotes AE leukemogenicity through enhancing AE transcriptional activities and modulating the AE-governed DNA methylation landscape in AML cells; therefore HIF1alpha may be a vulnerable and druggable target in AE+ AML. This hypothesis will be tested by pursuing three specific aims: 1.) Dissect the mechanistic details of how HIF1alpha is critical for AE-driven leukemogenesis; 2) Determine the role of HIF1alpha in AE-dependent DNA methylation; 3) Test pharmacological targeting of HIF1alpha as a therapeutic option for AE+ AML. To pursue our aims, we will use innovative combinations of biological techniques with unique transgenic and patient-derived xenograft (POX) mouse models, as well as innovative integration of aberrant HIF1alpha signaling and epigenetics in understanding and treating AE+ AML. The proposed research is significant, because it will disclose new genes/mechanistic pathways that are necessary for AE leukemogenicity, identify the therapeutic biomarkers, and discover new medicinal agents for AE+ AML. Further, it will thoroughly investigate the epigenetic and oncogenic role of HIF1alpha in cancer. The proximate expected outcomes are to demonstrate HIF1alpha-epigenetics crosstalk in defining AE-initiated transcriptional regulation and leukemia pathogenesis, and to establish the feasibility of using HIF1alpha inhibitors to enhance the therapeutic index of the existing treatment regimens. The results will have an important impact because they will advance our understanding of AE+ AML molecular pathology, aberrant epigenetics in leukemia and the oncogenic functions of hypoxia-independent HIF1alpha signaling in cancers. The findings will also lay the groundwork to develop newer strategies to better target AE+ AML.

NIH Research Projects · FY 2024 · 2020-09

Pre-exposure Immunologic Health and Linkages to SARS-COV2 Serologic Responses, Endothelial Cell Resilience, and Cardiovascular Complications: Defining the mechanistic basis of high risk endotypes. Tim Chan David A. Zidar Abstract The objective of this proposal is to understand the immunologic foundations of heart disease which can occur as a result of COVID19. Cardiac impairment, when it develops is often fatal, and our hypothesis is that the maintenance of endothelial function is critical to surviving the protracted nature of COVID19 pneumonia, especially in those with reduced or delayed antibody responses. Our first aim will be to analyze those differences in immune function which pre-date infection but appear to impact the risk of fatal COVID. This will be done by enrolling those at high risk for developing COVID19 (frontline healthcare workers), and performing serially assessments of their immunologic function if they develop COVID. We will specifically investigate the mechanisms that link pre-infection inflammatory pathways to protective serologic responses and symptom severity and recovery. Our second aim will be to perform in vitro experiments to assess the requirements for endothelial cell dysfunction and infectivity. We will compare various inflammatory and cardiovascular stimuli which seem to play a role in promoting COVID19-related cardiovascular complications. Our third aim is to characterize immune cells, endothelial cells, and cardiomyocytes in heart tissue from those with COVID19-induced left ventricular dysfunction. Using single cell sequencing techniques, we will determine cellular and molecular signatures that characterize the microenvironment of the COVID19-affected heart, compared to appropriate controls. Our conceptual model is that pre-existing immune dysfunction 1) reduces the efficiency of neutralizing antibody responses, and 2) in conjunction with cardiovascular disease risk factors, induces endothelial downregulation/depletion of nodal regulators which protect against inflammatory insults. This renders endothelial cells unable to withstand COVID-specific stimuli. Once completed, this study will provide the necessary information to improve the identification of those at risk for COVID-related heart disease and develop rationale approaches to improve the improve survival in the setting of COVID.

NIH Research Projects · FY 2025 · 2020-09

Abstract Kidney transplantation elicits powerful B cell responses that generate antibodies specific for the transplant donor. B cell responses to allogeneic HLA (panel reactive antibodies, PRA) and to the transplant donor (donor- specific antibodies, DSA) correlate with risk of rejection and graft loss and thus serve as diagnostic indices of allo-immunity. B cell responses that generate DSA initiate the most vexing types of rejection (antibody- mediated rejection, ABMR) and chronic ABMR and hence much interest exists in development of therapeutics that specifically target B cells. Some B cell responses however may correlate with or even promote graft survival (immune regulation, accommodation, operational tolerance). Although there has been extensive investigation of the specificity and function of the blood DSA hardly anything is known about the properties of donor-specific B cells. The research we propose will identify for the first time key characteristics of donor-specific B cell responses in kidney transplant recipients to determine what properties associate with and potentially contribute to stable graft function, as opposed to rejection. As a first objective, donor-specific B cell responses will be studied by determining Heavy and Light chain Ig sequences of donor- specific B cells by single cell sequencing and their phenotypic signature by RNA-seq . We will determine key properties of Ig, such as clonal diversity, extent of somatic hypermutation or Ig independent properties such as production of cytokines in recipients with stable function or rejection. As a second objective, donor-specific Ig sequences obtained by NGS will be studied prospectively over periods > a year (up to two years). We will determine whether clonotypes expand and diversify in anticipation of rejection as would be expected of T cell dependent responses; and how donor-specific sequences change following rejection treatment. We will also determine if in recipients with stable grafts, donor-specific B cells encode Igs with distinct properties. As a third objective, we will conduct the first investigation of key properties (e.g. maximum binding, avidity, direct activation of donor cells and antigen specificity, where possible, confirmed using graft tissues) of recombinant monoclonal DSA on donor cells to determine whether these properties could explain differences in pathogenicity. The research might reveal novel approaches to assessing risk and new targets for biological intervention. The research might also inspire rational approaches to modifying B cell responses in transplant recipients in a personalized manner with the goal of promoting protective responses and antagonizing pathogenic ones.

NIH Research Projects · FY 2024 · 2020-09

The molecular epidemiology of lung cancer in Africa is almost completely unknown, yet molecular profiling and institution of targeted therapy is now standardly used to influence care in patients with actionable mutations. HIV- infection is associated with a markedly increased risk of lung cancer, but its association with lung cancer and how it influences disease outcomes is not well studied. The capacity to correctly diagnose lung cancer on radiological images, tissue specimen acquisition, and subsequent histologic confirmation compounds the problem of under and misdiagnosis of lung cancer in Africa, especially with the prevalence and often presumptive clinical diagnosis of tuberculosis infection. In this consortium proposal, we therefore seek to address the above stated problems by 1) establishing national lung cancer diagnostic referral networks in Uganda and Tanzania, which will allow for improved detection of lung cancer and, hence decrease the lung cancer burden; 2) investigating the role of HIV-1 infection on the incidence and outcomes of lung cancer in the region; 3) determining the molecular profile of lung cancer in East African countries in order to facilitate advocacy for scale- up of targeted therapies (including immune-based therapies); 4) building capacity for lung cancer diagnosis and care in terms of improving imaging, pathology, radiology and tissue acquisition and clinical care of lung cancer 5) creating a lung cancer research collaboration that will build capacity through mentoring of junior researchers for future leadership in the field. At the end of this project, we envision an African-led lung cancer research network, clinical expertise for sample acquisition and care, improved pathology and radiology diagnostics services, a lung cancer screening program, and a large biorepository that should support lung cancer research in the region for many years to come.

NIH Research Projects · FY 2024 · 2020-09

Abstract The duration and nature of humoral immunity to SARS-CoV-2 (CoV2) infection need to be better understood. While most studies have focused on the immune response in patients with clinical illness, little is known about the antibody response to CoV2 immediately after exposure and before the onset of illness, especially in asymptomatic individuals. It is important to understand how this impacts long- term immunological memory. This supplement aims to extend the objectives of the original grant to address these gaps in our knowledge by prospectively following household contacts compared to clinical cases of CoV2 to determine innate and adaptive immune events associated with early viral exposure. We have conducted a comprehensive evaluation of samples from these patients, including RNA sequencing of peripheral blood cells, proteomic analysis of oral secretions (the site of initial CoV2 replication), measurement of viral load using N protein levels, and CoV2-specific IgA and IgG in blood and saliva. We have identified innate and adaptive signatures associated with viral clearance and among household contacts that may be associated with their failure to be infected or, if infected, fail to develop clinical illness. We have identified CoV2 antigens, notably the N protein that is expressed on CoV-2 infected cells, which are targets of ADCC. Antibodies generated by the N protein can similarly kill infected cells with various variants of concern, suggesting a potential variant- transcending target. We have also tracked the humoral immune response in a long-term cohort of nursing home residents to the CoV-2 mRNA vaccine and subsequent booster responses. This supplement will continue these ongoing studies and allow their completion.

NIH Research Projects · FY 2024 · 2020-09

Alzheimer's disease (AD), the most common form of dementia, affects 10% of the population aged 65 and older, and up to 50% of people over 85 years old1,2. Patients with AD suffer from a decline in episodic memory with inability to recall the recent past and eventual loss of long-term memories, loss of cognitive function and changes in personality. The major goal of this proposal is to define pathways for the clearance of amyloid-β (Aβ), whose aggregation into amyloid plaques is a hallmark of AD. Increasing evidence indicates an important role of astrocytes, the most abundant cell type among glial cells in the brain, in Aβ clearance. This proposal will focus on a potentially novel regulatory mechanism for astrocytic Aβ uptake by LRP4 (LDLR-related protein 4), an LDLR-like protein that is abundantly and specifically expressed in astrocytes. We and others have demonstrated that LRP4 is critical to the formation of the neuromuscular junction (NMJ) where it serves as a receptor for agrin, a factor from motoneurons. The agrin- LRP4 interaction activates the transmembrane tyrosine kinase MuSK to induce AChR clustering and NMJ formation. Results of our preliminary studies, some of which were published, suggest that LRP4 may play a role in regulating AD pathogenesis. First, LRP4 was selectively expressed in astrocytes in the hippocampus and prefrontal cortex (PFC), both AD-vulnerable regions. Its level in astrocytes was higher than those of LDLR and LRP1, both of which have been implicated in Aβ uptake. Second, LRP4 was reduced in postmortem brain tissues of AD patients. In accord, deleting LRP4 augmented Aβ plaques and AD-associated deficits in 5xFAD mice, an AD mouse model. Third, Aβ uptake was impaired in LRP4 mutant (mt) astrocytes. These results suggest a critical role of LRP4 to astrocytic Aβ uptake. What is the underlying mechanism(s) by which LRP4 regulates Aβ uptake? LRP4 is a single transmembrane protein with a large extracellular domain (ECD). Interestingly, we found, in addition to agrin, LRP4 can interact with ApoE, whose ε4 allele (ApoE4) impairs Aβ clearance. Further pilot studies indicate, first that agrin stimulation could increase Aβ uptake by astrocytes, in a manner dependent on LRP4. LRP4 could interact with agrin, which has been shown to associate with Aβ. Therefore, we posit that LRP4 serves as a receptor for ApoE- and agrin-mediated Aβ uptake. Second, LRP4 could interact with the prorenin receptor (PRR), an auxiliary protein of v-ATPase that is critical to maintaining endolysosomal pH, and is required for its down-regulation. LRP4 deficiency increased PRR levels and caused hyper-acidification of endolysosomes. Therefore, we postulate that PRR upregulation may be a mechanism of LRP4 deficiency. Finally, the agrin-LRP4 interaction could stimulate MuSK in astrocytes. We wondered whether MuSK activation is necessary for agrin-promotion of Aβ uptake. Based on these results and considerations, we hypothesized that LRP4 is critical to Aβ uptake by astrocytes via multiple mechanisms. In this proposal, we will determine whether LRP4 deficiency correlates with AD development in AD patients and expedites AD-like pathology in AD mice; determine whether LRP4 regulates astrocytic Aβ uptake by interacting with ApoE and PRR; and investigate the roles of the agrin- LRP4 pathway in Aβ uptake by astrocytes. We hope that the proposed studies will not only establish novel cellular functions of astrocytic LRP4 in regulating Aβ uptake, but also shed new light on AD pathogenesis. Such knowledge is prerequisite to the development of new therapeutic strategies for AD.

NIH Research Projects · FY 2025 · 2020-09

PROJECT SUMMARY/ABSTRACT Stroke is a neurological condition of increasing burden in many low- and middle income countries (LMICs) that is associated with high morbidity and mortality. Africa is particularly hard-hit by stroke due to rapid population growth, expanding lifespan, industrialization, adoption of harmful western diets, and greater prevalence of risk factors such as hypertension and diabetes. The situation is likely to worsen as projections for Africa note an increase of 10.8% in incident stroke cases. In spite of these daunting projections, stroke can be a preventable disorder. Evidence-based interventions such as life-style changes and management of risk factors can greatly reduce stroke burden. In sub-Saharan Africa (SSA) there are few widely used and effective approaches. Given resource constraints, the ideal approach needs to be practical, low-cost, widely scale-able, and acceptable to the end-consumer. Written in response to PAR-18-835: Global Brain and Nervous System Disorders Research across the Lifespan, the proposed project builds upon promising mixed-methods pilot work and capacity-building. In a recently completed Fogarty International Center project, the study team developed a curriculum-guided self-management program, TargetEd MAnageMent (TEAM) intended to reduce stroke risk in Uganda. Pilot results found excellent program retention over a 6-month period (87.5 %) and reduced stroke risk factors, including decreased blood pressure, improved serum lipid profiles, and improved glucose control among individuals with diabetes. The overall goal of this 2-phase, 5-year research project is to test an intervention intended to reduce stroke risk factors in Ugandans at high risk for stroke. The project will evaluate whether a patient -centered approach that uses peer educators (patients with relevant lived experience) and community health workers to co -deliver the intervention may reduce stroke burden. To enhance future dissemination potential of the TEAM approach, the project will include both effectiveness and implementation elements. In Phase 1 (months 1-12), we will refine the TEAM intervention for content and process guided by stakeholders (patients/family, clinicians, administrators) in the local context. In Phase 2 (months 13-60) we will conduct a prospective, randomized effectiveness-implementation trial of TEAM vs. enhance treatment as usual (ETAU). 246 participants will be randomized at baseline to receive either TEAM (N= 123) or ETAU (N=123). The project will also build research capacity in stroke risk reduction and facilitate the establishment of an infrastructure to facilitate futures scale-up. Taken together, the proposed project has substantial public health importance. It will provide the prerequisite outcomes data, training, and infrastructure needed to help with reducing stroke burden in Uganda and other countries in SSA.

NIH Research Projects · FY 2025 · 2020-09

TECHNICAL ABSTRACT The skeleton is a preferred organ for cancer dissemination from various tumors malignancies. The main objective of this research proposal is to understand the function of the Bone Marrow microenvironment, and specifically of its perivascular components, in the establishment of skeletal metastasis. The studies that are proposed aim to test the innovative hypothesis that Mesenchymal Stem Cells (MSCs), as perivascular cells (pMSCs), function as gatekeepers controlling tumor cell invasion to the bone. This new hypothesis provides clinically relevant information for therapeutic strategies that innovately aim at reducing the engraftment of circulating cancer cells by closing the gate through which the metastatic cell transit into the normal bone. We propose that the inhibition of one or combination of tumor cells and pMSCs specific genes would prevent or attenuate the metastatic process in vivo. To experimentally dissect the various cellular and extracellular matrix components controlling extravasation, we have designed and validated a unique in vivo extraskeletal humanized bone marrow niche-mimicking platform (humanized Ossicle). This platform is centered around the use of a porous, calcium phosphate ceramic into which human bone marrow-derived MSCs (hBM-MSC) are loaded and then implanted into the back of immune deficient mice. Bone is fabricated onto the walls of the ceramic by hBM-MSCs anchored at these locations and, at the centers; the host-derived blood vessels have pMSCs within the marrow space. We have documented that when melanoma is injected into the blood stream of the mouse, the bones become black (melanin), as is the bone in habitat. If we manipulate the expression of molecules are we propose involved in the process, such as CXCL12 or MCAM in the pMSCs, the mouse bone is black and the habitat is white. This black-and-white result sets the stage for Specific Aim 1 where we dissect the participation of pMSCs and the perivascular basement membrane in driving Skeletal Metastasis. In Specific Aim 2, we propose to determine the participation of HSC-niche ligand molecules expressed on cancer cells in driving SM. We have established a baseline using melanoma, which has the highest rate of lethality once it metastasizes into bone. This proposal will also be focused on the study of molecular mechanisms using breast cancer cell lines. A logical extension of this platform is to provide a predictive diagnostic for the patients' control over metastasis for different osteotropic cancers. In addition to the significant direct clinical impact, this proposed work is expected also to provide the platform for future projects addressing the roles of pMSCs that may influence other skeletal metastasis, such as regulation of local antitumor immune response, cancer cells dormancy and tumor angiogenesis, and to serve as template for the study of other osteotropic malignancies (prostate and lung cancer), thus broadening the significance of the findings and conclusions.

NIH Research Projects · FY 2024 · 2020-08

Understanding the process of neural degeneration is important in order to understand the responses of the nervous system to injury and disease. A common model used in such studies is the distal segment of the sciatic nerve after nerve transection or crush. Following such a lesion, Wallerian degeneration occurs in which the distal segment of the nerve fragments and degenerates, and the resulting axonal and myelin debris are cleared away. Interestingly, while this process occurs rapidly in the peripheral nervous system (PNS), it is extremely slow in the central nervous system (CNS), and, partly because of this, regeneration is generally ineffective in the brain and spinal cord. It is widely believed that inflammatory macrophages (mφs) derived from blood-borne monocytes are required for the phagocytosis of axonal and myelin debris. Immune cells enter injured tissue in response to chemotactic cytokines or chemokines. A major population of monocytes infiltrates into the distal nerve after axotomy in response to the chemokine CCL2, which acts on monocytes via the chemokine receptor CCR2. In mice in which the gene for CCR2 is knocked out, CCR2+ monocytes do not enter the nerve. It was, therefore, very surprising when we found that the clearance of both myelin and axonal protein is normal in Ccr2 knockout mice. We subsequently found that an important reason for the normal clearance appears to be phagocytosis by neutrophils, an immune cell not previously implicated in Wallerian degeneration. In fact, although neutrophil actions in the CNS are beginning to be examined, for example in models of multiple sclerosis, there are almost no studies on their actions in the PNS. Our preliminary evidence using the neutrophil-depleting antibody anti- Ly6G suggests, but does not prove, that neutrophils directly phagocytose and metabolize myelin. We will examine this hypothesis by examining the clearance of myelin proteins by western blotting before and after neutrophil depletion and by co-labeling tissue with Oil Red O, a stain for myelin metabolites, and with cell type specific neutrophils antibodies. We have shown that after axotomy two neutrophil attracting chemokines are induced in the sciatic nerve, CXCL1 and CXCL2. To determine whether these chemokines and their receptor, CXCR2, are involved in neutrophil infiltration into the nerve, we will use neutralizing antibodies, pharmacological antagonists, and knockout mice. We will investigate also whether the involvement of neutrophils in Wallerian degeneration is important for subsequent regeneration. “Wallerian-like” degeneration occurs in several demyelinating neuropathies, and we will examine whether neutrophils play a role in this phenomenon. We will use a mouse model for Guillain Barré syndrome based on our recent finding that neutrophils enter into the sciatic nerve in this model. Following up on our unexpected findings of normal clearance of myelin in Ccr2 knockout mice, our experiments will examine the role of neutrophils in Wallerian degeneration and in a demyelinating disease. Given the known importance of Wallerian degeneration for nerve regeneration due to the removal of myelin proteins, our studies will suggest ways of improving regeneration in the PNS and perhaps in the CNS.

NIH Research Projects · FY 2025 · 2020-08

A Multi-Omics Approach to Examine Symptoms and Medication Adherence in Women with Breast Cancer Breast cancer is most prevalent in postmenopausal women, and 3.4 million U.S. women are survivors. Most women with breast cancer are postmenopausal at the time of diagnosis, and at least 70% of tumors are hormone receptor positive (HR+). Aromatase inhibitors effectively prevent BC recurrence, and current standard includes adjuvant aromatase inhibitor (AI) therapy in a once daily standard dose regimen for a minimum of five years. However, AI adherence is a significant problem. Up to one third of women do not fill their initial AI prescription, adherence to AIs averaged 48% in the first year, and adherence decreases in subsequent 2-5 years. AI-attributed symptoms are the leading reason for not adhering to AI regimens and a major barrier to AI adherence. Moreover, AI-related symptom type and severity are highly variable among women. The source of symptom variability is unknown. The etiology of symptoms experienced during AI therapy may have biological underpinnings, yet little is known about factors in AI (anastrozole, letrozole, exemestane) absorption, distribution, metabolism, and elimination (ADME) pathways and the resulting symptoms. Symptoms and adherence, especially their relationship to each other, have not been well-characterized temporally. Further, potential biologic mechanisms related to AI absorption, distribution, metabolism, and elimination (ADME) for AIs have not been fully characterized. The dissertation project (F99) will examine temporal patterns of AI symptoms and adherence and their relationship over the first 18 months of AI therapy. It will also explore the role of genotypic (ADME) and phenotypic factors in symptoms experienced and AI adherence. The postdoctoral project (K00 phase) will incorporate two additional molecular methods—microbiomics and exosomics. They will be described and their potential role in AI symptoms experienced and adherence will be explored. The purpose of the proposed F99/K00 training and research is to utilize a biobehavioral, multi-omics approach to gain a deeper understanding of the complex web of AI symptoms and adherence, including the temporal variability among women and the interplay between symptoms and adherence. It will provide insight into potential biological mechanisms by describing molecular and phenotypic characteristics associated with symptoms and adherence. Ultimately, this research will inform future symptom management and adherence interventions and their timing.

NIH Research Projects · FY 2024 · 2020-08

Meiosis is the keystone of reproductive success in sexually reproductive organisms as failure to successfully complete meiosis results in aneuploidy, a leading cause of developmental diseases and miscarriages. Successful chromosome segregation is facilitated by using DNA recombination to form crossovers (COs) between homologous chromosomes. Not all recombination events result in a CO however and the majority are repaired as noncrossovers (NCOs). Thus, the decision whether to form a CO or NCO is a critical regulatory point during meiosis that is poorly understood. Despite having a clear picture of the genetic requirements for a crossover to form, it is completely unknown why some breaks are chosen to be repaired as crossovers and others as noncrossovers. The parent grant of the current equipment supplement addresses this critical knowledge gap by using heterozygous chromosome inversions in Drosophila melanogaster as a model. Heterozygous inversions pose an interesting problem for the meiotic program. Meiosis requires that homologous chromosomes recognize and use each other as templates for precise repair during recombination, yet inversions disrupt the synteny between homologs. There are two downstream effects: COs are suppressed locally on the inversion chromosome, and a checkpoint is activated, which leads to a genome-wide increase in COs on non-inverted chromosomes. We have shown that these responses are mediated through changing the CO vs. NCO decision. In this equipment supplement, we are requesting funds to purchase the Imaris image analysis software from Oxford Instruments. We currently have the microscopes we need (including a Leica Stellaris 5 confocal owned by us and housed in our lab, and access to a Leica TCS SP8 gated STED microscope housed in the CWRU Light Microscopy Core), but we do not have access to Imaris. Along with the purchase of the software, we are also requesting funds for 3 years of the maintenance plan that will include unlimited technical training and support for applications specific to our use case. Purchase of this software will generate data critical for analyzing the structure of the synaptonemal complex at inversion breakpoints and insight into defects in the structure that alter the CO/NCO decision, a high- priority goal of the parent grant. Importantly, the methods we are proposing have been successfully carried out by other Drosophila meiosis labs multiple times; we will be applying those methods to our own system.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY Following stroke, numerous impairments develop that affect walking ability. We lack a clear understanding of what characterizes these impairments and their relation to impaired walking ability. For example, we applied exoskeletal knee flexion to assist those with reduced knee flexion post-stroke. The assistance improved knee flexion but surprisingly worsened compensatory motions such as hip abduction. Preliminary analysis indicated that the assistance elicited hyperexcitable quadriceps reflex activity. Additionally, the quadriceps response was abnormally coordinated with hip abductor muscles only in post-stroke individuals. These results question traditional beliefs regarding which motions are compensatory and which are due to neural impairment. Thus, the critical gap to restoring functional gait post-stroke is characterizing the interconnection between biomechanical issues and neural impairments. Our novel approach is to combine state-of-the-art methods in biomechanics and neurophysiology to develop cause-and-effect models of the interconnection between gait kinematics, hyperexcitable reflex activity and abnormal coordination. The clinical significance is the groundwork for targeted interventions such as reflex modulation and neurally intelligent exoskeletons that interact with the impaired spinal circuitry. It will be shown in our preliminary work that we have years of experience characterizing post-stroke gait impairments using reflex stimulation, robotics and computational methods that uniquely qualify our group for this project. The objective of this proposal is to establish the biomechanical and neurophysiological mechanisms underlying pathological gait post-stroke. In Aim 1, we determine whether hip abduction is a compensation for reduced knee flexion by comparing people with stroke to induced similar walking patterns in healthy individuals. Muscle activation patterns and intralimb coordination will provide evidence towards fundamental differences in neural control after stroke. In Aim 2, we use reflex neurophysiological methods to probe post- stroke individuals’ reflex coordination patterns. We expect to find that hyperactive quadriceps reflexes are associated with impaired knee flexion. We additionally expect to show the interrelation between reflex activity in the quadriceps and the abductors as predicted by our models representing abnormal coordination. In Aim 3, we use reflex conditioning protocols during gait based on sophisticated paired peripheral nerve stimulation techniques. These methods will provide evidence of the spinal mechanisms at the root of abnormal reflex behavior that likely underlie impaired gait post-stroke. Together this comprehensive testing procedure incorporates biomechanics and neurophysiology to reveal new knowledge of post-stroke gait impairment. These results will have broad impact on our understanding of neuromuscular impairments and enable the development of targeted therapies for treatment.

NIH Research Projects · FY 2024 · 2020-07

OVERALL PROGRAM PROJECT: ABSTRACT Cell death pathways have historically been characterized by the proteases that become activated during cell fate decisions. Apoptosis has been defined by initiator and effector caspase activation and, largely due to the reagents available; cell death decisions were initially thought to be binary – caspase-dependent (apoptosis) or caspase-independent (all others). Much like other binary distinctions – like Th1 versus Th2 T cells or M1 versus M2 macrophages, which have undergone substantial expansion in the past decade, cell death decisions and their consequences are now recognized to be much more variables. In fact, even the mechanisms of cell death have been refined. They are no longer categorized by the proteases which activate the cascades but rather, by the effector mechanism. Necroptotic cell death is caused by activation of the pore-forming protein, MLKL. Secondary necrosis is caused by activation of the pore-forming protein, DFNA5 (Gasdermin E/GSDME), and pyroptosis is caused by activation of the pore-forming protein, Gasdermin D (GSDMD) This last category of cell death - pyroptosis - is one of the most immunologically important forms of cell death. Not only does pyroptosis kill the cell, but also GSDMD activation allows the release of IL-1β. This cytokine is among the most important in acute and chronic inflammation. Its secretion is implicated in rare genetic inflammatory diseases such as Familial Mediterranean Fever as well as more common diseases such as myocardial infarction, Alzheimer's disease, Crohn's disease and Ulcerative Colitis, Multiple Sclerosis and many others. With the discovery of the inflammasome in 2002 and its subsequent study, it became clear that the amyloid-like activation of these inflammasome complexes drove pro-IL-1β cleavage and IL-1β release from the cell, but also pyroptosis. Missing from this biochemistry, though, was the cell biology underlying IL-1β release. How is IL- 1β recognized by caspases and other proteases? If IL-1β is not released through a secretory ER-Golgi mechanism, how is trafficked out of the cell? How is this release coordinated with pyroptosis and other regulated cell death pathways that are operative in different populations of immune effector cells. This Program Project aims to utilize cell physiology, biochemistry, structural biology, and mouse and human disease models to unravel these important questions.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY/ABSTRACT Chronic musculoskeletal pain (MSKP) is a significant problem in many women with hormone receptor- positive early stage breast cancer receiving treatment designed to prevent cancer recurrence by blocking estrogen production via systemic inhibition of the aromatase enzyme. MSKP interferes with functional status, adherence to therapy, and increases utilization of health care resources. Unfortunately, very little is known about the molecular mechanisms underlying MSKP related to a decline in estrogen in women with breast cancer. Evidence supports that dysregulation of adrenergic function is present in women with MSKP. In fact, both β-antagonists and α2-agonists have been investigated as treatment for MSKP pain disorders, and evidence from our group as well as others support a role for pain inhibition via α1-antagonists. The purpose of this K99R00 proposal is to provide the applicant with the necessary training and research experience to examine the dynamic RNA transcriptome and DNA methylome to identify genes and biological pathways that are involved in development of cancer pain. The purpose of the proposed K99 study is to generate knowledge about MSKP development within the context of declining estrogen in women with breast cancer. The K99 phase of this project capitalizes on data and blood samples generated through an ongoing project that recruits postmenopausal women newly diagnosed with early stage breast cancer who will receive aromatase inhibitor therapy (R01CA196762, the EPICC study). The K99 also benefits from an ongoing project generating DNA methylation data from blood samples of the women in the EPICC study (R01CA221882). The specific research aims for the K99 study are to (1) test the hypothesis that 6 months of decreased estrogen leads to changes in gene expression and this is related to changes in the pain phenotype in women with breast cancer; and (2) test the hypothesis that 6 months of decreased estrogen leads to changes in DNA methylation and this is related to changes in the pain phenotype in women with breast cancer. The K99 training plan leverages the superb research-intensive environment and resources at the University of Pittsburgh to ensure the applicant’s development of (a) proficiency in omics (particularly transcriptomics and epigenomics), (b) competency in analysis and bioinformatics of omics data, (c) proficiency in pain mechanisms, and (d) knowledge and skills for professional career development. The specific aims of the R00 study are to (1) identify changes in the regulation of genes that are associated with variability in cancer pain based on cancer type; (2) identify changes in the regulation of genes that are associated with variability in cancer pain based on cancer therapy; and (3) explore the potential mediating and moderating effects of sex on the relationship between gene regulation and pain. This research generates the knowledge needed to develop a research program focusing on developing a profile for predicting individuals at risk for chronic pain development that includes demographic, clinical, and omics data.

NIH Research Projects · FY 2024 · 2020-07

Project Summary/Abstract Lung cancer (LCa) is the second most common malignancy in the US with about 222,500 new cases and 155,900 deaths each year. HIV-infected individuals and AIDS patients have increased relative risk of LCa by 250% after controlling for other potential risk factors. LCa in HIV+ people under antiretroviral therapy (ART) is diagnosed at younger ages than those in the general population. This trend in the ART era is implicitly attributed to prolonged life span and aging of the population. The success of new approaches to control lung tumorigenesis in the population is contingent to identify HIV-specific mechanisms that facilitate the tumor progression and metastasis. HIV-infected T cells release a variety of immunologically active exosomes, small vesicles of endocytic origin, to influence intercellular communication and material transfer at both local and distant sites, thus potentially contribute to enhanced risk for tumorigenesis. Our preliminary studies have found that exosomes secreted by HIV-infected T cells and purified from the blood of lung cancer patients of people living with HIV (PLWH) significantly stimulated lung cancer cell proliferation. HIV-associated exosomes induced MAP kinase ERK1/2 activation via interaction with epidermal growth factor receptor (EGFR) and the toll like receptor 3 (TLR3). Mechanistically, the HIV trans-activation response (TAR) element RNA, which exists in excess of other HIV RNAs in exosome from HIV-infected T cells, is responsible for enhanced cancer cell proliferation and proto-oncogene expression. We have established a bone marrow transplant (BMT) mouse model in which lethally-irradiated FVB mice were grafted with syngeneic bone marrow cells of Tg26 HIV- transgenic mice. Growth and metastasis of allografts LCa cells were significantly enhanced in grafted mice containing HIV+ bone marrow cells compared with that in non-grafted control mice. However, reconstitution of circulating immune cells and bone marrow remained the same between two groups of mice, suggesting that TAR RNA-bearing exosomes from reconstituted HIV+ immune cells may promote LCa progression in the BMT model. Taken together, these data lead us to hypothesize that TAR RNA-bearing exosomes from HIV-infected immune cells promote lung cancer growth and progression and that controlling release of the exosomes or directly targeting the TAR RNA may serve as an adjuvant for prevention and treatment of lung cancer in HIV- infected individuals. To test this hypothesis, we will first delineate the mechanism by which HIV-1-infected T- cell exosomes stimulate LCa growth and progression in vivo using the BMT model. The potentials for therapeutic intervention of LCa promotion by HIV will be examined through inhibition of exosome production and neutralization of TAR RNA. Finally, we will comprehensively examine the HIV-positive exosomal membrane proteins for interaction with EGFR and HIV-specific cargo components in the circulation using our novel EV-omics approach. Completion of the proposed studies will shed light on the mechanisms underlying HIV-mediated promotion of LCa and lay a foundation for therapeutic intervention of non-AIDS-defining cancers.

NIH Research Projects · FY 2025 · 2020-07

PROJECT SUMMARY Evidence-based psychotherapies for posttraumatic stress disorder (PTSD) and depression consistently produce strong, clinically meaningful effects for many individuals. However, these interventions also have significant dropout rates, a large minority of individuals continue to have debilitating symptoms, and even those who respond may be vulnerable to relapse upon future stressors. More efficient and mechanistically precise interventions are needed. Consistent with the cross-cutting theme of studying the role of the environment in the NIMH Strategic Plan, the etiologic role of exposure to destabilizing, stressful life events is common to both PTSD and depression. Not only do they share common distress-related triggers, symptoms, and maintaining processes, but they also commonly co-occur (upwards of 60%). Current PTSD and depression treatments typically focus on their respective disorders rather than on common processes that maintain psychopathology; and, importantly, they do not explicitly target positive adaptive processes associated with resilience. Decades of experimental studies, prospective studies, and psychotherapy trials have identified interconnected maladaptive and adaptive processes associated with persistent psychopathology after stressful, destabilizing events. These maladaptive processes include: 1) unproductive event processing; 2) avoidance; and 3) reward sensitivity and processing deficits. These processes prolong negative mood, interfere with adaptive coping and processing of emotional material, and increase sensitivity to future stressful life events. PATH (Positive Processes and Transition to Health) directly targets these maladaptive processes while also teaching parallel adaptive skills (constructive processing, approach, and positive emotion processing and reward seeking). Six, 90-min sessions target individuals who have experienced a destabilizing life event and have persistent stressor-related symptoms. PATH utilizes life event processing (revisiting, meaning making), focusing repeatedly on an identified destabilizing life event, positive life events, and future events as a framework to identify maladaptive processes and teach constructive processing skills. In a small open trial (R61, N = 45), we will examine whether PATH engages the key targets of unproductive processing, avoidance, and reward deficits. Next, we will conduct a randomized trial of PATH (R33, N = 135), comparing PATH to a Progressive Muscle Relaxation, seeking to replicate changes in the targets in a larger sample and examine PATH's impact on stressor-related psychopathology (PTSD, depression). PATH, a brief and focused treatment that targets key psychological processes common to PTSD and MDD, has the potential to reduce dropout, improve treatment engagement and outcomes, identify potential treatment mechanisms, and ultimately reduce the costly human and economic burden of stressor-related psychopathology.