Ohio State University

NIH Research Projects · FY 2025 · 2021-06

Project Summary/Abstract Sepsis is the most common cause of death in many intensive care units and represents a major burden to the US health care system. Despite advances in intensive care technology and mechanical ventilator support, pharmacological options for sepsis are limited, which reflects an insufficient understanding of host-dependent mechanisms that underlie this pathophysiological disorder. A wealth of evidence from recent clinical and experimental sepsis studies indicates that a prolonged immunosuppressive status, due to profound cell death and dysfunction of lymphocytes, is a critical determinant of sepsis-elicited mortality. Therefore, restoration of lymphocyte cell survival and functions by blocking immune inhibitory molecule(s) may represent a promising therapeutic regimen for treating sepsis. In this Proposal, we aim to study the role and mechanism of a previously unrecognized immune inhibitory molecule called SUSD2 (sushi domain containing 2) in promoting sepsis-induced immunosuppression. Through an unbiased gene profiling assay, our previous study has identified a cell surface molecule SUSD2 whose high expression correlated with an immunosuppressive phenotype in an experimental cancer model. In this Proposal, we observed elevated Susd2 expression in T lymphocytes in experimental septic animals and patients with sepsis compared to non-septic controls. Genetic deletion of SUSD2 (Susd2−/−) resulted in a significantly improved animal survival and attenuated apoptosis of T lymphocytes in the cecal ligation and puncture (CLP)-induced polymicrobial sepsis model. Mechanistically, our preliminary studies discovered an inhibitory effect of SUSD2 on interleukin-2 receptor (IL-2R) signaling, a well- established pathway essential for T cell survival and effector functions. The goal of the proposal is to examine the causal effect of SUSD2 on cell death and dysfunction of T lymphocytes in microbial sepsis. We hypothesize that 1) elevated SUSD2 expression leads to diminished IL-2-dependent cell survival and effector functions in T lymphocytes, resulting in a sustained immunosuppressive state and worse disease outcome in sepsis; 2) enhanced activation of STAT5 (signal transducer and activator of transcription 5) and BATF (basic leucine zipper ATF-like transcription factor) signaling maintains cell survival and effector functions in Susd2−/− T cells post sepsis; 3) SUSD2 blockade reverses sepsis-induced cell death and dysfunction of T lymphocytes. Single-cell RNA sequencing analysis of circulatory immune cells will be performed to examine the inhibitory effect of SUSD2 on T cell response post sepsis at the single-cell level. We will test whether treatment with a neutralizing anti-SUSD2 antibody reverses dysfunctional T cells isolated from septic patients. Results of these studies will provide both experimental and clinical evidence to support a promoting function of SUSD2 on sepsis-induced immunosuppression, which can potentially lead to the development of new approach for sepsis treatment.

NIH Research Projects · FY 2025 · 2021-06

Project Abstract Tumor-associated macrophages (TAMs), a major component of the tumor stromal mass, demonstrate great phenotypic heterogeneity and diverse functional capabilities under the influence of local tumor microenvironment (TME). These TAMs generally display an anti-inflammatory, M2-type phenotype and can facilitate tumor growth by promoting angiogenesis, invasion and metastasis, as well as immune evasion. However, it remains largely undefined exactly how these TAMs regulate anti-tumor immune responses within the TME. The objective of this application is to understand the role of TAM-derived PD-L1/siglec-15 in inducing intratumoral CD8 T cell dysfunction, to delineate mechanisms underlying PD-L1/siglec-15 upregulation in TAMs as well as to develop novel strategies to promote intratumoral CD8 T cell infiltration and function in favor of enhancing anti-tumor immunity. The long-term objective of this project is to understand signals required for functional polarization of TAMs within the TME, and its contributions to immune cell dysregulations, cancer development and progression, which may lead to the development of novel cancer therapeutic strategies.

NIH Research Projects · FY 2025 · 2021-06

ABSTRACT Legionella pneumophila is an infectious bacterium that causes Legionnaire’s disease and remains a major cause of morbidity and mortality in the immunocompromised individuals such as the elderly and cancer patients. Macrophages are the main immune cells that can clear Legionella after activation of the canonical Nlrc4/Naip5 inflammasome. Caspase-11 is a component of the non-canonical inflammasome and mice lacking caspase-11 allow significant Legionella replication in their macrophages. Down-regulation of the homologous caspase in human macrophages increases their permissiveness to Legionella. Our preliminary data show that caspase-11 is necessary for fusion of the Legionella-containing vacuole with the lysosomes via a mechanism that requires the polymerization and depolymerization of actin. Using state-of-the-art techniques including 3D confocal microscopy and high resolution SIM-S microscopy, RNA seq and proteomics analyses, we identified new molecules that are cleaved by caspase-11 and regulate restriction of Legionella in macrophages. This proposal will investigate the molecular mechanisms leading to caspase-11-mediated clearing of Legionella infection.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY Despite high vaccination coverage, pertussis outbreaks caused by the gram-negative obligate human pathogen Bordetella pertussis (Bp) are observed in many countries. Pertussis resurgence correlates with the switch in the 1990s from whole cell vaccines (wPV) which elicit long-lived Th1/17 immune responses, to acellular vaccines (aPV) which elicit Th1/2 skewed immune responses. Furthermore, aPV do not prevent nasal colonization or transmission of Bp. Current aPV are comprised of 1-5 bacterial proteins that were selected for their roles in pathogenesis and ability to elicit antibodies. In contrast, wPV present an undefined large number of antigens. The combination of limited antigenic diversity and Th2 skewed immune profile is a likely explanation for the incomplete protection provided by aPV. Recent studies including that from our laboratory also show that circulating Bp strains (CBp) from globally diverse countries have absent/reduced expression of current aPV antigens, suggesting that aPV may be significantly less effective against CBp strains. There is increasing recognition that CD4+ T cell responses are critical for long-lived protective immune responses that clear the entire respiratory tract. However, it is not clear that current aPV antigens are optimal CD4+ T cell targets. We will use state-of-the-art mass spectrometry, bioinformatics, and phenotypic and functional assays to identify proteins expressed by CBp that are processed and presented on Class II histocompatibility antigens of humans and mice, and that stimulate CD4+ T cell responses. This foundational data set will be coupled with a prime-pull vaccination strategy and a Th1/17 skewing adjuvant developed in our laboratory, to determine the immunogenicity and protective efficacy of newly defined antigens to create a next-gen aPV. Specific Aim 1: Define the set of naturally derived Bp peptides presented on MHC II and recognized by CD4+ T cells. We will identify the Bp antigens from circulating Bp strains (CBp) that are expressed on human and murine Class II, and use proliferation and flow cytometry assays to determine which antigens are recognized by CD4+ T cells of wPV-immunized individuals and convalescent mice. Specific Aim 2: To test the immunogenicity and protective efficacy of novel antigens against circulating Bp strains and their role in pathogenesis. We will test the immunogenicity and protective efficacy of the novel proteins using a murine model of Bp infection. We will create deletion mutants of the novel proteins in CBp to determine their role in pathogenesis and colonization of the respiratory tract. IMPACT: Our integrated approach to identify, test, and leverage novel Bp antigens will permit the rational design of next-gen aPVs that elicit long-lasting protection in the respiratory tract, prevent nasopharyngeal carriage and thereby reduce the spread of the disease pertussis.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Microorganisms residing in the intestinal lumen have a significant impact on brain function and behavior. Perturbation of microbe-gut-brain communication is believed to be involved in the pathogenesis of well-known gut-brain disorders such as irritable bowel syndrome (IBS) and related functional GI disorders. However, there is lack of understanding of the precise microbial mechanisms and the cellular pathways that allow gut microbes to communicate with the brain. To address this critical knowledge gap, the applicant has pioneered the zebrafish system for the study of microbe-gut-brain communication. Using in vivo real-time measurements of cell activity in zebrafish, the applicant’s recent research revealed that specific gut bacteria directly activate specialized sensory cells in the intestine epithelium, enteroendocrine cells (EECs), through the receptor transient receptor potential ankyrin A1 (Trpa1). Microbial, pharmacological, or optogenetic activation of Trpa1+EECs directly activates enteric neurons and stimulates vagal sensory ganglia. Preliminary studies identified a distinct subset of bacterial derived tryptophan catabolites as novel agonists that potently activate Trpa1. The objective of the proposed research is to determine the precise molecular mechanism by which enteric bacteria activate the EEC- vagal sensory pathway to modulate brain activity. The central hypothesis is that bacterial secreted tryptophan catabolites stimulate Trpa1 in EECs to activate vagal sensory neurons through a novel EEC secreted signal peptide, pituitary adenylate cyclase activating polypeptide (Pacap). To test this, the applicant will first use molecular microbiology and zebrafish gnotobiotic approaches to define the microbial pathway and mechanism that activates EEC Trpa1 signaling. Second, the applicant will use in vivo vagal calcium imaging, optochemical and genetic manipulation to identify the specific subtype of EECs that transmit enteric bacterial information to the vagus. Finally, the applicant will use pharmaceutical, genetic and cell transplantation approaches to define the EEC signaling peptide that transmits bacterial information from the gut lumen to the vagus. The proposed research is expected to make a significant new contribution to our understanding of the molecular mechanisms and cellular pathways by which enteric bacteria communicate with the brain. The interdisciplinary experimental approach together with the comprehensive career development plan will extend the applicant’s training from gastroenterology into vagal and brain physiology as well as molecular microbiology. A diverse team of established investigators at Duke University and UNC Chapel Hill, with expertise ranging from host-microbe interaction to gut-brain physiology to bacteriology, will oversee the applicant’s career development during the award period by contributing intellectually to her research training, providing mentorship, and offering career advice. This 5-year career development award will provide the applicant with the necessary professional and scientific skills to be an independent and successful microbiota-gut-brain scientist.

NIH Research Projects · FY 2025 · 2021-05

Summary Aminoacyl-tRNA synthetases (ARSs) establish the rules of the genetic code, whereby each amino acid is attached to a cognate tRNA. Errors in this process lead to mistranslation, which can be toxic to cells. Recent studies suggest that the selective forces exerted by cell-specific requirements and environmental conditions potentially shape quality control mechanisms. Approximately half of the ARSs possess a proofreading (or editing) function to hydrolyze mischarged aa-tRNAs and evidence that non-proteinaceous amino acids pose the greatest threat to fidelity is beginning to emerge. Early work in the Musier-Forsyth lab in the field of translational quality control focused on our discovery of Class II prolyl-tRNA synthetase (ProRS) editing. This led to a detailed mechanistic understanding of the novel bacterial ProRS posttransfer editing domain (INS) and the demonstration that the INS domain, when purified on its own outside the context of the ARS, was fully functional in tRNA deacylation. We subsequently discovered that single-domain INS homologs are widespread in Bacteria and in recent years, our focus in this area has turned almost entirely to understanding the function of these INS-like domains in tRNA editing. However, many open questions regarding the physiological function of these putative trans-editing proteins remain. The overarching goal of the research described in this MIRA application is to uncover the specific functions of a growing family of trans-editing proteins known as the INS superfamily. This diverse yet universally conserved family now has a solid and accumulating in vitro structure-function knowledge base, which strongly supports a role in maintaining translational fidelity. Our knowledge of the broader physiological roles of these proteins, especially in eukaryotes, is still in its infancy and is just beginning to reveal wider roles than previously anticipated. This major gap will be addressed in this work. While classical knock- down screens that only define essential versus non-essential genes do not immediately identify editing domains as essential, the strong conservation of these domains implies they play important, and in most cases still undiscovered, roles in cell survival and competitiveness. Proposed studies are designed to address some of the many open questions with regard to both physiological trans-editing functions and potential moonlighting functions of the INS superfamily. These domains are largely unexplored in eukaryotes, including a novel sub- family cluster that is encoded in many unicellular eukaryotic pathogens. The therapeutic potential of trans-editing domains has not been exploited and represents another major gap in the field that we hope to address by our planned studies. In the long term, combining drugs that target novel translational fidelity mechanisms along with known ribosome-targeting protein synthesis inhibitors such as aminoglycosides, may results in more effective therapeutic strategies.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY/ABSTRACT Hematopoiesis is a cellular developmental process that is controlled by a cell-intrinsic developmental program and cell-extrinsic factors from the microenvironment. The vascular niche present in the bone marrow and other hematopoietic organs provides the physical space and signals necessary for the proper regulation of this process. Hematopoietic stem cell (HSC) phylogenetic and functional diversity are restricted in patients who receive chemotherapy or are recipients of hematopoietic stem cell transplantation (HSCT), leading to clonal hematopoiesis and cytopenias which are significant sources of morbidity and mortality in these patients. At present, very little is known about the role of the microenvironment in regulating HSC diversity. We hypothesized that mechanisms exist within the vascular niche that can be modulated to allow it to support a more phylogenetically and functionally diverse population of HSCs. We performed a genetic screen in barcoded GESTALT zebrafish and found that dysregulated expression of prkcda, the zebrafish PKC-delta homolog, increased the number of HSC clones contributing to hematopoiesis by more than 50%. Phenotypic analysis using single cell RNA-seq demonstrated the presence of a novel population of immature neutrophils and expansion of erythroid precursor cells. Single cell ATAC-seq analysis showed that chromatin accessibility at the promoter of the native prkcda locus is reduced specifically in vascular niche cell populations. These data led us to hypothesize the existence of a specific epigenetic program within the vascular niche that regulates expression of factors supporting hematopoiesis and thereby maintains the capacity of the niche to support HSC phylogenetic and functional diversity. In Aim 1, we will use Cut and Run analysis for specific epigenetic factors to characterize the vascular niche in human endothelial cell cultures. Multimodal single cell RNA-seq/ATAC-seq will be used in adult and embryonic zebrafish to identify niche cell populations and cis-acting DNA elements near genes important for supporting hematopoiesis. In Aim 2, a CRISPR screen will be used to identify specific transcription factor binding sites in human vascular niche cells lying proximal to and controlling transcription of key genes involved in niche function, including CXCL12, ANG1, and KITLG. A panel of zebrafish CRISPR mutants will be made to study the function of selected enhancer elements in vivo. Changes in niche function will be assayed in vitro by co-culture with HSPCs and in vivo by GESTALT barcoding and scRNA-seq. This work will provide new mechanistic understanding of how the capacity of the hematopoietic niche to support a diverse population of HSCs is regulated and may lead to new therapies that improve hematopoietic recovery after chemotherapy or transplantation.

NIH Research Projects · FY 2025 · 2021-05

ABSTRACT With the rapid acceleration of the aging population and repeated failures to find a pharmacological cure for Alzheimer’s disease (AD), it is of paramount importance to identify modifiable physical attributes that are most likely to attenuate cognitive and neural decline in older adults. Individual studies have demonstrated that mobility, cardiorespiratory fitness, and muscle strength/power are associated with cognition among older adults. However, because these physical attributes have been studied in isolation, it remains unknown which of these attributes are most critical for successful cognitive aging and brain maintenance (keeping the brain young). Our long-term goal is to develop a precision medicine model of cognitive aging; that is, to identify which physical attributes are associated with specific cognitive functions and implement individually tailored exercise programs to optimize those cognitive abilities among older adults. Our overall objective in the current proposal is to directly examine differential contributions of mobility, cardiorespiratory fitness, and muscle strength/power metrics to current cognitive abilities, brain health, and longitudinal cognitive decline. Our central hypothesis is that these physical attributes account for unique variance in cognition, but that their relative predictive abilities will differ within specific cognitive domains and neural networks. Using gold-standard assessments of mobility, cardiorespiratory fitness, muscle strength/power, cognition, and brain structure and function (magnetic resonance imaging; MRI) in young, middle-aged, and older adults, we will pursue the following aims: 1) Determine the contribution of physical attributes to cognition, with a specific emphasis on episodic memory and executive function, among older adults. 2) Determine the contribution of physical attributes to cortical thickness (T1-weighted MRI), white matter microstructure (diffusion-weighted MRI) and brain function (functional MRI) among older adults. 3) Identify which physical attributes predict cognitive decline over a 2.5-year period in older adults and whether polygenic risk scores for AD moderate the association between physical attributes and cognitive decline. We will examine which modifiable physical attributes, including functional aspects of the motor system, predict cognitive decline among older adults. Outcome data from this proposal will impact the development of lifestyle interventions for optimization of cognitive performance among older adults, as the study will provide critical knowledge to optimize future exercise intervention studies aimed at mitigating age- and Alzheimer’s disease-related cognitive and neural decline. Moreover, we will examine whether physical attribute-brain-cognition associations are age dependent. The current proposal is well-suited for the mission of the National Institutes of Aging, as we propose to examine modifiable physical attributes that will maximize high quality-of-life years and independent functioning (via maintenance of cognitive and brain health) prior to mortality.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Gammaherpesvirus infections are associated with a number of malignancies that include B-cell lymphoproliferative diseases, gastric carcinoma, nasopharyngeal carcinoma. Immunodeficient individuals such as HIV patients are more susceptible to γ -herpesviruses-associated cancers. It generally takes several months to years for cancer to arise after primary infection with the virus. This observation highlights the multistep process of carcinogenesis that could only be achieved by viral persistent infection. EBV and KSHV are γ - herpesviruses that establish latent infection in lymphoid cell, which is maintained for the rest of the host’s life. Intermittent reactivation of these viral reservoirs will lead to more viruses production and spreading. Although there are antiviral drugs that specifically target lytic replication of γ -herpesviruses, they do not eliminate those latent viruses that could serve as a risk factor for viral-associated cancers. Our group recently showed that HIV infection leads to activation of Polo-like kinase 1 (PLK1), a proto- oncogene, in B and T-cell lymphocytes. PLK1 is a serine/threonine kinase that controls G2-M cell cycle progression and is frequently overexpressed in wide range of cancers. Its inhibitors have been developed as promising cancer therapy. We further discovered that PLK1 is involved in both regulation of EBV/KSHV latency and survival of their cellular reservoirs in the host. Protein knockdown as well as chemical inhibition of PLK1 was able to reactivate latent EBV/KSHV and promote cell death of γ -herpesvirus-reactivated B lymphocytes. These results expose a new viral mechanism that can describe how co-infection of γ -herpesviruses renders HIV patients an increased risk to cancers, despite the fact that HIV itself is not oncogenic. Our investigations into this topic will be accomplished by three separate aims that include: (1) Investigation of molecular mechanism of PLK1 activation by HIV1 in EBV/KSHV-infected lymphocytes. (2) Investigation of how PLK1 regulates EBV/KSHV latency and maintenance of their cellular reservoirs. (3) Inhibition of PLK1 as novel means to eradicate EBV/KSHV persistent infection by eliminating their cellular reservoirs. Collectively, our proposed studies will contribute to the understanding of latency in HIV and gammaherpesviruses infections that underlie various viral-associated cancers. This project will also help to identify new molecular target for curing HIV and EBV/KSHV infections and their-related malignancies.

NIH Research Projects · FY 2025 · 2021-05

SUMMARY Triple negative breast cancer (TNBC) is a heterogeneous, complex, and aggressive breast cancer subtype. TNBC patients respond poorly to chemotherapy, leading to high mortality rates and a worsening prognosis. RNAi therapeutics, including siRNA and miRNA, have shown tremendous potential for TNBC cancer therapy. Developing a safe targeted delivery system with endosomal avoidance of the payload is crucial in terms of realizing the full potential of RNAi therapeutics and could revolutionize clinical treatment of TNBC. Our group has demonstrated that delivery of anti-miR21 to TNBC can efficiently inhibit TNBC cell proliferation (Shu D, et al. ACS Nano. 2015, 27:9731; Yin H, et al. Shu D. Mol Therapy. 2019, 27:1252). We have also recently reported the use of RNA nanoparticle orientation for decorating exosome surfaces with targeting ligands to deliver siRNA loaded exosomes for TNBC treatment. We proved that exosomes can be utilized as nanocarriers to deliver siRNA to TNBC cancer cells very efficiently to inhibit cancer growth (Pi F, Binzel D et al. Nat. Nanotechnol. 2018;13:82). Preliminary data from the investigation with KB cell models in vitro has shown the mechanism behind the high efficiency of cancer inhibition to be the cytosolic delivery of siRNA via exosomes without endosomal trapping (Zhen Z, et al. J Control Release. 2019,311:43). In this study, we will investigate the targeting and delivery mechanisms of RNAi to TNBC cells by exosomes displayed with TNBC specific ligands for targeted delivery. It’s our goal to select a lead TNBC therapeutic candidate to move towards potential clinical translation. We will combine the targeting and drug delivery capabilities of RNA nanotechnology and exosomes for targeted delivery of RNAi to cell cytosols without endosomal entrapment. We will construct and evaluate the multi-functional exosomal RNA nanoparticle complexes harboring targeting ligands including RNA aptamers (EGFRapt, or alternatively, CD133apt, CD44apt, and nAHRsapt) or chemical ligand (Methotrexate) and tumor suppressing RNAi therapeutics (siRNA, suppressor miRNAs and anti- oncogenic miRNA). We will further investigate the pathways of internalization and intracellular trafficking in addition to investigating whether the payloads in the exosome are delivered to cytosol via fusion or to the endosome via endocytosis. In depth studies of the release and cellular processing of RNAi cargo loaded into exosomes will be completed. We will investigate the PK/PD parameters, delivery mechanisms, antitumor efficacy, and safety of the therapeutic RNA nanoparticles in order to select a lead candidate for preclinical translation. The preclinical studies will give a clear understanding of the viability of exosome-RNA nanoparticle complexes for TNBC therapy and provide data to move towards an Investigational New Drug (IND) application that will facilitate advancement to clinical trials.

NIH Research Projects · FY 2026 · 2021-05

Summary Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are an exciting and promising new class of anticancer drugs, which have been approved by the FDA for recurrent ovarian cancer with BRCA1 or BRCA2 mutations, and as maintenance therapy after frontline therapy for platinum sensitive ovarian cancer regardless of BRCA mutation. PARPi selectively kill BRCA1/2-deficient cancer cells through synthetic lethality. However, patients receiving PARPi eventually develop cancer progression, and acquired PARP inhibitor resistance remains a clinical hurdle. One of the mechanisms underlying acquired PARPi resistance is the restoration of DNA repair capacity, mainly through the secondary mutations of BRCA1/2. Our preliminary study has demonstrated that PARPi can enhance the Aldehyde dehydrogenase (ALDH) activity in ovarian cancer cells, mainly through inducing expression of ALDH1A1, an isoform of the ALDH family. In addition, we also found that ALDH1A1 is able to augment the microhomology-mediated end joining (MMEJ), one of the pathways for repairing DNA double-strand breaks (DSBs), enhance the expression of DNA polymerase θ (Pol θ), a key player in the MMEJ pathway, as well as reduce the sensitivity of BRCA2 mutated ovarian cancer cells to PARPi. Based on this scientific premise, we generate a hypothesis that PARPi-induced overexpression of ALDH1A1 enhances MMEJ via increasing the expression of Pol θ, and promotes cell survival after PARPi treatment in HR-deficient cancer cells, eventually leading to acquired PARPi resistance. Consequently, inhibition of ALDH1A1 should be able to synergize with PARPi in treating HR-deficient EOC, and reverse resistance to PARPi in HR-deficient EOC. The main objective of this proposal is to determine a novel mechanism that contributes to PARPi resistance in BRCA1/2-mutated EOC cells, and test the efficacy of targeting this mechanism in preventing and reversing PARPi resistance in these cells. Two specific aims are proposed to test this hypothesis and achieve our goal. In specific aim 1, we will determine the mechanism underlying PARPi-induced augmentation of MMEJ in HR-deficient EOC cells and its contribution to PARPi resistance. In specific aim 2, we will determine the therapeutic potential of an ALDH1A1 inhibitor, NCT-505, in preventing and reversing PARPi resistance in BRCA1/2-mutated EOC in vitro and in vivo. It is our expectation that at the conclusion of this project, we will have demonstrated a new mechanism contributing to the development of PARPi resistance in HR-deficient EOC. We will have also shown the therapeutic potential of an ALDH1A1 inhibitor in treating these patients.

NIH Research Projects · FY 2025 · 2021-05

Close to 45,000 Americans die from suicide each year and rates have been steadily rising for the last two decades. There is an urgent need for better access to effective suicide prevention strategies that are highly transportable across medical settings. Data from the current team indicates that a single session of crisis response planning (CRP) reduces suicide behavior by 76%; however, it is unknown how CRP works, and for whom, curtailing strategy optimization and widespread implementation. Clinicians speculate that CRP works by strengthening emotion regulation capabilities and reducing stress reactivity; however, this hypothesis has never been tested and no prior study has identified neural mechanisms and predictors of changes in suicide risk following intervention. Related, there are no objective brain-based markers of ‘reduced’ suicide risk to inform clinical decision making and guide high-risk patients to needed services. To address these gaps and ultimately improve suicide prevention efforts we seek to identify brain-based mechanisms and predictors of changes in suicidality following a single session of CRP in a cohort of adults with active suicidal intent. We will take an innovative and comprehensive approach by probing stress reactivity and emotion regulation neural circuits in the context of a clinical trials design. Specifically, we will combine sources of information and simultaneously collect assessments of neural function, psychophysiology (i.e., startle eyeblink potentiation), behavior and self-report during functional magnetic resonance imaging (fMRI) before (Time 1) and after (Time 2) randomization to a single, one-hour session of CRP or standard suicide risk management (control). A small group of volunteers with no history of suicide ideation or intent will be included for comparison. Using ecological momentary assessment (EMA) technology, acute changes in suicidality following intervention will be assessed twice daily for the first week. Monthly online clinical surveys will also be administered, and at 6-months post- intervention, the entire multimodal assessment battery will be re-administered (Time 3). This innovative, multilayered, longitudinal design will allow for a well-controlled test of how a single session of CRP acutely changes suicide risk (Aim 1). The study will also address whether the acute effects of CRP are sustained over time and how neural function influences long-term changes in suicidality (Aim 2). Lastly, we will conduct sophisticated analyses to integrate data across ‘units of analysis’ and functional domains to test whether there are reliable prognostic indicators of CRP intervention success (Aim 3). Findings from this study will provide critical new knowledge regarding how an ultra-brief session of CRP works and for whom, while uncovering a mechanistic signal of reduced suicide risk. In addition, consistent with the mission of the NIMH BRAINS program, the award will facilitate the launch of the PI’s innovative translational program of research and pave the way for a series of large-scale studies aimed at identifying neurobiological targets for resolving suicide risk and testing whether interventions with effective target engagement can enhance suicide prevention outcomes.

NIH Research Projects · FY 2025 · 2021-04

The project, "The economic and social impact of pandemic mitigation policies: A cross-country analysis of macro events," will explore the economic and social effects the mitigation policies and information environment that the pandemic spawned. We will link those policies to data from ongoing household-based panel studies from 10 countries and rich administrative data from an eleventh. We will exploit the substantial intra and inter-country temporal and geographic variation in non-pharmacological intervention policies induced by the pandemic disease. That variation, coupled with pre-pandemic baseline levels or long-running trends in the outcomes we will study, will identify the effects of the mitigation policies.

NIH Research Projects · FY 2025 · 2021-04

Abstract Acute Graft-versus-Host Disease (aGVHD) occurs due to donor T cell alloreactivity against host tissues and is the major cause of non-relapse mortality after allogeneic stem cell transplantation (alloSCT). Approximately 50% of patients are not responsive to front-line corticosteroid therapy and deemed “steroid-refractory” with limited to no effective standard therapies. Our long-term research goal is to identify and evaluate innovative approaches to improve patient outcomes by preventing and/or abrogating aGVHD toxicity post-alloSCT. The proposed research presents pioneering clinical and translational approaches to target epigenetic regulation of inflammatory mediators via Bromodomain and Extraterminal (BET) domain inhibition using novel non- benzodiazepine structured PLX (51107 and 2853) to prevent and mitigate aGVHD. PLX boasts improved pharmacokinetic and tolerability profiles to benzodiazepine-scaffolded BET inhibitors. We observed that BET inhibition with PLX results in potent suppression of T cell proliferation and pro-inflammatory cytokine secretion of IFN-g, IL-6, and TNF-α without affecting T cell viability. Our data also demonstrate that BET inhibition with PLX significantly downregulates transcription of T cell costimulatory genes, major inflammatory cytokines, and cell-cycle regulators. Importantly, we identified that BET inhibition decreases T cell proliferation and dampens inflammation independent of STAT-1. Thus, we targeted JAK/STAT blockade with the recently FDA approved JAK1/2 inhibitor ruxolitinib and observed synergistic effects of dual BET and JAK1/2 inhibition on T cell proliferation. We hypothesize that BET inhibition with PLX is a feasible, effective strategy to mitigate T cell mediated aGVHD inflammation as a single agent. Further, we propose preclinical analyses to assess the synergistic mechanisms and tolerability of dual BET/JAK1/2 inhibition. Aim 1 of the proposed research tests our hypothesis in a Phase 1b/2 proof-of-principle clinical trial for patients with steroid-refractory aGVHD with single agent PLX51107. Correlative studies are designed to assess 1) response to therapy and 2) immune reconstitution of T cell subsets. In Aim 2, we will use preclinical models of aGVHD to test the hypothesis that BET inhibition results in improvement in aGVHD survival by downregulating Th1 and Th17 pathogenic T cell responses while maintaining Treg mediated tolerance. We observed a very strong reduction in expression of Th1/Th17 pro-inflammatory genes such as IFN- g, IL-17 and IL-2 as well as co-stimulatory molecule CD40L with PLX treated T cells in vitro. We propose to analyze the effects of PLX on individual T cell subsets and their implications in aGVHD pathogenesis in vivo. Aim 3 will 1) test the hypothesis that dual BET and JAK1/2 inhibition will reduce GVHD and prolong duration of treatment response; and 2) assess the effects of combination therapy on Treg and effector T cell function. These experiments will determine the future applicability of T cell subset directed therapy for aGVHD as well as inform the development of future clinical trials combining BET and JAK1/2 inhibition for aGVHD prevention or treatment indications.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT Chemotherapy induced peripheral neuropathy is a common dose-limiting toxicity that can reduce therapeutic effectiveness and impact quality of life for cancer patients. The overarching goal of this research is to determine the molecular basis of chemotherapy-induced peripheral neuropathy to support the development of targeted therapies to prevent and treat this toxicity. The proposed studies are based on a reverse translational pharmacogenetic approach that uses genetic association findings to implicate critical pathways in peripheral neuropathy. Recent genetic association and functional validation findings support a role for sphingosine-1- phosphate (S1P) signaling in chemotherapy-induced neurotoxicity, which are consistent with previous studies in rodent models. The studies proposed in this application will extend these findings and address a significant gap in our knowledge of S1P signaling in target cells for toxicity, peripheral sensory neurons. The central hypothesis that will be tested is that modulation of S1P signaling in peripheral sensory neurons by microtubule targeting agents plays a critical role in their neurotoxicity. A human induced pluripotent stem cell derived sensory neuron model of chemotherapy neurotoxicity (iPS-SNs) will be employed for all studies. Pharmacological and genetic approaches will be used to modulate S1P signaling and interrogate chemotherapy toxicity linked to this signaling pathway. The three aims are complementary and address discrete functions of S1P. The first aim will investigate whether microtubule targeting agents alter sphingolipid metabolism in sensory neurons and will link specific S1P receptors to cytoskeletal changes. The studies proposed in the second aim will focus on Rho GTPase signaling downstream of S1P receptors and will establish the S1P signaling axis that is critical for chemotherapy-induced changes in neurite structure and the development of retraction bulbs. The third aim will use scRNA-seq and sc-ATACseq to elucidate whether paclitaxel-induced changes in gene expression in iPS-SNs involve S1P effects on chromatin accessibility. The ability of fingolimod, a multiple sclerosis therapy that targets S1P receptor signaling and is currently being tested for prevention and treatment of paclitaxel-induced peripheral neuropathy, to protect against chemotherapy-induced neurotoxicity will be examined. Collectively, these studies will reveal molecular mechanisms underlying the axon degeneration that occurs in sensory neurons in response to microtubule targeting agents and elucidate novel mechanisms for neuroprotection with fingolimod.

NIH Research Projects · FY 2025 · 2021-04

Summary Listeria monocytogenes is a facultative intracellular pathogen responsible for the life-threatening disease listeriosis. Although Lm produces numerous virulence factors, the secreted pore-forming toxin LLO is indispensable for pathogenesis. LLO is secreted at all stages of the Listeria intracellular life cycle and binds to cholesterol to form transmembrane pores. This virulence factor perforates the membrane of the Listeria- containing endocytic vacuoles to release the bacterium into its replicative niche, the cytosol. It was recently established that LLO also perforates the host cell plasma membrane, which promotes host cell invasion. It remains to elucidate how infected cells maintain viability despite perforation of their plasma membranes and how this low-grade perforation impacts the course of Listeria infection. The work performed in Aim 1 will establish novel mechanisms that maintain viability of infected cells despite perforation of their plasma membranes by LLO. Preliminary studies, via screening of a large siRNA library, led to the identification of novel families of host proteins that were not previously known to repair the plasma membrane of toxin-perforated cells. The Aim 1 studies will establish the mechanisms of action of these novel proteins. Specifically, they will determine the role plasma membrane depolarization plays in organizing calcium-dependent lysosome exocytosis, leading to the release of cytoprotective cathepsins on the cell surface. Aim 1 will also establish a new role for the septins, a family of cytoskeletal proteins, in plasma membrane repair. These studies will employ high-speed and super- resolution microscopy to analyze the molecular assemblies that orchestrate plasma membrane repair. The Aim 2 studies will establish the impact of plasma membrane perforation by LLO on Listeria intracellular survival and the innate immune response of antigen-presenting cells. We showed that transient plasma membrane perforation by LLO triggers Ca2+ influx-dependent activation of conventional PKCs on the endosomal network, a signaling event that is critical for Listeria phagosome escape. Aim 2 will identify the PKCs effectors by SILAC- based quantitative proteomic approach and how they contribute to the release of Listeria into the cytosol. Plasma membrane perforation also causes K+ efflux, which is known to activate the NLRP3 inflammasome. Aim 2 will dissect the role of low-grade plasma membrane perforation in the maturation of antigen presenting cells to enhance T cell immunity, in vitro and in vivo. This work is expected to broadly impact the development of vaccines and novel therapeutic strategies for a wide range of diseases in which pore-forming toxins are employed by pathogens.

NIH Research Projects · FY 2025 · 2021-04

Spinal cord injured (SCI) individuals have significantly reduced lifespans compared to the general population, a statistic that has not changed in 30 years. One reason is “endocrine, metabolic and nutritional diseases”, which are increasing at alarming rates in the SCI population (2019 Models Systems report). This is evidenced by SCI individuals having increased prevalence of Metabolic Syndrome (MetS), the complications of which put them at a higher risk for diabetes, cardiovascular disease and stroke compared to the general population. A central feature of MetS and contributor to morbidity is hepatic pathology in the form of non-alcoholic steatohepatitis (NASH), a severe form of nonalcoholic fatty liver disease (NAFLD). NASH includes hepatic lipid accumulation (steatosis) and inflammation, which in turn cause hepatocyte damage and release of pro-inflammatory mediators. NASH likely facilitates subsequent systems-wide pathology after SCI. Experiments in this proposal are designed to identify mechanisms that initiate and sustain NASH in acute and chronic SCI. Focus will be on increased sympathetic input to the liver and consequent intracellular changes in the liver that drive inflammation and fat accumulation. We hypothesize that excess sympathetic input to the liver after SCI initiates pro-inflammatory cascades involving TNFa and NFkB, which in turn initiate hepatic fat accumulation and prolonged inflammation. We will use transgenic mouse technology as well as unbiased - omics approaches to comprehensively identify cellular changes in the liver induced by SCI that drive “neurogenic” NASH and subsequent features of MetS. Our long-term goal is to identify therapeutic targets that can interrupt the dysfunctional spinal cord/liver axis after SCI to restore liver homeostasis and improve overall metabolic health.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT Susan Yoon, PhD, MSW, is a social scientist whose overarching career goal is to prevent substance use and improve health outcomes among vulnerable, at-risk youth by identifying key mechanisms for risk and resilience to adolescent substance use following child maltreatment. This K01 Mentored Research Scientist Development Award will provide Dr. Yoon with rigorous training and systematic mentored research experiences that will accelerate her successful transition to an independent investigator in the field of adolescent substance use research. A team of highly qualified and committed mentors, who are prominent researchers in their respective fields, will guide and supervise her K01 research and training activities. The proposed career development plan encompasses four hands-on training goals to improve her knowledge and skills in: 1) adolescent substance use research with vulnerable youth; 2) the use of contextual/spatial (i.e., activity space) data; 3) the application of the resilience framework; and 4) advanced statistics, including person-centered analysis, multi-level modeling, and spatial analysis. During the K01 award period, Dr. Yoon will obtain and apply these new skills and knowledge to address important questions about the complex associations among child maltreatment, youth activity spaces—refers to areas or places a person visits in daily routine—and adolescent substance use. Child maltreatment and adolescent substance use are two serious, tightly connected public health concerns. Despite the well-established link between these phenomena, the underlying mechanisms and moderators (protective factors) of this link remains elusive, significantly hampering our ability to effectively prevent adolescent substance use. Thus, the proposed study aims to: 1) examine how distinct patterns of longitudinal maltreatment experiences are associated with different patterns of adolescent substance use trajectories; 2) investigate the mediating effects of risky attributes within youth's activity spaces on the association between child maltreatment and adolescent substance use; and 3) determine the extent to which protective attributes within youth's activity spaces buffers the impact of child maltreatment on substance use during adolescence. To address the study aims, Dr. Yoon will employ creative integration and analysis of diverse data sources, including data linkage between administrative child welfare records and geo-coded youth spatial exposure data. The study also introduces theoretical innovation through the integration of resilience theory and novel spatially-situated protective factors to understand resilience to substance use following child maltreatment. This study directly addresses NIDA's research priorities and strategic goals of supporting research to understand the complex interactions of environmental, behavioral, and social factors influencing drug use trajectories. Findings will offer valuable insight into key contextual mechanisms and protective factors that can be targeted in interventions to prevent substance use among high-risk, vulnerable youth.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT: Traumatic brain injury (TBI) can lead to significant neuropsychiatric problems and neurodegenerative pathologies that develop with time after injury. These issues may be propagated by neuroinflammatory processes that continue well after the initial injury. Pt.3 We have reported that diffuse TBI in mice leads to “microglial priming” within cortical and hippocampal regions, in which the microglia remain in a sensitized state and are highly inflammatory following an immune challenge 30 days post injury (30 dpi). In this application, we show a distinct phase transition from acute (8-24 h) to sub-acute (7 d) and then to chronic (30 d) cortical-inflammation/microglia priming after TBI. Acutely, there was an inflammatory response after TBI that evolved into a subacute phase 7 dpi that was dominated by interferon (IFN) type I signaling. IFN responses are activated by cell distress and damage to promote an immune response that can prime innate immune cells, including microglia. We provide evidence of cortical neuronal damage 7 dpi with corresponding microglial activation. Pts.3&6 Single cell RNA seq (scRNAseq) of the cortex 7 dpi shows unique clusters of microglia, trauma-associated, that are influenced by IFNs. These microglia are involved in dendritic remodeling and suppression of neuronal homeostasis. At 30 dpi, there was cognitive impairment (associated with HPC & CTX), reduced network connectivity, and increased immune reactivity of primed microglia. Microglia are critical in these processes because microglial elimination (CSF1R antagonist) prevented TBI-induced neuroinflammation and IFN signaling, attenuated dendritic atrophy, and improved network connectivity. Thus, we hypothesize that increased interferon signaling is critical in promoting microglial priming and chronic neuroinflammation, dendritic remodeling, and cognitive decline. To address this, three aims are proposed using a midline fluid percussion injury in mice. In Aim-1, we will eliminate microglia to determine the influence of microglia on other CNS cells in the cortex and hippocampus acutely, sub- acutely, and chronically after TBI. ScRNAseq will be used to determine the transcriptome signature of microglia over time and in parallel with astrocytes, oligodendrocytes, and neurons at 3 these critical times after TBI. The focus will be determining which cells express IFNs and IFN receptors, and how they respond to increased IFN signaling with TBI. In Aim-2, we will determine if IFN signaling is critical in chronic neuroinflammation, pathology, cognitive decline, and microglial priming after TBI. Here, we will attenuate IFN signaling at the levels of IFN-a/b receptor activation (IFNαRKO, Mgl-IFNαRKO) and IFN production (STINGKO) to determine the extent to which these interventions ameliorate neuroinflammation, pathology, and microglial priming. In Aim-3, we will determine if TBI-induced microglial priming, chronic neuroinflammation, and cognitive decline 30 dpi are reversed by forced microglia turnover. We will remove microglia (CSF1R antagonist) when IFN responses are highest 7 dpi, and allow for microglial repopulation to 30 dpi. Completion of these aims will provide new insight on the IFN pathway that appears to be critical in the transition from acute to chronic inflammation mediated by microglia after TBI.

NIH Research Projects · FY 2025 · 2021-03

Underage drinking causes tremendous burden, and there is an urgent need to understand who is vulnerable, and why/how, to facilitate accurate detection and prevention. Exposure to interpersonal trauma (e.g., physical or sexual abuse) is a well-established risk factor for alcohol problems in youth; however, the developmental mechanisms through which trauma leads to alcohol use are unclear. In addition, millions of youth experience interpersonal trauma but only a subset develop alcohol use disorder (AUD). Identifying these high-risk youth, and the neurobiological mechanisms contributing to their risk, can pinpoint objective early intervention targets and aid in AUD prevention. Data from the Early Stage Investigator (ESI) PI suggests that youth with interpersonal trauma exposure and high levels of aversive reactivity to unpredictable threats (U-threat; temporally unpredictable and/or ambiguous) are at the greatest risk for the development of alcohol problems in young adulthood. To date, however, a mechanistic test of this hypothesis has never been conducted in a high-risk, trauma-exposed sample. The overarching goal of the study is to therefore validate a novel neurobiological risk phenotype for alcohol problems in trauma-exposed youth using multimodal U-threat reactivity data. Leveraging our unique access to high-risk youth in the central Ohio area, we will recruit a cohort of 200, 16-19 year olds, with and without a history of interpersonal trauma exposure (125 with trauma and 75 without) and conduct multimodal (neural function, physiology, behavior, and self-report) assessments of U-threat reactivity and alcohol use. Six months after baseline we will conduct a second multimodal lab assessment of U-threat reactivity. Every 3 months post-baseline we will assess youths’ alcohol use and relevant psychiatric symptoms and behaviors via online survey (up to 24-months). This innovative, longitudinal design will allow for a well-controlled test of whether interpersonal trauma exposure and neurobiological reactivity to U-threat synergistically interact to predict escalations in alcohol use in youth (Aim 1). The study will also address whether reactivity to U-threat is a stable risk factor for alcohol problems or a modifiable target that corresponds to changes in drinking behaviors over time (Aim 2). If reactivity to U-threat does indeed track behavior, it can be used as an objective, mechanistic target for future ‘Fast-Fail’ treatment studies. Lastly, given our multi-layered approach, we will conduct innovative analyses to integrate data across ‘units of analysis’ (brain, physiology, behavior, self-report) to predict the onset of alcohol problems in trauma- exposed youth and develop a new and replicable predictor with the highest accuracy performance (Aim 3). Findings from this work will provide critical new knowledge aiding in the identification of youth most at-risk for alcohol problems. The study will also validate a mechanistic, neurobiological risk model that identifies targets for intervention during a peak developmental risk window for alcohol use to ultimately help reduce the burden of AUD in this high-risk population.

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY For this proposal we intend to develop a novel vaccine to prevent and possibly treat Human T cell leukemia virus type-1 (HTLV-1) associated diseases. HTLV-1 is a human retrovirus that is the causative agent of a malignant T CD4+ cell lymphoproliferation referred to as Adult T cell leukemia/lymphoma (ATLL), as well as several inflammatory disorders with the most problematic being human myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 infection is endemic in many areas around the world including southern Japan, the southern United States, central Australia, the Caribbean, South America, equatorial Africa, and the middle East. Over 10 million people may be infected worldwide. It is estimated that approximately 5% of HTLV-1 positive individuals will develop ATL, and 2% HAM/TSP. Seropositive rates in certain areas reach 20–40% among people aged over 50 years. With millions affected worldwide, HTLV-1 is a major problem in endemic communities and remarkably, there are no effective vaccine or treatment options to prevent ATL or HAM/TSP afflicted individuals. Given this, aim to develop and test the efficacy of a novel vaccine to prevent HTLV1-mediated disease. Aim 1: To evaluate the immunogenicity, in immunocompetent murine models, including mice with a humanized immune system (NSG™-SGM3) VSV-based vaccine vectors that express the HTLV-1 glycoprotein and regulatory proteins TAX and HBZ (VSV-gp62-∆HT). The ability of our candidate vaccine to generate neutralizing antibodies to the glycoprotein will be analyzed, as well as the production of cytotoxic T cells (CTLs) to gp62, TAX and HBZ. Aim 2: We aim to compare whether our vaccine can be used to prevent HTLV-1 transformation associated disease. This approach will include establishing whether VSV-gp62-∆HT can prevent the establishment of HTLV- 1-assocated leukemia/lymphoma in NSG™-SGM3 mice. Our objectives are to collate sufficient information to warrant the consideration of a variety of Phase I trials to prevent HTLV-1 -associated disease.

NIH Research Projects · FY 2025 · 2021-02

Project Summary The acute respiratory distress syndrome (ARDS) is a deadly condition characterized by the rapid onset of hypoxemia and respiratory failure. The mainstay of therapy for ARDS patients is supportive care with mechanical ventilation (MV). Although life-saving, mechanical ventilation can exacerbate lung injury and even cause de novo injury, known as ventilator induced lung injury (VILI). VILI arises from mechanical forces during MV including excessive stretch (volutrauma), excessive pressure (barotrauma), and injury due to repeated collapse and reopening of lung units (atelectrauma). The molecular mechanisms by which these mechanical forces exacerbate lung injury remain poorly understood. Clinicians try to prevent VILI by monitoring airway pressures and using low tidal volumes, but injury persists even when these parameters are in a “safe” range. Currently, there are no pharmacologic therapies to prevent or treat VILI in patients with ARDS. mTORC1 is a central regulator of cell growth and lipid metabolism. In contrast to canonical activation of mTORC1 under favorable growth conditions, we recently discovered that mTORC1 is activated in lung epithelial cells following injurious mechanical ventilation. We also found that pharmacologic mTORC1 inhibition prevents lung injury during mechanical ventilation. We hypothesize that mTORC1 activation plays a central role in mediating VILI and represents a novel therapeutic target in ARDS. We will determine the mechanisms by which mTORC1 inhibition prevents VILI using mice with mTORC1 inactivation in type I and type II alveolar epithelial cells as well as novel in vitro models of mechanical ventilation in the human lung. In Aim 1 we will identify how mTORC1 activation induces surfactant dysfunction during ventilator induced lung injury. In Aim 2 we will identify the mechanisms by which mTORC1 regulates epithelial membrane repair following injurious mechanical ventilation. In Aim 3 we will use clinically relevant 2-hit models that utilize mechanical ventilation following lung injury from sepsis or influenza pneumonia to test the efficacy of mTORC1 inhibition to prevent VILI in ARDS. Our studies will provide an in-depth understanding of how mTORC1 activation impairs surfactant function and membrane repair during VILI and will identify novel drug targets for patients with ARDS.

NIH Research Projects · FY 2025 · 2021-02

SUMMARY/ABSTRACT Immune checkpoint inhibitors (ICIs) have transformed the management of patients with metastatic non-small cell lung cancer (NSCLC). Unfortunately, over 50% of patients do not respond to these therapies. Combination strategies with chemotherapy-ICIs show progress, but long-term responses remain rare, pointing to the role for other tumor-associated mechanisms affecting functionality of immune cells. Adenosinergic signaling has recently emerged as a powerful immuno-metabolic regulator within the tumor microenvironment (TME) exploited by tumors to promote their growth and suppress immunity. Preclinical studies on interference with adenosine generation or signaling through A2A and A2B adenosine receptors (A2BAR) have demonstrated efficacy in relieving this immunosuppression by reducing stress in the TME and decreasing expression of key adenosine-generating enzymes, thereby enhancing efficacy of immune checkpoint inhibition. A2BAR blockade in particular enhanced anti-tumor immunity through both a reduction in myeloid-derived suppressor cell differentiation and an enhancement of the capacity of dendritic cells to evoke anti-tumor T cell responses. These findings provide strong rationale for clinical applications of A2BAR antagonists in combination with current ICIs. To determine whether disruption of A2BAR signaling has the potential to improve upon single agent PD-1 immunotherapy, we propose a phase Ib clinical trial testing the A2BAR antagonist PBF-1129 in combination with nivolumab in patients with metastatic NSCLC. The primary objective of the clinical study is to evaluate the safety and tolerability of combination PBF-1129 with nivolumab; preliminary evidence of efficacy will be evaluated in an expansion cohort. Analysis of pre- and on- treatment blood and tumor samples will be conducted to evaluate the correlation between and immunological parameters and adenosine generation and signaling, and to evaluate the efficacy of PBF-1129 in targeting adenosine-mediated immunosuppression. Finally, we intend to further elucidate mechanisms of metabolic TME and immune regulation by adenosine in pre-clinical cancer models and test the combined PBF-1129/anti-PD-1 approach to ameliorate metabolic TME using a novel imaging modality. Together, we expect that A2BAR antagonist treatment combined with nivolumab will be a safe, effective approach targeting different mechanisms of immunosuppression and tumor growth in metastatic NSCLC patients, that we will uncover immunological profiles reflective of adenosinergic signaling disruption in these patients, and that we will demonstrate the utility of a novel combined imaging approach for evaluation of adenosine targeting in the TME.

NIH Research Projects · FY 2025 · 2021-02

Project Summary: Lung cancer is the leading cause of cancer deaths in both men and women. Non-small cell lung cancer (NSCLC) accounts for large majority of lung cancer diagnoses, and novel treatment strategies for this disease are urgently needed. ATR and its downstream effector CHK1 are key component of replication stress (RS) response that specifically deal with the stalled replication forks during DNA replication and are critical for cell survival under RS. Inhibitors targeting ATR and CHK1 are currently being tested in clinical trials. However, only limited efficacy has been observed when those agents are combined with standard therapy. Identifying the new synergistic conditions that render cells sensitive to ATR/CHK1 inhibitors will be key to improving the efficacy of these agents. Squalene epoxidase (SQLE), an enzyme controlling cholesterol biosynthesis by converting squalene to oxidosqualene in endoplasmic reticulum (ER), is frequently overexpressed in human cancers, including lung cancer. High expression of SQLE is associated with poor prognosis. Our recent genome-wide loss of function screen discovered that SQLE reduction led to enhanced sensitivity to a CHK1 inhibitor. Thus, the goal of this application is to determine whether SQLE inhibition sensitizes NSCLC cells to ATR and CHK1 inhibitors and whether high SQLE-expressing NSCLC cancer can be specifically targeted by the combined inhibition of SQLE and ATR/CHK1. Our preliminary data suggest that SQLE inhibition by shRNA knockdown causes RS and activates ATR/CHK1 activation. In addition, SQLE inhibition lead to increased protein expression of WIP1, a phosphatase that suppress the activity of ATM, a master DNA damage response protein controlling DNA repair. We hypothesize that SQLE inhibition leads to increased RS by suppressing DNA repair, rendering cells sensitive to ATR and CHK1 inhibition. Thus, co-administration of an SQLE inhibitor and an ATR or CHK1 inhibitor could synergistically suppress tumor growth. To test our hypothesis, three Specific Aims are proposed. Specific Aim 1 will interrogate the mechanism by which SQLE inhibition leads to increased WIP1 expression. Specific Aim 2 will determine whether SQLE inhibition leads to increased RS by impairing DNA repair, particularly homologous recombination, a major repair pathway that prevents and antagonizes RS. Specific Aim 3 will assess the efficacy of combined SQLE inhibition and ATR/CHK1 inhibition in suppressing tumor cell growth using in vitro assays as well as cell line-based and patient-derived xenograft (PDX) models of NSCLC. If successful, our studies will reveal a new synthetic lethal interaction between inhibition of SQLE and ATR/CHK1 and will have a significant impact on improving the survival of lung cancer patients by identifying novel therapeutic approaches.

NIH Research Projects · FY 2025 · 2021-01

Project Summary/Abstract Cortical inhibitory GABAergic interneurons (INs), which develop intricate local circuits, critically regulate higher- order brain functions by balancing and shaping neuronal activity. Consistent with its indispensable role in normal brain functions, malformation/malfunction of the inhibitory system is implicated in a wide array of brain disorders such as schizophrenia, autism, and epilepsy. Despite their importance, the molecular mechanisms underlying the wiring of IN local circuits remain largely unknown. Cortical INs comprise diverse cell types that are defined by morphology, physiology, and gene expression. Notably, different IN subtypes also show distinct synaptic specificity at laminar/cellular as well as subcellular levels. Although subtype-specific synaptic connectivity is considered a critical property of INs to ensure functional diversity of the inhibitory system, the molecular mechanisms underlying IN synaptic specificity remains poorly understood. The objective of this proposal is to determine the molecular mechanisms by which IN subtypes establish layer/cell type- and subcellular domain-specific synapses. To achieve this goal, we will perform a series of experiments using chandelier cells (ChCs), which exclusively innervate axon initial segments (AISs) of layer-specific pyramidal neurons (PNs). The ChC is known to critically regulate PN spike generation and has been implicated in schizophrenia and epilepsy. Besides their functional significance, the stereotypy of their synaptic organization make ChCs an attractive model to study the molecular mechanisms for IN synaptic specificity. Our preliminary data has shown that: (1) IgSF11 proteins that are known to bind with each other are expressed in both ChCs and layer-specific target PNs, (2) Gldn proteins that are known to bind to AIS-enriched proteins, NF186, are preferentially expressed in ChCs, (3) IgSF11 in ChCs plays an essential role in their presynaptic development, (4) Gldn and NF186 appear to play a role in initiating ChC synapses, and (5) IgSF11 that is free from the Gldn-NF186 system appears not to induce ChC synapses. Based on our findings, we propose to test the hypothesis that the layer-specific synaptogenic action and the subcellular domain-specific recognition mediated through IgSF11 homophilic interactions and Gldn-NF186 interactions, respectively, cooperatively determine ChC synaptic specificity. We will pursue the following specific aims to test our hypothesis. In Aim 1, we will determine the role of the IgSF11 homophilic interaction between pre- and postsynaptic neurons in layer-specific synapse formation by ChCs. In Aim 2, we will determine the role of Gldn and NF186 in ChC synapse formation on AISs. In Aim 3, we will determine the regulatory role of NF186/Gldn in gating IgSF11 signaling to induce ChC presynaptic boutons at AISs. Upon completion of this study, we will gain not only important insights into molecular mechanisms for IN wiring but also a clue to developing therapeutic strategies to functionally repair disordered/damaged brains.