University Of Arizona

NIH Research Projects · FY 2025 · 2022-07

Project Summary Accidental overdoses fatalities define the opioid epidemic despite research efforts for alternative means to address Opioid Use Disorder [1] and alternative chronic pain therapies [2]. The Centers for Disease Control report over 400,000 lives lost since 1999 and more than 130 people lose their life to an accidental overdose daily [3]. This number will continue to climb with excessive utilization of prescription opioids and the presence of stronger synthetic opioids in the illicit market [4-6]. A potentially fatal opioid overdose can be successfully reversed with naloxone, mu opioid receptor antagonist, [7], but with the number of pain patients rising above heart disease and diabetes [2] and the increased presence of fentanyl in the illicit opioid market, adequate distribution of naloxone to reverse an overdose before oxygen deprivation occurs remains difficult [8]. Attempts to reduce opioid overdoses with novel opioid-like compounds have been largely unsuccessful [9]. Therefore, it is essential that unexplored therapeutic strategies to prevent opioid-induced respiratory depression be discovered. Fatal opioid overdoses are typically attributed to respiratory depression, during which neurons within the preBötzinger complex (pBc) in the brainstem that control reflexive inspiration are inhibited. Our pilot data suggest that 1) the pBc contains the components of the endocannabinoid system, including the cannabinoid receptor 2 (CB2R), 2) CB2R activation by endogenous cannabinoid system (ECBS) lipids is critical to normal respiration control, and 3) exogenous application of a CB2R agonist mitigates morphine induced respiratory depression. The current proposal will build upon these findings, using behavioral pharmacology, whole body plethysmography, imaging, molecular biology, analytical chemistry, and gene-editing to test the hypothesis that endogenous cannabinoid levels in the pBc are reduced during opioid induced respiratory depression (OIRD) and that administration of a brain penetrant CB2R agonist will mitigate OIRD. Aim 1 will test CB2R agonism as a strategy to mitigate to OIRD. Each aim contains rationally designed studies that include sex differences through inclusion of male and female mice, application to both acute and chronic use of medicinal and recreational opioids (fentanyl, oxycodone, and heroin), and multiple chemical classes of CB2R agonists to prevent and reverse OIRD. Aim 2 will determine levels of endocannabinoid lipids, enzymes, and receptors in the preBotzinger complex (pBc) during OIRD. Successful completion of proposed studies will serve to enhance our knowledge of the role of ECBS within the pBc in during normal respiration and during OIRD and validate targeting the CB2R as a safe treatment therapeutic to reduce opioid overdoses.

NIH Research Projects · FY 2026 · 2022-07

Liposome, composed of a lipid bilayer comprising phospholipids (PL) and sterols such as cholesterol (Chol), has been extensively used for packaging and delivery of therapeutic agents due to its intrinsic biocompatibility and biodegradability. While most approved liposomal nanotherapeutics can improve pharmacokinetics (PK) and reduce systemic toxicities, improvements in therapeutic efficacy and overall survival are disappointing, underscoring the urgent need for enhanced therapeutic delivery. Chol plays a critical role in fortifying membrane packing and reducing bilayer fluidity and permeability by promoting the liquid condensed state in lipid membranes, enhancing bilayer rigidity and strength. Lipid bilayers with high levels of Chol are generally more stable than those without or with less Chol. However, under the physiological environment, Chol is rapidly extracted from the bilayer by biomembranes and serum proteins, which jeopardizes bilayer stability and results in premature content leakage, fast blood clearance and unwanted adverse effects, leading to suboptimal clinic efficacy. In addition, although enhanced permeability and retention effect allows nanotherapeutic accumulation to the periphery of diseased tissues, intracellular internalization and tissue penetration remain inefficient due to the tenacious resistance imposed by high interstitial fluid pressure and dense extracellular matrix, compromising the therapeutic outcome. These phenomena present formidable barriers for lipid bilayer-based therapeutic delivery. To tackle these key challenges, the overall vision of my research program is to establish a stabilized lipid bilayer with improved physicochemical properties that can further improve drug delivery and selectively fortify intracellular uptake and infiltration at target sites. We have established a Chol-derived PL via covalently attaching Chol to a PL with varied stimuli-responsive linkages. Via systemic structure activity relationship studies, we demonstrated that Chol-derived PL blocked Chol transfer, prevented payload leakage, prolonged circulation time, and augmented efficacy in treating lung inflammation, Alzheimer’s disease, lymphoma, pancreatic and triple negative breast cancer models, which were linker chemistry dependent. For the next five years, the goals of this proposal are to 1) unravel the underlying mechanisms and principles on how the structural alterations of a sterol-modified PL bilayer that forms liposome but cannot shuttle between biomembranes will affect drug and gene delivery via substituting Chol with other membrane sterols; and 2) establish a universal ultra pH-sensitive charge-reversal delivery platform to boost the cellular uptake and tissue penetration efficiency via incorporating an intelligent build-in cationization mechanism that selectively triggers effective adsorption-mediated endocytosis and transcytosis at diseased tissues. Completing these studies will provide fundamental and functional correlations of bilayer properties with therapeutic delivery, enable us to establish a set of design rules governing the optimal interactions between lipid bilayer and encased drugs, and provide a paradigm-shifting toolbox to advance the drug delivery technologies, facilitating clinical translation of treating human diseases.

NIH Research Projects · FY 2024 · 2022-07

SUMMARY Late stages of cancer metastasis involves the seeding of cancer cells at distant organs and the lethal outgrowth of seeded cells. In 2019, virtually all 31,620 prostate cancer (PCa) deaths in the United States were attributed to metastasis. Epithelial Mesenchymal Transition (EMT) is important for physiological development; however, EMT promotes migration, invasion, and seeding in multiple cancer types. In contrast, EMT inhibits proliferation and tumor outgrowth. SNAI1 and Twist1 are major EMT drivers that promote cancer cell seeding and inhibit cell proliferation and tumor outgrowth, in non-PCa. SNAI1 expression is detected in invasive primary and metastatic PCa. In contrast, the androgen receptor (AR) is a major driver of PCa cell proliferation and tumor outgrowth. Of note, AR activates, but SNAI1 represses, transcription of Cyclin D1 (a cell cycle driver). Both bind to an overlapping region of the ccnd1 (Cyclin D1) proximal promoter, suggesting direct competitive regulation of Cyclin D1 transcription. Given that outgrowth occurs at the end of the metastasis cascade, EMT is likely to occur transiently, early in PCa patients. In fact, vimentin (a marker of EMT) is highly expressed in PCa circulating tumor cells; however, vimentin is lowly expressed once seeded PCa cells grow into detectable metastases. While EMT and AR signaling have been independently investigated in PCa, how EMT and AR coordinate PCa metastasis remains unclear. Adhering to the transient nature of EMT in clinical PCa, a TetON-SNAI1 expression system was created, whereby transient expression of SNAI1 can be turned ON/OFF with doxycycline. Preliminary data shows that SNAI1 induces a mesenchymal morphology in PCa cells, suppresses cell growth, and upregulates mRNA of EMT markers such as vimentin and NPR2. However, AR signaling suppresses SNAI1’s ability to upregulate vimentin and NRP2 and downregulates Twist1 mRNA, independent of SNAI1 expression. Altogether, these data suggest that AR and SNAI1 have opposing roles in regulating EMT and proliferation; and are consistent with the late sequence of events that occur during PCa metastasis: i.e., seeding followed by outgrowth. Therefore, I hypothesize that SNAI1 mediates an anti-proliferative EMT-like invasion program to promote PCa seeding; subsequently, AR antagonizes SNAI1-mediated cyclin D1 repression to restore proliferation and promote metastatic outgrowth. The following aims are designed to address the central hypothesis. Aim1: Determine how AR impacts SNAI1’s ability to promote PCa invasion and metastatic seeding. Aim2: Determine how SNAI1 and AR regulate PCa proliferation. Aim3: Determine extent of AR and SNAI1 co- expression and activity in clinical PCa samples. Patients with aggressive PCa are treated with standard anti AR- signaling therapies; but will develop resistance within 2 years. Given the limited treatment options available for this group of patients, further study of SNAI1 and AR will help develop research models that are more clinically accurate and provide deeper insights for future therapeutic strategies.

NIH Research Projects · FY 2025 · 2022-07

Alzheimer's disease and related dementia (ADRD) constitutes a growing health crisis. Equally, chronic metabolic diseases such as type 2 diabetes (T2D) are increasing, because of the prevalence of obesity and other risk factors. T2D is a risk factor for ADRD and both T2D and ADRD share common causal mechanisms: insulin resistance; impaired glucose metabolism; inflammation; dyslipidemia; and impaired cholesterol mobilization. The APOE4 allele is the greatest genetic risk factor for AD. ApoE4 is poorly lipidated and lipidation of apoE, required for stability and positive function, is controlled by the ATP-binding cassette transporter ABCA1. Deletion of ABCA1 in FAD mouse models exacerbates pathology and behavioral deficits; and rare human loss-of-function mutations in ABCA1 increase ADRD risk. ABCA1 is a gene product of liver X receptor (LXR); however, induction of lipogenesis in the liver (steatosis and triglyceride elevation) by LXR agonists has hindered progress. A nonlipogenic ABCA1-inducer (NLAI) would address multiple causal factors in T2D and ADRD, including APOE4 risk in AD. We have optimized a phenotypic drug discovery strategy for NLAIs, yielding hit series that enhanced cholesterol mobilization, attenuated inflammation, and improved biomarkers of glucose metabolism. One hit and an early lead derived from it (CL2-57), increased ABCA1 and APOE, without upregulating lipogenic genes. CL2- 57 administered orally in the high-fat diet (HFD) model of obesogenic T2D, attenuated insulin resistance, reduced weight gain, and from full metabolomic analysis improved biomarkers and lipid profiles. Aim 1: To optimize NLAIs. Phenotypic optimization will be driven by reporter assays (induction of ABCA1 in CCF cells, with minimal effects on SREBP1c in HepG2 cells) and secondary assays in the testing funnel validated in development of CL2-57. In silico and in vitro predictors of oral/brain bioavailability and SAR will guide optimization. Validation by PCR/immunoassay will extend to ABCG1/APOE and FAS/SCD1 in cell cultures. Aim 2: In vivo PK/PD and safety. NLAI treatment of mice for 3 days ± LPS is sufficient to assess target engagement and pharmacodynamics in the liver and brain with a safety readout (no triglyceride elevation nor neutropenia) suitable to define PK/PD and dosing. Aim 3: In vivo efficacy will be measured A) in 5xFAD mice (Aβ, cognition, and disease-associated microglia) and B) in HFD-treated mice (WT, hAPOE3-KI, and hAPOE4-KI) to identify APOE genotype specific interactions with HFD and NLAI treatment in vivo and ex vivo in astrocytes and neurons. These mouse models will establish the efficacy of an NLAI development lead. Aim 4: Pretox and target deconvolution will be used to identify CYP liabilities and any off-targets that will inform future safety pharmacology. The following Milestones are proposed: #1 lead NLAI meeting in vitro TPP with brain bioavailability; #2 orally bioavailable lead NLAI that shows dose-dependent target engagement in the brain without lipogenesis, elevated triglycerides or neutropenia; #3 nomination of a development lead that meets the in vivo TPP with data on targets and CYPs.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT Despite decades of research into the immunopathology of heparin-induced thrombocytopenia (HIT), an unpredictable, life-threatening, immune-mediated adverse reaction to heparin treatment, a fundamental knowledge gap regarding the cause of HIT remains. The inability to predict HIT represents a considerable liability associated with heparin, which is given to 12 million individuals or one third of all hospitalized patients every year. Because the exact cellular and molecular mechanisms underlying HIT have yet to be identified, including intrinsic immune cell roles and the difference between pathogenic and non-pathogenic antibodies, there is an essential need to apply alternative approaches to understand the biological basis for HIT and to identify clinically implementable biomarkers. The PI’s central hypothesis is that immunogenomic variation impacts HIT pathogenesis and can be used to differentiate non-pathogenic and pathogenic PF4/heparin antibodies, which are produced by immune cell populations intrinsic to HIT. Based on strong preliminary data that constitute the largest genome-wide association study (GWAS) for HIT, the working hypothesis is that the presence of the HLA-DRB3*01:01 allele combined with proliferating Vβ 5.1 family T-Cell receptor (TCR) clonotypes in Th2 cells predisposes patients to pathogenic PF4/heparin antibody production, and these antibodies result in high HIT risk in patients with the ABO O blood group. We will pursue three Specific Aims (SAs) to test the central hypothesis: (SA1) Determine the role of ABO variation in HIT; (SA2) Determine the influence of genomic variation on PF4/heparin antibody production; and (SA3) Identify intrinsic immune cell involvement in HIT. In SA #1, deep ABO sequencing data will be utilized in our large cohort of functional assay-confirmed HIT patients to elucidate the role of ABO variability in HIT. In SA #2, we will perform several GWAS to determine genetic influences of PF4/heparin antibody production, including diverse populations, and differentiate genetic influences on pathogenic versus non-pathogenic antibodies. In SA#3, we will sample paired cell populations during the acute phase of HIT and after HIT resolution and conduct focused single cell (sc) studies to determine proliferation of TCR clonotypes and activated cell populations using sc-RNA-TCR-CITE-seq. Our studies overcome major limitations of previous genomic studies of HIT by incorporating large, diverse cohorts, a PF4/heparin antibody- positive case group, and functional assay confirmation of HIT cases. This work is technically and conceptually innovative as it leverages a sc-RNA-TCR-CITE-seq approach, utilizes large, unique HIT cohorts, and advances an original model of HIT immunopathogenesis. We expect to provide mechanistic insights into HIT pathology and advance a framework for clinical translation of HIT biomarkers with direct application to other adverse drug reactions. The proposed studies leverage large-scale biological data to distinguish patients pre-disposed to HIT, potentially shifting clinical practice from treatment to prevention through biomarker-guided heparin treatment.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Oral squamous cell carcinoma (OSCC) is the sixth most common human cancer worldwide. Approximately 30% of the oral premalignant lesions (OPLs) progress to OSCC, a process that may have a multifocal origin and can be promoted by carcinogens such as those found in tobacco. Our long-term goal is to identify the genetic alterations that promote high risk of progression to OPLs and to determine how those alterations modulate the response of OPLs to preventive strategies. The TP53 gene (also known as p53) and CDKN2A are the most frequently mutated genes in oral cancer, also found altered in OPLs. p53 GOF mutations and genomic alterations that result in loss of the CDKN2A gene associate with “cold” immune microenvironments in OPLs and OSCCs, with high risk of progression to carcinoma, and with extremely poor outcomes in OSCC patients. We hypothesize that the early appearance of mutations in p53 and CDKN2A inactivation modulate the oral tissue microenvironment and predispose OPLs to progress to OSCC. To test this hypothesis we will study mouse models that develop OPLs upon exposure to the tobacco-surrogate 4NQO, in the presence of p53 and/or CDKN2A mutations. Patients with high-risk OPLs could benefit from preventive strategies designed to block the malignant progression of OPLs. However, previous attempts with different chemopreventive agents have not been successful. Recently, immune checkpoint blockade with antibodies directed at programmed cell death protein 1 (PD-1) has been shown to improve the survival of patients with advanced OSCC in clinical trials, confirming the importance of the immune system in containing progression of invasive tumors. Moreover, our previous studies, confirmed by multiple independent groups, demonstrated that anti-PD-1 antibodies can also prevent the progression of OPLs to OSCC, in a 4NQO mouse model for oral carcinogenesis. Our preliminary studies indicate that the p53 and CDKN2A status of the OPLs may determine the response to anti-PD-1-mediated immunoprevention. In this proposal, we will assess the long-term benefits of anti-PD-1-mediated oral cancer prevention, to determine whether PD-1 blockade, administered in a preventive setting, can confer survival benefits, and to assess how p53 and CDKN2A mutations affect the sustained response to PD-1 blockade. To overcome resistance to anti-PD-1 we hypothesize that reactivation of p53 in OPLs carrying p53 mutations sensitizes the oral lesions to anti-PD-1. Our mouse models will allow us to test this hypothesis in vivo.

NIH Research Projects · FY 2025 · 2022-06

Infections with human papillomaviruses (HPVs) are the most common sexually transmitted infection in the US. In addition to causing cervical and other anogenital cancers and a rising incidence of head and neck cancer, HPVs are responsible for an estimated 5% of cancers worldwide. Importantly, persistent infection, and not an acute infection is the primary risk factor for (cervical) cancer development. Thus, understanding the virus-host interplay that promotes or restrict viral persistence has important implications for HPV biology and human cancers. Until recently, it has been challenging to study the immediate early events of the HPV lifecycle following infection and how these events contribute to initial genome amplification during the establishment phase and long-term viral persistence. We used a single-cell genomics approach to identify cellular factors involved in viral infection and persistence. Our preliminary data identify protein arginine N-methyltransferase 1 (PRMT1) as an important factor upon viral infection of primary human cervical cells. PRMT1 regulates several cellular functions that may be relevant to the HPV lifecycle. We demonstrate that PRMT1 inhibition leads to a highly dysregulated splicing pattern of the viral genes. Mechanistically, we identify a dramatic increase in m6A modifications of viral mRNA, specifically in introns. These data implicate RBM15, a regulatory subunit of the m6A methyltransferase complex. We hypothesize that PRMT1 regulates RBM15 controlled m6a deposition on viral mRNA to temporally regulate alternative splicing throughout viral infection. We will (1) Determine the importance of asymmetric protein dimethylation throughout the viral lifecycle. (2) Determine the role for RBM15 in the PRMT1 mediated regulation of m6a methylation of viral mRNA. (3) Determine the effects of m6a methylation on viral mRNA splicing. PRMT1 inhibition leads to dysregulated viral splicing. This proposal addresses a critical gap in our knowledge relating to how HPV splicing is regulated for optimal infection, establishment, and long-term persistence. We identify PRMT1 as a regulator of viral splicing and hypothesize that this occurs through targeted m6A deposition on viral introns. Since PRMT1 is an established druggable target, these results may lead to novel therapeutics for HPV-induced cancers.

NIH Research Projects · FY 2026 · 2022-06

PROJECT SUMMARY/ABSTRACT (revised) Mal-adaptations to stress often leads to impaired social behavior, a shared domain across many neuropsychiatric conditions. While it is well-established that environmental factors, such as stress, play an etiology role, the brain mechanisms, particularly the role of specific neural circuits mediating the maladaptive responses, remain to be elucidated. Chronic social defeat stress (cSDS) in mice is a highly relevant, validated model to study brain circuit mechanisms related to stress. cSDS has been shown to induce morphological and functional changes in multiple brain regions including the medial prefrontal cortex (mPFC), which contains heterogeneous cell types and is interconnected with other limbic brain regions. Using recently developed neuronal activity reporter mouse lines, termed fosTRAP and TRAP2, the applicants’ laboratories have determined that acute (aSDS) and chronic (cSDS) social defeat stress activate distinct populations of projection neurons in the mPFC. The goal of this study is to determine whether specific circuit connectivity is pertinent to stress-induced social impairment and if novel therapeutic intervention strategies could be devised to selectively target the relevant circuits to combat the social deficits and alleviate off target effects. The expertise of two laboratories (Ferguson- mouse behavior, molecular biology and bioinformatics; Qiu-neurophysiology and functional circuit mapping, optogenetics) will be combined to test the hypothesis that cSDS-induced, impaired social behaviors are encoded within a specific neural circuit in the mPFC. The PIs will employ a TRAP2 reporter mouse line to gain genetic access to the mPFC neurons that are activated by cSDS, followed by investigation of: Aim 1) whether disrupted synaptic homeostasis selectively occurs in NAc-projecting mPFC neuronal populations that are activated by the cSDS, and whether disrupted synaptic homeostasis occurs selectively in the mice susceptible to social impairment. In Aim 2, this team will use targeted optogenetic manipulation of neural activity in the cSDS-activated mPFC projection neurons that also selectively express excitatory or inhibitory opsins. The investigators will test the novel hypothesis that optogenetic inhibition of this specific neuron ensemble during the cSDS confers resilience, while repeated activation of these neurons leads to susceptibility to social impairments. Aim 3 will investigate potential dysregulated gene networks in cSDS-activated mPFC neurons and to what extent such changes depend on SIRT1, a major human genetic risk factor for depression, through an innovative combination of cSDS in TRAP2 and RiboTag mice and bioinformatics analyses on RNAseq data collected from both male and female mice. Impact: Successful completion of these aims could reveal a paradigm-shifting practice in circuit-based therapeutics aimed at restoring prefrontal synaptic homeostasis and could establish a specific corticolimbic circuit as a lead intervention target for preventing the development of stress induced circuit pathology, which is otherwise not possible by previous studies examining an indiscriminate wholesale population of PFC neurons.

NIH Research Projects · FY 2026 · 2022-05

Project Summary Ventral striatal dopamine progressively rises as rodents navigate toward spatially distant rewards, a surprising recent finding that was not anticipated by temporal difference learning models of dopamine function. Ramping dopamine release in the ventral striatum reflects the value and proximity of goals, scaling by the value of the reward and stretching or compressing in different environments to span the distance between start and goal locations. Activity in ventral tegmental area (VTA) dopamine neurons also ramps up as animals approach goals, but the strength and persistence of ramping activity in dopamine neurons depends on both the use of an internal model of goal proximity and on the amount of task experience. Ramping activity in dopamine neurons appears immediately when naïve mice run toward newly-discovered rewards in spatial environments, and ramps gradually fade away over several days of training if sensory information about reward proximity is available. When an internal representation of progress toward the goal is required, however, robust ramping activity in dopamine neurons persists indefinitely. These findings suggest the hypothesis that ramping activity in VTA dopamine neurons depends on the use of brain regions that represent an internal model of the environment and current progress toward goals. We propose to test this overarching hypothesis by addressing the following specific aims: 1) Test the hypothesis that neural activity in the ventral hippocampus contributes to ramping activity in midbrain dopamine neurons. 2) Compare the evolution of ramping activity in VTA dopamine neurons with the evolution of ramping dopamine release in the ventral striatum. 3) Characterize neural activity in VTA GABA neurons during spatial and non-spatial navigation to rewards. Understanding the neural mechanisms that underlie ramping activity in dopamine neurons will have relevance for the neural basis of cognitive control, goal-directed behavior, perseverance, and spatial learning. Our findings will provide valuable information relevant for mental disorders associated with dysfunction in these abilities including attention deficit hyperactivity disorder, obsessive compulsive disorder, depression, and addiction.

NIH Research Projects · FY 2026 · 2022-05

The use of immune checkpoint inhibitors (ICIs), alone or in combination with other cancer treatments is increasing dramatically with immune-related adverse events (irAEs) common (90%) during ICI treatment. Most irAEs are symptomatic and symptom self-management with timely reporting of moderate or severe symptoms to HCPs may reduce irAE severity by early recognition and management, resulting in fewer treatment interruptions and unscheduled health services. Using a sequential multiple assignment randomized trial (SMART) design, we will initially randomize 286 diverse survivors (30% Hispanic) who are within 12 weeks of starting ICIs and who also have elevated psychological distress to an Automated Telephone Symptom Management (ATSM) or to an active control condition. ATSM consists of weekly telephone symptom monitoring using the PRO-CTCAE items by an automated voice response technology. Participants are referred to a printed Handbook with information about symptoms, evidence-based self-management strategies, and when to report symptoms to HCPs. ATSM automatically sends a weekly symptom summary to HCPs. Active control survivors will receive automated symptom monitoring only with reports sent to HCPs. Survivors in ATSM whose psychological distress is still elevated for 2 consecutive weeks during weeks 2-8 (non- responders) will be randomized for the second time to add TIPC for 8 weeks or continue with ATSM alone. We hypothesize adding TIPC will improve self-efficacy for symptom self-management, including communication with HCPs and increase social support resulting in lower indices of psychological distress, other PRO-CTCAE symptoms, clinician-documented irAES (primary outcomes), and unscheduled health services use and ICI treatment interruptions (secondary outcomes). With total intervention time of 16 weeks, all survivors will be interviewed at baseline and week 17 post-intervention, and electronic health record data will be extracted for the participation period. Specific aims: Aim 1. Determine if primary and secondary outcomes over weeks 1-17 are lower (better) in the group created by the first randomization: the adaptive intervention that begins with ATSM with the need-based addition of TIPC vs. active control group. Aim 2. Among those not responding to ATSM on psychological distress during weeks 2-8 who enter the second randomization, determine: a) if primary and secondary outcomes over weeks 8-17 are lower (better) in TIPC+ATSM vs. ATSM alone group; b) the extent to which the effects of adding TIPC to ATSM on primary and secondary outcomes are mediated by increased social support, self-efficacy for symptom management and for communication with HCP. Aim 3. Explore which baseline characteristics of the survivor, cancer, and cancer treatment are associated with optimal primary and secondary outcomes resulting from three supportive care options: 1) symptom monitoring only with automated reports to HCPs (active control); 2) ATSM alone for 16 weeks; or 3) addition of 8 weeks of TIPC to ATSM if no response on psychological distress during weeks 2-8.

NIH Research Projects · FY 2026 · 2022-04

Abstract Pancreatic islet dysfunction is a signature feature in the pathogenesis of Type 2 Diabetes and can stem from developmental adaptations to placental insufficiency (PI) and fetal growth restriction (FGR). We have identified significant reductions in insulin production and secretion that persist in offspring with FGR. Our efforts to elucidate programming mechanisms in FGR islets indicate that reductions in normal, constitutive nuclear factor kappa B (NFκB) activity negatively affects insulin secretion. Additionally, our preliminary findings associate depressed NFκB activity with hypoxia-induced MALAT1 expression because this long intergenic non-coding (linc) RNA binds NFκB to prevent activation. The guiding premise of this project is that low fetal oxygen and glucose concentrations from PI cause β-cell dysfunction during development. Therefore, we plan to correct oxygen and glucose concentrations in FGR fetuses during PI and show improvements in insulin secretion and β-cell proliferation. Foundational experiments demonstrate that combined supplementation of oxygen and glucose to the fetus with PI-induced FGR improves insulin secretion, but the underlying cues that cause persistent β-cell failure are undiscovered. We hypothesize that correction of oxygen and glucose concentrations in the PI-FGR fetus to normal, control fetal values will prevent β-cell dysfunction by enhancing β-cell proliferation and insulin secretion through the restoration of constitutive and physiological NFκB activity. Furthermore, fetal oxygen and glucose correction will resolve programmed deficiencies in β-cells of FGR lambs. We have adapted our fetal sheep model of PI-FGR to test a supplemental mixture of oxygen and glucose in a controlled, in utero environment. Preliminary experiments with five days of oxygen and glucose correction lowered norepinephrine, increased insulin, and restored glucose-stimulated insulin secretion (GSIS) in islets, demonstrating its suitability as a model to test whether the capacity to reverse PI ameliorates β-cell failure. In Aim 1, we will evaluate the combined effect of oxygen and glucose correction to improve β-cell function in fetuses and neonates with PI- induced FGR. In Aim 2, we will determine limitations in GSIS that result from lower constitutive NFκB activity due to MALAT1 overexpression in FGR islets. By alleviating hypoxemia and providing glucose, a major nutrient for β-cell responsiveness, we expect that insulin secretion and β-cell proliferation will increase and programming mechanisms causing β-cell dysfunction will return to normal. Impact of these experiments will be high, as they will provide fundamental new knowledge about the reversibility of β-cell dysfunction in fetuses with PI-induced FGR. In addition, our experiments will define the unique roles for NFκB regulation in β-cells from FGR fetuses that cause developmental adaptations that persistently lower insulin secretion. We also will gain new insight on the reversibility of a distinct islet-programming mechanism when fetal oxygen and glucose is corrected, which is expected to improved short- and long-term outcomes in individuals with FGR.

NIH Research Projects · FY 2026 · 2022-04

Abstract: The severity and incidence of T2DM is directly related to hepatic lipid concentration. Even before β- cell failure ensues, the severity of non-alcoholic fatty liver disease (NAFLD) is positively associated with hyperinsulinemia and insulin resistance. The hepatic vagal nerve plays a key role in glucose homeostasis affecting both pancreatic insulin release and insulin sensitivity. Acutely eliminating hepatic afferent signaling stimulates insulin release and decreases skeletal muscle glucose clearance, simultaneously resulting in hyperinsulinemia and insulin resistance. Conversely, acutely stimulating the hepatic afferent nerve inhibits insulin release and improves glucose clearance. Until recently there was no evidence for a hepatokine that signaled to the vagal nerve to alter glucose homeostasis. We have established that hepatic lipid accumulation dose- dependently increases hepatic production and release of γ-aminobutyric acid (GABA), an inhibitory neurotransmitter. Our data proposes that hepatocyte produced GABA stimulates insulin release and decrease skeletal muscle glucose clearance by altering activity of the hepatic vagal nerve. To establish therapeutic potential, we have shown that liver GABA transaminase knockdown decreases liver GABA release, restoring insulin sensitivity and normo-insulinemia in diet-induced obese mice. Through clinical trials, we have highlighted the translational impact of potentially targeting hepatic GABA signaling. In clinical samples, we have shown that hepatic GABA-transaminase mRNA expression is positively correlated with serum insulin and HOMA-IR. In these same clinical samples, we have shown that glucose disposal during a hyperinsulinemic euglycemic clamp is positively associated with mRNA expression of GABA re-uptake transporters and negatively associated with mRNA expression of GABA exporters. We propose 3 Aims focused on our central hypothesis that GABA is a hepatokine that can help explain the link between hepatic lipid accumulation and hyperinsulinemia and insulin resistance in obesity. Aim 1: Assess how obesity, lipids, diacylglycerol, ceramides, and downstream signaling affect direction of flux through the GABA shunt and transport of GABA across the plasma membrane. Aim 2: Assess the glucoregulatory response to exacerbating hepatic GABA production in lean mice or limiting hepatic GABA production in obese mice. Aim 3: Assess the glucoregulatory response to knockout (loss) and adenoviral induced overexpression (gain) of hepatic GABA transporters in lean and diet-induced obese mice. Impact: Validation of GABA as a novel hepatokine that affects serum insulin and insulin sensitivity in obesity will provide new therapeutic targets to treat this disease.

NIH Research Projects · FY 2025 · 2022-04

PROJECT SUMMARY This proposal is aimed at developing immune cell engineering approaches for the monitoring and treatment of T cell-mediated autoimmune diseases such as Type-I diabetes (T1D). The native 5-module receptor complexes that drive T cell responses to peptide antigens embedded in MHC molecules have evolved through an iterative process over the last ~435 million years to optimize T cell responses to a broad range of challenges. Using a biomimetic approach, we have developed a 5-module chimeric antigen receptor (5MCAR) and reported that cytotoxic T cells expressing 5MCARs (5MCAR-CTLs) can specifically target and kill autoimmune CD4+ T cells that mediate T1D in preclinical mouse models, resulting in significantly reduced disease incidence in 5MCAR-CTL-treated mice. Our proof-of-concept work supports the idea, echoed by the RFA to which we are responding, that engineered immune cell-based immunotherapies hold promise for the treatment of autoimmune diseases. Also as noted in the RFA, this area of investigation is in its infancy and requires support of exploratory engineering approaches to reach its full potential. Our foundational work provides us with a novel platform from which to continue the development of novel biomimetic engineering approaches for detecting and eliminating autoimmune T cell responses. Our goals here will be to develop novel applications using our 1st generation 5MCAR technology and to iterate on the 5MCAR platform through the engineering and testing of 2nd generation 5MCARs. Specifically, we will: 1) determine if using 5MCARs to redirect central memory CD8+ T cells, stem cell memory CD8+ T cells, or Tregs are most effective at preventing T1D in preclinical models; 2) use 5MCAR-CTLs as sentinels that can trafficking throughout the body and report on the presence of pathogenic autoimmune CD4+ T cells prior to clinical symptoms; and, 3) engineer and test 2nd generation 5MCARs that more closely mimic their native counterparts with the goal of improving 5MCAR performance. When completed, the proposed work will provide valuable pre-clinical data for the therapeutic potential of biomimetic 5MCAR-CTLs and 5MCAR-Tregs that are engineered based on evolution’s blueprint.

NIH Research Projects · FY 2026 · 2022-04

Bioactive lipids, such as ceramide and its downstream metabolites, sphingosine, and sphingosine-1-phosphate (S1P), mediate critical biologic responses, including inflammation and cancer1. Thus, the enzymes regulating lipid metabolism are intriguing therapeutic targets. The long-term goal of this project is to define the role of the bioactive sphingolipid metabolizing enzyme, acid ceramidase (AC), in colitis and colitis-associated cancer (CAC) and determine whether targeting this enzyme could serve as a novel anti-inflammatory/anti-CAC therapy. The PI’s laboratory has an established track record of expertise in sphingolipid metabolism and function2,3. Our recent work has begun to uncover a specific and very unique role for myeloid (Mye) AC in colitis and CAC. Using dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM)/DSS-induced CAC in murine models, we found that AC expression is increased in the inflammatory infiltrate but not in the colon epithelium. Similarly, we observed increased AC expresion in tissue macrophages in humans with colitis and colon cancer. In conditional knockout mice deletion of AC in myeloid cells, (Mye AC cKO), but not intestinal epithelial cells, decreased immune infiltrate and protected mice from colitis and CAC. Moreover, we found that our AC-specific inhibitor, LCL521, attenuates inflammation in a chronic colitis model (IL10 deficient mice). Finally, our newest preliminary data using bone marrow derived macrophages (BMDMs) from Mye AC cKO mice strongly hint that Mye AC may be required for inflammatory responses in these cells. Together these data suggest that Mye AC plays a large role the development of colitis and CAC. Based on our substantial preliminary data, we hypothesize that loss of Mye AC activity is protective against colitis and CAC by modulating colonic inflammatory infiltrate, and that targeting Mye AC may result in novel disease-modifying therapy in colitis and CAC. This hypothesis will be tested by the following Specific Aims: Specific Aim 1. Establish that Mye AC cKO protects from chronic colitis and CAC in vivo. Specific Aim 2. Determine the mechanisms by which loss of Mye AC protects from chronic colitis in vivo and probe these mechanisms in cells. Specific Aim 3. Advance pharmacologic inhibition of AC as a novel colitis and CAC target. The significance of these studies lies in the unique role of AC as the ceramidase that is clearly important in colitis and CAC, and the potential for AC as a novel therapeutic target. Identifying the mechanisms by which AC regulates chronic colitis and CAC, with specific focus on Mye AC, is a crucial first step in the design of novel therapies targeting this pathway. In addition, the studies that target AC in specific mouse models of colitis and CAC, will allow us to begin to translate our studies into clinical therapeutic approaches in the very near future.

NIH Research Projects · FY 2026 · 2022-03

ABSTRACT There is strong and consistent evidence for an association between obesity and post-menopausal breast cancer; however, the exact mechanisms are not clear. Follicle stimulating hormone (FSH) increases with menopause, concomitant with increased adiposity, particularly visceral adipose tissue (VAT). Recent preclinical data suggests that FSH may drive the deleterious shift in adiposity, independent of estrogen (E2). Further, FSH receptors are now known to be expressed in breast tumors. To date, there have been no large- scale human investigations of these FSH-adiposity-breast cancer associations. Using the Women's Health Initiative (WHI), a long-term national epidemiologic study of postmenopausal women, we will test the hypothesis that FSH drives adiposity postmenopausally, both cross-sectionally and longitudinally. Large subsets of WHI participants completed repeat dual energy x-ray absorptiometry (DXA) scans, and therefore have available robust measures of adiposity (including a novel measure of VAT) and banked serum from the randomized hormone therapy trials (N=1400) and the observational study (OS; N=1499). Second, using a case-control design, we will test the hypothesis that higher baseline FSH levels associate with increased risk of breast cancer using adjudicated cancer incidence data spanning >25 years follow-up (N=785 cases; N=2510 non-cases). Further, we will determine whether adiposity mediates the FSH-breast cancer risk associations. For all analyses, we will account for exogenous HT use (conjugated equine estrogen ± medroxyprogesterone acetate or reported HT use in the OS), endogenous estrogen (E2), and adipose-derived hormones that directly or indirectly regulate FSH (e.g. leptin). Our study is the first comprehensive epidemiologic investigation of FSH and obesity to measure and study FSH levels in the years prior to breast cancer diagnosis to assess the potential role of FSH in breast cancer. This study, which includes rich hormone and body composition data will enable immediate translation to more precise breast cancer prevention interventions aligned with new medications in the pipeline or currently in use for other cancers and osteoporosis, such as FSH and FSH receptor modulators, upstream GnRH antagonists (e.g. goserelin), and estetrol (E4).

NIH Research Projects · FY 2026 · 2022-02

ABSTRACT The COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused profound socioeconomic challenges for humankind. Antiviral agents blocking SARS-CoV-2 viral replication that complement vaccination are urgently needed to stop the current pandemic and to avoid potential future outbreaks. The papain-like protease (PLpro), an essential cysteine protease that regulates viral replication and host immune sensing, is a promising antiviral target against SARS-CoV-2. However, the rapid development of potent PLpro inhibitors has been hindered by limited draggable interactions at the active site due to restricted P1 and P2 sites with glycine recognition. To address these challenges, we have investigated novel, druggable binding sites, distal to the active site, using structure-guided design and X-ray crystallography. These efforts led to a series of 2-phenylthiophene-based inhibitors with low nanomolar potency. Crystal structures revealed that these potent SARS-CoV-2 PLpro inhibitors engage with a novel ligand-binding site, the “BL2 groove”, leading to slower off-rates, improved binding affinities, and low micromolar antiviral potency in SARS-CoV-2-infected human cells. Moreover, these inhibitors showed good microsomal stability and in vivo exposure after intraperitoneal (IP) administration. Building on these encouraging preliminary data, we propose in this project to further optimize and develop these novel PLpro inhibitors to achieve in vivo antiviral efficacy. We propose: Aim 1) to optimize our lead PLpro inhibitors for improved potency and drug- likeness properties using structure-guided design; Aim 2) to evaluate and triage PLpro inhibitors based on biochemical, ADME, and antiviral assays; Aim 3) to assess the PK/PD profile of top inhibitors and to establish in vivo antiviral efficacy. Completion of the research will lead to small molecules suitable for development as drug candidates to treat SARS-CoV-2.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY/ABSTRACT Women are at greater life-time risk for Alzheimer’s disease (AD). One potential factor contributing to greater life- time risk of AD is the midlife menopausal endocrine aging transition when multiple AD risk conditions can emerge and which are consistent with prodromal / preclinical features of the disease. While estrogen or hormone therapy administered when menopausal women are symptomatic could reduce risk of AD, the fear of breast cancer leads many women to forego this approach. An innovative alternative to estrogen therapy is to target estrogen action in brain while avoiding estrogen-associated proliferation in breast tissue. To achieve that goal, we propose Phase 2 clinical development of “PhytoSERM”, a selective estrogen receptor beta (ERß) modulator that promotes estrogenic action through ERß in brain while inhibitory in reproductive tissue. PhytoSERM is a rationally designed formulation of 3 phytoestrogens (each are Generally Recognized as Safe by the FDA). Our earlier NIA supported PhytoSERM Phase 1b/2a clinical trial determined that PhytoSERM was safe and well-tolerated, exhibited predictive pharmacokinetics in peri- and postmenopausal women and identified responder phenotype (https://clinicaltrials.gov/ct2/show/NCT01723917). Proposed herein is a Phase 2, double-blind, randomized, placebo-controlled, parallel-group, clinical trial to determine efficacy of PhytoSERM in symptomatic peri- and post-menopausal women. Primary objectives are to determine safety and efficacy of PhytoSERM to sustain brain glucose metabolism as determined by 18F-FDG- PET because the menopausal transition is accompanied by reduction in cerebral metabolic rate of glucose, which correlates with menopausal symptoms and progression of AD biomarkers later in life. Secondary objectives will determine efficacy of PhytoSERM on: 1) cognitive function, 2) frequency and severity of vasomotor symptoms and 3) changes in sleep quality and mood symptoms. Tertiary objectives are to determine impact of PhytoSERM on exploratory MRI outcomes including 1) gray matter volume in AD-vulnerable regions, 2) white matter fiber integrity by diffusion tensor imaging, (3) intrinsic connectivity measured by resting state functional MRI, 4) cerebral blood flow determined by arterial spin labeling (ASL) and 5) blood-based biomarkers relevant to AD risk. The Phase 2 PhytoSERM clinical trial addresses multiple strategic directions of the National Institutes on Aging’s 2020-2025: Aging Well in the 21st Century ref Specifically, Goal C-3 to: “Develop effective interventions to maintain health, well-being, and function and prevent or reduce the burden of age-related diseases” and “Conduct clinical studies / translation of new interventions to the clinical setting.” Goal D-4: Translate basic discovery into effective treatment and/or prevention strategies for AD/ADRD and” Goal F-4: Support research on women’s health.” PhytoSERM clinical trial also contributes to achieving the National Alzheimer’s Disease Project Act (NAPA) to effectively prevent and treat AD by 2025 Goal 1B. PhytoSERM addresses a critical unmet need in women’s health to reduce risk of Alzheimer’s in later life.

NIH Research Projects · FY 2026 · 2022-02

Project Summary/Abstract Autophagy is frequently upregulated in cancer cells under metabolic stress to recycle cellular components for protein and ATP synthesis to promote cell survival. Based on its important roles in maintaining cell viability and inducing resistance to radiation and chemotherapy, inhibition of autophagy has become a viable therapeutic approach that has been evaluated in clinical trials. However, there is a need to identify predictive biomarkers to enable selection of patients that may best respond to autophagy inhibitors. Our preliminary data demonstrates that the mTORC1 regulator REDD1 controls sensitivity to autophagy inhibition suggesting that cancers with significant REDD1 levels, such as renal cell carcinoma (RCC), are hypervulnerable to this therapeutic approach. Our major goal is to investigate the mechanisms that control sensitivity to autophagy inhibition in RCC cells to optimize its potential clinical application. In Aim 1, we will determine the role of REDD1 as a regulator of RCC pathogenesis and sensitivity to autophagy inhibition. In Aim 2, we will investigate the mechanistic link between PIM1 inhibition and upregulation of REDD1 with a focus on endoplasmic reticular stress. In Aim 3, we will evaluate the impact of clinically-relevant autophagy inhibitor-based combinations for RCC therapy.

NIH Research Projects · FY 2026 · 2022-02

The University of Arizona (UA) is a research-intensive institution with a strong record of supporting diverse students. It is an American Indian and Alaska Native-Serving Institution (AIANSI) and was recognized as a Hispanic-Serving Institution (HSI) in 2018. The goal of the Initiatives for Maximizing Student Development (IMSD) program is to build our under- represented (UR) students’ sense of belonging, self-efficacy and science identity. To meet this goal, IMSD trainees enroll as a cohort in a one-unit success course in the first year, and engage in individualized and cohort-based professional development and career readiness activities. UA IMSD will utilize a new Annual Review to create a personalized academic and research plan for each trainee and mentor. IMSD mentors will engage in inclusive mentoring workshops to support their ability to work with their trainees. IMSD scholars are immersed in a tiered mentoring system and receive support from the IMSD PI/Co-Is, staff, faculty, their dissertation advisor, the Director of Graduate Studies of their program, and peer mentors. NIH funding will be used to support program staff, first year student stipends, the success course, and social/professional development opportunities. In recent years, IMSD has provided opportunities to other UR biomedical sciences students who would benefit from the program. In this proposal, we will create an engaged community of biomedical research scholars comprised of 12 NIH- funded IMSD trainees and additional UR graduate students in the biomedical sciences. Furthermore, IMSD leadership has been expanded to include campus leaders with faculty appointments in diversity, administration, and education who will serve as mentors and role models. We seek to develop IMSD trainees who will be scientists, researchers and leaders in their field. Our goal is to be the premier institution in the Southwest for supporting diverse students in the Biomedical Sciences.

NIH Research Projects · FY 2025 · 2022-01

PROJECT SUMMARY/ABSTRACT Hypertension stimulates cardiac fibroblast (CF) expansion, activation, and excess extracellular matrix (ECM) production. Although there are no approved treatments for cardiac fibrosis, angiotensin converting enzyme inhibition (ACEi) limits CF activation and ECM accumulation. Recent findings from the laboratory of the PI demonstrate that resident CFs, once considered functionally homogeneous, consist of physiologically distinct populations that differentiate to diverse phenotypes in response to pressure overload. The premise for this application is based on these findings in which hypertensive rats were transiently treated with an ACEi prior to single cell RNA sequencing on resident CFs. Pre-treatment with ACEi shifts CF subpopulations to generate homeostatic CFs with a reduced capacity for fibrosis. This effect persists after treatment is stopped, indicating memory is retained. The proposed studies will reveal the mechanisms by which CF subpopulations shift to determine how to reprogram CFs to display a homeostatic, less fibrogenic phenotype. Following ACEi, homeostatic CFs comprise the largest subpopulation of resident CFs and are the least fibrogenic. Trajectory analysis revealed a gateway CF subpopulation that is the immediate precursor to activated CFs, and this gateway cluster was the most depleted by ACEi. Gateway CFs were defined by high expression of Spp1, encoding for the protein osteopontin, which induces several pro-fibrotic genes and represents a critical target candidate to maintain the activated CF pool. ACEi altered expression of epigenetic genes, indicating changes in chromatin structure may drive the persistent shift from gateway to homeostatic CF subpopulations. These compelling preliminary results led to the central hypothesis: transient reduction in angiotensin II signaling alters CF memory to protect against left ventricle (LV) fibrosis by fibroblast subpopulation-specific reprogramming of chromatin structure to shift an osteopontin-producing gateway subpopulation toward a homeostatic subpopulation with low fibrogenic capacity. To test the hypothesis, the following specific aims are proposed: Aim 1) elucidate the degree to which reduction in angiotensin II signaling mediates the persistent shift in resident CF physiology that protects from future fibrosis; Aim 2) determine the impact of chromatin structural modification on shifting the gateway cluster toward the homeostatic cluster; and Aim 3) ascertain the degree to which reduction in osteopontin mediates the shift to a less fibrogenic phenotype. In this application, the research team uses a multidisciplinary approach employing in vivo and in vitro methodologies to test the hypothesis. Successful completion of these experiments will determine whether reduction in angiotensin II signaling mediates the expansion of a subset of homeostatic CFs that renders the LV resistant to fibrosis. It is expected that the key drivers regulating the shift from a gateway to a homeostatic subpopulation of CFs will be identified. Impact: These anticipated findings will have a positive impact in developing CF-targeted therapies for the treatment and prevention of fibrotic remodeling that underlies heart disease.

NIH Research Projects · FY 2026 · 2022-01

A central problem in ovarian cancer is late diagnosis, which causes the 5-year survival rate to plummet below 50%. Ovarian cancer symptoms are vague and nonspecific, and current screening is generally not effective. Because ovarian cancer is so deadly, risk-reducing salpingo-oophorectomy (RRSO) is often recommended for women at high risk; however, RRSO has fertility and health consequences. It is now believed that ovarian high-grade serous carcinoma (HGSC) may begin in the fallopian tubes (FTs) as serous tubal intraepithelial carcinoma (STIC), and that precancerous changes are detectable before metastasis to the ovary and peritoneal cavity occurs. Our preliminary data indicate that there are significant changes in serum protein biomarkers in HGSC cases 12-84 months prior to diagnosis. Further, we have also shown that changes occur in multispectral fluorescence image markers of normal and cancerous ovaries and FTs, and that we can build a thin falloposcope suitable for traversing the uterus and FT for imaging and cell collection. We will address the unmet clinical need for a minimally invasive test for STIC and early (stage I/II) ovarian cancer. Currently, no methods enable the detection of ovarian HGSC with a lead time of more than 12 months. Overall, our work will meet the need to detect aggressive cancers at the earliest possible stage. Our initial target population is women at high risk for ovarian cancer who wish to delay or avoid RRSO. We will combine blood screening for protein markers with a minimally invasive falloposcopy for optical imaging and FT cell collection. Our procedure will be tested in a study of women at high risk undergoing bilateral salpingo- oophorectomy with hysterectomy, which will enable us to obtain and compare test results to gold standard histology. The specific aims are to: 1) Develop and validate biomarkers that detect STIC and early epithelial ovarian cancer. We will improve upon our existing cut-off based algorithm with newly-discovered markers as well develop a velocity-based biomarker algorithm. The algorithm that detects disease 12-84 months prior to diagnosis will be confirmed in an independent, blinded set of clinical blood samples. 2) Develop endoscopic imaging and pathomics markers. We will improve our prototype falloposcope system with higher resolution multispectral imaging and improved cell collection ability. We will develop imaging and karyometric markers from the FT images and the cells collected, and perform a pilot in vivo study. 3) Develop an actionable clinical strategy for early detection of epithelial ovarian cancer. A study will be performed in women at high risk who are planning a RRSO. Those who test positive from our blood test developed in Specific Aim 1 will have their tissue undergo a falloposcopy. Imaging and pathomics data will be used to develop a classifier, which will be compared to gold standard histology findings of normal FT, STIC, or occult HGSC.

NIH Research Projects · FY 2026 · 2021-12

Phase separation control of transcription in Ewing sarcoma Eighty-five percent of Ewing sarcomas are caused by translocations between the genes EWS and FLI1, creating a powerful oncogene – EWS-FLI1. The remainder are caused by translocations involving an EWS homologue, FUS. This sarcoma is an aggressive primary bone tumor affecting 1 in 300,000 people annually with more than 80% of tumors occurring in adolescents, making it the second most common pediatric bone cancer. Half of known translocations identified in sarcomas involve one of a family of three proteins – FUS, EWS, and TAF15, known as the FET family of proteins. Our work over the last few years has contributed to expanding the model for FET protein regulation of transcription. FET proteins control transcription in an organizational capacity. FET proteins undergo an assembly process called phase separation, which is the process thought to drive formation of non-membrane bound organelles, also called granules. In this proposal, we highlight our accomplishments isolating an EWS-FLI1 associated granule from cells. These granules have considerable similarity to those we have discovered to associate with RNA polymerase II (RNA Pol II) during transcription, which we refer to as transcription granules. This proposal aims to establish a model that EWS- FLI1 fusion protein seed aberrant granules that incorporate EWSR1 and other FET proteins to alter transcription and promote tumorigenesis. Our proposed work will investigate basic molecular mechanisms by which EWS-FLI1 can contribute and drive pediatric sarcoma pathology. Our proposal involves three aims. (1) We will determine the binding partners that comprise fusion protein granules for EWS-FLI1. (2) We will investigate EWS-FLI1 and EWSR1 activities to bind nucleic acids, phase separate, and alter transcription through effects on nucleic structures. Finally, (3) we will investigate how changes to DNA structure and chromatin are linked to aberrant phase separation by EWS- FLI1, EWSR1, and additional remodeling factors.

NIH Research Projects · FY 2025 · 2021-12

Diets high in fat have been linked to obesity, and a high fat diet (HFD) has been implicated in altering intestinal function, including inflammation; however, the contribution of specific HFDs and their predominant fatty acids (FAs) on key pathophysiologic processes is largely unknown. Studies with trans-fats, lard, olive oil, fish oil, plant- based fats (corn oil, soybean, etc.) have demonstrated that specific sources of FAs exert extremely diverse effects in disease, most commonly in cardiac and metabolic disease. Therefore, the long-term goal of this proposal is to dissect the complexity of components and effects of specific dietary FAs on gut pathobiology and determine the mechanisms involved. Specifically, we aim to define the specific roles of two ceramide-generating enzymes, ceramide synthases (CerS) 5 and 6, in the regulation of myristate-induced activation of inositol requiring enzyme 1α (IRE1α) and define their critical roles in intestinal homeostasis and pathophysiology. IRE1α is a critical branch in the unfolded protein response (UPR) or ER stress and is an evolutionarily conserved signaling mechanism to overcome stress in the ER, resulting in a pause of new protein synthesis and induction of chaperone proteins to restore normal ER function. Functionally, ER stress and IRE1αv have been linked to alterations in intestinal homeostasis and inflammation. Specifically, ER stress genes are upregulated in intestinal epithelial cells (IECs) from a murine experimental colitis model (IL10-deficient)1 and in IECs or biopsies from inflammatory bowel disease (IBD) patients1,2. IRE1α, as well as other effectors of ER stress, can be initiated by several triggers including high fat diet (HFD) or treatment with saturated FAs, as well as exogenous and endogenous sphingolipids3-5. These important clues in the literature, together with our studies on bioactive sphingolipids in intestinal inflammation, led us to investigate the role of specific dietary FAs in sphingolipid metabolism and their role and mechanism(s) of action in intestinal ER stress. Our very novel observations demonstrate that the C14 saturated FA myristate (rich in milk based diets), but not palmitate (C16 saturated FA), increases ER stress, as well as inflammation in IECs in culture and in mouse intestinal tissues in vivo6. Mechanistically, ceramide synthases 5 and 6 (CerS 5 and 6) are required for the activation of IRE1α branch of the UPR, inducing splicing of XBP1 and expression of IL66. Importantly, these data now lead us to the hypothesis that CerS5/6 and the generation of C14 ceramide regulate IRE1α in the intestine and that this may mediate specific responses in intestinal pathobiology (specifically intestinal inflammation). To test this hypothesis, we propose the following specific aims: Specific Aim 1. Determine the role of CerS5/6 in the regulation of ER stress, specifically IRE1α, in intestinal epithelial cells and intestinal organoids. Specific Aim 2. Define the role of CerS5/6 in ER stress-mediated intestinal pathobiology in vivo.

NIH Research Projects · FY 2026 · 2021-12

Project Summary Atherosclerosis is a narrowing of arteries caused by plaque buildup. Dyslipidemia, especially elevated low- density lipoprotein cholesterol, is a major risk factor for atherosclerotic plaque formation. Current therapies for atherosclerosis focus on lowering cholesterol. However, conventional lipid lowering therapies (such as statins) are associated with obvious side effects. Developing more specific and efficient treatments are needed. Fibroblast growth factor 1 (FGF1) has been widely studied for its therapeutic benefits in cardiovascular disorders primary utilization of its mitogenic functions. Recently, FGF1 was shown to exert an unexpected metabolic activity by regulating adipose remodeling and glucose homeostasis, demonstrating a potential for treatment of metabolic syndrome. However, wild-type FGF1 (FGF1WT) induced hyperproliferation can lead to increased tumorigenic risk; this becomes the primary obstacle for its widespread application. To reduce this risk, we recently engineered a partial FGF1 agonist carrying triple mutations of the heparin-binding sites (FGF1ΔHBS), which abolished proliferative potential, but maintains full FGF1WT metabolic activity. Notably, chronic treatment of db/db mice with FGF1ΔHBS almost completely reversed diabetes-associated NAFLD. These findings suggest that FGF1ΔHBS is a potentially safe and efficient therapeutic approach for treatment of metabolic syndrome. Although characterization of the metabolic functions of FGF1 is ongoing, little is known about its roles in atherosclerosis. A small case observation found an increased FGF1 expression in neovascularized and macrophage-rich regions of plaque, implying a potential pathological role of FGF1 in human atherogenesis. However, whether and how FGF1 plays beneficial or detrimental roles in atherogenesis remain unexplored. We recently examined the impact of FGF1 administration on the pathogenesis of atherosclerosis in ApoE-KO mice and found that FGF1ΔHBS markedly ameliorated atherosclerotic phenotypes without significant proliferative potential in liver. Furthermore, FGF1 treatment reduced cholesterol levels in the blood, liver and intestine, but increased cholesterol contents in feces. These preliminary data indicate that FGF1 regulation of cholesterol homeostasis in both liver and intestine is responsible for its protection from atherosclerosis. The liver is a major site for cholesterol biosynthesis while the intestine maintains cholesterol homeostasis by mediating intestinal absorption of dietary and biliary cholesterol. Therefore, we hypothesize that non-mitogenic variant FGF1ΔHBS prevents atherosclerosis by inhibiting hepatic cholesterol synthesis and suppressing intestinal cholesterol absorption without risks of hyperproliferation. We will test the hypothesis in three specific aims: 1) Determine the roles of FGF1 in the development of atherosclerosis; 2) Determine the effects and mechanism of FGF1 on hepatic cholesterol biosynthesis; 3) Determine the effects and mechanism of FGF1 on intestinal cholesterol absorption. This project will provide fundamental evidence for FGF1ΔHBS acting at the hepatocytes and intestinal enterocytes as a novel approach for the prevention of atherosclerosis in future clinical studies.

NIH Research Projects · FY 2025 · 2021-09

SUMMARY/ABSTRACT: Overall Project The strategic vision of the Precision Aging Network (PAN) is to develop the essential scientific knowledge to understand the discrepancy that currently exists between cognitive healthspan and human lifespan. We must reveal the neural mechanisms that 1) account for optimal brain performance in old age resulting in healthy cognitive function, and 2) those that underlie decline in brain function leading to age-related cognitive impairment (ARCI), Alzheimer’s disease (AD), or Alzheimer’s disease-related dementias (ADRD). The ultimate goal of the PAN is to develop not only a strong scientific foundation for the essential knowledge needed to match cognitive healthspan with human lifespan, but also to leverage big data approaches that apply precision medicine concepts to prolong optimal brain function. To achieve this goal of sustaining optimal cognitive function in old age, and to extend quality of life for people across levels of risk for ARCI, AD, or ADRD, we maintain that methodologies such as those developed and implemented in the PAN will be required. Although ‘chronological age’ is consistently associated with increasing incidence of disability, including chronic brain disorders such as AD and ADRD, the exact mechanistic relationships between ‘biological age’ and decline in brain function is not known. The number of people now living with some form of dementia is estimated to be 50 million worldwide, which is expected to double every 20 years. Because of the enormous heterogeneity in brain and cognitive function among individuals in their 70s, 80s and 90s, the urgent challenge for science, medicine and healthcare providers is to discover interventions that are individually effective in delaying or preventing ARCI, AD, or ADRD. Untangling the complex relationship between age and cognitive performance requires a strategy that includes the study of very large, diverse, well-characterized and longitudinally sampled populations. This will require ‘big data’ but also the means to translate the massive amounts of information gathered into ‘smart data’ or ‘knowledge’. This demands radically different conceptual models. Currently, no single approach adequately identifies the means to modify personal aging trajectories for improved brain health in individuals. The approach proposed in PAN is designed to overcome obstacles of earlier methods. The focus is on how to distinguish the various combinations of age, sex, genetics, race-ethnicity, health, lifestyle choices and environmental factors that influence brain drivers that increase susceptibility to dysfunction, as well as those factors that increase brain protection and resistance against dysfunction. The fundamental principle of the precision medicine approach is to ’individualize’. This will enable strong and specific predictions for each person to close the gap between cognitive healthspan and human lifespan. The root of this concept is in the teachings of Hippocrates, who said – “It is more important to know what sort of person has a disease than to know what sort of disease a person has.”