Grants

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Microorganisms residing in the intestinal lumen have a significant impact on brain function and behavior. Perturbation of microbe-gut-brain communication is thought to underlie the increased visceral sensitivity and anxiety that occur in disorders of gut-brain interaction (DGBI) like irritable bowel syndrome (IBS). Recent studies suggest that enterochromaffin cells (ECs), a group of enteroendocrine cells in the intestinal epithelium that secrete serotonin, sense microbial irritants. EC activation drives visceral pain and anxiety, suggesting a critical role of EC in mediating the pathophysiological development of IBS. However, the cellular signaling and neuronal circuitry by which EC activation drives anxiety remain largely unknown. Whether and how gut microbiota modulates ECs to promote anxiety also remains unclear. Using in vivo real-time measurements of cell activity in zebrafish, our recent research revealed that specific gut bacteria secrete tryptophan metabolites indole and indole-3-aldehyde (I3A) to directly activate ECs through the receptor transient receptor potential ankyrin A1 (Trpa1). Using optogenetics, gut-brain in vivo imaging, and behavioral analysis, new preliminary data from our laboratory demonstrated that the Trpa1-expressing ECs directly connect and communicate with vagal sensory fibers. Stimulating Trpa1-expressing ECs activate a subset of vagal sensory neurons that express Parvalbumin 7 (Pvalb7). Stimulating Trpa1-expressing ECs with microbial irritants promotes anxiety. This proposal will test the central hypothesis that indole and I3A secretion by enteric irritant bacteria activates the Trpa1+EC- Pvalb7+vagal sensory neuron pathway to promote anxiety. Three Aims were proposed. Aim 1 will use zebrafish and bacterial genetic manipulation to determine molecular mechanisms that promote the formation of EC-vagal synaptic connections and test the hypothesis that microbial irritant signals promote Trpa1+ECs to form synaptic connections with Pvalb7+vagal sensory neurons. Aim 2 will use optogenetics, vagal calcium imaging, and novel genetic reporter lines to image real-time EC-vagal sensory neuron communication in vivo to determine the precise molecular and signaling mechanisms that mediate Trpa1+EC-Pvalb7+vagal sensory neuron communication. Aim 3 will use genetics, optogenetics, brain calcium imaging, and behavioral analysis to determine the key brain region that is activated by the Trpa1+EC-Pvalb7+vagal sensory pathway and underlies microbial irritants-induced anxiety. The proposed project is expected to provide a mechanistic understanding of how microbial irritants perturb EC-vagal communication to alter brain activity and promote anxiety in a microbial, molecular, and cellular-specific manner. The results are expected to provide evidence for new therapeutic approaches that target EC-vagal circuitry to treat DGBIs associated with microbial dysbiosis.

NCI - National Cancer Institute

PROJECT SUMMARY B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer and remains a leading cause of childhood cancer death. Over 10% of B-ALL cases have point mutations in PAX5, a gene that encodes a key transcription factor for B-cell development. PAX5 mutations are the signature lesions in two recently identified B-ALL subtypes: PAX5alt, characterized by diverse genetic alterations in PAX5, and a subtype defined by PAX5 P80R mutation. While PAX5alt patients, particularly adults, have a significantly worse prognosis compared to PAX5 P80R patients, the underlying mechanisms driving these differences remain poorly understood. Both subtypes frequently acquire additional genetic alterations, such as wild-type PAX5 allele deletion and Ras pathway mutations, which further contribute to disease progression. Despite advances in chemotherapy, many patients experience suboptimal outcomes and poor quality of life, emphasizing the need for improved therapies. Our preliminary studies show that PAX5 P80R leukemic cells have increased sensitivity to Dexamethasone (Dex) treatment, likely due to high dependence on glucose uptake and glycolytic activity. These findings suggest that metabolic alterations contribute to the differential clinical responses observed between the two PAX5-driven subtypes. To systematically dissect these mechanisms, we will use orthogonal experimental models (e.g., B- ALL cell lines, mouse leukemia models, and human B-ALL samples) alongside multi-omics platforms, such as RNA-seq, CUT&Tag, ATAC-seq, END-seq, whole-genome sequencing, and single-cell analyses in this project. We hypothesize that rather than loss of function, PAX5 mutants can exert dominant effects in B-ALL initiation through novel PAX5 binding activities and gene transcription activation, which may block normal B-cell differentiation and give rise to pre-leukemic cells; once additional genetic lesions are acquired, PAX5-mutant clones can progress to overt B-ALL. We aim to (Aim 1) identify the transcriptional and metabolic alterations induced by PAX5 mutants, particularly their different effects on Dex treatment. Additionally, we will (Aim 2) investigate the stepwise acquisition of genetic lesions that drive leukemic transformation, with a focus on the pre-leukemic cells and their transition to overt leukemia using the Pax5 P80R mouse model. Finally, we will (Aim 3) study the role of aMEGF10, a gene significantly overexpressed in PAX5 P80R B-ALL, and assess the therapeutic potential of targeting the novel aMEGF10-SYK signaling pathway. In summary, this project will provide comprehensive insights into the molecular features of PAX5-mutant B-ALL subtypes, reveal novel mechanisms of leukemogenesis and treatment responses, and identify potential therapeutic targets. These findings are expected to improve patient risk-stratification and lead to the development of more effective targeted therapies with lower toxicity for PAX5-mutant B-ALL.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary/Abstract Evolution is one of the most powerful forces influencing the success and failure of life. Successful adaptation to one’s environment dictates the survivability of one’s genetic lineage. This is especially true for pathogenic bacteria, which must adapt to many stresses to successfully colonize and infect their host, replicate, and spread to a new host. Despite its importance, there is a fundamental gap in our understanding of how evolution influences bacterial pathogenesis. This proposal’s objective is to use an experimental evolution approach to determine the effects of evolution on the pathogenesis of the bacteria Klebsiella pneumoniae. K. pneumoniae frequently colonizes the gut, where it predominantly behaves as a commensal; however, gut colonization is the primary risk factor for K. pneumoniae disease and colonizing strains cause at least 80% of infections in colonized individuals. Thus, the gut is an important reservoir for infectious K. pneumoniae, which begs the question: how does evolution impact K. pneumoniae gut colonization and infection? This question is of high public health importance, as the prevalence of multi-drug-resistant K. pneumoniae and hypervirulent K. pneumoniae strains increases. Moreover, these strains are converging into dangerous multi-drug resistant, hypervirulent variants of K. pneumoniae. This necessitates a deeper understanding of K. pneumoniae gut colonization and infection. The central hypothesis of this proposal is that measurable, repeatable evolutionary events will enhance Kp gut fitness and modulate fitness during infection in a microbiome-dependent manner. We will test this hypothesis using two approaches: 1) we will assay K. pneumoniae evolution in the gut using a replay experiment; 2) Measure the impact of gut-derived mutations on K. pneumoniae pathogenesis. Using this approach, we will address the following questions: 1) does gut microbial community structure drive evolutionary outcomes?, 2) are K. pneumoniae strains experimentally evolved in the gut are fitter than their ancestors?, 3) does evolution in the gut enhance or restrict the ability of K. pneumoniae to colonize a novel host?, 4) does serial passage in the gut lead to reduced fitness during infection? The work in this proposal is innovative, as it requires a multidisciplinary approach to integrate experimental evolution with model systems that are directly relevant to human health. Completing this work will have a sustained positive impact through the identification and characterization of the mechanisms that underpin K. pneumoniae success in the gut and their impact on infection. This will help guide the development of decolonization interventions to reduce the risk of infection in individuals colonized by K. pneumoniae and potentially other opportunistic gut colonizers.

NIAID - National Institute of Allergy and Infectious Diseases

SUMMARY HIV and Mycobacterium tuberculosis (Mtb) are among the world’s deadliest infections, and co-infection is particularly devastating because each pathogen accelerates the progression and severity of the other. Notably, for reasons that are not well understood, people living with HIV (PLWH) remain at elevated TB risk despite effective antiretroviral therapy (ART) and viral suppression. Our goal is to define how HIV-TB co-infection, even with virologic control, impairs Mtb immunity. Macrophages and CD4+ T cells are central to the pathogenesis of both diseases; Mtb infects lung macrophages and depends to CD4+ T cells to prevent disease progression, while HIV infects both macrophages and CD4+ T cells. Macrophages can harbor latent HIV proviruses, which persist despite treatment. Both pathogens reprogram macrophage metabolism, which is intimately linked to antimicrobial functions. However, the impact of co-infection on macrophage immunometabolism and Mtb control remains unclear. In addition, although antiretroviral treatment (ART) can restore CD4+ T cell counts to normal ranges, the T cells often remain dysfunctional and exhibit signs of exhaustion. We unite leading HIV and TB investigators and leverage unique cellular and animal models of HIV latency. We hypothesize that, despite ART and virologic suppression, PLWH experience macrophage immunometabolic reprogramming that enhances TB susceptibility and that ART-restored CD4+ T cells are dysfunctional and fail to enhance microbicidal properties of macrophages. Using dual-reporter human macrophage models and ex vivo studies of PLWH and controls, we will profile antimicrobial and immunometabolic responses of Mtb-infected macrophages, and we will test the contribution of exhausted CD4+ T cells to macrophage dysfunction. A long-standing obstacle to studying HIV- TB coinfection has been the absence of a small animal model. We will use a novel humanized mouse model that recapitulates human lung immune cell populations and HIV infection dynamics, including active viral replication, latency establishment, and viral suppression with ART. We will assess how untreated and treated HIV infection affects Mtb pathogenesis, assessing pathogen burden, immunometabolic responses, and cellular infection patterns, using spectral flow cytometry, scRNAseq and metabolic profiling. These studies will clarify the molecular basis of immune dysfunction during HIV-TB co-infection and inform on host-directed therapies to restore immune function and improve outcomes.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary/Abstract The SLC4A2 (or Anion Exchanger2, AE2) is a ubiquitous membrane transporter that mediates the sodium (Na+)- independent and electroneutral exchange of chloride (Cl−) and bicarbonate (HCO3−) ions, and participates in the regulation of intracellular pH (pHi). AE2-deficiency in humans has been linked with the development of Primary Biliary Cholangitis (PBC), which is a chronic cholestatic liver disease associated with autoimmune phenomena. Supporting these findings, mice with whole-body deletion of SLC4A2 develop a spontaneous and progressive autoimmune cholangitis that resembles the human PBC. However, the cell intrinsic requirement of SLC4A2 in immune cells and/or biliary epithelial cells and the mechanisms underlying the loss of tolerance against biliary epithelial cells in the absence of SLC4A2 are unknown. Here, we aim to dissect the role of SLC4A2 in the development of liver inflammation and autoimmune cholangitis using SLC4A2 conditional knockout (cKO) mice. To this end, we have generated tissue-specific SLC4A2-cKO in the liver (i.e., Slc4a2fl/flAlbCre+ mice) as well as in T cells (i.e., Slc4a2fl/flCd4Cre+ mice) to study the requirement of this membrane transporter in immune tolerance in the liver. We aim to: 1) characterize the immune cell composition in the liver and lymphoid organs of Slc4a2fl/flCd4Cre+, Slc4a2fl/flAlbCre+ mice and their control littermates, and ii) study the alterations that occur in T cells and liver cells in the absence of SLC4A2. Investigating the mechanisms underlying the autoimmune cholangitis and liver inflammation caused by SLC4A2 deficiency in mice will be an important step forward to better understand the etiopathogenesis of PBC and to the development of future therapeutic approaches to treat these patients.

NCI - National Cancer Institute

Project Summary Bladder cancer (BLCA), the sixth most common cancer in the United States and 9th overall among all cancers, is rarely diagnosed in individuals aged <40 years. The Cancer Genome Atlas (TCGA) described the genomic landscape of BLCA patients and noted that >60% of cases have alterations in subunits of SWItch/sucrose non- fermentable (SWI/SNF) nucleosome remodeling complexes, such as SMARCB1, also known as integrase interactor 1 (INI-1). Presently, the response rate of BLCA to standard-of-care therapies is relatively low, and therefore, the identification of novel metabolic pathway-mediated druggable targets is urgently needed. Our preliminary data demonstrate that SMARCB1 loss expression has a significantly worse prognosis in patients with BLCA. Orthotopically implanted SMARCB1-knockout (KO) xenograft shows enhanced tumor growth and metastasis. While attempting to identify the metabolic landscape mediated by SMARCB1 loss BLCA, we have identified altered methionine metabolism, nucleotide synthesis, and glutathione metabolism. Our current proposal will seek to define further the metabolic pathway by SMARCB1 loss in BLCA progression as well as immunometabolism and determine whether the key pathways can be therapeutically targeted for the treatment of BLCA. In aim 1, Determine the metabolic alteration by SMARCB1-loss BLCA tumor cells and tumor infiltrating immune cells. In aim 2, Trace the key metabolic pathways in SMARCB1 loss metastatic BLCA. At the level of basic biology, this project aims to understand the metabolic rewiring mediated by SMARCB1 loss in BLCA progression as well as immunometabolism and metastasis by utilizing cutting-edge high-resolution mass spectrometry techniques to profile the metastatic mediated tumors cells and immunometabolism harboring SMARCB1 loss. This will further examine the pathway-specific metabolic regulation non-invasive liquid diet delivery of stable isotopes into mouse models, which will lay the foundation to test the therapeutic strategy in the future.

NIA - National Institute on Aging

PROJECT SUMMARY Alzheimer’s disease (AD) is a fatal, progressive neurodegenerative disease affecting a total of 6.5 million Americans over the age of 65, with that number growing each year as our population ages. There is currently no prevention nor cure for AD, and only a handful of drugs that slow, but do not stop, the disease’s progression. The AD brain is marked by accumulation of amyloid β plaques, tau neurofibrillary tangles, and lipid droplets. Recent studies report brain and whole-body metabolic dysfunction in AD, to the extent that glucose insensitivity, insulin resistance, and metabolic hormone dysregulation are nascent hallmarks of AD. Additionally, metabolic syndromes such as type 2 diabetes, obesity, and hypertension are significant risk factors for the development of AD. The contemporary understanding of AD as a metabolic disorder has sparked a growing interest in metabolism-based therapy. One such therapy, the ketogenic diet (KD), a high-fat/low-carbohydrate diet, is increasingly being studied in the context of AD and has shown promise in preclinical and clinical trials. However, the potential of the KD as a treatment for AD is marred by variable efficacy, low compliance, and side effects outweighing benefits. The molecular mechanisms by which the KD ameliorates AD pathology are unclear and warrant further study, and potential mechanisms include inhibiting carbohydrate metabolism, increasing fat metabolism, and altering amino acid (AA) metabolism. My F99 phase (Aim 1) of this proposal will focus on β- hydroxybutyrate (BHB), the primary ketone body produced by KDs, which can act on the body in two main ways: metabolism for energy and signaling. I will disentangle these molecular features of BHB (1.1) in vivo via transgenic mouse models and (1.2) in vitro via orthogonal validation of candidate pathways identified in neuronal tau interactomics. My K00 phase (Aim 2) will expand to AAs, specifically looking at isoleucine (Ile) restriction and dissecting the effects of Ile on protein synthesis (2.1), signaling (2.2), and metabolism (2.3) in the context of AD and brain aging. The unifying goal of this F99/K00 proposal is to use the KD as a starting point for the development of novel metabolic therapies for AD and brain aging by (1) identifying the bioactive components of the KD that are necessary and sufficient for its benefits in AD and brain aging and (2) disentangling the convergent and divergent mechanisms of action of dietary metabolites in AD and brain aging. This F99/K00 proposal will enable my long-term goal of establishing an independent lab that applies pharmacological, genetic, and mass spectrometry approaches to dissect the molecular underpinnings of metabolic interventions in AD and brain aging.

NHLBI - National Heart Lung and Blood Institute

Abstract: The fight-or-flight response is a critical regulator of heart function and major target of cardiovascular therapeutics for heart failure, arrhythmias, and ischemic heart disease. In response to stress, catecholamines released by the sympathetic nervous system stimulate β-adrenergic receptors on cardiomyocytes, resulting in increased heart rate and contractility. Although existing medications targeting upstream components of this pathway are effective therapeutics for cardiovascular disease, they often cause undesirable non-cardiac side effects. Rad, a small RGK-family GTPase and calcium channel inhibitor protein, has emerged as a downstream mediator of β-adrenergic signaling and a promising target for cardiac-specific therapeutics. In response to β-adrenergic stimulation, Rad is phosphorylated by protein kinase A (PKA), resulting in disinhibition of Cav1.2 and an increase in the L-type calcium current responsible for excitation-contraction coupling. However, the molecular mechanism by which Rad phosphorylation results in Cav1.2 current enhancement remains poorly understood. The objective of this proposal is to elucidate the physiological significance of Rad phosphorylation in regulating Cav1.2 function in the cardiac fight-or-flight response to inform the development of novel cardiac-specific therapeutics. To do this, we propose an innovative approach combining chemical biology and electrophysiology to achieve precise temporal and spatial control of Rad phosphorylation. By encoding the unnatural amino acid caged-serine, a derivative of serine that is “decaged” to natural serine by a brief pulse of near-UV light, at phosphorylation sites S273 and S301, we will achieve real-time, site-specific, light-mediated control of Rad phosphorylation during electrophysiological recordings of Cav1.2 currents. In Aim 1, we will employ a heterologous cell culture system to quantify the distinct contributions of S273 and S301 phosphorylation to Cav1.2 current enhancement upon forskolin-induced PKA activation. In Aim 2, we extend these studies to ex vivo cardiomyocytes isolated from Rad-knockout mice, enabling us to examine Rad phosphorylation in the context of native cardiac β-adrenergic signaling pathways. By elucidating the molecular mechanisms of Rad-mediated Cav1.2 regulation, this study will offer critical insights into the cardiac β-adrenergic response. These findings will lay the foundation for developing next- generation, cardiac-specific therapies targeting Rad phosphorylation for the treatment of cardiovascular diseases. The successful completion of this work will provide comprehensive training in biomedical research techniques, supporting the fellowship applicant's long-term goal of achieving a career as a physician-scientist dedicated to advancing the understanding and treatment of cardiovascular disorders.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY This project aims to uncover the molecular mechanisms that regulate membrane protein complex assembly at the human endoplasmic reticulum (ER) membrane. While significant progress has been made in understanding membrane protein insertion and folding, little is known about how thousands of membrane proteins find their correct binding partners to assemble into functional complexes of defined stoichiometry. Based on our preliminary data and published work, we hypothesize that assembly is highly regulated, and we plan to uncover the physiological roles of the underlying regulatory mechanisms using the large family of oligomeric voltage- gated ion channels (VGICs) as model complexes. VGICs fulfill many essential functions and for example mediate excitation-contraction coupling in heart and muscle cells, and trigger hormone and neurotransmitter release in secretory and neuronal cells. Mutations that impair VGIC biogenesis or function cause severe cardiological, neuropsychiatric, and neurodevelopmental diseases, emphasizing a critical need to better understand the assembly and quality control pathways that control their cellular levels. Our approach combines genetics, cell biology, and structural biology to determine the physiological role and mechanism of action of two poorly characterized VGIC assembly factors and to identify and characterize novel VGIC assembly factors. We will begin by advancing our understanding of voltage-gated calcium channel assembly by dissecting a potential novel chaperone function of the ER membrane protein complex (EMC). Using fluorescent calcium channel reporter cell lines and function-separating mutations, we have shown that EMC’s novel assembly function is required for calcium channel assembly in human cells. By reconstituting early calcium channel assembly events using an in vitro translation and ER insertion system, we will analyze how the EMC protects nascent channels from promiscuous interactions, aggregation and premature degradation to facilitate assembly. Additionally, we have developed selective nanobody inhibitors to investigate the physiological relevance of EMC’s novel assembly factor function in human iPSC-derived cardiomyocytes. These selective tools and assays now enable us to explore EMC’s assembly client spectrum using mass spectrometry. In parallel, we will investigate the assembly and quality control of other VGICs, such as potassium channels, using genome-wide genetic screens and characterize candidate factors using our established pipeline. This research will yield critical insights into the regulatory processes governing membrane protein assembly and identify potential therapeutic targets for VGIC- related diseases.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Despite being an established RCT-supported therapy, the adoption of deep brain stimulation (DBS) for Parkinson’s disease (PD) remains relatively low. While partly due to being a surgical therapy, adoption is further hindered by limited clinical benefit, off-target side-effects, and the burden of generator replacement surgery or battery charging. These limitations are at least in part attributable to the use of relatively simplistic fixed high- frequency stimulation. Recent optogenetic and computational modeling work has shown that finely tuned intermittent burst-patterned pallidal DBS can differentially modulate specific cell types and produce motor benefits that endure beyond stimulation cessation. Our team’s pilot study in people with Parkinson's disease (PwPD) comparing conventional and burst-DBS in globus pallidus internus (GPi) showed equal efficacy and tolerability, motivating further investigation. A mechanistic understanding of the physiological underpinning of therapeutic benefits of burst-DBS is critical to optimizing and ultimately facilitating translation of pallial burst DBS for PwPD, thereby addressing the limitations of fixed high-frequency DBS. While early results are encouraging, questions remain about the relevance and optimal location and burst-DBS frequency for long-lasting benefits in the human basal-ganglia-thalamo-cortical (BGTC) motor network. We will utilize our team’s expertise in intracranial physiology and PD to analyze BGTC circuits through recordings of single-neuron and evoked potential dynamics, local field potentials (LFP), electrocorticography, and network physiology. We focus on the GPi because its main sources of input are the two basal ganglia structures implicated in preclinical work (striatum and globus pallidus externus), both of which are underactive in circuit theories of PD. We address our hypothesis through 3 aims. AIM 1: Identify the optimal location and frequency for GPi burst-DBS through functional circuit mapping: We will define human-specific settings and location for producing the most potent antiparkinsonian modulations of BGTC neurophysiological PD readouts in response to intraoperative burst-DBS. AIM 2: Validate LTP effects of the optimized burst-DBS paradigm and derive brain-behaviour relationships: We will critically assess spatiotemporal neural features to demonstrate that burst DBS produces beneficial forms of LTP in the BGTC circuit, and to provide proof-of-principle of sustained benefit on a short timescale in response to intraoperative burst-DBS. AIM 3: Demonstrate subacute neural corelates of personalized burst-DBS benefit & relapse dynamics: We will leverage sensing-enabled DBS devices to evaluate acute (during burst-DBS) and enduring (beyond stimulation cessation) antiparkinsonian therapeutic effects and neurophysiology of personalized burst-DBS in the outpatient setting, in order to determine whether LFP and/or cortical recordings can be used as functional proxies to track enduring motor benefits and as biomarkers for closed-loop burst-DBS. Our work has the potential to improve therapy by providing a biological foundation for future large scale clinical trials assessing long-term outcomes of optimized GPi burst-DBS.

NEI - National Eye Institute

Project Summary: For millions of years, sensory systems have compensated for sensory generated through body movements. This is epitomized in the primate visual system which must account for rapid shifts in eye position three times a second and massive blink-related disruptions to retinal illumination every few seconds. Amazingly, primate visual experience is stable across these frequent and extensive disruptions. Little is known about the neural mechanisms underlying this visual continuity. Visual continuity, at minimum, requires two neuronal processes. First, a process by which ongoing perception is maintained during the retinal disruption; second, a process by which retinal disruptions are prevented from reaching perceptual thresholds. Behavioral work in the 1980s found that sensitivity to luminance changes, independent of eyelid closure, dramatically decreased if the change co- occurred with a blink. This phenomenon was coined blink suppression. Although little physiological evidence has been found for either mechanism of visual continuity, each offers a unique approach to interrogate the neuronal mechanism of perception: blink suppression as a means to preclude input from perception, and cortico-thalamic loops as a means of maintaining perception independent of visual input. In the first aim of this proposal, I will establish a blink suppression paradigm for use in animal models. This paradigm will be valid if behavioral performance is worse when an eyelid independent luminance change is paired with a blink compared to when unpaired. I will then record at multiple levels of the visual hierarchy while animals perform the task. I hypothesize that a copy of the blink motor command – a corollary discharge – alters early visual cortical activity in a such a way that it aligns blink-induced activity with aspects of visual activity that do not contribute to perception. In the second aim of this proposal, I will determine the contribution of cortico-thalamic loops to visual continuity. I will record from pulvinar neurons with known projections to V1 and V4 by using optogenetic tagging. I predict that pulvinar and V4 will more strongly modulate one another during blinks and that this activity underlies visual continuity. In the final aim of this proposal, I will causally test the necessity and sufficiency of blink corollary discharge to produce visual continuity. I will use optogenetics to inhibit and excite the lateral premotor cortex (LPMC), the likley origin of blink corollary discharge. I will inhibit LPMC on trials where a blink occludes an external stimulus or stimulate LPMC when an external stimulus changes in luminance. I predict that trials with an inhibition of LPMC will result in monkeys reporting their own blinks as a luminance change, and trials with a stimulation of LPMC paired with an external luminance change will result in monkeys failing to detect the luminance change. By leveraging a novel blink suppression paradigm and powerful analytical methods to extract activity patterns related to perception, I will delineate the neuronal mechanisms of perception and visual continuity.

NIA - National Institute on Aging

Project Abstract Healthy aging may be a case of mind over matter. Experiments in systems ranging from worms to mice have established that neural states, which humans often associate with the feelings and motivations behind our behaviors, may be as influential as physical experiences in promoting a long and healthy life. One major motivational drive for animals is hunger, which promotes feeding. Feeding can be generated by the physiological need to consume nutrients as well as the hedonic properties of food. While brain circuits and mechanisms that regulate feeding have been described, it is unclear how they contribute to the generation of motive forces that drive feeding, and ultimately impact aging. Based on visually identified and quantified behaviors exhibited by hungry flies, we have found that flies exhibit distinct and measurable hunger drives that can be homeostatic (i.e., need-based) or hedonic (i.e., pleasure-based). These can be distinguished, at least in part, using sophisticated feeding behavior metrics that have shown homeostatic feeding is best represented by the number of feeding events on a protein rich food, while hedonic feeding is characterized by the duration of feeding events on a highly palatable food. We have discovered specific regions of the fly brain that influence feeding duration on palatable food but not the number of protein food events, suggesting that they are involved specifically in hedonic feeding. While the direct manipulation of homeostatic hunger has been shown to extend lifespan, preliminary data from our laboratory indicate that hedonic drive shortens fly lifespan. To understand how hedonic feeding is generated by the brain and manipulate this hedonic perception, we will determine how the identified hedonic neural circuitry encodes a hedonic hunger state in Aim 1. For Aim 2, we will identify how different levels of hedonic perception influences aging in Drosophila. The proposed study will provide key insight into how the brain weighs environmental cues to drive hedonic feeding behavior and how hedonic feeding impacts lifespan in various environmental contexts. Additionally, we will manipulate these cues to determine the cellular mechanisms through which feeding neural states can affect healthy aging.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

SUMMARY Alcohol Use Disorder (AUD) is a global public health issue for which more effective treatments are urgently needed. While it is widely appreciated that social context is a powerful modulator of human alcohol self- administration. this factor has been largely ignored in preclinical AUD research. The development of an alcohol self-administration model which allows experimental control over social context and tight control over alcohol self-administration stands to reveal new information about the neurobiological bases of AUD. Specifically, the present proposal seeks to experimentally determine the nature of the interaction between social interaction and self-administered alcohol (i.e., complimentary vs. substitute goods) using a simple-to-implement preclinical AUD model that allows the study of social-context as a factor regulating alcohol drinking behavior. Further, we seek to determine how this interaction is changed by the development of alcohol dependence, during which we hypothesize alcohol reinforcement transitions from being a mainly-positive reinforcer to a mainly- negative reinforcer. Additionally, we will determine how male and female rats differ in terms of social context control over alcohol self-administration behavior. We also propose to study the inverse relationship of this interaction; how alcohol self-administration, alcohol dependence, and biological sex shape the nature and quality of social interaction. To begin to dissect the neurobiological basis of the interaction between social context and alcohol self-administration behavior, we propose to combine several neurohistochemical techniques to determine the brain regions which contain neurons strongly activated during alcohol-seeking behavior in social vs. non-social contexts (i.e., express the immediate early gene cFos). Further, we seek to determine how this brain-wide activity maps onto the neurocircuitry of the prototypical social neuropeptide, oxytocin (i.e., to co-detect neurons which synthesize the oxytocin peptide and/or which express the oxytocin receptor). Finally, we propose to leverage a combination of novel AAV technologies, optogenetics, and pharmacology, to test the functional relevance of neurons of the brain oxytocin neurocircuitry to control alcohol self-administration behavior in social vs. non-social contexts. This highly innovative research proposal is also highly feasible given the expertise and technologies offered by this unique team of researchers. Successful completion of this research project is expected to have a high impact and a sustained impact on preclinical AUD research in broad terms, by 1. providing a means to systematically examine the neurobiology of AUD from the largely ignored perspective of social context, 2. Identifying the neurobiological basis for the control of social context over alcohol consumption, and 3. Determine a causal role for neurons of the brain oxytocin system in the modulation of alcohol consumption across social vs. non-social contexts. Ultimately, by providing new information about the neurobiology of AUD, this project stands to move the field toward the generation of novel candidate treatments for AUD which have the best chance to successfully make the leap across the pre-clinical to clinical translational divide.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Follicular B cells have two developmental pathway options upon activation: the canonical germinal center pathway and the extrafollicular pathway. The germinal center is a key microanatomical structure in which B cells undergo stringent selection and mutation, resulting in a pool of B cells with high-affinity for the initiating foreign antigen and little to no autoreactive B cell clones. On the other hand, B cells that enter the extrafollicular pathway do not undergo stringent selection and therefore can result in not only the activation of foreign antigen-specific B cells but also self-reactive B cell clones. It has been demonstrated, by our lab and others, that a unique type of extrafollicular B cell has the capacity to produce both protective and pathogenic antibodies. This B cell compartment, composed of B cells that co-express Tbet and CD11c, plays a key role in the protection against many bloodborne pathogens but also has been implicated in the progression of autoimmune diseases. While antibodies from Tbet+CD11c+ B cells are mutated, these cells, in the context of acute infection, do not enter the germinal center reaction. It is unknown whether or not Tbet+CD11c+ B cells have the capacity to enter the germinal center during differing inflammatory environments, and whether the Tbet+CD11c+ B cell compartment is comprised of two independent pools, one with protective functions or one with pathogenic functions, or if the compartment is comprised of polyreactive Tbet+CD11c+ B cells. We hypothesize that Tbet+CD11c+ B cells have germline BCR sequences that allow for the recognition for both viral and self-antigen and that, due to a decrease in selection pressure experienced by extrafollicular B cells, Tbet+CD11c+ B cells that recognize both types of antigens are able to expand and participate in the immune response regardless of the initiating stimulus and inflammatory environment. My first aim is to uncover the basis for Tbet+CD11c+ autoreactivity during acute viral infection. To determine this, I will sequence viral antigen-specific Tbet+CD11c+ B cells, reexpress their immunoglobulin (Ig) sequences, and test their reactivity to self-antigen. I will also revert their sequences back to germline, reexpress the germline Ig sequences, and determine if the autoreactivity is retained. My second aim is to uncover how the autoreactive B cell pool differs under unique inflammatory environments. To this end, I will determine the contribution of Tbet+CD11c+ B cells to the autoreactive pool during chronic viral infection and chronic inflammation in the absence of virus using flow cytometry, determining antibody specificities, and investigating if Tbet+CD11c+ B cells contribute to the germinal center reaction during chronic inflammation using transgenic mice models and single-cell B cell receptor signaling. If successful, my project will uncover a key relationship between protective and pathogenic B cells. Moreover, my project will help elucidate how the behavior of extrafollicular B cells is influenced by its surround environment.

NIAID - National Institute of Allergy and Infectious Diseases

CD21-CD23- CD11c+ Tbet+ Age Associated B Cells or ABCs, expand in primary immune and peripheral tissues during chronic conditions such as aging, autoimmune diseases, atherosclerosis, and obesity. These ABCs largely produce IgG2a/c autoantibodies which exacerbate autoimmunity and metabolic dysfunction, positioning them as a promising therapeutic target for a broad array of inflammatory and infectious diseases. Furthermore, ABCs have recently been found to inhibit germinal center responses to conventional antigens such as those in most effective vaccines, which could explain the poor vaccine outcomes in elderly, autoimmune, and obese patients. As such, this suggests that blocking ABCs may improve vaccine responses across a broad array of platforms. ABCs expand in vitro and in vivo in response to a combination of TLR agonists, cytokines like IFNg and IL-21 as well as CD4+ T cell help, but the nature and coordination of signals which mediate ABC expansion and effector function remain inadequately understood. We recently discovered that ABC cells express unexpectedly high levels of class B scavenger receptor and fatty acid translocase CD36. Even more interesting, expansion of ABCs during obesity or in response to TLR7 inflammatory stimulation requires CD36, suggesting a role for CD36 in stimulation or maintenance of this unique B cell subset during chronic inflammation of obesity and autoimmunity. CD36 is capable of a myriad of different roles, including innate signaling, efficient uptake of antigen and metabolic reprograming, any one of which could be contributing to ABC activity. For example, CD36 mediated fatty acid uptake and subsequent PPAR-beta signaling support mitochondrial oxidative phosphorylation over glycolysis. In addition to mediating lipid uptake for metabolic regulation, CD36 also binds long chain fatty acids including oxidized phospholipids and pattern associated molecular patterns (DAMPS/PAMPS) such as apoptotic cells and amyloid proteins which could be presented by MHC II to engage helper T cells. CD36 engagement also triggers intracellular signaling cascades leading to innate activation and secretion of inflammatory cytokines. This project aims to combine unique mouse strains engineered to lack CD36 selectively in B cells with inflammatory in vivo mouse models to determine the relative contribution of CD36 to mediating antigen uptake and presentation, innate immune activation, and metabolic engagement by ABC cells during obesity or autoimmune disease. Elaborating a better understanding of signals employed by CD36 during activation of ABCs will allow targeted clinical interventions to slow their inflammatory contribution to disease and also improve immune responses to vaccination or infection for patients with obesity, autoimmune disease, or related chronic inflammatory conditions.

NCI - National Cancer Institute

ABSTRACT Prostate cancer remains the second leading cause of cancer-related mortality among men in the United States, with neuroendocrine prostate cancer (NEPC) representing an aggressive and lethal subtype that emerges from castration-resistant prostate cancer (CRPC). NEPC often develops following long-term androgen deprivation therapy or androgen receptor signaling inhibitors (ARSIs), such as enzalutamide, and is characterized by poor prognosis and a low 5-year survival rate. Understanding the molecular mechanisms driving NEPC progression is essential to developing novel therapeutic approaches. Carnitine Palmitoyltransferase 1C (CPT1C), known for its role in regulating brain energy homeostasis, is upregulated under stress in various cancers, promoting tumor adaptation and aggressiveness. Emerging evidence from our lab indicates that CPT1C expression is significantly elevated in NEPC and correlates with neural lineage markers. Functional studies reveal that CPT1C inhibition reduces NEPC growth and arrests the cell cycle. These findings suggest that CPT1C plays a pivotal role in NEPC and is a promising therapeutic target. This project hypothesizes that CPT1C regulates neural lineage plasticity, tumor aggressiveness, and therapy resistance in advanced prostate cancer. Aim 1 focuses on elucidating CPT1C’s role in prostate cancer progression, with a particular emphasis on neural lineage plasticity, resistance to enzalutamide, and its interactions with lipid metabolic proteins. Aim 2 investigates the mechanisms underlying CPT1C protein regulation, including its interaction with the chaperone complex, and evaluates CPT1C turnover pharmacologically in both in vitro and in vivo models. Aim 3 leverages RNA bioengineering technologies to validate CPT1C as a target in advanced prostate cancer models, using liposome-polyethylenimine nanocomplexes for targeted delivery. The primary goal of this project is to dissect novel mechanisms of neural lineage plasticity and develop new therapeutic strategies for NEPC by targeting CPT1C. We anticipate that the results of this study will define the role of CPT1C in the development, progression, and therapy resistance of NEPC. These findings will provide a foundation for developing BioRNA/CPT1C-siRNA as a potential therapeutic approach for NEPC, offering the promise of significantly improving the quality of life for prostate cancer patients.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Lung development requires a tight interplay between the pulmonary epithelium and the surrounding mechanical environment, including the airway smooth muscle (ASM) and the thoracic cavity. Defects in the mechanical environment of the embryonic lung, such as congenital diaphragmatic hernia (CDH), are associated with pulmonary hypoplasia leading to significant mortality and morbidity. Therefore, it is necessary to investigate the role of the mechanics on lung development. The central objective of this proposal is to characterize the effects of the mechanical environment on the spatial and temporal patterns of branching morphogenesis in the lung. Mechanical forces have been thought to play a role in branching morphogenesis of the embryonic lung for decades, but a detailed understanding has been limited by the experimental challenge of simultaneously manipulating and observing lung development in a physiologically relevant setting: studies in vivo are limited by the inaccessibility of the lung in the embryo, while those in culture restrict lung explant growth to a non- physiological, flat surface. Nonetheless, recent innovations in microfluidic approaches support physiologically relevant lung development ex vivo and enable tracking of branching morphogenesis under various applied pressures. Studies using these approaches have revealed a tight coupling between transpulmonary pressure, ASM contractions, and the rate of lung branching. The overall hypothesis of this proposal is that branching in the lung is driven by a pressure-dependent ‘clock’ of ASM contractions. To test this hypothesis, I will combine a mouse model of CDH, whole-mount immunostaining, quantitative imaging and analysis, and a microfluidic culture model to track the development of lungs under various mechanical perturbations. First, I will determine whether the mechanical stimulus of ASM contraction is sufficient for inducing lung branching events. Second, I will define the progression of lung development in a mouse model of CDH and assess the role of local changes in pressure on branching rates in lungs cultured ex vivo. Collectively, the findings of this proposal will provide detailed insight into the function of ASM contractions and compressive forces in a physiologically relevant setting amenable to fine perturbations. These results will have a broad impact on understanding the physical forces that sculpt lung development and will provide original insights that will help guide strategies for treatment of prenatal lung hypoplasia. The activities proposed here will enhance my ability to conduct experiments and data analysis and to communicate research to a broader audience. These activities will also offer opportunities to teach and mentor emerging scientists. I will leverage the analytic skills acquired during my undergraduate training and expand my aptitude in bioengineering and mechanobiology by making use of the world-class expertise and extensive resources that my supervisor (Dr. Celeste Nelson) and Princeton University have to offer. These activities will help me become an independent researcher and will provide a robust foundation for establishing my own research direction investigating the determinants of multiscale coordination in tissue morphogenesis.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

PROJECT SUMMARY/ABSTRACT During pre-implantation mammalian development, cells must make their first critical fate choice to specify the trophectoderm (which forms the extra-embryonic placenta) or inner cell mass (which develops into the embryo proper). To achieve this lineage commitment, the embryo coordinates the major mechanical changes of compaction and polarization with critical signaling programs that drive cell fate. The role of Yap mechanotransduction in guiding trophectoderm fate beginning at the 8-cell stage has been well-studied. However, trophectoderm development is not driven by Yap signaling alone, but also requires activation of the Notch pathway. While other developmental contexts provide insight into how Notch could interact with mechanics and Yap to guide cell fate decisions, the role of Notch in mouse pre-implantation lineage determination remains unknown. This proposal will elucidate how Notch integrates with Yap and mechanics to guide trophectoderm specification and pre-implantation embryogenesis. I will investigate the central hypothesis that Notch lateral inhibition starting at the 2-cell stage primes a subset of cells toward the trophectoderm lineage; during polarization, Notch signaling could stabilize the apical domain to reinforce Yap signaling and maintain trophectoderm identity through blastocyst morphogenesis. Historically, live cell dynamics of pre-implantation embryo development have been challenging to study due to phototoxicity. However, recent advancements in light-sheet microscopy, biosensors, and optogenetics allow us to visualize and perturb signaling dynamics with unprecedented spatiotemporal resolution. I will harness this high-resolution imaging system to determine (1) how Notch signaling relates to embryo mechanics and (2) how Notch and Yap signaling dynamics specify and maintain trophectoderm fate. For the completion of these aims, I will gain training in advanced microscopy, quantitative developmental biology, scientific communication, teaching, and mentorship. The expertise from my sponsor, Dr. Orion Weiner, an expert in signaling dynamics and cellular mechanics, and support from the F31 NRSA will ensure that I receive the mentorship and resources necessary to complete my proposed research and training plan. My work will reveal how the embryo integrates different modes of signaling with mechanics to guide cell fate decisions. A better understanding how pre-implantation development is regulated will help advance Assisted Reproductive Technologies and ameliorate infertility.

NIA - National Institute on Aging

Summary Protein aggregation and deposition in the brain is a striking feature of several neurodegenerative disorders including frontotemporal dementia (FTD), which accounts for up to 15% of dementias. There are currently no disease-modifying treatments for FTD, due, in part, to the lack of disease models that accurately recapitulate the complex pathology of the disease. FTD-Tau accounts for 30–50% of FTD cases and is pathologically identified by the presence of insoluble Tau aggregates, which are more prone to appear in the presence of Tau mutations. Mitochondrial function is disrupted in diseases attributed to aggregate-prone proteins, and this disruption might activate a crosstalk with the nucleus and cytoplasm through signaling pathways including several transcriptional, translational and post-translational programs (mitochondrial stress response). This project seeks to address the role played by succinylation and SUMOylation, two post-translational modifications (PTMs), in the mitochondrial-nuclear communication mediated Tau toxicity, in the context of FTD. SUMOylation consists in the covalent and reversible attachment of an 11 kDa SUMO (Small Ubiquitin-like MOdifier) to a target protein. There are three known SUMO paralogs in vertebrate brains, SUMO1-3, which are primarily localized in the nucleus but can also be found in the cytoplasm and associated with mitochondrial proteins. SUMO2, the most expressed, has been found to be neuroprotective in conditions of cellular stress. Succinylation is mediated by metabolic intermediates generated within the mitochondrial tricarboxylic acid (TCA) and has been linked to neurodegeneration. Intriguingly, Tau can be SUMOylated and succinylated, under certain conditions, but little is known about the effects of these PTMs. We found that i) mitochondrial respiratory chain (RC) enzymes, particularly succinate dehydrogenase (SDH, which converts succinate to fumarate in the TCA cycle), are impaired in brain extracts of the PS19 mice, which express P301S Tau; ii) SUMO2 is displaced from the nucleus of PS19 neurons; iii) succinylation is increased and SUMO2 levels and conjugation are reduced in FTD patients iPSC-derived neurons and in PS19 mice; iv) overexpression of SUMO2 rescues RC defects and reduces the aggregation of mutant Tau (mTau) in PS19 mice; v) several mitochondrial pathways are altered in excitatory neurons of PS19 mice. Thus, we hypothesize that in FTD, 1) dysregulation of succinylation plays a role in mTau- induced toxicity; 2) mTau toxicity is counteracted by SUMO2; 3) hypersuccinylation occurs at the detriment of neuroprotective SUMO2ylation, further aggravating Tau toxicity. By using patients’ iPSCs-derived neurons expressing mTau, their isogenic controls, and PS19 mice, we will investigate 1) the causal relationship between Tau-induced mitochondrial dysfunction and alterations of retrograde mito-nuclear signaling through succinylation, 2) whether aberrant crosstalk between succinylation and SUMOylation drives Tau aggregation and toxicity, 3) Tau-induced cell-specific transcriptional programs mediated by altered succinylation and SUMO2ylation, and whether SUMO2 counteracts mitochondrial dysfunction by relocating to mitochondria.

NIA - National Institute on Aging

Project Summary Alzheimer’s disease (AD) is a progressive neurodegenerative disease affecting more than 50 million people worldwide, but the mechanisms involved in disease development remain poorly understood. Genome-wide association studies have uncovered numerous loci associated with disease, but identifying causal variants and genes remains a challenge. Studies have revealed that AD risk variants are enriched in active enhancers of human monocytes, macrophages, and microglia, suggesting many of these variants act by disrupting gene expression specifically in myeloid cells. In addition, many genes implicated in AD risk are highly expressed in myeloid cells, strongly implicating these cell types in the etiology of AD. By integrating human genetics with epigenomic and transcriptomic datasets from human myeloid cells, we identified a myeloid cell enhancer containing an AD-associated functional variant on chromosome 11, and two putative target genes of this enhancer, embryonic ectoderm development (EED) and phosphatidylinositol binding clathrin assembly protein (PICALM). EED is an essential subunit of the polycomb repressive complex 2 (PRC2) known to function in regulation of gene expression and clearance behavior in mouse microglia, but it remains relatively unstudied in the context of AD in human cells. PICALM is an adaptor protein known to function in endolysosomal pathway and autophagy, but its role in microglia remains unknown. The overall goal of this proposal is to understand how our nominated AD risk enhancer influences the expression of its two target genes PICALM and EED, and to test the hypothesis that these two potential causal genes regulate microglia functions downstream of TREM2. In Aim 1, I will determine the role of the candidate AD risk enhancer in regulating the expression of PICALM and EED and human microglial cell function by combining CRISPR gene editing in human induced pluripotent stem cells (iPSCs), microglial differentiation protocols, and xenotransplantation methods involving direct injection of microglia precursor cells into the mouse brain. I will delete the candidate enhancer region and perform transcriptomic and epigenetic profile of edited microglia via RNAseq and ATACseq in addition to microglia functional assays. To assess the in vivo consequences of deleting the risk enhancer in microglia, I will transplant the edited microglia into the brains of wildtype and 5xFAD humanized mice and characterize transcriptomic profile of these microglia via snRNAseq analysis and perform immunohistochemistry. In Aim 2, I will investigate how PICALM and EED regulate human microglia function, namely TREM2 mediated efferocytosis, or phagocytic pathway. I will determine the subcellular localization of PICALM and EED using biochemical and imaging techniques and test the role of these two genes in microglial functions downstream of TREM2 signaling such as actin rearrangement, phagosome formation, and downstream kinase signaling. My proposed aims will help understand the mechanisms in which the causal variants and target genes drive the AD risk, and enhance our understanding of PICALM and EED in microglia and in AD.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Angiosarcomas are aggressive soft tissue sarcomas arising in endothelial cells with a poor prognosis and inadequate treatment options. Due to the rarity of the disease, there are limited resources for studying angiosarcoma and therefore little progress in identifying novel therapeutics. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and have emerged as potential therapeutic targets in disease. MiRNAs undergo a series of enzymatic processing steps culminating with DICER1 cleavage to generate mature miRNAs. The miRNAs are then loaded into Argonaute (Ago) silencing complexes where the miRNA direct the complex to target mRNA transcripts for post-transcriptional repression. Global miRNA repression is a hallmark cancer. However, most cells require some expression of DICER1 and miRNAs for survival. Studies in mouse models of angiosarcoma have demonstrated that the endothelial biallelic deletion of Dicer1 and loss of miRNAs leads to angiosarcoma development. While most cells require DICER1 expression for survival, endothelial cells can not only survive, but this leads to transformation and tumorigenesis. This implicates the importance of miRNAs in repressing tumorigenesis in endothelial cells. However, it is unclear how miRNA loss leads to transformation or which tumor suppressing miRNAs contribute. This finding motivated a CRISPR- CAS9 loss of function screen with a miRNA focused gRNA library to identify critical tumor suppressing miRNAs. Results from this screen identified novel miRNA candidates that have not been interrogated in angiosarcoma. With this preliminary data, this proposal addresses the hypothesis that miRNAs are critical tumor suppressors in angiosarcoma and understanding their roles in tumorigenesis will lead to the identification of novel therapeutic interventions. In Aim 1, the functional consequences of the novel tumor suppressing miRNA candidates will be evaluated in normal cells and angiosarcoma. Aim 2 will test the repurposed FDA-approved antibiotic enoxacin for its potential to enhance miRNA processing as a therapeutic in angiosarcoma and as a tool to understand further understand miRNA functions. Completion of these studies will provide insights into the critical miRNA involved in repressing tumorigenesis, define their regulatory network, and determine the therapeutic relevance of enoxacin enhancement of miRNAs in angiosarcoma.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

The acquisition and selection of recurrent, somatic mutations in hematopoietic stem cells (HSCs), termed clonal hematopoiesis (CH), has emerged as a significant risk for the development of myeloid neoplasia and mortality. Mutant HSCs can undergo clonal expansion by outcompeting wild-type HSCs and clonal evolution upon the acquisition of additional genetic alterations. Exposure to cytotoxic therapy is a significant risk factor for developing CH, and CH that arises after such exposures is more likely to carry mutations in genes that regulate the DNA damage response (DDR), with TP53 and PPM1D being the most common. PPM1D encodes a serine/threonine phosphatase that suppresses the DDR and p53 activation. Accordingly, PPM1D is mutationally activated in CH whereas loss-of-function mutations occur in TP53. Despite seemingly convergent functions, there are important biological and phenotypic differences between PPM1D- and TP53-mutant CH which suggests that PPM1D and p53 may drive HSC clonal changes through distinct processes. In addition to directly inactivating p53 via dephosphorylation, PPM1D can regulate the DDR upstream of p53 and changes in cell cycle, senescence, and apoptosis downstream of p53. Though PPM1D-mutant CH is more common and expands more rapidly in both aging and cytotoxic therapy-exposed individuals, TP53-mutant CH confers a significantly higher risk of malignancy. Accordingly, PPM1D- and TP53-mutant clonal evolution are associated with different co-mutation and karyotypic features. Using our genetically modified mouse models (GEMMs), we discovered that mutations in PPM1D and TP53 drive HSC expansion in different ways and hypothesize that the distinct phenotypes of PPM1D- and TP53-mutant CH are due to differences in the activation of the DNA damage response and p53-dependent cellular programs in the setting of cytotoxic therapy and accumulation of genetic alterations. To test this hypothesis, advance mechanistic insights, and guide therapeutic efforts we propose two aims. In Aim 1 we will quantify differences in how Ppm1d activation and Trp53 inactivation alter the immunophenotypic composition of the hematopoietic stem and progenitor cell pools (HSPCs) of our GEMMs at baseline and after genotoxic stress. In Aim 2 we will determine the effects of Ppm1d activation and Trp53 inactivation on the HSPC cell states and transcriptional programs under homeostatic conditions and after a DNA damaging insult. Completing these aims will define the distinct ways PPM1D and p53 shape the immunophenotypic and transcriptional landscape of hematopoiesis and highlight the cellular states and transcriptional programs that may underlie clonal changes. In so doing, we seek to improve prognostication and treatment strategies for CH and clonal evolution. .

NCI - National Cancer Institute

PROJECT SUMMARY Mitochondrial apoptosis is regulated by the pro- and anti-apoptotic members of the BCL-2 family, whose protein interactions determine whether a cell will live or die in response to distress. Cancer cells hijack the pathway, overexpressing anti-apoptotic members to suppress the pro-apoptotic members, thereby ensuring cellular immortality. Indeed, apoptotic suppression is a cardinal feature of oncogenesis and chemoresistance. BAK is one of two essential executioner proteins of the BCL-2 family and resides in a latent state in the mitochondrial outer membrane. When triggered by stress signals, BAK undergoes a conformational transformation, self-associates, and permeabilizes the mitochondrial outer membrane, resulting in the release of apoptogenic factors that commit the cell to apoptosis. How full-length BAK self-associates into an oligomer remains a mechanistic mystery and is the focus of my F32 application. The Walensky laboratory reported the generation of the first stable and homogeneous oligomeric species of full-length BAX, the cytosolic homolog of BAK, and characterized it through a battery of structure-function analyses. In contrast to BAX, which undergoes a cytosol to mitochondrial translocation upon triggering, BAK is constitutively localized in the mitochondria as a membrane-embedded protein. As such, BAK has been challenging to express in full-length form, and with sufficient purity and yield to embark on parallel studies. Encouraged by the success of generating and characterizing a full-length BAX oligomer, I applied key learnings to produce a stable, full-length oligomeric species of BAK (BAKO) for the first time, setting the stage for my postdoctoral studies on dissecting the structure and function of BAKO. My goal is to generate fresh insight into the structure-function mechanism of BAK-mediated mitochondrial apoptosis and leverage the molecular details of the oligomeric interfaces to inform new opportunities for enhancing BAK oligomerization and apoptosis induction in treatment-resistant cancer. Thus, I aim to (1) develop and characterize nanobodies that bind to oligomeric BAK and (2) harness BAKO-binding nanobodies to determine the structure of oligomeric BAK and the binding interfaces critical to its membrane- permeabilizing function. To accomplish my research aims, I will undertake a multidisciplinary workflow that incorporates a nanobody discovery platform, protein engineering, biochemical assays in model membranes and mitochondria, a series of structural methods, and mechanistic analyses of apoptosis in cancer cells. In pursuing this research plan, which includes a series of alternative approaches, I aim to both characterize the execution-phase of BAK-mediated apoptosis and uncover novel and potentially druggable surfaces for therapeutic benefit in cancer. I am excited to be pursuing a comprehensive postdoctoral training program in the laboratories of Dr. Loren Walensky at the Dana-Farber Cancer Institute and Dr. Andrew Kruse at Harvard Medical School, and look forward to developing as an independent and innovative scientist at the intersection of biochemistry, structural biology, and cancer biology.

NCI - National Cancer Institute

Project Summary The Wnt signaling pathway has a peculiar molecular architecture: it relies on two large and highly disordered scaffold proteins, APC and Axin, that assemble into a protein machine called the Destruction Complex (DC) which regulates the interactions between pathway kinases, CK1α and GSK3β, and the transcriptional effector, β-catenin. Mutations in either of the scaffold genes drive oncogenesis across a variety of deadly cancers (e.g. colorectal, gastric, breast, melanoma, osteosarcoma) as well as diseases resulting from tissue dysbiogenesis such as Hypertrophic Cardiomyopathy (affecting 1:500 people) and Congenital Hypertrophy of the Retinal Pigment Epithelium (affecting 1-2% of populations of European ancestry). Despite the significance, we still do not grasp the answers to basic questions regarding how the DC works–how is it organized, how do mutations in these scaffolds lead to its dysregulation, and how should we treat scaffold mutations? In fact, there are currently no FDA approved drugs that target the Wnt pathway. We propose a suite of experiments to determine the structure-function relationship of the DC, taking into account our recent finding that the DC is a dynamic, biomolecular condensate. In our previous study we integrated advanced techniques for dissecting condensates, including endogenous CRISPR tagging, super resolution microscopy, optogenetic reconstitution, and partial differential equation models. These techniques enabled the detailed description of how changes in the organization of these condensates can both accelerate and inhibit Wnt signals, which suggests how changes in DC scaffolds can mechanistically lead to changes in Wnt signaling fidelity. Since then, we have used proximity labelling to identify other potential DC regulators as well as observed aberrant DC morphologies that are caused by known colorectal cancer-causing mutations in APC. Here we seek to (Aim 1) understand how DC-resident proteins affect its structure and function and (Aim 2) understand how the DC is remodeled by oncogenic mutations in APC. We employ a combination of fundamental biophysical experiments, geared towards uncovering the organizing principals of the DC, along with directly-translatable lead generating experiments, to pinpoint therapeutic targets specific to mutant APC cells. Overall, this research will significantly advance our understanding of Wnt pathway regulation and its perturbation in cancer, potentially leading to precise treatment options with fewer off-target effects that have plagued the failed attempts to drug the Wnt pathway as well as enhancing our overall comprehension of condensate biology in disease contexts.