Weill Medical Coll Of Cornell Univ

NIH Research Projects · FY 2026 · 2021-05

Mitochondria play essential roles in cell biology because are central hubs of most metabolic pathways. They are not only essential for energy conversion, but also for the biosynthesis and catabolism of virtually all cell constituents. Mitochondrial dysfunction causes havoc in all cells, but especially in those cell types that are highly dependent on mitochondrial energetic and metabolic functions, such as neurons and glia. Genetic alterations of the mitochondrial proteome, which includes more than 1000 proteins, encoded by both the nuclear and the mitochondrial genomes, result in primary mitochondrial disorders. These diseases, for which there is currently no effective treatment, result in severe and often fatal neurodegeneration. Mitochondrial dysfunction also plays a role in the pathogenesis of many age-related neurodegenerative disorders, such as Alzheimer and Parkinson disease and ALS. Therefore, addressing therapeutically the consequences of mitochondrial dysfunction could have a profound impact on the treatment of many human disorders. A major challenge in devising effective treatments for mitochondrial encephalopathies is our limited understanding of the ramifications of the effects of mitochondrial dysfunction. The conventional view that these disorders are caused simply by energy failure is inadequate, as it is becoming increasingly clear that mitochondrial dysfunction affects much more than just ATP generation and leads to an extensive rewiring of cell metabolism. An exciting new development in the field is the observation that various types of mitochondrial dysfunction activate transcriptional and metabolic responses that involve multiple stress signaling pathways. We and others have identified a “mitochondrial integrated stress response” (mtISR) in diverse genetic forms of mitochondrial disorders, suggesting that mtISR is strongly associated with mitochondrial diseases and a potential pathogenic common denominator. We postulate that, while in the short term these responses may be compensatory, if sustained and unresolved, they become maladaptive and causes imbalances of key metabolites, which may be more detrimental than the energy defect itself. While we now fully appreciate these maladaptive mechanisms in peripheral tissues, such as muscle and heart, very little is known about them in the CNS affected by mitochondrial encephalopathies. A deeper knowledge of the characteristics and the consequences of the mtISR in the CNS is needed to understand its pathogenic significance and develop targets therapeutic strategies. Our research group has a long-standing commitment to investigating the pathogenic mechanisms of mitochondrial diseases and we have accumulated over two decades of expertise in studying the mechanisms of mitochondrial encephalopathies and mitochondrial dysfunction in neurodegeneration. In this R35 application, we focus on fundamental gaps in knowledge on the mtISR in mitochondrial encephalopathies by studying disease models that recapitulate human diseases. We will use a series of approaches, both established and technologically innovative, to generate a blueprint of the metabolic rewiring in the diseased CNS and identify targets potentially responsive to therapeutic modulation.

NIH Research Projects · FY 2025 · 2021-05

ABSTRACT Despite recent progress in immunotherapy (checkpoint blockade and adoptive T cell transfer), most patients with solid tumors still do not respond or subsequently develop acquired resistance to therapy. Our group and others have described an immune resistance mechanism mediated by the metabolic dysregulation of Tryptophan (Trp) catabolism through the Kynurenine (Kyn) - aryl hydrocarbon receptor (AHR) pathway. The production of Kyn and signaling through the AHR suppresses CD8+ and CD4+ effector T cells and enhances the generation of immunosuppressive cell types, including FoxP3+CD4+ T cells (Tregs), myeloid-derived suppressor cells (MDSCs) and M2-polarised tumor-associated macrophages (TAMs) - cells which play a critical role in limiting anti-tumor immunity. We propose to image signaling activity through the IDO/TDO-Kyn- AHR pathway, in order to optimize the timing (scheduling) of combination drug treatment (treatments targeting this pathway along with immune based therapies). In this proposal, we plan to: use imaging to better understand signaling through the Trp–Kyn-AHR pathway in the tumor microenvironment, by monitoring AHR transcriptional activity using dual reporter systems. We have successfully developed a DRE (dioxin responsive enhancers)-AHR reporter system in order to: 1) quantify the kinetics of engagement of the AHR upon in vitro stimulation with different agonists/antagonists and its correlation with phenotypic changes in different components of the TME: cancer cells, macrophage and T cells; 2); to monitor the dynamic of activation of the AHR pathway in vivo using a biosensor system during tumor progression in IDO/TDO-expressing cancer models 3) to evaluate the in vivo dynamics of AHR activation after response to therapeutic interventions (PD-1/CTLA-4 blockade, T cell therapy) in the same models 4) design therapies combining the inhibition of the Trp-Kyn-AHR axis with immune therapy based on reporter assays of the AHR activity over time; and 4) evaluate the potential for clinical translation. This approach addresses an unmet need and the proposed strategy is strongly supported by 4 experts in the field and our recent publication in Nature Communication– see letters of support.

NIH Research Projects · FY 2025 · 2021-05

ABSTRACT Unless COVID overtakes it, tuberculosis is likely to keep its grip on its grim record of being the leading infectious cause of human death. Humans are the only known natural host and transmitting reservoir for the causative agent, Mycobacterium tuberculosis (Mtb). This means that person-to-person transmission through air is essential for Mtb's survival as a species. Despite multifaceted efforts to reduce TB's transmissibility, TB's reproductive number, Ro, remains among the highest of frequently-lethal infectious diseases. Aerogenic transmission is a stage in Mtb's life cycle that must have been subjected to strong evolutionary pressures, yet our knowledge of Mtb's transmission biology is sorely lacking. The problem has been nearly inaccessible to basic-science study for want of suitable technologies and animal models. This Program Project proposes to lay a basic-science foundation for potential new transmission blocking interventions by bringing a synergistic combination of investigators and disciplines together for a collaborative attack that mobilizes genome-wide screening under transmission-relevant conditions, characterizes Mtb's metabolomic, lipidomic and biochemical responses to those conditions, introduces and improves animal models, and uses aerosol physics as a guide and tool. Project 1 will identify genes that Mtb requires to survive the transitions between major states that the pathogen encounters en route to, during and after aerosol transmission. Project 2 will identify conserved, essential metabolic programs in Mtb that have evolved in response to transmission-related stresses, such as changes in humidity and gas composition. Project 3 builds on the recent discovery of cough-inducing lipids produced by Mtb to characterize an even more potent tussive lipid as a virulence factor and to develop a model of cough-based transmission among guinea pigs. Project 4 characterizes the physical and rheological properties of respiratory fluids relevant to TB transmission and uses that information to control the mechanical generation of physiologically relevant, respirable aerosols of Mtb. Core A ensures the efficient flow of information, personnel and materiel among these interconnected units, while Core B develops a mouse model of simulated transmission using the aerosolization device and settings of Project 4 and applies that model to confirm which genes Mtb depends on to survive aerosol transmission to a new host.

NIH Research Projects · FY 2025 · 2021-05

Triple negative breast cancer (TNBC) are typically aggressive cancers with shorter median time to relapse and death than other breast cancers. Because these cancers are defined by the absence of a target, identification of tailored therapies has been challenging. However, immune therapy shows important promise in TNBC, including the first FDA approval for immunotherapy in breast cancer and favorable response data for the addition of immunotherapy to neoadjuvant chemotherapy. Recent evidence suggests that tumor molecular characteristics may provide clues to both the different etiology and prognoses for TNBCs. Gene expression studies revealed that TNBCs are heterogeneous and composed of finer subtypes, defined in part by immune response signatures. It has been hypothesized that the patients’ immune response plays an important role in determining tumor progression. Further, sequencing studies have identified a set of genes that are frequently mutated in breast tumors and several mutational signatures that reflect distinct mutagenic processes, and may have etiologic implications. The mutational signatures that have been identified in TNBCs are distinct from the more common luminal breast cancers, highlighting the need for research specific to this subtype. We propose to perform whole exome sequencing (WES) of matched tumor and germline DNA samples from 400 TNBC patients from four prospective cohort studies, the Nurses’ Health Study, Nurses’ Health Study II, Cancer Prevention Study II, and Cancer Prevention Study 3. In combination with our existing rich database of germline GWAS, breast cancer risk factors, and tumor immune signatures, we are well positioned to better understand how genetic and nongenetic risk factors influence breast tumor mutational signatures, immune response and prognosis. We will assess the association of genetic and nongenetic risk factors with tumor mutational profiles (Aim 1), and tumor immune profiles (Aim 2). We will also examine the association between immune response signatures and tumor mutation profiles (Aim 3). In exploratory analyses, we will evaluate whether a possible joint effect of somatic mutational signatures and immune response signatures are associated with breast cancer-specific survival (Aim 4). Knowledge from this study will be extremely valuable in developing prevention strategies and treatment targets for these aggressive tumors. To complete these aims, we have assembled a multidisciplinary team of experts in breast cancer epidemiology, genetic epidemiology, statistical genetics, bioinformatics, immuno-oncology, and tumor genomics. The Principal Investigators have extensive experience with the cohort resources and have worked collaboratively for over thirteen years. We have also partnered with the B-CAST and AMBER consortia to create a large and diverse repository of WES data from TNBCs, which will enable us to both replicate our results and compare mutational profiles and their associations with prediagnostic and clinical factors in European-ancestry and African American women.

NIH Research Projects · FY 2025 · 2021-05

Triple negative breast cancer (TNBC) patients present a formidable clinical challenge, as they exhibit higher rates of metastatic recurrence, and respond poorly to available targeted therapies. Given the clinical significance, it is imperative to develop effective targeted anti-metastatic therapies for the treatment of TNBC. Metals serve as co-factors for tumor promoting pathways and targeting metals to disable these pathways has emerged as a potential anti-cancer therapeutic strategy. Copper, an essential trace element functions as a catalytic cofactor for a host of metalloenzymes/proteins that contribute to tumor angiogenesis and metastasis. In addition, increased copper uptake by malignant cells has led to the development of copper-specific chelators as therapeutic agents. In a potentially transformative discovery, we have identified within the primary tumor, a discrete population of highly metastatic OCT4+ SOX2+ cells. We show that these metastatic cells have significantly elevated levels of intracellular copper and exhibit increased sensitivity to copper deficiency. We have also shown that copper depletion alters the architecture of extracellular matrix in the metastatic lungs of both mouse and human. Our central hypothesis is that copper contributes to two key aspects of metastasis: cancer cell intrinsic metabolic pathways that directly contribute to metastasis; and the “pre-metastatic niche” that supports colonization, and outgrowth of disseminated metastatic tumor cells. To test this hypothesis, we will use pre-clinical models to elucidate cellular and molecular mechanisms of oral copper chelator tetrathiomolybdate (TM) and leverage banked samples from our completed phase II TM clinical trial to develop collagen remodeling products as biomarkers of TM response. The central goal of this proposal is to understand the mechanistic basis of copper depletion as a viable treatment approach against TNBC metastasis. We expect to make significant advances through the following aims. Aim 1 will establish the direct link between copper-mediated metabolic reprograming and metastasis in TNBC and dissect key cellular and molecular mechanisms, and Aim 2 will investigate how copper deficiency reprograms the extracellular matrix in the distal metastatic organs to generate an inhospitable microenvironment to impair metastasis and develop clinically valuable biomarkers of response to TM. Fundamental discoveries from these integrated pharmacological and genetic investigations has the potential not only to advance TM into larger randomized trials, but to identify new copper pathways for the development of novel therapeutic strategies against TNBC.

NIH Research Projects · FY 2026 · 2021-04

Project Summary/Abstract Neurologic diseases affect as many as one billion people worldwide and are a major cause of disability and human suffering. Current standard of care imaging (contrast-enhanced MRI) is extremely limited to detect many neurological and neurodegenerative diseases. MR spectroscopic imaging (MRSI) has a great potential to supplement routine clinical MRI for clinical conditions including brain neoplasms, neonatal and pediatric disorders (hypoxia-ischemia, inherited metabolic diseases, and traumatic brain injury), demyelinating diseases, and infectious brain lesions. Gadolinium contrast agents have incomplete clearance, and repeated use of contrast-enhanced imaging has recently received an FDA warning due to brain accumulation. MRSI does not use contrast material and has no/minimal risk for patients. 3D encoded MRSI methods provide high sensitivity per unit time and unit volume. Presurgical and radiation treatment planning will greatly benefit from full 3D information, ideally with isotropic resolution. Echo-planar spectroscopic imaging (EPSI) based 3D whole-brain MRSI have been on most scanner platforms, and are probably the most commonly used fast MRSI techniques to date. The primary limitation of 3D MRSI has been magnetic B0 field inhomogeneity, which broadens lineshapes and diminishes spectral quality in about 40% of the brain (e.g., mesial temporal lobe, inferior frontal cortex, medial frontal gyrus, brainstem, and cerebellum). This limits the ability to evaluate critical brain regions such as mesial temporal lobe (MTL) and orbitofrontal cortex (OFC), which have pivotal roles across neurologic disorders. Recently, we introduced a radically novel concept called Unified Coil (UNIC), which includes innovative decoupling methods to bring the distance between separate shim and RF loops to zero millimeters. Both RF and shim coils are at a close proximity to the target organ for maximized RF SNR and shimming. Physical law implies that the only effective way to shim local inhomogeneous field (as in MTL/OFC) is by placing size-matched shim coils which generate opposite high-order field to counteract the inhomogeneous field. Our hypothesis is that UNIC will dramatically increase brain volume coverage and allow true metabolic evaluation of the entire brain using 3D MRSI. This will enable broader applications in patient management with various neurological disorders. The proposed study will prototype the first UNIC head coil (Aim 1), optimize the technique in shimming performance and hardware complexity (Aim 2), and assess the technique quantitatively in improving brain coverage of 3D MRSI (Aim 3). Successful completion of this study will largely resolve the longstanding B0 inhomogeneity issue in whole brain. Such coils can be widely used to benefit the entire MRSI community by advancing B0 shimming technology. It will help catalyze the widespread clinical acceptance of MRSI.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT Clonal hematopoesis (CH) defined as the presence of acquired mutations detectable in peripheral blood of normal healthy individuals without hematologic malignancies (HM) has been well characterized by sequencing population based cohort studies. The presence of CH is associated with >12-fold risk of eventual HMs. More data are needed to delineate disease specific and mutation specific risk as well as factors that might shape the evolutionary trajectory from CH to HM. We have demonstrated in our prior work that participants in the WHI who developed AML were four times more likely to harbor a mutation a median of 9.6 years before the onset of AML compared to controls (70% vs. 30%, OR 4.0, 95% C.I. 2.5-6.3) with mutations in TP53, IDH1/2, and spliceosome genes being highly associated with increased risk of AML and rarely present among controls. The long-term goal is to identify both mutational and cell-extrinsic factors that contribute to the development of HM and thus provide the basis for future clinical trials of HM interception and prevention. Published data indicate the ability of metabolic factors and inflammation to influence the expansion of CH. Our central hypothesis is that mutational, inflammatory and metabolic factors that predict the development of HM can be prospectively identified, thus enabling improved risk assessment. We will utilize peripheral blood samples collected at baseline from the Women’s Health Initiative (WHI) cohort that prospectively followed 168,808 women for a median of 10.8 years. All cancer outcomes were adjudicated by central review. Our specific aims will determine the following: (1) the risk of baseline pre-HM mutations and development of specific HM among participants in the WHI. We will select 400 cases of HM (200 chronic lymphocytic leukemia (CLL) and 200 cases of multiple myeloma) along with age matched 400 controls that did not develop HM during WHI follow up (2) To determine the impact of metabolic and inflammatory abnormalities in promoting CH expansion and impacting the progression from CH to HM. Our study is significant because there is no known intervention strategy to prevent or delay the progression of CH to HM and in general, prospective, randomized, controlled trials of prevention strategies require many years of follow-up to reach definitive conclusions. Our study will establish individuals at highest risk of HM based on mutational, inflammatory and metabolic factors and provide grounds for monitoring people individuals with CH at highest risk of HM. Moreover, these data will provide novel insight into intervention strategies to prevent the onset of HM. The proposed research is innovative in investigating mutational and metabolic as well as inflammatory factors that impact the progression of CH to HM using long term data from a large cohort of women.

NIH Research Projects · FY 2025 · 2021-04

SUMMARY Melanosome pH controls pigmentation and affects skin cancer risk; however, the signaling pathways that affect this important pigment mechanism are poorly understood. The Melanocortin 1 Receptor (MC1R), through transmembrane adenylyl cyclase (tmAC)-defined cAMP signaling pathways, has an important role in pigmentation, and affects skin cancer risk by activating the expression of key pigment synthesizing enzymes. But whether MC1R signaling affects melanosome pH has remained unclear. We recently identified a new cAMP signaling pathway in melanocytes, defined by the soluble adenylyl cyclase (sAC), that regulates melanosome pH. Whereas elevation of tmAC-dependent cAMP increases eumelanin by upregulating key pigment enzymes (e.g., tyrosinase), a reduction in sAC-dependent cAMP also increases eumelanin by inducing the alkalization of melanosome pH and enhancing tyrosinase activity. Thus, our overarching hypothesis is that sAC and tmACs regulate distinct cAMP signaling cascades in melanocytes and function in concert to control pigmentation. What remains unclear are the upstream and downstream mechanisms that control sAC-dependent regulation of melanosome pH and pigmentation. In our first Aim, we will use human primary melanocytes expressing either wild type MC1R or MC1R polymorphisms along with Mc1re mouse melanocytes and a novel Mc1re (e/e) conditional sAC knockout mouse to determine the interplay between MC1R signaling and sAC-dependent control of melanosome pH and pigmentation. In our first Aim, we will also assess whether bicarbonate, a known stimulator of sAC that has been linked to melanin synthesis, affects melanosome pH and pigmentation in human and mouse melanocytes in a sAC-dependent manner. In Aim 2, we will determine how sAC regulates melanosome pH and pigmentation. Our preliminary data suggests that sAC activates the cAMP effector protein exchange protein activated by cAMP (EPAC), which then stimulates the melanosome ion channel two-pore channel 2 (TPC2). Using genetic and pharmacological methods in mouse and human melanocytes, we will establish which EPAC isoforms and melanosome channels are required for sAC-dependent control of melanosome pH. Finally, our preliminary data suggest that sAC inhibition rescues the defective melanosome pH and tyrosinase activity in Oca2 deleted mouse melanocytes both in vitro and in mice. Thus, pharmacological sAC inhibitors are potential therapeutics for oculocutaneous albinism type 2. We will further explore this therapeutic possibility with a new conditional sAC knockout Oca2-/- (p/p) mouse model. Overall, the experiments in this proposal will systemically examine the cAMP dependent signaling cascades that regulate melanosome pH and pigmentation. The proposed studies will establish new models that will overcome limitations in our investigation of cAMP signaling in pigmentation, will provide greater insight into the cAMP-dependent mechanisms that control melanosome pH, and may lead to new therapeutics for diseases of pigmentation.

NIH Research Projects · FY 2026 · 2021-04

The program will strengthen patient-oriented HIV clinical investigation training of physician-scientists and other exceptional clinicians in Mwanza, in northwestern Tanzania. The program builds upon two decades of collaboration and capacity building by the partner institutions. The primary research institution in Tanzania will be the Mwanza Intervention Trials Unit (MITU), which is a unit of Tanzania’s National Institute for Medical Research (NIMR). MITU was established in 2006 as a collaboration between NIMR and the London School of Hygiene and Tropical Medicine (LSHTM) to strengthen HIV interventional research in Tanzania. MITU collaborates closely with the Weill Bugando School of Medicine, which is also located in Mwanza. Weill Bugando was established in 2003 to train physicians for northwestern Tanzania. MITU and Weill Bugando have collaborated with Weill Cornell Medical College since their founding. This research training program aims to fill a critical gap in Mwanza: the need for Tanzanian physicians with rigorous training in patient-oriented HIV clinical investigation. We use the NIH definition of clinical investigation as research which directly interacts with individual patients or clusters of patients in Phase I, II, or III clinical trials, pragmatic trials, or formative epidemiologic or behavioral research in preparation for clinical trials. The goals of the proposed training program are: 1.To increase the number of clinical investigators at MITU and thereby increase institutional capacity for HIV clinical investigation 2. Establish Weill Bugando/MITU as a sustainable training center for HIV clinical investigation. The ultimate goal is to prevent new HIV infections, provide effective HIV care, and improve HIV outcomes in Tanzania and East Africa. We will provide long-term training to fifteen outstanding clinician scientists to conduct patient-oriented HIV clinical investigation. We will primarily train physicians but will also train exceptional other clinicians (PharmD, DMD). We will train 10 PhDs, and 5 MS degree candidates. Tanzanian trainees will benefit from participation in 17 active research projects in five synergistic areas of HIV investigation including HIV prevention and vaccine research, implementation of HIV testing and treatment, women’s health, HIV related co-infections, and HIV and cardiovascular disease. We will also strengthen the PhD program in clinical investigation at Weill Bugando by introducing 4 new graduate courses in clinical investigation. Weill Cornell is committing institutional funds so that 4 of the best PhD graduates can have 2-year post-doctoral appointments as MITU research scientists after their Fogarty training. At the end of the 5-year training program, MITU will have a cadre of clinical investigators and a greater depth and breadth of externally funded HIV patient-oriented research. MITU/Weill Bugando will be a training hub for HIV clinical investigators in East Africa.

NIH Research Projects · FY 2025 · 2021-03

SUMMARY A stem-mesenchymal phenotype of the tumor epithelium, and its associated immunosuppressive and desmoplastic stroma, are fundamental characteristics of the most aggressive and poor survival type of colorectal cancer CRC. However, the molecular and cellular mechanisms driving this process are still far from clear. This proposal stems from a series of recently published and unpublished observations in my laboratory that identify the two atypical PKCs (aPKC; PKC and PKC/) as novel tumor suppressors acting in concert to prevent this aggressive form of CRC. Thus, the simultaneous loss of both aPKCs in the intestinal epithelium (in a new DKOIEC mouse line) results in highly mesenchymal adenocarcinomas with a reactive and strongly immunosuppressed stroma. Both aPKCs are significantly downregulated in mesenchymal/stromal/ immunosuppressive CRC human patients who have the most unfavorable prognosis. Our unpublished preliminary data demonstrate that intestinal epithelial cells (IECs) deficient in PKC/ (or both aPKCs) upregulate the stem cell receptor CD44, concomitant with the downregulation of Lgr5+ intestinal stem cells, suggesting the appearance of a new type of tumor initiating cells (TICs). Inhibition of CD44+ in tumor organoids demonstrate its requirement for growth and supports its physiopathological relevance. Consistently, inhibition of one of the key CD44 stromal ligands (hyaluronan) in vivo abrogates the mesenchymal phenotype of DKOIEC tumors inhibiting the immunosuppressive response and restoring immunosurveillance. We hypothesize that the upregulation of a new type of CD44+/Lgr5- TICs by the loss of PKC/ is central in the development of the aggressive type of CRC. The upregulation of the MAP kinase cascades, together with the identification, in a series of unbiassed approaches, of the transcription factor KLF4 as a potential critical intermediary between PKC/ and CD44 expression, led us to hypothesize that the activation of ERK/JNK by PKC/ deficiency triggers AP1 and, concurrently, induces the degradation of KLF4; both actions cooperate to drive CD44 expression and the mesenchymal phenotype of very aggressive CRC. Therefore, in this proposal, we will determine the role of CD44 in the aggressive/mesenchymal type of CRC (Aim 1), as well as the molecular mechanisms whereby PKC/ regulates CD44 expression and function in this process (Aim 2). The successful completion of the proposed studies will create a new paradigm of significance and impact that will contribute to a more comprehensive understanding of the mechanisms driving the poor prognosis mesenchymal type of CRC, which will be key for the design of new therapeutic targets for this type of aggressive neoplasia.

NIH Research Projects · FY 2025 · 2021-03

Summary Abstract The overall vision for our research is to discover novel mechanisms by which histone and non-histone proteins on DNA, i.e. chromatin, regulate genomic processes and aging. In particular, we strive to integrate different fields, such as the role of chromatin in genome stability and the role of chromatin in aging. Using a combination of biochemistry, structural biology, molecular genetics in budding yeast, tissue culture and genome-wide approaches, we have discovered that chromatin is disassembled and reassembled during not only gene expression and DNA replication but also during DNA double-strand break repair. We have revealed the mechanistic bases for these events and their key impact on these genomic processes. In more recent years, we have expanded the questions that we address beyond chromatin – for example uncovering novel mechanistic bases of aging and discovering new ways to extend lifespan. Similarly, inspired by our recent finding that chromatin structure reduces the processing of DNA double-strand breaks to single-strand DNA (termed DNA end resection), we have devised innovative CRISPR/Cas9 gRNA library screening approaches to identify novel activities that regulate DNA end resection during DNA double-strand repair. Most of the cells in the human body are in G0/G1-phase and it is critical that excessive DNA end resection does not occur in these cells. If it were to occur, it would block DNA repair by the only pathway that is used to repair DNA double-strand breaks in G0/G1-phase cells, namely non-homologous end joining (NHEJ), and it would result in translocations and deletions from the ensuing homology-mediated repair. Indeed, the extent of DNA end resection is the critical decision point in the choice between using the NHEJ or homologous recombination (HR) pathway for repairing DNA double-strand breaks. We propose that mechanisms must be in place that limit excessive DNA end resection in G0/G1-phase cells to prevent HR, yet enable sufficient DNA end processing of un-ligatable DNA ends to allow NHEJ-mediated repair. The proteins and pathways that regulate the extent of DNA end resection in G0/G1-phase cells are currently unknown. Thus, a major goal of this program is to discover the machinery and mechanisms that regulate DNA end resection in G0/G1-phase cells. We are uniquely positioned to do this, based on our expertise, novel genetic screening approach and compelling preliminary data. Another critical, yet poorly understood, aspect of genome maintenance is how gene expression is “shut-off” in the vicinity of a DNA lesion to prevent collisions between the transcription and DNA repair machinery. Similarly, it is crucial that transcription is restarting after DNA double-strand break repair, but the mechanism is unknown. We have recently discovered some of the proteins involved using our novel assays and genetic screens, so the second major goal of this program is to discover the fundamental mechanisms of transcriptional shut-off and restart around DNA double-strand breaks.

NIH Research Projects · FY 2025 · 2021-03

Abstract Niche-derived growth factors (GFs) are essential for establishing and maintaining mammalian spermatogonial stem cells (SSCs) in the adult testis. In culture, however, optimal conditions for expansion of pure populations of self-renewing SSCs remain elusive. We recently found that mouse SSCs possess unexpectedly robust mechanisms to counter-regulate GF signaling by inducing expression of negative feedback regulators (NFRs), including members of the Spry and Dusp gene families. Abrogation of specific NFRs in SSCs led to increase ERK signaling, decreased expression of stem cell-associated genes in vitro and loss of stem cell activity upon transplantation, suggesting that excessive GF-dependent signaling is detrimental to SSC function and may favor differentiation. The cellular alterations that occur following perturbation of NFRs are unknown. However, our data imply that SSCs are programmed to limit ERK signaling within a narrow physiological range. This proposal addresses the mechanisms by which NFRs restrict GF signaling in SSCs, prevent unscheduled differentiation, and enable long-term propagation of SSC clones. Using genetic mouse models, SSC culture, transplantation analysis, and a newly-developed real-time biosensor for ERK activity, we address the following questions: (1) Which NFRs are required for SSC self-renewal? (2) How does ablation of NFRs result in loss of stem cell activity? (3) How do NFRs control the dynamic intracellular signaling cascades downstream of GF receptors? And (4) At which nodes in the ERK pathway do NFRs exert their actions? In addition to providing a rational basis to improve SSC culture systems, these studies will also identify novel adult SSC markers and shed light on mechanisms by which cross-talk between the niche and SSCs balances self-renewal and differentiation in vivo.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY/ABSTRACT The goal of this research is to develop cardiac quantitative susceptibility mapping (QSM) for non-invasive meas- urement of blood oxygen saturation, towards the long-term objective of improving early diagnosis, therapeutic decision-making, and clinical outcomes for patients with pulmonary hypertension (PH). PH is a progressive and life shortening disorder affecting ~10% of adults over age 65. Given that PH can be irreversible in its later stages, early diagnosis and physiologic monitoring are critically important. Impaired oxygenation of the lungs and heart chambers (cardiac oxygenation) is a key manifestation of PH that impacts symptoms and clinical outcomes. Increased pulmonary arterial pressure in PH impairs pulmonary oxygen exchange, decreasing delivery of oxy- genated blood to the left heart. Systemic cardiac output is often compromised in PH, resulting a larger differential blood oxygen saturation between the left and right heart. Invasive catheterization (cath) is currently used to measure cardiac oxygenation but entails procedural risks, ionizing radiation exposure, and is impractical for early diagnosis and serial monitoring - a non-invasive method to accurately measure blood oxygenation would be of substantial utility. MRI is well suited for PH assessment as it enables integrated evaluation of pulmonary anat- omy, pressure, as well as cardiac function and remodeling - blood oxygenation is a key gap in MRI evaluation of PH. This gap stems from limitations in current pulse sequence technology rather than fundamental MRI physics. It is well known that deoxygenation changes the magnetic susceptibility of blood. These changes have tradition- ally been probed using a magnitude property of the MR signal: the transverse relaxation time (T2). However, this requires patient-specific calibration that is difficult in clinical practice. In contrast, QSM relies on the phase of the MR signal to directly measure susceptibility and thus cardiac oxygenation. We have obtained highly encouraging preliminary data for QSM measurement of cardiac blood oxygenation, with close agreement between QSM and oxygenation measured invasively. We have identified key challenges for developing cardiac QSM, including motion suppression and prolonged scan times. The current research proposes to develop an accelerated cardiac QSM method, and to test QSM in relation to oxygenation on invasive cath, as well as effort tolerance and clinical prognosis. Study Aims are as follows: (1) Develop accelerated cardiac QSM using free-breathing acquisition and optimized reconstruction. (2) Test accelerated and current cardiac QSM among PH patients in comparison to T2-based cardiac oxygenation and the reference standard of invasive cardiac catheterization. (3) Determine whether cardiac QSM stratifies clinical severity and predicts PH disease progression. The expected outcome of this research is a non-invasive method for measuring cardiac oxygenation – a critically important marker in PH that currently relies on invasive testing. Given the increasing prevalence and therapeutic options for this serious condition, non-invasive oxygenation assessment by cardiac QSM holds broad significance towards the goal of early diagnosis, therapy optimization, and improved clinical outcomes for millions of patients with PH.

NIH Research Projects · FY 2025 · 2021-01

ABSTRACT The ability of mammalian cells to elicit inflammation is central to many processes including embryogenesis, wound healing, tissue regeneration, and cancer metastasis. A major source of inflammatory signaling is the aberrant presence of double-stranded (ds) nucleic acids in the cytoplasm. Mammalian cells have evolved high- ly conserved mechanisms to detect cytosolic nucleic acids as an anti-viral defense. In normal cells, cGAS (cy- clic GMP-AMP synthase) and its downstream signaling effector STING (stimulator of interferon genes) have been proposed as essential mediators of type I interferon (IFN) signaling and downstream immune activation. We have shown however, that in cancer cells with chromosomal instability (CIN), there is no evidence of type I IFN signaling despite the presence of cytosolic DNA and constitutive activation of cGAS and STING. Instead, cancer cells rewire their signaling downstream of STING to selectively suppress IFN signaling and enable oth- er pro-metastatic pathways such as NF-κB. Three important pieces of evidence bring into question the essen- tiality of the cGAS-STING pathway in promoting anti-tumor immunity and suggest heretofore unappreciated redundancies and context dependence of nucleic acid sensing in cancer: 1) chromosomally unstable cancer cells retain IFN-responsiveness to cytosolic dsRNA. 2) Cancer cells with CIN can still elicit a robust, anti-tumor immune response to cytosolic dsDNA, in a manner independent of cGAS-STING and type I IFN. 3) Expression of nucleic acid sensors and downstream inflammatory pathways is highly variable across tumor subpopulations and metastatic cell states – in which a continuum of stem-like to more committed epithelial progenitors is ob- served. Together, these findings challenge the current view that cGAS-STING signaling is the universal media- tor of inflammation in response to cytosolic dsDNA. Herein, we aim to understand functional redundancies and interactions across cytosolic nucleic acid sensing pathways and how their transcriptional outputs vary with tu- mor cell differentiation status. We will systematically interrogate key nucleic acid sensors and their downstream effectors in three syngeneic mouse models characterized by increased metastatic potential and high levels of CIN. We will experimentally manipulate CIN rates to identify cytosolic nucleic acid-dependent, but cGAS- STING-independent mechanisms of immune activation (Aim 1). We will then couple high-throughput single-cell sequencing with combinatorial CRISPR-mediated gene inactivation of key cytosolic nucleic acid sensors and effectors in metastasis-initiating stem cells distinguished by SOX2 expression, versus their more differentiated counterparts, to map the cell state-specific regulatory logic of this pathway (Aim 2). Unraveling the context- dependence of this extremely important and versatile signaling cascade has the potential to transform our thinking about chronic inflammation in cancer and to reveal therapeutic vulnerabilities in chromosomally unsta- ble cancer cells that are otherwise resistant to cGAS-STING signaling.

NIH Research Projects · FY 2025 · 2020-12

ABSTRACT In response to large numbers of senior center clients who suffer untreated depression and the dearth of geriatric mental health providers, we have partnered with senior center stakeholders to simplify Behavioral Activation (BA) to match the skill set of lay volunteers (“Do More, Feel Better”; DMFB). The lay delivery model: 1. makes use of existing volunteer resources that can address the insufficient workforce; and 2. has potential for being an acceptable and sustainable delivery model. However, the capacity of this model to engage the same target (increased activity) and to yield comparable clinical outcomes as professionally-delivered interventions is yet to be determined in a fully-powered trial. This Collaborative R01 proposes fully powered randomized effectiveness trial testing the effect of DMFB in comparison to professionally-delivered BA (MSW BA) on increased activity level (target) and decreased depressive symptoms. The specific aims are to: 1. Test the effectiveness of DMFB, in comparison to MSW BA, for depressed (PHQ- 9>10 and Ham-D>14) older adults (>60) on increasing overall activity level (target) and reducing depression symptoms; and 2. test whether increased activity level predicts greater reduction in depression severity and whether increased activity's impact on depression is non-inferior across conditions. Client participants will be a total of 288 older (>60 years) non-psychotic, non-demented individuals with elevated depressive symptoms from 6 Seattle, 6 New York City, and 6 Tampa area senior centers serving economically and ethnically diverse communities. Eligible clients will be randomized within senior center to either DMFB (n=144) or MSW BA (n=144). Two lay volunteers and 2 MSWs per center will provide the intervention. Our proposal responds to the 2012 IOM report which highlighted the dearth of mental health providers for older adults and the need to develop a workforce of nontraditional providers. DMFB is a streamlined BA intervention that has high potential for sustainability by making use of an untapped volunteer resource and supervision infrastructure within senior centers. Our findings will identify effective interventions for an underserved and difficult to engage population, our partners in aging services would be pleased to implement either delivery format of BA to activate depressed older adults.

NIH Research Projects · FY 2024 · 2020-12

Abstract Cardiovascular disease is the leading cause of death globally. Cardiovascular magnetic resonance (CMR) is routinely used in the clinical management of cardiovascular disease and is the standard method for the assessment of cardiac function and myocardial tissue properties. Today, most clinical CMR studies are still conducted on 1.5T scanners because of their availability and robustness in executing CMR protocols. In general, 3.0T provides higher SNR, spatial resolution, and reduced scan time than 1.5T. However, the increased B0 field strength also poses technical challenges for CMR. A major challenge is the susceptibility or off-resonance artifacts due to worsened B0 field inhomogeneity. In the last two decades, steady-state free precession (SSFP) has revolutionized CMR on 1.5T because it boosts SNR and contrast to noise ratio (CNR) markedly over gradient-echo based acquisitions. However, its routine use on 3.0T has been inconsistent despite continuously improving B0 homogeneity and shimming capabilities. Therefore, gradient-echo based acquisitions are still routinely used, such as for cine imaging, cardiac relaxometry, and coronary MRA, negating the major advantages of 3.0T for CMR. Due to the distance of standard whole-body shimming coils from the target organ, they only provide shimming capabilities up to the second-order spherical harmonic (SH), and are incapable of shimming higher-order localized field variations such as those present near the heart-lung interface. This remains an unmet challenge. In this project, we will apply a novel unified shim-RF coil technique to overcome the primary limitation in 3.0T CMR. We will develop a unified shim-RF coil and validate its safety and high-order shimming capability (Aim 1). We will develop a respiratory motion-resolved B0 field mapping technique based on our low-rank Multitasking framework and real-time shim circuits to allow dynamic shimming for free breathing CMR (Aim 2). We will then validate the technology in human subjects on 3.0T (Aim 3). Successful completion of this project will enable robust SSFP CMR on 3.0T, a major technical challenge, which allows reliable high-resolution, high- SNR CMR, including but not limited to cine and coronary MRA. This novel cardiac shimming system has the potential to have a major impact in accelerating the clinical adoption of 3.0T CMR.

NIH Research Projects · FY 2025 · 2020-12

The regulation of energy storage and utilization in adipocytes is a dynamic process that influences overall energy homeostasis. Adipocytes store nutrients in lipid droplets as triglycerides (TG), and mobilize them as needed. While these cells respond to sympathetic signals by increasing TG lipolysis to release free fatty acids (FFA) and glycerol, they also reabsorb FFA for re-esterification as triglycerides or alternatively for oxidation. Activation of b-adrenergic receptors and downstream cyclic AMP signaling not only increases lipolysis, but also promotes fatty acid oxidation at the expense of re-esterification, although the underlying mechanisms remain poorly understood. We hypothesize that catecholamines direct fatty acids for oxidation through regulation of signal transducer and activator of transcription 3 (STAT3) and suppression of glycerol-3-phosphate acyltransferase 3 (GPAT3). Our preliminary data demonstrate that STAT3 specifically undergoes Ser727 phosphorylation at the lipid droplet in response to stimulation of b-3 adrenergic receptors and activation of lipolysis in adipocytes. The pool of lipid droplet-associated STAT3 binds to and inhibits GPAT3, effectively suppressing GPAT3-catalyzed re-esterification, to promote fatty acid oxidation. Adipocyte-specific Stat3 KO mice exhibit normal rates of lipolysis, but manifest a specific defect in lipolysis-driven oxidative metabolism, resulting in reduced energy expenditure and increased adiposity on high fat diet. The experiments outlined in this proposal are designed to expand insights into this novel function of STAT3, determining its metabolic consequences and delineating the mechanism of action. Aim 1 will focus on the stimulation of STAT3 phosphorylation by catecholamines in vivo, delineating the signaling pathway and specific kinase(s) responsible for the critical STAT3 Ser727 phosphorylation event. The interaction of STAT3 with GPAT3 will be investigated in aim 2 using co-immunoprecipitation and in vitro binding assays. Additionally, the mechanism by which STAT3 interaction results in suppression of GPAT3 activity will be investigated using in vitro GPAT activity assays and proteomic approaches. Finally, in aim 3, the physiological relevance of this novel regulatory pathway in the development of obesity in males and females will be examined. Additionally, non-phosphorylatable STAT3 S727A mutant adipocytes and mice will be employed to determine the in vivo role of this phosphorylation site. These studies will provide a more complete understanding of the regulation of lipolysis-driven oxidative metabolism, and will improve our understanding of this energy expending pathway in white adipose tissue. This may lead to the development of new therapeutic approaches to curtail obesity and the devastating metabolic diseases with which obesity is associated.

NIH Research Projects · FY 2024 · 2020-12

PROJECT SUMMARY The zoonotic transmission of viral pathogens from animals to humans contributes to the majority of emerging infectious diseases, which pose a substantial and increasing threat to human health. While host innate immunity—namely the production of Type I Interferon (IFN) and the subsequent production of Interferon Stimulated Genes (ISGS) with antiviral activities—plays an important role in determining viral tropism and limiting the cross-species transmission of viruses, much work remains on identifying and characterizing ISGs limiting viral tropism. Our lab has determined that while the human immunodeficiency virus (HIV-1) and non-human primate simian immunodeficiency viruses (SIV) are able to at least partly evade IFN-mediated defenses in cells from their cognate host, they remain exquisitely sensitive to such defenses in cells from unnatural hosts. Since HIV-1 and HIV-2 both arose from the cross-species transmission of diverse SIV species, the human and primate immunodeficiency viruses are a rich model system for studying the relationship between host innate immunity and viral adaptation. In order to identify human genes inhibiting SIV infection, we have modified a recently described high-throughput, CRISPR-based screening assay to identify human ISGs with activity against SIVmac239. Our preliminary data suggests that our model system is working as designed, as had identified a number of candidate genes that inhibit SIVmac infection in human cells. In Aim 1, we will determine the mechanism of action of novel ISGs with confirmed activity against SIVmac239. We will perform stepwise mechanistic studies to dissect the point of inhibition in the viral lifecycle. We will additionally perform co- immunoprecipitation and co-localization studies to identify viral components and cellular cofactors relevant to mechanism of inhibition. In Aim 2, we will use our established pipeline to screen for ISGs inhibiting diverse SIV strains. Preliminary data strongly suggests that we will be able to directly use the system we have developed to screen diverse strains of SIV. We will assess multiple SIV strains for sensitivity to IFN and screen those that are sensitive for inhibitory ISGs. We hypothesize that there will be both conserved and distinct inhibitory factors targeting the various SIV species. Combined, these approaches will reveal novel restriction factors that inhibit diverse SIVs in human cells, providing insight into the evolutionary adaptations that HIV-1 and HIV-2 made in order to successfully colonize humans.

NIH Research Projects · FY 2025 · 2020-12

Project Summary In the cerebral cortex, gamma-aminobutyric acid (GABA)ergic interneurons are the major source of inhibition. Interneuron dysfunction is strongly associated with autism and childhood epilepsy. We demonstrated that environmental influences such as electrical activity are fundamental for the maturation of GABAergic circuits. However, the identity of the activity patterns controlling interneuron development remains poorly understood. The long-term goal of this research is to uncover how early interneuron dysfunction leads to lasting neuropathologies. The objective of this proposal is to reveal the signaling pathways underlying activity- dependent development and to assess how perturbations in this process lead to aberrant brain function. To this end, we will use the murine barrel cortex as a well-established model for the study of activity-dependent circuit maturation. We will focus our studies in cortical interneurons since our previous work indicates that these neurons are exquisitely sensitive to environmental perturbations in the neonate. In the near term, this proposal is aimed at investigating the role of specific interneuron subtypes in regulating the emergence of early activity patterns (Aim 1). In addition, this project will determine the calcium-dependent signaling pathways for the functional maturation of interneuron networks. We will study the role of Cacna1c, a gene encoding for the Cav1.2 subunit of L-type calcium channels. Mutations in this gene are strongly associated with Timothy syndrome and other neurodevelopmental disorders (Aim 2). Finally, we will assess how developmental defects in interneuron number lead to abnormal brain activity during development and impaired behavior in the adult (Aim 3). With respect to the outcomes, our work is expected to identify basic mechanisms fundamental for the emergency of a healthy balance in the number of excitatory and inhibitory neurons. In addition, these results are expected to have a significant translational impact because they will expand our mechanistic knowledge on how mutations in the CACNA1C gene, strongly associated with autism, bipolar disorder, schizophrenia and Timothy syndrome, may lead to behavioral abnormalities frequently observed in these patients.

NIH Research Projects · FY 2024 · 2020-09

Project Summary In the cerebral cortex, gamma-aminobutyric acid (GABA)ergic interneurons are the major source of inhibition. Interneuron dysfunction is strongly associated with autism and childhood epilepsy. We demonstrated that environmental influences such as electrical activity are fundamental for the maturation of GABAergic circuits. However, the identity of the activity patterns controlling interneuron development remains poorly understood. The long-term goal of this research is to uncover how early interneuron dysfunction leads to lasting neuropathologies. The objective of this proposal is to reveal the signaling pathways underlying activity- dependent development and to assess how perturbations in this process lead to aberrant brain function. To this end, we will use the murine barrel cortex as a well-established model for the study of activity-dependent circuit maturation. We will focus our studies in superficial circuits since our previous work indicates that these circuits are exquisitely sensitive to environmental perturbations in the neonate. In the near term, this proposal is aimed at investigating the role of specific interneuron subtypes in regulating the emergence of long range connectivity (Aim 1). In addition, this project will determine the role of GABAergic inputs for the functional maturation of pyramidal networks. We will study the role of Gabrb3, a gene encoding for the beta3 subunit of GABAA channel. Mutations in this gene are strongly associated with Angelman syndrome and ASD (Aim 2). Finally, we will assess how developmental defects in early GABAergic signaling lead to abnormal brain activity in cortico-cortical pathways during development (Aim 3). With respect to the outcomes, our work is expected to identify basic mechanisms fundamental for the emergency of a healthy E/I balance. In addition, these results are expected to have a significant translational impact because they will expand our mechanistic knowledge on how mutations in the GABRB3 gene may lead to behavioral abnormalities frequently observed in ASD patients.

NIH Research Projects · FY 2024 · 2020-09

SUMMARY Aging in mammals is complex, with hallmarks including reduced propensity for stem cell self renewal, deficiencies in DNA repair, reduced responses to growth stimulating hormones and nutrients, metabolic disruption and increased susceptibilities to the onset of diseases. The origins of the deficits in self renewal, self repair, and metabolic homeostasis are central questions in the aging field. Arguably, no single factor can be identified that provides a causative effect. We have focused upon deficits in NAD+ metabolism as a potentially pleiotropic effector leading to downstream dysfunctions in cellular, tissue and organism health. Processes such as senescence, which can more readily arise from genetic factors such as defects in DNA repair genes (e.g. Werner and Bloom Syndromes) present an interesting opportunity to further investigate the role of NAD+ deficiency, given that genetically altered fibroblasts are commercially accessible as potential tools in this regard. We propose to generally characterize how aging affects NAD+ metabolism in progeroid cells, and in aged mice. Specifically, we will characterize NAD+ biosynthetic potential as well as rates of NAD+ turnover. Moreover we will assess how cells respond to pharmacologic interventions that increase NAD+ biosynthesis in order to determine if these interventions mitigate age-dependent phenotypes in these fibroblast cells. These studies will provide a deeper view of how NAD+ decline occurs in cells and tissues, and if some cells and tissues are more susceptible to this decline than others and why. A second part of the application focuses on the discovery of a novel NAD+ enhancer called dihydronicotinamide riboside (NRH), which can raise NAD+ concentrations from 3- 10 fold in mammalian cells. Preliminary data shows that NRH uses a novel mechanism of action,wherein it is converted to NMNH, independent of the known kinases Nrk1 or Nrk2, leading to biosynthesis of NAD+. In mice this compound increases NAD+ concentrations many-fold over control in most tissues. This application investigates its mechanism of action in fibroblasts and in mice to elucidate a novel biosynthetic pathway to NAD+ with translational potential for treatment of disease. Thus, in the latter part of the application, we characterize NRH effects in aged mice and ascertain if it can induce mitochondrial biogenesis. We provide studies to characterize its effects using metabolomics approaches. Finally we test NRH to treat a model of metabolic syndrome and to characterize the effect of age on disease and treatment outcomes. The objectives of the grant are accomplished via 3 specific aims: 1. To characterize NAD+ homeostasis in cells and mice as a function of age. 2. To elucidate effect and metabolic pathway of NRH in fibroblasts and mice. 3. To determine the effect of NRH on aged mice in altering NAD+ metabolism, mitochondrial biogenesis and mitigation of a model of metabolic syndrome. The accomplishment of the objectives will provide new understanding of how NAD+ is a key factor in aging and whether next generation NAD+ precursors can alter phenotypes that are hallmarks of aging.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Inflammatory bowel diseases, which include both ulcerative colitis and Crohn's disease, are estimated to affect 3 million Americans, and the number of people living with IBD continues to rise. Currently available medications are costly, ineffective for some patients, and associated with serious risks including opportunistic infections, bone marrow suppression, hepatic inflammation, pancreatitis, and cancer. Thus, there is an urgent need to improve our understanding of modulators of intestinal inflammation and repair in order to identify novel therapeutic targets for the treatment of IBD. Innate lymphoid cells (ILCs) are a relatively-recently characterized family of immune cells that are enriched at barrier surfaces and modulate inflammation in response to cytokine and microbial signals. In particular, group 2 ILCs (ILC2s) sense alarmins and cytokines such as IL-25, IL-33, and TSLP, can be activated by the nervous system, and produce type 2 cytokines that promote anti-helminth immunity and allergic inflammation. Furthermore, our lab has shown that ILC2s also exert tissue-protective functions via secretion of the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG), resulting in amelioration of tissue damage following intestinal injury. In new preliminary studies presented here, we show that expression of the neuropeptide, neuromedin U (NMU), is increased during intestinal inflammation in mice, and lack of endogenous NMU results in more severe disease in a model of chemical-induced intestinal damage and inflammation. Conversely, therapeutic administration of NMU results in upregulation of ILC2- derived AREG and ameliorates chemical-induced intestinal damage. Furthermore, similar to in inflamed murine intestines, NMU expression is also elevated in IBD patient biopsies, and the receptor for NMU is detected on human colonic ILCs. Based on our new preliminary data, we hypothesize that enteric neuron-derived NMU activates the tissue-protective functions of ILC2s. We propose to generate a detailed understanding of how NMU mediates tissue protection in both murine models of intestinal inflammation and human IBD. In Aim 1, we will test the hypothesis that during intestinal injury and repair, expression, cellular sources, and spatial pattern of NMU expression are altered. We will also test the role of endogenous enteric-derived NMU in maintaining tissue homeostasis. In Aim 2, we will employ novel reporter mice to directly test the cellular and molecular mechanism by which NMU mediates tissue protection. In Aim 3, we will define the NMU-NMUR1 axis in the healthy human intestine and determine how alterations in NMU-NMUR1 signaling correlate with clinical and endoscopic measures of IBD disease activity. In addition to uncovering fundamental and novel neuropeptide biology and their unique roles in IBD, these studies will provide preclinical justification for development of novel therapeutics to target this pathway.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT Candidate. Dr. Gunisha Kaur is an anesthesiologist and medical anthropologist with a background in basic neuroscience research, two decades of fieldwork with torture survivors, and clinical expertise in pain management, an anesthesia sub-specialty. She has spent the past five years conducting research on pain after torture. She has demonstrated that evaluators using only the United Nations Istanbul Protocol (UNIP, global standard for medical assessment of torture survivors) detected and treated pain 16% of the time as compared to a pain specialist (gold standard) who identified pain in 85% of subjects. Her use of a validated pain screen accurately captured all of the pain diagnoses when employed. Dr. Kaur hypothesizes that the novel application in this population of a validated pain screen, similar to those used for posttraumatic stress disorder (PTSD) and major depression (MD), can augment the UNIP and improve its sensitivity for pain from approximately 15% to 90%, as compared to the gold standard. She further proposes an evidence-based somatic pain treatment model, adapted to feedback from torture survivors on acceptability and practicality. Career Development Plan. Dr. Kaur's career goals are to become an independent physician-scientist focused on the diagnosis and treatment of chronic pain in refugee torture survivors. For this training she will: 1. Acquire instruction in quantitative methods of pain research, 2. Gain skills in the conduct of complex, mixed-methods clinical trials, 3. Develop expertise in advanced ethics of clinical research with vulnerable populations. Dr. Kaur will develop these skills through mentorship, coursework, and implementation of her research project. Environment. The proposed research and training will take place at Weill Cornell Medicine through the Weill Cornell Center for Human Rights, one of the most established academic human rights centers in the US. Research. An estimated 87% of torture survivors (27 million people) worldwide suffer chronic somatic pain related to trauma mechanism, such as brachial plexopathy from suspension by upper extremities or lumbosacral plexus injury from leg hyperextension. However, a vast majority of this pain is likely undiagnosed by providers who use only the standard evaluation protocol of torture survivors, the United Nations Istanbul Protocol. Without evidence-based diagnostic tools and treatment guidelines for use by general providers, the complex clinical presentation of torture survivors results in somatic pain being confounded by concurrent psychiatric illness such as PTSD or MD. Rehabilitation from complex trauma is possible, but it requires considering somatic pain a substantial component of pathology. In Aim 1, a blind comparison to gold standard study with 100 torture survivors will compare the diagnosis of pain using the standard UNIP versus the UNIP with the novel application of a validated pain screen. Aim 2 will evaluate the acceptability of somatic pain treatment using qualitative interviews with 30 torture survivors. Aim 3 will assess the feasibility of recruiting and retaining subjects in somatic pain treatment, to inform the design of a subsequent clinical trial.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Twenty percent of US women with breast cancer also have diabetes mellitus (DM) and face a 50% higher risk of death up to 10 years after cancer diagnosis compared to non-diabetic women with breast cancer. A possible explanation is that DM receives less attention during cancer treatments because patients, oncologists, and primary care providers (PCPs) prioritize cancer care over DM management. Oncologists may focus on cancer care (rather than DM management), patients may not see their PCPs in the period after cancer diagnosis, and PCPs may not feel comfortable managing DM in the context of chemotherapy regimens that frequently affect glucose control. Given these concerns, identifying a provider who could effectively manage DM may be an attractive solution. Nurse practitioners (NPs) have been successfully integrated into many oncology care teams to support general cancer care. Separately, NPs have been shown to successfully manage DM in various non- cancer settings. However, no study has determined whether a NP on the oncology care team can effectively manage DM during chemotherapy for breast cancer. I hypothesize that a NP trained in DM care and embedded in the oncology team can effectively manage DM during this acute phase of breast cancer care. The objective of this study is to engage stakeholders to develop and implement a NP-led intervention to manage DM for women receiving chemotherapy for incident non-metastatic breast cancer. To accomplish this, I propose the following research aims: 1) elicit the perspectives of patients, NPs, oncologists, and PCPs about barriers to and facilitators for a NP managing DM during chemotherapy, 2) develop a NP-led intervention to manage DM during chemotherapy, and 3) conduct a pilot study to implement the intervention and assess implementation outcomes (reach, acceptability, appropriateness, feasibility, fidelity). Findings from this pilot study will lay the groundwork for a multi-site, randomized trial testing the effectiveness of this NP-led model. As a PhD-trained health services researcher focused on cancer outcomes and health disparities, I have gained quantitative research expertise. I now seek to expand my research skills to include implementation trials. To accomplish this goal, I will pursue mentorship and training in qualitative research methods, healthcare delivery, and in stakeholder-engaged intervention design, implementation, and evaluation in real-world settings. I will be mentored by two nationally-recognized clinician scientists, Dr. Monika Safford and Dr. Lisa Kern. Together, we designed a career development plan for me to gain skills through coursework and reading that is then solidified through experiential learning by carrying out my research aims. This Award will enable me to advance toward my long-term goal of becoming an independent health services researcher working at the intersection of cancer care, primary care, and health equity to improve patient outcomes and reduce health disparities.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY / ABSTRACT Clonal blood differentiation through the acquisition of somatic mutations result in abnormal accumulation of blood components and clinically manifest as myeloid disorders. The study of how these somatic mutations perturb the differentiation trajectories in human hematopoiesis is often challenged by the admixture of normal hematopoietic cells with the neoplastic cells that cannot be distinguished by cell surface markers. To overcome this limitation, we developed a novel single-cell multi-omics Genotyping of Transcriptomes (GoT) platform that directly links somatic genotypes with transcriptomes of thousands of single cells. Thus, GoT enabled the comparison of mutant and wildtype cells within the same sample in the context of progenitor identities, thereby turning the co-mingling of mutant and wildtype hematopoiesis from a limitation to an advantage. As proof of principle, GoT was applied to CD34+ progenitor cells from patients with calreticulin- mutated myeloproliferative neoplasms (MPN), revealing key pathways that were aberrantly activated in the mutant cells, such as a robust unfolded protein response in the megakaryocytic progenitors, on the one way, and NF-KB pathway in stem cell-enriched populations, on the other. Overall, GoT revealed that the transcriptional impact of calreticulin mutations is highly variable as a function of progenitor identity – which bears significant implications for therapy by enabling the discovery of targetable pathways specific to the earliest stem cells. Thus, to demonstrate the cell identity-dependency across other key driver mutations, as a fundamental concept in myeloid disorders, I will apply GoT to thrombopoietin receptor-mutated progenitor cells and to clonally-diverse cells from MPN samples (Aim 1). Next, in order to define cell extrinsic determinants of somatic mutation impact, I will determine the immune niche interactions with calreticulin-mutant and wildtype progenitor cells, as well as the impact of immunomodulatory therapy on these interactions (Aim 2). Finally, I will test the hypothesis that the cell’s epigenome precedes the cell identity-dependency of somatic mutation effects, by developing and applying a novel single-cell platform that integrates somatic genotyping with chromatin accessibility states of progenitor cells (Aim 3). Thus, I will define the genetic, epigenetic, transcriptional and environmental factors that culminate in the clinical output of somatic mutations in human hematopoiesis. These studies will, therefore, unveil not only fundamental concepts in clonal hematopoietic differentiation but also specific targets for therapeutic intervention.