University Of California, San Francisco

NIH Research Projects · FY 2025 · 2023-01

SUMMARY/ABSTRACT: The use of autologous gene modified cells that are resistant to HIV infection to reduce viral reservoir size and delay or prevent HIV recrudescence is on the forefront of HIV curative science. However, many current gene modification approaches focus solely on reducing CCR5 expression on hematopoietic stem cells or mature CD4 T cells. These approaches may have limited impact in people with HIV (PWH) that have virus able to use other coreceptors for entry, and only provide a single layer of protection against infection in vivo. As a result, the AMC #097 study, “A Phase I Study of Stem Cell Gene Therapy for HIV Mediated by Lentivector Transduced, Pre-Selected CD34+ Cells: A Trial of the AIDS Malignancy Consortium,” was implemented to combine multiple anti-HIV genes into a single lentiviral vector that is designed to block HIV-1 infection at different stages of the HIV-1 life cycle providing strong pre-integration inhibition of HIV-1 infection using a lentiviral vector that including a CCR5 shRNA, a chimeric human-macaqueTRIM5α restriction factor, and a HIV TAR decoy. This strategy has been shown to prevent infection in vitro and in vivo, and 12 PWH requiring autologous SCT for lymphoma have already received gene modified stem cells through AMC097. One participant stopped ART outside study protocol following SCT and experienced post-treatment control. Whereas the primary endpoints of the study are to evaluate the safety of such an approach, support is urgently needed to perform in- depth HIV reservoir analyses and to implement assays to determine if gene modified cells become infected following transplantation with or without analytical treatment interruption. We will: (1) test the hypothesis that autologous SCT with gene modified stem cells simultaneously targeting different stages of the HIV-1 replication cycle will lead to blood and gut tissue expansion and maintenance of a transduced CD4+ T and other immune cells resistant to HIV-1 infection; (2) test the hypothesis that SCT with gene modified stem cells will reduce the size of the HIV-1 reservoir and residual HIV-1 transcriptional activity, and lead to post-treatment HIV control following withdrawal of ART, and (3) determine if gene modified cells become infected prior to and following cessation of ART a novel duplexed single-cell-droplet (scdPCR) assay with the ability to simultaneously detect cellular HIV-1 DNA or RNA and the integrated lentiviral vector or chimeric TRIM5α transcriptional activity.

NIH Research Projects · FY 2026 · 2023-01

Project summary: Uveal melanoma (UM) accounts for approximately 5% of all melanomas and is the most lethal type of melanoma. 50% of UM patients develop metastasis, mostly to the liver, an invariably lethal complication, which currently cannot be effectively treated. Despite dramatic successes in other melanoma subtypes, immune checkpoint blockade, and targeted therapies have been largely ineffective in metastatic UM and there is an urgent need to identify effective therapies. UM lacks mutations in BRAF, NRAS, NF1 and KIT common in cutaneous melanomas and is genetically defined by mutations in the Gaq signaling pathway. We discovered that RasGRP3, a Ras-guanyl nucleotide exchange factor (RasGEF), is dramatically overexpressed in UM compared to other melanoma subtypes and cancers and links the constitutively activated Gaq pathway to the MAP- kinase pathway. RasGRP3 is also directly activated by oncogenic Gaq signaling via mechanisms that partially depend on PKC. We hypothesize that RasGRP3 is a therapeutic target in UM and seek to understand the mechanism behind its marked upregulation to identify alternative targets for therapy. Our preliminary data implicate protein kinase D (PKD) downstream of PKC to be directly involved in RasGRP3 regulation. Our data also demonstrate that PKD is involved in the adaptive resistance that undermines the efficacy of MEK inhibitors. As a druggable kinase, PKD thus is a possible therapeutic target in UM. In this proposal, we will evaluate both RasGRP3 and PKD as therapeutic targets using newly developed genetically engineered and xenograft models of UM metastatic to the liver and investigate the underlying mechanism of RasGRP3 upregulation in UM (Aim 1 and 2). In Aim 3, we will dissect the mechanism underlying the adaptive resistance to MEK inhibition, which represents a key bottleneck limiting the therapeutic efficacy of MEK inhibition, to identify rational therapy combinations that overcome this resistance and improve the therapeutic efficacy of MEK inhibition in the setting of metastatic UM.

NIH Research Projects · FY 2026 · 2022-12

PROJECT SUMMARY: Intestinalization of the esophagus, termed Barrett’s esophagus (BE), is thought to develop in response to chronic acid and bile reflux and carries great clinical significance because it is the precursor to esophageal adenocarcinoma (EAC). The incidence of BE is quite high, estimated to be found in at least 1:100 people. While relatively few with BE progress to cancer there is great importance to being able to detect and treat those at risk of progression as EAC is an aggressive cancer with potential for early spread. Efforts to screen for high-risk disease in those with BE have, to date, not been very successful. Therefore, there is profound need to define the process by which BE progresses into EAC, to develop biomarkers to diagnose early progression and assess progression risk in BE tissues as well as to develop novel therapies for treatment. The objective of this R01 proposal is to investigate the ability of biomarkers to identify BE patients at high risk of progression. Using results from two externally funded genomic studies in non-dysplastic BE (NDBE) and BE with low-grade dysplasia (LGD) and previously published results, we will compare biomarkers and determine an optimized combination for risk stratification in two prospective cohorts including patients with NDBE or LGD. We will then validate our risk stratification assay in an independent US cohort. Finally, we will compare our genomic biomarker panel results in paired biopsy and brush samples. A unique collaboration between the PI Dr. Stachler, expert gastroenterologist (Dr. J Bergman), a talented computational biologist (Dr. CZ Zhang), and an expert biostatistician (Dr. K Zwinderman) along with key collaborators allows a truly innovated study to be performed. This will be accomplished using an unprecedented collection of clinically derived samples, a highly optimized targeted sequencing panel, and novel computational approaches that allow a wide array of information to be determined in a cost effective, clinically relevant manor. Aim 1: Identify a set of genomic biomarkers highly predictive of progression in biopsies from a prospective cohort of patients diagnosed with NDBE and LGD and assess whether the addition of methylation-based biomarkers improves stratification. For clinical implementation all biomarkers should be compared head to head in order to determine an optimized combination of biomarkers for risk stratification. Aim 2: Validate the risk stratification assay in a multi-institutional cohort of patients with a baseline diagnosis of NDBE or LGD. For clinical implementation, it is vital to validate any risk stratification assay on completely independent cohorts looking at clinically relevant time points. Aim 3: Determine if brush-based sampling devices improve biomarker detection over standard endoscopic biopsies. Broad sampling of the BE epithelium may allow for increased rates of detection for genomic or methylation biomarkers, therefore we will determine if samples from a brushed based device can better risk stratify patients compared to standard biopsies.

NIH Research Projects · FY 2026 · 2022-12

The quantal nature of synaptic transmission depends on the transport of neurotransmitter into synaptic vesicles (SVs), an activity driven by a H+ electrochemical gradient (∆µH+). In contrast to relatively stable ionic gradients across the plasma membrane, ∆µH+ and other ions including Cl- fluctuate with the exo- and endocytosis of SVs. Vesicle filling requires coordination with these changing conditions and hence regulation of transport. In contrast to the SV uptake of most transmitters that relies primarily on the chemical component of ∆µH+ (∆pH), uptake of the principal excitatory transmitter glutamate depends predominantly on membrane potential. The vesicular glutamate transporters (VGLUTs) also exhibit unusual properties, including allosteric regulation by lumenal H+, cytosolic and lumenal Cl- and an associated Cl- conductance. We hypothesize that these mechanisms coordinate glutamate flux with different steps in the exo- and endocytic recycling of synaptic vesicles. The long-term objective of this proposal is to understand how these properties of the VGLUTs contribute to excitatory neurotransmission. The strategy is to determine how these mechanisms regulate VGLUT activity, and use this information to characterize their physiological role. This program takes advantage of our previous work identifying these regulatory mechanisms, assays we developed to study them, recent structural information and VGLUT knockout neurons that we can use to test rescue by mutants. Aim 1: Elucidate the mechanism and physiological role of pH in vesicular glutamate transport. The requirement for allosteric activation of the VGLUTs by lumenal H+ suggests a mechanism to prevent tonic efflux of glutamate across the plasma membrane that would degrade the quantal signal. We recently identified a single residue that confers the pH requirement of vesicular glutamate transport. We will now use this information to determine how pH regulates glutamate transport and how this regulation influences excitatory transmission. Aim 2: Determine how Cl- allosterically regulates vesicular glutamate transport. We recently found that an extensive cytoplasmic interaction network influences the allosteric regulation by lumenal pH on the other side of the SV membrane, suggesting that the alternating access involved in glutamate transport depends on the balance in strength between cytoplasmic and lumenal gates. We hypothesize that Cl- also affects the two gates, either directly or indirectly. We will thus determine how the cytoplasmic interaction network and lumenal residues contribute to allosteric regulation of glutamate flux by cytoplasmic and lumenal Cl-. Aim 3: Aim 3: Determine how lumenal Cl- affects glutamate storage and release. Removal of extracellular Cl- prevents recovery from the synaptic depression that normally follows strong stimulation. To determine whether this reflects a requirement for the efflux of lumenal Cl- mediated by a VGLUT-associated conductance, we will rescue VGLUT1/2 double knockouts with mutants lacking the conductance, and monitor the effects on glutamate release and SV pH.

NIH Research Projects · FY 2026 · 2022-12

Project Summary A normal and robust immune system relies on the development of cells with diverse repertoire of antigen recognition but that remain tolerant of self-tissues. Breakdown of this immune tolerance can give rise to autoimmune disease, and hence multiple mechanisms are in place to ensure immune self-tolerance. The thymus is a critical site for the development and education of T cells to promote tolerance to self through expression of tissue specific antigens (TSAs) by specialized medullary thymic epithelial cells (mTECs). The transcriptional regulators Aire and Fezf2 are known to act within mTECs to promote the expression of thousands of TSA self- antigens for the purpose of removing developing self-reactive T cells in a process known as negative selection. Aire and Fezf2 are shown to be required for some, but not all TSA genes expressed in mTECs, suggesting additional transcriptional regulators are required for the full repertoire of the observed TSA expression in mTECs. Furthermore, recent work has highlighted the complexity of mTECs with at least four mTEC populations: Aire+ mTECs, Ccl21a mTECs, Late/Post Aire mTECs, and tuft cells. However, there is limited knowledge about the developmental regulation and progenitors of these heterogeneous mTEC subsets. We have identified the transcription factor Ikaros (Ikzf1) as a novel regulator of mTEC composition and function, with deletion of Ikaros in mTECs causing a reduction of Aire+ mTECs and an expansion of tuft cells. Moreover, Ikaros-deficient mTECs have a defect in TSA gene expression resulting in specific signs of autoimmunity. Interestingly, mutations in IKZF1 have been linked to human autoimmune diseases. Thus, we hypothesize a novel role for Ikaros in mTEC lineage development, TSA expression and central tolerance, and that defects in mTEC function could contribute to the autoimmunity seen in humans with IKZF1 mutations. We propose to test our hypothesis and increase our knowledge of this novel mTEC transcriptional regulator through the following specific aims: (1) Identify the stage(s) of mTEC development at which Ikaros modulates mTEC function, (2) Interrogate the molecular mechanism of Ikaros function in mTECs, and (3) Determine if TEC specific deletion of Ikaros affects T cell tolerance and autoimmunity. To complete these aims, we have developed a unique and powerful set of genetic tools and mouse reporter lines that allow us to perturb Ikaros function in mTECs. We will use flow cytometry to investigate mTEC and thymic immune cell phenotypes, combined scRNA-seq/scATAC-seq to analyze gene expression and chromatin accessibility, and supplement with mechanistic studies in vitro in cell lines. Our long-term goal is to increase our understanding of the regulation of mTEC development and function in central tolerance, and how alterations can lead to a break of tolerance and autoimmunity.

NIH Research Projects · FY 2026 · 2022-12

ABSTRACT The type and pattern of immune cell infiltrate in breast cancer is of growing clinical importance as they associate with response to therapy and are the specific target of immunotherapy. `Cold' cancers that lack infiltrating T cells exhibit pronounced transforming growth factor β (TGFβ) activity and predict poor outcomes in breast cancer patients. However, the factors that influence the genesis of the type of tumor immune microenvironment (TiME) have yet to be defined. We found that radiation-preceded breast cancers in women treated with radiation therapy for Hodgkin's lymphoma are significantly enriched for TiME devoid of lymphocytes and rich in myeloid cells, TGFβ and cyclooxygenase 2. We used a Trp53 null mammary chimera model to determine the factors underpinning of this unexpected difference. Tumors with an immunosuppressive TiME lacking lymphocytes arose only in irradiated mice, even when the transplant was not irradiated, indicating host biology was key, as well as in mice lacking functional adaptive immunity, pointing to a role for innate immunity. Strikingly, transient aspirin treatment before cancer developed blocked the development of cold tumors. We hypothesize that systemic inflammation provokes the development of tumors with immunosuppressive, cold TiME. Chronic low- level inflammation from aging, obesity, stress and chronic syndromes following viral infection is common. Here we will test the specific hypothesis that inflammation-induced TGFβ during carcinogenesis alters tissue-resident myeloid cells to promote the genesis of cancers with an immunosuppressive TiME. AIM 1 will use state-of-the- art analysis of cytokines and immune characteristics that correlate with the development of tumors with cold TiME using a novel biobank of blood, plasma, bone marrow, spleen, and nonmalignant mammary glands and their associated cancers as a function of inflammation or anti-inflammatory aspirin conditions at 4-, 8- and 18- months post-treatment. The relevance of these findings will be tested by immunoprofiling women with breast cancer. AIM 2 will use parabiosis to test whether factors circulating during systemic inflammation contribute and use macrophage depletion and a mouse in which myeloid cells cannot signal through TGFβ to test whether circulating TGFβ elicits monocyte activation to promote the development of cold TiME. AIM 3 will analyze the resulting high-content data using deep learning and bioinformatics methods to identify tumor subtypes and to infer key events. The main goal of our study is to test the innovative hypothesis that inflammation-induced TGFβ promotes cold tumors by altering tissue-resident myeloid cells during carcinogenesis. Our proposal to conduct systematic, high content analysis and modeling of the mechanisms by which breast cancers develop with an immunosuppressive TiME is highly significant in view of the growing clinical importance of the TiME.

NIH Research Projects · FY 2024 · 2022-10

PROJECT SUMMARY/ABSTRACT It has long been known that physiological stress during gestation can lead to the alteration of stress and metabolic responses over multiple generations. Such information must be transmitted through the germline; however, the mechanistic effects of physiological stress on the germline and, more broadly, whether stress- related changes occur at the level of the germ cells themselves remain unexplored. The maternal hypothalamic- pituitary-adrenal axis is activated upon gestational stress and its downstream effectors are glucocorticoids, a class of steroid hormones, which are released into the bloodstream then bind to the ubiquitously expressed Glucocorticoid Receptor (GR). While stress-induced levels of glucocorticoids are known to impair oocyte competence in adult women, their impact on the fetal oocyte remains largely unexplored. Interestingly, GR has been shown to downregulate the activity of Heat Shock Factor 1 (HSF1). While HSF1 is well characterized for its role in proteostasis, it is also known to regulate chromosome architecture in the oocyte during meiosis. This proposal will investigate how oocyte intrinsic HSF1 activity and maternal gestational stress influence fetal oocyte development. By using genetic mouse models, in vivo models of increased GR activity, whole ovary clearing, three-dimensional imaging, and quantitative analysis, this proposal will (1) establish a comprehensive, three-dimensional spatiotemporal map of HSF1 expression, localization, and activity in the fetal ovary and test whether HSF1 plays a germ cell-intrinsic role in embryonic meiosis and (2) identify the relationship between GR and Hsf1 in the fetal oocyte and the consequences for both embryonic meiosis and oocyte growth and maturation. Improving our limited understanding of the long-term consequences of global increased GR activity on the developing germline is crucial as the potent pharmacological agonist of GR, dexamethasone, is routinely administered during pregnancy to stimulate lung maturation when premature birth is a concern. Additionally, elucidating the mechanistic effects of physiological stress in the oocyte will support our understanding of the consequences for vulnerable populations who have a higher risk of exposure to stress- inducing environmental and socioeconomic conditions. To successfully complete the work outlined in this F31 proposal and to achieve my career goals, I have chosen to perform the proposed work in the laboratory of my Sponsor, Dr. Diana Laird, at the University of California, San Francisco. The complementary expertise of my Co-Sponsor, Dr. Marco Conti, and Collaborator, Dr. Aditi Bhargava, as well as the support provided by this F31 Fellowship assure I will receive the technical training and mentorship to complete my pre-doctoral research and contribute to the field of reproductive biology.

NIH Research Projects · FY 2025 · 2022-09

Abstract Refractive errors are a major cause of vision loss worldwide, and the rising prevalence of myopia and associated blinding conditions is a significant public health concern. Regulation of ocular axial growth is critical for normal refractive development to ensure that a focused image falls directly on the retina. Our goal is to decode the molecular and genetic program that governs ocular axial growth. Ocular growth is driven by an intrinsic, genetic process during prenatal and postnatal development (vision-unadjusted) and by a postnatal, vision-guided process, emmetropization, thought to interact with intrinsic ocular growth such that the eye's axial length matches its optical power. Enhanced intrinsic ocular growth and defective emmetropization are thought to cause a mismatch between ocular axial length and optical power, leading to myopia. Ocular axial growth relies on signals from the retina to the sclera to promote extracellular matrix remodeling and ocular elongation. However, the mechanisms by which the signals translate to ocular axial growth remain elusive. Our studies suggest that PRSS56, a secreted serine protease, is a component of the intrinsic machinery that supports ocular axial growth. However, it is not known whether Prss56 has a direct role in emmetropization. We propose to uncover the molecular and cellular processes underlying PRSS56-dependent refractive development and associated errors and assess the role of PRSS56 in vision-guided ocular growth. Despite evidence that altered expression of PRSS56 affects ocular axial length, the factors that regulate its expression and mediate its effect are not known. The Wnt-mediated pathway is associated with myopia pathogenesis, and we have found that Prss56 responds to Wnt signaling agonists. In Aim 1, we will elucidate the link between Wnt and Prss56 by modulating WNT activity in genetic mouse models and studying the effect on the retinal expression of PRSS56 and ocular growth (Aim 1.1). We will also determine, in conditional mouse models, whether retinal pigment epithelium–localized Serpine3—which we identified as a candidate mediator of PRSS56-dependent growth—helps relay PRSS56-dependent signals that support ocular growth (Aim 1.2). In Aim 2, we will characterize the function of PRSS56 to guide the identification of its substrate(s) and targeted therapies. In Aim 3, we will test the role of PRSS56 in emmetropization and PRSS56-dependent regulation of ocular axial growth by temporarily inactivating PRSS56 in conditional mutant mice and using experimental paradigms that induce axial elongation in response to visual blur or optical defocus. The proposed studies will provide a molecular and genetic framework to understand the mechanisms of ocular growth and guide us to potential therapeutic targets to manage myopia.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY We propose to extend the California Lupus Epidemiology Study (CLUES), which has established a racially and ethnically diverse cohort of over 450 individuals with systemic lupus erythematosus (SLE). The CLUES cohort was launched from the successful California Lupus Surveillance Project, which established the incidence and prevalence of SLE in San Francisco County. Individuals identified through the surveillance effort were invited to participate in the longitudinal CLUES cohort. The study currently includes i) extensive clinical data, including physician-assessed measures of SLE disease activity, medical history, SLE manifestations, and outcomes such as damage; ii) biologic specimens and data, including genetic, epigenetic, gene expression and environmental exposure information; and iii) data from structured interviews with participants covering sociodemographics, healthcare access and gaps, symptoms, disability, and a wide variety of patient-reported outcomes. This exceptionally broad and deep data collection has catalyzed a wide spectrum of SLE research studies, ranging from the examination of clinical and patient-reported outcomes in SLE to studies of DNA methylation and gene transcription across racially and ethnically diverse populations. Through data collection, analyses and dissemination, the overarching aim of the CLUES cohort is to advance our understanding of the epidemiology, biology, natural history, and outcomes of SLE, particularly among diverse racial, ethnic and socioeconomic groups. In the renewal period, our aims are 1) to continue longitudinal data collection on CLUES participants, including comprehensive patient-reported data, 2) to enhance and maintain a state-of-the-art biospecimen repository and provide access to this valuable resource to investigators, and 3) to conduct two special projects, one examining the metabolome during and after flares to gain insight into mechanisms of SLE disease activity, and one examining the natural history of disability across the activity spectrum, including how flares may affect disability trajectories. The overall project leverages outstanding institutional resources and builds on the proven track-record of the investigators in building a successful administrative and management infrastructure for CLUES, developing and maintaining longitudinal cohort studies, and creating effective systems for sharing clinical data and biospecimens. CLUES is unique because there are very few population-based studies of Asians or Hispanic-Americans with SLE, two groups that are disproportionately affected by the disease and who comprise a significant and growing proportion of the U.S. population. In addition, we continue to address disease flares, an area that is ripe for research and central to advancing knowledge about the natural history, outcomes, biologic mechanisms of SLE and related health disparities.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ ABSTRACT The goal of the proposed NIAAA K23 Mentored Patient-Oriented Research Career Development Award is to support Dr. Brittany Bryant in gaining the knowledge, skills, and expertise required for the successful transition from clinical practice to building a career as an independent clinical researcher focused on improving treatment options for Black justice-involved youth (JIY). Black JIY are overrepresented in the criminal justice system due to institutional racism and discrimination. The General Strain Theory posits that high levels of ethnic and racial discrimination (ERD), paired with elevated levels of witnessing or experiencing police brutality, places Black JIY at unique risk of increased alcohol and other drug use (AOD) as a means of coping with stressful events. Black JIY report engaging in comorbid alcohol and cannabis use to achieve temporary respite from discrimination- induced stress. While JIY engage in higher rates of comorbid alcohol and cannabis use compared to non-justice- involved youth, overall, Black JIY are less likely to be diverted to substance use treatment programs, less likely to engage in care after being released on probation, less likely to benefit from substance use treatment in terms of reduced risk of recidivism, and are more adversely affected by early onset of AOD activity than White JIY. To date, no known adolescent substance use treatment directly addresses ERD, the increased risk of AOD due to ERD, or explicitly presents tools on how to appropriately respond to AOD and discrimination distress among Black JIY. To address this problem, Dr. Bryant’s research program aspires to improve the understanding of 1) the impact of ERD on adolescent AOD within the Black JIY population; 2) culturally adapt the evidence-based Cannabis Youth Treatment Series (CYT) using the ADAPT-ITT model to address ERD, reduce AOD, and reduce recidivism with input from Black JIY, parents, and community members; and 3) pilot the culturally adapted intervention to assess feasibility, acceptability, and changes in substance use and discrimination distress. For the duration of the award, Dr. Bryant will 1) increase her record of scholarly publications; 2) expand knowledge and skills in mixed methods research and applied research methods; 3) develop expertise in effectively conducting community-based research; and 4) obtain training in effectively culturally adapting evidence based interventions. The training and mentorship afforded to Dr. Bryant will enhance her career development and long- term goals of becoming an independent community-based clinical researcher focused on improving treatment options, policies, and procedures to decriminalize substance use and prioritize diverting Black JIY to substance use treatment. Moreover, the proposed project aligns with NIAAA’s strategic plan to 1) prioritize the development of culturally appropriate interventions to prevent or reduce alcohol misuse and related consequences, 2) improve the implementation, accessibility, and use of alcohol treatment tailored to the needs of individuals, and 3) raise awareness about the effects of alcohol on health and well-being, while supporting an underrepresented woman of color trained in AOD research.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Unintended pregnancies put women at risk of poor maternal and child health outcomes. In India, half of all pregnancies are unintended and younger women have the highest risk of having an unmet need for contraception. New evidence suggests that young, newly married women in this setting desire to postpone pregnancy, yet have low use of contraception and high rates of unintended pregnancy, and desire reproductive health knowledge. Young women in India also have low empowerment, and desire training in life skills that could be used to help them gain status and opportunity in their households and communities. Few rigorously designed and tested interventions have focused on women’s empowerment (including life skills and health- related) and unintended pregnancy, especially among young, married women in India. This proposal tests the impact of a combined life skills and health empowerment intervention for young married women on avoiding unintended pregnancy, and other health and empowerment outcomes. The intervention, DAMINI, is currently on-going in villages in Uttar Pradesh, India, and the goal of this proposal is to conduct a two-arm randomized cluster study of (1) DAMINI (life skills and health empowerment); and (2) the standard of care health education and access to contraceptives provided by community health workers. This approach will focus on recently married women aged 18-25 years who are at risk of pregnancy but do not want a pregnancy at the time of enrollment and will follow them for two years post-intervention through in-person surveys. DAMINI is delivered in groups and we will work in 40 intervention and 40 control village clusters; with 1 group per cluster and 6-7 women in each cluster/group (total N=520). We will also collect longitudinal triadic in-depth interviews with newly married women, their husbands, and mothers-in-law to understand changing norms and pathways of impact. The first aim is to determine the effectiveness of DAMINI on the risk of having an unintended pregnancy, compared to health education alone. Survival analysis of time to pregnancy (primary outcome) will be used to analyze the impact on unintended pregnancy. Given potentially different factors affecting parous vs. nulliparous women, we will model the effect of DAMINI on them separately, and include parity specific measures such as sex of previous births and duration since marriage.The second aim is to assess the effectiveness of DAMINI on intermediary outcomes related to life skills and health empowerment, compared to health education alone, using the same methodological approach. Finally, to inform future implementation research, we will use an effectiveness-implementation hybrid design to conduct a mixed methods process evaluation with participants and their households and DAMINI and community leaders. This will also include an economic evaluation, specifically a cost effectiveness analysis. If proven effective, it is anticipated that this intervention could be integrated into existing self help groups and scaled up in India (and other parts of South Asia) to improve women and children’s health, long-term economic and empowerment related outcomes.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY Research into effective strategies to reduce unintended pregnancy and short interval births, and to mitigate associated adverse maternal and neonatal health outcomes, has been hampered by poor conceptualization and measurement of pregnancy intentions. Until recently, no validated psychometric measures had been available to prospectively capture the range and complexity of feelings many women have about pregnancy, including ambivalence and uncertainty. Reliance on simplistic measures and retrospective data have left serious scientific gaps in our understanding of the causes, and health consequences, of unintended pregnancy and short interpregnancy intervals (IPIs). Scholars have expressed an urgent need for improved measures and longitudinal study designs, citing their importance to rigorous investigations of the role of pregnancy intentions on the causal pathway to unintended pregnancy, short IPIs, and adverse health outcomes. This proposal addresses these critical scientific and measurement gaps using longitudinal data from population-based surveys, fielded to over 12,000 women, aged 18-44, across nine US states. These surveys include the Desire to Avoid Pregnancy (DAP) scale, a novel instrument developed by our team using cutting-edge, rigorous psychometric methods. The instrument reconceptualizes “intentions” as “preferences” and captures the diversity of considerations and feelings women have about a potential future pregnancy. Using three years of data, we will examine the predictive relationships between DAP scores, contraceptive use, incident pregnancy, and birth outcomes. We will use structural equation models to investigate the extent to which DAP scores mediate relationships between the personal and social contexts of women’s lives and pregnancy and birth outcomes, addressing confounding that has hindered prior investigations. Critically, analyses will for the first time apply prospective pregnancy preference data to investigate whether the elevated risk of negative maternal and birth outcomes from short IPIs results from physiologic mechanisms, or the contexts in which these frequently unintended pregnancies occur. We will then capitalize on the DAP’s strengths to develop novel measurement tools that expand its utility. First, we will use item response theory (IRT) to develop a shorter DAP version that maintains construct validity, facilitating the integration of rigorous, person-centered measures into future research, surveillance, and clinical care. Then, we will use IRT to establish evidence-based thresholds on the scale, defining standardized groupings of pre-pregnancy preferences (e.g., “undesired” or “less preferred”), and addressing the urgent need for alternative measures to “unintended pregnancy” that capture if women attain their reproductive preferences. A better understanding of how pregnancy preferences are formed, and how unintended pregnancy contributes to adverse maternal and neonatal outcomes – as well as the contributions of our novel instruments to future science – can guide the field toward interventions that can more successfully promote reproductive autonomy and improve maternal and child health.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Many factors contribute to diagnostic errors, but key among them are foundational issues in healthcare: complex and fragmented care systems, the limited time available to providers trying to ascertain a firm diagnosis, and the work systems and cultures that support or impede improvements in diagnostic performance. While approaches to identifying diagnostic errors exist, few studies have linked identification of underlying systemic and structural causes of errors to existing quality improvement programs in hospitals. Even fewer have applied resilience theories or positive deviance approaches to characterize the features of cases where the diagnostic process is optimal and then use those findings to frame health system improvement. This application builds directly on our currently funded study - Utility of Predictive Systems in Diagnostic Errors (UPSIDE) - which is defining risk factors, underlying causes, and prevalence of diagnostic errors among patients admitted to hospitals participating in our 55-hospital research collaborative, the Hospital Medicine Reengineering Network (HOMERuN). UPSIDE has developed reference standard approaches to adjudication of diagnostic errors, defined factors associated with errors, and created collaborations with our sites and national organizations, providing a uniquely powerful opportunity to transform how diagnostic process evaluation programs can be used to improve patient safety. The overall goal of this Center is to turn our highly successful multicenter network into a diagnostic error learning health system that will integrate diagnostic error assessments into existing quality and safety programs, provide support and expertise needed to reduce diagnostic errors, and catalyze scientific, personnel, and infrastructure changes which will last beyond the duration of this grant. To achieve our overall goals, we will: 1) Implement a case review infrastructure which can accurately identify diagnostic errors and characterize diagnostic processes among patients suffering inpatient deaths, ICU transfers, or rapid-response team calls taking place at hospitals associated the Hospital Medicine Reengineering Network; 2) To develop site-level audit and feedback and group-wide benchmarking reports of error rates, diagnostic process faults, diagnostic process resilience features and use these data to frame collaboration between existing safety and quality programs at our sites; 3) To use our data and collaborative model to develop and pilot test interventions based on highest priority findings; and 4) Develop understanding of our program’s reach, adoption, implementation, and maintenance, as well feasibility and initial experience with pilot interventions. This project will establish a learning health system which can achieve excellence in diagnosis as an ongoing part of care, a system which can be a model for others as well.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY ABSTRACT: This project uses cutting-edge legal epidemiology techniques to examine population-level maternal and child health (MCH) impacts of pregnancy termination policies in the U.S., including identifying which groups experience adverse consequences and the conditions that buffer consequences. Pregnancy termination in the U.S. is common, with about one out of five pregnancies ending in a termination. Historically, most research about public health impacts of pregnancy termination policies focused on the relationship between legal status of pregnancy termination and maternal morbidity and mortality related to illegal versus legal pregnancy termination. In the U.S. today, however, most pregnancy termination policies do not make all pregnancy terminations illegal; rather, they create barriers to women being able to terminate their pregnancy. Most states have at least one such policy and some states have had some pregnancy termination policies for more than 40 years. Recently, though, the number, strength, types, and co-occurrence of such policies have dramatically changed; some of these policies and policy combinations have contributed to a decrease in the number of pregnancy terminations. The next few years could bring even more drastic changes in these policies. Such policy changes could have public health impacts on women who continue their pregnancies and give birth – particularly on MCH outcomes such as maternal morbidity and mortality, infant mortality, and adverse birth outcomes. Methodologically rigorous research about impacts of being unable to obtain a pregnancy termination on subsequent MCH has been conducted at the individual-level. Yet, only very limited research, much of it with notable methodological limitations, has been conducted at the population-level. This limits our understanding of population-level impacts of these policies. In this project, we assess whether state-level pregnancy termination policies from 2005-present affect MCH outcomes. This study involves state-of-the art coding of our exposure, including policy adoption and effective dates. Outcomes include births and changes in composition of births, as well as MCH outcomes (maternal morbidity and mortality, preterm birth, low birthweight, and infant mortality). Outcome data will come from insurance claims data, vital statistics data, and the Pregnancy Risk Assessment Monitoring System. We will use both epidemiologic and econometric data analysis approaches to allow for causal interpretation of findings. Findings will provide key evidence to prepare public health and health care systems to care for groups of women and children most affected by pregnancy termination policies, as well as identify other state-level policies and characteristics that might help buffer any adverse MCH impacts of pregnancy termination policies.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Research linking unintended pregnancy with increased risk of adverse maternal and child health outcomes is fraught with significant conceptual and scientific limitations, including cross-sectional designs, residual confounding, and simplistic, retrospective assessment of pregnancy intentions. A critical gap in our scientific knowledge thus remains: Are the adverse maternal health outcomes associated with unintended pregnancy and childbearing due to unintended pregnancy itself or rather contextual factors that are associated with increased risk of unintended pregnancy? The proposed research is the first to apply state-of-the-art methods and theory to the measurement of pregnancy intentions to rigorously address this long-standing scientific gap. This prospective study follows a cohort of 2,200 non-pregnant women over one year, measuring preferences about a possible pregnancy using a new, robust instrument: the Desire to Avoid Pregnancy (DAP) scale. This validated psychometric scale captures a continuum of cognitive, affective, and practical considerations about pregnancy and childbearing, moving beyond conceptually limited “intending” and “not intending” labels. Participants experiencing incident pregnancies – and a matched subset of non-pregnant women with similar pregnancy preferences – are then followed for an additional three years to measure mental and physical health outcomes during pregnancy and after birth. This innovative design positions us to address three aims. In Aim 1, we will identify the time-varying and invariant contextual factors in women’s lives that shape their preconception pregnancy preferences, such as relationship factors and financial stability, and test how preferences predict incident pregnancy. In Aim 2, among women who experience a new pregnancy, we will examine the relationship between prospectively assessed pregnancy preferences and maternal health outcomes, accounting for temporally important confounding factors (from Aim 1). In Aim 3, we will investigate how the relationship between pregnancy and adverse health outcomes differs by pregnancy preferences. Specifically, with our matched design, we will compare health outcomes among pregnant women to those of women who best represent their counterfactual: women who had similar pregnancy preferences yet who did not experience pregnancy. Thereby, we emulate a hypothetic trial in which women, conditional on confounders, are randomly assigned to pregnancy, allowing us to examine whether the negative health effects of pregnancy are amplified among those who most desire to avoid pregnancy. Achieving these aims will contribute new insights into the health repercussions of unintended pregnancy for women and elucidate the degree to which adverse outcomes stem from the intention status of pregnancies or underlying social and contextual factors. These insights are critical to developing appropriately focused maternal morbidity prevention efforts. Robust data on health consequences of attaining one’s preferred pregnancy outcomes are also needed to inform interventions around contraceptive access and reproductive autonomy.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Macromolecules fluctuate between different structural states of a conformational ensemble. One of the major effects of ligands and mutations is to change the relative stability of these different states. However, most of our structural biology modeling revolves around a paradigm of distinct and singular structures. Our major goal is to move beyond static images of biological macromolecules, while retaining the ability to interrogate the resulting models to improve ligand design and mutational engineering. We are also interested in creating experimental methods to perturb the relative populations of these conformations, using temperature or chemical perturbation to bring them into the window where they can be observed and modeled. In two previous grants supported by NIGMS, we have focused three primary technologies: 1) ensemble modeling, where alternative conformations present in X-ray (and now, increasingly, cryoEM) density maps are explicitly identified and refined as a conformational ensemble or multiconformer model; 2) multitemperature crystallography, where the temperature of X-ray data collection is shifted, while avoiding radiation damage, to change the relative balance of different populations; 3) model validation, where the density at specific points is quantified to support or falsify modelling. We have applied these paradigms broadly and collaboratively, with a commitment to open methods and software. Two major foci have been: 1) ligand discovery using combinations of multitemperature crystallography and empirical X-ray fragment screening (most notably to identify new ways to allosterically inhibit the phosphatase PTP1B); 2) protein mutational engineering (most notably in the context of protein design and in understanding the relationship between conformation dynamics and catalysis). With MIRA support, we will continue our computational developments to further improve cryoEM modeling of alternative conformations, to perform large scale test of the effects of ligand binding on protein conformational heterogeneity, to improve validation and comparison of distinct ensemble model types, and to quantify density signals for alternative conformations, hydrogens, and modifications. In parallel, our experimental work will focus on the structural basis of new ligands to counter antibiotic resistance and on defining the conformational landscape of the oligomeric enzyme glutamine synthetase. Our experimental work provides an important testbed for new computational innovations and ways to validate the importance of newly modeled alternative conformations. MIRA support will also enable us to conduct our research in a transparent and open manner, dedicating ourselves further into early data disclosure (e.g. preprints and posts on our website) and data reuse (e.g. deposition of primary diffraction and EM data), which are already paying dividends by enabling other researchers. In summary, our research will create robust experimental and computational methods to access conformational ensembles and provide avenues to exploit conformational heterogeneity for useful ends.

NIH Research Projects · FY 2025 · 2022-09

Life expectancy in the United States (U.S.) is approximately 78 years—notably lower than other high-income counties. Several factors contribute to this lower life expectancy, including elevated rates of chronic diseases, obesity, and inadequate access to healthcare services. An important but often underexamined dimension is the environment within which people live, work, and play. Research has consistently shown that our surrounding environment can profoundly influence health outcomes and affect premature mortality. Yet, few studies have investigated the interactive contributions of area-based factors on differences in mortality across population groups. We propose to investigate the impact of multiple domains of area-level factors on mortality for adults in the Multiethnic Cohort (MEC) Study using novel, composite measures at the geographic level of city, town, or Census designated place. Composite measures for distinct population groups will include indicators from the domains of housing; education; employment; healthcare; traffic; tobacco, alcohol and unhealthy food outlets; and environmental hazards as these factors have been shown to influence health behaviors and chronic diseases. Specifically, we aim to examine the impact of these composite area-based measures on all-cause mortality (Aim 1) and causespecific mortality (Aim 2), across population groups in the MEC and estimate years of life lost due to these composite measures. In addition, we will use causal inference methods to investigate whether individual-level health behaviors (smoking, diet, physical activity, alcohol use), comorbidities (diabetes, heart disease, cancer) and social support mediate the associations between composite area-based measures and mortality, and investigate these associations across subgroups defined by sex, life stage, and education. We will seek guidance from a multidisciplinary External Advisory Board and regional Community Advisory Board in the development of composite area-based measures, interpretation of study findings, and dissemination of the study measures and results. The findings from the proposed study will provide key evidence of the specific interacting area-based factors that should be targeted to improve the lifespan for population groups in the U.S. The impact of this research is high as it will have translational relevance in providing empirical evidence for community stakeholders, policy makers, and implementation scientists to direct interventions for reducing premature mortality.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract Cutaneous squamous cell carcinoma is a form of skin cancer, originating from keratinocytes, that kills an estimated 8000 people per year in the United States. Compared to other cancers with similar incidences, death tolls, and/or economic burdens, our understanding of the genomic landscape and genetic evolution of cutaneous squamous cell carcinoma is limited. The overarching goals of this grant are to define the driver mutations in cutaneous squamous cell carcinomas, to delineate the genetic evolution of cutaneous squamous cell carcinomas from precursor lesions, and to establish the earliest genetic alterations occurring in pre- neoplastic keratinocytes. Towards these goals, in aim1, we will identify driver mutations in exome and genome sequencing data covering cutaneous squamous cell carcinoma. This data will be aggregated from publicly available sources as well as new sequencing data from our own institution, collectively comprising the largest analysis of cutaneous squamous cell carcinoma sequencing data to date. In aim2, we will sequence cutaneous squamous cell carcinomas and the remnant precursor lesions from which they arose. Most cutaneous squamous cell carcinomas are discovered adjacent to benign precursor lesions known as actinic keratoses, and here, we will analyze patient-matched lesions to reveal the mutations driving the transition from the benign to the malignant state. In aim3, we will elucidate the earliest somatic alterations occurring in individual keratinocytes from normal skin. Deep sequencing of normal skin has shown that keratinocytes can form patches of related cells, sometimes harboring mutations known to drive cutaneous squamous cell carcinoma, but no studies have truly genotyped keratinocytes at single-cell resolution, leaving gaps in our knowledge of the incipient events that occur in pre-neoplastic keratinocytes. We have developed an innovative workflow to call mutations in individual skin cells with high specificity and sensitivity, permitting us to catalog, for the first time, the genomic alterations in human skin at single-cell resolution. Similar lines of genomic studies have proven fundamental in advancing our understanding of other cancers by revealing therapeutic points of intervention, biomarkers to measure disease progression, biomarkers to estimate disease likelihood, and guidance on the best prevention tactics. Here, we will address these gaps in knowledge for cutaneous squamous cell carcinoma. Taken together, completion of these studies will realize longstanding goals within the field of dermatology, which will pave the way for new treatment strategies and new preventions strategies to alleviate the public health burden posed by cutaneous squamous cell carcinoma.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Neonatal stroke is an important cause of death and disability, and diagnosis is often delayed. There is insufficient knowledge regarding repair mechanisms that occur in response to focal ischemia-reperfusion injury that is the most common cause of early stroke. Angiogenesis, fibrosis, and perivascular cell repopulation occur in close proximity, with paracrine signaling supporting endothelial cell interactions that are vital for repair. Modulating this neurovascular niche may be a potential target for enhancing outcomes after ischemic injury in the developing brain. Erythropoietin and cell-based therapies have emerged as promising delayed treatment strategies for stroke, although the mechanism of their benefit is still not entirely clear. It is likely that dynamic release of pro-angiogenic growth factors and activation of signaling pathways downstream of erythropoietin receptor have differential effects on endothelial cell subtypes in distinct brain regions and at different time points after injury. In addition, the defined role of local fibrosis in injury progression and repair following early focal brain injury is unknown. Effectively inducing long-term, functional angiogenesis requires understanding and mimicking mechanisms that occur in the developing brain. Our objectives are to understand local angiogenesis and fibrosis in ischemic and peri-infarct regions following focal ischemia-reperfusion injury in the developing brain, and to determine the mechanisms of regeneration and repair with delayed erythropoietin by focusing on the vascular response. In Aim 1, we will test the hypothesis that endothelial tip cells at the vascular front are critical for angiogenesis following neonatal stroke, and that delayed erythropoietin will enhance angiogenesis and alter endothelial cell-subtype gene expression profiles to promote tip cell programs. In Aim 2, we will quantify fibroblasts and perivascular cells in the ischemic core and peri-infarct penumbra in the acute, subacute, and chronic stages after stroke and determine how erythropoietin signaling impacts local fibrosis and repair. Finally, in Aim 3, we will determine and modify specific signaling pathways to test the hypothesis that dynamic endothelial cell signaling modulated by erythropoietin is crucial for promoting local angiogenesis following focal brain injury. This will determine critical, modifiable pathways important for injury progression and repair following neonatal stroke. Our primary hypothesis is that delayed erythropoietin treatment will promote vascular growth and remodeling, reduce subacute fibrosis and astrocytic proliferation in the ischemic core, and enhance perivascular signaling to improve histological and functional outcomes after neonatal stroke. Together, these three aims will explore the roles of specific cellular subtypes and pathways in recovery after focal brain injury, with the broader goal of optimizing therapeutic strategies to improve long-term outcomes after a common cause of full-term brain injury that currently has no therapy.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Strict glycemic control is critical for optimal type 1 diabetes (T1D) outcomes but remains challenging for youth from historically marginalized ethnic groups, in whom hemoglobin A1c (A1c) levels are consistently the highest in the nation. In California, Latinx youth are the largest marginalized ethnic group with T1D, but they experience less optimal glycemic control than their White counterparts. Continuous glucose monitoring, insulin pumps, and automated insulin delivery systems can assist with achievement of target A1c levels, but Latinx youth have lower rates of diabetes device use compared to White youth. Virtual peer groups (VPGs) can improve patient engagement and diabetes self-care in Latinx youth, and preliminary data suggest that VPGs may increase the use of diabetes technology in this population. Jenise Wong, MD PhD, and Jennifer Raymond, MD MCR, propose this project grant with the objective to increase the adoption and sustained use of diabetes devices in Latinx adolescents with T1D by engaging them and their families in the development and evaluation of a VPG intervention designed to improve technology use. This project will engage multiple stakeholders (patients, families, health care workers) from three clinic populations in California (Children’s Hospital of Los Angeles, University of California Davis, and University of California San Francisco) and result in the DREAM (Device use Reimagined through Education And Mentorship) program. In collaboration with a multidisciplinary team of pediatric endocrinologists, psychologists, and public health experts, the researchers propose a project with the following aims: (1) partner with stakeholders to adapt a virtual peer group model to promote the initiation and continued use of diabetes technology among Latinx adolescents with T1D, (2) evaluate the feasibility and acceptability of DREAM in a single arm, pragmatic trial, and (3) assess the effect of DREAM on clinical and patient-centered outcomes. These aims will be addressed in a formative phase and a clinical trial phase. In Phase 1, the research team will address Aim 1 by engaging stakeholders in focus groups and semi-structured interviews to obtain qualitative data that will directly inform the design of the DREAM VPGs and the clinical trial. A subset of stakeholders will form Advisory Councils who will provide longitudinal input during the clinical trial. In Phase 2, the research team will conduct a single arm, pragmatic trial of the DREAM intervention in 120 Latinx youth aged 13 to <18 years with T1D, recruited from three pediatric diabetes clinic populations in California. To address Aim 2, the researchers will collect qualitative feedback from focus groups and quantitative data on VPG feasibility, appropriateness, and acceptability as primary outcomes. Preliminary data on clinical outcomes of A1c and hospital utilization, and patient-centered outcomes will be assessed to address Aim 3. The results of this study will result in a novel, culturally appropriate, VPG model to increase use of diabetes technology for Latinx youth with T1D that may be further adapted and disseminated for other age groups, geographic settings, or racial/ethnic populations.

NIH Research Projects · FY 2026 · 2022-09

Lysosomal storage disorders (LSDs) are severe diseases arising from mutations in critical enzymes and collectively have an estimated incidence of 1 in 5,000 to 1 in 5,500 live births. Patients with LSDs are at increased risk of serious perinatal morbidity and mortality, with some not even surviving to birth. The current treatment for pediatric patients, enzyme replacement therapy (ERT), is limited by three aspects: the progressive development (sometimes in utero) of organ-specific manifestations, the development of anti-ERT antibodies, and the inability of ERT to cross the blood-brain barrier to address neurologic effects. Thus, there is an unmet medical need to develop more effective therapies for patients with LSDs, starting before birth. In a mouse model of mucopolysaccharidosis type 7 (MPS7), we showed that in utero ERT (IUERT) followed by postnatal ERT improved survival, crossed the blood-brain barrier, ameliorated disease, and induced tolerance to the ERT. Based on these results, we obtained an IND to perform a first-in-human, non-randomized, single site phase 1 clinical trial of IUERT and seek funding to support this clinical trial. Since each individual LSD is rare, but they share similar pathophysiology, we have included eight different LSDs (and their specific ERT) under this protocol: MPS Types 1, 2, 4a, 6, and 7, Infantile-onset Pompe Disease (IOPD), Neuronopathic Gaucher (Types 2 and 3), and Wolman disease. We will enroll 10 maternal-fetal pairs for infusion of the ERT via the umbilical vein every 2-4 weeks, starting after 18 weeks of gestation. We will evaluate the safety and feasibility of this prenatal therapy, as well as the efficacy of ERTs in resolving fetal manifestations (if present) and improving long-term outcomes including neurologic and cardiac function, mobility, and growth. (Aim 1). We will also examine the pharmacokinetics and pharmacodynamics of IUERT by evaluating enzyme trough levels throughout gestation, as well as levels of disease-specific lysosomal accumulations before and after birth (Aim 2). Finally, we will evaluate whether in utero exposure to the recombinant enzyme will induce tolerance, as determined by lack of anti-drug antibodies and generation of enzyme-specific regulatory T cells (Aims 3). In the past year, our team has successfully treated a fetus with IOPD (whose two previous siblings had severe cardiomyopathy and suffered perinatal demise); this patient was born at term after multiple prenatal enzyme infusions and has normal cardiac function. We have assembled a multidisciplinary team and partnered with several experts on biochemical analyses for LSDs. Since we anticipate identifying fetuses based on a known family history, we have also been collaborating with multiple national and international patient advocacy groups to include patients and families in the design and execution of this trial. We conducted a parent survey to evaluate their attitudes and found that the majority of respondents would choose to enroll in a phase I clinical trial for fetal ERT for a future pregnancy affected by an LSD. Ultimately, we seek to improve the options available to families and patients with LSDs.

NIH Research Projects · FY 2025 · 2022-09

PROJECT 3 SUMMARY/ABSTRACT Breast cancer is a heterogeneous disease, with different subtypes likely arising from distinct precursor cells in the normal breast. What remains unknown is how we can target distinct precancerous cell types to prevent or intercept breast cancers in high-risk populations in a personalized manner. We previously combined detailed single-cell analyses of histologically normal breast tissues from patients with inherited mutations in BRCA1 and BRCA2 to identify aberrant cell types enriched in these cancer-prone tissues. This proposal seeks to develop models to identify new targets for breast cancer prevention in diverse high-risk states, and to help determine who would benefit from these interventions. This will be performed by combining advances in organoid culturing with single-cell RNA sequencing, mass cytometry, and multiplexed immunofluorescence studies. First, organoids will be generated from the breast tissue of patients at increased risk of developing breast cancer based on the presence of imaging-based markers, focusing on background parenchymal enhancement (BPE) on MRI as an indicator of global risk of developing invasive cancer. Tissue-based and organoid-based techniques will be used to determine the cell types enriched and pathways deregulated in this disease state. Second, the tissue environment of women with DCIS in the setting of BPE who demonstrate response and nonresponse to endocrine therapies (in Project 4) will be evaluated. Third, high-risk states including young women who developed triple-negative breast cancer before the age of 40 will be evaluated for potential cancer prevention targets and deregulated pathways at the tissue level, including by the development of T cell-organoid co-culture systems to model immune surveillance. Finally, candidate prevention/ intervention strategies will be assessed in patient-derived organoid models of high-risk tissues to identify potential compounds for a future adaptive platform trial for breast cancer prevention (Project 4 aim 4). The project lead, Dr. Rosenbluth, is a breast medical oncologist with a research background in cell and cancer biology and with expertise in 3D culture models of cancer prevention. An expert team has been assembled for this project including Dr. Laura Esserman, an internationally recognized expert in breast cancer research, Dr. Laura van 't Veer, world renowned molecular biologist and inventor of MammaPrint, and Dr. Funmi Olopade, a leader in clinical cancer genetics and breast cancer prevention, as well as additional collaborators and experts in aspects of breast cancer research and in adaptive platform trials.

NIH Research Projects · FY 2024 · 2022-09

Project Summary/Abstract The detailed structure of the beta cell niche, and that of the islet in general, remains poorly understood; this is particularly the case for human islets. Islet structure appears heterogeneous across the pancreas, and whether conserved structural features exist among islets is unknown. A detailed understanding of the organizational principles of islets would advance our ability both to reconstitute stem-cell derived islets as a cure for type 1 diabetes (T1D) and to block the progression of events that lead to the loss of beta cells during the progression of diabetes. Therefore, the goal of this proposal is twofold: first, to identify and experimentally validate the critical organizational principles of the islet in general and the beta cell niche in particular, and second, to leverage these organizational principles to engineer more functional islets as a cure for T1D. Towards the first goal, we have developed a custom, semi-automated, 3D imaging and analysis pipeline that permits quantification of the statistical properties of the beta cell niche at sub-micron resolution and across hundreds of individual beta cells. Preliminary analyses of healthy mouse and human islets revealed that (1) in both species beta and delta cells maintain at least one physical contact with a source of basement membrane, whereas alpha cells do not, and (2) beta cells in engineered islets that contact sources of vascular basement membrane have dramatically elevated insulin expression. We hypothesize that beta cell contact with basement membrane is a conserved element of islet structure that must be incorporated into engineered islets to optimize beta cell function. Towards the second goal, we have demonstrated that reconstituting stem cell- derived beta cells into pseudo-islets in a manner that maximizes their contact with basement membrane improves their response to glucose by at least two-fold in vitro and further extends their functionality in vivo. Building on these preliminary findings, we first aim to dramatically expand this analysis across tens of thousands of individual cells in human and mouse islets, incorporating all endocrine cell types along with immune cells, vascular cells, and nerves. This will result in the first quantitative assessment of the endocrine cell structural niche that acknowledges the structural heterogeneity of islets and aims to identify conserved structural motifs. Second, we aim to determine if conserved features of the beta cell niche are necessary and sufficient for optimal beta cell function. We will test this hypothesis using in vitro reconstituted islets, primary human islets cultured ex vivo, and engineered human islets transplanted into mice in vivo. Finally, we will use genome editing techniques to test the necessity of specific pericyte-derived basement membrane molecules for glucose homeostatic function in engineered islets. Taken together, our study will provide the first quantitative structural blueprint for the pancreatic islet, will identify features of the beta cell niche that are conserved and divergent across humans and mice, and will demonstrate a strategy for reconstituting more functional human tissues from stem cells that uses a structural blueprint to guide tissue engineering.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract This proposal’s long-term goal is to take advantage of the biology of the NR4A family of orphan nuclear hormone receptors to selectively manipulate antigen-specific T cell responses in immune-mediated diseases. NR4A members (NUR77, NURR1, and NOR1) are encoded by three genes (Nr4a1-3, respectively) that are rapidly induced by antigen (Ag) stimulation in lymphocytes. Although they are thought to function as ligand- independent, constitutively active transcription factors, small molecule agonist and antagonist ligands have been developed, rendering them druggable. We and others have shown that NR4A TF expression scales with the intensity of Ag stimulation, and are highly upregulated in thymocytes undergoing negative selection, in regulatory T cells (Tregs), as well as in self-reactive, anergic, or exhausted T cells. Due to an overlapping expression pattern and considerable structural homology in their DNA-binding domain, the NR4A TFs exhibit profound functional redundancy; thymic deletion of multiple - but not individual - NR4A family members (Nr4a1 and Nr4a3 >> Nr4a2, which is minimally expressed) results in severe Treg deficiency and a “scurfy-like” disease that phenocopies Foxp3-/- mice. Consequently, it has not been possible previously to unmask additional redundant functions of this family during thymic selection and in conventional mature CD4 T cells (Tconv). This represents a major gap in our knowledge that limits full therapeutic exploitation of these factors. Recently, we took advantage of both conditional genetic and bone marrow chimera strategies in order to preserve Treg homeostasis. Unexpectedly, mixed chimeras harboring both WT and Nr4a1-/- Nr4a3-/- (DKO) bone marrow rapidly develop anti-nuclear autoantibodies (ANAs) and a systemic inflammatory disease despite a replete Treg compartment of largely WT origin. This disease differs qualitatively from that seen in germline DKO mice with Treg-deficiency, and is B cell-extrinsic. We show that negative selection of DKO thymocytes is profoundly impaired in a cell-intrinsic manner. Consistent with escape of self-reactive T cells into the periphery, DKO CD4 Tconv cells expressing phenotypic and transcriptional markers of anergy accumulate in these chimeras. However, these self-reactive DKO cells nevertheless exhibit exaggerated proliferation and IL-2 production, suggesting that functional anergy is defective. We therefore hypothesize that the NR4A family play cell-intrinsic, but redundant, roles in both central and peripheral CD4 T cell tolerance. To test this hypothesis: In our first aim, we propose to define the role of the NR4A family in thymic negative selection in the face of both ubiquitous and tissue-restricted antigens, and to define the transcriptional targets that contribute to this function. In our second aim, we propose to isolate NR4A contributions to peripheral T cell tolerance, and to canonical features of mature CD4 T cell anergy - including impaired signal transduction, cytokine production, and proliferation. We will use both bulk and single cell genomic approaches to define the mechanism by which the NR4A family regulates the transcriptome and epigenetic profile of naïve and tolerant CD4 T cells.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Use of bisphosphonate (BP) medications for the prevention of fractures is declining, partially due to patient and provider fears about the occurrence of atypical femur fractures (AFF), which are clearly associated with long- term bisphosphonate use. Many individuals are choosing to not use these medications at all rather than risk having this rare outcome. This decision may leave individuals at high risk of morbidity and mortality. Balancing the risk of typical osteoporosis-related fractures, such as hip or vertebral fractures, with the risk of the much rarer AFF, is an important component of decision-making by physicians and patients about medication use. Further, an understanding of which subgroups of women may be at greater or lesser risk of AFF will be useful when making clinical decisions about initiation of medication, duration of treatment, and use of drug holidays. The proposed study will address these issues by combining individual-level data from three large, population-based cohort studies with radiographically verified AFFs, comprehensive longitudinal medication exposure, data harmonized definitions of other covariables, and centrally coordinated statistical programming. We will be focusing on two Specific Aims. In Aim 1, we will examine the risks of long-term use of BP for the prevention of AFF by determining the independent effects of BP treatment and drug holidays on AFF risk, including the potential interplay between pre-holiday duration of treatment and duration of holiday. In novel exploratory analyses, we will determine the effects of re-initiation of BPs after a drug holiday. We will also evaluate the relationships between AFF risk and the use of other fracture prevention medications (e.g., intravenous BPs, denosumab and SERMs), other potential risk factors (e.g., physical activity, bone mineral density (BMD), race), and comorbidities. In Aim 2, we will use the pooled data from our 3 cohorts to develop and validate predictive models incorporating patterns of long-term BP treatment, drug holidays and clinical risk factors, to comprehensively model the expected fracture protection and potential harms for individual patients and evaluate the group-level balance between AFF risk and osteoporosis-related fracture prevention. These models will allow clinicians to quantify individualized risk, balancing the benefits and harms of bisphosphonate treatment accounting for other risk factors. Development of these predictive tools and addressing important gaps in the scientific evidence related to BP treatment, drug holidays, and re-initiation of BP or other anti-osteoporosis medications will have an immediate positive impact on clinical practice and patient care by encouraging and improving the optimal use of osteoporosis medications.