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Chevron Foundation

Supports STEM education, workforce development, and community vitality in regions where Chevron operates.

NIEHS - National Institute of Environmental Health Sciences

Project Summary/Abstract N6-methyladenosine (m6A) is the most abundant messenger RNA (mRNA) modification and also occurs on chromatin-associated RNA in mammals. m6A significantly affects messenger RNA (mRNA) processing, export, localization, translation, and stability, thus playing critical roles in a diverse range of biological processes including stress responses. We identified the first two m6A RNA demethylases, FTO and ALKBH1, in 2011 and 2013 respectively, revealing that m6A RNA methylation is reversible and can broadly affect gene expression. Our previous research also revealed upregulation of FTO in human and mouse keratinocyte cells upon exposure to arsenic. The upregulated FTO may mediate mRNA m6A demethylation and affect cellular homeostasis, DNA damage response, and tumorigenesis induced by arsenic exposure and UV irradiation. We have recently discovered an additional layer of RNA m6A methylation regulation: we identified prevalent m6A methylation of mammalian promoter-associated RNA (paRNA), enhancer RNA (eRNA), and RNA transcribed from transposable elements (repeat RNA), which we collectively termed chromatin-associated regulatory RNAs (carRNAs). We showed that these carRNA m6A modifications could be recognized by binding proteins (readers), which recruit either the nuclear degradation exosome complex to degrade the methylated RNA or histone modification proteins to alter local histone mark deposition, thereby modulating the chromatin state and downstream transcription. Our preliminary results suggest that nuclear-localized FTO in keratinocyte cells also mediates carRNA demethylation, which can induce chromatin state and transcription changes in response to arsenic stimulation. In Aim 1, we plan to identify the exact carRNA and carRNA m6A sites affected by FTO and uncover the underlying regulatory pathways that contribute to stress response and tumorigenesis in response to arsenic exposure and UVB irradiation. We have also discovered carRNA m6A demethylation by ALKBH5, which was not previously known, in mouse embryonic stem cells (mESCs). ALKBH5 undergoes SUMOylation in cells treated with reactive oxygen species (ROS), which inhibits its demethylation activity and protects cells from DNA damage. In Aim 2, we plan to establish carRNA m6A demethylation by ALKBH5 and elucidate the underlying chromatin regulation pathways and mechanisms using mESCs as the mode system. We will also investigate how ROS-induced ALKBH5 SUMOylation may affect the chromatin state and DNA damage response in mESCs and keratinocyte cells under H2O2 treatment or arsenic exposure. Collectively, the proposed studies will determine the epitranscriptomic-epigenetic interactions in environmental stress responses and tumorigenesis for the first time. New mechanistic insights and new molecular targets for improving human health may emerge.

NIA - National Institute on Aging

ABSTRACT Our broad research goal is to understand chromatin regulatory mechanisms in nuclear and epigenetic programs and how these mechanisms are deregulated in aging and disease. A fundamental mechanism for regulating chromatin involves the reversible modification of histones by chemical moieties such as acetyl-, methyl-, and phospho- groups. These different histone marks are linked to discrete chromatin states and regulate the accessibility of DNA to transacting factors. In budding yeast, histone deacetylation by the chromatin silencing factor Sir2 prevents genomic instability and aging, and in mammals, de-regulation of histone acetylation is linked to cellular senescence and aging-related pathologies from neurodegeneration to cancer. Here, we focus on the mammalian Sir2 family member SIRT7, a chromatin regulatory, highly selective, lysine deacetylase enzyme. Previous studies reported that loss of SIRT7 function in mice leads to genomic instability, shortened lifespan and aging-related phenotypes including fatty liver, cardiac disease, and hematopoietic stem cell dysfunction. This project will study new roles of SIRT7-dependent histone deacetylation in chromatin regulatory mechanisms that are deregulated in aging-associated metabolic pathologies. It employs biochemical, cellular, and genomic approaches, and leverages SIRT7 knockout (SIRT7-KO) mice and new mouse models in which SIRT7 is overexpressed (SIRT7-OE). A central hypothesis is that SIRT7 protects against aging and metabolic disease processes and attenuates metabolic pathologies when overexpressed in mice. The project also hypothesizes that a novel histone substrate of SIRT7, H3K36, contributes to functions of SIRT7 in aging and metabolic pathways. Little is known about acetylation of H3K36, but di-methylation of H3K36 (a histone modification linked to gene regulation) is implicated in many human cancers, developmental disorders, and recently, metabolic disease. We hypothesize that deacetylation of H3K36 by SIRT7 is coupled to methylation by lysine methyltransferase (KMT) enzymes. We will test the model that a key mechanistic function of SIRT7 is to clear H3K36 acetylation from large swaths of DNA across the genome to enable methylation by KMTs, and that such a H3K36 acetyl-methyl switch mechanism is essential for preventing aging-associated gene expression reprograming in metabolic tissues.

NIH

Significance to VA: Type 2 diabetes (T2D) is a chronic, adult-onset metabolic disease with no cure to date. Alarmingly 1 in 4 (25%) Veterans are diabetic, significantly higher percent compared to civilians. Diabetes is the leading cause of renal and cardiovascular diseases, blindness, and amputations in Veterans. The annual mortality rate of Veterans with diabetes is nearly twice the rate compared to Veterans without diabetes (VA Fact Sheet 2019). The predominant risk factors for T2D advent in Veterans are military exposures and PTSD. Relevance to VA Military Exposures Research Program Mission: Vietnam War (VW) Veterans were exposed to Agent Orange (AO). Evaluating the effects of military exposures, it is established that tetra- chloro-dibenzo-p-dioxin (TCDD), contained in AO caused T2D pathology. T2D is a presumptive condition in VW Veterans. In the recent Global War on Terrorism (GWOT) conflicts, 85% of military personnel were exposed to open burn pits which emitted large amounts of TCDD. Measuring TCDD levels in the blood involves a long, cumbersome procedure requiring specific equipment and highly trained personnel, thereby deterring routine evaluation of TCDD exposures. Further, little is known about the chronic metabolic outcomes, specifically T2D, in GWOT Veterans. There is an immediate and dire need to identify Veterans with increased susceptibility to advent of T2D due to service-related TCDD exposures, independent of their genetic makeup, aging, or obesity. The proposal fills this gap with our overarching goal to establish a rapid, blood genetic test to identify Veterans vulnerable to chronic health sequelae of T2D, years after TCDD exposures. Innovation and Impact: We have established that low GAS5 levels are causal to T2D (US Patent Nos. 10,724,097; 11,214,835, 11,278,521. All with VA-asserted rights). The data demonstrates that GAS5, a noncoding regulatory RNA, is the genetic target of TCDD in humans, which unknown thus far. The project will establish GAS5 as a surrogate biomarker of TCDD-exposure related advent of T2D in Veterans. Importantly, to integrate into clinic, we developed a rapid blood droplet test for reproducible, consistent measurements of GAS5 levels requiring minimal technical expertise. Successful completion of the proposal will establish GAS5 as a surrogate biomarker of TCDD-exposure related advent of T2D in Veterans. This determination will significantly reduce the advent of T2D as the most preventable cause of death in US Veterans. Specific Aims: Our compelling data shows TCDD decreases GAS5. We hypothesize that low GAS5 levels in the blood correlate with past military associated TCDD exposures and the advent of T2D in Veterans. Specific Aim 1: Longitudinal study evaluating the correlation of blood GAS5 with TCDD levels in Veterans. Specific Aim 2: Determine the TCDD-regulated molecular mechanisms affecting GAS5 in human adipocytes. Methodology: For SA1, we will leverage our existing JAHVA Research Biospecimen Repository and obtain de-identified blood samples (IRB determination: Not Human Subject Research). The levels of TCDD, GAS5 will be measured and correlated to T2D by a sample-blinded biostatistician. For SA2, we will use human adipose stem cells differentiated to adipocytes. We will elucidate the underlying molecular and cellular mechanisms to understand TCDD-regulation of GAS5 levels, thereby providing unequivocable support complementary to SA1. NPC86, a therapeutic which increases GAS5, will be used as a molecular tool. Path to Translation/Implementation: We have developed a minimally invasive, minimal technical expertise requirement and ease-of-use, rapid (2-hour turn around) blood droplet test to measure GAS5 levels that can be easily incorporated into clinic. The results can be evaluated along with the Veterans’ TCDD exposure history to implement a personalized, time-sensitive treatment plan to prevent the advent of T2D.

NCI - National Cancer Institute

SUMMARY. Regulating the timing of meals and snacks to re-align the body’s circadian clock and improve metabolic health is emerging as a promising approach for cancer prevention in early animal and small clinical studies. Yet, a major barrier to studying meal timing and cancer is that large population studies rarely measure meal timing, which makes it impossible to conduct epidemiological studies of meal timing and cancer risk on a large-scale and across individuals with different biological and environmental characteristics and varied meal timing practices. Although obesity and its related metabolic dysregulation are important risk factors for at least 13 cancer types, weight management is notoriously difficult in the long-term. Behavioral strategies are needed that can improve metabolic risk factors for cancer but that do not necessarily rely on weight loss. Herein, we propose to discover and then externally validate novel objective biomarkers of meal timing practices, then test their relationship with incidence of breast (BC), endometrial (EC), and colorectal (CRC) cancers, three of the most prevalent obesity-linked cancers. Our central hypothesis is that meal timing is associated with perturbations in blood metabolomic profile, and with obesity-related cancer incidence in free-living humans. We will test our hypothesis with unique data from large-scale cohorts with validated measures of meal timing and sleep and longitudinal metabolomics data measured on the same metabolomics platform to facilitate data harmonization. In Aim 1, we will Identify biomarkers of meal timing patterns using a discovery and external validation design in the Cancer Prevention Study 3 Diet Assessment Sub-study (DAS) (n=750) and the Interactive Diet and Activity Tracking in AARP (IDATA) Study (n=718) and measure their association with risk of obesity-related cancer in the Cancer Prevention Study 2 (CPS-2, 782 BC matched sets; 517 CRC matched sets with 16-year follow-up time) and CPS-3 cohorts (1695 BC patients, 1983 controls, 3-year follow-up). In Aim 2, we will examine whether there is large-scale, real-world evidence that meal timing patterns are associated with obesity-related cancer risk among 185,000 US adults in the CPS-3 cohort. The proposed study will answer critical, outstanding questions about which meal timing practices are associated with cancer- relevant metabolic factors and risk of obesity-related cancers in a real-world scenario and identify objective biomarkers of meal timing behaviors that will facilitate large-scale investigations of meal timing and cancer risk at the population level. These meal timing biomarkers could also be used to assess response to meal timing interventions in clinical studies. Following successful completion of this project, we plan to apply the resulting biomarker profiles to study meal timing and cancer risk across international cohorts in the Consortium of Metabolomics Studies (COMETS). Epidemiological research stemming from our study findings will be vitally important prior to issuing public health guidance on meal timing for cancer prevention.

NIA - National Institute on Aging

Project Summary: In older adults with mild dementia due to Alzheimer’s disease (AD), difficulties with instrumental activities of daily living stem not only from memory loss, but also from co-occurring depressive symptoms (DEP). DEP + AD is highly prevalent and major driver of disability in older adults, yet most patients with this combination of symptoms do not derive meaningful benefit from current treatments. There is a pressing need to develop safe, multi-component interventions designed to maximize functional capacity in this vulnerable population by simultaneously targeting memory and DEP. It is now well established that depressive and memory symptoms arise from altered function within specific brain circuits, and, that different types of transcranial electrical stimulation (tES) can target these brain circuits to provide symptomatic relief to this population. Parallel lines of preliminary evidence specifically suggest that transcranial direct current stimulation targeting left prefrontal cortex (tDCSPFC) can improve depressive symptoms in people with major depressive disorder, whereas transcranial alternating current stimulation targeting the left angular gyrus (tACSAG) at gamma frequency (40Hz) can improve episodic memory in people with moderate dementia. At the same time, technological advances now allow researchers to: (1) study and optimize multi-channel stimulation montages (i.e., electrode placement and current parameters) using individualized electric field (E-field) modeling; (2) target multiple brain networks simultaneously within a single montage; (3) implement active-sham protocols that enhance double-blinding and control for potential peripheral stimulation effects; and (4) deliver stimulation at home utilizing a remotely monitored caregiver-led administration that has been found to be safe and effective, while increasing treatment access, relieving patients of travel burden, and limiting geographical selection bias. Building upon these preliminary studies and advances, we propose to conduct a phase II, randomized, sham-controlled, double-blinded, parallel-arm trial. The first aim of this study is to assess the separate and combined effects of tACSAG at gamma frequency and tDCSPFC on memory, depressive symptoms, and instrumental activities of daily living in DEP + AD. The second aim is to determine how much of the clinical effects on memory on the one hand, and DEP on the other hand, depend upon the amount of the E- field induced respectively in the left angular gyrus and the left dorsolateral prefrontal cortex, as assessed by individualized E-field high-resolution modelling. The third aim will assess intervention-related changes in brain activity using resting-state EEG. This project aims to demonstrate that tACS and tDCS can be effectively integrated into a single, scalable home-based intervention to maximize functional gains in older adults with mild dementia due to AD + DEP. It will also provide insights into the specificity of brain network-symptom mapping and lay the ground for the development of a highly scalable personalized optimization of stimulation interventions in future trials.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY/ABSTRACT (30 lines or less) The premise for the proposed research stems from precedence in other diseases, such as cancer, cardiovascular, neurodegenerative, and most relevant to the current proposal, autoimmune diseases, in which circulating extracellular vesicles (cEVs) play a role in pathophysiology and are important biomarkers for early detection. However, little is known concerning the role of cEVs in human type 1 diabetes (T1D). Our overall hypothesis is that cEVs have the potential to be used as biomarkers for early pre-disease detection of T1D, based on their distinct molecular and functional phenotype in T1D and pre-disease stages, compared to healthy or low risk individuals. Our specific aims are: 1) To identify the distinct protein and RNA cargo unique to cEVs at different stages of T1D disease development; and 2) To investigate the effect of cEVs from subjects at different stages of disease progression on immune and beta cell phenotypes and elucidate the functional relevance of their distinct molecular cargo. To address these aims, we have assembled a team of investigators with highly relevant expertise and techniques. We propose to use longitudinal samples from The Environmental Determinants of Diabetes in the Young (TEDDY) study of children with T1D associated genetic risk to identify critical timepoints and underlying mechanisms mediating, A) the earliest stages of pathogenesis preceding AAb appearance of the first islet autoantibody, and B) the period of seroconversion from single to multipe AAb+ or remaining single AAb+ and C) the period after multiple AAb appearance with a highly variable rate of progression to hyperglycemia. We have the expertise and technical ability to isolate cEVs from plasma, perform proteomic and RNAseq analysis on EVs, and perform immune and beta cell related functional assays. Our Research Plan is to generate cEVs from donors at different stages of T1D disease progression, to identify the uniquely packaged protein and RNA cargo from these cEVs, to evaluate the effects of the cEVs on immune cell functional phenotype and islet health and elucidate the functional relevance of the distinct molecular cargo targets. These studies will yield novel mechanistic insights into early disease pathogenesis and identify potential novel biomarkers for T1D initiation and progression.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT In this grant cycle, Rutgers Cancer Institute had an unprecedented opportunity to expand clinical research to a significant number of additional hospitals of the RWJBarnabas Health (RWJBH) system. This unified and organized expansion was enabled by 1) Dr. Libutti’s roles as Cancer Center Director and Senior VP for Oncology Services of RWJBH; 2) Dr. Hochster’s roles as Rutgers Cancer Institute Associate Director for Clinical Research and Director of Oncology Research for RWJBH; and 3) development of a system-wide academic health system between Rutgers and RWJBH effective 01/01/2019 through a Master Affiliation Agreement. In this newly integrated and unified “one-site” model, Rutgers Cancer Institute has an unprecedented number of patients at its disposal for clinical research activities to impact the catchment areas' priorities and needs positively. The Office of Human Research Services (OHRS) serves as a centralized research administration, housing all administrative tasks at Rutgers Cancer Institute while providing oversight and direction for each clinical oncology research operation site throughout RWJBH. OHRS is responsible for working with Rutgers and system-wide clinical investigators to manage the business, clinical, and regulatory functions of all phases of pediatric and adult oncology cancer clinical trials throughout the health system. Our model is “one site with one CTMS, one IRB, and one EMR’, which is currently operational. The organizational structure, which reports to Drs. Libutti and Hochster is comprised of 158 FTEs with ten distinct offices divided and based on specialization within functional areas such as clinical operations, quality assurance, and regulatory affairs. In parallel with the growth of Rutgers Cancer Institute, screening, enrollment, and the number of available NCTN clinical trials increased significantly with the integration of RWJBH health system sites into OHRS. To date, the number of open trials has more than doubled (52 vs. 24 in 2019), and enrollments in NCTN clinical trials have increased by more than 500% (662 vs. 132). Minority enrollment has steadily increased from 29.9% in 2019 to 41.3% in 2024 over the grant period. During the current grant period, therapeutic trial accrual has risen by more than 400% (552 vs.132). In recent years, Rutgers investigators have held prominent roles within ECOG-ACRIN, NRG, and Alliance. Evidence of our success in scientific contributions is reflected in our investigators' significant leadership roles, memberships in key group studies, and authorship of numerous publications stemming from NCT trial participation. Moreover, Rutgers consistently mentors and engages young investigators in clinical trial research, as demonstrated by the recent appointment of two junior investigators who have assumed leadership roles in ECOG-ACRIN clinical trials.

National Institutes of Health

<p>The National Institute of Allergy and Infectious Diseases (NIAID) seeks to advance its mission through the&nbsp;continued&nbsp;support of the Clinical Trials in Organ Transplantation in Children and Adults (CTOT-CA) program.&nbsp;The goal of the CTOT-CA is to improve the outcome of organ transplantation through clinical trials with mechanistic studies. The CTOT-CA will support multicenter clinical trials, with associated studies of immune mechanisms, in heart, lung, kidney, liver, and intestinal transplantation. The program will also support clinical trials of non-hematopoietic cellular transplantation as replacement therapies; vascularized composite tissue transplantation; and transplantation of bone marrow or mesenchymal stem cells, or of immunologically active cells, as adjuncts to organ transplantation. Grant authorities that allow NIAID to forecast this opportunity are as follows: Sections 301 and 405 of the Public Health Service Act as amended (42 USC 241 and 284) and under Federal Regulations 42 CFR Part 52 and 2 CFR Part 200.&nbsp;&nbsp;</p>

National Institutes of Health

The National Institute of Allergy and Infectious Diseases (NIAID) seeks to advance its mission through the continued support of the Clinical Trials in Organ Transplantation in Children and Adults (CTOT-CA) program. The goal of the CTOT-CA is to improve the outcome of organ transplantation through clinical trials with mechanistic studies. The CTOT-CA will support multicenter clinical trials, with associated studies of immune mechanisms, in heart, lung, kidney, liver, and intestinal transplantation. The program will also support clinical trials of non-hematopoietic cellular transplantation as replacement therapies; vascularized composite tissue transplantation; and transplantation of bone marrow or mesenchymal stem cells, or of immunologically active cells, as adjuncts to organ transplantation. Grant authorities that allow NIAID to forecast this opportunity are as follows: Sections 301 and 405 of the Public Health Service Act as amended (42 USC 241 and 284) and under Federal Regulations 42 CFR Part 52 and 2 CFR Part 200.

NCI - National Cancer Institute

Project Summary Allogeneic stem cell transplantation (allo-SCT) is the only potentially curative therapy for AML. However, relapse of AML post-transplant remains the major cause of failure and continues to be a significant challenge. Understanding the mechanism behind post-transplant relapse is crucial for developing effective prevention and treatment strategies. Studies suggest that relapse after allo-SCT is multifactorial, involving the expansion of residual leukemia, potentially with new driver mutations. Immune escape mechanisms enable leukemic cells to evade the graft-versus-leukemia effect, in addition to T cell exhaustion and dysregulation. One strategy to prevent relapse is maintenance or pre-emptive therapy with anti-leukemic agents, with a low risk of graft failure and good tolerability after allo-SCT. To address this, we have conducted multiple clinical trials and are currently running a phase II trial evaluating venetoclax (Ven) with azacitidine (AZA) posttransplant, (NCT04128501). In this trial, patients receive this combination either for maintenance or to eradicate minimal residual disease (MRD) after transplant. Additionally, we are collecting longitudinal blood and bone marrow samples from participating patients as part of this study. In this proposed study, our aims are as follows: 1) To characterize the mutational landscape at diagnosis, post-transplant, and at relapse in AML patients receiving venetoclax and azacitidine after allogeneic SCT, utilizing single cell DNA sequencing to explore relapse mechanisms 2) To define T cell evolution in AML patients receiving venetoclax and azacitidine post- transplant, and the immune mechanisms that eradicate MRD with this combination. We have unique advantages in this research. Current data on post-transplant genetics mainly derive from next generation sequencing, whole exome sequencing, or flow cytometry, all with limitations. High throughput single-cell genomic analysis enables the detection of mutation cooccurrences within individual cells and determination of the order in which mutations appear. Post-transplant milieu cannot be analyzed solely by disease-specific genetic alterations but also immune-related changes. Given the association of T cell clonal emergence and immunotherapy response, we will also analyze T cell receptor repertoires and immune profiles post-transplant. This will help determine whether a specific subgroup benefit from Ven and AZA, particularly for patients with impending relapse, such as those with MRD detection. As a summary, in this exploratory grant, we will focus on mutational landscape and T-cell clonality to assess responders to Ven and AZA in the post-transplant setting. If successful, our project will provide critical information to identify which patients are more likely to benefit from Ven and AZA for disease prevention and guide tailored post-relapse treatments based on individual disease and immune-related findings.

NCI - National Cancer Institute

PROJECT SUMMARY Anaplastic thyroid cancer (ATC) primarily affects elderly individuals and has a dismal prognosis compared to other thyroid cancer subtypes. One of the hallmarks of ATCs is their admixture with myeloid cells, primarily macrophages. The recent development of combinatorial treatment with BRAF and MEK inhibitors led to improved outcomes in Class 1 BRAF-mutant ATC patients. Although their initial response to MAPK inhibition is substantive, responses are not durable, leading to a median overall survival (OS) of ~15 months7. With aging, somatic mutations in hematopoietic stem and progenitor cells (HSPCs) promote clonal expansion over non- mutant HSPCs. When this is present in the absence of malignant transformation it is termed clonal hematopoiesis (CH). CH mutations arise most frequently in epigenetic modifier genes, such as DNMT3A and TET2. CH is associated with an increased risk of atherosclerotic cardiovascular disease and other diseases associated with aging. We showed in a pan-cancer analysis that CH, in particular CH with putative driver mutations (CH-PD), is associated with adverse outcomes in solid tumor patients, including those with ATC. The specific interactions between CH leukocytes and tumor cells in the TME and their impact on therapeutic response remain uncharted. We find that TET2-mutant CH is enriched in the TME of patients with ATC and other solid tumors. We developed syngeneic immunocompetent mouse models of concurrent Tet2-mutant CH and orthotopically implanted BrafV600E-driven ATC to explore the mechanisms involved. Using single cell-CITE-RNASeq we found that Tet2- mutant macrophages selectively infiltrate mouse BrafV600E-mutant ATC and cause resistance to BRAF-MEK inhibition through overexpression of Tgfβ-family ligands. Importantly, inhibition of the effects of Tgfβ at three distinct nodes restores sensitivity to MAPK pathway inhibition, opening a path for synergistic strategies to improve outcomes of patients with ATCs and concurrent CH. The mechanisms by which Tgfβ activation render ATCs insensitive to MAPK inhibition remain to be defined. We will investigate whether macrophage Tgfβ ligand overproduction induces resistance to MAPK inhibitors through cancer cell autonomous mechanisms and/or by its immune suppressive effects and use genetic approaches to nominate the key Tgfβ ligands responsible for treatment resistance. The mechanisms delineating how DNMT3A CH leads to worse outcomes in solid cancers in general, and ATC in particular, have eluded explanation. We will determine whether Dnmt3a-CH affects ATC biology and response to therapy in mice and test the hypothesis that this is driven by the infiltrating mutant myeloid population. Finally, we found that CH-PD is associated with worse OS in patients with ATC, but the effect of individual CH genes has not been established. We will determine whether specific CH genotypes impact OS in ATC and if this manifests at low CH variant allelic fractions (VAF) using a high sensitivity assay. We will also investigate whether advanced thyroid cancers of any type with a high tumor-to-blood CH VAFratio (i.e. CH mutant cell enrichment in the tumor as compared to blood) have worse clinical outcomes.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ ABSTRACT Coagulopathy manifesting as disseminated intravascular coagulation (DIC) with associated bleeding and venous thromboembolism (VTE) is a common complication in patients with acute myeloid leukemia (AML), occurring in 10-20% patients at diagnosis and within the first 30 days of treatment. With improvements in supportive care, the overall survival of AML patients has improved, but early mortality from DIC remains a major challenge. While DIC is extensively studied in acute promyelocytic leukemia (APL), which is a small and distinct subgroup of AML, mechanisms of DIC and subsequent bleeding and VTE risk are not clearly understood in non-APL AML (referred to as AML from here on). Majority of the studies examining DIC in AML have focused on the tissue factor pathway and there is limited understanding of the role of endothelial dysfunction and the contact pathway of coagulation in coagulopathy of AML. Examining novel biomarkers in these pathways may explain the mechanisms of bleeding, and high VTE risk due to central venous catheters in AML patients. Clonal hematopoiesis (CH) refers to the acquisition of somatic mutations in driver genes in the hematopoietic stem cells that lead to increased risk of leukemia, cardiovascular disease including venous thromboembolism (VTE) and hemorrhagic stroke in the general population. A select group of mutations known as “adverse risk clonal mutations” (ACMs) in TP53, ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, and ZRSR2 genes are associated with poor prognosis in AML. DIC is often observed in patients with high-risk AML, which is characterized by high white count, lactate dehydrogenase and blast%; these features are also seen in individuals who harbor FLT3-ITD mutation and other ACMs. The overall objectives of this proposal are to harness the resources from the University of Alabama at Birmingham (UAB) AML cohort (PI: Dr. Gangaraju) of 761 patients that have next generation sequencing data for clonal mutations to: (1) Examine the association between ACMs and DIC at AML diagnosis, and between ACMs and 30-day bleeding and VTE risk, and (2) Identify novel biomarkers of endothelial dysfunction and contact pathway of coagulation associated with ACMs at AML diagnosis and with 30-day bleeding and VTE risk in AML. This cohort has rich phenotypic data on demographics, comorbidities, labs at AML diagnosis and validated outcomes of interest including bleeding and VTE. A sub- group of patients provided plasma samples for biomarker studies, and the study team has expertise in measurement of the biomarkers proposed in this application. Investigating the mechanistic pathways of DIC, VTE and bleeding risk in AML aligns with NHLBI’s mission to understand the mechanisms of these complications in cancer. This will be the first step towards achieving my long-term goal of identifying AML patients at increased risk of bleeding and VTE, and examining strategies to provide safer and efficacious anticoagulation in these complex patients.

U.S. National Science Foundation

The Collaboratory to Advance Mathematics Education and Learning (CAMEL) for K-12 initiative aims to advance mathematics learning and education through purposeful collaboration that draws on the interdisciplinary Science of Learning (including neuroscience; cognitive, developmental, and social sciences; computer science; machine learning; engineering; and education research), deep experiences in education practice and teaching, and innovations in the use of data science, AI and technology. Through an agreement with philanthropic partners, including the Walton Family Foundation (WFF), CAMEL consists of two phases. Phase I invites proposals for the creation of new research networks to support the generation of high value datasets that aim to advance math learning and education. These research networks must include researchers who study the basic science of learning, education practitioners, and data scientists. The high value dataset may come from either generating a new dataset or by increasing the value of an existing dataset. Phase II, open only to awardees of Phase I, establishes a collaboratory, which is a socio-technological platform that prioritizes community-building and capacity-building to sustain collaborative efforts to advance math learning and education in K-12. Expanding Participation in STEM, NSF Priorities and Gold Standard Science: NSF prioritizes cutting-edge discovery science and engineering research, advancing technology and innovation, and creating opportunities for all Americans. NSF has established priorities set forth by Congress, the administration and the NSF director to promoteNSF's mission. Proposers should review the list ofNSF prioritiesand are encouraged to align their proposals with them, where appropriate. NSF also expects the highest standards of scientific rigor, integrity and adherence to appropriate tenets ofGold Standard Sciencein proposals, as appropriate for the field of science and research modality.

Rockwell Collins Foundation

Funds STEM education, community development, and workforce readiness in aerospace industry communities.

NIGMS - National Institute of General Medical Sciences

Summary Leukocyte motility is critical for immunology, inflammation, and hemostasis. Immune cells exchange molecular information by direct contact, enabled by motility within secondary lymphoid organs. In inflammation, neutrophils crawl into sites of infection after adhering to blood vessel walls. Hematopoietic stem and progenitor cells (HSPCs) migrate into to bone marrow after adhesion to the blood vessel wall under flow. In work largely funded by NIGMS, the Hammer laboratory has worked to understand the chemo- mechanics of leukocyte migration for over two decades. We have used traction force microscopy (TFM), in which we measure the forces exerted by cells during motility by monitoring the defection of fiduciary beads embedded within an elastic polyacrylamide gel. We have used this method to measure the traction forces of neutrophils and macrophages, as well as many other cell types. With the technique of TFM in hand, we are now positioned for significant breakthrough in our molecular understanding of traction stresses during leukocyte motility, owing to the development of methods to delete or alter intracellular components within a cell. Of specific interest is the fascinating phenomenon of upstream migration, in which leukocytes migrate against the direction of flow on surfaces presenting intercellular adhesion molecule-1 (ICAM-1). Using CRISPR-Cas9, we now are able do a directed screen of molecules that have been implicated in cell migration, and specifically, upstream migration, to understand precisely how these molecules contribute to the generation of traction forces in leukocytes. We will use two cultured cells lines – KG1a cells (a model HSPCs) and HL-60 cells (a model neutrophil) – that allow us to compare the role of different intracellular effector molecules in cell motility to establish universal mechanisms. We will conduct a directed screen of a limited but important set of effector molecules which have been implicated in upstream migration and leukocyte motility, such as cytoskeletal regulators and Rho-GTPases. Our elucidation of traction stresses will be complemented by immuno-fluorescent staining of the spatial distribution of adhesion receptors and actin cytoskeleton to provide information about cell organization. This MIRA is organized in three projects. In Project 1, we will perform traction mapping of KG1a cells during upstream migration. We will then screen candidate controllers of upstream migration using CRISPR-Cas9 and then measure how deletion affects both directional motility and traction stresses. In Project 2, we will use CRSIPR-Cas9 to screen a family of motility modulators and study their effect on HL-60 cell chemokinesis and chemotaxis. In Project 3, we will use TFM to measure the traction stresses of HL-60 cells during upstream migration and when upstream migration is reversed through CRISPR-Cas9 deletion. Then, we will measure the correlation between upstream migration of HL-60 cells and trans-endothelial migration on HUVEC monolayers.

NIH

Abstract Amyotrophic lateral sclerosis (ALS) is a devastating and fatal neuromuscular disease that places extreme financial, physical, and emotional burdens on affected Veterans and their caregivers. Currently, ALS remains without a cure and is commonly diagnosed at advanced disease stages when treatments are ineffective. Though the loss of motor neurons is a disease hallmark, it is now clear that various tissues and cells play a role in the pathogenesis and progression of ALS. As such, multi-factorial therapeutic targeting is an increasingly necessary approach for developing and testing ALS treatments. This therapeutic gap represents an unmet need in ALS. My CDA-2 funding has supported pre-clinical animal studies to assess and optimize the therapeutic potential of the secretome, or conditioned culture medium, from adipose-derived stem cells (ASCs). We and others have determined that systemic mesenchymal stem cell (MSC) secretome therapy, including ASC secretome, imparts neurologic, survival, and other benefits in the mSOD1G93A mouse model of ALS. Preliminary studies have also revealed a significant ASC-CM-mediated reduction in key serum inflammatory biomarkers, which hints at a possible mechanism of its action. However, when given before, through and into late stages of disease, the benefits facilitated by this secretome therapy eventually declines. As such, a clinically relevant adjunctive treatment could enhance the therapeutic benefits of ASC secretome therapy. Trans-spinal direct current stimulation (tsDCS) represents an approach to potentially improve the efficacy of ASC secretome long-term. For the proposed study, we will treat mSOD1G93A ALS mice with ASC secretome derived from ASCs grown using our patented optimized culture approach for neurological disease treatment and test whether regular tsDCS extends the therapeutic benefits in ALS. Our hypothesis is that combining daily systemic ASC secretome therapy and weekly tsDCS beginning at symptom onset will synergistically slow disease progression and extend the therapeutic benefits of ASC secretome. Our long-term goal is to not only extend life, but also improve quality of life for ALS-afflicted Veterans and their families. SPECIFIC AIM 1. Demonstrate whether regular trans-spinal lumbar electrical stimulation supplemented with systemic ASC secretome therapy improves functional and survival outcomes in mSOD1G93A mice. The goal of this Aim is to determine whether weekly tsDCS combined with systemic ASC secretome or control medium therapy ameliorates motor dysfunction and prolongs lifespan in mSOD1G93A mice. As part of a modified Neuroscore we will assess limb extension, grip strength, and locomotor function to assess the onset and progression of symptoms. Overall survival and lifespan from symptom onset will also be analyzed. SPECIFIC AIM 2. Evaluate whether combined tsDCS and ASC serum therapy impart histological motor neuron survival and neuromuscular junction (NMJ) innervation benefits. To test the hypothesis that our proposed therapy could impact the underlying neurodegenerative characteristics differentially from outward behavioral and functional measures such as neurological function, quality of life, and survival, we will perform histologic analysis of lumbar spinal cord and hindlimb muscle to assess MN survival and NMJ innervation. As MN and NMJ are typically fully degenerated at humane end-stage (the endpoint in Aim 1), we will treat a separate cohort of mice with ASC secretome, control medium, and weekly tsDCS between 70-90 days of age, a period we have documented secretome effects on moderate stages of neuromuscular degeneration therapy. Following sacrifice at 90 days, lumbar spinal cord and gastrocnemius muscle will be sectioned and immunolabeled for MN quantification and NMJ innervation, respectively.

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NHLBI - National Heart Lung and Blood Institute

Modified Project Summary/Abstract Section Hypertension (HTN) control continues to be suboptimal in the US among men and at-risk populations living in socioeconomically disadvantaged communities. These differences stem in large part from adverse circumstances including poor access to care. Strategies to mitigate the effect of poor access to care on HTN control include the use of community health workers (CHWs) to link people to care and navigate health systems. With their tremendous reach, barbershops can serve as a platform to screen men for HTN and navigate them to care and other support to improve HTN control. The effect of a CHW-delivered barbershop-based linkage implementation program on HTN control is unknown. To address this research-to-practice gap we will build on our successful CLIP program (which focused on HTN prevention) to develop and implement the Community-to-Clinic Linkage Implementation Program to Control Hypertension in Barbershops (CLIP HTN). We hypothesize that integrating CLIP HTN into trusted community spaces with CHWs will improve early detection, increase linkage to care, and enhance long-term sustainability of HTN control efforts. The Specific Aims are: Aim 1: Identify barriers and facilitators of CLIP HTN adoption in barbershops using the Consolidated Framework for Implementation Research (CFIR) to develop a context-specific implementation strategy. Aim 2: Compare in a cluster RCT of 22 barbershops among 440 men with uncontrolled HTN, the effect of barbershop facilitation (BF; n=11 barbershops) vs a self-directed control (n=11 barbershops) on BP reduction (primary outcome), HTN control, linkage to care, and adoption of CLIP HTN (secondary outcomes) at 12 months. Aim 3: Compare the effect of BF strategy vs. a self-directed control on sustainability and cost-effectiveness of CLIP HTN at 18 months (secondary outcome). Methods: Data will be analyzed through a mixed methods approach to assess implementation, effectiveness, health outcomes, and economic viability. Impact: CLIP HTN leverages community-based resources to improve access to care to control HTN. This scalable model can transform HTN control efforts nationwide.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Protozoan parasites, together with helminths, account for approximately 25% of all human infections worldwide, with humans hosting more than 70 species of protozoa. Within this group, apicomplexan parasites cause severe diseases including malaria, opportunistic infections, and foodborne outbreaks that impose significant health risks and socioeconomic burdens in both resource-limited settings and developed nations, including the United States. In this project, we will investigate apicomplexan phosphoethanolamine N-methyltransferases (PMTs), essential enzymes required for phosphatidylcholine biosynthesis but absent from mammalian genomes. We will comparatively characterize the molecular mechanisms of apicomplexan PMTs from multiple human and animal apicomplexan parasites including Plasmodium, Cyclospora, Toxoplasma, and Eimeria. This project will integrate comparative genomics, enzymology, biophysics, and mutagenesis to elucidate the mechanistic features of apicomplexan PMTs, including catalytic mechanisms, kinetic parameters, and ligand- recognition determinants (Aim 1). We will also utilize artificial intelligence and machine learning (AI/ML)- supported structural analysis with complementary computational and experimental validation to determine high- resolution structures and map conserved versus variable ligand-binding regions (Aim 2). The resulting structure- function relationships will identify potentially druggable binding pockets for selective small-molecule inhibitor development and provide foundational knowledge to enable both broad-spectrum and lineage-specific antiparasitic therapeutics. Furthermore, this proposed research integrates well-established methodologies with advanced instrumentation accessible to graduate and undergraduate researchers, including students in the Course-based Undergraduate Research Experience (CURE), thereby advancing biomedical research while strengthening innovative STEM education at Kennesaw State University (KSU).

NHLBI - National Heart Lung and Blood Institute

Project Summary Hematopoietic stem cells (HSCs) sustain blood production throughout an organism’s lifetime. They are clinically significant as the key therapeutic component of bone marrow transplants for treating a multitude of hematological and non-hematological disorders. However, HSC transplantation is limited by scarcity of compatible donors. Despite significant efforts, generating large numbers of bona fide HSCs in vitro from induced pluripotent stem cells (iPSCs) remains a major challenge. This inability likely stems from an incomplete understanding of the developmental cues necessary for HSC specification. In vertebrate embryos, HSCs are specified from hemogenic endothelium lining the ventral wall of the dorsal aorta (DA) and require signals from the surrounding microenvironment. Previous work in our laboratory demonstrated that neural crest cells (NCC) migrate to the DA and direct HSCs specification in zebrafish via locally secreted or displayed signals. NCCs of the trunk differentiate into sympathetic neurons, which secrete catecholamines. In mice, catecholamines are required for HSC maturation. However, inhibition of catecholamine production in zebrafish did not affect HSC specification, pointing to the idea that an earlier phase of HSC specification directed by NCCs is independent of catecholamines. Although HSC programming is highly conserved across vertebrates, the requirement for NCCs in specifying HSCs has not yet been examined in mouse, and the cues provided by NCCs for HSC specification remain elusive. My preliminary data in mouse reveal that NCCs migrate into proximity of the DA during the HSC specification window and are found closely associated with nascent HSCs locating them at the right time and place to provide inductive signals for HSC specification. Interestingly, preliminary data in mouse and zebrafish show that NCCs do not contact the DA directly, except via filopodial extensions. To determine whether NCCs mediate HSC specification in mouse, I will map the association of migrating NCCs to the DA and emerging NCCs by lineage tracing and specifically ablate NCCs before they reach the DA with an inducible diphtheria toxin receptor mouse model. Finally, I will determine if NCCs mediate HSC specification through filopodial communication with the DA by generating a novel optogenetic zebrafish line permitting tissue specific knock out of Myosin X, required for filopodia formation and maintenance, in living animals and visualize any loss of HSCs using advanced live imaging technology.

NCI - National Cancer Institute

PROJECT SUMMARY Acute myeloid leukemia (AML) is the deadliest blood cancer characterized by the abnormal function of hematopoietic precursor cells. Leukemic cells acquire metabolic adaptations that initiate transformation or promote the growth of transformed cells. These metabolic alterations can be exploited for diagnosing, monitoring, and treating cancer, but broad strategies to target reprogrammed metabolism are lacking. The challenges are two-fold: 1) identifying metabolic dependencies that are specific to cancer cells, and 2) developing metabolic interventions that selectively target malignant cells while sparing normal tissues. We discovered that BCAT1, the first enzyme catalyzing branched-chain amino acid (BCAA) metabolism in the cytosol, is aberrantly activated in myeloid leukemia. This contrasts with its mitochondrial isozyme BCAT2, which is required for hematopoietic stem cell (HSC) function but dispensable for leukemogenesis. While BCAT1 activation is common in cancer, its oncogenic mechanisms remain elusive, raising a fundamental question about how BCAA metabolism is reprogrammed to meet the metabolic needs of cancer-initiating cells. In preliminary studies, we developed new mouse models, including BCAT1 and BCAT2 conditional knockout and knockin overexpression mice, to analyze the role of BCAT isozymes in hematopoiesis and leukemia. BCAT1 inactivation had no effect on hematopoiesis, whereas its overexpression enhanced HSC self-renewal and accelerated AML development. In contrast, BCAT2 is required for HSC function, but its overexpression had no effect on hematopoiesis. In compartment-specific genetic reconstitution assays, BCAT1-mediated effects on HSCs and leukemogenesis are compartmentdependent, and its cytosolic activity is necessary for producing S-adenosylmethionine (SAM), a universal methyl donor. These findings explain the BCAT1-associated DNA hypermethylation phenotypes in AML patients and reveal a new mechanism of metabolic compartmentalization in regulating the leukemic epigenome. Building on this new framework, this project aims to dissect the functional and mechanistic roles of compartmentalized BCAA metabolism in epigenetic regulation and AML pathophysiology. We hypothesize that BCAT1 promotes myeloid leukemogenesis by controlling compartment-specific BCAA metabolism and SAM-dependent epigenetic processes. We will test this hypothesis by establishing the functional roles of compartmentalized BCAA metabolism in myeloid leukemia and elucidating the mechanistic roles of BCAT1 in controlling SAM-dependent epigenetic processes. While we will prioritize human-based approaches such as AML cell lines and patientderived xenograft (PDX) models, mouse models will be used to assess metabolic regulation and function in vivo. Together, these studies will not only elucidate the mechanistic basis of ‘compartment-specific’ function for BCAT isozymes but also establish a new paradigm for compartment-dependent metabolism-epigenetics crosstalk in leukemia biology. The concepts, tools, and resources from this study will be broadly applicable to developing strategies to target the metabolic liabilities of cancer-initiating cells in hematological cancers and beyond.

NIH

Significance to VA: Defects of the craniomaxillofacial (CMF) skeleton are among the top reasons for Veterans seeking reconstructive surgery. CMF defects commonly occur in Veterans due to active combat as well as civilian life for etiologies spanning trauma, stroke, cancer, and cerebral aneurysms. Among CMF defects, the most devastating is the skull defect due to the necessity of an intact skull for cerebral protection, normal neurologic functioning, as well as psychological well-being and vocational opportunities, particularly when the defects are large and visible. The current clinically available materials for cranial defect reconstruction are limited by donor site morbidity for autologous bone and complications and cost for alloplastic materials, thereby providing an opportunity to develop strategies targeting skull regeneration. Innovation and Impact: This project introduces a novel anisotropic composite material consisting of nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) and a phosphate-eluting hydrogel (aMCGPh) to enhance osteogenesis and skull regeneration. This approach builds on extensive prior data on the base material MC-GAG combined with the observation that augmenting phosphate ion delivery in a temporospatially controlled manner can improve osteoprogenitor differentiation via the sodium phosphate cotransporters PiT-1 and PiT-2. The innovative materials-only strategy eliminates the need for growth factors or preloaded progenitor cells, offering a safe, cost-effective, and scalable option for cranial defect repair. Successful outcomes would provide Veterans with an off-the-shelf regenerative solution available at point-of- care in the operating room. Specific Aims 1. To evaluate the in vitro effects of aMCGPh on osteogenic differentiation of primary human mesenchymal stem cells (hMSCs). Objective: Characterize the contributions of phosphate dosage, diffusion, and mechanical properties of the respective hydrogels on the two aMCGPh variants on osteogenic gene expression and cell-mediated mineralization compared to MC-GAG. 2. To assess the in vivo safety and efficacy of aMCGPh in a rabbit calvarial defect model. Objective: Evaluate in vivo bone healing, biomechanics, inflammation, vascularization, and local and systemic safety of aMCGPh variants for calvarial regeneration. Methodology: Aim 1 will evaluate two dosages of aMCGPh with two different distances for phosphate diffusion, the necessity of PiT-1 and PiT-2 using knockdown studies, and the importance of mechanical properties of the hydrogel component to assess the impact of the phosphate-eluting hydrogel depot on hMSC osteogenic differentiation measured using gene expression, protein expression, and matrix mineralization studies. Aim 2 will assess efficacy of aMCGPh on in vivo rabbit cranial defect healing using radiographic, biomechanical, histologic studies as well as determine safety using bloodwork and distant tissue necropsy. In vivo comparison groups will include: unreconstructed defects, autologous bone-reconstructed defects, MC- GAG-reconstructed defects, and two dosages of aMCGPh-reconstructed defects. Path to Translation/Implementation: Our proposed studies are unified in the goal of delivering a useful, regenerative material for a common surgical problem in Veterans. Our preliminary work has outlined a promising composite material for skull defects, aMCGPh, for which we have already obtained a provisional patent (VA Invention ID 2023-139; Provisional Patent No. 63/503,824). This project will generate preclinical safety and performance data in preparation for an Investigational Device Exemption application to the FDA.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT The goal of this project is to develop tools to determine the mechanisms by which single amino acid variants affect myosin structure and function at multiple scales. Major challenges have been that: 1) a limited number of human β-myosin structures; 2) the computational expense (CPU time) to simulate the dynamics of such a large protein, and 3) difficulty in obtaining tissue from patients with disease causing variants. To overcome these challenges, we developed new structure-based, dynamics models of the chemo-mechanical cycle of β-myosin. These models are generated from new human crystal and cryo-EM structures, augmented by known structures available from bovine muscle. Our Molecular and Brownian Dynamics models contain F-actin structure to study how its interactions with β-myosin affects signaling between the nucleotide binding pocket, the actin-myosin interface, converter domain and other regions of myosin. These simulations are augmented by our novel inter- protein pathway analysis based on graph theory. We will use stochastic-kinetic models of sarcomeres to simulate contraction and relaxation with variable amounts of myosin variants. We will use gene edited human inducible pluripotent stem cells (hiPSCs) to validate our results. Our culture conditions result in myofibrils expressing predominantly β-myosin and demonstrate kinetics of adult myofibrils. We will purify myosin from these hiPSC- CMs for biochemical kinetic measures of the chemo-mechanical cycle. Each of these mechanical states results from structural changes in myosin and its association with actin. Additional studies will be performed at the level of myofibrils to study how mutations affect contractile function. We will also use single molecule, super-resolution to study high resolution structural changes in thick filaments from hiPSC-CMs, and Molecular Dynamics models of the Interacting Heads Motif structure of myosin on the thick filament backbone. This platform will be used to predict the effect of a selected group of variants of uncertain significance (VUSs).