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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 Cognitive Neuroscience (CogNeuro) program seeks to fund proposals that can advance our understanding of the neural mechanisms underlying human cognition and behavior. Funded proposals typically relate precise and rich quantifications of physiological responses and behavior in ways that advance theory (Intellectual Merit). Funded proposal also typically strengthen the field through, for example, outreach, mentoring the next generation of diverse cognitive neuroscientists, and/or increasing awareness and utilization of the research the field produces (Broader Impacts). <span style="text-decoration: underline;">Intellectual Merit</span> In general, successful proposals provide a theoretical motivation and consist of a series of experiments/analyses that test the differential predictions of that theory; they go beyond quantifying physiology during certain tasks and behaviors. Research topics considered for funding include but are not limited to: perception, recognition, categorization, implicit and explicit memory, working memory, attention, language, decision-making, and social reasoning. Commensurate with the inherently multidisciplinary nature of the field and the limitations of any single technique, a wide variety of physiological methods are considered, including but not limited to: neuroimaging (e.g., fMRI, EEG, MEG), non-invasive stimulation (e.g. TMS, tDCS), lesion analysis, intracranial recording, optogenetics, genetics, optical imaging, computational modeling, and pharmacological interventions in both human and non-human primates and other animal models. The program is particularly interested in proposals that achieve or enable convergence across multiple techniques. Critically, proposals will be returned without review if they are focused on: 1) either behavior or physiology and lack a specific link between them, 2) understanding clinical populations, 3) non-human animals without a clear benefit to our understanding of humans. <span style="text-decoration: underline;"></span> <span style="text-decoration: underline;">Broader Impacts</span> In general, successful proposals make impacts beyond traditional academic routes, such as publishing research or teaching undergraduate courses. Strong broader impacts can be quite varied but will typically involve specific efforts strengthening the field and/or increasing its visibility that leverage the characteristics of the institution, department, and/or researcher. Consider the following non-exhaustive examples: 1) STEM education and outreach, particularly in underserved communities, 2) directly involving undergraduates and high-school students in research, 3) making tools and applications for the general public, 4) science journalism or advocacy. These efforts can, but need not, directly relate to the proposed research. It is also encouraged to include budget for these efforts if warranted. <span style="text-decoration: underline;">Mentoring Plans</span> Strong mentoring plans generally go beyond inclusion in standard lab activities and incorporate specific ideas for forwarding the careers of young scientists and trainees that leverage the setting and content of the proposed research. <span style="text-decoration: underline;"></span> <span style="text-decoration: underline;">General Directions</span> Prior to the development of a full proposal, investigators are strongly encouraged to submit a one-page summary of the proposed research to a program director to evaluate its appropriateness for the CogNeuro program. Please contact the director early enough to allow for revisions and incorporation what may be extensive feedback. The summary should include an overview of your research and statements of intellectual merit and broader impacts, the two NSF review criteria. See the Merit Review Fact Sheet for more important facts about the NSF merit review process. Please read the NSF Proposal &amp; Award Policies &amp; Procedures Guide (PAPPG) carefully, as it will be strictly adhered to even if other directives or communications appear to contradict it. Currently, the average standard/CAREER award size is $175,000-$225,000 per year for 3-5 years. Awards in excess of $1.0 million are exceptionally rare and almost always multidisciplinary. Please be judicious in your requests, understanding the realities of the limited funding available for all proposals. See the Listing of Active Cognitive Neuroscience Awards for additional award information. Declined proposals are ineligible for resubmission until a minimum of one year has passed since the due date of their initial submission, unless specifically allowed by the PO in the feedback received during the decline process. This moratorium allows investigators the time required to digest the results of the merit review and revise their proposal accordingly. A proposal that has not been substantially revised will be returned without review as per the PAPPG. PIs are strongly encouraged to submit the Single Copy Document titled &ldquo;List of Suggested Reviewers&rdquo; with their full proposal. Sharing of data and other materials is an expectation for funded research. Please consult the NSF Dear Colleague Letter: Effective Practices for Data for more details. Interested in talking with a program director? Send a one-page description of the proposed research to sbe-cogneuro@nsf.gov.

U.S. National Science Foundation

The Cognitive Neuroscience (CogNeuro) program seeks to fund proposals that can advance our understanding of the neural mechanisms underlying human cognition and behavior. Funded proposals typically relate precise and rich quantifications of physiological responses and behavior in ways that advance theory (Intellectual Merit). Funded proposal also typically strengthen the field through, for example, outreach, mentoring the next generation of diverse cognitive neuroscientists, and/or increasing awareness and utilization of the research the field produces (Broader Impacts). Intellectual Merit In general, successful proposals provide a theoretical motivation and consist of a series of experiments/analyses that test the differential predictions of that theory; they go beyond quantifying physiology during certain tasks and behaviors. Research topics considered for funding include but are not limited to: perception, recognition, categorization, implicit and explicit memory, working memory, attention, language, decision-making, and social reasoning. Commensurate with the inherently multidisciplinary nature of the field and the limitations of any single technique, a wide variety of physiological methods are considered, including but not limited to: neuroimaging (e.g., fMRI, EEG, MEG), non-invasive stimulation (e.g. TMS, tDCS), lesion analysis, intracranial recording, optogenetics, genetics, optical imaging, computational modeling, and pharmacological interventions in both human and non-human primates and other animal models. The program is particularly interested in proposals that achieve or enable convergence across multiple techniques. Critically, proposals will be returned without review if they are focused on: 1) either behavior or physiology and lack a specific link between them, 2) understanding clinical populations, 3) non-human animals without a clear benefit to our understanding of humans. Broader Impacts In general, successful proposals make impacts beyond traditional academic routes, such as publishing research or teaching undergraduate courses. Strong broader impacts can be quite varied but will typically involve specific efforts strengthening the field and/or increasing its visibility that leverage the characteristics of the institution, department, and/or researcher. Consider the following non-exhaustive examples: 1) STEM education and outreach, particularly in underserved communities, 2) directly involving undergraduates and high-school students in research, 3) making tools and applications for the general public, 4) science journalism or advocacy. These efforts can, but need not, directly relate to the proposed research. It is also encouraged to include budget for these efforts if warranted. Mentoring Plans Strong mentoring plans generally go beyond inclusion in standard lab activities and incorporate specific ideas for forwarding the careers of young scientists and trainees that leverage the setting and content of the proposed research. General Directions Prior to the development of a full proposal, investigators are strongly encouraged to submit a one-page summary of the proposed research to a program director to evaluate its appropriateness for the CogNeuro program. Please contact the director early enough to allow for revisions and incorporation what may be extensive feedback. The summary should include an overview of your research and statements of intellectual merit and broader impacts, the two NSF review criteria. See the Merit Review Fact Sheet for more important facts about the NSF merit review process. Please read the NSF Proposal &amp; Award Policies &amp; Procedures Guide (PAPPG) carefully, as it will be strictly adhered to even if other directives or communications appear to contradict it. Currently, the average standard/CAREER award size is $175,000-$225,000 per year for 3-5 years. Awards in excess of $1.0 million are exceptionally rare and almost always multidisciplinary. Please be judicious in your requests, understanding the realities of the limited funding available for all proposals. See the Listing of Active Cognitive Neuroscience Awards for additional award information. Declined proposals are ineligible for resubmission until a minimum of one year has passed since the due date of their initial submission, unless specifically allowed by the PO in the feedback received during the decline process. This moratorium allows investigators the time required to digest the results of the merit review and revise their proposal accordingly. A proposal that has not been substantially revised will be returned without review as per the PAPPG. PIs are strongly encouraged to submit the Single Copy Document titled List of Suggested Reviewers with their full proposal. Sharing of data and other materials is an expectation for funded research. Please consult the NSF Dear Colleague Letter: Effective Practices for Data for more details. Interested in talking with a program director? Send a one-page description of the proposed research to sbe-cogneuro@nsf.gov.

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.

Comcast NBCUniversal Foundation

Supports digital equity, STEM education, and community development programs in communities served by Comcast 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).

Consolidated Edison Foundation

Supports clean energy education, STEM programs, and environmental sustainability in the greater New York City area.

NIMH - National Institute of Mental Health

PROJECT SUMMARY/ABSTRACT___________________________________________________________ Neurodevelopmental disorders (NDDs) like autism, intellectual disability, and ADHD are a global health concern. Progress in understanding the causes of NDDs has been slow and treatment options for core symptoms are limited, partially because these brain disorders currently do not have well-defined biological signatures of pathology that can be utilized in experimental neuroscience paradigms or targeted for intervention. There has been considerable recent progress in identifying statistically significant, high-confidence NDD genetic risk variants, including the heterozygous loss of the 16p11.2 chromosomal region (16pdel). Emerging evidence suggests that 16pdel impacts key prenatal neurodevelopmental processes, though the exact disease mechanisms remain elusive. Patients exhibit neuroanatomical features that could potentially be linked to dysregulated response to signal transduction pathway stimulation. Here, we will investigate how 16pdel fetal brain cells respond to key developmental signaling pathway stimulations at the molecular and cellular levels to identify potential dynamic context-dependent disease mechanisms. To address this question, we will leverage a novel in vitro human stem cell-based platform we developed called the "cell village," which enables large-scale, high-throughput analysis of molecular and cellular responses to cell-extrinsic stimuli across diverse neurotypical control and 16pdel patient donor cell lines. This approach minimizes technical variation and allows for systematic exploration of genetic and cellular variability in a controlled uniform environment. By integrating multi-omic datasets, our study aims to uncover the mechanistic links between gene expression, cellular phenotypes, and pathway activation in 16pdel brain cells in a dish. The proposal is structured around three specific aims: First, we will define the nature and dynamics of signaling dysfunction in 16pdels as hypersensitive, hyperresponsive, and/or hyperactivated in response to stimulation. Second, we will decipher the epigenetic mechanisms underlying pathway dysregulation by investigating chromatin accessibility, methylation patterns, and 3D genome organization in first-of-their-kind multi-omic villages. This aim seeks to determine if 16pdel cells are epigenetically primed for aberrant response to signaling pathway stimulation. Third, we will determine the cellular consequences of dysregulated signaling in 16pdels with a particular focus on neurogenesis and morphogenesis mechanisms. By elucidating the molecular mechanisms driving signal transduction pathway dysregulation in 16pdel fetal brain cells, as well as the impact on cellular functions critical for proper neurodevelopment, this research could illuminate the fundamental neurobiology of a high-confidence genetic risk factor associated with complex NDDs and nominate future targets for intervention, thus improving outcomes for patients and their families.

NCATS - National Center for Advancing Translational Sciences

Project Summary INTS12 is a chromatin interacting subunit of the mammalian Integrator complex that binds to accessible chromatin and controls various aspects of transcription such as elongation and termination. INTS12 interacts with negative elongation factors as well as RNA Polymerase II and regulates its pausing and release into productive elongation. Studies on INTS12 biology in various mammalian systems such as hematopoietic tissues is lacking despite ample evidence of its presence and likely contributions to hematopoietic development and disease. For instance, INTS12 is highly expressed in early stage erythroid progenitor cells during ex vivo erythroid differentiation of human CD34+ hematopoietic stem cells, and it is significantly reduced in the rare hemolytic anemia Congenital Dyserythropoietic Anemia Type IV (CDA IV; CDAN4), which affects young children and renders them transfusion dependent. This proposal will address our core hypothesis that INTS12 function is important for transcription regulation during normal human red cell development and reduced INTS12 levels in CDA IV red cells contributes to ineffective erythropoiesis due to disrupted RNA Polymerase II elongation control. To achieve this, a newly established erythroid progenitor cell line called BEL-A that can be expanded indefinitely, and differentiated to mature erythroid cells will be used. Aim1 will focus on INTS12 chromatin binding in normal wild type (WT) and CDA IV mutant BEL-A cells, and this will be correlated with existing unpublished chromatin accessibility and transcription factor binding data from erythroid stage-matched WT and CDA IV BEL-A cells. Further, the effect of depleting or overexpressing INTS12 on red cell development in WT and CDA IV will also be characterized. In Aim2, we will explore INTS12 functions in transcription by assessing the impact of INTS12 perturbations such as depletion and overexpression on nascent gene expression and RNA Polymerase II occupancy. These data will be correlated with INTS12 occupancy determined from Aim1, and any alterations in nascent transcription leading to changes in RNA Pol II pausing and elongation due to perturbed INTS12 levels will suggest that INTS12 contributes to altered transcription regulation in CDA IV. Transcription elongation by RNA Polymerase II is highly regulated and involves many transcription co- factors and epigenetic mechanisms, some of which are chemotherapy targets for certain hematological malignancies. Further, INTS12 interacts with chromatin using a conserved PHD domain that is being investigated as a potential chemotherapy target using derivatives of a class of compounds known as Amiodarones. The knowledge harnessed from this proposal will thus enable future investigations into INTS12 and Integrator complex biology in erythropoiesis, as well as translational studies on the potential for INTS12 as a therapeutic target in CDA IV patients.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Hematopoietic stem cells (HSCs) reside in specialized microenvironments in the bone marrow (BM) that comprise multiple cellular constituents, including nerves, with vital regulatory functions. Our previous studies established that the nociceptive nervous system is an essential BM niche component controlling HSC mobilization from the bone marrow into the peripheral blood. Our new preliminary resutls reveal that aging is associated with a progressive loss of nociceptive innvervation in the bone marrow, and that supplementaiton of nociceptive nerve-derived CGRP attenuates the expansion and BM HSCs in aged mice, restores their engraftment ability, and reverses the myeloid-biased differentiation. Based on these findings, we hypothesize that nociceptive nerve-derived CGRP is a critical rejuvenating signal for aged HSCs, and that targeted chemogenetic activation of BM nociceptive neurons using DREADDs can be harnessed to reverse HSC aging in vivo. In the Specific Aim 1, we will determine whether CGRP administration reverses bona fide aging hallmarks and identify the downstream molecular programs that mediate this rejuvenation. In Specific Aim 2, we will investigate whether local activation of BM nociceptive neurons using DREADDs rejuvaentes old HSC in vivo. Together, these proposed studies will shed light into the critical functions of an under-appreciated component of the HSC niche and uncover novel therapeutic strategies for age-associated hematopoietic diseases.

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

PROJECT SUMMARY Proper embryonic development requires the precise orchestration of a series of well-conserved developmental steps in both space and time. Defects in the temporal unfolding of these steps can give rise to congenital diseases. Surprisingly, the speed of embryonic development is highly species-specific. For instance, among mammals, the duration of embryogenesis ranges from 15 days in mice to 116 days in elephants. Understanding how these species-specific developmental rates are set could have significant translational potential, including accelerating stem cell differentiation for cell therapies or decelerating cellular processes to combat tumor growth and aging. To precisely measure developmental speed across species, we rely on the segmentation clock, a molecular oscillator whose period tightly correlates with the duration of embryogenesis. We have recapitulated the segmentation clock in vitro using mouse and human pluripotent stem cells, therefore enabling cross-species comparisons. Using this experimental platform, we recently found that a key factor regulating developmental speed is the cytosolic NAD+/NADH ratio. This parameter was elevated in mouse cells (fast-developing) compared to human cells (slow-developing). Moreover, experimentally lowering the NAD+/NADH ratio slowed down the segmentation clock, whereas its increase resulted in acceleration. Here we propose to identify the mechanisms that give rise to differential cytosolic NAD+/NADH ratios between species. Cytosolic redox balance is known to be regulated by NADH shuttling systems that oxidize NADH in the cytosol and transfer the reducing equivalents to the mitochondria. Two primary NADH shuttles exist: the glycerol-3-phosphate (G3P) and the malate-aspartate (MA) shuttle. Preliminary data suggest that the G3P shuttle is more active in mouse embryonic cells than in human cells due to differential expression of shuttle enzymes. We hypothesize that species-specific expression of G3P shuttle components in mouse and human embryonic cells drives differences in G3P shuttle activity, thereby establishing distinct cytosolic NAD+/NADH ratios and contributing to the disparity in developmental speed between these two species. In Aim 1, we will directly test the role of NADH shuttles in establishing species-specific NAD+/NADH ratios by comparing shuttle fluxes between mouse and human embryonic cell types, and by manipulating the expression of shuttle components. In Aim 2, we will investigate the regulatory mechanisms underlying species-specific expression of NADH shuttle components, particularly GPD1L, a key enzyme in the G3P shuttle which is expressed in mouse cells but not human cells. By identifying the cis- and trans-regulatory elements controlling species-specific GPD1L expression, we will begin to uncover the genetic basis for differences in developmental speed between species. Overall, the proposed research will provide critical insights into species-specific developmental timing and enhance the utility of human stem cells for therapeutic applications by devising novel strategies for the acceleration of developmental rate.

NCI - National Cancer Institute

PROJECT SUMMARY In cancer and chronic infection, T cells develop into a dysfunctional state called exhaustion because of persistent antigen stimulation. Exhausted T cells upregulate immune checkpoints, display dysregulated metabolism, and progressively lose effector function and the ability to persist and develop immune memory. T cell exhaustion hinders the clearance of pathogens and malignant cells by the immune system and limits the effectiveness of immunotherapies such as chimeric antigen receptor (CAR) T cell therapy. Therefore, understanding how T cells adapt to chronic antigen receptor signaling is critical for developing more effective immunotherapies. A stem-like CD8 T cell subset has been identified in chronic infections and cancers. Stem-like CD8 T cells mediate long-term immunity by self-renewal and replenishing other CD8 T cell subsets. Stem-like properties in T cells are essential for the efficacy of immunotherapies. We have identified key transcription factors that regulate the differentiation of stem-like CD8 T cells. However, the molecular program underlying the adaptation of stem-like CD8 T cells to chronic antigen receptor signaling is incompletely understood. Our recent findings show that the redox sensing KEAP1-NRF2 pathway is critical for the differentiation of stem-like CD8 T cells and adaptation of CD8 T cells to chronic antigen receptor signaling. In the proposed study, we will determine how the KEAP1-NRF2 pathway regulates the adaptation of CD8 T cells to persistent antigen receptor signaling through preventing TCR hyperactivation and promoting metabolic fitness. Our study will shed important new light on the development of more potent and efficacious immunotherapies for cancers and chronic infections. Furthermore, KEAP1 and NRF2 have been extensively investigated as potential drug targets for chronic diseases. Therefore, our findings hold significant promises for informing interventions that aim at modulating this pathway.