Fred Hutchinson Cancer Center

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT This proposal presents a five-year research career development program focused on the study of T-cell reconstitution following hematopoietic stem cell transplantation (HSCT) and how graft-vs-host disease (GVHD) can be shaped by clonotypic response to microbiota. The candidate is currently an Instructor of Medicine at the University of Washington and Research Associate at the Fred Hutchinson Cancer Research Center. This proposal builds on the candidate’s previous research and clinical experience by providing advanced training in two domains of expertise represented by his mentor team of Dr. Geoffrey Hill (GVHD in HSCT) and Dr. Philip Bradley (computational modeling of the T-cell response). The proposed experiments and didactic work will provide the candidate with a unique set of cross-disciplinary skills that will enable his transition to independence as a physician scientist in T cell mediated immunity in HSCT. T-cells play a fundamental role in the pathogenesis of GVHD which remains a major barrier for the successful application of HSCT for a wide range of benign and hematologic malignancies. GVHD involvement of the gastrointestinal (GI) tract remains a major cause of morbidity and mortality. The composition of the GI microbiome is associated with onset and severity of GVHD, though current understanding of how specific microbial species contribute to GVHD pathogenesis remains largely correlative with limited mechanistic insights. While the microbiome acts as a major source of cognate antigen for T cells, little is known about how anti-microbial TCRs may contribute to pathology in this setting. Part of the difficulty in parsing out the alloreactive response from immune surveillance on a clonal level includes the vast combinatorial diversity of αβ TCRs, the high prevalence of low copy number TCRs in any given donor pool, and sampling limitations. The foundation of this proposal is based on preliminary studies using a novel computational algorithm to identify expanded donor TCRs that are not constrained by donor and host genetics, but rather appear to be influenced by commensal microbes. How exactly these anti-microbial TCRs might function in the post-transplant context and its physiological relevance to GVHD are questions that this proposal begins to address. More specifically, the aims of this proposal are to 1) Validate computationally identified anti-microbial CD4+ TCRs in an scRNA seq platform and define cellular phenotypes in relation to compartment localization, 2) Reconstruct computationally identified CD4+ TCRs and determine antigenic specificity through genomic screening, and 3) Dissect the functional role of identified anti-microbial CD4+ TCRs on the propagation of acute graft-vs-host disease. The scientific objective of this proposal is to examine the drivers of clonotypic T-cell expansion following allogeneic stem cell transplant and assess how the pathogenesis of GVHD is coupled to microbial surveillance.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Immunotherapy using CAR-T cells has induced dramatic responses in hematological malignancies, but extending this success to more common epithelial cancers, which cause the greatest mortality, has proved more challenging. In a phase 1 trial we conducted, CAR-T cells targeting the tumor-associated antigen ROR1 induced responses in patients with chronic lymphocytic leukemia but became terminally exhausted in breast and lung cancer patients, indicating that strategies to preserve CAR-T function are needed to achieve efficacy in solid tumors. Recent work has demonstrated that a subset of PD-1+Tcf1+ “precursor” exhausted cells (Tpex) is critical for mediating successful tumor responses to immunotherapy. Tpex retain a stem-like capacity for self-renewal, proliferation, and the ability to differentiate into effector-like cells. Interestingly, Tpex preferentially co-localize with antigen-presenting cells (APCs) and reside in tumor-draining lymph nodes (tdLN), where they may be protected from factors in tumors that drive terminal exhaustion, such as more chronic antigen stimulation, mTOR activation, and hypoxia. Unlike conventional T cells, CAR-T cells are activated by intact antigen expressed on tumor cells, not by peptide/MHC complexes expressed on both tumor cells and APCs. This design restricts CAR- T activation exclusively to tumors, where preservation of stem-like qualities may be impaired relative to tdLNs. To test this, we developed a Kras/p53 (KP) autochthonous model of ROR1+ lung cancer that recapitulates the dysfunction of ROR1 CAR-T cells we observed in patients. Using this model, we found ROR1 CAR-T cells did not accumulate in tdLNs and showed faster attrition of Tpex and terminal exhaustion in tumors compared to tumor-specific T cells (TCR-T) that were cultured identically prior to infusion. Tcf1+ CAR-T cells showed greater proliferative capacity, stem-like self-renewal, and tumor control compared to Tcf1- CAR-T cells, suggesting that strategies to preserve Tcf1+ CAR-Ts could significantly improve efficacy. Interestingly, CAR-T cells in tdLNs formed a small reservoir of “pre-exhausted” PD-1-Tcf1+ central memory cells that we could increase by vaccination and that may be able to generate more polyfunctional effectors than Tpex, which are epigenetically committed to producing hypofunctional effectors. We hypothesize that insufficient activation of CAR-T cells by APCs in tdLNs impairs maintenance of stem-like Tpex reservoirs, resulting in less durable tumor control, and that using vaccination to increase reserves of Tcf1+ CAR-T cells in LNs will overcome this barrier. In this proposal, we will use the KP model to determine: 1) whether CAR-T are impaired in maintaining a stem-like Tcf1+ reservoir relative to TCR-T cells and whether this is due to insufficient activation by APCs; and 2) whether activating CAR- T cells with endogenous APCs in LNs via a novel vaccine platform will improve numbers of Tcf1+ CAR-T cells, tumor control, and response to PD-L1 blockade. Our results will reveal whether CAR-T exhaustion in solid tumors is driven in part by impaired Tpex formation and will evaluate a novel vaccine platform to overcome these defects that can be translated to the clinic to enhance the efficacy of CAR-T cells in patients with solid tumors.

NIH Research Projects · FY 2025 · 2023-06

Project Summary/Abstract (Overall Component, RFA-CA-22-040) We propose to develop a blood-based test whose indicated use is to complement mammography in the early detection of breast cancers. Although mammography saves lives through early detection, it is imperfect. Approximately one in seven breast cancers goes undetected despite screening mammography, and interval cancers that manifest within a year of a normal mammogram remain a vexing problem, especially (although not exclusively) for the >27 million women in the United States with heterogeneously or extremely dense breasts at high risk for interval cancers. We propose to develop a blood test that could be used as an adjunct to mammography to improve early detection by improving sensitivity and/or specificity of mammography. Using a novel biomarker discovery approach leveraging human-in-mouse breast cancer patient-derived xenograft models and state-of-the-art mass spectrometry methods, we prioritized 162 candidate breast cancer protein biomarkers for validation studies. Our BCC will perform EDRN Phase 2 biomarker validation studies of our prioritized 162 candidate plasma protein biomarkers of breast cancer. We have an experienced multidisciplinary team (including two junior investigators) with a strong track record of productive collaboration and representing expertise in clinical oncology, cancer biomarkers, pathology, CLIA/CAP/GLP assays, epidemiology, radiology/breast imaging, cancer screening, ‘omics data generation, and biostatistics. Our team includes 2 industry partners, encompasses 3 CLIA laboratories, and can provide expertise and access to multiple quantitative platforms in a CLIA/CAP/GLP environment to support EDRN Network Collaborations for biomarker validation studies with other BCCs. The Biomarker Development Laboratory will contribute to the biomarker validation studies by: (i) developing qualified reagents & methods for quantifying 162 candidate protein biomarkers, (ii) procuring plasma biospecimens (compliant with EDRN PRoBE study design) for phase 2 biomarker validation studies, (iii) delivering plasma aliquots to our BRL CLIA labs in a blinded fashion, and (iv) analyzing EDRN phase 2 validation data generated by the BRL, providing statistical, epidemiological, and breast imaging expertise to set and evaluate performance metrics to ensure biomarkers are adequate to provide clinical utility for early detection. The Biomarker Reference Laboratory will contribute to the proposed validation studies by: (i) validating a CLIA- compliant standard operating procedure for an immuno-MRM assay to quantify up to 162 candidate protein biomarkers of early-stage breast cancer that will serve as the basis for our biomarker validation studies, (ii) performing phase 2 biomarker validation studies, and (iii) providing reference laboratory support for the EDRN network (e.g., mass spectrometric analyses, flow cytometry, NextGen sequencing and ELISA assays. The Administrative Core will support all aspects of the BCC, including managing all administrative and project management aspects of the BCC as well as managing all center logistics and communication.

NIH Research Projects · FY 2026 · 2023-06

ABSTRACT In Zimbabwe, the burden of cancer among people with HIV is increasing. Consistent engagement in HIV care is critical to reducing morbidity and mortality among people with HIV in general, and continuation of ART through cancer treatment is essential for people with HIV who have cancer. While HIV primary care is decentralized and highly accessible, specialty care, including for cancer treatment, is limited to a few major hospitals. This results in unique barriers to concurrent engagement in HIV and cancer care continua for Zimbabwe’s largely rural population. While guidelines for treatment of people with HIV and cancer in low- and middle-income settings recommend integration of cancer and HIV care, there is a limited evidence base for how to accomplish this successfully in these settings. There is an important gap in provision of continuous HIV care when people with HIV in Zimbabwe receive a concurrent diagnosis of cancer. The goal of this project is to address that gap by identifying barriers to HIV care engagement specific to people with HIV receiving cancer treatment, and devising, refining, and testing strategies to address those barriers. In Aim 1, we will use questionnaires and routinely collected clinical data to measure disruptions to HIV-related care over time among cancer patients with HIV. In Aim 2A, we will use a mixed-methods approach including questionnaires and in-depth interviews to quantify and contextualize patient-identified barriers and facilitators to consistent engagement in the HIV care continuum during cancer treatment. In Aim 2B, we will first prioritize barriers based on ubiquity and strength of impact, then convene a group of experts and key stakeholders in HIV and cancer healthcare and research to develop a set of strategies to address the highest priority barriers. Strategies will be implemented and refined one at a time using a rapid cycle improvement process, with a final set of 2-3 refined strategies combined and piloted together in Aim 3. In Aim 3, we will measure the acceptability of the package of strategies to patients and hospital staff as our primary outcome. Secondary outcomes will include uptake, experience of barriers and facilitators to HIV care engagement, and disruptions to HIV and cancer care. We will compare secondary outcomes in Aim 3 participants to Aim 1 participants in a quasi-experimental pre-post study design. These aims constitute the mentored research component of the candidate’s career development plan. In parallel with this research, the candidate will pursue additional training in implementation science, qualitative methods, and the design and evaluation of clinical care interventions, supported by an exemplary team of experienced HIV and cancer investigators in the United States and Zimbabwe. The proposed work will address a critical gap in provision of clinical care for people with HIV and cancer in Zimbabwe, and substantially expand Dr. Montaño’s methodological expertise, providing the training, experience and data for her transition to independence. The high burden of HIV and cancer, decentralized primary care infrastructure coupled with limited access to specialty cancer treatment, and low cost of doing research make Zimbabwe an ideal setting to conduct this formative study. However, the lessons learned from this research will have broad application, including in rural settings in the US, where limited access to specialty care often results in similar barriers to healthcare engagement and burden on patients seeking care via siloed and geographically dispersed health systems.

NIH Research Projects · FY 2026 · 2023-06

Project Summary/Abstract Among the several ongoing revolutions in biological research, two (the ability to visualize increasingly complex biomolecular machines and assemblages and the ability to create novel protein structures and corresponding functions) represent the foundation of our laboratory's research program. For many years, we have divided its activities between (i) determining the structures, mechanisms and biological roles of gene targeting proteins and base modifying enzymes, and (ii) employing protein engineering to create new protein folds, assemblages, and functions, and to test our understanding of protein form and function. We now plan to renew and expand upon our research mission by address several questions and problems in the fields of nucleic acid enzymology, molecular recognition, and protein engineering. The first two projects will build upon our experience studying and engineering homing endonucleases (also called `meganucleases'). In doing so, we will answer questions surrounding the evolution, recognition mechanisms and engineerability of (i) sequence-specific microbial RNA endonucleases and (ii) eukaryotic retrotransposons. We plan to leverage our basic studies of these systems to create new tools for transcriptomic analyses and genome engineering, respectively. The third project addresses a current challenge in protein engineering: the accurate design of biomolecular interfaces that rely heavily on stereospecific hydrogen-bond networks to facilitate the balance of affinity, specificity and reversibility that is a hallmark of biomolecular interactions and regulation. To do so, we will create and then couple together protein- protein and protein-small molecule binding functions through the creation of novel synthetic ligand-induced protein multimerization systems. The project will contribute to the field of molecular design and engineering by optimizing strategies for the accurate design of stereospecific hydrogen bond networks for that facilitate protein-protein and protein-ligand recognition and binding.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY/ABSTRACT Colorectal cancer (CRC) remains one of the leading causes of cancer-related deaths around the world. Many environmental risk factors and over 200 genetic risk variants have been identified for this complex, multifactorial disease. However, despite the strong biological rationale for the importance and abundance of gene-environment (GxE) interactions, the extent to which environmental risk factors (broadly defined here as lifestyle, diet, obesity, drug use and intermediate biomarkers) modulate genetic risk factors is poorly understood. To achieve the promise of precision prevention, we need to gain a deeper understanding of GxE interactions in CRC risk. Understanding which modifiable risk factors modulate genetic risk, which is fixed, provides biological insights and actionable targets for new prevention intervention strategies. To accelerate the discovery of GxE interactions in CRC risk and to take an important next step towards translation, we propose a comprehensive innovative approach that combines single-cell multi-omics data, individual-level harmonized epidemiological and clinical data, and genome-wide data from large, well-characterized, study populations, with novel computational and statistical approaches. Dramatic improvements in single-cell multimodal omics technologies, combined with new computational tools based on powerful deep-learning modeling approaches now allow us to predict the impact of genetic variants on gene regulation in a cell-type-specific holistic manner. Because simultaneously measured single-cell gene expression (scRNA-seq) and chromatin accessibility (scATAC-seq) data for normal colorectal mucosa tissue is lacking for samples from different population groups with detailed assessment of environmental risk factors, we propose in Aim 1 to generate such data for 50 individuals. This resource, together with other single cell multi-omics compendia for colorectal tissue (like HTAN), will be leveraged to develop functional prediction scores for genetic variants across the genome. In Aim 2, we will use these functional prediction scores to boost statistical power for discovery of novel GxE interactions. We will perform genome-wide GxE scans in over 230,000 CRC cases and controls across genetic ancestries, and key environmental risk factors, including obesity, diabetes, smoking, alcohol, drug use, dietary factors and intermediate biomarkers linked to metabolic dysregulation and chronic inflammation. To expand the number of key risk factors we can evaluate, we will utilize existing genetic instruments. In Aim 3, we will comprehensively characterize and translate GxE interactions. To do so, we will stratify GxE findings by clinical factors, including age of onset, demographics, and tumor subtypes. Additionally, we will incorporate GxE interactions and genetically predicted biomarkers in a comprehensive trans-ancestral risk prediction model to improve prediction and provide actionable information to reduce CRC risk. Our advisors have stressed the importance of including the interplay between genetic and environmental risk factors in risk prediction modeling to enhance the acceptance of risk prediction models.

NIH Research Projects · FY 2026 · 2023-06

This application focuses on Fbw7, an E3 ubiquitin ligase and tumor suppressor, and on its substrate c-Myc (hereafter, Myc), an oncogenic transcription factor widely implicated in human cancers. E3 ligases mark protein substrates for degradation through ubiquitin conjugation. Fbw7 recognizes a network of proteins with crucial roles in proliferation, differentiation, metabolism, and apoptosis. Fbw7 substrates include important oncoproteins (e.g., cyclin E, Notch, Myc, Jun) and thus Fbw7 mutations promote tumorigenesis by deregulating its oncogenic substrates. The Fbw7 pathway therefore has broad implications for cancer biology and for the development of new therapeutic strategies. We will address important and unresolved aspects of this pathway in two general areas. The first involves how phosphorylation and Fbw7 control Myc stability and activity, in both normal cells and in cancers. The second area will address new paradigms in the ways that Fbw7 recognizes substrates and how these interactions may underlie Fbw7 mutations in cancers. This proposal may thus impact many areas of research related to Myc and the Fbw7 pathway. Fbw7 binds substrates after they are phosphorylated within motifs termed degrons, that typically contain two phosphorylated residues that interact with Fbw7. The first two Aims are focused on Myc regulation by phosphorylation of a newly discovered Myc T244 degron that acts in concert with the canonical Myc T58 degron to bind Fbw7 dimers. Aim 1 will study how T244 degron phosphorylation is regulated in normal and tumor cells and whether hierarchical Myc T244 degron phosphorylation controls Myc stability, as well as how the T58 and T244 degrons are coordinately regulated by mitogenic and oncogenic signaling pathways. Aim 2 will study the functions of the T244 degron in normal cells and tumorigenesis and how it cooperates with the T58 degron. This will be accomplished through physiologic knockin models, in human cells and mice, to create engineered Myc mutations that ablate Myc degron phosphorylations. Aim 3 will study how Fbw7 dimers interact with substrates and how these interactions underlie Fbw7 mutations and their functions in cancers. This includes determining the extent to which two separate degrons are required for degradation across the Fbw7 substrate network. Identifying these new degrons may lead to entirely new pathways that control the degradation of critical substrates and that may be abnormal in cancer cells. Tumors often have heterozygous Fbw7 missense mutations that dimerize with wt-Fbw7, and the hypothesis that these mutations specifically stabilize oncogenic substrates that require two degrons will be tested. Finally, the Myc T244 degron binds Fbw7 through a novel mode that involves Fbw7 R689, a tumor hotspot, and the role of R689 in dimer-dependent selective substrate recognition will be studied, as well as similar possible functions for other Fbw7 missense mutations.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY Alaska Native people have the highest incidence and mortality rates of colorectal cancer (CRC) globally, with rates that are more than double those observed in the general U.S. population (incidence: 89.0 vs. 38.6/100,000; mortality: 39.6/100,000 vs. 13.8/100,000). These high rates cannot be explained by access to screening, as dedicated efforts to increase CRC screening have resulted in screening rates in Alaska Native people that are comparable to the US average. These disparities have persisted for over 40 years and are of noted concern to community members and Tribal health leaders alike, making this a priority research area for Alaska Native people. A key knowledge gap is the lack of sizable and comprehensive studies characterizing the molecular features of colorectal tumors in Alaska Native patients. Accordingly, we propose an integrative approach to better understand CRC tumorigenesis and molecular subtypes in this high-risk population, and to evaluate whether various tumor and tumor microenvironment features contribute to disease progression. Specifically, we aim to deeply characterize the mutational, transcriptional, and cellular landscapes of CRC tumors and their immune microenvironments in 500 Alaska Native patients and link these characteristics to CRC-specific mortality. We will build on our existing collaboration between the Alaska Native Tribal Health Consortium (ANTHC) and the Fred Hutchinson Cancer Center. We will capitalize on ANTHC’s unique biorepository (linkable to comprehensive medical records) that contains tumor tissue samples of the majority of Alaska Native CRC patients diagnosed in Alaska. Pilot data generated using biospecimens from this resource and utilizing the same platforms as proposed here demonstrate high-quality data and ensure that IRB and tribal approvals are in place to enable rapid execution of the proposed work. To identify clinically meaningful population differences, we will compare molecular profiles with those from non-Hispanic White CRC patients using available data. In Aim 1, we will perform whole-exome sequencing of CRC tumors and paired normal samples to identify driver genes and clinically actionable mutations particularly relevant to the Alaska Native population. To gain etiological insight, we will analyze mutational signatures that may be linked to environmental exposures and identify germline mutations in high-penetrance CRC risk genes to investigate their role in the increased CRC risk in Alaska Native people. In Aim 2, we will apply two complementary cutting-edge spatial profiling technologies to fully characterize the tumoral immune response and discover mechanisms of immune evasion. We will perform spatial whole transcriptome profiling of CRC tumors using Nanostring’s GeoMx Digital Spatial Profiling technology. We will use Akoya Biosciences’ PhenoCycler platform for spatially resolved single immune cell phenotyping. We expect that insights gained through this work can inform the development of high-value, novel therapeutic strategies. In Aim 3, we will leverage these molecular data to develop novel predictors of CRC-specific mortality that could help guide clinical decision making, improve outcomes, and reduce long-standing disparities.

NIH Research Projects · FY 2025 · 2023-05

Project Summary/Abstract The HIV Vaccine Trials Network (HVTN) has experienced significant challenges recruiting participants in early-stage HIV vaccine clinical trials. Formidable barriers to participation in HVTN preventive HIV early phase trials, including lack of HIV public urgency, numerous study visits, long-term follow-up, vaccine induced seropositivity and HIV stigma, have resulted in significant challenges enrolling participants into these trials. We will co-refine an existing media campaign, develop, test and evaluate a multilevel communication strategy to address some of these crucial concerns and strengthen our reputation of trustworthiness in marginalized communities disproportionately affected by HIV to increase their enrollment into early-stage HIV vaccine trials. Over the next 7 years, the HVTN will focus on the early phase trial program to move the field forward and to identify products efficiently and effectively for later efficacy testing. This will require the development and deployment of evidence-based strategies to combat misinformation and to strengthen community engagement. Our team and the clinical research sites in our Network have forged longstanding community partnerships with diverse stakeholders. During our phase 3 clinical trials for the US government-funded COVID-19 vaccine clinical trials we developed new strategies to supplement our community engagement program: (1) a consumer-driven marketing and advertising campaign; (2) virtual town halls and listening sessions; and (3) the use of expert panels to review protocols and provide guidance and direction. We will build on this work to inform our HIV strategy. We assembled an experienced team of diverse researchers to accomplish the following aims: 1) Identify components of an initial media campaign that impact HIV vaccine trial registration and develop a multilevel communication strategy; 2) Evaluate the impact of the multilevel communication strategy on Phase I HIV vaccine trial registration; and 3) Determine if the multilevel communication strategy generates public engagement with vaccines and vaccine trials. This project will be the first large-scale communication study conducted in the United States to improve community awareness of and engagement with HIV vaccine trial research. It will assess misinformation and engagement with this strategy to inform current and future HIV vaccine trials recruitment and is expected to have a positive health impact by establishing robust strategies to improve diversity, equity, and inclusion in Phase 1 preventive HIV vaccine trials.

NIH Research Projects · FY 2026 · 2023-05

Project Summary/Abstract Nowhere are racial disparities in cancer survival rates as striking as in hematopoietic cell transplantation (HCT) because both the patient and the donor contribute to survival outcomes. HLA genetic features are ancestry- informative. When ancestry-specific features are accounted for, survival disparities are diminished but major gaps still exist between African, Hispanic, Asian and Caucasian American transplant patients. Relapse of the blood cancer remains the chief cause of transplant failure; however, the role for the NKG2 axis, the major anti- tumor pathway, in relapse and survival after HCT is ill-defined. The unmet need is to identify the NKG2 ligand/receptor immunogenetic factors involved in relapse and which account for disparities in survival. If these factors were known, then prospective risk-assessment of the patient’s germline and optimized donor selection could diminish or even abolish survival disparities across US populations. We have elucidated the survival disparities between HCT patients of African, Hispanic, Asian and Caucasian American ancestry, and have identified key ancestry-informative NKG2 ligand and receptor variants that impact gene expression and survival. We propose to identify ancestry-specific NKG2 ligand and receptor missense and regulatory variation that account for survival in each race, and examine the impact of optimal features across races to minimize or abolish survival disparities in HCT. The specific aims are to 1) identify mechanisms that underpin NKG2 ligand/receptor expression variation in diverse races; 2) define NKG2 ligand/receptor ancestry-informative variation for survival in each race, and 3) define survivorship disparities with ancestry-specific “ideal” immunogenetic characteristics. The goals will be achieved through systematic analysis of NKG2 ligand/receptor variation to identify functional variants, followed by large-scale genotyping of related donor, unrelated donor and cord blood transplant patients and donors and identification of ideal features that inform survival within each race. The extent to which survival disparities may be diminished or even abolished through transplantation of patients and donors with ideal characteristics will be defined. The information from this project will increase the success of transplantation for all patients in need of this life-saving therapy.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY/ABSTRACT The long-term objective of our research is to learn and understand genetic mechanisms underlying initiation and progression of type 1 diabetes mellitus (T1DM), and to develop effective screening, diagnosis, preventive, and treatment strategies in the fight against T1DM. In this project, our focus is on genetic associations with the progression from seroconversion to the onset of T1DM, more specifically, discovering novel Conformational Regulatory Segments (CRS) in HLA/KIR/FcGR/IGHG genes that are responsible for the disease progression; CRS refer to both regulatory and functional elements, including peptides, nucleotides or other regulatory elements within or between genes. The HLA/KIR/FcGR/IGHG genes are highly polymorphic and Next Generation Targeted Sequencing (NGTS) will be used as extended genetic polymorphisms go under the radar of GWAS. Discovering and characterizing progression-associated CRS within HLA/KIR/FcGR/IGHG genes would help us to understand the genetic mechanism of the disease progression, and further to develop effective prevention and treatment remedies to slow or even revert progression to clinical diagnosis. In 2018, we received a bridge funding from NIDDK to carry out a pilot study of HLA genes in the disease progression, based on Diabetes Prevention Trial-1 (DPT-1). Our pilot study produced an important finding of two specific residues β57 and (-18β), within HLA-DQB1, in which β57 was structurally known to play an important role in antigen recognition and the residue (-18β) located in the signal peptide. In support of this application, we obtained clinical and genetic data from another multi-center international Oral Insulin Prevention Trial (TN07) and were able to replicate the genetic association with β57. We were able to partially replicate the association with the residue (-18β), even though DQ genotyping at intermediate resolution did not cover the residue (-18β) located in the signal peptide. To build on this preliminary result, this proposal has two specific aims: Aim 1 is to estimate the penetrance of DQB1* β57, with or without (-18β), to the progression from the precisely determined seroconversion to T1DM onset, using The Environmental Determinants of Diabetes in the Young (TEDDY). TEDDY is a prospectively conducted birth cohort and has frequent measurements of autoantibodies so that seroconversion time can be precisely determined. Aim 2 is to carry out mechanistic investigation of DQB1 CRS as well as additional CRS in HLA/KIR/ FcGR/IGHG genes, leveraging extensive functional data collected in TEDDY. Specifically, we will assess how discovered CRS associate with longitudinally measured autoantibody levels (GADA, IAA, IA-2A, ZnT8A), the β-cell function (glucose, C-peptide, OGTT) and HbA1c levels, and will investigate how CRS associate with cis- and trans-gene expressions using the longitudinal RNAseq data in a TEDDY case-control study. Results from this project will gain significant insights into genetic mechanism underlying the disease progression and will impact the translational research of preventative and therapeutic methods/strategies against T1DM.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY Latently infected CD4+ T cells harboring integrated and replication-competent HIV genomes persist during ART and are the main obstacle to HIV eradication. In most people with HIV (PWH), the reservoir is extremely stable with a half-life of over 3 years. All prior attempts to significantly reduce its size or accelerate its decay have failed. Using samples from participants in the MERLIN clade B primary HIV infection cohort (Lima, Peru), we observed 5 to 10-times faster decay of the HIV reservoir in individuals initiating ART during the first 3 months of infection compared to those randomized to start ART later, suggesting that HIV-infected cells in people treated early are more susceptible to elimination. Differences in the half-life of the reservoir, which are maintained during at least the first 4 years of ART, offer a unique opportunity to identify mechanisms that could be harnessed to reduce the reservoir in all PWH on ART. In this project, we propose to unravel the cellular and viral features responsible for the rapid clearance or long-term persistence of individual HIV reservoir cells. We will test the hypothesis that the capacity of reservoir cells to persist for prolonged periods is driven by a combination of cellular and viral features, which differ between early and late treated individuals and result in differential reservoir decay. We will take advantage of the unique MERLIN cohort to study HIV reservoir cells in 12 participants who initiated ART less than 3 months after HIV acquisition (early ART: rapid decay) and 12 participants who deferred treatment for 6 months (late ART: slow decay). We will study the early, intermediate and late reservoirs, using cryopreserved leukaphereses (collected at 1 & 2-3 years of ART) and newly collected leukapheresis from the same continually- suppressed participants at >7 years of ART. In Aim 1, we will test the hypothesis that intrinsic cellular features of HIV reservoir cells underlie differences in reservoir decay. We will employ a single cell approach to identify pro-survival factors associated with reservoir stability (Bcl-2, TCF-1, FOXO3A etc.) or that may protect infected cells from immune clearance (ligands of immune checkpoint molecules, Serpin B9, TGF-β). We will also evaluate the clonality of the reservoir with the hypothesis that clonal expansions of intact genomes will be more common in late treated participants. In Aim 2, we will test the hypothesis that specific viral characteristics also contribute to the persistence of HIV-infected cells and will be gradually enriched over time on ART. To determine if specific viruses are selected against during therapy, we will use an assay that couples integration site sequencing with HIV transcription assessment, to determine the proportion of reservoir cells that are transcriptionally active. We will use a novel approach to reconstruct molecular viral clones from the latent reservoir, and will measure the fitness of these viruses and functionally assess their HIV proteomes. In this way, we will determine if the reservoir is gradually enriched in proviruses encoding functional Nef and Vpu, which cause escape of antigen presentation and impede CTL-mediated killing as well as neutralization and ADCC by autologous antibodies. Results from this study will identify cellular and viral mechanisms that can be targeted to accelerate decay of the HIV reservoir.

NIH Research Projects · FY 2026 · 2023-04

Title: Dormancy-dependent determination of hematopoietic stem cell fate from hemogenic endothelium Project Summary/Abstract: Hematopoietic stem cells (HSCs), uniquely defined by their simultaneous capacity for multilineage blood cell formation and life-long self-renewal, represent a valuable resource for both the treatment and study of blood and immune disorders. Methods to generate HSCs de novo, such as from pluripotent stem cells (PSCs), are of great interest, as they could significantly enhance availability of HSCs for research and therapeutic purposes. However, reproducible protocols to produce functional HSCs from PSCs have been largely elusive. This likely reflects our incomplete understanding of the molecular programs required for HSC specification during embryonic development. The primary objective of this project is to address this critical barrier by identifying the unique factors necessary to impart HSC fate from the embryonic precursors to hematopoiesis, hemogenic endothelium (HE). Toward this goal, we will leverage an innovative ex vivo vascular niche platform that supports the development of murine embryonic HSCs, combined with integrated single cell techniques, to uncover the distinctive molecular properties of HE that can acquire functional HSC fate in vitro. In initial studies using this approach, we determined that HSC-competent HE possess a transcriptional signature uniquely characterized by relative metabolic and mitotic dormancy associated with decreased MYC target gene activity. We further determined that HSC emergence in vitro is dependent on niche-derived chemokine, CXCL12, and that its receptor, CXCR4, is expressed on HSC-competent HE. Based on these studies, we hypothesize that the dormant state of HSC-competent HE functions to delay hematopoietic differentiation and establish self- renewal programs essential for HSC specification, which will be tested in Aim 1. We further hypothesize that CXCL12-CXCR4 signaling reinforces dormancy and self-renewal programs in HE, supporting their transition to functional HSCs in the vascular niche, which will be tested in Aim 2. Our broader objective is to determine whether modulation of signal pathways promoting dormancy during HE specification from murine and human PSCs can enhance functional HSC development in vitro, which will be pursued in Aim 3. Success in these studies would provide new insight into the molecular mechanisms orchestrating HSC genesis and facilitate progress toward the long-term goal of de novo generation of HSCs from PSCs for therapeutic applications.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY/ABSTRACT Tens of thousands of otherwise deadly cancers are cured worldwide each year by hematopoietic stem cell transplantation (HCT), but unfortunately over one in ten patients will develop a viral lower respiratory tract infection, with almost half of these patients succumbing to the infection. Without an intact immune system in the first few months after transplant, these life-threatening infections offset the benefit derived from potentially life- saving transplant. Over half of these infections are caused by four viruses: RSV, HMPV, HPIV3, and HPIV1, none of which currently have any pharmacologic interventions for treatment or prevention after HCT. Although adults are universally infected with these viruses in childhood, HCT recipients lose their immunity, making them vulnerable to severe complications. Passive immunization with monoclonal antibodies (mAbs) represents a strategy to reduce the risk of these infections. While several anti-RSV mAb candidates have progressed through clinical trials, their use is limited to infants in whom RSV is responsible for virtually all cases of lung infection. However, the clinical efficacy of these mAbs is expected to be substantially lower in HCT patients because other important viruses like HMPV, HPIV3, and HPIV1 contribute significantly to disease. To fill this clinical gap for HCT patients, we have discovered two cross-neutralizing mAbs: one that targets both RSV and HMPV and another that targets both HPIV3 and HPIV1. Together, these mAbs could be combined to simultaneously protect against the four viruses that cause most lung infections after HCT. To test efficacy, we will administer these mAbs prophylactically and therapeutically to immunocompetent and immunocompromised animals. We will also test the pharmacokinetics of these mAbs with modifications designed for increased half-life and lung bioavailability, such that a single dose could bridge the entire period of vulnerability after transplant. Another often neglected pitfall in bringing novel antibody therapies to the bedside is the potential for resistance. Recent failed clinical trials of anti-RSV mAbs have shown that the emergence of escape variants can cripple clinical development. How to predict success or failure during the preclinical phase before candidates progress into clinical trials is an important question, and the answers could save massive amounts of resources, effort, and time. To fill this knowledge gap, we have developed an innovative approach called deep mutational scanning that provides a comprehensive picture of the viral mutational landscape, allowing an unprecedented preclinical evaluation of resistance. Since the two cross-neutralizing mAbs described in this proposal bind to conserved epitopes, these and similar mAbs may have a high barrier of resistance. To prepare for and counter resistance by future viral variants, we will leverage predictions from our complete mutational maps to identify next- generation mAbs, allowing us to stay a few steps ahead of viral evolution. These novel cross-neutralizing mAbs and the innovative and rigorous approaches we have developed to vet them could provide a new standard of care for HCT patients and inform the design and testing of other candidates with the greatest chance for success.

NIH Research Projects · FY 2026 · 2023-03

Project Summary/Abstract Antiretroviral-based HIV prophylaxis is highly effective at preventing acquisition of HIV, yet there are many implementation challenges. Population-based effectiveness studies seek to evaluate real-world impact of preventive interventions. There is, however, a major limitation in our current ability to measure effectiveness of preventive interventions at the population-level due to its requirement of resource-extensive longitudinal testing in a closed cohort. HIV recency assays (assays that provide information on the timing of HIV acquisition) offer resource- efficient estimates of incidence. However, the utility of such assays is currently limited due to lack of precision. In addition, designing efficacy trials to evaluate new HIV preventive interventions is increasingly challenging when effective prevention agents exist. To fill these gaps, we will advance statistical methodology to measure HIV incidence and develop a new trial design to assess efficacy of an HIV preventive intervention. Specifically, we will extend existing methods for estimating HIV incidence using recency assay data to accommodate covariate effects on assay properties, temporal trends in HIV incidence, and to estimate HIV incidence with increased precision. We will also develop a new class of HIV prevention efficacy trial design termed the ‘augmented active-controlled design’ which will leverage additional information to infer HIV incidence absent intervention, i.e. ‘counterfactual placebo’ HIV incidence. To extend and develop these methods, we will define a statistical framework; define approaches to estimating and drawing inference about parameters given the data; derive and compare analytic properties of the inferential methods; evaluate performance in simulation studies; and apply the methods to real data to generate new scientific insights. These novel methods have direct application to evaluating the impact of HIV preventive interventions in population-based effectiveness studies and randomized controlled efficacy trials, and will be applicable to the study of other infectious diseases. 1

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY/ABSTRACT Cytomegalovirus (CMV) infection is a frequent and life-threatening complication that significantly limits the successful outcome of allogeneic hematopoietic stem cell or bone marrow transplantation (referred to hereafter at BMT). We have recently developed long-desired preclinical (murine) models of CMV reactivation after BMT and show for the first time, that humoral immunity is critical to prevent viral recrudescence. We now demonstrate that either the transfer of immune T cells or strain-specific sera (containing IgG antibody) prevents viral reactivation, viremia, end-organ viral replication and CMV disease during graft-versus-host disease (GVHD). We plan to utilize our unique, but well-established preclinical models to define the impact of protective antibody on the temporal and spatial characteristics of CMV reactivation, dissemination and antigen presentation. Subsequently we will define the interplay of T cell and humoral responses that are required for the durable control of CMV reactivation after BMT. Finally, we will develop clinically tractable pathways to enhance CMV immunity whilst preventing GVHD and associated inflammation, targeting IL-6 inhibition. Our proposed studies will provide critical insights into the immune requirements for effective and long-term control of CMV in recipients with GVHD and/or clinically relevant immune suppression and inflammation that will allow us to optimize CMV immunity in clinical BMT to improve patient outcomes.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY To maintain health, the host must avoid generating inflammatory responses to beneficial gut bacteria, while retaining the ability to respond to pathogens. Maintaining this balance is particularly complicated in early life as many of the mechanisms that serve to promote tolerance to resident commensal microbes in adults are either absent or not yet established during this period. We recently identified maternal antibodies as key regulators of host-microbiota mutualism in neonates. Specifically, we found that in addition to IgA, healthy mothers generate microbiota-reactive IgG antibodies, which are transmitted via breastmilk and coat bacteria in the neonatal gut. In comparison with control offspring, neonates that do not receive these maternal isotypes harbor increased numbers of commensal bacteria in gut draining lymph nodes, mount inappropriate, microbiota-driven CD4 T- dependent immune responses, and suffer increased morbidity when subjected to a chemical form of colitis. Building from these exciting findings, this proposal seeks to understand the mechanisms by which maternal antibodies regulate nascent host-microbiota interactions in neonates. Specifically, we will determine the antigen specificities and the effector mechanisms (e.g., complement activation or Fc receptor ligation) required for distinct maternal IgG isotypes to restrain neonatal adaptive immune responses to beneficial gut bacteria. Additionally, we will define the signaling pathways and cell types required to trigger dysregulated adaptive immune responses in offspring that do not receive breastmilk antibodies. We will employ innovative approaches to achieve these goals by leveraging fostering and optimized infant feeding approaches with transgenic mouse models and multi-parameter flow cytometry. These studies are significant because they address key gaps in our knowledge regarding how favorable relationships between the host and resident microbiota are established in early life. Additionally, our work will also advance our understanding of the mechanisms by which breastfeeding promotes health. We expect that the insight gained from this research will significantly aid in our ability to manipulate host-microbiota interactions and mucosal immunity during early life, regardless of mode of nutrition.

NIH Research Projects · FY 2026 · 2023-02

Project Abstract/Summary • Our framing hypothesis is that the combined constraints of peptide processing, HLA allelic specificity, and peptide editing generate a significant “funnel effect”, where the peptidome constitutes only a tiny percentage of all possible peptides obtainable from a cell’s proteome and the ligandome constitutes only a small percentage of all peptides from a cell’s peptidome. Our research focus is understanding how the interplay of differential sequence recognition at each step ultimately informs functional presentation of a highly restricted subset of all possible proteome-derived peptides – and how to identify the most “translatable” pHLA targets from ligandomes. • We will rigorously test this hypothesis using our allele-specific mass-spec platform for peptide discovery, ARTEMIS, to identify relevant, HLA-restricted peptides from Mesothelin and high-risk HPV E6/E7 oncoproteins. Identified peptides will be validated and characterized biochemically. Cell-surface displayed peptide arrays from E6/E7 and Mesothelin will be used to determine the magnitude of the “funnel effect” of peptide processing on peptidome composition. Patient T cell responses to identified Mesothelin peptides will be mapped using state- of-the-art methodologies. Unusual peptides and alternate binding conformations will be characterized crystallo- graphically. We will assess viral immunoevasion mechanisms, HLA-G presentation and receptor interactions, and the presentation of glycosylated peptides. Deliverables include mapping the full Mesothelin and E6/E7 presentomes across multiple alleles and presentation modalities, ranked for future clinical exploitation as immu- notherapy targets. • Significance: From a basic science perspective, assaying cellular peptidomes and ligandomes is fundamental for understanding how the immune system responds to infections and cancer, immunoevasion mechanisms, the recognition rules of MHC proteins, the mechanistics of antigen processing/editing, and how self is defined. From a translational perspective, validated HLA-restricted peptides represent targets for diagnosis and therapy, through therapeutic vaccination, engineered T cell-based adoptive immunotherapies, and antibody-based recog- nition of specific, disease-associated peptide/HLA complexes Our proposed studies advance both objectives by elucidating the underlying molecular principles governing peptide processing and presentation through inte- grated studies of an extended set of classical and non-classical human HLA class I proteins and, in the process, identify specific pHLAs useful for future clinical applications.

NIH Research Projects · FY 2026 · 2023-01

ABSTRACT OVERALL The main goal of our IPCAVD Program grant is to evaluate in humans self-amplifying mRNA (saRNA) vaccines that express two HIV-1 Env-derived protein immunogens that activate and initiate the maturation of VRC01-class B cell receptors (BCRs). The first immunogen, 426c.Mod.Core, was specifically designed to bind with high affinity to the unmutated (germline, gl) forms of those BCRs as they are expressed on the surface of naïve B cells. The second immunogen, HxB2.WT.Core, although unable to bind germline VRC01-class BCRs, binds the VRC01-class BCRs that became activated by 426c.Mod.Core and have accumulated some somatic mutations. As a result, the boost immunization with HxB2.WT.Core furthers the maturation of the VRC01-class antibodies elicited by the 426c.Mod.Core. These observations were made with the adjuvanted recombinant (rec) forms of these two immunogens. As mRNA- based vaccines are less costly and more easily GMP-manufactured that rec proteins, we believe that they will accelerate the preclinical and clinical evaluation of HIV-1 Env-derived immunogens. Here, we propose to first compare preclinically the VRC01 B cell and antibody responses elicited by these two Env immunogens when delivered by saRNA vaccines to those elicited by the corresponding adjuvanted rec proteins. And then, if the results are promising, the saRNA vaccines expressing the two immunogens will be GMP-manufactured for clinical evaluation. As the 426c.Mod.Core adjuvanted rec protein will be evaluated clinically (phase I) in the spring of 2022 (HVTN301) and the HxB2.WT.Core rec protein is currently being GMP manufactured for a follow-up phase I clinical evaluation in 2023, we will be in a unique position to compare the VRC01 B cell and antibody responses elicited by humans immunized with these two HIV-1 Env-derived immunogens when delivered as adjuvanted rec proteins and as expressed by saRNA vaccines. To accomplish our goals in this IPCAVD grant we will take advantage of our expertise in immunogen-design and testing, expertise in the analysis of B cell and antibody responses elicited by vaccination and during infection, our ability to rapidly sequence BCR genes using high through put technologies, the availability of appropriate animal models, our expertise in saRNA vaccine technology, our unique expertise in conducting clinical testing of HIV-1 vaccines, the existing collaboration among the participating groups and the documented expertise of the participants to successfully manage complex Programs.

NIH Research Projects · FY 2026 · 2023-01

ABSTRACT Chromatin remodeling and protein synthesis are tightly regulated processes that impact gene expression and cellular phenotypes. However, it is unknown to what extent these two regulatory mechanisms may be linked or independent in controlling normal epithelial physiology and disease states. We have uncovered a new functional relationship between the chromatin remodeler ARID1A and mRNA translation elongation. This link is involved in maintaining cellular fitness in the context of bladder carcinogenesis and represents a new tumor suppressive mechanism we call transcriptional-translational conflict. Loss of ARID1A triggers a simultaneous increase in oncogenic transcripts, but also inhibition of the translation elongation factor eEF2, which results in a reduction in protein synthesis and prevents cancer pathogenesis. However, this process can be reversed by restoring translation elongation, which enables the efficient synthesis of oncogenic mRNAs and cancer progression. This finding provides a unified gene expression model which explains why ARID1A is a context specific tumor suppressor. Importantly, ARID1A deficient tumors retain a sensitivity to pharmacologic inhibition of translation elongation initiation. Recently, our laboratory has developed and characterized new in vitro and in vivo models of both human and murine ARID1A deficient bladder cancer where we can also toggle translation elongation. We have used these models to discover a critical link between ARID1A and the process of mRNA translation elongation that is vital for urothelial homeostasis and bladder cancer progression. We hypothesize that transcriptional-translational conflict in urothelium lacking ARID1A is mediated through decreased eukaryotic elongation factor 2 (eEF2) activity, which when reversed unleashes a poised druggable oncogenic program sufficient to drive cancer progression. Our long-term objective is to utilize state-of-the-art mouse models and primary organoid systems, whole transcriptome polysome profiling, and patient derived xenografts to definitively investigate the fundamental link between ARID1A and protein synthesis regulation in a highly relevant population of bladder cancer patients. To do so, we will address the following aims: 1) Determine the relevance and mechanism of transcriptional-translational conflict in urothelial cell transformation and carcinogenesis; and 2) Elucidate how gene expression parity enables cancer progression and represents a context specific therapeutic vulnerability. This research will help us gain a deeper understanding of the biology of bladder cancer and open a new paradigm for treating patients with lethal disease. Our work is particularly important for the progress of precision medicine because it seeks to mechanistically tie a highly prevalent bladder cancer genotype (ARID1A loss) to a new treatment modality that holds therapeutic promise for bladder cancer patients.

NIH Research Projects · FY 2026 · 2022-12

Project Summary/Abstract The multitude of candidate cancer biomarkers being discovered across various laboratories hold great potential to enhance the practice of precision medicine. However, it is a long and challenging process – often culminating in failure – to rigorously develop and validate these biomarkers before they can be used in clinical practice. In particular, phase III, IV, and V biomarker validation studies are expensive and time- consuming to conduct; it is essential to carefully design and analyze these studies and to make the most efficient use of the specimens collected. Motivated by our collaborative work on biomarker development for cancer early detection, this proposal seeks to develop cutting-edge statistical tools for analyzing phase III and IV biomarker studies in order to accelerate the biomarker development process. The methods proposed in Aim 1 target the selection of primary endpoints and inference procedures to accommodate potential overdiagnosis when assessing screening efficacy in phase IV trials. The methods proposed in Aim 2 enable the combination of phase IV samples with phase III samples in phase III biomarker development. The methods proposed in Aim 3 integrate information from heterogeneous study cohorts (which differ in screening modalities and eligibility criteria) when estimating design parameters for biomarker clinical utility trials. Our statistical methods will have immediate applications to analysis of data from two cancer applica- tions: i) the New Onset Diabetes (NOD) Cohort study and the Early Detection Initiative (EDI) study for pancreatic cancer early detection, and ii) five low-dose CT (LDCT) screening cohorts and the Prostate, Lung, Colorectal, and Ovarian Cancer Screening (PLCO) trial for lung cancer screening. Moreover, the developed methodology will have broader application in other phase III and IV cancer biomarker studies and will be valuable for advancing the NCI Early Detection Research Network (EDRN)'s current priority in designing biomarker clinical utility trials. All statistical programs and algorithms developed in this proposal will be made freely available to the public.

NIH Research Projects · FY 2026 · 2022-11

PROJECT SUMMARY/ABSTRACT Modification of autologous T cells with chimeric antigen receptor (CAR) molecules was first proposed nearly 30 years ago as a therapy for people living with HIV. Since then, CAR-T cells have emerged as a potent and highly successful therapy for liquid tumors, while HIV-specific CAR-T cells have only begun to show efficacy in large animal models and clinical trials. Based on our longstanding interest in CAR-T cell therapies for HIV, we posit three primary barriers that limit the curative potential of this approach. First, low levels of HIV-1 antigen at the cell surface (namely Env protein), especially during antiretroviral therapy (ART), render latently infected cells nearly invisible to CAR-T cells and other virus-specific immune effectors. Second, the wealth of knowledge regarding cellular trafficking of the viral Env protein has yet to be thoroughly applied in the context of Env- dependent HIV cure strategies. Third, CAR-T cell persistence and function wane over time, prior to complete clearance of the latent HIV reservoir. Our groundbreaking preliminary data outlines a path to overcome these limitations. We recently reported findings in four rhesus macaques that were infected with an HIV-like virus, suppressed by ART and then infused with virus-specific CAR-T cells containing the CD4 extracellular domain (CD4CAR). To expand these potent antiviral effectors in vivo, animals were next boosted with an irradiated cell line stably expressing HIV-1 Env. Following ART treatment interruption (ATI), viral control was observed in 2 of 4 animals, consistent with robust and Env-dependent expansion of CD4CAR-T cells. The central goal of this proposal is to increase the potency and feasibility of this approach. In AIM 1, we will transition our Env boosting strategy from an immortalized cell line to an FDA-approved mRNA lipid nanoparticle (mRNA-LNP) platform, analogous to the Moderna and Pfizer/BioNTech vaccines for SARS-CoV-2. Env immunogens will be optimized for CD4CAR T cell interactions and developed as Env mRNA-LNP vaccines. In AIM 2, we will use CRISPR- Cas9 gene editing to extend the durability and function of CD4CAR-T cells. We will compare a series of CAR products that carry inactivated immune checkpoint alleles, which we hypothesize will support more durable function and efficiently clear persistent viral reservoirs. In AIM 3, we will benchmark Env mRNA-LNP and immune checkpoint gene editing strategies in our vetted nonhuman primate (NHP) model of HIV gene therapy. These experiments will feature a powerful competitive repopulation study design, providing critical information on basic CAR biology that cannot be gathered in clinical studies. Together, these aims build on what we believe to be the most promising anti-HIV cell and gene therapy approach reported to date. Our unique and highly informative NHP model of HIV persistence and CAR-T cell therapy will fill in critical gaps in knowledge regarding CAR-T cell safety and function in limited antigen environments, and facilitate clinical translation both in developed and developing nations. The lessons we learn from these studies will be applicable not only as a curative therapy for HIV-1, but for a range of diseases such as solid tumors where CAR-T cell therapies must be similarly augmented.

NIH Research Projects · FY 2025 · 2022-09

SUMMARY Colorectal cancer (CRC) affects ~145,000 people/year in the US and is the 3rd most common cause of cancer related deaths. CRC arises from early lesions that are pre-cancerous; these early lesions are colon adenomas and serrated sessile lesions (SSL). Colon adenomas account for 80-85% of the CRC precancerous lesions and progress to CRC via an early adenomaàadvanced adenomaàCRC sequence. In light of the well characterized clinical natural history of adenomas, we plan to study them as early lesions and to determine the mechanisms involved in the formation and progression of early precancerous lesions. Notably, only a few early adenomas will progress to advanced adenomas (AA) and even fewer will progress to CRC. Our group and others have shown that mutations alone are not sufficient to cause adenoma initiation and/or progression in the majority of cases. There are likely multiple adenoma nonautonomous mechanisms that cooperate with the DNA alterations in the adenomas to cause progression, and these mechanisms are likely operative in discrete subsets of affected individuals. We and others have observed alterations, such as tissue senescence, high cancer driver gene mutation loads, aberrant DNA methylation patterns, and dysbiotic gut microbiomes, in the normal colon of people with advanced adenomas and CRC patients. We have termed normal colons with these features “primed colons” and propose that these features are plausible mechanisms that affect adenoma initiation and progression. Based on these observations and our prior studies, we hypothesize that early lesion progression requires a suite of hallmark behaviors and that these behaviors are induced by adenoma autonomous factors (e.g. cancer driver gene mutations) and adenoma nonautonomous factors from the “primed colon” or adenoma microenvironment. Our proposed studies will integrate basic and translational cancer research Projects to iteratively examine the direct causal relationships and interactions of adenomas, the colon “primed” microenvironment, and host- systemic factors as “co-organizers” of adenoma initiation and/or progression. The Specific Aims are: Aim 1) To determine the adenoma cell autonomous molecular factors that distinguish nonadvanced adenomas from advanced adenomas and that regulate nonadvanced adenoma progression. (Projects 1 and 2) Aim 2) To determine the adenoma nonautonomous factors from the “primed” colon and from the adenoma microenvironment that associate with advanced human colon adenomas and regulate adenoma progression. These factors will include the following “primed” colon states: 1. senescence state; 2. cancer driver gene mutation burden; 3. gut microbiome state; 4. colon methylome, and 5. colon immune activity state. (Projects 1-3) Aim 3)To determine how adenoma autonomous and nonautonomous factors from the adenoma microenvironment and the “primed” colon cooperate to drive adenoma formation and progression.(Projcts 1-3)

NIH Research Projects · FY 2025 · 2022-09

The past decade has seen substantial progress in the discovery of microbiome biomarkers associated with human health and diseases. However, despite the exciting prior work, we currently still lack an understanding of the mechanism by which the gut microbiome impacts human health. An outstanding challenge is how to integrate microbiome and other -omics data types generated in microbiome multi-omics profiling studies to elucidate microbial functional pathways. Unfortunately, available statistical methods for integrative analysis do not adequately address the analytical challenges specific to microbiome data. Microbiome data are compositional, zero-inflated, high-dimensional, and highly structured where samples are related by ecologically defined distances and taxa are related by their phylogeny. We propose to use our expertise in network analysis and high-dimensional statistical inference to tackle these challenges unique to microbiome data analysis. Our overall objective is to develop rigorous statistical methods that yield reliable and powerful inferences relating microbial functional pathways with host health conditions. The proposed methodologies include a novel inference procedure for joint analysis of microbial and metabolomic networks (Aim 1), a novel method for joint dimensionality reduction which incorporates prior biological knowledge about the relationships between samples and between variables (Aim 2), and a powerful framework for jointly associating microbiome and other -omics data types with health outcomes (Aim 3). We will develop efficient and easy-to-use software tools for the proposed methods (Aim 4). This work is innovative and significant, because it will provide systems biology insights into the role of the microbiome and has the potential to make a major impact on the identification of novel microbiome biomarkers. Successful completion of this proposal will generate important shared analytical tools, including new methodologies for integrative analysis and their user-friendly software tools. With these analytical tools, the longer-term goal of this project is to hasten the discovery of microbiome- based therapeutic targets and contribute to the development of microbiome-based targeted therapies.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Cellular differentiation is a fundamental biological process through which complex multi-cellular organisms develop from single-cell embryos and maintain tissue homeostasis throughout life. Cells integrate signals from the microenvironment and transmit them to downstream transcriptional regulators, which execute the expression and chromatin changes to define phenotypic state transitions in differentiation trajectories. Elucidating the principles of how cells choose their fate, and the path they take to get there, is a major challenge in the field. Single-cell (sc) RNA sequencing technologies are revolutionizing our understanding of the cellular spatio- temporal trajectories that shape differentiation. The emergence of additional high throughput, multimodal technologies such as paired RNA&ATAC-seq, scCUT&Tag and spatial technologies provide unprecedented opportunities to extract mechanistic insights into the lineage decisions that underly differentiation trajectories. This proposal aims to exploit this enormous potential by developing sophisticated new algorithms that integrate single-cell measurements to model and interpret complex biology. Through analysis of multiple single-cell RNA- seq datasets, we demonstrate that phenotypic asymmetries are a pervasive feature of lineage decisions. We will develop algorithms to unravel the mechanisms that drive lineage decisions and the underlying asymmetries in three broad research directions. We will investigate the role of: (i) enhancer priming and transcriptional regulation, (ii) open and heterochromatin dynamics, and (iii) cell communication in shaping differentiation trajectories. Our studies will lead to novel insights surrounding cell-autonomous and non-autonomous mechanisms engaged by cells as they navigate the phenotypic landscape. Successful completion of this research will provide a robust mechanistic basis to delineate normal differentiation events, decipher dysregulation of these mechanisms in disease, understand repurposing of differentiation mechanisms in wound healing and regeneration, and reconstruct differentiation processes in vivo and ex vivo to unlock the therapeutic potential of cell engineering.