Beckman Research Institute/City Of Hope

NIH Research Projects · FY 2025 · 2023-09

SUMMARY/ABSTRACT Image guided radiotherapy (IGRT) or systemically administered radionuclide therapy are attractive approaches that can perturb the tumor microenvironment (TME) so that targeted immunotherapy can convert a largely immunoresistant into an immunosusceptible tumor. Although this approach has been clinically explored using high or low or high plus low dose IGRT plus untargeted checkpoint immunotherapy, further improvements are warranted and require testing in appropriate clinical studies and preclinical models. In this application we propose three aims to test our hypothesis that IGRT and or targeted alpha therapy (TAT) followed immediately by immunocytokine (ICK), a form of targeted immunotherapy, will lead to increased tumor infiltration of IFN+ CD8s (Teff) and decreased Foxp3+ CD4s (Treg). This hypothesis is supported by two studies targeting CEA, a major marker of solid tumor malignancies (eg. colon and breast), in CEA transgenic mice that are immunocompetent, express CEA in normal tissues, and are tolerant to CEA. We now propose to test this hypothesis in two clinical trials, aims 1 (IGRT + ICK) and 2 (TAT), and perform immunocorrelate studies for Teff and Treg cells, among others, on pre- and post-therapy biopsies. Aim 2 is a TAT only study as a prelude to a third clinical trial combining TAT plus ICK, that will be initiated at the end of the project period. In aim 3, we will refine our animal studies to determine TME changes immediately after sequential therapy for both IGRT plus ICK or TAT plus ICK, by a combination of PET imaging for CEA, CD8s and stromal cells by fibroblast activated protein (FAPI), along with flow and IHC analysis of tumors, LNs and spleen. We expect the results of aims 1 and 2 to further guide aim 3, and the results of aim 3 to guide future trials that incorporate IGRT and/or TAT plus ICK in CEA positive malignancies.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY: Background: Acute myeloid leukemia (AML) is one of the most aggressive types of hematopoietic malignancies with various genetic alterations. Ten-Eleven Translocation 2 (TET2), an enzyme involved in DNA demethylation, is deleted or mutated in 15-20% of AML patients. Those patients with TET2 deficiency are poorly responsive to currently available therapeutic regimens, leading to more adverse outcomes than patients with other AML subtypes. Thus, it is urgent to identify new therapeutic target(s) and develop novel effective approaches to treat TET2-deficient AMLs. Tet2 loss in mice facilitates the self-renewal of hematopoietic stem cells (HSCs) and leukemic stem/initiating cells (LSCs/LICs). The LSCs/LICs reside in a specialized microenvironment called “niche” in the bone marrow (BM) to support their survival and self-renewal. There are several critical gaps in our current knowledge of the molecular mechanism underlying LSC/LIC homing and of the role of TET2 deficiency in the BM microenvironment. Meanwhile, evidence is emerging to support a novel function for TET2-mediated oxidation of methyl-5-cytosine (m5C) in RNAs, including messenger RNA (mRNA). However, it is unknown whether and (if so) how TET2-mediated RNA m5C demethylation contributes to leukemogenesis. Our preliminary study showed that Tet2 deficiency stimulates the Tetraspanin 13 (Tspan13)/C-X-C motif chemokine receptor 4 (Cxcr4) axis to facilitate AML homing/migration into the BM microenvironment, giving rise to increased LSC/LIC self-renewal and fast leukemogenesis in vivo. Tet2 deficiency-mediated increase of mRNA m5C modification in Tspan13 is recognized by Y-box binding protein 1 (YBX1), which in turn stabilizes Tspan13 transcript and increases its expression, thereby activating the Cxcr4 signaling. Pharmacological inhibition of CXCR4 suppresses LSC/LIC homing into the BM microenvironment and shows a synergistic effect with hypomethylating agents in killing TET2-deficient AMLs. These results lead to our central hypothesis that TET2-mediated mRNA m5C demethylation is involved in reprogramming BM microenvironment. Guided by strong preliminary data, we propose three Specific Aims to test our hypothesis: (1) Determine the definitive role of TET2 in the homing of LSCs/LICs into BM microenvironment; (2) Characterize the mRNA m5C-dependent and functionally essential targets of TET2 and decipher the molecular mechanisms underlying the role of TET2 in LSC/LIC homing and self-renewal; and (3) Assess the therapeutic potential of targeting the TET2/CXCR4 axis in high-risk TET2-deficient AMLs. Overall, our proposed studies will substantially advance our understanding of the fundamental biology of TET2-mediated epitranscriptomic changes in BM microenvironment and may result in the development of novel effective approaches to treat AMLs with TET2 deficiency. Thus, our project is of high novelty and significance in both basic research and translational medicine.

NIH Research Projects · FY 2024 · 2023-09

With the majority of Americans supporting legalization of medical cannabis, the number of patients asking their clinicians about medical cannabis has also significantly increased. Many clinicians, however, feel that they have inadequate knowledge about the efficacy, side effects, and abuse potential of medical cannabis. One national survey of oncologists reported that only 30% felt sufficiently informed to make recommendations regarding medical cannabis use, even though 80% regularly conducted conversations about medical cannabis with patients. A study from Washington State found 21% of patients with cancer surveyed had used cannabis in the past month. Despite the common belief among many patients that herbal therapies such as cannabis are inherently safe, the evidence is growing that greater caution is needed with cannabis as it may have potential direct side effects and may lead to interactions between herbs such as cannabis and medications. Thus, an important knowledge gap exists regarding the use of cannabis by patients with cancer during active cancer treatment, and this prospective cohort study is proposed to address these questions. This study aims to assess the prevalence and patterns of cannabis use among patients with cancers of the lung and multiple myeloma during active treatment as well as inquire about the communication patterns regarding cannabis use with their treating medical team. In order to reduce the complex variables that exist between the types of cancers and treatments, we will study two unique cancer populations – non-small cell lung cancer (NSCLC) and multiple myeloma (MM), with each cohort studied separately. These patients provide a homogenous population of patients with cancer that have generally standardized treatment regimens. Cancer patients currently receive platinum-based chemotherapy either before (neoadjuvant) or after (adjuvant) receiving surgery together with a PD-1 inhibitor (a type of immune checkpoint inhibitor (ICI), ex. nivolumab). Newly diagnosed MM patients are recommended to receive induction therapy with lenalidomide, bortezomib, and dexamethasone (RVd) for three to six months followed by an autologous stem cell transplant (ASCT). These two cohorts provide unique opportunities to study the impact of cannabis use in both a solid tumor and hematologic cancer during chemotherapy. For the NSCLC cohort, we will also assess the effect of cannabis on immunotherapy efficacy. For the MM cohort, we will assess the effects of cannabis during ASCT. Concurrently, we will survey oncology healthcare providers about their perceptions, education, and practice patterns regarding cannabis use by patients. Data collection will include information about all the medications including prescription, over-the- counter, herbs, and supplements in order to assess for the prevalence of potential medication interactions with cannabis. We hypothesize that a substantial proportion of patients receiving cancer treatment are using cannabis (~20%) and that there are different patterns of cannabis related benefits and harms based on cancer type and treatment as well as patient demographics including socioeconomic factors.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Patients diagnosed with glioblastoma (GBM) have a median overall survival of less than two years even after receiving multimodal therapies. Multiple factors account for this treatment resistance including: 1) Inability of therapies to cross the blood-brain barrier to reach invading cells; 2) GBM’s molecular heterogeneity and overlapping escape mechanisms that overcome targeted therapies; 3) Evasive mechanisms that render GBMs resistant to immunotherapy. Therefore, there is an unmet need for GBM treatment approaches that address multiple resistance mechanisms. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily which was discovered as a transmembrane receptor for the products of nonenzymatic glycation and oxidation of proteins. RAGE is expressed by glioma cells and is activated by its ligands present in GBM tumor microenvironment (TME). Activation of RAGE stimulates multiple signaling pathways that promote GBM progression. Recently, we demonstrated that genetic ablation of intracellular RAGE in gliomas inhibited multiple oncogenic pathways that not only regulated glioma growth and invasion, but also, improved the efficacy of immunotherapies by promoted an immunologically “permissive” TME. We also discovered that RAGE ablation in TME enhances the efficacy of immunotherapy. Based on these observations, we propose to evaluate RAGE inhibition as a multifaceted therapy for GBM. Our central hypothesis is that RAGE inactivation will not only suppress oncogenic pathways that are important for GBM growth and invasion, but also, enhance responses to immunotherapy. Three independent aims are proposed. Aim 1 will determine the mechanism of RAGE ablation on enhancing the anti-tumor immune responses in syngeneic mouse GBM models. Findings from this Aim will uncover novel strategies that could enhance immunotherapy efficacy in these resistant tumors. Aim 2 will measure the synergistic effects of small molecule RAGE inhibitors with immunotherapy. In this Aim, we will perform the pre-clinical studies to optimize the dosing regimen of RAGE inhibitors for future GBM clinical trials. Finally, Aim 3 will Identify mechanisms of immunotherapy resistance to RAGE ablation. This Aim will identify the mechanisms by which RAGE ligands such as S100A9 attenuate tumor immune responses. Success of any of these aims, which are supported by compelling preliminary data, is expected to lead to the development of novel and critically needed GBM therapies.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract The outcomes for patients with large B-cell lymphoma (LBCL) that are relapsed or refractory to frontline therapy remain quite poor. While anti-CD19 chimeric antigen receptor (CAR19) T-cells have emerged as a promising treatment option for this group of patients, over half of these patients still go on to exhibit disease progression. The mechanisms through which resistance to CAR19 T-cell therapy develops, and factors predictive of poor outcomes, have not yet been well characterized. In this proposal we seek to elucidate these mechanisms with the ultimate goal of informing the design of improved immunotherapies that will result in better outcomes for patients with LBCL. Novel methods to profile tumor-derived cell-free DNA from the blood plasma of patients, also referred to as circulating tumor DNA (ctDNA), have unlocked significant opportunities to monitor treatment response and study tumor biology both prior to and after therapy. We recently applied one such method called Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq), a targeted sequencing approach for ctDNA detection and profiling, to a cohort of patients with LBCL undergoing therapy with the CAR19 platform axicabtagene ciloleucel (axi-cel). We found that ctDNA levels prior to and following CAR19 T-cell infusion were predictive of response, and also identified several genes involved in B-cell lineage commitment and determination of the tumor immune microenvironment that were recurrently altered in patients who developed progressive disease. In this proposal we will build on this prior data by validating our findings in an independent cohort of patients undergoing therapy with an alternate CAR19 platform (Aim 1). We will then assess the downstream effects these alterations have on tumor phenotype and the tumor microenvironment. By integrating gene expression data and cutting-edge immunophenotyping and computational tools, we will resolve the components of the intratumoral immune milieu to gain a better understanding of how different immune cell populations contribute to response and resistance (Aim 2). Finally, we will employ a novel organoid tissue culture system to directly assess how alterations in these genes effect the interaction between CAR19 T-cells and tumor cells in the context of an intact immune microenvironment (Aim 3). Ultimately, we hope that these studies will have implications not only in development of improved CAR19 platforms for lymphoma, but also in the improvement of immunotherapies for other cancer types as well. This proposal will be carried out at the Stanford University School of Medicine, under the mentorship of Ash Alizadeh, MD/PhD. Through completion of this proposal, I will gain the relevant experience in bioinformatics and tumor immunology to successfully launch a career as an independent investigator focused on developing and translating new immunotherapies for patients with lymphoma.

NIH Research Projects · FY 2025 · 2023-09

Project Summary The circadian clock confers temporal control to metabolic pathways, and its disruption leads to insulin resistance and obesity. Skeletal muscle plays a critical role in nutrient metabolism and protein homeostasis. We and others demonstrated that the muscle-intrinsic clock regulates skeletal muscle development, growth, and metabolism. Despite the extensive studies of circadian regulation in glucose and lipid metabolism, there is a current knowledge gap regarding clock function in protein metabolism that determines muscle mass. In addition, although circadian misalignment is prevalent in a modern lifestyle, potential circadian etiologies underlying muscle wasting and impaired metabolic capacity remains unknown. We have identified a novel clock-driven temporal control of PI3K-Akt-mTORC1 signaling in skeletal muscle that is independent of feeding-induced activation. Surprisingly, clock disruption mimicking shiftwork resulted in progressive muscle atrophy accompanied with impaired PI3K-Akt signaling and elevated protein turnover. Furthermore, mechanistic studies revealed circadian clock transcriptional control of the Insulin/Igf-1-PI3K-Akt-mTOR signaling cascade. These findings, together with prior research support a hypothesis that that the muscle-intrinsic clock confers temporal control in PI3K-Akt-mTOR cascade to drive protein metabolism and insulin sensitivity, and this mechanism underlies circadian disruption-induced muscle atrophy and insulin resistance. The overarching goal of this project is to comprehensively define this newly discovered clock-PI3K-Akt-mTOR regulatory axis in muscle nutrient homeostasis and muscle mass regulation. Specifically, we will leverage our unique clock modulation models with multi-omics approaches to comprehensively define the molecular mechanisms responsible for and the physiological significance of the clock-Akt-mTOR regulatory axis in protein metabolism, insulin sensitivity and muscle mass maintenance. More importantly, we propose to test genetic and pharmacological clock-augmenting interventions to counteract muscle anabolic and metabolic deficits induced by clock disruption. The outcome of this proposal may uncover a circadian etiology underlying impaired metabolic capacity in sarcopenia and provide the mechanistic basis for clock-targeting interventions.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY: Liver cancer is estimated to afflict 42,230 individuals and result in approximately 30,230 deaths in the US in 2021. The incidence rates for liver cancer have more than tripled since 1980, making it the 5th leading cause of cancer- related deaths in the US. Approximately three-fourths of liver cancer cases are hepatocellular carcinoma (HCC). As for all cancers, detection of HCC at an earlier stage is critical to elicit the best chance of a cure. Early detection of HCC has significant potential to reduce mortality rates, due to the significant efficacy of local treatments for early-stage disease vs. systemic therapy for advanced-stage cancers. Although surveillance of patients at high- risk for HCC (e.g., those with chronic hepatitis or liver cirrhosis [LC]) is widely performed, the population of patients with HCC without viral etiologies is increasing because of insufficient screening for HCC. At present, alpha-fetoprotein (AFP) is the most widely used blood tumor marker; and hepatic ultrasound is a low-cost imaging method for surveillance of HCCs. However, both approaches have limited sensitivity and specificity for detecting early-stage HCC, highlighting the imperative need to develop robust biomarkers for the early detection of HCC. Accumulating evidence indicates that dysregulation of microRNAs (miRNAs) occurs in all human cancers, including HCC. As biomarkers, miRNAs are more resilient than mRNAs, and are frequently deregulated even in the earliest stages of neoplasia. Furthermore, the recent discovery that cancers actively excrete small extracellular vesicles, called exosomes, has brought additional enthusiasm to this burgeoning translational research topic. While exosomes are considered to reflect their respective cells-of-origin, their use in biomarker research has been hampered due to lack of standardized protocols for their isolation and purification, use of cell line-derived, but not patient-derived specimens for biomarker discovery; and lack of biomarker discovery in cancer-derived exosomes from matched tissues and plasma specimens. Furthermore, despite the perception that exosomal-miRNAs (exo-miRNAs) may be superior to circulating cell-free miRNAs (cf-miRNAs), no studies have undertaken an effort to directly compare these two types, to support or negate their superiority as disease biomarkers. In this proposal, we will address these concerns by undertaking the following Specific Aims. Aim 1: Discover candidate cf-miRNA and exo-miRNA biomarkers using small RNA-Seq in matched tissue and plasma from patients with early-stage HCC vs. controls. Aim 2: Develop a biomarker panel composed of cf-miRNAs and exo-miRNAs for the identification of patients with HCC in an independent cohort. Aim 3: Clinically validate the optimized panel of non-invasive plasma miRNA biomarkers in a large prospective cohort of patients with HCC. If successful, this proposal will provide molecular characterization of cell-free and exosomal miRNAs as liquid biopsy biomarkers, which may allow early-detection of HCC using a non-invasive, and inexpensive assay.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT Our overall goal, which is fully responsive to PAR-20-271, is to develop a selective and effective inhibitor of the multi-functional DNA repair enzyme exonuclease 1 (EXO1) that can be used both as a research tool (chemical probe) and as a pre-clinical starting point toward the development of a potential cancer therapeutic drug. There is no EXO1-specific small molecule inhibitor listed in the Chemical Probe Portal or other literature. We will achieve our goal through discovery research, from implementing a primary high-throughput screen (HTS) that we have already developed, to validating hits via a well-developed “critical path” of secondary assays, to performing early hit-to-lead optimization via purchase of commercially available analogs of validated chemical scaffolds and limited focused medicinal chemistry. EXO1 represents a druggable target, as it contains functionally essential exonuclease activity for double-strand break response and repair (DSBRR) for processing of stalled replication forks, which are critical pathways by which cells counteract endogenous DNA damage and replication stress. Compared to normal cells, cancer cells carry a significantly higher burden of double-strand breaks and replication stress, which generates a therapeutic window for treating cancer. To exploit this, current therapeutic approaches primarily target proteins acting in repair pathways or in checkpoint signaling pathways controlling repair. Many cancer cells are already defective in DSBRR; thus, EXO1 inhibition will cause cancer cell-specific cell death through a synthetic lethality mechanism. Furthermore, EXO1is will display greater specificity than currently used PARP inhibitors, because PARPs participate in a wide array of other cellular processes, whereas EXO1 does not. Our group was the first to clone the human EXO1 gene and to characterize its biochemical properties. We have expressed and purified the full-length and active EXO1 enzyme at scale, developed a robust fluorescence-based enzyme inhibition assay, and performed a pilot HTS in our own core facility. Thus, in collaboration with the Prebys Center of Sanford Burnham Prebys Medical Discovery Institute, we are well positioned to 1) identify inhibitors of EXO1 exonuclease by performing HTS of a well-curated ~320,000 compound library; 2) validate hits for potency and selectivity; 3) perform “structure-activity relationship (SAR)-by-catalog” and limited focused medicinal chemistry and benchmark absorption, distribution, metabolism, and excretion (ADME)/pharmacokinetic (PK) characterization of best probes; and 4) determine the mode of action (MOA) and biological effects of validated EXO1i candidate probes. All of our Aims are responsive to and within the scope of PAR-20-271. The development of novel EXO1is will not only allow us to provide a critical tool (i. e. chemical probe) to test mechanistic insights into the replication-repair interface but will also support development of a novel chemotherapeutic drug that blocks both upstream DNA replication steps and the downstream DSBRR pathway, with the potential to induce clinical synthetic lethality in breast cancer and other DSBRR-deficient cancers.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT The gut microbiota consists of a community of diverse microbes and has many effects on human (patho)physiology. Microbiome composition has been associated with many diseases, but causal inference is often lacking. Preclinical and clinical studies have demonstrated that the intestinal microbiota can regulate innate and adaptive immunity, including T cell and antitumor immunity after allogeneic hematopoietic cell transplantation (allo-HCT) and checkpoint blockade. My lab has focused on the role of gut microbiota in outcomes of allo-HCT and immunotherapy. For example, we showed that microbiota composition undergoes significant and frequent changes during allo-HCT and that lower intestinal microbiota diversity is associated with increased mortality. We also found that dominance by certain species, most frequently Enterococcus, is associated with lethal graft-versus-host disease (GVHD); that exposure to certain antibiotics is associated with worse outcomes following allo-HCT and chimeric antigen receptor T cell (CART) therapy; and that hematopoietic reconstitution is associated with the presence of beneficial flora. These studies have been translated into clinical trials using autologous fecal microbiota transplant, administration of defined bacterial consortia, and antibiotic stewardship to spare and/or restore the commensal flora. The overarching hypothesis of this proposal is that the intestinal microbiome is an important modulator of innate and adaptive immunity in the setting of cancer immunotherapy. While immunotherapies are curative in some recipients, improving their efficacy and abating toxicities are unmet needs in oncology. The major goals are to improve cancer immunotherapy by targeting the intestinal microbiome based on preclinical and clinical studies. Examples of our ongoing and planned studies include: a) development of a new pipeline for microbiome analysis, b) preclinical and clinical projects regarding intestinal microbiome and CART, c) new techniques to analyze the effects of diet and drugs on the intestinal microbiome, and d) preclinical and clinical studies regarding immune modulation by bile acids, as an example how we study the mechanisms by which the intestinal microbiome can modulate immunity and cancer immunotherapy. We have organized a multicenter global consortium to collect fecal samples (funded separately from this application) along with a novel multi-omic approach to integrate patient, microbiome, and tumor profiling modalities using a computational platform (MSK-MIND) for data harmonization and machine learning. These investigations will be performed via perpetual dialogue between work with mice and humans: human studies enable us to observe correlations, develop hypotheses, and test therapeutic strategies; animal studies enable us to establish or refute causal relationships between microbiota and host immunology and to obtain mechanistic insights. These data will inform the future development of clinical trials to test therapeutic strategies to enhance efficacy and decrease toxicity in patients receiving cancer immunotherapy, such as CART and allo-HCT.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY There has been little or no long-term improvement in outcomes for patients with treatment-refractory acute myeloid leukemia (AML). The recently developed image-guided radiation strategy, total marrow and lymphoid irradiation (TMLI) delivers high radiation doses to major leukemia reservoirs while sparing normal tissues. Although TMLI prior to allogeneic hematopoietic cell transplantation (HCT) improved patients’ 2-year overall survival (OS) rate from <10% to 48%, relapses remained common. Such radiation resistance is a consequence of both intrinsic cancer cell properties and the extrinsic influence of the tumor microenvironment. It was previously demonstrated that radiation-induced cell death causes the release of danger signals recruiting Toll-like Receptor- 9 (TLR9)-positive myeloid cells, which jump-start tumor vascularization and regrowth. These cancer-promoting, rather than immunostimulatory, effects are mediated by TLR9-mediated secretion of cytokines such as IL-6, thereby leading to activation of Signal Transducer and Activator of Transcription 3 (STAT3). STAT3 is a multifaceted oncogene and a central immune checkpoint regulator operating in AML cells as well as in tumor- associated myeloid cells in patients. However, it remains an elusive target, with no FDA-approved direct small molecule STAT3 inhibitors. To overcome this challenge, we previously developed a strategy to deliver oligonucleotide STAT3 inhibitors, such as siRNA or decoy DNA, specifically into commonly TLR9-positive AML and normal myeloid cells. The nuclease-resistant, second-generation CpG-STAT3 decoy inhibitor (CSI-2) injected intravenously showed efficacy in targeting STAT3, suppressing leukemia cell survival and/or inducing immune responses against moderate burden of human and mouse AML in vivo. The hypothesis is that combining the immunostimulatory CSI-2 strategy with conditioning TMLI treatments will improve treatment efficacy against AML even at high burden (>50% leukemic blasts in the bone marrow) by providing time for the generation of adaptive T-cell driven immune responses. The preliminary results demonstrated that the TMLI regimen can improve uptake of CSI-2 by AML, thereby reducing leukemia-initiating potential, augmenting AML immunogenicity, and thereby inducing potent CD8+ T cell-mediated antileukemic immune responses. We propose to: 1. elucidate the molecular mechanisms of TMLI/CSI-2 effect on AML cell differentiation; 2. optimize TMLI to maximize the effect on leukemic bone marrow vascular structure, CSI-2 delivery, leukemogenic potential, and immunogenicity; 3. assess the efficacy and cellular mode-of-action of the TMLI/CSI-2 combination treatment compared to either treatment alone in human or mouse AML models in humanized or syngeneic mice, respectively. The overarching goal of this interdisciplinary proposal is to produce a clinically relevant, effective, and safe combinatorial radiation-immunotherapy for patients with relapsed/recurrent AML, representing the highest unmet need in cancer therapy.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Early interventions for high-risk B-cell malignancies, including diffuse large B cell lymphoma (DLBCL), Burkitt's Lymphoma (BL), and B-cell acute lymphoblastic leukemia (B-ALL), remain an urgent clinical need. Development of such interventions requires a deep understanding of the molecular mechanisms underlying the evolution, i.e., initiation, establishment, and sustenance, of these malignancies. B-cell malignancies are initiated >5-fold more frequently in patients suffering from refractory autoimmune B-lymphoproliferative disorders such as systemic lupus erythematosus (SLE), making SLE a relevant disease model to study initiation of B-cell neoplasms. The hormone prolactin (PRL) is known to exacerbate the symptoms of SLE, enhance survival of lymphoid cells, and promote the expression of the protooncogenes MYC and BCL2 in these cells. Whether PRL contributes to evolution of B-cell malignancies was unknown. PRL receptors (PRLRs) are type I cytokine receptors that have long (LF), intermediate (IF, only in humans) and short (SF) isoforms generated by alternative splicing. Increased expression of the LF/IF relative to the SF PRLRs on cells leads to cell proliferation and survival, whereas increased expression of SFs relative to LF/IF inhibits proliferation, promotes differentiation, and induces apoptosis. We hypothesized that PRL, by signaling specifically through the pro-proliferative and anti-apoptotic LF/IFPRLR, promotes the malignant transformation of B cells, and establishes and sustains the growth of overt B-cell malignancies. To test our hypothesis, we measured changes in B cells in vivo in SLE- and DLBCL/BL- prone mouse models and in vitro in human B-cell malignancies after specifically knocking down expression of the LF/IFPRLR using a non-toxic splice modulating oligomer (SMO). The LFPRLR SMO prevents the synthesis of the LFPRLR in mice and the LF/IFPRLR in humans without affecting the SFPRLRs. Knockdown of LFPRLR reduced the numbers of pathogenic B-cell subsets in SLE- and DLBCL/BL-prone mice and lowered the risk of B-cell transformation in SLE-prone mice by downregulating expression of the activation-induced cytidine deaminase (AID) enzyme, whose overexpression we previously showed, drives the evolution of B-cell neoplasms. We found that overt human B-cell neoplasms aberrantly express autocrine PRL and sometimes only the LF/IFPRLR. Knockdown of LF/IFPRLR in overt B-cell malignancies reduced cell viability, downstream STAT3 activation, and expression of MYC and BCL2. Our preliminary findings warrant detailed studies of molecular pathways underlying the disturbances in B cells downstream of LF/IFPRLR in SLE-prone mice that are vulnerable to transformation (Aim 1), in mice with pre-malignant B-cell clones prone to overt B-cell malignancies (Aim 2), and in overt human B-cell neoplasms (Aim 3). Our research will solidify isoform-specific suppression of the production of LF/IFPRLR as a therapeutic strategy in SLE that concurrently lowers the incidence of B- cell malignancies in these patients (Aim 1), in people with pre-malignant and indolent B cells who are vulnerable to developing aggressive B-cell malignancies (Aim 2), and in overt B-cell neoplasms (Aim 3).

NIH Research Projects · FY 2026 · 2023-07

Lung cancer is responsible for the most cancer-related deaths in the United States, and Lung adenocarcinoma (LUAD) is the major histologic subtype. LUAD presents clinically with four major histologic subtypes (lepidic, acinar, papillary and solid), has variable presentation of EGFR and Kras mutations depending on ethnicity, age, and sex, and can be subclassified into four separate categories based on genome-wide DNA methylation profiles. To date, there is little connection between these widely disparate manifestations of LUAD besides their effects on overall patient survival. There is evidence in mouse models to suggest that the majority of LUAD arise from surfactant protein c (Sftpc)-positive alveolar epithelial type 2 (AT2) cells, and that Scgb1a1-positive club cells can also contribute a fraction of LUAD cases. However, it is unknown if LUAD can arise from AT1 cells, the other major epithelial cell type in the distal lung that covers 95% of the alveolar surface. AT1 cells were historically thought to be terminally differentiated. However, we have recently developed a Gramd2-driven CreERT2 mouse model that specifically activated the KrasG12D oncogenic driver in AT1 cells, and found that AT1 cells can serve as a cell of origin for LUAD with predominantly papillary histology and distinct transcriptomic signatures, including increased transforming growth factor beta (TGF-β)-mediated epithelial to mesenchymal transition (EMT). This is in contrast to AT2 cell-specific Sftpc-driven KrasG12D, which resulted exclusively in lepidic LUAD and was enriched for VEGF-mediated angiogenesis. Therefore, we hypothesize that LUAD, as it is currently defined, may actually be a collection of at least 4 adenocarcinoma subtypes that arise in the distal alveolar compartment from different cells of origin, and that the great variation we see in LUAD presentation and clinical outcome can be explained in part by which cell type LUAD arises in. However; several questions remain. We do not know if the oncogenic driver in AT1 cells influences histologic presentation. We will therefore (Aim 1) characterize Gramd2-CreERT2 driven EGFR mutations, the other major oncogenic driver in LUAD. It is also possible that induction of KrasG12D in AT1 cells results in disrupted tumor microenvironments that stimulate AT2 cells; we will therefore (2) perform GFP+ lineage tracing to determine in vitro and in vivo cell contributions to tumor formation. We will also establish the translational implications of our prior research (Aim 3) and utilize inhibitors of TGFβ that have succeeded in preclinical models but failed in clinical trials to determine if cell of origin influences response to therapy in both mouse models and unique human patient LUAD cohorts. Understanding the connection between cell of origin and clinical presentation will allow for enhanced patient stratification, improved assessment of best therapeutic outcomes, and potential reclassification of LUAD into multiple cancer types.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Objective: We will develop an advanced photoacoustic computed tomography (PACT) breast imaging system capable of detecting anatomical and functional changes in breast cancer treated with neoadjuvant therapy (NAT). Significance: NAT improves outcomes in breast cancer patients by increasing the likelihood of breast conservation, providing important prognostic information, and enabling adaptive therapy such as change in systemic treatment and de-escalation of surgery in exceptional responders. As such, identification of responders enables personalized cancer treatment. Challenges: Current breast imaging does not sufficiently detect breast cancer treatment response. Standard of care (SOC) breast imaging technology either assesses anatomical details or metabolic function, not both. In addition, ionizing radiation, exogenous contrast agents, and patient perceived discomfort and inconvenience usually restrict imaging frequency required for timely evaluation of response. For example, although dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is considered the SOC in breast imaging, this test is limited by the need for intravenous heavy metal contrast, duration of study, patient discomfort, and high resource costs while delivering only moderate accuracy in detection of NAT response. As such, there is no reliable,non-invasive, cost-effective imaging method to identify treatment response.PACT is an emerging technology with great potential to address these problems by imaging both function and anatomy without exogenous contrast. Solutions: Capitalizing on our experience and success in building two PACT breast imaging systems, we propose the construction and clinical testing of an innovative PACT imaging system that integrates the two previous systems to enable both anatomical and functional imaging. The Dual Mode PACT (DM-PACT) will combine dual-sided light delivery, large-view detection aperture, and dense acoustic sampling for rapid functional and high-resolution anatomical imaging to assess treatment-related responses in breast cancer. The imaging features generated by the DM-PACT will be first characterized and correlated with the histopathological results of the resected breast cancer specimens from patients treated with NAT. A diagnostic model using the imaging features will be trained and tested in a larger group of breast cancer patients treated with NAT. We will compare the performance of DM-PACT with the performance of SOC DCE-MRI in treatment response discrimination. The success of this project will result in imaging technology that directs response- driven, personalized breast cancer treatment plans.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Sickle Cell Disease (SCD) is the most common hematologic disorder affecting millions of people worldwide. Allogeneic hematopoietic cell transplantation (HCT) is the only curative treatment but is associated with a significant risk of treatment-related organ toxicities and graft failure. While myeloablative total body irradiation (TBI) conditioning facilitates full engraftment, it is associated with higher organ toxicities. Conversely, reduced intensity conditioning is associated with less organ toxicities, but the risk of graft rejection is higher, and partial chimerism achieved with RIC cannot resolve the hematological abnormalities in most cases. Therefore, a conditioning regimen that can increase chimerism while lowering regimen-related toxicities is an unmet need to treat SCD. We recently successfully treated our first SCD patient with image-guided total marrow irradiation (TMI), delivering 6 Gy of radiation to the bone marrow (BM) while limiting the vital organ dose to 0-2 Gy, followed by matched donor HCT, which resulted in full chimerism without adverse events. Yet, the exact mechanism of the enhanced donor chimerism after TMI is not clear; emphasizing the need for preclinical studies. We have developed the first preclinical 3D image-guided TMI bone marrow transplant (BMT) model, using immunocompetent C57BL/6 mice and humanized homozygous BERK sickle mice (SS). Initial work suggests TMI-based bone marrow targeted dose escalation is feasible in the SCD mice model supporting increased chimerism and reduced organ damage. In collaboration with Janssen Pharmaceuticals, we will test a thrombopoietin (TPO) mimetic drug (TPOm, aka JNJ-26366821), which is a fully synthetic, PEGylated TPO receptor, c-MPL agonist peptide. The safety of TPOm has been proven in Phase I clinical trials and is phase II ready. Our preliminary data in a mouse model suggest post-BMT TPOm administration can enhance HSC regeneration, mitigate radiation damage, and support vascular regeneration. We hypothesize that the TMI- based dose escalation will facilitate engraftment (higher homing and chimerism) with minimal toxicities to other organs, resulting in improved pathophysiology of SCD. We further hypothesize that TPOm intervention along with TMI will improve BM vascular recovery, HSC expansion, and engraftment. To test the hypothesis, we will Optimize donor cell homing, engraftment, and expansion after BMT in a rodent SCD model using the following sub-aims. We will identify the optimal TMI dose to enhance donor chimerism and full engraftment which will reduce hemolysis, anemia, and tissue damage. We will investigate how TMI-based differential dose escalation activates SDF-1 gradient to support increasing donor cell homing to the bone marrow. Investigate if adding TPOm can further improve engraftment by augmenting BM vascular recovery and donor HSC expansion. The overarching goal of our interdisciplinary proposal is to produce a clinically relevant, effective, and safe TMI dose escalation alone or in combination with TPOm to improve chimerism and vascular regeneration while lowering regimen-related toxicities after HCT for SCD.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY/ABSTRACT Preemptive use of antivirals (PET) to control cytomegalovirus (CMV) viremia in hematopoietic cell transplant recipients (HCT-R) was a therapeutic advance balanced by elevated toxicity. Newer drugs such as letermovir (Prevymis) have lower toxicity with increased efficacy. FDA approval of Prevymis was for 100 consecutive days (d) of prophylaxis which reduced CMV reactivation by ~2fold in high and low risk HCT-R. The antiviral effect waned after 18 weeks without a survival benefit. We co-developed with NCI, a modified vaccinia Ankara (MVA) vaccine named Triplex expressing CMV immunodominant antigens. We published a safety study in healthy volunteers showing strong immunogenicity of the vaccine (NCT1941056), which preceded a published successful placebo-controlled and randomized Phase 2 trial (NCT2506933) of CMV-positive (P) HCT-R with either CMV-Positive (CMV-P) or CMV-Negative (CMV-N) HCT donors (HCT-D). The Phase 2 trial met its primary endpoint of reduced CMV reactivation in the vaccine arm by 50% with accelerated reconstitution of protective CMV immunity. Triplex outcomes can be improved; one approach is to vaccinate the immunocompetent HCT-D as demonstrated by our impressive preliminary results from an ongoing pilot study (NCT3560752) which showed that all HCT-R (N=12) receiving stem cells from vaccinated matched related donors (MRD) were protected from requiring PET. We hypothesize that Triplex injection of HCT-D will initiate protective immunity by transfer of expanded CMV-protective T cells as a component of the stem cell infusion to the HCT-R, preceding dosing with Prevymis, thereby eliminating its need. In Aim 1, we propose a Phase 2 randomized placebo-control trial (in centers not prescribing Prevymis for MRD-HCT) with eligibility of CMV-P HCT-R with MRD (intermediate risk) undergoing T-cell replete HCT for hematologic malignancy. CMV-P HCT-R will be randomized to receive stem cells from HCT-D receiving a single injection of Triplex or placebo identical to the pilot trial. This trial will show that a single HCT-D vaccination is sufficient to replace 100d of Prevymis to prevent PET usage in MRD-HCT-R. In the 180d trial period we will assess PET usage, measure CMV-specific CD8/CD4 T cells with the goal of associating frequency, memory phenotype, and gene expression with protection against reactivation leading to viremia or disease. To extend Triplex benefit to haploidentical HCT-R treated with post-HCT cyclophosphamide (PTCY) who are at high risk for CMV reactivation, in Aim 2 we propose a two-stage (Phase 1b/2) trial to choose an optimal Triplex vaccine strategy that promotes effective immune reconstitution with minimal CMV reactivation. All HCT-D will be vaccinated once with Triplex, and all HCT-R will be boosted with 3 Triplex injections on d28, 56, and 100. The initial open-label Phase 1b segment will either have patients abstain from Prevymis, or given 21d-100d of prophylaxis. The vaccination regimen with the least usage of Prevymis that still results in no increase in reactivation compared to standard Prevymis will be selected for follow-on randomized Phase 2 segment of vaccination with reduced or no Prevymis dosing compared to standard of care Prevymis with no vaccination.

NIH Research Projects · FY 2026 · 2023-04

ABSTRACT The long-term goal of this project is to define the molecular mechanisms of an error-prone, stress-induced Okazaki fragment maturation (OFM) pathway by which cancer cells counteract replication stress and survive. Replication stress is a hallmark of cancer cells and has been considered the Achilles' heel for cancer treatment such as radio- and chemotherapy. Under elevated temperature stress, yeast cells mutant for flap endonuclease 1 (FEN1 in humans or RAD27 in yeast) activate DNA damage response pathways to block cell proliferation and induce cell senescence and death; however, a subpopulation of cells can overcome these barriers and escape otherwise lethal conditions. Genome-wide mutations and rearrangements have been suggested as a major molecular mechanism that drives this evolution. However, how such spontaneous mutations are acquired in cells under replication stress is a long-standing question. Recently, we identified an error-prone, 3' flap OFM pathway that is activated in response to stress to support cell survival and fuel cellular evolution; its induction leads to genome-wide mutagenesis and suppression of restrictive growth temperature-induced lethality, a process mimicking that of cancer cells acquiring drug resistance. This led us to a model in which OFM can go in two ways, which may dictate the fate of cells, including human cancer cells: a 5' flap-based, error-free process or an alternative 3' flap-based, stress-induced, and error-prone process. However, key components that drive such flap dynamics remain undefined. The objectives of the proposed project are to define the key enzymes that catalyze 3' flap formation and cleavage in mammalian cells and to provide proof of concept that suppressing alternative 3' flap OFM can prevent drug resistance in human cancer cells. Further preliminary data gathered to support this grant application show that 3' flap OFM is conserved in both yeast and human cells. We observed that anti-cancer EGFR tyrosine kinase inhibitors activated the ATM/CHK2 DNA damage checkpoints in human lung cancer cells. Using yeast genetic screening, we identified Pif1 (PIF1 in humans) and Sgs1 (BLM and WRN in humans) as helicases for 5' to 3' flap transformation and Rad1 (XPF in humans) and Mus81 (MUS81 in humans) as 3' nucleases for 3' flap cleavage, in addition to the 3' nuclease activity of Pol . Therefore, our central hypothesis is that unprocessed 5' flaps in mammalian cells activate ATM/ATR and CHK1/2 signaling to recruit and stimulate PIF1, BLM, and WRN and/or other helicases for transforming 5' flaps into 3' flaps for nucleolytic degradation by 3' nucleases including Pol , XPF, and/or MUS81, and that blocking the 3' flap OFM pathway will suppress DNA mutations and thus prevent drug resistance. To test this, we will: i) determine the roles of helicases PIF1, BLM, and WRN in 3' flap formation and induction of alternative OFM; ii) define the functional distribution of 3' nucleases Pol , XPF, and MUS81 in processing 3' flaps with or without secondary structures during 3' flap OFM; and iii) define the extent to which stress-activated ATM/CHK2 signaling induces 3' flap OFM and mutations to support cancer cell survival and promote drug resistance.

NIH Research Projects · FY 2026 · 2023-04

Within the US, the incidence of diabetes is at epidemic proportions. At the same time, technological advancements are offering promising strategies to therapeutically ameliorate diabetes. The Arthur Riggs Diabetes & Metabolism Research Institute (AR-DMRI) at City of Hope (COH) is uniquely poised as a center of translational research excellence with a long-standing record of success in diabetes research. As such, the COH AR-DMRI has a profound commitment to training the next generation of talented scientific leaders with the skills needed to develop novel technologies and translate them into the clinic. The internationally recognized experts Drs. Debbie Thurmond and Rama Natarajan formalized this commitment by bringing together research mentors from 3 themes: Prevention/Risk/Biomarkers/Omics, Metabolism, and Diabetes Therapy to create the (PROMT) Predoctoral T32 Training Program. The goal of the T32 program is to support outstanding predoctoral students with analytically intensive science education with distinct perspectives to become scientific leaders in diabetes research in line with the NIDDK mission. The PROMT program will admit 2 predoctoral trainees/ year to participate in research across the translational science pipeline. PROMT will provide an unprecedented experience for predoctoral trainees and is predicted to strengthen their commitment to research careers that drive real change in clinical practice. Eligible candidates will be recruited through the Irell & Manella Graduate School of Biological Sciences (IMGS) at COH. After 2 years of IMGS core curriculum, PROMT students will commit to engage in PhD dissertation research and T32 program activities for 2 years. A 3rd year of PROMT training is available by competitive renewal. Trainees will select from among 16 Mentors and 17 Co-Mentors/Clinical Partners that comprise a collaborative group of faculties with varying degrees of interests from basic, translational to clinical science. Research interests of the mentors include islet biology, epigenetics, genomics and metabolomics, adipose and muscle metabolic dysregulation, and human islet transplantation, among others. Program highlights include robust institutional support (Years 1 and 2) and unique research resources on campus, including 3 GMP-compliant manufacturing facilities, and the Helford Clinical Research Hospital. The AR-DMRI is home to the NIDDK-sponsored Integrated Islet Distribution Program (IIDP) and the Human Islet Research Network (HIRN). The T32 curriculum focuses on diabetes and diabetes-related metabolic diseases, with an emphasis on responsible research conduct and reproducibility. Laboratory research, coursework, and translational research internships will be enhanced by regular institutional journal clubs, seminars, and symposia, and a yearly PROMT retreat. The PROMT structure includes an Executive Committee, Internal Advisory Board, External Advisory Board, Curriculum Committee, and Recruitment Committee. The AR- DMRI is in a period of rapid growth, adding new departments and top-level faculty and state-of-the-art facilities, and is pleased to welcome PROMT T32 students into our rigorous training environment.

NIH Research Projects · FY 2026 · 2023-03

Background. Despite the significant success of recent therapies, Acute Lymphoblastic Leukemia (ALL) remains the second leading cause of childhood death. The discordance between therapeutic improvements and poor outcomes is partially caused by the difficulty of salvaging relapsed disease. While outcomes have improved in de novo treatment, dismal rates of overall survival (less than 25%) were observed in relapsed subtypes of ALL. The clinical armamentarium for treating relapsed or refractory T-ALL must be supported with new options. Strategy. While most of T-ALL cases exhibit gain-of-function mutations in Notch signaling, therapies against Notch have not fulfilled their clinical promise. To identify alternative targets for new therapies, we propose to define how cell-intrinsic oncogenic events integrate with external signals from the microenvironment. Recent studies point to the functional impact of “stromal” signals in leukemia biology. However, one significant gap in this knowledgebase is how microenvironmental factors become essential for leukemogenesis and maintenance. Preliminary results. According to our results in primary T-ALL cells, activating mutations in Notch failed to saturate Notch signaling: Notch signal strength increased when T-ALL cells encounter Notch ligands within the microenvironment – e.g. interleukin 7 (IL-7). The increased strength of Notch signaling correlated with increased surface expression of IL-7Rα by direct transcriptional activation of the IL-7Rα promoter, resulting in T-ALL hyper- responsiveness to IL-7. IL-7 also induced the cell cycle regulator SKP2, activated STAT5, and (surprisingly) STAT3. Primary T-ALL cells showed persistent STAT3 activation and our results suggest STAT3 deletion impairs T-ALL leukemogenesis. Hypothesis. These data support significant interplay between oncogenic factors and the microenvironment. According to our hypothesis, interplay between microenvironmental signals (IL-7), Notch signaling, SKP2, and STAT3 form a reciprocal positive feedback loop that is essential for T-cell leukemogenesis; this axis also compensates for the action of standard therapies in relapsed and refractory disease. Approach. To test this, we propose: 1) To determine the temporal requirement for STAT3 deletion in initiation, progression, and relapse in T-ALL by using a model of inducible genetic deletion of STAT3 in combination with a model of Notch-induced T-ALL. 2) To map how T-ALL development is affected by Notch/IL-7/STAT3/SKP2 signaling circuitry by using overexpression and gene silencing approaches to define the reciprocal regulation of Notch, STAT3, and SKP2. 3) To identify the impact of inhibiting Notch/STAT/SKP2 circuitry in relapsed T-ALL by testing both pre-clinical and clinical inhibitors of STAT signaling and SKP2 inhibitors in pre-clinical PDX models of T-ALL. Impact. Successful completion of this proposed work will: 1) define how cooperation between oncogenic signaling and the microenvironment affects therapy of relapsed and refractory T-ALL; 2) build a foundation for validating new molecular targets in relapsed and refractory T-ALL; 3) provide a proof-of-principle for an alternative strategy in which entire molecular circuits are considered during the development of therapies.

NIH Research Projects · FY 2025 · 2023-02

Project Summary: Allogeneic hematopoietic cell transplantation (Allo-HCT) can cure hematological malignancies, but this treatment causes graft-versus-host disease (GVHD), an immune response of donor cells against the recipient. Anti-inflammatory corticosteroid medications can often control GVHD, but in some cases, GVHD is resistant to this treatment, especially when it affects the intestinal tract. The goal of this project is to understand the mechanisms that cause steroid-resistant (SR) gut GVHD and to develop effective approaches to prevent or treat SR-Gut-GVHD. Results with a newly established murine model have demonstrated that SR-Gut- GVHD is induced by prolonged administration of corticosteroids. Corticosteroid treatment induces expansion of IL-22-producing Th/Tc22-like cells, IL-22-dependent abnormalities in gut microorganisms, and reduced numbers of anti-inflammatory CX3CR1hi mononuclear phagocytes (MNP) that regulate T cell expansion and control bacterial translocation. Information from our preliminary studies and other investigators suggests that corticosteroid treatment inhibits the polyamine-hypusine pathway in T and MNP cells and regulates the fidelity of T cell lineage differentiation and the expansion of pro- and anti-inflammatory MNP cells. Our studies also suggest that steroid treatment increases Ceacam-1 expression by intestinal epithelial cells, thereby increasing bacterial transcytosis and unfavorably altering the balance between proinflammatory CX3CR1lo MNP and anti- inflammatory CX3CR1hi MNP cells. These changes trigger a feedforward pathogenic loop consisting of expanding IL-22-producing Th/Tc22-like cells, IL-22-dependent dysbiosis, and increased numbers of proinflammatory CX3CR1lo MNP with decreased numbers of regulatory CX3CR1hi MNP, leading to full-blown SR-Gut-GVHD. To test this hypothesis, this application proposes 3 aims. Aim 1 will determine whether corticosteroid inhibition of the polyamine-hypusine pathway in T cells leads to expansion of Th/Tc22-like cells with lineage infidelity. Aim 2 will determine whether corticosteroid inhibition of polyamine-hypusine pathway in CX3CR1+ MNP cells augment expansion of proinflammatory CX3CR1lo MNP with reduced numbers of anti- inflammatory CX3CR1hi MNP in response to challenge by dysbiosis. Aim 3 will determine whether observations in murine models reflect the pathogenesis of SR-Gut-GVHD in humans and whether reversal of abnormalities in the polyamine-hypusine pathway and whether blocking bacterial interaction with Ceacam-1 on intestinal epithelial cells by anti-Ceacam-1 mAb prevents and reverses SR-Gut-GVHD. Relevance: By elucidating in-depth mechanistic understanding of the pathogenesis leading to SR-Gut-GVHD in a well characterized murine model and by assessing the relevance of the experimental results in colon tissue from patients with SR-Gut-GVHD, results of this project could identify potentially effective approaches for translational testing in humans.

NIH Research Projects · FY 2026 · 2023-01

Modified Project Summary/Abstract Section In the United States, nearly 10% of the population (more than 30 million) has type 2 diabetes (T2D) and this number is growing not only in older adults but also in children, teens, and young adults. The alarming and continued increase of T2D incidence, especially among young Americans, creates an urgent need for new therapeutics and interventions. One of the underlying conditions leading to T2D is the dysfunction of beta cells. While discovering the molecular changes in beta cells leading to their dysfunction has been a focused effort in the field, less is known about how the microenvironment, such as secreted factors from exocrine pancreas, may also contribute to pathogenesis. Trefoil factor family peptides (TFF1, TFF2, and TFF3) are unique secreted proteins in that they contain six cysteine residues that form three intramolecular disulfide bridges, which makes them more stable and resistant to proteases and other harsh conditions. Trefoil factors have been shown to play essential roles in maintaining the integrity of gut and lung epithelium, and in repairing epithelial cells after injury. Trefoil factors are expressed in the pancreas, but their roles are not well understood. We have preliminary data revealing that Tff2 is expressed by the exocrine pancreas and the knockout of this gene in the pancreas leads to beta cell dysfunction in adult mice. However, the molecular and cellular mechanisms by which Tff2 exerts on beta cells are unknown. Our central hypothesis is that Tff2 protects against the damaging effects of diabetogenic stress. We will use our pancreas-specific knockout mouse model of Tff2 and primary human tissues/cells for our studies. In Aim 1, we will examine how the lack of Tff2 in the murine pancreas affects beta cell dysfunction in aged mice and under diabetogenic conditions. In Aim 2, we will clarify the distribution patterns of trefoil factor family proteins and their receptors in human pancreatic tissues and study the crosstalk between exocrine and islet cells via TFF2 by using in vitro model systems. We will use innovative tools to test novel hypotheses on the actions about trefoil factors within a translation-focused institutional environment at City of Hope. This work will positively impact diabetes research by addressing a promising candidate secretory factor for diabetes prevention/treatment.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Machine learning has the potential to transform pathologic diagnosis and to address very limited accessibility of expert pathology in low-income countries. Routine histology images of solid tumors contain an immense number of visual features that can be extracted and processed by artificial intelligence tools like machine learning, which excels at basic image analysis tasks such as tumor detection. In addition, machine learning can also predict clinically relevant features directly from histology images including microsatellite instability and immune features that independently predict prognosis response to therapy. This large, multicultural, racially and ethnically diverse study uses images of whole slides from routinely collected clinical specimens and applies computational pathology methods and digital spatial expression profiling to quantifiably improve CRC diagnosis, prognosis and predictive models together with clinical, epidemiologic and genetic data. The study goals will be accomplished through three specific aims. In Aim 1, we will apply novel machine learning algorithms from whole slide images to reproducibly identify MSI, histopathologic and immune features of colorectal cancer in racially/ethnically diverse populations. We will study H&E slides from 6,751 CRC cases, digitizing existing slides from 5,551 CRC cases and 1,200 new cases of CRC with contemporaneous clinical and epidemiologic data. Then, we will apply deep learning methods to accurately identify histopathologic features and immune characteristics of CRC. We will use a robust training validation, and testing design (70%/15%/15%) to ensure the rigor and reproducibility of our findings. In Aim 2, we will test whether machine learning algorithms that predict MSI and immune features related to CRC prognosis improve with the addition of clinical, epidemiologic, and germline genetic data. We will use machine learning statistical methods to test whether algorithms developed in Aim 1 improve prediction of overall survival and response to therapy with the addition of supplemental information beyond whole slide digital images. Finally, in Aim 3, we will compare the information derived from digital spatial profiling of expressed proteins in colorectal tumors with the information derived from Immunoscore quantification of lymphocyte populations at the tumor center (CT) and the invasive margin (IM), and explore whether these measures improve the models developed in Aims 1 and 2 in a subset of samples. We will perform GeoMx digital spatial profiling of 56 proteins expressed in 150 Stage I-III TNM colorectal cancers to compare the performance of digital spatial profiling to Immunoscore, a scoring system relying exclusively on expression patterns of CD3+ and CD8+ T cells. This study takes advantage of pathologic, epidemiologic, clinical, immunologic and germline genetic data from racially/ethnically diverse CRC patients from California, Detroit, New York, Florida, Puerto Rico, Israel and Spain. Our overarching goal is to improve the efficient diagnosis of colorectal cancer with clinically impactful immune profiles.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT (30 lines or less) The premise for the proposed research stems from precedence in other diseases, such as cancer, cardiovascular, neurodegenerative, and most relevant to the current proposal, autoimmune diseases, in which extracellular vesicles (EVs) play a role in the pathophysiology and are important biomarkers for early detection. However, in human type 1 diabetes (T1D), little is known concerning EVs in cellular communication. Our overall hypothesis is that autocrine-paracrine interactions, mediated through EVs, between the islets and islet-infiltrating immune cells in the pancreas contribute to the development and progression of T1D. Our specific aims are: 1) to assess the functional impact of islet-infiltrating T-cell derived EVs (T-EV) from T1D and autoantibody+ (Aab+) donors on islet health and on distinct immune cell populations; 2) to investigate the contribution of stressed or T1D islet-derived EVs (I-EV) on immune cell phenotype and islet health; and 3) to decipher the differential protein cargo in T-EV and I-EV from T1D and Aab+ donors. To address these aims, we have assembled a team of investigators with highly relevant expertise, techniques and unique resources of cell lines and tissue samples. From 36 human tissue donors with T1D or Aab positivity, we have >600 T cell lines grown directly from the individual islets of pancreata. We have the expertise and technical ability to isolate EV from islets (I-EV) and from islet-infiltrating T cell lines (T-EV) from T1D donors. Our preliminary data indicates I-EV and T-EV have both paracrine and autocrine effects on islet health and immune cell phenotype. Our Research Plan is to generate T-EV and I-EV from donors with T1D of distinct durations or positivity for islet autoantibodies, to evaluate their effects on islet health and immune cell function, and to determine the uniquely packaged protein cargo from these EVs whose molecular composition reflects the pathophysiologic state of the disseminating cell. These studies will yield important information concerning the communication between immune cell populations and islets via EVs in the pathogenesis of T1D, and potential biomarkers or therapeutic targets for T1D.

NIH Research Projects · FY 2025 · 2022-09

Abstract Widespread use of serum prostate-specific antigen (PSA) screening results in 50% of new cases of prostate cancer being diagnosed with low-grade localized disease. Standard of care for these cases is to defer immediate treatment in favor of active surveillance (AS), a low-toxicity management which entails close monitoring with PSA tests, repeat prostate biopsies and multi-parametric MRI (mpMRI). Around 30% of men on AS progress or electively undergo definitive treatment within two years of diagnosis. Efficacious low-cost, low-toxicity strategies to limit exit from AS are needed to reduce over-treatment and improve health-related quality of life (QOL). Accordingly, in this proposal we will test whether and how exercise alters prostate cancer molecular landscape. Using a two-arm RCT design, 122 men initiating AS (e.g., inactive, diagnostic biopsy <6 months) will be randomly allocated (1:1 ratio) to exercise therapy intervention (n=61) or usual care (plus general physical activity advice; n=61) for 12 months. Exercise therapy will consist of home-based, treadmill walking five days each week at 65-75% of their personal baseline exercise capacity. All sessions will be implemented using TeleMed-X, a novel telemedicine platform pioneered in our program. Our central hypothesis is that exercise therapy will reduce the number of nimbosus features to collectively inhibit clinical progression. This central hypothesis is tested in three distinct, but complementary aims: Aim 1. Determine effects of exercise therapy on molecular nimbosus hallmarks; Aim 2. Determine effects on the radiologic and pathologic nimbosus hallmarks; and Aim 3. Delineate the precision of estimating rates of clinical progression at 18 months.

NIH Research Projects · FY 2025 · 2022-09

Abstract Cancer is the second most common cause of death in developed nations, and incidence is rising among developing nations. An estimated 70% of cancers are attributable to “modifiable” risk factors, including obesity, chronic inflammatory diseases, and poor diet, all of which have been associated with increased oxidative stress. These are not themselves “carcinogenetic”, but they are thought to act as “cancer promoters”, increasing the probability of developing cancer. With advances in whole genome sequencing and development of computational techniques to examine the cancer genome, we can now use mathematical profiling of somatic mutational profiles (termed mutational signatures) to identify potential causes underlying a given tumor (e.g., smoking versus UV light). It remains unclear, however, if there is a mutational signature that is a biomarker of cumulative lifetime exposure to reactive oxygen species (ROS)-mediated DNA damage and if this correlates with cancer-associated lifestyle factors. Here, we will utilize cutting-edge multi-omic profiling and molecular biology and computational tools to better understand the contribution/mechanism of oxidative stress as a cancer promoter. To examine the correlation of ROS mutational signature levels and inflammation-related cancer risk factors, we will perform whole genome sequencing and mutational signature analysis of a large cohort of colorectal tumors from patients with detailed, longitudinally collected lifestyle data (e.g., smoking, caloric intake, red meat intake, exercise level, etc.) collected by the Molecular Epidemiology of Colorectal Cancer study. We will also evaluate whether accumulation of ROS-generated mutations is biased toward CTCF binding loci and whether chromosomal architecture is modified by exposure to carcinogens or cancer- associated processes, thereby mediating unique “epigenomic signatures”. These aims will also provide data that can be used in the development of two novel computational tools for the analysis of cancer driver mutational signatures and epigenomic signatures of carcinogen exposure. Finally, we will test the molecular and clinical benefit of intermittent fasting during daily radiation therapy based on the hypothesis that lifestyle factors could modulate susceptibility to ROS mutagenesis. Patient- reported quality of life and clinician-reported adverse events, as well as molecular assay for tissue-specific levels of ROS-associated DNA damage, will allow us to assess whether intermittent fasting can reduce normal tissue toxicity. Successful completion of the proposed research will provide a comprehensive examination of the epidemiology and mechanism of ROS-mediated DNA damage in human cancers and demonstrate the safety and potential efficacy of intermittent fasting as a clinically translatable and easily adaptable approach to reducing both acute and chronic side effects associated with radiotherapy.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Primary central nervous system lymphoma (PCNSL) is a rare hematologic maligancy in which non-Hodgkin lymphoma (NHL) initially presents in the central nervous system (CNS). Therapeutic options for PCNSL are limited; standard of care high-dose methotrexate-containing regimens have been unchanged for over 40 years, and are not curative in most patients. Chimeric antigen receptor (CAR) T cell therapy targeting CD19 (CD19- CAR T cells) is a powerful form of immunotherapy that has an established safety profile when delivered intravenously (IV) to treat patients with NHL. Our clinical platform for manufacturing CD19-CAR T cells at City of Hope (COH) has been evaluated in a series of phase 1 clinical trials for B cell acute lymphoblastic leukemia (ALL) and for NHL. To date, all previous and ongoing CD19-CAR T cell trials have infused the CAR T cell product IV. We have preliminary evidence that IV-administered CD19-CAR T cells can be detected in the CNS and have anti-tumor activity in treating patients with PCNSL. However, the efficacy of IV CAR T cell therapies for patients with PCNSL is limited, possibly due to poor trafficking of CAR T cells from blood to CNS that may result in reduced activity against PCNSL compared to systemic NHL. In phase 1 trials at COH, locoregional delivery of CAR T cells to treat CNS malignancies such as glioblastoma has led to improved outcomes. Studies in our animal models show improved disease response, durability and resistence to tumor rechallenge using intracerebroventricular (ICV)- vs IV- delivered CD19-CAR T cells in xenograft mouse models of CNS lymphoma. Thus, to optimize the efficacy of CD19-CAR T cells and improve the outcomes of patients with PCNSL, we propose to administer CD19-CAR T cells via ICV delivery. We hypothesize that ICV-delivered CD19-CAR T cells will be safe and demonstrate high anti-PCNSL activity. In Specific Aim 1, we will conduct a phase 1 clinical trial to assess the safety and activity, and determine the recommended phase 2 dose (RP2D) of ICV delivered CD19-CAR T cells in participants with PCNSL. In Specific Aim 2, we will conduct a series of correlative studies to assess mechanisms of toxicity, CAR T cell persistance, trafficking to the peripheral blood, immune cell phenotype, and effect on tumor. We plan to examine the effects of ICV-delivered CAR T cells on normal CD19+ B cells in the peripheral blood to determine whether CAR T cells traffic from the CNS to the blood, as we expect based on our preclinical animal models of PCNSL. Activity against normal B cells in the blood would indicate that ICV delivery could be a viable treatment option for patients with secondary CNS lymphoma, who have both CNS and systemic disease. Successful completion of the proposed clinical trial and correlative studies will expand therapeutic options for patients with PCNSL and could inform the design of a potential subsequent clinical trial to evaluate the safety and efficacy of treating patients with secondary CNS lymphoma.