Beckman Research Institute/City Of Hope

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Therapeutic resistance is a major contributor to high lethality in pancreatic ductal adenocarcinoma (PDAC). A prominent feature of PDAC is desmoplasia, the formation of fibrous tissue that not only plays a critical role in reducing drug perfusion but also in limiting anti-tumor immune cell infiltration and function. The fibrous tissue is primarily composed of the extracellular matrix (ECM) components hyaluronan (HA) and collagen (CN). Previous methods to target these major ECM components have caused severe systemic side effects in patients and, thus, finding a safe, effective approach to disrupt the PDAC ECM and improve drug delivery remains a critical unmet need. Our long-term goal is to develop tumor-specific, microbial-based agents that express functional ECM- degrading enzymes. This novel strategy will remediate tumor desmoplasia, minimize systemic toxicity, and maximize the penetration and efficacy of therapeutics against primary PDAC tumors, as well as distal metastases. The objective of this proposal is to determine the utility of attenuated Salmonella typhimurium (ST)-based agents, engineered to express the ECM-degrading enzymes hyaluronidase (ST-HAse) and collagenase (ST-CNase), in triggering collapse of dense tumor stroma and in enhancing therapeutic efficacy in clinically-relevant models of PDAC. The rationale underlying this proposal is that successful completion of these studies will identify a feasible, tumor-targeting approach to ameliorate desmoplasia in PDAC, which will enable anticancer agents to achieve their greatest therapeutic effects. Our central hypothesis is that degrading both HA and CN in PDAC using tumor-specific ST vectors will induce the greatest stromal collapse, ultimately leading to enhanced penetration and efficacy of therapeutic treatment. This central hypothesis will be tested in relevant models of PDAC by pursuing three specific aims: (1) Determine the effect of dual ST-HAse/CNase treatment on the antitumor efficacy of standard-of-care chemotherapy; (2) Determine the impact of dual ST-HAse/CNase treatment on efficacy of immune checkpoint blockade therapy; and (3) Develop and characterize recombinant STs expressing HAse and CNase under tumor-inducible promoters. The use of tumor-colonizing ST and tumor-inducible bacterial promoters to express ECM-degrading enzymes is an innovative strategy to limit the effects of stromal degradation to tumor tissues. Furthermore, simultaneously degrading HA and CN will result in greater tumor permeability than targeting either component alone. The results of this work will have a significant impact for PDAC patients, because it is predicted to yield an agent(s) that can, in the short-term, be optimized for manufacturing and Investigational New Drug (IND)-enabling studies and, in the long-term, become a first-in-class, microbial-based agent used to significantly improve drug permeability of desmoplastic primary and metastatic tumors that are inaccessible to conventional therapy.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Patients with recurrent epithelial ovarian cancer (EOC) have a poor prognosis with a post-relapse median survival of approximately 30 months and limited therapeutic options, thus presenting a fundamental unmet medical need. Progress in immunotherapy across a broad range of tumor types provides hope that immunological approaches may improve outcomes for patients with EOC. Particularly, a type of immunotherapy called chimeric antigen receptor (CAR) T cell therapy retrains the immune system to target cancers by recognizing specific cancer markers. EOC presents several challenges to effective CAR T cell immunotherapy, including poor tumor site infiltration, activation, inadequate function and persistence of these T cells within the harsh peritoneal tumor microenvironment. Additionally, there are a lack of effective CAR T cell targets on the surface of advanced EOC tumor cells. Our goal is to develop effective therapies against metastatic EOC, with a specific focus on regional delivery of CAR T cell therapies to treat peritoneal metastasis. TAG72 is highly over- expressed in EOC and other solid tumors with little or no expression in normal tissues, making it an ideal target for CAR T cell therapy. Our team at City of Hope has developed and completed laboratory testing of a TAG72- targeting CAR T cell therapy. Our preclinical data also supports superior anti-tumor activity when TAG72 CAR T cells are administered regionally by intraperitoneal delivery versus systemically by intravenous delivery, likely due to direct and immediate antigen CAR T cell access to tumor cells. The hypothesis is that regionally- administered TAG72-CAR T cells will be safe and mediate anti-tumor effects, which will be assessed in the following specific aims: 1) Evaluate safety and feasibility of regional intraperitoneal delivery of TAG72-CAR T cells in patients with advanced EOC in a phase 1 clinical trial; 2) Assess CAR T cell-mediated immune landscape changes that may indicate therapeutic response or resistance; and 3) investigate pathways of tumor resistance and CAR T cell-induced tumor evolution. Our program has incorporated an innovative use of pre-conditioning regimens to our solid tumor CAR T cell therapies, regional routes of CAR T cell administration, and a fully- optimized TAG72-CAR construct. These features aim to improve the potency and selectivity of targeting TAG72+ tumors while potentially minimizing immune responses that limit persistence and/or function of TAG72-CAR T cells. This approach is significant in that it will expand our therapeutic portfolio for EOC and other solid tumors.

NIH Research Projects · FY 2025 · 2022-08

Project Summary Alzheimer’s disease (AD) is the most common form of dementia in the elderly and there is no cure for this disease. The molecular and cellular mechanisms underlying AD pathogenesis remains to be elucidated to develop effective therapies for this disease. Many mouse models have been generated for AD research and these models provide important insights to aid our understanding of the pathological basis of the disease. However, because there are significant species differences between mouse and human neural cells, establishing human disease modeling platforms is needed to complement studies in animal models to better understand AD. Human induced pluripotent stem cells (hiPSCs) have been widely used for disease modeling since the development of the iPSC technology. hiPSCs have been used to model various aspects of AD. Because hiPSCs and their derivatives have been considered phenotypically young, hiPSC-derived cells have been used to model early events of AD. Direct reprogramming is another type of reprogramming that converts one type of somatic cells into another without going through the iPSC stage that involves extensive epigenetic modifications, thus enabling generation of human cells that possess key elements of cellular aging. Therefore, directly reprogrammed cells derived from patient somatic cells would allow us to model age-related pathologies of AD. ApoE4 is the strongest and the C allele of the CLU rs11136000 SNP is the third strongest genetic risk factor for AD. The objective of this proposal is to define the effect of the CLU rs11136000 SNP alone or together with ApoE4 on the risk to AD and uncover molecular and cellular mechanisms underlying the effect, using human cellular models generated from hiPSCs or through direct reprogramming. We will use gene-edited isogenic cells to define the effect of CLU SNP in combination with ApoE isoform. In addition, because hiPSC-derived cells and directly reprogrammed cells derived from the same donors have the same genetic background but different cellular aging status, they represent isogenic cellular platforms that will enable us to specifically study the effect of cellular aging. These isogenic models will allow us to recapitulate age-associated phenotypes and uncover novel pathological mechanisms underlying AD. Because both CLU and ApoE are highly expressed in astrocytes, we propose to define the effect of the CLU SNP alone or together with ApoE using astrocytes. We hypothesize that CLU modulates AD pathologies in an ApoE isoform- and age-dependent manner. Therefore, we propose following Specific Aims: Aim 1: To derive astrocytes with different APOE/CLU genotypes and cellular aging status from hiPSCs or through direct reprogramming. Aim 2: To define the effect of APOE and CLU variants on AD pathogenesis using astrocyte-neuron or astrocyte-OPC co-cultures. Aim 3: To determine the relationship of APOE/CLU genotypes and aging with gene expression change in human brains. The proposed studies will help to define the roles of the CLU SNP in the development of age-associated AD pathologies, to uncover mechanisms underlying AD pathogenesis, and to design novel therapeutic strategies for AD.

NIH Research Projects · FY 2026 · 2022-07

Today there are more than 85,000 EPA-registered synthetic chemicals, but only 10% have been tested for carcinogenicity in animal studies. Of those tested, ~200 have shown evidence of causing cancer and/or an increase in mammary tumors. However, few of the chemicals have been evaluated in human studies, and results in humans have been inconclusive. Our study will focus on chemical body burden of per- and poly-fluoroalkyl substances (PFAS), and concomitant risk of developing invasive breast cancer during the menopausal transition. These “forever chemicals” are pervasive, enduring, and of high public interest, yet studies of their possible relationship to breast cancer have been limited. We hypothesize that elevated body-burden levels of these endocrine-disrupting chemicals (EDCs) will increase the risk of developing invasive breast cancer, and that alterations in DNA methylation (DNAm) and the breast microenvironment are mechanisms that link these chemical exposures and breast cancer. We will test this hypothesis using a three-pronged approach. In Specific Aim 1, we will conduct a prospective study of women in the American Cancer Society Cancer Prevention Study-3 (CPS-3) to assess the association between body burden of PFAS during the menopausal transition and subsequent development of invasive breast cancer. We will measure PFAS levels in plasma samples collected 1 to 7 years before invasive breast cancer diagnosis in 1000 CPS-3 participants and 1000 matched, cancer-free CPS-3 participants, all between 40 and 57 years of age at blood draw. In Specific Aim 2, studying the same participants, we will measure DNAm using Infinium MethylationEPIC BeadChips and conduct an epigenome- wide association study (EWAS) to identify DNAm changes associated with levels of PFAS in the cancer-free participants. We then will determine the association between the PFAS-associated DNAm changes and risk of developing breast cancer. This valuable DNAm data also can be used later for other outcomes, including exposure to other EDCs. In Specific Aim 3, we will determine the direct effects of PFAS on tissue- and molecular- level states associated with susceptibility to cancer initiation in genomically-well-characterized primary human breast mammary epithelial cells (HMECs). Identifying these mechanisms in primary breast cells is critically important, as PFAS mechanisms of action generally differ by tissue. We will use 2-D cultures to determine the effects of short-term exposures and 3-D cultures to define the effects of protracted chemical exposures on changes in epithelial lineage consistent with accelerated aging and age-related molecular changes in genome methylation, lineage-specific transcription, and cytokeratin proteins. Results from this multi-disciplinary approach will advance our understanding of the effects of PFAS exposures on risk of developing breast cancer during an important window of susceptibility. Ultimately, we hope results from our proposed project will identify an integrated biological signature of environmental exposure, deliver mechanistic insights into breast cancer development from EDCs, and inform future studies for prevention strategies to reduce or mitigate exposures.

NIH Research Projects · FY 2025 · 2022-07

Project Summary / Abstract In the last decade there has been an explosion of resolved high-resolution structures of G-protein coupled receptors (GPCRs) and their complexes with several G-proteins, collectively known as transducer proteins. GPCRs are dynamic proteins, and exist in multiple functional conformational states. Comparisons of three-dimensional structures of the inactive and active states of GPCRs have led to identification of residue pair distances that show distinct changes upon activation. Such residue pairs are known as “activation microswitches”. Molecular Dynamics (MD) simulations is an attractive tool for identifying (i) the residue pairs that are critical to GPCR activation, (ii) residue pairs involved in allosteric communication from the ligand binding site to the G protein coupling site, and (iii) residue pairs in the GPCR:G protein interfaces that contribute to their coupling strength and selectivity. While our ability to generate long time scale dynamics trajectories have increased exponentially, the results of MD simulations have largely been analyzed using prior knowledge of the GPCRs. There is a critical need for adopting the unbiased, data-driven, systems biology tools to analyze long time scale MD trajectories data to mine knowledge on the residue motions that provide information on allosteric communication network in GPCRs. Our overarching goal in this grant is to apply Bayesian Network (BN) modeling, an interpretable machine learning methodology, to the MD simulation trajectories data on GPCR:G protein complexes in order to identify the residues in various GPCR structural regions that contribute to ligand selectivity. Network-centered approaches have not been used, so far, to analyze high-dimensional residue pairs MD simulation data. BN modeling, in particular, has attractive properties (interpretability, probabilistic nature of the data representation, statistical validation, tools for topology comparison and analysis) that presently deployed secondary MD simulation data analysis methods, such as principal component analysis (PCA) of residue pairs, lack. We propose to use BN modeling with large scale MD trajectories (multiple short and multiple long trajectories) of inactive state GPCRs and fully active state GPCR:G protein complexes to (i) identify the activation microswitches, the residue pairs that show large scale conformational changes upon activation, and (ii) outline the residue network involved in the allosteric communication from the agonist binding site to the G-protein coupling sites. We will also (iii) identify the residues in the GPCR:G protein interface that contribute to selectivity in coupling to specific family of G proteins (Gs, Gi and Gq). In aim 2, we will (iv) dissect time-correlated events using BN series and dynamic BN (DBN) models to delineate and scrutinize the residue networks that lead to large scale transitions. Importantly, the deliverables will include an unprecedented “toolkit” (algorithms + software) incorporating system biology tools for analyzing MD simulation trajectories data that are generalizable to any protein complexes.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Administrative Supplements on Long-Term Cancer Survivorship. While childhood leukemia and lymphoblastic lymphoma therapies have achieved remarkable cure rates, they have created a growing population of survivors facing persistent treatment-related neurocognitive, academic, and related functional challenges into adulthood. To address these long-term effects, we developed and have iteratively refined a high-intensity intervention program (HIP) that, unlike traditional child-directed interventions (which have not yielded long-lasting improvements), takes a parent-directed approach, equipping parents with evidence-based tools to support their children’s learning and academic success. The parent randomized controlled trial (R01CA261793) evaluates our fourth iteration of HIP: HIP-eHealth, which delivers parent training remotely from a single site (City of Hope) and guides parents in supporting their children’s academic progress through use of the award-winning online learning platform IXL. In the parent study, survivors of childhood leukemia and lymphoblastic lymphoma and their parents (target: N = 166 dyads) are randomized to HIP-eHealth or a lower-intensity, single session mimicking usual care provided to survivors of pediatric brain tumors (LIP). HIP-eHealth has shown promising early results, with high completion rates and positive parent reports of increased self-efficacy and improved child academic outcomes. However, preliminary data from 133 currently enrolled parent/child dyads has revealed unexpected challenges emerging from the introduction of IXL. IXL’s curriculum-based assessments in Math and English Language Arts have revealed that 56% of children randomized to HIP-eHealth are performing at least two grades below their actual grade level. Such substantial academic gaps cannot be effectively closed without sustained practices extending well beyond active intervention, after external support concludes and families transition to independence. Unfortunately, many parents have required far more study team support than expected to facilitate consistent IXL engagement. Understanding the factors influencing sustained, independent parental engagement vs. reliance on external support is crucial for optimizing intervention effectiveness and ensuring long-term academic gains. In this project, we will conduct semi-structured interviews with 30 parent/child dyads (N = 60 total participants) who completed HIP-eHealth, strategically sampling families who required high study team support vs. those who demonstrated independent engagement in IXL. In Aim 1, we will identify parental beliefs and contextual factors influencing parents’ sustained engagement in their children’s academic support. In Aim 2, we will examine child perspectives on intervention components and factors contributing to continued use of academic strategies and practices post-intervention. This research will provide essential context for interpreting parent study findings and transforming our descriptive observations of variable parent-child engagement patterns into actionable insights critical for scaling HIP-eHealth effectively.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with dismal prognosis. A near-universal oncogenic driver of PDAC is the constitutive activation of the small GTPase protein Kras, which induces multiple downstream signaling cascades that together facilitate rapid cell proliferation, metastasis and therapeutic resistance. To surmount the high energy demands of these activities, Kras also triggers metabolic adaptations to promote nutrient scavenging from extracellular sources, such as through macropinocytosis. Macropinocytosis is a process by which extracellular material is non-specifically engulfed and then degraded in lysosomes to produce end-products utilized by tumor cells for biosynthesis. This process essentially confers resistance to a myriad of anabolic inhibitors. Syndecan-1 is a heparan sulfate proteoglycan (HSPG) upregulated on the surface of cells that serves as the key mediator of macropinocytosis in PDAC and other Kras-driven cancers that includes bladder, lung, prostate, colon and breast. In addition to mediating macromolecular transport, HSPGs can be found in the tumor extracellular matrix (ECM) binding to and regulating the interaction of numerous signaling molecules (e.g. growth factors and cytokines) with their cognate receptors. The pro-tumorigenic activities of HSPGs are exquisitely regulated by enzymatic modification of their heparan sulfate (HS) moieties. Mammalian heparanases employ hydrolytic cleavage of the beta-(1,4)-glycosidic bond between glucuronic acid and glucosamine to promote the release of growth factors and enzymes involved in ECM remodeling, invasion and metastasis. In contrast to mammalian heparanases, bacterial heparinase III (HepIII) depolymerizes HSPGs through a unique beta-elimination mechanism that cleaves at the alpha-(1,4)- glycosidic bond. Various studies have confirmed HepIII modification of HSPGs suppresses neovascularization, macropinocytosis, tumor growth and metastasis. However, the inability to restrict HepIII activity to tumor tissue has long prohibited its use as a therapeutic agent. Using attenuated, tumor-targeting Salmonella typhimurium (ST) vectors, we have developed the first recombinant ST expressing functional HepIII (ST-HepIII) through a tightly regulated, inducible promoter. We have confirmed the ability of ST-HepIII to suppress high-affinity HS interactions, macropinocytosis, and growth of Kras-mutant tumors. In this application, we will: 1) Determine the impact of ST-HepIII treatment on metabolite availability and metabolic-associated gene pathways in vivo; 2) Determine anti-tumor efficacy of anabolic inhibitors in combination with ST-HepIII; and 3) Develop and characterize recombinant STs expressing HepIII under tumor-inducible promoters for greater clinical feasibility. Completing these aims will allow us to develop a novel class of tumor-targeting agents capable of suppressing a metabolic process essential to the survival of PDAC and other Kras-driven cancers. Our agents may be used to counteract acquired resistance to standard-of-care therapies that target cooperative anabolic processes and positively impact survival for patients with difficult-to-treat cancers.

NIH Research Projects · FY 2025 · 2022-05

ABSTRACT N6-methylation (m6A) is one of the most abundant endogenous modifications in eukaryotic mRNA. Accumulating evidence suggests that dynamic m6A RNA methylation has significant roles in multiple biological processes, and tumorigenesis by introducing another layer of post-transcriptional regulation of gene expression. Acute myeloid leukemia (AML), one of the most common types of acute leukemia, is a hematological malignant disease. In AML patients, gene mutations and genomic rearrangements often occur in hematopoietic stem/progenitor cells (HSPCs), and turn HSPCs into leukemia stem cells (LSCs), which play a central role in the development and maintenance of AML. To date, there is not an effective targeted therapy available for AML. The roles of m6A modification and its associated machinery in the pathogenesis of AML and the maintenance of LSCs/LICs remain elusive. As a nuclear m6A RNA reader that has been identified, YTHDC1 recognizes nuclear m6A-sites and acts as a critical mediator of nuclear m6A. YTHDC1 has unique roles in the regulation of nuclear RNA splicing, alternative polyadenylation, nuclear export and decay. However, its role and function in AML has not been reported yet. Our preliminary studies showed that YTHDC1 is overexpressed in human AML. We found that YTHDC1 is required for MLL-AF9-induced leukemogenesis and the survival of LSC/LIC. Thus, we hypothesize that YTHDC1 upregulation leads to post-transcriptional deregulation of a set of genes that are required for the maintenance of LSCs, thereby contributing to the pathogenesis of AML. In this proposal, we will determine 1) the role of YTHDC1 as a nuclear RNA reader in the initiation, development and maintenance of AML by regulating LSC self-renewal and survival; and 2) the underlying molecular mechanisms that mediate the role of YTHDC1 in the pathogenesis of AML. We will employ both genetic murine models as well as patient-derived xeno-transplantation (PDX) models to investigate the role of YTHDC1 in the pathogenesis of AML in vivo, and will combine transcriptome and epitranscriptome analysis to identify the key downstream targets and associated downstream pathways that mediate the role of YTHDC1 in leukemogenesis. In addition, we will elucidate molecular mechanisms by which YTHDC1 epigenetically controls the expression of its direct targets in AML cells. Our studies will define the unrecognized role of YTHDC1 in the AML development/maintenance and regulation of LSC/LIC self-renewal, quiescence and survival, and clarify the underlying mechanisms of YTHDC1. Thus, success of our project will provide new insights into the complicated mechanisms underlying epitranscriptomic regulation of leukemogenesis.

NIH Research Projects · FY 2025 · 2022-04

PROJECT SUMMARY B-cell acute lymphoblastic lymphoma (B-ALL) is a neoplasm of B-cell lymphoid precursors that typically affects children, but occurs in adults as well. While intensive multi-agent chemotherapy is highly effective in the pediatric population, outcomes remain poor in adults and high-risk patients. The recent introduction of blinatumomab and CD19-directed CAR T therapy has transformed the care of patients with relapsed and refractory (r/r) B-ALL. However, an increasing number of reports describe a high rate of post-treatment relapse due to acquired resistance to these immunotherapies, notably via down-regulation or loss of CD19 surface expression. CAR T cells that were recently developed against another target, CD22, showed similar shortcomings with relapses associated with diminished antigen expression. The search for alternative targets is therefore essential to overcoming antigen escape. To address this issue, we have developed CAR T cells against a new B-ALL target, the B-cell activating factor receptor (BAFF-R). BAFF-R is a marker of B cells that is also functionally expressed in B-ALL, including in patients with CD19-negative relapse. Although one of BAFF-R’s ligands (BAFF) has been successfully targeted for the treatment of autoimmune diseases, we are the first to have developed BAFF-R CAR T cells that are effective against B-cell malignancies in vivo, including in CD19-negative mouse models. Because BAFF-R is a critical regulator of B-cell function and survival, we can expect that the tumor’s ability to escape therapy by down-regulation of BAFF-R will be limited. While we anticipate clinical efficacy of BAFF-R CAR T cell therapy, dual targeting of CD19 and BAFF-R can prevent the emergence of resistance and improve clinical outcomes. Therefore, we are also developing bispecific CD19- BAFF-R CAR T cells that we aim to rapidly translate from the bench to the bedside. In Specific Aim 1, we will evaluate BAFF-R CAR T cell therapy in a first-in-human clinical trial in patients with r/r B-ALL who are ineligible for or have failed prior CD19-directed therapy. The trial, currently open at City of Hope, will use our TN/MEM- derived manufacturing platform, which yields a naïve/memory T-cell enriched T cell product and has shown remarkable efficacy and tolerability in B-ALL. We will conduct extensive correlative studies using cutting-edge technologies, such as assessing the kinetics of the CAR T cells and that of diverse cytokines, therapeutic effect, and potential mechanisms of relapse and antigen escape. In Specific Aim 2, we will establish the therapeutic efficacy of bispecific CD19-BAFF-R CAR T cells against mixed (CD19-negative + BAFF-R- negative) B-ALL tumor models that mimic the heterogenous tumor phenotype leading to resistance. We will also perform extensive testing of cGMP-grade bispecific CD19-BAFF-R CAR T cells that is required prior to submission of an IND application to the FDA. Our proposal addresses the urgent need for new therapeutic options in patients with r/r B-ALL and could also benefit patients with other B-cell malignancies.

NIH Research Projects · FY 2026 · 2022-03

PROJECT SUMMARY (ABSTRACT): Title: The role and therapeutic potential of IGF2BP2 in MLL-rearranged leukemia. Background: N6-methyladenosine (m6A) modification is the most abundant internal modification in eukaryotic messenger RNAs (mRNAs) and plays roles in many normal bioprocesses. Evidence is emerging that the aberration in m6A modification and the associated machinery also plays important roles in various types of cancers. Acute myeloid leukemia (AML) is one of the most common forms of hematopoietic malignancies with various cytogenetic and molecular abnormalities. The mixed-lineage leukemia (MLL)-rearranged (MLLr) AML is a common and fatal subtype of AML, which accounts for 5%-10% of “de novo” AML cases and 10%-15% of therapy-related leukemia (t-AML) cases. MLLr AML patients are associated with poor outcomes, with a 5-year overall survival (OS) rate of ~30%. Therefore, there is a critical unmet medical need to develop improved therapeutics for MLLr AML treatment. The leukemia stem/initiating cells (LSCs/LICs) are considered to be the root cause for the treatment failure and relapse of AML. Collectively, it is critical to better understand the molecular mechanisms underlying MLLr AML pathogenesis and LSC/LIC self-renewal, which may lead to the development of improved novel therapeutic strategies to treat MLLr AML. Our preliminary data showed that IGF2BP2, which encodes an m6A reader, is specially overexpressed in MLLr AML and its increased expression is associated with a poor prognosis in AML patients. Our preliminary functional studies suggest that IGF2BP2 likely plays a critical oncogenic role as an m6A reader in promoting MLLr AML pathogenesis. IGF2BP2 is also expressed at a significantly higher level in MLLr LSCs/LICs compared to healthy hematopoietic stem/progenitor cells (HSPCs) and bulk MLLr AML cells, implying a role of IGF2BP2 in MLLr LSC/LIC self-renewal. In addition, we have developed a potent inhibitor (namely CWI1-2) that targets IGF2BP2 directly and exhibits high anti-leukemia efficacy as shown in our preliminary studies. Hypothesis: IGF2BP2 plays an essential role in MLLr AML pathogenesis and LSC/LIC self-renewal, and that pharmacological inhibition of IGF2BP2 can lead to effective treatment of MLLr AML. Specific Aims: 1) Determine the role of IGF2BP2 in MLLr AML pathogenesis and LSC/LIC self-renewal; 2) Decipher the molecular mechanism underlying the role of IGF2BP2 in MLLr AML; and 3) Assess the therapeutic potential of pharmacologically targeting IGF2BP2 in treating MLLr AML. Potential Impact: Our proposed studies are of high novelty and high significance in both basic research and translational medicine, which will substantially advance our understanding of the biology of MLLr leukemia, and may also result in the development of effective novel therapeutics for the treatment of MLLr leukemia.

NIH Research Projects · FY 2025 · 2022-03

Project Summary Hypertensive and ischemic heart diseases are the two most important risk factors of heart failure. In response to elevated blood pressure, the heart manifests hypertrophic growth to ameliorate ventricular wall stress. This once adaptive response may decompensate and progress into heart failure. On the other hand, myocardial infarction causes significant structural damage of the heart. The common clinical practice to treat cardiac ischemia via restoration of coronary arteries leads to additional reperfusion injury. Insults from ischemia and reperfusion together significantly weaken the pumping function of the heart. Despite extensive interests and urgent clinical needs, our understanding of the mechanisms for heart failure development remains limited. Pathological cardiac remodeling is a common route of both hypertensive and ischemic heart diseases. In response to either elevated demand or cardiac damage, the heart mounts an acute reaction to compensate for the loss of cardiac contractility. Under persistent stress, however, decompensation occurs and heart failure develops. Previous studies have shown that metabolic alteration precedes most if not all other changes during pathological cardiac remodeling. However, the contribution and mechanism of metabolic remodeling in heart failure is still elusive. Preliminary results here show that de novo pyrimidine biosynthesis is acutely and significantly augmented in the heart in response to pressure overload, preceding structural and electrophysiological alterations. Moreover, Cad (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) as the rate-limiting enzyme of this pathway is strongly induced. On the other hand, de novo pyrimidine biosynthesis is also upregulated by reperfusion after ischemia. Based on previous findings and these pilot data, a hypothesis of pyrimidine biosynthesis in pathological cardiac remodeling has been formulated. Both gain-of and loss-of- function mouse models have been generated that will be employed to test 1) the role of Cad and de novo pyrimidine biosynthesis in pressure overload-induced cardiomyopathy, 2) the role of Cad and de novo pyrimidine in cardiac ischemia/reperfusion-caused pathological cardiac remodeling, and 3) the feasibility of using a Cad inhibitor to arrest heart failure development under hypertensive and ischemic heart disease conditions. In vitro experiments using primary cardiac myocyte culture will be performed to corroborate the in vivo tests. Elucidation of the role of de novo pyrimidine biosynthesis during pathological cardiac remodeling and heart failure will advance our understanding of the pathophysiology of hypertensive and ischemic heart diseases and pave a way for novel, more effective therapeutic design.

NIH Research Projects · FY 2026 · 2022-02

Project Summary/Abstract Cytomegalovirus (CMV) is one of the most important causes of infectious complications following hematopoietic cell transplantation (HCT). Letermovir is an effective antiviral and was recently approved for prophylaxis to prevent CMV reactivation after HCT. Graft-versus-host disease (GVHD) prophylaxis has advanced alongside CMV prophylaxis, but the effects of novel GVHD therapies on infectious complications and immune reconstitution patterns are unclear. Current data suggest that modern GVHD prophylaxis with post-transplantation cyclophosphamide and sirolimus may offer an immunologic advantage over traditional prophylaxis with calcineurin inhibitors. In this proposal, Dr. Zamora will prospectively examine how these GVHD prophylaxis strategies influence CMV-specific T-cell and humoral immunity after HCT, and how these immunological changes can affect overall clinical outcomes. In the first aim of this proposal, Dr. Zamora will examine the effects of viral, host, and transplantation factors, including GVHD prophylaxis, on polyfunctional CMV-specific cellular immune reconstitution. He will use advanced analytical methods to compute polyfunctional T-cell responses and compare differences in immune responses between GVHD prophylaxis regimens. Dr. Zamora will also compare the accuracy of these analytical methods, versus traditional methods of measuring polyfunctionality, in predicting late clinically significant CMV infection after HCT. Furthermore, he will study the immunologic influence of regulatory T cells on the development of polyfunctional T-cell immune reconstitution after HCT and investigate whether this may be affected by the presence or absence of CMV reactivation. Historically, humoral immunity was not felt to be important in CMV prevention after HCT; however, recent animal studies have challenged this notion. Therefore, in the second aim Dr. Zamora will characterize factors influencing functional CMV-specific humoral immune reconstitution after HCT, using state-of-the-art neutralizing antibody and cell-to-cell spread inhibition assays. He will also evaluate the kinetics of CMV-specific antibody responses at the epitope level, using a novel serological profiling technology that can detect antibody responses to thousands of pathogen epitopes (VirScan). Dr. Zamora will investigate the associations of CMV-specific humoral immunity, as measured by these novel immune platforms, with the prevention of late clinically significant CMV reactivation after HCT. Dr. Zamora aims to define CMV-specific T-cell and humoral immune reconstitution kinetics in the current era of advanced GVHD prophylaxis regimens and effective antiviral prophylaxis. This study has the potential to define immunologic parameters to optimize CMV prophylaxis strategies and provide the basis for novel immunotherapy and immune monitoring approaches.

NIH Research Projects · FY 2025 · 2022-02

Summary The late stages of the mammalian pregnancy are accompanied with increased insulin resistance due to the increased glucose demand of the growing fetus. Therefore, as a compensatory response, in order to maintain the maternal normal blood glucose levels, the beta cells mass expands leading to increased insulin release. Beta cell proliferation, beta cell neogenesis, and decreased beta cell apoptosis, are believed to be major contributors for beta cell adaptive response during pregnancy and defects in this adaptive response can lead to gestational diabetes mellitus (GDM). My preliminary results indicate that Nrf2 is required for adaptive beta cell expansion in adult mice during overnutrition, and that Nrf2 levels are upregulated in beta cells of pregnant mice. Despite multiple studies describing Nrf2 protective effects on beta cells in Type 2 and Type 1 diabetic models, no study has ever uncovered the role of Nrf2 in the expansion of beta cell mass during pregnancy. Does in vivo loss- or gain-of-Nrf2 function affect beta cell proliferation, survival and mass in pregnant mice? Does this potential alteration persist in the early post-partum period? Does in vivo loss- or gain-of-Nrf2 function affect insulin secretion in pregnant mice? Does in vivo loss- or gain-of-Nrf2 function affect glucose homeostasis in pregnant mice? Which genes are upregulated and downregulated in islets during pregnancy in response to in vivo loss or gain of Nrf2 function? Which are the Nrf2 target genes in islets during pregnancy? Do human beta cells also require Nrf2 for pregnancy-driven proliferation? These important questions about the physiological role of Nrf2 in beta cells during pregnancy need to be answered to advance our knowledge and find therapeutic means to treat GDM. We hypothesize that Nrf2 is necessary for beta cell expansion during pregnancy and that disruption of Nrf2 expression or function leads to GDM. We believe Nrf2 can serve as a potential therapeutic target for treating GDM. We will test our hypothesis by completing the following specific aims: 1) To determine the role of Nrf2 on the expansion of beta-cell mass during pregnancy. 2) To uncover the mechanisms by which Nrf2 regulates beta-cell mass expansion during pregnancy. 3) To test if Nrf2 is necessary for pregnancy-mediated adaptive human beta cell proliferation in vivo. These studies will provide insight into how Nrf2 promotes expansion of functional beta-cell mass during pregnancy and will provide a crucial basic platform for designing and testing novel therapeutic strategies for the treatment of GDM.

NIH Research Projects · FY 2025 · 2022-01

PROJECT SUMMARY Recent studies have revealed pervasive transcriptional regulation through alternative promoters in normal tissues and cancer. How the majority of alternative promoters contribute to tumor formation, diagnosis or treatment remains unknown. Tet1 was first identified as an MLL partner in AML. It belongs to the Tet (ten- eleven translocation) family of proteins (Tet1/2 and Tet3), which oxidize 5-methylcytosine (5mC) into 5- hydroxymethylcytosine (5hmC), and other oxi-mC intermediates, thereby facilitating DNA demethylation. Interestingly, we found that a short isoform of TET1 (referred to as TET1-S), which contains a catalytic domain but lacks the N-terminal CXXC domain, and is under control of an alternative promoter, was expressed in human bone marrow (BM) cells. Additionally, TET1-S was upregulated in BM cells from Myelodysplastic Syndrome (MDS) patients as compared to healthy individuals. We showed that TET1-S is expressed in BM cells at a much higher level compared with TET1-F. However, its role in the hematopoietic system is unknown. Based on our preliminary results, we hypothesize that Tet1-S plays an important role in the maintenance of hematopoietic stem cells (HSCs) and its upregulation contributes to the development of MDS by disrupting normal function of HSCs and hematopoiesis. To test this hypothesis, we will determine 1) the oncogenic role and underlying mechanisms of Tet1-S in the pathogenesis of MDS, 2) whether Tet1-S is required for the development of MDS, and 3) the molecular mechanisms by which Tet1-S regulates gene expression in HSPCs and erythroid progenitor cells. We will employ multiple genomic approaches to systematically analyze the progressive effects of Tet1-S overexpression on 5mhC/mC distribution, chromatin accessibility and gene expression in hematopoietic stem/progenitor cells. Our work will provide new insights into the distinct role of TET1-S upregulation in the pathogenesis of MDS as well as its specific role and mechanisms in maintaining epigenetic landscapes and gene regulation in HSPCs.

NIH Research Projects · FY 2026 · 2022-01

Chronic clonal blood disorders such as myeloproliferative neoplasms (MPN) and chronic phase (CP) chronic myelogenous leukemia (CML) may over time transform, respectively, into secondary (s) acute myeloid leukemia (AML) and blast crisis (BC) CML, which are poorly responsive to currently available therapies, including allogeneic stem cell transplantation. Thus, the availability of novel and more effective treatments is a true unmet need for these patients. MicroRNAs (miRNAs) are small non-coding RNAs that target messenger RNAs and regulate the corresponding protein levels. MIR142, encoding miR-142, is a highly conserved “gene”, expressed at high levels in hematopoietic cells and is involved in the development and function of myeloid, lymphoid and megakaryocyte- erythroid progenitors. MIR142 has been found mutated and/or downregulated both in lymphoma and AML. Furthermore, miR-142 knock-out (KO) causes impaired hematopoiesis in zebra fish and mice, with expansion of hematopoietic stem and progenitor cells (HSPCs) and decreased hematopoietic output. We recently demonstrated that miR-142 KO in mouse models with clonal myeloproliferative disorders (MPDs; i.e., FLT3-ITD+ MPN or CP CML) prompts transformation into an AML-like disease and confers a significantly shorter survival to these animals. Our data support a role of miR-142 deficit in deregulation of the metabolism of clonal hematopoietic stem cells (HSCs), with a switch to higher levels of oxidative phosphorylation (OxPhos) via increased fatty acid oxidation (FAO); these changes likely play a key role in the transformation of clonal HSCs into leukemic stem cells (LSCs). We demonstrated that rescue of miR-142 deficit with a novel miR-142 mimic compound (CpG-M-miR-142) reduced OxPhos levels and viability of LSCs, decreased LSC burden and activity and prolonged survival of treated BC CML mice. Thus, the central hypothesis of this proposal is that the understanding of the cellular and molecular basis of miR-142 downregulation and its impact on the transformation of clonal MPD into aggressive AML-like disease will allow us to design and optimize novel treatments to compensate for the miR-142 deficit and prevent and cure MPD transformation. We propose the following Specific Aims (SAs): SA#1: To define the role of miR-142 deficit in the sAML/BC CML transformation. SA#2: To dissect the molecular mechanisms through which miR-142 deficit contributes to sAML/BC CML transformation. SA#3: To investigate the pharmacokinetic (PK), pharmacodynamic (PD) and therapeutic impact of a synthetic CpG-M-miR-142 that will rescue miR-142 deficit in sAML/BC CML.

NIH Research Projects · FY 2026 · 2022-01

Pancreatic cancer remains among the most lethal of solid tumors, due to late diagnosis and a high probability of metastatic spread. Effective new systemic treatments are needed in order to improve outcomes in patients. Targeted radionuclide therapy has demonstrated effectiveness cancer therapy, notably with the success of 177Lu- dotatate in neuroendocrine tumors including those of the pancreas. Prostate Stem Cell Antigen (PSCA) is upregulated in 60-80% of pancreatic adenocarcinomas, making it a promising target for antibody-directed therapy. An engineered antibody fragment, the A2 scFv-Fc2 DM has been specifically designed for optimized delivery of therapeutic radionucides in pancreatic cancer. It is based on a humanized, high-affinity anti-PSCA antibody, and contains Fc mutations engineered to foster rapid blood clearance via the hepatobiliary route. As a result, radiation dose to key organs/tissues (bone marrow and kidney) is minimized, enabling effective delivery of an alpha- or beta-emitting radionuclide to tumors. In Aim 1, biodistribution studies will be undertaken in mouse models, in order to confirm the expected tumor targeting and hepatic clearance. Formal dose estimations will be made for the scFv-Fc2 DM radiolabeled with either 177Lu or 225Ac for therapy. Aim 2 will explore the potential efficacy of the anti-PSCA scFv-Fc2 DM in mouse models of pancreatic cancer, including subcutaneous xenografts of human pancreatic tumor cells, a syngeneic model of KPC-PSCA tumors in huPSCA knock-in mice, and patient-derived pancreatic tumor xenograft models. The relative efficacies and toxicities of the alpha-emitter 225Ac and beta-emitter 177Lu will be analyzed in order to prepare for future clinical therapy studies. In Aim 3, clinical production and conjugation of the anti-PSCA scFv-Fc2 DM will be performed, testing conducted, and an IND filed. Finally, in Aim 4 we will conduct a first-in-human imaging study using 64Cu-DOTA-anti-PSCA scFv- Fc2 DM in patients with advanced pancreatic cancer, to evaluate the targeting, and clearance properties and potential radiation dose delivery of this novel engineered antibody fragment. Results from this clinical immunoPET study will be central to guiding future development of a radioimmunotherapy agent that can be implemented in a theranostic approach to pancreatic cancer.

NIH Research Projects · FY 2026 · 2021-12

Project Summary/Abstract Triple-negative breast cancer (TNBC) is characterized by the lack of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, all of which are important therapeutic targets. TNBC is the most difficult-to-treat subgroup of breast cancers and is resistant to many current cancer therapies. The present situation of poor prognosis with limited therapy options in TNBC emphasizes an urgent need for more effective therapeutics. The ability to escape from the surveillance by the immune system is regarded as one of the essential hallmarks of cancer cells. Recent exciting discoveries have identified many important signals and mechanisms mediating cancer cell immune evasion. Immunotherapies have been developed to target these signals, revolutionizing the treatment of a variety of human cancers. Tumor-associated macrophages (TAMs) represent the major components of the tumor microenvironment in TNBC. Recent studies demonstrate that the blockade of a “don’t eat me” signal CD47 leads to direct phagocytosis of living cancer cells by macrophages, and significantly inhibits the engraftment of various malignant hematopoietic and solid tumor cells in mice that lack T, B, and NK cells, indicating a critical role of macrophages in cancer immunosurveillance. Targeting TAMs in the tumor microenvironment represents a new class of promising cancer immunotherapy. While inducing anticancer functions of TAMs holds considerable promise for cancer treatment, there are several barriers that need to be overcome to achieve desired efficacy for treating TNBC. In preliminary studies, we found that TAMs can be reprogrammed by small molecule antineoplastic compounds to induce their phagocytic ability against TNBC cells. However, the underlying molecular mechanisms regulating the reprogramming of macrophages remains unclear. The overall objective of the proposed study is to understand the underlying mechanisms of macrophage-mediated immunosurveillance in TNBC and to develop strategies to effectively treat TNBC by exploiting tumoricidal roles of TAMs, with a combination of in vitro and in vivo preclinical TNBC models. In Aim1, we will assess the efficacy of reprogramming macrophages in TNBC treatment by using metastatic TNBC models and chemotherapy-resistant patient-derived xenograft models. In Aim2, we will study the molecular mechanisms by which macrophages are reprogrammed by dissecting the functions and roles of Pattern Recognition Receptor signaling pathways in macrophage reprogramming and characterizing TAM subgroups in TNBC tumors. In Aim3, we will determine the effects of targeting macrophage cell surface molecular machinery on activating TAMs for TNBC treatment. Successful completion of the proposed studies should shed light on the basic principles of cancer cell immune evasion and inspire the development of novel therapeutics for TNBC treatment.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Brain aging is characterized by reduced cognitive capacities, learning and memory. The exact mechanisms of brain aging remain elusive. Because aging is the greatest risk factor for major debilitating neurodegenerative disorders, including Alzheimer's disease (AD), it is important to uncover mechanisms underlying brain aging in order to develop effective therapies for age-related neurodegenerative diseases. AD is the most common neurodegenerative disorder and a leading cause of disability and death. However, the precise mechanisms underlying AD pathogenesis remains to be elucidated. Although many transgenic mouse models have been generated for AD research and these models are important for our understanding of the pathological basis of the disease, it is increasingly recognized that there are significant species differences between mouse and human neural cells. Therefore, there is an urgent need to establish human disease modeling platforms to complement studies in animal models for AD research. Direct reprogramming is a cellular reprogramming technology, which allows direct conversion of one type of somatic cells, such as fibroblasts, into another type of somatic cells, such as neurons. It has been shown that direct reprogramming enables generation of human neurons that possess key elements of cellular aging. Therefore, directly reprogrammed cells could provide a human cellular platform for us to model brain aging and age-related late-onset diseases, such as late-onset AD (LOAD). The objective of this proposal is to develop human age-relevant glial cellular models using direct reprogramming technology, in order to recapitulate age-associated phenotypes in brain aging and neurodegeneration and uncover novel underlying mechanisms. Increasing evidence suggests that astrocytes play important roles in brain health and pathogenesis of neurodegenerative diseases. Therefore, we propose to establish cellular models for brain aging and AD using astrocytes directly reprogrammed from fibroblasts of aged subjects and LOAD patients and co-cultures of astrocytes with other brain cell types, including microglia, oligodendrocytes, and neurons. We hypothesize that cellular aging regulates astrocyte function and cell-cell interactions to modulate brain aging phenotypes and LOAD pathologies. Accordingly, we propose the following Specific Aims:Aim 1: To generate age-associated astrocytes through direct reprogramming and evaluate how cellular aging regulates astrocytic functions. Aim 2: To determine whether and how astrocytic cellular aging modulates neuroinflammation and neuronal phenotypes. Aim 3: To determine whether and how astrocytic cellular aging regulates OPC properties and myelination. The proposed studies will likely help to define roles of glial cellular aging in brain functional deterioration during aging and uncover the underlying mechanisms, which could lead to the development of novel strategies to maintain brain health and reduce risk for AD. The knowledge gained from this study could help us to design novel therapeutic strategies for AD.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Multiple myeloma (MM) is an incurable blood cancer affecting approximately 83,000 people in the United States. Although treatments for MM have improved significantly in recent years, the vast majority of patients experience multiple relapses and ultimately succumb to complications of the disease. For the last 12 years, I, the Research Specialist, have devoted my work toward contributing to improving outcomes for cancer patients. Since 2014, I have worked in the laboratory of Unit Director Flavia Pichiorri, where we exclusively specialize in MM. I am now involved with three National Cancer Institute–funded studies. 1) Radioimmunotherapy/CAR T cells: The use of therapeutic isotopes such as the beta emitter 177Lu or the alpha emitter 225Ac have increased the specific killing of cancer cells, but side effects are clinical concerns. Although CAR T cells may reduce tumor burden, patients remain at risk of relapse. We predict that the use of CD38-directed radioimmunotherapy and CS1-directed CAR T cells will result in more durable remissions while lowering the dose for each agent, with decreased side effects and enhanced immunomodulation. 2) Reolysin: Reolysin, an oncovirus, shows only modest activity in MM patients, likely because of resistance mechanisms to oncoviruses. We observed that carflizomib, a second generation proteasome inhibitor, showed synergic killing of MM cells in vitro when combined with Reolysin. We found that carfilzomib facilitates Reolysin infection through modulation of the antiviral response of the microenvironment. We also discovered that HDAC inhibitors can reduce the surface expression of an important adhesion receptor and upregulate the expression of an oncovirus receptor. 3) Overcoming IMiD research in myeloma: To overcome disease resistance to immunomodulatory drugs (IMiDs) such as lenalidomide (Len), combinatorial therapies may be necessary. I have investigated the safe, orally available drug leflunomide (Lef), which has been used for rheumatoid arthritis for the last 20 years. We demonstrated that Lef directly inhibits several kinases, including the PIM family of serine/threonine kinases in MM cells, negatively affecting c-Myc protein levels, which are commonly upregulated in MM. With this information, we reasoned that a suitable drug to use in combination is the immunomodulatory drug lenalidomide (Len). The completion of the projects’ goals will rely in large part on my qualifications, and the results thus far point to my value as a Research Specialist in the cancer research community.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY - OVERALL The overall goal of this Glioblastoma (GBM) Therapeutics Network (GTN) U19 application from City of Hope, Translational Genomics Research Institute, and University of Alabama at Birmingham is to develop superior treatments for patients with GBM, the most common and aggressive primary brain tumors in adults. Effective treatments remain elusive and patients are rarely cured with standard therapies. This GTN U19 application embodies a unique combination of approaches designed to significantly advance the treatment of patients with GBM by addressing tumor heterogeneity, blood-brain barrier penetration, and the immunosuppressive GBM tumor microenvironment. The three proposed research projects will translate therapeutic agents from preclinical development, through IND-enabling studies, and into phase I clinical studies in adult patients with GBM. Each project is based on novel molecular preclinical studies with small-molecule inhibitors and immunomodulatory agents that use signature-guided assessment and treatments. Specific goals of the projects are: Project 1. Develop and clinically test an engineered oncolytic herpes virus expressing a full- length anti-CD47 monoclonal antibody for treatment of GBM. Project 2. Develop and clinically test tasquinimod as an adjunct to enhance the efficacy of anti-GBM immunotherapies administered peri-operatively. Project 3. Develop and clinically test a molecular “signatures of vulnerability” guided treatment of GBM with neddylation inhibitor pevonedistat. In addition, this U19 application proposes strategies that will address major barriers in drug development by incorporating two innovative research tools: 1) intracerebral microdialysis to rationally select appropriate systemically administered therapies for testing in GBM patients and 2) next generation exome and transcriptome sequencing to identify molecular “signatures of vulnerability” that can guide appropriate patient selection for clinical trial enrollment. These analytical capabilities will enable us to quantify CNS drug penetration and dissect genomic heterogeneity in tumor and stromal cells in the proposed clinical trials. Also, two of the proposed projects leverage City of Hope’s GMP facilities to manufacture biological agents and small molecules that will be tested in adult GBM patients for the first time. In summary, the innovative projects and shared resources cores in this application combine our strengths in basic, translational, and clinical research in a highly collaborative setting that promotes the sharing of ideas, results, resources, and clinical populations to develop effective treatments for GBM. If successful, data generated by these studies have the potential to transform the treatment of adult GBM patients by introducing new agents that circumvent tumor heterogeneity and immunosuppression.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT This application is in response to the PAR-19-250: “EnvironmentalInfluences on Aging: Effects of Extreme Weatherand Disaster Events on Aging Populations .”Air pollutants are especially detrimental for aging populations; exposure to air pollution as measured by ambient particulate matter (e.g., PM2.5) has been linked to diseases that increase with age, including cardiovascular diseases (CVD) and stroke. Older women (who outnumber men 3:2) are particularly susceptible to CVD endpoints and to stroke risk in particular; stroke risk in women doubles immediately following menopause. Extreme weather events such as wildfires and prolonged drought in the last decade alone have adversely affected air pollution exposure in states such as California. A number of metropolitan counties in the state have particulate pollution levels above federal and state ambient standards, and during extreme weather events, these levels rival that of the worst cities in the world. Our overall study objective is to evaluate the intersection of extreme weather events and air pollution in an aging female population who are at increased risk for CVDs, and in particular, at peak susceptibility for stroke. In Aim1, we will evaluate the acute effects from wildfire events by ascertaining stroke events within geographically affected areas based on satellite imagery. Elevated PM2.5 exposure estimates resulting from a wildfire event in the affected areas will be associated with hospitalizations (including emergency room visits) from stroke and CVD. In Aim 2, we will determine the role of specific PM2.5 components in stroke risk and mortality. Employing complementary satellite- and source-based approaches, we will identify key sources of PM2.5 and its chemical constituents that are attributable to stroke. Leveraging 25-years of follow-up from the study population, this aim will permit us to delineate components of air pollution from drought and wildfire events versus other (e.g., transportation, industrial) exposures. For both aims, select CVD endpoints (e.g., myocardial infarction) will also be explored. In Aim 3, we will evaluate the association between PM2.5 exposure with serum immune markers among 2000 participants whose exposures reflect a cross-section of exposure during the 2015 drought and wildfire season. This aim will inform the purported biologic underpinning for stroke and CVD risk, by key PM2.5 sources and constituents. To successfully accomplish these aims, we will leverage longitudinal data from the California Teachers Study, a geographically-defined population-based cohort study of 133,479 women whose 25 year follow-up spans: (a) key periods of stroke and CVD susceptibility in women, and (b) elevated levels of air pollution exposure in California where there have been prolonged periods of drought and numerous wildfires.

NIH Research Projects · FY 2025 · 2021-09

Abstract Combining cyclin-dependent kinase (CDK) inhibitors with endocrine therapy improves outcomes for metastatic estrogen receptor positive (ER+), HER2 negative, breast cancer patients. However, the value of this combination in potentially curable earlier stage patients is variable. Our preliminary results examined the evolutionary trajectories of early stage breast cancer tumors using single cell transcriptomic profiling of serial tumor biopsies from a clinical trial of preoperative endocrine therapy alone (letrozole) or in combination with the cell cycle inhibitor ribociclib. Resistant tumors with accelerated loss of estrogen signaling show up-regulation of the JNK pathway, while those that maintain estrogen signaling during therapy show potentiation of CDK4/6 activation consistent with ERBB4 and ERK signaling up-regulation. Cell cycle reconstruction identified that tumors cells can reactivate during combination treatment, indicating stronger selection for a proliferative state. We hypothesize that resistance to CDK4/6 inhibition in earlier stage breast cancer is driven by JNK MAPK pathway stimulation and reactivation of the cell cycle through promotion of CDK6 expression or decreased cell cycle inhibitor function. In Aim 1, we will use a new mechanistic model of CDK4/6 regulation by cell cycle Inhibitors and Promoters (CIP) that couples estrogen and JNK signaling with cell cycle progression to measure the mechanisms driving cell cycle activation in a series of isogenic cell lines sensitive and resistant to CDK4/6 and endocrine inhibitors and in patient tumor cells. This analysis will reveal how distinct signaling pathways contribute to cell cycle reactivation during estrogen, CDK4/6 and JNK inhibition treatments and provide signatures of each resistant mechanism across cell types, over time and between systems. Aim 2 leverages our collection of patient tumors from the FELINE clinical trial to discover the intracellular and intratumoral resistance mechanisms driving proliferation. Fundamental resistance mechanisms will be measured in over ~300,000 patient cells from 360 tumor samples using single cell RNA sequencing data already in hand to identify core intracellular signaling states that act alone or in concert to drive proliferation. Next, the population of cells within each tumor will be analyzed to quantify intratumoral heterogeneity and how resistant populations differ in growing or shrinking tumors during drug treatment. Applying CIP to project proliferation across patient tumor cells will allow prediction of inhibitor strategies that most effectively block intracellular and intratumoral proliferation. Lastly, Aim 3 will apply a series of JNK pathway drugs with clinical potential to design and test treatment strategies that maintain durable inhibition of proliferation in ER+ cancer cells. Iterative feedback between mathematical models and patient/experimental data serves to provide a deep understanding of cell cycle regulation and mechanisms of dysregulation leading to resistance. Together, these experiments will reveal the balance between estrogen and alternative mediated JNK signaling, and their roles in resistance and provide a guide for therapeutic regimes with more durable control of cancer cell proliferation.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY/ABSTRACT Approximately 10% of new cancer diagnoses in the United States are hematological malignancies that include a spectrum of blood cancers and related disorders. In chronic lymphocytic leukemia (CLL) and myeloid neoplasms, somatic hotspot mutations frequently occur in five RNA splicing factors (SFs): SF3B1, SRSF2, U2AF1, ZRSR2, and genes encoding the U1 snRNA. These mutations drive aberrant splicing of mRNAs to promote leukemogenesis. Recently, a novel class of RNAs called circular RNA (circRNA) was found to be aberrantly expressed in many types of liquid tumors. Unlike mRNAs that form through normal splicing to produce linear RNAs, circRNAs are produced through backsplicing that results in RNA circularization. Because of advances to detection and annotation methods, circRNAs are now known to possess functional and clinical significances, suggesting a novel role in cancer biology. However, to date, their precise role in hematological malignancies, namely leukemia, remains undefined. The long-term goal is to investigate the functions and therapeutic potentials of circRNAs. Moreover, the role of SF mutations (SF3B1, SRSF2, U2AF1, ZRSR2, and genes encoding the U1 snRNA) in the aberrant expression of circRNAs remains unknown. Thus, the overall objective is to link SF mutations to aberrant circRNA expressions. To this end, I hypothesize that mutations in SF3B1 and other SFs such as SRSF2, U2AF1, ZRSR2, and genes encoding the U1 snRNA upregulate circRNA abundance to promote leukemogenesis. The rationale for this research is that linking SF mutations to aberrant expressions of circRNAs would define a novel regulatory axis, unlocking new therapeutic opportunities for treating leukemia. Preliminary data from CLL patient B cells and cell lines showed that mutant SF3B1 promoted aberrant expression of circRNAs in biological important molecular pathways such as protein transport and cell cycle regulation. To identify circRNAs critical for cell survival, I have validated the emergent CRISPR CasRX technology as a tool for screening circRNAs. I have also prototyped an experimental workflow for validating the functions of these circRNAs. Additionally, to further determine the role of SF mutations on circRNA biogenesis, cell lines with somatic mutations for SF3B1, SRSF2, U2AF1, ZRSR2, and genes encoding the U1 snRNA have been established. Finally, I have engineered cell-based and minigene-based reporter systems to investigate the mechanisms of backsplicing. Using this collection of tools, I propose the following aims: to identify and validate the functional impact of SF mutation-associated circRNA in leukemia (Aim 1) and to determine the mechanism of backsplicing (Aim 2). I expect the findings from this proposal will define a novel regulatory axis linking SF mutations and circRNAs aberrant expressions to leukemogenesis.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY More than 44,000 new cases of Kaposi sarcoma (KS) are reported globally each year, 84% of which occur in Africa. This and other Kaposi sarcoma-associated herpesvirus (KSHV)-induced malignancies predominate in people with acquired or iatrogenic immunodeficiencies. Although KSHV can be detected in other human body fluids, its frequent detection in saliva in groups both with and without risk of sexually transmitted infections (e.g., children) suggests that the oral cavity is the site of primary acquisition. However, the mechanism of KSHV oral transmission in vivo, particularly the critical viral envelope glycoproteins (gps) required for viral entry, remains unresolved. Several KSHV–host interactions have been identified, but all prior experiments were performed in vitro and have not been validated in vivo due to prior lack of an appropriate animal model. Through collaboration with the Wisconsin National Primate Research Center, our laboratory has access to the common marmoset (Callithrix jacchus, CJ), a recently developed KSHV non-human primate model that is susceptible to KSHV oral infection, and under immunosuppression acquires KS-like skin lesions. The objective of this application is to elucidate the minimum gps required to initiate primary oral infection in vivo, as a prerequisite to selecting key gps for developing an effective prophylactic vaccine candidate. This application builds on Dr. Ogembo’s recently completed NCI K01 CA184388-05 research on KSHV entry mechanisms and vaccine development. Recently, we showed that in vitro, the KSHV glycoprotein gH/gL is essential for viral infection of epithelial, endothelial, and fibroblasts cells, but not B cells. Notably, we and others have also shown that both monoclonal and polyclonal Abs to KSHV glycoproteins gB, gH/gL, and gpK8.1, can neutralize KSHV infection of diverse permissive human cells in vitro. Building on this success, we generated KSHV deletion mutants lacking the four glycoproteins thought to be critical for viral entry (gB, gH/gL, gpK8.1) and various monoclonal antibodies specific to these gps. In this project, we will use human ex vivo samples and the CJ KSHV model to test the hypothesis that gB and gH/gL are critical for KSHV in vivo oral transmission. The premise of our proposal is built on strong evidence that 1) KSHV can infect CJ, which develop KS-like skin lesions, and 2) Abs against the KSHV glycoproteins gB and gH/gL can neutralize KSHV infection in vitro and ex vivo. Furthermore, the permissiveness to KSHV infection of human cells ex vivo and CJ makes these platforms ideal to test the KSHV gp requirements for infection. Successful completion of the proposed study will elucidate the minimum KSHV gps required for primary infection in ex vivo and in vivo models, advancing our long-term goal of defining the initial steps in KSHV infection of humans and the role of antibodies in protecting against the early steps of KSHV transmission. This will ultimately inform design and development of prophylactic vaccines that can prevent KSHV infection and its associated cancers.

NIH Research Projects · FY 2024 · 2021-08

SUMMARY: Circulating Extracellular Vesicles in the Pathogenesis of Type 1 Diabetes Type 1 diabetes (T1D) is an autoimmune disease, characterized by death, dedifferentiation or dysfunction of functional beta cells. However, precise molecular events that initiate beta cell loss and dysfunction or mediate autoimmunity is a major gap in knowledge that remains unaddressed. Emerging evidence suggests that circulating extracellular vesicles (EVs), known mediators of intercellular microcommunication, may play important roles in the pathogenesis of T1D. However, their precise function and molecular contents, especially in the initiation of beta cell loss and dysfunction in humans are largely undefined. Here, we hypothesize that circulating EVs in T1D, through their distinct molecular characteristics, are cytotoxic to beta cell health, thus contributing to the pathogenesis of T1D, with the potential to serve as biomarkers for early disease diagnosis. Our preliminary data suggests that EVs, but not EV-depleted fraction in the humoral factors in T1D are cytotoxic, particularly to human beta cells, but not to alpha cells. Specifically, EVs from T1D subjects, but not from control subjects, induce human beta cell death suggesting that circulating T1D-EVs are key components contributing to humoral cytotoxicity. Therefore, characterizing T1D EVs at various stages of the disease and control could identify important biomarkers for early detection of the disease that correlate with beta cell loss and dysfunction. Our research plan has the following Specific Aims: AIM 1: To determine the functional effects of circulating EVs from pre-disease to late stage T1D subjects on human beta and alpha cell health. We will characterize circulating EVs from the plasma of T1D subjects at i) early (1-5 years), ii) late (>10 year) and iii) pediatric (<18 year) stages and compare them with i) autoantibody positive non-T1D, ii) T2D (control for secondary effects such as hyperglycemia) and iii) appropriate age, ethnicity, and sex-matched healthy control non-diabetic subjects for their effect on human beta and alpha cell health. AIM 2: To establish the functional role of circulating EVs in the pathogenesis of T1D in vivo using a rodent model. To provide proof-of-concept, we will administer circulating EVs from diabetic female NOD mice to non- diabetic controls and test T1D disease progression in the recipient (Aim2A); we will inhibit EV secretion in young pre-T1D female NOD mice and test T1D disease progression and quantify beta cell death and function (Aim2B). AIM 3: Investigate the molecular mechanisms, cargo composition and cargo function of human T1D- EVs. We will address the differential function of T1D EVs in beta cell cytotoxicity by investigating their uptake mechanisms, (Aim3A); compare EV cargo in T1D and control subjects (Aim3B); examine the function of differentially expressed EV-RNAs (Aim3C). Our proposed research is clinically relevant and will profoundly change our understanding of the progression of T1D. If successful, our study has the potential to identify new biomarkers and therapeutics for T1D.