NIH Research Project Grant

For this proposal we intend to develop a novel vaccine to prevent and possibly treat Human T cell leukemia virus type-1 (HTLV-1) associated diseases. HTLV-1 is a human retrovirus that is the causative agent of a malignant T CD4+ cell lymphoproliferation referred to as Adult T cell leukemia/lymphoma (ATLL), as well as several inflammatory disorders with the most problematic being human myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 infection is endemic in many areas around the world including southern Japan, the southern United States, central Australia, the Caribbean, South America, equatorial Africa, and the middle East. Over 10 million people may be infected worldwide. It is estimated that approximately 5% of HTLV-1 positive individuals will develop ATL, and 2% HAM/TSP. Seropositive rates in certain areas reach 20–40% among people aged over 50 years. With millions affected worldwide, HTLV-1 is a major problem in endemic communities and remarkably, there are no effective vaccine or treatment options to prevent ATL or HAM/TSP afflicted individuals. Given this, aim to develop and test the efficacy of a novel vaccine to prevent HTLV1-mediated disease. Aim 1: To evaluate the immunogenicity, in immunocompetent murine models, including mice with a humanized immune system (NSG™-SGM3) VSV-based vaccine vectors that express the HTLV-1 glycoprotein and regulatory proteins TAX and HBZ (VSV-gp62-∆HT). The ability of our candidate vaccine to generate neutralizing antibodies to the glycoprotein will be analyzed, as well as the production of cytotoxic T cells (CTLs) to gp62, TAX and HBZ. Aim 2: We aim to compare whether our vaccine can be used to prevent HTLV-1 transformation associated disease. This approach will include establishing whether VSV-gp62-∆HT can prevent the establishment of HTLV1-assocated leukemia/lymphoma in NSG™-SGM3 mice. Our objectives are to collate sufficient information to warrant the consideration of a variety of Phase I trials to prevent HTLV-1 -associated disease.

Contraceptive patterns are changing and the implications for ovarian cancer risk are unknown. Oral contraceptives (OCs), which are an established protective factor for ovarian cancer, are being used less frequently and intrauterine device use (IUD) is on the rise. However, the impact of changing contraceptive patterns on ovarian cancer incidence is unclear. While most studies of IUDs and ovarian cancer risk suggest an inverse association, some studies report no association or even a slight increase in risk. Differences between these studies could be attributable to IUD type, timing, or molecular features of tumors including histotypes. Preliminary data from the New England Case Control (NEC) Study suggest the association between IUDs and ovarian cancer risk vary by histotype, with a non-significant decrease in risk of low-grade serous and clear cell tumors but not for other histotypes. Furthermore, we observed an increased risk of ovarian cancer with low tumor stromal CD163 expression, reflecting heme scavenger receptor expression decreased M2-type macrophage infiltration. These observations suggest the impact of IUD use on ovarian cancer risk may differ by subtype and subgroup, but larger samples sizes are needed. Inflammation is known to play a role in ovarian cancer pathogenesis, and IUDs exert their physiologic effect through local inflammation. However, IUD-associated inflammation may be accompanied by an immune response that could lead to clearance of premalignant cells or local infection. An appreciation of how ovarian cancer risk varies by expression of immune markers within tumors may inform the biologic mechanisms possibly involved (e.g. CD3, CD8, CD4, CD69, FOXP3, CD163). Here we propose to evaluate the association between IUD use and ovarian cancer risk in 17 case control studies and 7 cohort studies with a total of more than 20,000 cases that collected, with varying degrees of detail, data on IUD type and timing of use (e.g. age at use and before/after first birth), as well as detailed histologic data critical for distinguishing ovarian cancer subtypes. Importantly, our proposed research includes a case-control study of African American women enrolled between 2010-2015, reflecting recent contraceptive trends and increasing diversity. The inclusion of cohort studies with updated contraceptive use data and a case-control study which recently completed enrollment will provide information on the contemporary contraceptive use. Furthermore, we will utilize unique resources and innovative platforms to examine the potential mechanisms through which contraceptive choice influences ovarian cancer risk. In more than 3,000 cases with detailed contraceptive data, we will simultaneously measure a panel of immune markers by multiplex immunofluorescence which shows co-localization of marker expression. Both the high prevalence and modifiable nature of contraceptive use make this an important public health question with a significant impact on the population burden of ovarian cancer.

Oral squamous cell carcinoma (OSCC) is the sixth most common human cancer worldwide. Approximately 30% of the oral premalignant lesions (OPLs) progress to OSCC, a process that may have a multifocal origin and can be promoted by carcinogens such as those found in tobacco. Our long-term goal is to identify the genetic alterations that promote high risk of progression to OPLs and to determine how those alterations modulate the response of OPLs to preventive strategies. The TP53 gene (also known as p53) and CDKN2A are the most frequently mutated genes in oral cancer, also found altered in OPLs. p53 GOF mutations and genomic alterations that result in loss of the CDKN2A gene associate with “cold” immune microenvironments in OPLs and OSCCs, with high risk of progression to carcinoma, and with extremely poor outcomes in OSCC patients. We hypothesize that the early appearance of mutations in p53 and CDKN2A inactivation modulate the oral tissue microenvironment and predispose OPLs to progress to OSCC. To test this hypothesis we will study mouse models that develop OPLs upon exposure to the tobacco-surrogate 4NQO, in the presence of p53 and/or CDKN2A mutations. Patients with high-risk OPLs could benefit from preventive strategies designed to block the malignant progression of OPLs. However, previous attempts with different chemopreventive agents have not been successful. Recently, immune checkpoint blockade with antibodies directed at programmed cell death protein 1 (PD-1) has been shown to improve the survival of patients with advanced OSCC in clinical trials, confirming the importance of the immune system in containing progression of invasive tumors. Moreover, our previous studies, confirmed by multiple independent groups, demonstrated that anti-PD-1 antibodies can also prevent the progression of OPLs to OSCC, in a 4NQO mouse model for oral carcinogenesis. Our preliminary studies indicate that the p53 and CDKN2A status of the OPLs may determine the response to anti-PD-1-mediated immunoprevention. In this proposal, we will assess the long-term benefits of anti-PD-1-mediated oral cancer prevention, to determine whether PD-1 blockade, administered in a preventive setting, can confer survival benefits, and to assess how p53 and CDKN2A mutations affect the sustained response to PD-1 blockade. To overcome resistance to anti-PD-1 we hypothesize that reactivation of p53 in OPLs carrying p53 mutations sensitizes the oral lesions to anti-PD-1. Our mouse models will allow us to test this hypothesis in vivo.

There is abundant evidence that obesity confers increased risk for at least 13 forms of cancer. The incidence of breast, colon, and liver cancer are all increased in obese populations, and the epidemiologic evidence for the obesity-breast cancer connection is particularly strong. One in eight women will be diagnosed with breast cancer during their lifetime. Breast cancer incidence increases approximately 10-fold for women over the of age 60, compared to age 50 or younger. This increase in breast cancer risk is associated with an increase in obesity. Indeed, obesity increases the risk of triple-negative breast cancer in premenopausal women and estrogen receptor positive breast cancer in postmenopausal women. A rarer form of inflammatory breast cancer is dramatically increased (up to 5-fold) in both groups. More importantly, obesity shortens disease-free survival in both pre- and postmenopausal women. Patient mortality in breast cancer is primarily caused by distant metastases. Obesity at the time of diagnosis is associated with increased risk of distant metastasis and mortality. Studies in rodents have confirmed these relationships, showing that dietary-induced obesity and high-fat diets lead to increased incidence and growth of tumors in oncogene and carcinogen-induced breast cancers. Despite this body of correlative evidence, the mechanisms of obesity-induced breast cancer risk remain poorly understood. One possibility is that the obesity causes insulin resistance in the liver and compensatory elevation in circulating insulin to control glucose levels. At the same time, other tissues, including tumors, may not be insulin resistant and so are exposed to increased insulin signaling. Indeed, we have shown that reducing insulin resistance by treating with omega-3 fatty acids reduces breast cancer growth in mice. We have also shown that time-restricted feeding (TRF) versus unrestricted feeding of a high-fat diet improves insulin resistance despite sustained obesity and equal caloric intake. Furthermore, we showed that TRF inhibited obesity-driven breast tumor growth and corrected tumor circadian rhythms, and that the TRF impact on tumor growth was mediated by reducing insulin levels. A number of important questions remain unanswered. Firstly, how does insulin drive tumor growth? Is it a direct effect on the tumor cell, or on the microenvironment? Secondly, does correction of the circadian rhythms in the tumor cell by TRF contribute to the reduced tumor growth? Thirdly, how do nutrients and insulin entrain the circadian clock in tumors? Due to the link between obesity, insulin resistance and breast cancer in pre- and postmenopausal women, and the translational potential of time-restricted feeding, we will investigate the effect of deleting the insulin receptor, mTORC1 signaling, or components of the circadian clock in tumor cells to test whether loss of these signals alters tumor growth in vivo and the response to TRF. We will also test whether TRF enhances chemotherapy to inhibit tumor growth. Accumulating evidence from TRF-related clinical studies support the translational relevance of our proposal. Translational, mechanistic findings from these studies will impact on breast cancer prevention and therapy.

NA

This proposal focuses on the divergent effects of the two major dietary forms of vitamin K on breast cancer. K vitamins act as cofactors for gamma-glutamyl carboxylase (GGCX), which post-translationally introduces γcarboxyglutamate residues into proteins. Although most of the 17 known γ-carboxylated proteins function in coagulation and bone homeostasis, the presence of GGCX in most tissues (including mammary gland) suggests more extensive physiological roles for vitamin K. We have demonstrated that triple negative breast cancer (TNBC) cell lines express GGCX and produce γ-carboxylated proteins in response to vitamin K1 (phylloquinone), the major dietary form. In TNBC cells, K1 treatment enriches for the stem cell marker aldehyde dehydrogenase 1 (ALDH1) and promotes mammosphere formation. These data suggest that K1 sustains GGXC mediated γcarboxylation to drive aggressive breast cancer phenotypes. Through analysis of genomic cancer datasets, we find that ~25% of breast tumors express GGCX and the vitamin K oxidoreductase (VKOR) genes required for its activity. Patients with such tumors have poorer survival than those whose tumors do not express these genes at high levels. Patients with this subtype of tumor would be candidates for therapies that limit K1 availability and/or inhibit GGCX. Surprisingly, we found that vitamin K2 (menaquinone-4), another naturally occurring form present in diet, does not stimulate γ-carboxylation or stem cell phenotypes in TNBC cells, but instead strongly suppresses cell growth, migration and energy metabolism. These provocative data indicate that K1 and K2 exert distinct effects on breast cancer cells, with K1 promoting and K2 suppressing aggressive phenotypes. We also found that expression of the vitamin K2 biosynthesis enzyme UbiA Prenyltransferase Domain Containing 1 (UBIAD1) is undetectable in TNBC, suggesting altered cellular handling of vitamin K. In Aim 1 we will dissect the effects of K1 and K2 in vitro, evaluate the role of UBIAD1 and conduct feeding studies to measure accumulation of K1 and K2 in TNBC xenografts and host mammary gland in relation to tumor growth. In Aim 2 we will determine whether deletion of GGCX from TNBC cells impacts γ-carboxylated protein synthesis and aggressive phenotypes in vitro and in vivo. Aim 3 will identify relevant γ-carboxylated GGCX substrate proteins that mediate the effects of K1. We anticipate that growth of tumors with high GGCX activity and low UBIAD1 will be stimulated by high dietary K1 and inhibited by high dietary K2. These findings would identify GGCX as an oncogene and the vitamin K pathway as a therapeutic target in a subset of patients with advanced breast cancer.

Ovarian endometriomas are deep endometriosis lesions on the ovary. Endometriomas are a unique form of endometriosis in that they do not respond to hormonal therapy and carry the highest risk of developing clear cell ovarian cancer. There is an urgent need to determine the unique pathogenesis of endometriomas to improve the lives of women. As an alternative to retrograde menstruation, the induction theory of endometriosis posits that a substance induces an adult cell to transdifferentiate into endometriosis, although the inductive substances or the cells which transdifferentiate into endometriosis have yet to be identified. AKA mice (Arid1aflox/flox; Krasflox-stop-flox-G12D; Amhr2Cre) spontaneously and reproducibly develop large, cystic endometriomas that recapitulate human endometriomas at the histologic and molecular level. As the genetic recombination lies in the granulosa cells of the ovary, AKA endometriomas do not develop by retrograde menstruation. The AKA mouse model allows for the cellular and molecular interrogation of the paradigmshifting induction theory of endometriosis using a rigorously reproducible and easily manipulatable model. Transcriptomic analysis of AKA endometriomas revealed enrichment in cellular senescence genes. Cellular senescence is defined as a permanent cell cycle arrest. Senescent cells exhibit a senescenceassociated secretory phenotype (SASP) and secrete high levels of pro-inflammatory molecules, similar to those found in the pelvic cavity of women with endometriosis. These results suggest that senescent cells in the AKA ovary secrete factors and induce endometriosis. As for the cells induced, granulosa cells exhibit the ability to transdifferentiate, a developmental process by which fully differentiated cells change into different fully differentiated cells. The role of senescence in endometriomas is conceptually novel, and the ability of granulosa cells to transdifferentiate into endometriosis through senescence signaling is a new paradigm. The central hypothesis is that the senescent microenvironment, mediated by Arid1a loss and oncogenic Kras, is critical for developing endometriomas through induction and transdifferentiation of granulosa cells. The objective of Aim 1 is to characterize the unique transcriptomic profile of the senescent cells and the endometriotic microenvironment using spatial transcriptomics. The objectives of Aim 2 are to identify the genetic changes (i.e., Kras G12D) required for senescence in granulosa cells, validate the expression of the endometriosis SASP (from SA 1), and establish SASP-mediated endometriosis transdifferentiation using primary murine granulosa cell cultures and a soluble Cre recombinase. The objectives of Aim 3 are to determine if senescence is essential for endometriosis and determine whether senotherapies restore ovarian function, fertility, and reduce endometrioma development and progression. Targeting senescence through senotherapies is critical to developing nonhormonal therapies, an urgent unmet need for endometriosis.

Novel metabolomic contrast probes for human lung cancer characterization We identified preliminary proof-of-concept metabolomic markers for human LuCa from paired tissue and blood serum samples from pre-symptomatic LuCA patients. The markers can be used as imaging probes for Luca characterization. To validate the efficacy of the markers for classifying LuCa, we propose to evaluate and enhance their capability as probes for LuCa detection and ultimately advance LuCa early-detection using serum metabolomic markers. The project’s goal will be achieved through tasks in the following three specific aims: 1) To evaluate the efficacy of tissue-serum LuCa MRS metabolomic probes identified in a successful preliminary project, by comparison with an additional 200 pairs of tissue and serum specimens and 200 serum samples from matched healthy controls, 2) To measure tissue-serum LuCa MRS metabolomic probes with mass spectrometry (MS) and MS imaging (MSI) to associate the probes with LuCa pathologies and identify serum MS LuCa probes, and 3) To test LuCa metabolomics probes using 200 serum samples collected before LuCa detection and evaluate LuCa metabolomic probe health- and cost-effectiveness as compared to existing advanced tests. The project’s goal is to identify a novel serum contrast probe able to contribute to LuCa early disease detection at asymptomatic stages to overcome persistent challenges currently faced in the LuCa clinic.

Patients with ulcerative colitis, a form of inflammatory bowel disease, face an increased risk of developing colorectal cancer. Although advances have been made in the therapeutic management of this disease, much less attention has been given to the development of cancer preventive strategies for this high-risk population. Ulcerative colitis patients often develop folate deficiencies that require supplementation with folic acid (FA), a synthetic form of folate. The effect of FA on risk for colorectal cancer remains unclear and the recent suggestion that FA supplements may be useful in preventing colitis-associated colorectal cancer is of great concern. Preliminary studies from this group provide the first evidence that FA supplementation (8 mg/kg diet) causes a dose-dependent increase in the formation of colorectal tumors in mice with AOM/DSS-induced colitis. Results from associated RNASeq and in vitro analyses suggest that FA promotes tumorigenesis by activating ERK and inducing NF-κB signaling in colonic epithelial cells with dysfunctional p53, the gatekeeping event in the development of colitis-associated cancers. The hypothesis of the proposed studies is that FA supplementation promotes UC-associated cancer in cells with dysfunctional/mutant p53, but not in cells with wild type p53, via an inflammatory pathway mediated by ERK and NF-κB. The mechanism by which FA induces tumor formation will be investigated in Aim 1 using CRISPR engineered isogenic human RKO colon carcinoma cells with varying p53 status (p53+/+, p53-/- and p53+/Mut). The impact of FA and dysfunctional p53 on activation of ERK, NF-κB signaling, as well as cell cycle progression will be evaluated. In vitro findings will be validated in Aim 2, where complimentary in vivo analyses will examine the combined effect of FA and mutant p53 on DSS-induced colitis-associated tumorigenesis and associated biomarkers in p53+/+ and p53+/515A (mutant) mice. In addition, the impact of high dose FA given prior to the induction of colitis will be examined. Tumor incidence and multiplicity, as well as degree of intestinal inflammation, will serve as primary endpoints of these studies. Based on the important contribution of intestinal microbiota to colitis and their ability to synthesize folate de novo, the impact of FA administration on the diversity and relative abundance of fecal and adherent bacteria within the intestine of p53+/515A mice with DSS-induced colitis will be examined in Aim 3. Mice will be treated with 5-aminosalicylic acid (5-ASA), a common maintenance therapy for ulcerative colitis, for the duration of FA exposure to recapitulate the clinical therapy of a patient following a diagnosis of colitis. The composition of the fecal and colon-adherent microbiomes, determined from 16S rRNA and metagenomic sequencing data, will be correlated with colonic inflammation, barrier function and tumor incidence/multiplicity. The results are expected to provide significant insight into the impact of FA supplementation on colitis-associated tumorigenesis and inform the first guidelines for the use of FA supplements by patients with ulcerative colitis.

In recent years, the incidence of colorectal cancer (CRC) under the age of 50 years has increased significantly and population-based screening is currently not offered to persons under 50 years. Consequently, persons with early-onset (<50 years) CRC (EOCRC) more frequently present with symptomatic disease at advanced stages (III/IV) resulting in greater loss of life in young cases. In 2020, the U.S. Preventive Services Task Force recommended the age for CRC screening by colonoscopy or fecal tests be reduced to 45 years. However, the uptake of screening by these methods in all screen-eligible populations is low, including in those <50 years who are genetically at high risk, so adherence in the asymptomatic population <50 years is also likely to be low. Also, over half of EOCRC occur in persons under <45 years of age, where no such screening would be offered. A minimally invasive, blood-based screening test for EOCRC would provide a cost-effective and patient-friendly option for triaging and identifying those warranting a follow-up colonoscopy, while increasing screening adherence in the younger population. Cancer-specific methylated DNA biomarkers are highly suited for population-based cancer screening via the detection of circulating tumor DNA (ctDNA) in blood plasma because they are more prevalent across patients with a given cancer type than tumor mutations and are more stable (nuclease-resistant) in plasma. The methylated SEPTIN9 plasma ctDNA test, “mSEPT9” (Epi proColon V2.0, Epigenomics), is FDA-approved for CRC screening in persons aged ≥50 years who decline colonoscopy and fecal tests. This test urgently needs to be assessed in persons <50 years to determine its suitability for the detection of EOCRC. Even so, a multi-marker test is likely to have superior sensitivity to a single-marker test. Thus, the objectives of our study are to identify and confirm a panel of methylated ctDNA markers for the plasma-based detection of EOCRC, and to compare the diagnostic performance of this panel to the mSEPT9 test in persons <50 years of age. In Aim 1, we will identify the most prevalent differentially-methylated (tumor vs. normal) DNA markers in CRC cases <50 years by performing deep Methyl-Seq across 4.2 million CpG sites in paired primary tumor vs. normal colon tissues from EOCRC cases, and leukocytes from healthy controls (to filter out non-specific markers). The identified markers will be validated in independent sample series. For the top-ranked markers, we will then develop methylation-specific real-time PCR assays with high analytical sensitivity and test these in pooled plasma from metastatic CRC cases (to select markers producing the strongest signals) and healthy controls (to eliminate any producing low-level non-specific signals) to finalize the marker panel. In Aim 2, we will evaluate the diagnostic performance characteristics of the mSEPT9 test vs. the multi-marker panel test in plasma from colonoscopy-verified CRC cases and controls <50 years. This study will yield a multi-marker methylated ctDNA panel with improved diagnostic performance over the single-marker mSEPT9 test for cost-effective, blood-based, CRC screening in asymptomatic persons <50 years of age.

The mechanisms that underlie the emergence of an obesity-associated increased risk in breast cancer after menopause are not fully understood. Increased adipose-derived estrogens certainly contribute to obesity-associated postmenopausal breast cancer, but additional mechanisms are also involved. Most women gain weight during menopause, and this is seen primarily as an increase in visceral adipose tissue (VAT) in the abdominal region. This in turn increases inflammation locally, systemically, and at distant sites such as subcutaneous adipose (SAT) in the breast, suggesting that changes in body composition during menopause could drive increased cancer risk. The long-term goal is to identify the mechanisms underlying obesity-associated tumor risk during menopause, and to use a precisionmedicine approach to develop interventions to effectively minimize this risk in women with obesity. The overall objective of this grant is to determine the functional role that menopausal VAT deposition plays in obesity-related breast cancers. The central hypothesis is that increased VAT deposition during menopause mediates breast tumor development and growth through increased production of adipokines, cytokines, and growth factors that signal systemically to metabolically activate a subset of tumor-promoting macrophages in the mammary adipose/breast. Using a well-characterized preclinical rat model of obesity and postmenopausal breast cancer combined with a syngeneic orthotopic transplant model and in vitro assays, the central hypothesis will be tested with the following specific aims: 1) Determine the functional contribution of menopause-induced visceral adipose accumulation on mammary tumor development; 2) Interrogate how modifying insulin resistance, VAT, and adiposederived signals alters macrophage activation, and the resulting impact on tumor development and growth after OVX. Preclinical findings will be confirmed in relevant clinical samples. The research proposed in this application is highly innovative as it will directly assess the biological role of visceral fat, inflammation, insulin resistance, and associated metabolic activation of macrophages in the tumor promotion process. Further, it will define a specific macrophage subtype that could be targeted to decrease obesity-associated cancers after menopause. This is significant as it will identify new targets for future pharmacological therapies and will provide the foundational platform for a precision medicine approach, using a clearly measurable target (decreased VAT), during a critical life stage (perimenopause/ menopause), to decrease cancer risk in women with obesity.

Squamous cell carcinoma of the head and neck (HNSCC) is a devastating, frequently disfiguring, and often fatal disease, expected to affect more than 53,000 people in the U.S. in 2020 and kill more than 10,000. Prevention of this terrible disease is critical. Cigarette smoking, alcohol consumption, and human papilloma virus (HPV) are well established major causes of HNSCC; only smoking and alcohol consumption are considered here. Cigarette smoking and alcoholic drinks are sources of multiple DNA adducts that are critical in the carcinogenic process. This proposal will establish a liquid chromatography-nanoelectrospray ionizationhigh resolution tandem mass spectrometry (LC-NSI-HRMS/MS) profile analysis of 12 oral cell DNA adducts that are likely causes of HNSCC. This was inspired by our recent analysis of DNA adducts in oral cells, in which we found levels more than 20 times higher in cigarette smokers than in non-smokers. These exciting results encouraged us to propose a profile analysis of important carcinogen-derived DNA adducts in oral cells according to the following specific aims: 1. Develop an LC-NSI-HRMS/MS profile analysis method for quantitation of 12 important and representative carcinogen and toxicant – DNA adducts in human oral cells and tissue. The adducts are derived from various carcinogens and DNA reactive compounds in cigarette smoke and alcoholic beverages. 2. Apply the profile analysis to oral cells from currently healthy individuals: a) 100 non-smokers who are non drinkers or light drinkers; b) 100 cigarette smokers who are non-drinkers or light drinkers; and c) 100 cigarette smokers who are moderate or heavy drinkers. Comparisons of adduct levels in groups a and b will identify adducts enhanced by cigarette smoking while comparisons of groups b and c will identify adducts that are enhanced by the combination of smoking and moderate or heavy drinking. 3. Test the longitudinal stability of the oral cell DNA adduct profile analysis over a 6 month period in 50 smokers who are non-drinkers or light drinkers. 4. A) Determine the DNA adduct profile in oral cells collected from 75 smokers with HNSCC and compare to that in 200 smokers without HNSCC recruited in Specific Aim 2 with the goal of identifying an adduct profile that is characteristic of HNSCC incidence. B) Compare the oral cell DNA adduct profile from part A of this aim to that in tissue, both normal and tumor, in a subset of 60 patients from part A who undergo surgery, to determine whether oral cell DNA adduct patterns are consistent with those in tissue. Our results will potentially identify individuals who are susceptible to HNSCC but are unable to quit smoking. Once identified, aggressive lifestyle and monitoring interventions in these subjects such as oral examinations 2-4 times per year can be initiated for prevention or early detection of this disfiguring and often fatal cancer.

Despite recent advances in cancer metabolism, whether and how nutritional interventions affect tumor development, metastasis and therapeutic response are still poorly understood. Thus, the goal of this study is to elucidate the effect of glutamine supplementation on tumor development and therapeutic responses, and eventually provide molecular evidence that nutritional interventions on cancer patients can inhibit tumor growth and sensitize tumors to treatments. Using metabolomic analysis, we and others have found that, compared to other amino acids, many solid tumor cells are situated in a glutamine poor environment in vivo. Interestingly, we found that glutamine deficiency in melanoma tumors resulted in cancer cell de-differentiation and resistance to treatment due to increased histone methylation levels. This finding further prompted us to test if increases in glutamine levels through dietary supplementation can be detrimental to tumor cells that have been well adapted to a low glutamine environment. Our preliminary data demonstrated that supplementation of glutamine in the diet is sufficient to increase tumoral α-ketoglutarate levels and leads to decreased histone methylation in melanoma patient-derived xenograft (PDX) tumors. Importantly, we found that high glutamine diet significantly hinders tumor growth and decreases expression of melanoma-associated oncogenes compared to control diet. In support with this, accumulating evidence from in vivo experiments demonstrate that glutamine is not an essential nutritional source to support TCA cycle and tumor growth. Thus, we hypothesize that dietary glutamine supplementation inhibits melanoma tumor growth and sensitizes tumor cells to current treatments via epigenetic reprogramming. In this proposal, we will 1) determine the effect of dietary glutamine supplementation on melanoma tumor growth, metabolism and oncogene expression in vivo; 2) determine the molecular mechanisms by which glutamine supplementation inhibits tumor growth; 3) investigate the effect of glutamine supplementation in response to BRAF/MEK inhibitors and immunotherapy for melanoma treatment. Despite many proven clinical benefits of glutamine supplementation to cancer patients, recent in vitro studies showing that tumor cells are avid glutamine consumers led to cautionary usage of dietary glutamine on cancer patients. Completion of the proposed studies will provide insight into glutamine driven epigenetic regulation and its effect on tumor growth. The results of these studies will reveal a novel therapeutic direction for using dietary glutamine supplementation to prevent tumor growth and enhance therapeutic responses without detrimental side effects.

In 2020, breast cancer has surpassed lung cancer as the most commonly diagnosed cancer in women. Compared to estrogen receptor (ER)-positive breast cancers, ER-negative (ER-) breast cancers have worse prognoses and no effective prevention strategies. In this study, we will explore new strategies for immunoprevention of ER- breast cancer. Inducing potent anti-tumor immunity for prevention of poorly immunogenic breast cancers has been highly challenging. Engagement and expansion of activated dendritic cells (DCs) could facilitate broad and efficient anti-tumor immunities. However, certain existing DC stimulators (e.g., agonists of toll-like receptors and STING) also triggered adverse immune responses. For cancer prevention, it is imperative to develop safe and effective approaches to boost DC immunity. To this end, we screened for dietary supplements that increase DC activities and identified natural vitamin E (VitE) as a stimulator of DC functions. Excitingly, we found that breast cancer patients who took VitE during immunotherapies had a significantly better survival rate and improved therapeutic response than patients who didn’t take VitE, suggesting that VitE administration may potentiate anti-tumor immunity. Indeed, systemic (oral) administration and local (at injection site together with cancer vaccines) delivery of VitE significantly prolonged tumor-free survival in ER- mammary tumor mouse models that didn’t respond to cancer vaccines alone. These data led us to hypothesize that VitE administration, via reinforcing DC activation and antigen presentation, enhances immunoprevention of ER- breast cancer by cancer vaccines. We will test whether VitE could enhance cancer vaccine-induced immune surveillance and prevent/delay tumor initiation/progression in genetic engineered mouse models of (HER2+ and basal-like subtypes) ER- mammary tumors and the CD34+ humanized mouse models (for prevention of human ER- breast cancers) (Aim1). As a proof of concept, we will primarily use a tripleantigen (tumor associated antigens neu/IGFBP-2/IGF-IR) peptide vaccine (TAVac) for proposed studies since TAVac has shown partial efficacy in delaying tumor progression in ER- mammary tumor mouse models. Importantly, corresponding DNA vaccines against human HER2/IGFBP-2/IGF-IR are currently under phase I/II clinical studies for prevention of HER2+ and HER2- breast cancer recurrence. To gain mechanistic insights into how VitE potentiates anti-tumor immunity, we will investigate i) the global effect of VitE on the immune cell landscape by mass cytometry (CyTOF); ii) the impact of VitE on DC and T-cell subset compositions, functionality and signaling networks; iii) major immunophenotype changes critical for VitE-enhanced immunoprevention; iv) how VitE prompts antigen processing/presentation in DCs and whether VitE functions through SHP1, a critical DC checkpoint (Aim2). Finally, we will test novel strategies to further improve the immunoprevention efficacy against ER- mammary tumors (Aim3). If successful, our strategies could be readily tested in future clinic trials for immunoprevention of breast cancer, particularly, for women at high risk for ER- breast cancer.

Intestinalization of the esophagus, termed Barrett’s esophagus (BE), is thought to develop in response to chronic acid and bile reflux and carries great clinical significance because it is the precursor to esophageal adenocarcinoma (EAC). The incidence of BE is quite high, estimated to be found in at least 1:100 people. While relatively few with BE progress to cancer there is great importance to being able to detect and treat those at risk of progression as EAC is an aggressive cancer with potential for early spread. Efforts to screen for high-risk disease in those with BE have, to date, not been very successful. Therefore, there is profound need to define the process by which BE progresses into EAC, to develop biomarkers to diagnose early progression and assess progression risk in BE tissues as well as to develop novel therapies for treatment. The objective of this R01 proposal is to investigate the ability of biomarkers to identify BE patients at high risk of progression. Using results from two externally funded genomic studies in non-dysplastic BE (NDBE) and BE with low-grade dysplasia (LGD) and previously published results, we will compare biomarkers and determine an optimized combination for risk stratification in two prospective cohorts including patients with NDBE or LGD. We will then validate our risk stratification assay in an independent US cohort. Finally, we will compare our genomic biomarker panel results in paired biopsy and brush samples. A unique collaboration between the PI Dr. Stachler, expert gastroenterologist (Dr. J Bergman), a talented computational biologist (Dr. CZ Zhang), and an expert biostatistician (Dr. K Zwinderman) along with key collaborators allows a truly innovated study to be performed. This will be accomplished using an unprecedented collection of clinically derived samples, a highly optimized targeted sequencing panel, and novel computational approaches that allow a wide array of information to be determined in a cost effective, clinically relevant manor. Aim 1: Identify a set of genomic biomarkers highly predictive of progression in biopsies from a prospective cohort of patients diagnosed with NDBE and LGD and assess whether the addition of methylation-based biomarkers improves stratification. For clinical implementation all biomarkers should be compared head to head in order to determine an optimized combination of biomarkers for risk stratification. Aim 2: Validate the risk stratification assay in a multi-institutional cohort of patients with a baseline diagnosis of NDBE or LGD. For clinical implementation, it is vital to validate any risk stratification assay on completely independent cohorts looking at clinically relevant time points. Aim 3: Determine if brush-based sampling devices improve biomarker detection over standard endoscopic biopsies. Broad sampling of the BE epithelium may allow for increased rates of detection for genomic or methylation biomarkers, therefore we will determine if samples from a brushed based device can better risk stratify patients compared to standard biopsies.

Understanding the causes and mechanisms underlying circadian rhythm disruptions that are associated with fatigue during cancer treatment remains unclear. This current deficiency means that successful cancer treatment falls short of its potential and prior quality-of-life remains elusive for patients. Our long-term goal is to improve debilitating behavioral sequelae in cancer patients, thus improving quality-of-life, other comorbidities, and mortality. Thus, the overall objective here is to establish the potential role of circadian disruption as a fundamental pathway by which chemotherapy promotes cancer-associated fatigue. Indeed, robust circadian rhythmicity of virtually all physiology is extremely well-conserved; desynchrony of these rhythms leads to negative health and behavioral consequences. The central hypothesis is that chemotherapy-induced inflammation inhibits SCN function leading to fatigue. The rationale for this work is that circadian circuitry disruption is an understudied, relevant pathway in psycho-oncology research that could elucidate mechanisms and new, rhythm-focused interventions. Three specific aims are proposed to test the central hypothesis using our novel breast cancer “survivor” mouse model. Aim 1 will determine the ability of the master clock to entrain after chemotherapy. Behavioral SCN rhythm adaptations to environmental challenges will be assessed. Aim 2 will identify the role of central inflammation in master clock disruptions after chemotherapy. The role of chemotherapy-induced neuroinflammation on SCN molecular and behavioral rhythms will be quantified. The potential resolution of fatigue will also be assessed. Aim 3 will determine the role of circadian disruption in chemotherapy-induced fatigue. Genetic and pharmacological SCN timing manipulations will precede a battery of behavioral assessments of the physical, motivation, and cognitive components of fatigue. In vivo and ex vivo circadian timing approaches combined with systems-, cellular-, and molecular-level analyses will pinpoint the effects of two regimens of chemotherapy on master oscillator circadian circuitry relevant to cancer-related behavioral comorbidities. The proposed research is conceptually innovative because using circadian approaches is new to psychooncology. It is also technically innovative by way of the superior translational model and the circadian genetic and pharmacological techniques planned. This research will result in essential new knowledge about how common cancer treatments affect the pacemaker, which is crucial to extensive downstream physiology and behavior (i.e., beyond fatigue). Results will provide much needed evidence to make circadian-based approaches standard in clinical practice, as well as inform the design of novel circadian-directed pharmacological and nonpharmacological interventions. This research is applicable to other cancers and in non-oncological populations treated with chemotherapy (e.g., stem cell transplant, lupus).

Childhood acute lymphoblastic leukemia (ALL) was uniformly fatal prior to the 1960s. Survival rates today approach 95%, making ALL one of medicine's great success stories. As the number of survivors across the US has increased, the focus of research has shifted to life after cancer. Research in ALL survivors has highlighted problems in executive functions, representing mental functions governed by the frontal lobes. Most patients will be cured prior to entering kindergarten, meaning that these neurocognitive problems potentially create a lifetime burden. Indeed, research in long-term survivors of ALL show that neurocognitive difficulties affect scholastic and vocational success, creating a profound and long-lasting burden on quality of life. While survivorship issues are well-documented, research in young patients undergoing treatment is lagging. Our plan to assess neurodevelopmental changes in patients during treatment will help pinpoint the timing and extent of neurotoxic exposures in children treated for ALL, providing tangible opportunities to implement strategies of remediation and prevention. The overall objective of this proposal is to identify markers of altered neurocognitive development in ALL patients. We recently piloted a prospective study where young ALL patients completed neurocognitive evaluations and non-sedated neuroimaging on two occasions occurring at a 6-month interval. Preliminary outcomes highlighted the importance of evaluating growth trajectories in gaining insights into the etiology of neurocognitive morbidity. In our pilot study decrements in executive functions (EF) were observed. We also observed that frontal white matter growth was substantially slower in ALL patients relative to peers. And finally, increased concentrations of neurofilament light, which is a marker of axonal damage, was associated with a slower rate of change in frontal white matter volume in ALL patients. Based on our groundwork results, we propose to evaluate early markers of abnormal neurodevelopmental trajectories in ALL patients undergoing active treatment. We will employ a longitudinal design where newly diagnosed ALL patients between the ages of 3-6 years old (n=30) will be compared to controls (n=30). Leveraging the power of a within-subject design, participants will be assessed on three occasions occurring at major treatment milestones (180 observations total). Using validated cognitive neuroscience paradigms, we will identify changes in discrete aspects of executive function for aim 1. Non-sedated structural and functional neuroimaging will be used for the work proposed under aim 2 to evaluate changes in brain volume, connectivity, and metabolism. Lastly, we will utilize ultrasensitive digital assays for quantifying neurochemical markers of brain injury in ALL patients. Results from this work will have impactful implications for understanding early neurodevelopmental changes in children undergoing treatment for ALL, providing a framework for subsequent studies linking early markers to neurocognitive outcomes in survivorship. Gaining insight into early neurodevelopmental change is invaluable for future efforts aimed at curbing neurotoxicity of cancer treatment.

Ovarian cancer is the 5th leading cause of cancer deaths in women in the U.S. Earlier detection is the key to increased survival for women with ovarian cancer, yet a screening tool that is both sensitive and specific enough for use in the general population has yet to be developed. In contrast, screening for cervical cancer by Pap tests has been routinely performed for over 50 years. In the liquid-based Pap test, cells are collected from the cervical opening and placed into an alcohol-based fixative and then examined for abnormal cells. Since ovarian cancer cells have been observed in Pap tests, ovarian cancer peptide biomarkers may also be present; yet Pap samples have not been rigorously examined for diagnostic peptides. Our central hypothesis is that proteins shed by ovarian cancer cells can be detected in the Pap test fixative and on cervical-vaginal swabs by Mass Spectrometry (MS)-based proteomics. The Pap test fixative and swabs are ideal for biomarker discovery since they are derived from a site proximal to the ovarian cancer (i.e. proteins may be secreted or shed from the tumor and flow through the fallopian tube into the uterus and out the cervical opening). Recently, ovarian cancer precursor lesions have been identified in the fimbria of the fallopian tube, strengthening the hypothesis that ovarian cancer proteins will be found in the lower genital tract, perhaps even at early stages. In preliminary studies, candidate biomarkers were successfully identified by MS-based proteomics in Pap test fixatives from women with ovarian cancer, and the levels of biomarker peptides were quantified in cases vs. benign and healthy controls, demonstrating the feasibility of this approach. The longterm goal of this project is to develop a noninvasive screening test that can be incorporated into a routine Pap test or a self-administered home test, so that women can be screened simultaneously for cervical and ovarian cancer. The objective of this study is to verify ovarian cancer biomarker peptides identified in Pap test samples and extend the results to cervical swabs, using our collection of biospecimens from over 600 patients and controls. Aim #1 will build on our preliminary studies to prioritize ovarian cancer biomarker candidates found in liquid-based Pap test samples by performing Tandem Mass Tag™ 11-plex isobaric labeling, 2D LC-MS/MS, and bioinformatics integration. In Aim #2, the most robust candidate biomarkers will be quantified by developing a targeted Selected Reaction Monitoring (SRM)-MS assay coupled with a multi-protein classifier, which will then be used to test hundreds of Pap test samples from women with ovarian cancer, other gynecologic cancers, and benign gynecologic conditions, as well as healthy women. In Aim #3, proteins eluted from cervical swabs will be tested to determine whether they are also detected by our SRM-MS assay. Results from this study may be extended to the early detection of ovarian cancer in women in high risk groups and eventually for screening the general population, thereby shifting the paradigm for how women are screened for ovarian cancer and improving survival rates for the >22,000 women diagnosed with ovarian cancer each year.