2022
Target antigen and T-cell exhaustion impact outcomes after CAR 19 and post-CAR relapse
Project Summary: This study aimed to identify whether relapse after CAR T-cell therapy is related to loss of expression of a target antigen (i.e. CD19, the target of CAR T-cell therapy in lymphoma) or due to a lack of T-cell fitness. First, we set up assays at Moffitt Cancer Center and the University of Miami to measure target antigen density using quantitative flow cytometry from patient lymphoma biopsies. In approximately one-third of biopsies we were able to obtain enough yield to measure target antigen density, the limitation being obtaining enough tumor cells at the time of the biopsy. We also showed that the quantitative flow cytometry may be run with similar results from fresh or viably frozen samples, simplifying the workflow. We are in the process of accumulating sufficient samples to judge the relationship between target antigen density and clinical outcomes. Second, we set up assays to measure T-cell fitness. We found that expression of CD39 in the apheresis T cells, which we show mark exhausted T cells, is associated with poor clinical outcomes after CAR T-cell therapy. These exhausted CD39+ T cells are more prevalent in the blood of patients with large tumor volume. Moreover, in a study of paired apheresis T cells and manufactured CAR T cells, the presence of CD39-high exhausted T cells in the apheresis led to production of poor-quality CAR T-cells. Together these results suggest that large tumors drive T-cell immunosuppression, which in turn leads to low CAR T-cell fitness and a propensity for relapse.
Trends and Disparities in Cervical Cancer Screening Uptake and Follow-Up Among Women in Florida
Project Summary: There are three main goals for this project: Aim 1: Estimate the prevalence of cervical cancer screening over the calendar year and establish patterns of adherence to recommended guidelines for follow-up among Florida women. Aim 2: Assess follow-up receipt and time to follow-up among women with an abnormal cervical cancer screening test result among Florida women. Aim 3: Evaluate factors independently associated with cervical cancer screening receipt, adherence, and follow-up among Florida women. The IRB approval for UF to receive the OneFlorida+ data was received in December 2023. UF received data from OneFlorida+ in February 2024. Currently, we are determining which women have received a cervical cancer screening. We have identified all codes relevant to cervical cancer screening and are considering the cohort of women who also had a primary care or women’s health visit. We are currently working on restricting to the women who had at least one visit with obstetrics and gynecology, family medicine, internal medicine, and general practice. Thus far, we have obtained very low estimates of the prevalence of cervical cancer screening. IRB approval for UF to share the dataset with University of Miami was received in July 2024. The data team is preparing a dataset of all variables received for women who received cervical cancer screening during the period. This will allow Dr. Schlumbrecht’s team to complete Aim 2. Dr. Schlumbrecht has committee personal funds to cover an additional 0.1 FTE for the biostatistician at the University of Miami to complete the analyses by FY25.
Establishing a Multi-Site HIV Oncology Research Program in Florida
Project Summary: Persons with HIV (PWH) diagnosed with cancer experience significantly elevated mortality and increased likelihood of disease relapse following cancer therapy. However, the role of HIV-related immune cell and inflammatory changes that occur in PWH while receiving cancer therapy remains unclear. To address these knowledge gaps, Moffitt Cancer Center and UM Sylvester Comprehensive Cancer Center (SCCC) initiated this study to 1) Expand the HOPE biospecimen resource to include PWH and solid cancers from SCCC; and 2) Describe the distribution of immune-related biomarkers (immune alterations, inflammation, and microbial translocation) in PWH diagnosed receiving systemic cancer therapy for solid cancers. A total of 20 HIV-positive cancer patients were enrolled in the study, who contributed 45 serum samples. Among the 18 enrolled Moffitt participants, seven participants contributed a baseline, during treatment, and post-treatment sample; six participants contributed a baseline and during treatment sample; and five patients contributed a baseline and post-treatment sample. At the University of Miami site, two patients contributed a baseline sample
2019
Determinants of Response to CAR-T Cellular Immunotherapy in Aggressive B-Cell Lymphoma
Project Summary: The overarching goal of this project was to determine whether CAR-T treatment resistance in B-cell lymphoma is driven by tumor cell extrinsic immunological factor and tumor cell intrinsic genomic variance between individual tumors. This hypothesis was tested through two specific aims: 1) to identify tumor microenvironmental and systemic immune determinants of response to CAR-T therapy in lymphoma [Moffitt], and 2) to determine genomic factors driving CAR-T treatment failures in lymphoma [Sylvester]. From these collective studies, we have identified cytokines from lymphoma patient samples refractory to CAR-T therapy that positively correlate with both toxicity and treatment response. Further RNA-Seq analysis on pre-treatment lymphoma patient samples revealed gene signatures rich in inflammatory and myeloid cell related genes that were associated with both CAR-T therapy toxicity and efficacy when correlated to post-treatment outcomes. A subset of lymphoma patient samples with matched germline DNA was sequenced (whole genome sequencing), which generated a genomic dataset that demonstrated novel markers of prognosis in DLBCL that associated with poor outcomes in response to CAR-T cells.
Epigenetic Basis of Glioblastoma Chemoresistance
Project Summary: The objectives were to elucidate the molecular basis for hypoxic downregulation of DNA mismatch repair (MMR) genes and to identify novel therapeutic modalities that would reverse the hypoxic repression and therefore increase the efficacy the ability to kill tumor cells with the DNA-damaging chemotherapeutic temozolomide. In glioblastoma cell lines, we found that 60% of 118 promoters with the collective gene ontology term of “DNA repair” exhibited alterations in nucleosome occupancy and/or DNA methylation, using an innovative targeted single-molecule footprinting method that we developed, fully optimized, filed a patent application, and have used to leverage progress and funding for additional projects. The results relevant to this FACCA project have demonstrated widespread epigenetic dysfunction of not only MMR pathways but all known cellular modes of DNA repair. Many of the promoters of these DNA repair genes exhibited strong evidence for binding of nuclear respiratory factor 1 (NRF1), which responds to oxidative stress induced by hypoxic growth within cells in the inner mass of solid tumors. Critical preliminary data showing that MLH1 expression is essential for temozolomide-induced tumor cell death was obtained in a mouse xenograft model using a human glioblastoma cell line with the MMR gene MLH1 knocked out by CRISPR/Cas9 gene editing. Deletion of both NRF1 binding motifs in the MLH1 promoter of a colon cancer cell line reduced transcription under normoxic conditions but completely impaired hypoxic repression, demonstrating that NRF1 mediates the hypoxic response. Furthermore, we determined that an additional transcription factor, NFY, binds to the MLH1 promoter and works in concert with NRF1 to maintain an open promoter that protects against DNA methylation.
Nuclear Envelope Defect and Generation of Micronuclei in Ovarian Cancer Development and Immune Therapy
Project Summary: The overarching goal of this project was to investigate the formation of micronuclei and aneuploidy in ovarian cancer. These studies found an important role of micronuclei in response to paclitaxel chemotherapy and found ZBP-1 binding to micronuclei Z-DNA is an important innate immunity signaling pathway in cancer. Further analysis of genetic alterations within pre-neoplastic ovarian lesions revealed simultaneous loss of LMNA (lamin A/C) gene and gain of point mutations in P53. Further analysis is ongoing to determine the relationship between the two genetic alterations. Furthermore, we determined that lamin A/C is a determinant of Taxol chemotherapy sensitivity and uncovered a cell death mechanism in chemotherapy, by nuclear membrane rupture.
Epigenetically Reversing BRAF Inhibitor Resistance in Melanoma by Vitamin C
Project Summary: The overarching goal of this project was to determine if vitamin C treatment is capable of overcoming resistance to BRAF inhibitors using in vitro and in vivo melanoma models and to define the molecular mechanisms through which vitamin C can re-sensitize BRAF inhibitor-resistant melanoma cells, given that vitamin C plays a role in epigenetic regulation by DNA demethylation. We have found that vitamin C treatment dose dependently re-sensitizes BRAF inhibitor (BRAFi)-resistant melanoma cells by decreasing the EC50 of vemurafenib treatment and that combination vitamin C + vemurafenib treatment both blocks proliferation and increases apoptosis in resistant cells. These studies demonstrated that vitamin C treatment restored 5-hydroxymethlcytosine levels in BRAFi-resistant cells, thus promoting an epigenetic phenotype characteristic of BRAFi-naïve melanoma cells. Further, RNA-Seq analysis in vitamin C-treated BRAFi-resistant melanoma cells revealed transcriptional changes that were enriched in downregulated genes within the MAPK pathway and were associated with re-sensitization.
2018
Role of Intestinal Microbiota in Lung Cancer Therapy
Project Summary: The overarching goal of this project was to determine the functional relationship between the immune response, intestinal microbiota, and repose to immune checkpoint inhibitors in patients with lung cancer. This goal and associated hypothesis that microbiota determines checkpoint inhibitor efficacy and toxicity by engaging host immune responses are supported by four specific aims. At the close of the project, microbiota DNA was sequenced from 62 fecal biospecimens obtained from patients with non-small cell lung cancer treated with a PD-1 checkpoint inhibitor. The dataset revealed that microbiota composition was not significantly different between responders and non-responders at treatment completion but were significantly different at baseline before immunotherapy. These differences were mainly upregulated genes within carbon fixation pathways (responders) and phosphotransferase pathways (non-responders). The group further demonstrated that colonization of Lewis lung carcinoma bearing mice with preserved microbiota from patients with lung cancer (responders and non-responders) resulted in improved response to checkpoint inhibitors only in mice colonized with bacteria from responders. Beneficial microbiota fell within two main microbial families (Lachnosiraceae and Oscillospiraceae), which suggests that only specific microbial families are associated with response to checkpoint inhibitors and highlights the potential for optimizing immunotherapy through provision of specific bacterial strains. Finally, the group demonstrated that the microbiome from lung cancer patients receiving immunotherapy and opioids is not more colitogenic than that from patients without opioid treatment.
FACCA 2018 Disparities Think Tank
Project Summary: The overarching goals of this project were to determine a common set of measures and sampling tool(s) to characterize each center’s catchment area; to explore opportunities to integrate identified measures with existing secondary data sources; and to evaluate potential challenges of wide-scale implementation of measures, sampling tool(s), and biospecimen process for assessing genomic ancestry. These activities have resulted in agreed-upon common measures and questionnaires tailored to the respective populations within each center’s catchment area. Each center collected questionnaire-based data and biospecimens as appropriate to the plan and conditions within their catchment areas.
2017
Preventative Vaccination Against Neoantigens in MRD and Premalignant Settings
Project Summary: The overarching goal of this project was to develop a broadly useful, clinically applicable, and cost-effective vaccination strategy against neoantigens that can be induced in future tumors in patients in remission or in high-risk individuals. This goal was supported by two specific aims to 1) develop formulations of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) lipid nanoparticles complexed with B cell mRNA or siRNAs and determine whether they can activate dendritic cells in situ, and 2) determine whether vaccination of mice with DOTAP mRNA or siRNA complexes can protect mice from a subsequent tumor challenge that was manipulated in situ to express specific neoantigens. These studies demonstrated feasibility of generating lipoplexes containing anti-TAP siRNA complexes and delivering such complexes successfully to dendritic cells. This resulted in successful downregulation TAP and concomitant downregulation of MHC Class I expression, suggesting that MHC levels can be used as a functional readout of TAP siRNA in dendritic cells. Further, in vivo vaccination with encapsulated TAP siRNA significantly inhibited growth of TAP-deficient RMA-S-expressing lymphoma cells in an immunocompetent mouse model.
Florida Increases Rates of Screening and Treatment of Hepatitis C Virus (FIRST HCV)
Project Summary: The overarching goal of this project was to develop and implement practice-based interventions to reduce the incidence of hepatitis C (HCV)-related hepatocellular carcinoma by 50% utilizing a state-wide electronic health record infrastructure to assess HCV screening and treatment throughout the state. The team established a statewide collaborative group that includes faculty from cancer centers and Veterans Affairs from around the state. The EMR-fed database was created and the collected data demonstrated that overall the percent of patients receiving an order for HCV testing was low across all birth cohorts and particularly fell short of CDC recommendations for a one-time test for baby boomers. The team also identified racial/ethnic and gender differences in likelihood of HCV test orders. In 2020, the Florida HCV working group stemming from this award was added to the Health and Human Services list of hepatitis elimination projects. The team is developing a multilevel intervention to increase HCV screening among adults living in Florida.
The Florida Pancreas Collaborative: A Partnership Dedicated to the Prevention and Early Detection of Pancreatic Cancer
Project Summary: The overarching goal of this project was to formally establish the Florida Pancreas Collaborative to promote early detection and prevention of pancreatic cancer. This goal was supported by two specific aims: 1) to prospectively recruit a multi-center cohort of patients newly diagnosed with intraductal papillary mucinous neoplasms (IPMNs) or other pancreatic conditions and generate a common biorepository that complements institutional resources, and 2) to evaluate the diagnostic performance of circulating miRNAs in distinguishing high-risk versus low-risk IMPNs using collected biospecimens. These efforts resulted in the recruitment of more than 1,300 eligible cases and more than 1,200 biospecimens (including blood and cyst fluid samples) from patients with IPMN.
2016
Targeting Mitotic Functions of TBK1 and CDK2 to Combat Cancer
Project Summary: The overarching goal of this project was to determine whether novel inhibitors of CDKs can induce mitotic catastrophe in lung cancer cells as single agents or in combination with TBK1 inhibitors. The study team generated lung cancer cell lines that are depleted of TBK1. These cells were subjected to double thymidine block then released with or without TBK1 inhibitor treatment accumulated in mitosis. Further, TBK1 was shown to colocalize with CDC20 on centrosomes, have increased colocalization of BUBR1 and CDC20, and have enhanced recruitment of BUBR1 to kinetochores. Additional mechanistic studies suggest that TBK1 facilitates mitotic progression through satisfying the spindle assembly checkpoint. The team also generated novel CDK inhibitors that lack a Michael Acceptor, which is known to produce off-target effects. These newly generated inhibitors retained activity against CDKs and induce aggregation of CDKs followed by proteasomal degradation. Finally, the team found that TBK1 does not alter CDK phosphorylation status or vice versa, suggesting that direct crosstalk between these kinases does not occur but that fidelity of their respectively activity influences mitosis by different mechanisms.
The Role of HoxBlinc INCRA in NPM1 Mutation-Mediated Pathogens of Myeloid Malignancies
Project Summary: The overarching goal of this project was to determine wither nucleophosmin 1 mutation activates HoxB gene-associated long intergenic non-coding RNA (HoxBlinc), leading to upregulation of Hox gene expression. We have reported that HoxBlinc recruits the Setd1a/MLL1 HMT complexes and differentially activated anterior HOXB gene expression, which play a critical role in maintaining the balance between self-renewal and differentiation of HSC/HPCs. In the one-year funding period by FACCA, we investigated the role of HoxBlinc in the regulation of normal hematopoietic stem cell function and the pathogenesis of acute myeloid leukemia. We successfully generated HoxBlinc transgenic mouse models and analyzed their hematological phenotypes. We also performed experiments to: 1) define the effects of HoxBlinc transgenic expression on HSC behavior in vivo; 2) determine whether overexpression of HoxBlinc alone is sufficient to cause myeloid malignancy in mice, 3) investigate the molecular mechanism(s) by which HoxBlinc regulate chromatin conformation and activate HOX gene expression in HSC/HPCs using high-throughput, genome-wide chromatin structure, and three-dimensional organization studies, and 4) explore whether HoxBlinc servea as an effective therapeutic target using MLL-rearrangement- and Npm1 mutation-driven myeloid malignancies.
Oncogenic Role of KSHV miRNAs in Cell and Animal Models of Kaposi’s Sarcoma
Project Summary: The overarching goal of this project was to determine whether KSHV-encoded miRNAs contribute to Kaposi’s sarcoma tumorigenesis in vitro and in vivo. The study team generated and validated recombinant KSHV virus with mutated miRNAs and then generated infected murine mesenchymal stem cells for testing. RNASeq analysis revealed remarkable similarities between the host transcriptomes of oncogenic and non-oncogenic infected mesenchymal cells and highlighted histones as the most differentially expressed genes in the KSHV lytic dataset. Further, the team demonstrated that HDAC inhibitors are able to potently induce KSHV lytic replication in infected cells. To characterize global chromatin changes in response to KSHV infection, the team has completed ChIP-Seq and is beginning bioinformatic analysis.
RAGE signaling through the inflammasome: novel combined inflammatory therapeutic targets in cancer
Project Summary: Myelodysplastic syndromes (MDS) are highly associated with both inflammation and aging. Our recent studies showed that an inflammatory pathway, mediated by S100A9 and its receptor CD33, activates myeloid-derived suppressor cells (MDSC). These cells then kill the stem cells in the bone marrow and cause MDS. Thus the development of drugs aimed at MDSCs may provide a unique opportunity to effectively treat MDS. Hence, the objective of this study was to collaborate with Drs. Lippman and Hudson at the Sylvester Cancer Center in Miami to complement our study with their findings on RAGE receptors, which also interact with S100A9 in solid tumors.
Defining and Targeting the Aberrant Chromatin Function in Uveal Melanoma
Project Summary: The overarching goal of this project was to determine if the combination of aberrant G protein signaling (specifically driving MEK signaling) and loss of the BAP1 tumor suppressor can lead to HDAC8 dependence for cell growth in uveal melanoma. The study team completed extensive experiments that demonstrated 1) uveal melanoma lines with combination GNAQ and BAP1 mutations form larger tumors than melanocytes only harboring GNAQ mutation in syngeneic mouse models. Additionally, BAP1 knockout was not sufficient to induce xenograft growth in vivo; 2) in vitro synthetic lethality screening of uveal melanoma lines identified several epigenetic regulators essential for cell survival including many genes coding for bromodomain proteins; 3) phosphoproteomic profiling of uveal melanoma cells treated with MEK, HDAC, and combination inhibitors revealed that MEK inhibition is associated with upregulation of specific tyrosine kinase receptors. Further activity-based proteomic analysis identified pathways enriched as a result of adaptive signaling in response to MEK inhibition including tyrosine metabolism, adhesion and cytoskeletal remodeling, apoptosis, and growth factor signaling; 4) pan-HDAC inhibition using Panobinostat synergizes with MEK inhibition using trametinib to induce apoptosis and prevents three-dimensional growth in colony assays and these phenomena are heightened in melanoma cells lacking BAP1; and 5) unbiased drug screening was conducted that identified compounds with activity against uveal melanoma cell line growth. Of these, compounds targeting HDACs had the most prominent effect in all cell lines tested. Combination HDAC, MEK inhibitor therapy suppressed adaptive AKT and YAP signaling, suggesting a viable strategy to prevent MEK inhibitor resistance. In vivo testing using xenograft mouse models demonstrated durable suppression of melanoma growth compared to either single agent alone.
The Effects of Immigration on Breast Cancer in Women of African Descent
Project Summary: The overarching goal of this project was to investigate epigenetic DNA methylation marks in a cohort of women of African descent with breast cancer to identify genetic markers associated with disparities in outcomes. The study team enrolled 89 newly diagnosed patients with breast cancer and obtained 80 germline samples, which were submitted for SNP and DNA sequencing analysis. These analyses identified differentially methylated DNA regions in Haitian, Haitian immigrant, and African American women with ER+ breast cancer. Moreover, pathway analysis identified common cancer pathways, including ENT, PI3K, DNA damage, and cell cycle among all three groups of women.
2015
Gut Microflora and Estrogens: A New Paradigm for Breast Cancer Risk Reduction
Project Summary: The overarching goal of this project was to assess the role of the microbiome in estrogen metabolism by examining the association of fecal bacteria with levels of urinary estrogen metabolites and with breast density. The study team developed procedures for identifying eligible women and for sample collection prior to initiating study recruitment in late 2015. The team measured estrogen metabolites in urinary samples from 167 women using metabolomic protocols developed at Moffitt. The team also assembled data on mammographic density for women enrolled at Moffitt. Similarly, the team collected, processed, sequenced, and profiled the microbiome in 167 stool samples. Findings indicated suggestive associations of gut microbiome alpha diversity with both urinary estrogen and breast density. Specific bacterial species were also implicated for both endpoints.
Role of GPCR-Androgen Receptor Crosstalk in Metastatic Castration-Resistant Prostate Cancer
Project Summary: The overarching goal of this project was to determine if the G-protein coupled receptor arginine vasopressin receptor-1a (AVPR1a) is a therapeutic target in castration-resistant prostate cancer (CRPC). The study team found that AVPR1a knockdown selectively reduced CRPC cell line proliferation compared to androgen dependent or non-tumorigenic prostate epithelial cells. Ectopic AVPR1a expression in androgen-dependent PC cells confers castration resistant growth in vitro and in vivo. Furthermore, treatment of CRPC cells with the AVPR1A ligand, arginine vasopressin (AVP), activates ERK and CREB, known promoters of PC progression. A clinically safe and selective AVPR1A antagonist, relcovaptan, blocks CRPC in three distinct mouse models representing: newly emergent CRPC, established CRPC in the prostate, and end-stage bone metastatic growth. In bone metastatic CRPC, treatment with relcovaptan significantly inhibits CRPC line growth within the bone and limits the extent of cancer-induced bone disease, suggesting that AVPR1a antagonists can directly suppress CRPC growth and progression, as well as preventing pathological bone disease resulting from metastatic spread. Based on these preclinical findings, repurposing AVPR1A antagonists is a promising therapeutic approach for CRPC.
Modeling the Patterns of Breast Cancer Early Metastases
Project Summary: The overarching goal of this project was to gather data to better model the patterns of early metastatic spread of breast cancer to provide additional evidence that adding surveillance imaging to standard follow-up care for high-risk breast cancer survivors increases overall and disease-free survival. The study team accrued 10 patients (target) in the prospective surveillance study and conducted data collection on more than 70 subjects in the retrospective outcomes analysis. In 2019, a student team member analyzed six datasets, processing them for nodule detection and sizing. The team identified one study subject with growth within a nodule over more than three timepoints that correlated with clinical follow-up scans monitoring the nodule for metastatic progression of breast cancer, suggesting that this strategy can directly measure the growth rate of metastatic breast cancer and increase reliable detection of early metastases in patients with breast cancer whose disease is in remission.
