CD200R Signaling in Melanoma Progression and Immunotherapy
Xue-Feng Bai and Lai-Chu Wu
Melanoma is the most dangerous type of skin cancer and the leading cause of death from skin diseases. Melanoma is characterized by early metastasis to distal organs, such as the lung, and the overall success of treatment in metastatic melanoma is limited. In our recent study we have found that melanoma cell expression of CD200, a cell surface glycoprotein, can significantly inhibit melanoma tumor formation and metastasis. Such inhibition of tumor formation and metastasis appears to be mediated by CD200 receptor (CD200R)-positive myeloid cells, since injection of B16 melanoma cells resulted in significantly accelerated tumor growth and metastasis in CD200R-deficient mice than in wild type mice. In addition, stimulation of CD200R in normal mice with an agonistic antibody in vivo dramatically inhibited metastatic B16 tumor foci formation in the lungs. Based on these observations, we hypothesize that CD200R signaling in myeloid cells inhibits melanoma tumor progression and that targeting CD200R is a useful immunotherapy of melanoma. To test this hypothesis, we will first generate CD200R-deficient Braf/Pten mice that develop conditionally inducible melanoma, to determine if lack of CD200R signaling in myeloid cells accelerates spontaneous melanoma formation and metastasis. Second, we will examine if triggering CD200R using an agonistic antibody is a useful approach for immunotherapy of spontaneous melanoma, and if so, determine what signaling pathway is involved. Information generated from these studies will lead to understanding the role of CD200R signaling in melanoma pathogenesis and immunotherapy.
CS-1 Targeted NK vs. T-cell Chimeric Antigen Receptor Therapy With or Without Elotuzumab
Jianhua Yu and Craig Hofmeister, MD
Patients with relapsed hematologic malignancies represent an unmet therapeutic need. Chimeric antigen receptor (CAR) therapy represents an intriguing option for patients with multiple myeloma (MM) -- CS1 is a MM-associated antigen highly and universally expressed on MM cells but minimally expressed on normal cells, and thus can be utilized to direct cytotoxic immune cells to specifically kill MM cells. CAR NK cells are novel effector cells whose killing may be enhanced by monoclonal antibodies, and these cells may avoid the potentially lethal side effects of CAR T cells such as cytokine storms. The humanized CS1 mAb, Elotuzumab, can enhance antibody-dependent cellular cytotoxicity of NK cells against MM cells and has progressed to phase III trials. CS-1 targeted CAR NK cells are largely unexplored and their combination with a monoclonal antibody may be synergistic. We have generated CS1-CAR NK cells to more effectively eradicate MM cells in vitro. We hypothesize that targeting CS1 by CAR cells alone or in combination with CAR T cells or Elotuzumab is a promising therapeutic strategy, and the combination will be synergistic. We will test this by investigating the effect of CS1-CAR NK cells alone or in combination with CS1-CAR T cells or Elotuzumab in vitro and in an in vivo model system. We believe these experiments will lay the foundation for a phase I trial for relapsed myeloma patients utilizing autologous CAR NK cells.
Tethered Cationic Lipoplex Nanoparticle (TCLN) Assay for Early Lung and Liver Cancer Detection and Surveillance via Extracellular RNAs in Exosomes and Circulating Tumor Cells
L. James Lee, Patrick Nana-Sinkam, Kalpana Ghoshal, Michael E. Paulaitis and Carl Schmidt
Lung cancer and liver cancer are two of the leading cause of cancer deaths worldwide. A patient-friendly early detection and surveillance method would substantially reduce the mortality in these serious diseases. ‘Liquid biopsy’ relying on detection of either circulating tumor cells (CTCs) or extracellular RNAs encapsulated in exosomes in patient blood samples has great potential to achieve this goal. But existing detection methods are far from perfection because CTCs are rare (~5-100 per 1 mL human blood) and exosomes are very small (<100 nm in diameter). Furthermore, none are able to simultaneously capture and detect both circulating exosomes and CTCs. We recently developed a simple and low-cost method ‘Tethered Cationic Lipoplex Nanoparticle (TCLN) biochip’ that, for the first time, may achieve this goal (a manuscript is under review in Nature Nanotechnology). In TCLN, molecular beacons (MBs) are pre-loaded in liposome nanoparticles to capture circulating exosomes and detect encapsulated extracellular RNAs from the patient blood or fluid sample without extra complicity. The in situ detection of target RNAs without diluting the sample leads to very high detection sensitivity not achievable by existing methods, e.g. qRT-PCR. The same biochip can also detect intracellular biomarkers in the captured CTCs by lipoplex internalization. Preliminary data in lung cancer cell line and patient blood samples using miR-21 and TTF-1 mRNA as biomarkers are very promising. TCLN can be extended to a multiplexing array design to allow the detection of many targets for RNA profiling. This new method may also be applied to other cancers and viral infection. In this proposal, we plan to evaluate its feasibility for early lung and liver cancer detection and surveillance. Our primary objectives are (1) to modify TCLN techniques so they can be used in flow cytometry and as a single-point detection method for clinical and point-of-care applications, (2) to compare target RNAs as biomarkers in tumor tissue and blood samples from MYC transgenic and miR-122 KO mouse models at different disease stages to evaluate the feasibility of using TCLN assay for early cancer detection, and (3) to identify target microRNA/mRNA biomarkers in lung and liver cancer and to conduct pilot studies using blood samples from well-defined lung and liver cancer patient groups. With data gathered from this project, we will submit two R01 or R33 proposals to NCI in two years and a P01 proposal in the future. This proposal is highly recommended by Nanoscale Science and Engineering Center (NSEC) at OSU. If funded, NSEC and the College of Engineering will match $50K/year for 2 years to allow the proposal team to pursue a broader scope of this challenging project.
STAT3 as a Mediator of Immune Suppression in the Pancreatic Cancer Stroma
Greg Lesinski and Michael Ostrowski
One important hallmark of pancreatic cancer (PCa) is the dense stroma consisting of activated fibroblasts termed ‘pancreatic stellate cells’ (PSC) that surround each tumor. This fibrotic material arises due to chronic inflammation in the pancreatic microenvironment. Although these stromal cells produce numerous factors supporting tumor growth, little is known regarding their interactions with immune cells within the tumor microenvironment. Our data shows that PSC secrete factors that promote differentiation of immune cells into myeloid derived suppressor cells (MDSC). We hypothesize that STAT3 signal transduction in the tumor-associated stroma is essential for promoting immune suppression in PCa. Our long-term goal is to develop a comprehensive research program aimed at understanding how the stroma influences local and systemic immune suppression in PCa. This knowledge will be instrumental for prioritizing the most relevant stromal targets that could be manipulated in the clinic to enhance the efficacy of immunotherapy for PCa. To test our hypothesis we will 1) develop mice with spontaneously arising, mutant KRAS-driven pancreatic cancer [Mist1(KRasG12D/+)] and STAT3-deleted stromal fibroblasts and monitor tumor incidence and immune biomarkers and 2) define the external stimuli responsible for STAT3 activation in PSC, and identify other downstream pathways that cooperate with STAT3 to promote PSC survival. These data will be essential for a future R01 application.
Novel Small Molecule Inhibitor of PHD3 Effects on Human Breast Cancer Metastasis and Migration on Nanoscale Variable Modulus Devices
Tim Eubank and John Lannutti
Better therapies for triple negative breast cancer patients are in critical need. Current options are surgery, taxane chemotherapies and radiotherapy. It is reported clinically that breast cancer patients whose tumors metastasize exhibit reduced desmoplakin (Dsp) expression. Dsp is the major protein component of the desmosome which regulates cell adhesion. Here, we introduce a novel small molecule inhibitor of PHD3, the prolyl hydroxylase that stabilizes HIF-2a, which we found augments Dsp mRNA and protein expression in PyMT and MDA-MB-231 tumor cells. Thus, we propose to study the ability of our novel PHD3 inhibitor to augment Dsp expression in human triple negative breast tumor cells implanted in SCID mice and reduce their migratory and metastatic ability. Key to this study will be the utilization of nanoscaled tools originating within the nationally known Nanoscale Science and Engineering Center (NSEC) at Ohio State. These tools will be used to quantify migration in response to chemotactic stimuli, differences in modulus representing age-based effects, gel-based barriers to cell migration to simulate invasion with and without embedded M2 macrophages, and DNA FRET-based ‘origami’ force sensors to potentially reveal local forces exerted by migrating tumor cells both in vitro and from tumor explants.
Insulin Receptor Splicing in Response to Hypoxia and Drug Resistance: A Pilot Study
Dawn Chandler and Peter Houghton
Alternative pre-messenger RNA (pre-mRNA) splicing has emerged as a predominant mechanism for proteomic diversity and gene expression. This diversity has been emphasized by recent work that elucidates a network of alternatively spliced genes in response to cell stimuli that are part of the tumorigenic process (DNA damage and Epithelial to Mesenchymal Transition, EMT, for example). Though the importance of alternative splicing in a number of cell processes has been recognized since its discovery, the mechanisms underlying its regulation remain poorly understood. Solid tumors are characterized by both transient and chronic hypoxia, and the ability for tumor cells to adapt to hypoxia is essential for tumor progression. We have shown recently that stimulation of vascular endothelial cell proliferation and angiogenesis induced by the major angiogenic factor, VEGF, is dependent upon insulin-like growth factor (IGF) signaling. Additionally, both human vascular endothelial cells and cells derived from pediatric sarcomas express predominantly the alternatively spliced form of the Insulin Receptor gene (IN-R), that has high affinity for IGF-2. Importantly, IN-R is spliced in response to hypoxia, and pediatric sarcoma cells secrete IGF-2. Consequently, IN-R splicing appears critical in progression of pediatric sarcomas both from acting as a receptor for autocrine growth of tumor cells, and for paracrine growth of vascular cells, and hence angiogenesis. The primary goal of this proposal is to understand the mechanisms and consequences of IN-R pre-mRNA splicing in response to hypoxia. This work will test the hypothesis that regulatory elements and splicing factors are modulated in response to hypoxia and are involved in the alternative splicing of IN-R to contribute to tumorigenesis. In order to determine the effectors of IN-R hypoxia-induced splicing, we have developed a novel in vitro splicing assay that models pre-mRNA splicing from cells that have undergone hypoxia. We will utilize the hypoxia-induced splicing assay to identify RNA sequences and their respective binding partners that are necessary for regulation of IN-R pre-mRNA splicing. Furthermore, we will use established mouse xenograft assays and novel antisense oligonucleotides (ASOs) to modulate IN-R splicing and determine the role of the spliced isoforms in tumor progression and as novel targets for therapeutic intervention.
PRMT5 Dysregulation as a Driver Event in Richter’s Transformation
Rosa Lapalombella and Robert Baiocchi
Richter's transformation (RS is a complication of B cell chronic lymphocytic leukemia (CLL) in which the leukemia changes into a rapidly proliferating diffuse large B cell lymphoma (DLBCL)1 with a poor prognosis despite the use of standard lymphoma therapy. The mechanisms underlying this transformation process remain to be clarified. PRMT5 is a type II methyltransferase that is highly expressed in lymphoma cell lines and primary tumors but not in normal resting or proliferating B cells. PRMT5 has been shown to induce transcriptional repression of cell cycle regulatory and tumor suppressor genes and is considered a driver event in both initiation and maintenance of cellular oncogenesis. Our preliminary data show that PRMT5 is variably expressed in CLL and overexpressed in patients with RS months prior to the morphologic transformation process where large B-cell lymphoma is noted. We hypothesize PRMT5 dysregulation to be both a driver event in CLL transformation to DLBCL and a potential therapeutic target for intervention. By using ChiP-Seq and RNA-Seq methods, we aim to provide insights of how PRMT5 dysregulation drive key cell growth and survival programs required for initiation and maintenance of the CLL and RS phenotype. This information will be vital to develop novel therapeutic approaches for RS, a currently incurable disease.
Gene Discovery Using a Drosophila Tumor Model
Amanda Simcox and Vicor Jin
The Ras signaling pathway is implicated in multiple cancers, correspondingly, there is an intense effort to identify Ras regulators and effectors. We have developed a conditional Ras-driven tumor cell model in Drosophila. When oncogenic RasV12 is expressed the cells proliferate, and when RasV12 is switched off the cells become dormant, but remain viable for weeks. Our goal is to use this model to discover new genes in the Ras pathway and to investigate tumor dormancy. RNAseq data comparing cells in the two states, RasON and RasOFF, has identified 363 conserved genes that are significantly induced by Ras expression and 300 conserved genes that are specifically expressed in dormant cells. Functional analysis of a subset of prioritized genes from each set will be conducted using RNAi. In addition to finding new genes in the Ras pathway, it is important to understand tumor cell dormancy because in this quiescent state the cells evade cancer therapies targeting proliferating cells. These dormant tumor cells, however, need to be killed too because later they can cause recurrence of the cancer.
The Role of the Epstein Barr Virus in NK-cell Lymphoma
Aharon G. Freud, Robert Baiocchi and Pierluigi Porcu
Extranodal NK-cell lymphoma, nasal type (ENKL) is an aggressive, uniformly EBV-positive extranodal non Hodgkin lymphoma (NHL) of natural killer (NK) cell origin that affects immune competent individuals in their 40-SOs. Even patients with disease localized to the nasopharynx typically experience early dissemination to other extranodal sites, despite aggressive chemo-radiation. The disease is rare in the US but is common in Latin America. While the mechanism of EBV-induced B-cell transformation in immunosuppressed patients has been well studied, very little is known about how EBV infects and transforms NK cells, what subsets of NK cells are targeted, what EBV genes are expressed in tumor cells, what are the EBV proteins recognized by the host T-cells, and how the latter affect outcome. Even less is known about the mechanisms of drug resistance that so greatly limit the efficacy of systemic chemotherapy in ENKL. Therefore, models to study viral pathogenesis, immune response, and drug therapy are needed. We hypothesize that EBV-positive NK lymphoma cells (EP-NK) circulate in the peripheral blood (PB) of ENKL patients and that these cells can be identified and purified to aid in the development of investigational models of ENKL. We also hypothesize that ENKL tumor and circulating lymphoma cells express EBV antigens that can be recognized by the host's T-cells. Our key objectives therefore are to develop models to study mechanisms of EBV-induced NK cell transformation in vitro and in vivo, and to characterize the host's immune response to ENKL for therapeutic purposes. Our group has a demonstrable collective expertise in the clinical and laboratory aspects of EBV-associated lymphomas, including ENKL, and in the biology of normal NK cells. The clinical resources to complete the proposed aims are available. Multiple vials of cryopreserved PB mononuclear cells (PBMC) from 8 OSU ENKL patients obtained at the time of detectable EBV viremia are available in the OSU LTB for specific aim 1 (purification of EP-NK cells) and specific aim 2 (study ofT-cell response). We have also partnered with investigators from the Institute Nacional de Enfermedades Neoplasicas (INEN, Peru') and with Dr. Natkunam (Stanford), who is internationally known for her work in ENKL and has an ongoing research partnership with colleagues in Guatemala. INEN will provide tissue blocks or slides and Dr. Natkunam will provide a tissue microarray (TMA) with >100 cases of ENKL for specific aim 2 (study of EBV gene expression in ENKL tissue). Genomic DNA from these samples will also be obtained for later experiments, which include next generation sequencing (NSG) of viral and tumor cell DNA and RNA and methylation profiling.