RAGE/Diaph1, Diabetes, and Kidney Disease: Mechanisms and Novel Therapeutic Strategies

In Year Three of the funded grant, we have substantial progress in the following critical areas: 1). As noted in the project narrative, we generated four different lines of mice to directly test the hypothesis that RAGE and DIAPH1 contribute to the pathogenesis of diabetes-associated nephropathy in the podocytes and/or in myeloid cells/macrophages. All of the mouse lines are now generated and largely completed (mice sacrificed) and samples being evaluation by Dr DAgati. There are no new pending mice to generate all are generated and on time course. 2). We have determined that the small molecule RAGE/DIAPH1 antagonist is best administered orally and that the RAGE antagonist survives the medicated chow pelleting, heating and irradiation. Our first data on treated vs. untreated male and female diabetic mice illustrates reduction in mesangial sclerosis, reduced thickening of the glomerular basement membrane and reduction in podocyte effacement in diabetic mice receiving RAGE229 medicated chow (vs vehicle). Additional mice are on study and time course at this time to complete the indicated enrollment.3). For transcriptomics and metabolomics/lipidomics assay, Dr. Ramasamy will be testing the macrophages from the mice through the time course and he has verified all of his experimental systems for the performance of the outlined studies. Dr. Ramasamy identifies substantial progress in the development and validation of metabolomics and lipidomics assays here at NYU and in transcriptomic data (all on macrophages) in order to understand detailed mechanisms of the role of these molecules in the diabetic kidney. Taken together, despite the >3 month shutdown due to COVID19 our work in Year 3 has been productive and we await tissue and other analyses, as above, to render final conclusions.

Exploit Dimethyl Fumarate to Uncover Druggable Vulnerabilities and Prevent Recurrence of ER+ Breast Cancers

The PI, Irida Kastrati, PhD, transferred from the University of Illinois at Chicago to a new institution, Loyola University Chicago (LUC).At LUC, the PI obtained a new IACUC approval and completed an HRPO review. An MTA was submitted and is pending approval for transferring PDXs from our collaborator Dr. Carol Sartorius at U. Colorado to our institution. Initiation of in vivo animal work is currently halted due to COVID-19 measures in Illinois.

TYK2 as a Biomarker and Therapeutic Target for NF1-Associated Malignant Peripheral Nerve Sheath Tumors

Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas that account for approximately 5% of all soft tissue sarcomas. These tumors occur at an increased frequency in patients with the Neurofibromatosis Type 1 (NF1) cancer predisposition syndrome, but also occur sporadically or as a secondary complication of radiation therapy. In the setting of NF1, MPNST arise from malignant transformation of a benign precursor lesion, a plexiform neurofibroma. Initial treatment for MPNST typically involves surgery and radiation with or without chemotherapy. However, despite aggressive therapy, the recurrence rate is high and the vast majority of people with these cancers will die within 5 years of diagnosis. Treatment for metastatic disease is limited to cytotoxic chemotherapy and clinical trials. As such, there is a pressing need to identify novel therapeutic targets. Prior work from our laboratory identified TYK2 as a gene mutated in a subset of MPNSTs. More recently, we have shown that genetic knockdown of TYK2 in both human and murine MPNST cell lines results in decreased tumor growth and increased cell death in vitro. Additionally, genetic knockdown of Tyk2 in murine MPNST cells resulted in decreased tumor burden in subcutaneous tumors and metastatic tumor models. Immunohistochemistry (IHC) for TYK2 was performed on 27 MPNST and 16 plexiformneurofibromas to evaluate TYK2 association with tumor type, overall survival, metastasis and therapeutic response. Additionally, similar to genetic knockdown, pharmacologic inhibition of TYK2 dose-dependently decreased the percent cell confluence and induced apoptosis over time in four MPNST cell-lines, as assessed by IncuCyte proliferation and apoptosis assays. In murine MPNST JW23.3 cells, incubation with TYK2 inhibitors reduced pSTAT3 levels, but not pERK or pS6K.

Targeting Fibroblast Growth Factor Receptors in Preclinical Patient-Derived Xenograft Models of CRPC

In this reporting period, the didactic coursework and training opportunities that were proposed for this project were completed. However, significant disruptions to research activities were encountered due to the ongoing COVID-19 pandemic response and ACURO approval delays. These factors led to the application and approval of a no cost extension to complete all proposed work. Nevertheless, all study approvals (IACUC, ACURO, HRPO) have been obtained and experiments using fibroblast growth factor receptor and androgen receptor inhibitors inpatient derived xenograft models are underway. The approved project objectives and scope will not change and the work to be completed in the extension period will include all experiments and analyses related to Specific Aim 1.

AXL-Targeting Antibody-Drug Conjugate as Novel Therapy for Triple-Negative Breast Cancer

We have identified AXL, a receptor protein tyrosine kinase (RTK), being highly expressed and activated (phosphorylated) in Triple Negative Breast Cancer. We have determined that AXL provides survival benefit to the tumor cells. We have also discovered a monoclonal antibody that is highly specific to AXL, and does not bind to other related receptor tyrosine kinases. We have also shown that the antibody internalizes and degrades the AXL receptor. We have humanized the antibody for clinical development. We have established high producer cell line and propagated in chemically defined medium. We thus propose to conduct the following studies using novel target and novel therapeutic. Specific aims are: Aim 1. To develop a humanized antibody-drug conjugate (ADC) that can effectively target the AXL membrane receptortyrosine kinase. Aim 2. To test the efficacy of the AXL-targeted ADC in preclinical animal models Aim 3. To develop a mass spectrometry-based method to effectively monitor AXL expression and activation in xenograft tissues and clinical samples.

Dysregulation of the PACT-mediated Crosstalk Between Protein Kinases PKR and PERK Contributes to Dystonia 16 (DYT16)

Currently, the available treatment options for dystonia are merely palliative and the drug development has not progressed significantly due to a lack of understanding about the involved molecular pathomechanisms. We investigated if PACT, the gene mutated in dystonia 16 (DYT16), causes a disruption in the normal regulatory crosstalk between PERK and PKR kinases leading to a loss of cell homeostasis after ER stress. Both PERK and PKR kinases phosphorylate eIF2 alpha and activate a downstream signaling pathway that allows recovery and survival after ER stress. The most significant finding during the last funding period was that PACT is a substrate of PERK kinase and PERK phosphorylates PACT in vivo after ER stress. This is a paradigm-shifting finding as it was previously unknown that PACT could participate and regulate both PKR and PERK pathways. The molecular etiology of DYT16 has now been conclusively shown to be a dysregulation of eIF2 alpha signaling. Thus, our research has uncovered a PACT-mediated novel regulatory pathway and laid the foundation for more in depth drug development to target PACT-PERKinteractions in future. In addition, it has added significant new knowledge about how cells respond to ER stress. In the brief period of 8 months we made significant progress included in this report, before the COVID-19 pandemic slowed down our work significantly.

TP53 Synthetic Lethal Screen in Organoid Avatars to Discover Novel Therapeutic Targets for Colon Cancer

This application addresses the FY18 PRCRP topic area of Colorectal Cancer. Military relevant risk factors such as ionizing radiation often produce cancers with mutations in TP53. This proposal seeks to address a gap in colorectal cancer treatment that has profound impact on the health and well-being of military service Veterans, their beneficiaries, and extends to the American public as a whole. TP53 mutations are common in colon cancers (50 ), and most often lead to resistance to chemotherapy. A systematic screen for synthetic lethal interactions between gene knockout, TP53 mutation, and chemotherapy such as Cisplatin will produce novel targets useful for treating a large fraction of colon cancers.Hypothesis: Three-way synthetic

Understanding the Role of Gene-Environment Interactions in the Degeneration of Human Dopaminergic Neurons in Parkinson's Disease

Gene environment interactions (GxE) are key to better understanding PD. We are testing pesticides for their effects "in the dish", and in the context of mutations in the synuclein and GBA genes that are also being analyzed in Dr Ritz's epidemiologic cohort. Our experiments will enable us to answer whether key genetic risk factors create sensitivities in patients to particular toxicants and we will ascertain whether gene-toxicant interactions play out specifically at the level of the dopamine neuron. To date, we have generated a unique set of reagents from patients with PD caused by synuclein and GBA mutations and observed differential effects to PD-linked pesticides and toxicants with respect to survival, neurite outgrowth and calcium signaling. From the PEG cohort data and leveraging agricultural pesticide application records (CA-PUR database (discussed below), 1974-2018), we have established long-term exposure profiles for over 200 widely used agricultural pesticides for 1,870 PD patients and population-based controls. Using this data for analysis, we have generated a list of 33 pesticide toxicants that are both significantly associated with PD (FDR less than 0.01) and have exposure in both alpha-syn SNPs and GBA variant carriers. This epidemiologic analysis agnostically highlighted, out of pesticides widely used agriculturally in California over the study period, the most significantly associated with PD. In synergy, the lab-based teams (Rubin and Khurana) will in the next phase of research test these epidemiologically derived pesticides in in vitro cell lines in the context of mutations in the synuclein and GBA mechanisms. Validated hits from these screens will then be used in conjunction with SNCA and GBA genetic data to assess GxE interactions with pathway relevant SNPs in the PEG study.

Characterization of a Novel Humanized Mouse Model of Neurofibromatosis

The purpose of this study was to characterize a mutant mouse that bears an NF1 mutation that is identical to one found in humans. No human samples exist to determine the nature of how this particular mutation affects the NF1 gene and protein. This mutation is particularly important to understand as it does not lead to the neurofibroma formation found in most patients, but it can lead to learning challenges, developmental delay and in some cases epilepsy. This study was able to determine that this particular mutant NF1 protein neither affects the most common pathways associated with the function of the NF1 protein nor leads to some inflammatory aspects of the disease. These are critical findings as they reveal that the protein has additional functions that are linked to a very specific region of the protein and that this mutation may be acting in very distinct ways from the effects of complete loss of the NF1 protein. This study also found a link between this mutation and programmed cell death in a region of the brain critical for learning and memory. This information will allow new studies to more clearly define the molecular basis for the neurological aspects of Neurofibromatosis with this new understanding bringing the potential for new therapeutic targets.

Macrophage Migration Inhibitor (MIF) Therapeutics for Neuroprotection and Prevention of Scar in Traumatic Retinal Detachment

Retinal detachment (RD) is a prevalent cause of blindness that is common after ocular injury to military personnel. Permanent vision loss occurs due to death of photoreceptors and formation of excessive scar tissue, known as proliferative vitreoretinopathy (PVR). There are no effective pharmaceuticals to prevent these problems. The inflammatory protein, macrophage migration inhibitory factor (MIF), is produced at high levels in RD and PVR, as well as in excitotoxic (NMDA-mediated) damage, which is important in blast injury. We tested the ability of different clinically-relevant MIF inhibitors to block photoreceptor death after NMDA damage in a chick excitotoxic retinal damage model. These inhibitors, ibudilast, AV1013, and CPSI-1306, are well tolerated in the eye, and treatment with the maximum dose of each drug does not show retinal toxicity. Ibudilast pretreatment significantly reduced the number of TUNEL positive cells in the retina after NMDA damage. CPSI-1306 also reduced TUNEL. AV1013 had no effect. Ibudilast and AV1013 also blocked epithelial mesenchymal transition in the invitro PVR model.

Targeting the Microbiome to Enable Immunotherapeutic Efficacy in Pancreatic Carcinoma

Our goal is to perform clinical trials in PDA patients testing strategies to modulate the microbiome to enhance the efficacy for immunotherapy. However, the optimal regimen has not been defined. We will perform experiments in mouse PDA models and in human organotypic systems which are designed to define the most efficacious microbiome modulatory regimens - either antibiotics or probiotics - to combine with immunotherapy. In Aim 1 we will test the immune-activating and tumor-protective effects of specific antibiotic and probiotic regimens in mouse models of PDA and an innovative microfluidic-based organotypic model derived from freshly resected human PDA. In Aim 2 we will determine the regimen that most effectively synergizes with immunotherapy. Aim 3 will encompass the first clinical trial in PDA targeting the microbiome as a strategy to enable immunotherapeutic efficacy. Collectively, these Aims will lead to a new treatment paradigm for PDA patients that targets the microbiome.

Understanding the Role of Gene-Environment Interactions in the Degeneration of Human Dopaminergic Neurons in Parkinson's Disease

Gene environment interactions (GxE) are key to better understanding PD. We are testing pesticides for their effects in the dish, and in the context of mutations in the synuclein and GBA genes that are also being analyzed in Dr Ritzs epidemiologic cohort. Our experiments will enable us to answer whether key genetic risk factors create sensitivities in patients to particular toxicants and we will ascertain whether gene-toxicant interactions play out specifically at the level of the dopamine neuron. To date, we have generated a unique set of reagents from patients with PD caused by synuclein and GBA mutations and observed differential effects to PD-linked pesticides and toxicants with respect to survival, neurite outgrowth and calcium signaling. From the PEG cohort data and leveraging agricultural pesticide application records (CA-PUR database (discussed below), 1974-2018), we have established long-term exposure profiles for over 200 widely used agricultural pesticides for 1,870 PD patients and population-based controls. Using this data for analysis, we have generated a list of 33 pesticide toxicants that are both significantly associated with PD (FDR less than 0.01) and have exposure in both alpha-syn SNPs and GBA variant carriers. This epidemiologic analysis agnostically highlighted, out of pesticides widely used agriculturally in California over the study period, the most significantly associated with PD. In synergy, the lab-based teams (Rubin and Khurana) will in the next phase of research test these epidemiologically derived pesticides in in vitro cell lines in the context of mutations in the synuclein and GBA mechanisms. Validated hits from these screens will then be used in conjunction with SNCA and GBA genetic data to assess GxE interactions with pathway relevant SNPs in the PEG study.

Targeting ART1, a Novel Immune Checkpoint, for the Treatment of Lung Cancer

We have found ART1 expressed in multiple human non-small cell lung cancer (NSCLC) cell lines and in mouse and human NSCLC tumors. ART1, an ARTC family mono-ADP-ribosyltransferase, functions extracellularly to target arginine-rich cell surface proteins on neighboring cells or target soluble proteins in the local tumor microenvironment. Following ART1 knockdown in murine immune competent lung cancer models, we note a highly significant increase in cytotoxic tumor infiltrating CD8 T cells and a subsequent decrease in tumor burden. Mono-ADP-ribosylation of the P2X7 receptor on subsets of CD8 T cells may induce receptor activation and apoptosis, a process described as NAD-induced cell death (NICD). ART1-induced mono-ADP-ribosylation of P2X7R could therefore allow cancer cells overexpressing ART1 to blunt the T cell immune response against them by inducing T cell apoptosis. Although little is known about ART1 in human cancer, its expression may be upregulated by ER stress and its enzymatic activity increased by release of NAD into the local microenvironment, both of which occur following cytotoxic cancer therapy, particularly radiation therapy. In the current application addressing the LCRP Area of Emphasis Identify innovative strategies for prevention and treatment of lung cancer, we therefore propose to evaluate the therapeutic efficacy of anti-ART1 antibodies which we have already developed and functionally optimized and to determine whether ART1 blockade has additive anti-tumor effects to immune checkpoint blockade and radiation therapy. We hypothesize that therapeutic inhibition of ART1 with an anti-ART1 monoclonal antibody will promote immune mediated rejection of lung cancer. Moreover, we hypothesize that ART1 inhibition combined with radiation therapy will demonstrate synergistic anti-tumor effects.

Biodegradable Cationic Nanoparticles as a Push Chemodrug Carrier and a Pull cfDNA Scavenger Against Breast Cancer Metastasis

The goal of this project is to develop a novel therapeutic approach for the prevention of breast cancer metastasis, by using nucleic acid binding nanoparticles (NABNPs) designed to deliver high payloads of cytotoxic drugs to tumor tissues, while simultaneously scavenging the pro-inflammatory cell-free DNA (cfDNA) that is released in the blood circulation as a result oftumor progression and/or destruction by chemotherapy. This is a collaborative research project conducted in partnership by two teams, supported by two distinct awards (BC180904, BC180904P1). Among the key aims of the research supported by this award (BC180904P1, partnering team) is the development of experimental models that enable the visualization and quantification of breast cancer dissemination in live animals, using non-invasive imaging techniques that can detect biox2;luminescent signals from live cancer cells, and compare them over time, in order to obtain longitudinal measurements of both the anatomical location and overall burden of metastatic tissue. During the first year of the award, we engineered multiple triple negative breast cancer (TNBC) cell lines with different lentivirus constructs, each encoding for both fluorescent (EGFP, ZsGreen) and bioluminescent reporters (different variants of Luciferase from the firefly Photinus pyralis). We then compared the performance of the various lentivirus constructs in terms of sensitivity and stability of expression over time, in both mouse and human TNBC cells, in vitro and in vivo. Our experiments led to the generation of 3 independent TNBC cell lines (1 murine, 2 human) expressing high levels of both ZsGreen and Luciferase (Photinus pyralis), and can be efficiently monitored in terms of longitudinal growth and spontaneous metastatic dissemination in live animals.

Understanding the Role of Gene-Environment Interactions in the Degeneration of Human Dopaminergic Neurons in Parkinson's Disease

Gene environment interactions (GxE) are key to better understanding PD. We are testing pesticides for their effects in the dish, and in the context of mutations in the synuclein and GBA genes that are also being analyzed in Dr Ritzs epidemiologic cohort. Our experiments will enable us to answer whether key genetic risk factors create sensitivities in patients to particular toxicants and we will ascertain whether gene-toxicant interactions play out specifically at the level of the dopamine neuron. To date, we have generated a unique set of reagents from patients with PD caused by synuclein and GBA mutations and observed differential effects to PD-linked pesticides and toxicants with respect to survival, neurite outgrowth and calcium signaling. From the PEG cohort data and leveraging agricultural pesticide application records (CAPUR database (discussed below), 1974-2018), we have established long-term exposure profiles for over 200 widely used agricultural pesticides for 1,870 PD patients and population-based controls. Using this data for analysis, we have generated a list of 33 pesticide toxicants that are both significantly associated with PD (FDR<0.01) and have exposure in both alpha-syn SNPs and GBA variant carriers. This epidemiologic analysis agnostically highlighted, out of pesticides widely used agriculturally in California over the study period, the most significantly associated with PD. In synergy, the lab-based teams (Rubin and Khurana) will in the next phase of research test these epidemiologically derived pesticides in in vitro cell lines in the context of mutations in the synuclein and GBA mechanisms. Validated hits from these screens will then be used in conjunction with SNCA and GBA genetic data to assess GxE interactions with pathway relevant SNPs in the PEG study.

MYCN Reprograms Neuroblastoma Metabolism

Despite current aggressive regimens, the majority of patients with MYCN amplification die due to drug-resistant disease, and further intensification ofchemotherapy will not significantly improve this outcome. We propose an entirely novel strategy to oppose MYCN oncogenic function in NB: by blockingthe metabolic reprogramming driven by MYCN. Based on our data and the recent literature, our guiding hypotheses are that: a) lipid metabolism is requiredfor NB tumorigenesis, and b) targeting MYCN-driven lipogenesis will effectively block NB tumor growth. We have demonstrated that lipid metabolism is aselective metabolic dependency of MYCN-driven tumors. MYCN drives both fatty acid (FA) synthesis and FA uptake to maintain NB cell survival. TargetingFA uptake effectively blocks NB in vivo tumor growth.