Summary Leptin acts on leptin receptor (LepRb)-expressing neurons throughout the brain, but the roles for many populations of LepRb neurons in modulating energy balance and behavior remain unclear. We found that the majority of LepRb neurons in the lateral hypothalamic area (LHA) contain neurotensin (Nts). To investigate the physiologic role for leptin action via these LepRbNts neurons, we generated mice null for LepRb specifically in Nts neurons (Nts-LepRbKO mice). Nts-LepRbKO mice demonstrate early-onset obesity, modestly increased feeding, and decreased locomotor activity. Furthermore, consistent with the connection of LepRbNts neurons with local OX neurons and the ventral tegmental area (VTA), Nts-LepRbKO mice exhibit altered regulation of OX neurons and the mesolimbic DA system. Thus, LHA LepRbNts neurons mediate physiologic leptin action on OX neurons and the mesolimbic DA system, and contribute importantly to the control of energy balance.

Diabetes and insulin resistance are associated with altered brain imaging, depression, and increased rates of age-related cognitive impairment. Here we demonstrate that mice with a brain-specific knockout of the insulin receptor (NIRKO mice) exhibit brain mitochondrial dysfunction with reduced mitochondrial oxidative activity, increased levels of reactive oxygen species, and increased levels of lipid and protein oxidation in the striatum and nucleus accumbens. NIRKO mice also exhibit increased levels of monoamine oxidase A and B (MAO A and B) leading to increased dopamine turnover in these areas. Studies in cultured neurons and glia cells indicate that these changes in MAO A and B are a direct consequence of loss of insulin signaling. As a result, NIRKO mice develop age-related anxiety and depressive-like behaviors that can be reversed by treatment with MAO inhibitors, as well as the tricyclic antidepressant imipramine, which inhibits MAO activity and reduces oxidative stress. Thus, insulin resistance in brain induces mitochondrial and dopaminergic dysfunction leading to anxiety and depressive-like behaviors, demonstrating a potential molecular link between central insulin resistance and behavioral disorders. A s life expectancy in humans has increased, we are faced with a worldwide epidemic of age-related diseases such as type 2 diabetes (T2D) and Alzheimer's disease (1). These parallel epidemics may not be coincidental. Indeed, studies have demonstrated an association between diabetes and a variety of brain alterations including depression, age-related cognitive decline, Alzheimer's disease, and Parkinson's disease (2, 3). In addition, individuals with both type 1 and type 2 diabetes have been shown to have a variety of abnormalities in brain imaging, including altered brain activity and connectivity by functional MRI (4, 5), altered microstructure by diffusion tensor imaging (6, 7), and altered neuronal circuitry in the striatum (8). Conversely, patients with Alzheimer's disease show signs of central insulin resistance with increased insulin receptor substrate (IRS) 1 serine phosphorylation in the brain and decreased insulin concentrations in the cerebrospinal fluid (9, 10). Furthermore, pilot clinical trials of intranasal insulin administered to individuals with Alzheimer's disease suggest decreased rates of cognitive decline (11).These observations in humans have been mechanistically supported by studies in rodents and cultured cells, which have shown that insulin receptor signaling in brain has an important role in central regulation of metabolism and may also be crucial for proper brain function (12)(13)(14). We have previously demonstrated that mice with insulin resistance in brain due to targeted deletion of the insulin receptor (NIRKO mice) develop hyperphagia, mild obesity, reduced fertility, and decreased counterregulatory response to hypoglycemia (15, 16). NIRKO mice also display glycogen synthase kinase 3 beta (GSK3-beta) activation, resulting in hyperphosphorylation of tau protein, a hallmark of e...

Introduction We explored whether integrin αvβ8 inhibition potentiates immune checkpoint blockade (ICB) in syngeneic orthotopic models of breast cancer. Integrin αvβ8 mediates cell type specific and tissue localized activation of TGFβ1/3 to regulate the immune system. For example, αvβ8 expressed on dendritic cells (DC) in the intestine has been shown to be a key mediator of tolerance, maintaining gut immunologic homeostasis. Methods Efficacy was evaluated in combination with anti-PD-1 in EMT6 and PyMT breast cancer syngeneic mouse models. A potent αvβ8 inhibitor was orally administered at 60mg/kg BID for 21 days. Anti-αvβ8 or non-isoform specific anti-TGFβ mAbs were dosed TIWx3 at 7 and 10mg/kg, respectively. Tumor volumes are presented as mean±SEM. Statistics were performed by t test, one-way ANOVA, or log-rank test. Flow cytometry and transcriptome analysis on bulk and single-cell levels were used to assess the mechanism of action in EMT6. Results A similar αvβ8 expression pattern on DC, macrophages and regulatory T cells (Treg) was observed in mouse models and human tumors. Combination of oral αvβ8 inhibitor with anti-PD-1 was efficacious in the primary ICB resistant EMT6 model and resulted in superior tumor regression during treatment (p=0.0003) and improved survival with 5/12 complete responders relative to 0/12 in anti-PD-1 alone. Across studies the αvβ8 inhibitor phenocopied the results obtained with αvβ8 and TGFβ mAbs (n=3 independent studies). Complete responders re-challenged 89 days after treatment with EMT6 or 4T1 tumors showed no EMT6 tumor growth, suggesting the combination induced long-term immunologic memory. Analysis of tumors by flow cytometry showed combination resulted in increased CD8 T cell infiltrates (p=0.0006), T cell activation (CD8+CD69+, p=0.0194) and IFN-γ expression (CD8+IFN-γ+, p=0.0021). Single cell transcriptomic analysis of lymph nodes showed that αvβ8 inhibition potentiated DC co-stimulation (Cd40, Cd83/6) and migration (Ccr7, Cxcl16, Ccl22). Moreover, combination treatment led to tumor infiltrated Treg dysfunction including downregulation of Ctla4, Il10 and Tigit, and upregulation of Ifn-γ. The observation of these anti-tolerance and pro-inflammatory signatures in DC and Treg has not been described previously. Anti-tumor efficacy was driven by immune-mediated mechanisms as confirmed by a CD8 depletion study. Efficacy was confirmed in PyMT breast cancer model. Conclusions An αvβ8 inhibitor in combination with anti-PD-1 showed efficacy in syngeneic mouse models, supported by increased T cell infiltrates and evidence of reduced tumor tolerance. These results show that an orally administered αvβ8 targeted inhibitor is a potent modulator of anti-tumor immune response acting across the immunologic synapse, and is a promising therapeutic approach to ICB refractory tumors. Citation Format: Natalia J. Reszka-Blanco, Vinod Yadav, Megan Krumpoch, Laura Cappellucci, Dan Cui, James E. Dowling, Elizabeth Gwara, Bryce Harrison, Dooyoung Lee, Fu-Yang Lin, Lia Luus, Meghan Monroy, Terence I. Moy, Eugene Nebelitsky, Qi Qiao, Andrew Sullivan, Dawn Troast, Blaise Lippa, Bruce Rogers, Adrian S. Ray. Inhibition of integrin αvβ8 enhances immune checkpoint induced anti-tumor immunity by acting across immunologic synapse in syngeneic models of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1559.

Background Integrins play a key role in facilitating immune cell trafficking throughout the body and represent an important receptor family for therapeutic intervention. In particular, the α4β7 integrin is a clinically validated target for the treatment of inflammatory bowel diseases (IBD), as exemplified by the humanised monoclonal antibody vedolizumab, which blocks the interactions between α4β7-expressing lymphocytes and its ligand MAdCAM-1. This blockade leads to the inhibition of these circulating lymphocytes from exiting the bloodstream and entering intestinal mucosal tissues resulting in a decrease in mucosal inflammation in patients. While oral inhibitors of the α4β7 integrin are advantageous over biologics, the efforts have been impeded by challenges to achieve desired selectivity and optimal DMPK properties. The aim of this study was to develop and characterise orally bioavailable small-molecule inhibitors of the α4β7 integrin and to determine their therapeutic potential. Methods Oral small-molecule inhibitors targeting the α4β7 integrin were discovered using Morphic Integrin Technology (MInT) platform. These small-molecule inhibitors were tested for potency and selectivity against a broad panel of integrin family members in multiple biochemical and cell-based functional assays in a ligand-competitive fashion. An acute PD assay with CFSE-labelled lymphocytes was developed to evaluate the activity of the small-molecule compounds in blocking lymphocyte trafficking to gut-associated lymphoid tissues in mice. The in vivo activity was also examined through changes in circulating α4β7+ CD4+ T memory cells in a relevant non-human primate model. Results Key drug candidate small molecules demonstrated over 1000-fold selectivity in vitro against a broad panel of integrin family members, including the α4β1 integrin. These compounds effectively blocked lymphocyte trafficking to mesenteric lymph nodes and Peyer’s patches in the gut in a dose-dependent manner, similar to an α4β7-specific antibody, in an acute gut-homing assay in mice. Additionally, these inhibitors also demonstrated effective occlusion of immune trafficking in a relevant non-human primate model. The lead compound has favourable DMPK properties, good oral bioavailability and is projected to have sufficient exposure in humans to effectively block α4β7-expressing immune cells in circulation. Conclusion Potent, selective, oral small-molecule inhibitors of α4β7 integrin have been discovered that demonstrate on-target, mechanistic efficacy in two animal models relevant to human IBD. It has the potential to be an effective and safe therapeutic in monotherapy as well as serving as a backbone for combination with other IBD drugs.

Objective: MORF-057 is an orally bioavailable, selective, and potent small molecule inhibitor of α4β7 integrin being developed for inflammatory bowel diseases (IBD) that is currently in phase 1 clinical testing. We have previously presented work that characterized its nonclinical pharmacologic profile. The current study integrates data to generate a pharmacokinetic (PK) and pharmacodynamic (PD) model of MORF-057. Methods: To determine in vivo potency, MORF-057 was tested in murine gut homing assays and the PD response was determined relative to the non-protein bound drug in mouse plasma. A cell adhesion assay (CAA) for α4β7 was refined to enable detection of picomolar-level sensitivity. Murine receptor occupancy (RO) assays for α4β7 and α4β1 were established under physiologic conditions, and MORF-057 was evaluated for its potency and selectivity in fresh mouse whole blood. These datasets were used to build and validate predictive models of PD response. Results: MORF-057 strongly inhibited the homing of α4β7hi cells to murine gut lymphoid tissues with an IC90 of 7.9 nM. MORF-057 showed high potency in CAA with an IC90 of 8.8 nM. Similarly, RO assays confirmed MORF-057 to be a highly potent inhibitor of α4β7 in mouse whole blood with an IC90 of 20.5 nM and over 1500-fold selectivity vs. α4β1. The predictive models built upon these datasets revealed a strong PK-PD relationship of α4β7 inhibitors in vivo. Conclusions: We observed consistently high potency of MORF-057 across multiple assay platforms. Integrated modeling based on these assays, particularly the RO assay, successfully predicted the PD response to MORF-057. These data begin to establish the relationship between PK, target engagement, and PD with MORF-057.