Relevant Murine Model Research Articles

Abstract P796: Integrated Analysis of Gait Parameters and Gene Expression Profiles in a Murine Model of Subarachnoid Hemorrhage

Objective: Gait variability analysis has been adopted in clinical settings to characterize the presentation of various neurological diseases. However, literature and practice lack a comprehensive murine model assessment of the gait deficits that result from subarachnoid hemorrhage (SAH). Further, correlations between gait parameters and the gene expression profiles associated with SAH have yet to be identified. The present study quantitatively assesses gait deficits through a clinically relevant murine model of SAH to determine associations between gait deficits and SAH-related gene expressions. Methods: A total of 159 dynamic and static gait parameters from the endovascular perforation murine model for simulating clinical human SAH were measured using the CatWalk system. Pearson’s correlation analysis was applied, and 88 genes associated with SAH were identified from the Ingenuity Pathway Analysis database to aid the investigation of the relationship between gait variability and gene expression profiles. Results: Eighty gait parameters and the mRNA expression levels of 35 of the 88 SAH-associated genes exhibited significant change in the SAH models (p < 0.05). Totals of 42 and 38 gait parameters correlated with the 35 SAH-associated genes positively and negatively with Pearson’s correlation coefficients of > 0.7 and < -0.7, respectively. p-SP1 453 expression in the motor cortex in SAH animal models displays a significant correlation with a subset of gait parameters associated with muscular strength and coordination of limb movements. Conclusion: This examination of gait variability and its strong correlation to gene expression profiles provides a quantitative and reliable assessment of the SAH model’s motor performance. This research provides valuable insights into the study of disease progression and offers novel therapeutic interventions in the murine modeling of SAH. p-SP1 453 expression could act as a biomarker to monitor SAH pathological development and a therapeutic target for SAH.

Antibiofilm Peptides: Relevant Preclinical Animal Infection Models and Translational Potential.

Biofilm-forming bacteria may be 10-1000 times more resistant to antibiotics than planktonic bacteria and represent about 75% of bacterial infections in humans. Antibiofilm treatments are scarce, and no effective therapies have been reported so far. In this context, antibiofilm peptides (ABPs) represent an exciting class of agents with potent activity against biofilms both in vitro and in vivo. Moreover, murine models of bacterial biofilm infections have been used to evaluate the in vivo effectiveness of ABPs. Therefore, here we highlight the translational potential of ABPs and provide an overview of the different clinically relevant murine models to assess ABP efficacy, including wound, foreign body, chronic lung, and oral models of infection. We discuss key challenges to translate ABPs to the clinic and the pros and cons of the existing murine biofilm models for reliable assessment of the efficacy of ABPs.

A Twin Disease Mouse Model to Assess Atherosclerosis Progression Following Lung Infection in ApoE Deficient Mice

Inflammation is a risk factor for atherosclerosis progression and plaque instability. Previous studies have demonstrated that hospitalisation for pneumonia is associated with increased short- and long-term risk of cardiovascular disease. Herein, we describe a clinically relevant twin disease murine model to study the impact of inflammation from acute bacterial pneumonia on atherosclerosis.

Protective and healing effects of apoptotic mimic-induced M2-like macrophage polarization on pressure ulcers in young and middle-aged mice

Pressure ulcers (PUs) have no cure and are of significant health and economic concern worldwide, owing to the increasing population of elderly individuals at high risk for PU and who have impaired tissue repair. Macrophages play a pivotal role in PU development and healing. Imbalances between M1 (inflammatory) and M2 (anti-inflammatory/reparative) macrophages result in delayed resolution of inflammation and wound healing. We hypothesized that M1-to-M2 macrophage polarization mediated by artificial apoptotic cell mimics, phosphatidylserine-containing liposomes (PSLs), would protect against PU formation and accelerate PU healing in young (2-month-old) and middle-aged (12-month-old) mice. We used a clinically relevant murine model of ischemia-reperfusion-induced PU. Middle-aged mice displayed the delayed wound healing associated with increased inflammation, decreased collagen deposition, reduced angiogenesis, and delayed wound closure relative to their younger counterparts. PSL treatment significantly inhibited PU formation and promoted tissue remodeling in both age groups. These effects were mediated by increased M1-to-M2 macrophage polarization, induced by the PSLs. Thus, this study suggests, for the first time, that PSL-induced M2-like macrophage polarization is a promising strategy to protect against PU formation and promote PU repair in human patients of all ages.

Development of new treatments with drug repositioning strategies: POEMS and Guillain‐Barré syndrome

AbstractPOEMS syndrome and Guillain‐Barré syndrome (GBS) are rare and potentially intractable peripheral neuropathies. This review considers the development of new therapeutic interventions for these disorders using drug repositioning strategies. The prognosis for patients diagnosed with POEMS syndrome was previously quite poor; however, therapeutic interventions based on those used successfully for the treatment of myeloma have significantly improved the prognosis of this disease. While many new drugs have been developed to treat myeloma, thalidomide is the only drug with a safety and efficacy profile that has been documented by the results of a randomized controlled trial. While POEMS syndrome is an extraordinarily rare disease, prospective controlled trials should be performed to establish evidence‐based therapeutic strategies. Similarly, GBS is an acute, immune‐mediated polyneuropathy. While many patients achieve full recovery, others go on to experience long‐lasting neurological sequelae; this latter group includes patients who were treated appropriately with immunoglobulins or plasmapheresis. Complement activation has been associated with severe nerve injury in patients diagnosed with GBS; indeed, inhibition of complement activation prevented disease‐associated paralysis and nerve injury in a relevant murine model. Eculizumab, an antibody that targets complement protein C5, is a potent inhibitor of complement activation that has been approved for the treatment of complement‐mediated disorders. A recent phase 2 clinical trial of eculizumab for GBS performed in Japan revealed that eculizumab may be effective for the treatment of patients with this disease. Further confirmation of this finding will be a major step forward with respect to the management of GBS.

Lentiviral Mediated Gene Therapy for Pyruvate Kinase Deficiency: A Global Phase 1 Study for Adult and Pediatric Patients

Introduction: Pyruvate Kinase Deficiency (PKD) is a rare inherited hemolytic anemia that is caused by mutations in the PKLR gene leading to decreased red cell pyruvate kinase (RPK) activity and impaired erythrocyte metabolism. The disorder is characterized by anemia, reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected individuals. PKD represents a significant unmet medical need as current therapies are palliative and limited to chronic blood transfusions, iron chelation therapy, and splenectomy. The side effects of these supportive treatments include iron overload, end-organ damage and increased infection risks. AG-348, an allosteric activator of RPK, is under evaluation in clinical trials, predominantly in less severely-afflicted transfusion-independent patients. Allogeneic hematopoietic stem cell transplantation (HSCT) has been performed in selected cases and resulted in transfusion independence, suggesting that the disorder may be reversed when an adequate level of hematopoietic stem and progenitor cells (HSPCs) harboring a corrected PKLR gene engraft in the bone marrow (BM). The therapeutic efficacy of allogeneic transplant is limited by the availability of a suitable donor and transplant-associated toxicities. Preclinical studies conducted in a clinically relevant PKD murine model have demonstrated the safety and efficacy of Lin- BM cells transduced with the therapeutic lentiviral vector, PGK-coRPK-WPRE, in ameliorating the PKD phenotype. More specifically, transplantation of transduced cells resulted in increased erythrocyte survival, decreased reticulocytosis, and improvement in the secondary manifestations of hemolytic anemia, including splenomegaly and hepatic iron overload. Based on compelling preclinical data, a global Phase 1 clinical trial RP-L301-0119 (clinicaltrials.gov#NCT04105166) is underway to evaluate the feasibility and safety of lentiviral mediated gene therapy in adults and pediatric subjects with severe PKD. Methods: 6 subjects with severe PKD (defined as having a history of severe and/or transfusion-dependent anemia despite prior splenectomy) will be enrolled in the Phase 1 study; the first 2 subjects will be adults (age ≥18-<50 years), followed by 2 older pediatric subjects (age ≥12-17 years) once initial safety has been established. Subsequently, 2 younger pediatric subjects (age ≥8-11 years) will be enrolled. Peripheral blood (PB) hematopoietic stem cells are collected on two consecutive days via apheresis after mobilization with granulocyte-colony stimulating factor (G-CSF) and plerixafor. CD34+ HSPCs are enriched, transduced with PGK-coRPK-WPRE lentiviral vector (LV), and cryopreserved. Following final release testing of the investigational product (IP), RP-L301, myeloablative conditioning with therapeutic drug monitoring (TDM)-guided busulfan is administered over 4 days. RP-L301 is then thawed and infused. Patients are followed for safety assessments, including replication competent lentivirus (RCL) and insertion site analysis (ISA), and for efficacy parameters including PB and BM genetic correction, decrease in transfusion requirements, clinically significant improvement in anemia, and reduction of hemolysis. Results: An adult female PKD subject (age 31 years) with significant anemia and transfusion requirement has received treatment as of July 2020. Mobilization and apheresis procedures were performed successfully and busulfan conditioning was administered at the target area under the curve (AUC). IP consisted of 3.9×106 CD34+ cells/kg body weight, with a mean vector copy number (VCN) of 2.73. Safety and preliminary efficacy results will be available at the time of presentation. Conclusions: Efficacy in pre-clinical models indicates promising potential for clinical gene therapy in severe PKDHematopoietic stem cell mobilization using G-CSF and plerixafor appears feasible and effective in adult PKD patientsIP was successfully manufactured to meet the required specifications for the Phase 1 clinical study and administered without short-term infusion related complications Disclosures Navarro: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: SN has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Sevilla:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company. Glader:Agios Pharmaceuticals, Inc.: Consultancy. Beard:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents & Royalties, Research Funding. Rao:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Schwartz:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents & Royalties, Research Funding.

Single-Cell Profiling of AKI in a Murine Model Reveals Novel Transcriptional Signatures, Profibrotic Phenotype, and Epithelial-to-Stromal Crosstalk.

Current management of AKI, a potentially fatal disorder that can also initiate or exacerbate CKD, is merely supportive. Therefore, deeper understanding of the molecular pathways perturbed in AKI is needed to identify targets with potential to lead to improved treatment. We performed single-cell RNA sequencing (scRNA-seq) with the clinically relevant unilateral ischemia-reperfusion murine model of AKI at days 1, 2, 4, 7, 11, and 14 after AKI onset. Using real-time quantitative PCR, immunofluorescence, Western blotting, and both chromogenic and single-molecule in situ hybridizations, we validated AKI signatures in multiple experiments. Our findings show the time course of changing gene expression patterns for multiple AKI stages and all renal cell types. We observed elevated expression of crucial injury response factors-including kidney injury molecule-1 (Kim1), lipocalin 2 (Lcn2), and keratin 8 (Krt8)-and of several novel genes (Ahnak, Sh3bgrl3, and Col18a1) not previously examined in kidney pathologies. AKI induced proximal tubule dedifferentiation, with a pronounced nephrogenic signature represented by Sox4 and Cd24a. Moreover, AKI caused the formation of "mixed-identity cells" (expressing markers of different renal cell types) that are normally seen only during early kidney development. The injured tubules acquired a proinflammatory and profibrotic phenotype; moreover, AKI dramatically modified ligand-receptor crosstalk, with potential pathologic epithelial-to-stromal interactions. Advancing age in AKI onset was associated with maladaptive response and kidney fibrosis. The scRNA-seq, comprehensive, cell-specific profiles provide a valuable resource for examining molecular pathways that are perturbed in AKI. The results fully define AKI-associated dedifferentiation programs, potential pathologic ligand-receptor crosstalk, novel genes, and the improved injury response in younger mice, and highlight potential targets of kidney injury.

Alterations of peripheral nerve excitability in an experimental autoimmune encephalomyelitis mouse model for multiple sclerosis

BackgroundExperimental autoimmune encephalomyelitis (EAE) is the most commonly used and clinically relevant murine model for human multiple sclerosis (MS), a demyelinating autoimmune disease characterized by mononuclear cell infiltration into the central nervous system (CNS). The aim of the present study was to appraise the alterations, poorly documented in the literature, which may occur at the peripheral nervous system (PNS) level.MethodsTo this purpose, a multiple evaluation of peripheral nerve excitability was undertaken, by means of a minimally invasive electrophysiological method, in EAE mice immunized with the myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide, an experimental model for MS that reproduces, in animals, the anatomical and behavioral alterations observed in humans with MS, including CNS inflammation, demyelination of neurons, and motor abnormalities. Additionally, the myelin sheath thickness of mouse sciatic nerves was evaluated using transmission electronic microscopy.ResultsAs expected, the mean clinical score of mice, daily determined to describe the symptoms associated to the EAE progression, increased within about 18 days after immunization for EAE mice while it remained null for all control animals. The multiple evaluation of peripheral nerve excitability, performed in vivo 2 and 4 weeks after immunization, reveals that the main modifications of EAE mice, compared to control animals, are a decrease of the maximal compound action potential (CAP) amplitude and of the stimulation intensity necessary to generate a CAP with a 50% maximum amplitude. In addition, and in contrast to control mice, at least 2 CAPs were recorded following a single stimulation in EAE animals, reflecting various populations of sensory and motor nerve fibers having different CAP conduction speeds, as expected if a demyelinating process occurred in the PNS of these animals. In contrast, single CAPs were always recorded from the sensory and motor nerve fibers of control mice having more homogeneous CAP conduction speeds. Finally, the myelin sheath thickness of sciatic nerves of EAE mice was decreased 4 weeks after immunization when compared to control animals.ConclusionsIn conclusion, the loss of immunological self-tolerance to MOG in EAE mice or in MS patients may not be only attributed to the restricted expression of this antigen in the immunologically privileged environment of the CNS but also of the PNS.

Soy Isoflavones and Gastrointestinal Health.

Soy isoflavones are known to have beneficial effects on several aspects of gastrointestinal physiological functions (contractility or motility, secretion, morphology, and barrier function). In this review, we discuss the effects of soy isoflavones on the overall gut function and inflammation and assess how these effects might be implicated in the treatment of several gut-related diseases. Soy isoflavones influence several key aspects of gastrointestinal health: improve basal intestinal secretion, alleviate inflammation, limit intestinal morphological damage, and improve epithelial barrier function in several clinically relevant murine models of gastrointestinal diseases. Dietary supplementation with isoflavones proves to be a key means to improve the overall gut function and health. Future mechanistic studies with isoflavone interventions will help treat clinically related diseases such as cystic fibrosis and inflammatory-related gut problems such as colitis and diabetes.

Immunomodulatory dose of clindamycin in combination with ceftriaxone improves survival and prevents organ damage in murine polymicrobial sepsis.

Sepsis is a life-threatening organ dysfunction resulting from inflammatory responses instigated by toxins secreted by bacteria. Immunomodulatory effect of clindamycin is earlier reported in a murine lipopolysaccharide (LPS)-induced sepsis model. There are no studies demonstrating the immunomodulatory effect of clindamycin in combination with ceftriaxone in a clinically relevant murine polymicrobial sepsis model induced by cecal ligation and puncture (CLP). Ceftriaxone is combined to control the bacterial growth. Following 3h of CLP challenge, Swiss albino mice were administered vehicle, ceftriaxone alone (100mg/kg, subcutaneously), and in combination with clindamycin at immunomodulatory dose (200mg/kg, intraperitoneally). Survival was assessed for 5days, and bacterial count and biochemical and physiological parameters were measured after 18h of CLP challenge. Ceftriaxone alone caused significant reduction in bacterial count in blood, peritoneal fluid, lung, liver, and kidney homogenate which was not further substantially reduced by ceftriaxone and clindamycin combination. Day 5 survival was greatly improved by combination compared with ceftriaxone alone which was also evident through marked drop in blood glucose, total white blood cell (WBC) count, and body temperature. The combination group significantly mitigated the cytokine (tumor necrosis factor (TNF)-α and interleukin (IL)-6) and myeloperoxidase (MPO) levels in plasma, lung, liver, and kidney of CLP-challenged mice, which further helped in significantly suppressing the elevated levels of liver and kidney function parameters. Clindamycin at immunomodulatory dose in combination with ceftriaxone attenuated organ damage and improved survival of septic mice by suppressing infection, inflammatory responses, and oxidative stress.

Gαs (GNAS) suppression of the p53 genomic‐stability checkpoint unleashes RAS‐driven oncogenesis

As one of the most frequently mutated oncogenes, mutant RAS has been well established to drive proliferative cell programs that are exploited by cancer. Interestingly, RAS, like many oncogenes, has been found in benign tumors that never progress to cancer. Although seemingly counterintuitive, RAS mutant tumors require an additional mutational insult to progress to malignancy. Using an unbiased, pan‐cancer analysis from patient sequencing data, we discovered that RAS mutations significantly co‐occur with mutations in GNAS, the gene encoding the Gαs subunit of G protein couple receptors (GPCRs). Given the established tractability of GPCRs as drug targets, we aimed to explore GNAS as a potential co‐driver in RAS mutant cancers. To first validate the ability of mutations in RAS and GNAS to cooperate to induce malignancy, we expressed the constitutive active mutations, KRASG12D and GNASR201C in the skin of adult mice. These double mutant mice rapidly developed skin papillomas, while mice with either mutation alone had no phenotype. Furthermore, when GNASR201C mice were treated with the chemical carcinogen DMBA, known to induce Hras mutations, mice rapidly developed squamous cell carcinoma in only 3 weeks. Analysis of the lesions revealed a surprising lack of mutations in the Trp53 tumor suppressor gene, which controls genomic stability. In vitro experiments revealed that activation of signaling through Gαs, or its downstream kinase PKA, was sufficient to block DNA damage‐induced p53 protein accumulation. Analysis of the underlying mechanisms demonstrated that MDM2, the cognate p53 E3 ubiquitin ligase, is a direct substrate of PKA, which phosphorylates MDM2 on S166 resulting in enhanced ligase activity. In vitro ubiquitination assays revealed that activation of Gαs or PKA increased MDM2‐mediated p53 ubiquitination and degradation. In patients, appendix cancer is the best example of the GNAS‐KRAS co‐driver state, with 70% of patients harboring this co‐mutation. Ongoing experiments are aimed at validating the mechanism of Gαs‐PKA regulation of MDM2‐p53 growth checkpoints in relevant murine and human intestinal organoid models. In summary, we have uncovered a significant co‐occurrence of RAS and GNAS mutations in cancer and demonstrated that these mutations cooperate to initiate carcinogenesis in vivo. Mechanistically, the cooperation of RAS and GNAS can be explained by the ability of Gαs to functionally repress p53, through PKA mediated phosphorylation of MDM2, releasing the normal brakes that check aberrant RAS‐driven growth.Support or Funding InformationDana Steffen was supported by the National Science Foundation Graduate Research Fellowship Program (NSF GRFP) and the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through an institutional training grant from the National Institute of General Medical Sciences (T32 GM007752). This study is supported in part by the San Diego NCI Cancer Centers Council (C3) Collaborative Translational Cancer Research Pilot Grant.

An ABC Transporter Drives Medulloblastoma Pathogenesis by Regulating Sonic Hedgehog Signaling

Mutations in Sonic hedgehog (SHH) signaling promote aberrant proliferation and tumor growth. SHH-medulloblastoma (MB) is among the most frequent brain tumors in children less than 3 years of age. Although key components of the SHH pathway are well-known, we hypothesized that new disease-modifying targets of SHH-MB might be identified from large-scale bioinformatics and systems biology analyses. Using a data-driven systems biology approach, we built a MB-specific interactome. The ATP-binding cassette transporter ABCC4 was identified as a modulator of SHH-MB. Accordingly, increased ABCC4 expression correlated with poor overall survival in patients with SHH-MB. Knockdown of ABCC4 expression markedly blunted the constitutive activation of the SHH pathway secondary to Ptch1 or Sufu insufficiency. In human tumor cell lines, ABCC4 knockdown and inhibition reduced full-length GLI3 levels. In a clinically relevant murine SHH-MB model, targeted ablation of Abcc4 in primary tumors significantly reduced tumor burden and extended the lifespan of tumor-bearing mice. These studies reveal ABCC4 as a potent SHH pathway regulator and a new candidate to target with the potential to improve SHH-MB therapy. SIGNIFICANCE: These findings identify ABCC4 transporter as a new target in SHH-MB, prompting the development of inhibitors or the repurporsing of existing drugs to target ABCC4.

Abstract B21: Synergistic antitumor immunity observed with combination FRα-targeting antibody-drug conjugate plus anti-PD-1 therapy is CD8+ cell dependent

Abstract Background: Immune checkpoint blockade, specifically targeting PD-1/PD-L1, is a promising approach for cancer therapy. However, for some common cancers, such as epithelial ovarian cancer (EOC), PD1/PDL1 blockade as monotherapy has a low response rate. In these less immunogenic cancer types, focus has turned to combining checkpoint inhibitors with cancer therapies that elicit immunologic cell death (ICD). Antibody-drug conjugates (ADCs), comprising a tumor-targeting antibody armed with highly potent small-molecule payloads, are a clinically validated anticancer therapy. Mirvetuximab soravtansine is an ADC comprised of a folate receptor alpha (FRα)-binding antibody linked to the potent tubulin-disrupting maytansinoid DM4 via a cleavable disulfide sulfo-SPDB linker and is currently being evaluated in combination with pembrolizumab (anti-PD1 antibody) in a phase 1b study (FORWARDII, NCT02606305). Here we report preclinical studies that provide a mechanistic understanding for the antitumor activity of combination therapy consisting of PD-1/PD-L1 blockade and an FRα targeting disulfide linked DM4 ADC. Methods: A chimeric anti-mouse FRα antibody (rmFR1-12) was generated and its specificity for murine FRα was assessed by ELISA and FACS. rmFR1-12 was conjugated to DM4 via a s-SPDB linker to generate rmFR-1-12-s-SPDB-DM4 ADC, which was quality checked using standard analytical methods. In vitro assays were used to assess cytotoxicity against FRα-positive cell lines, as well as the ability of rmFR1-12-s-SPDB-DM4 to induce ICD and engage cells of the immune system. In vivo studies performed in albino C57/B6 mice evaluated the antitumor activity of rmFR1-12-s-SPDB-DM4, anti-PD-1 or the combination therapy against ID8 tumors, a translationally relevant murine model of EOC. Results: rmFR1-12 binding was specific for FRα. The rmFR1-12-s-SPDB-DM4 ADC was cytotoxic against FRα-positive cell lines and induced ICD as measured by upregulation of ICD markers. In vivo studies using syngeneic ID8 tumors demonstrated that rmFR1-12-s-SPDB-DM4 or anti-PD-1 monotherapy treatment resulted in modest antitumor activity. In contrast, combination treatment demonstrated synergistic antitumor activity, inducing complete tumor regressions and increased survival over control or monotherapy regimens. Depletion of CD8+ cells abrogated the antitumor activity of the combination, whereas depletion of CD4+ cells had no impact. Finally, combination-treated mice generated a robust memory response when rechallenged with ID8 cells. Conclusion: Anti-FRα-s-SPDB-DM4 induces ICD in FRα-positive cell lines and demonstrates synergistic antitumor activity when combined with anti-PD1 in a murine EOC model. The antitumor immunity is dependent upon CD8+ cells. These findings support the clinical assessment of mirvetuximab soravtansine and pembrolizumab. Citation Format: L. Cristina Gavrilescu, Yulius Setiady, Ling Dong, Sharon Chicklas, Luke Harris, Jan Pinkas, Richard Gregory, Joe Ponte. Synergistic antitumor immunity observed with combination FRα-targeting antibody-drug conjugate plus anti-PD-1 therapy is CD8+ cell dependent [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B21.

Multi-omics: Differential expression of IFN-γ results in distinctive mechanistic features linking chronic inflammation, gut dysbiosis, and autoimmune diseases

Low grade, chronic inflammation is a critical risk factor for immunologic dysfunction including autoimmune diseases. However, the multiplicity of complex mechanisms and lack of relevant murine models limit our understanding of the precise role of chronic inflammation. To address these hurdles, we took advantage of multi-omics data and a unique murine model with a low but chronic expression of IFN-γ, generated by replacement of the AU-rich element (ARE) in the 3’ UTR region of IFN-γ mRNA with random nucleotides. Herein, we demonstrate that low but differential expression of IFN-γ in mice by homozygous or heterozygous ARE replacement triggers distinctive gut microbial alterations, of which alteration is female-biased with autoimmune-associated microbiota. Metabolomics data indicates that gut microbiota-dependent metabolites have more robust sex-differences than microbiome profiling, particularly those involved in fatty acid oxidation and nuclear receptor signaling. More importantly, homozygous ARE-Del mice have dramatic changes in tryptophan metabolism, bile acid and long-chain lipid metabolism, which interact with gut microbiota and nuclear receptor signaling similarly with sex-dependent metabolites. Consistent with these findings, nuclear receptor signaling, encompassing molecules such as PPARs, FXR, and LXRs, was detectable as a top canonical pathway in comparison of blood and tissue-specific gene expression between female homozygous vs heterozygous ARE-Del mice. Further analysis implies that dysregulated autophagy in macrophages is critical for breaking self-tolerance and gut homeostasis, while pathways interact with nuclear receptor signaling to regulate inflammatory responses. Overall, pathway-based integration of multi-omics data provides systemic and cellular insights about how chronic inflammation driven by IFN-γ results in the development of autoimmune diseases with specific etiopathological features.

Left ventricular response in the transition from hypertrophy to failure recapitulates distinct roles of Akt, β-arrestin-2, and CaMKII in mice with aortic regurgitation.

BackgroundAlthough aortic regurgitation (AR) is a clinically important condition that is becoming increasingly common, few relevant murine models and mechanistic studies exist for this condition. In this study, we attempted to delineate the pathological and molecular changes and address the roles of some potentially relevant molecules in an animal model of surgically induced AR.MethodsAR was induced by puncturing the aortic valve leaflets in C57BL/6J mice under echocardiographic guidance.ResultsAs early as 1 week following AR, the left ventricles (LV) displayed marked impairments in diastolic function and coronary flow reserve (CFR), as well as cardiac hypertrophy and chamber dilatation at both end-systole and end-diastole. LV free wall thickening and cardiomyocyte hypertrophy in LV were observed 2 weeks following of AR while a decline in ejection fraction was not seen until after 4 weeks. Nppa (natriuretic peptide A) and Nppb (natriuretic peptide B) increased over time, in conjunction with prominent Akt activation as well as slight CaMKII (Ca2+/calmodulin-dependent protein kinase II) activation and biphasic changes in β-arrestin-2 expression. Treatment of AR mice with Akt inhibition exacerbated the eccentric hypertrophy, while neither inhibition of CaMKII nor β-arrestin-2 overexpression influenced the response to AR.ConclusionsOur structural, functional, molecular and therapeutic analyses reveal that Akt, but not CaMKII or β-arrestin-2, plays a regulatory role in the development of LV remodeling after AR in Mice. These results may shed important light on therapeutic targets for volume overloaded cardiomyopathy.

Activation of β-Catenin Cooperates with Loss of Pten to Drive AR-Independent Castration-Resistant Prostate Cancer.

Inhibition of the androgen receptor (AR) is the main strategy to treat advanced prostate cancers. AR-independent treatment-resistant prostate cancer is a major unresolved clinical problem. Patients with prostate cancer with alterations in canonical WNT pathway genes, which lead to β-catenin activation, are refractory to AR-targeted therapies. Here, using clinically relevant murine prostate cancer models, we investigated the significance of β-catenin activation in prostate cancer progression and treatment resistance. β-Catenin activation, independent of the cell of origin, cooperated with Pten loss to drive AR-independent castration-resistant prostate cancer. Prostate tumors with β-catenin activation relied on the noncanonical WNT ligand WNT5a for sustained growth. WNT5a repressed AR expression and maintained the expression of c-Myc, an oncogenic effector of β-catenin activation, by mediating nuclear localization of NFκBp65 and β-catenin. Overall, WNT/β-catenin and AR signaling are reciprocally inhibited. Therefore, inhibiting WNT/β-catenin signaling by limiting WNT secretion in concert with AR inhibition may be useful for treating prostate cancers with alterations in WNT pathway genes. SIGNIFICANCE: Targeting of both AR and WNT/β-catenin signaling may be required to treat prostate cancers that exhibit alterations of the WNT pathway.

Immunomodulatory Effect of Doxycycline Ameliorates Systemic and Pulmonary Inflammation in a Murine Polymicrobial Sepsis Model.

Acute lung injury is an inflammatory condition developed after severe sepsis in response to excessive secretion of pro-inflammatory cytokines. Doxycycline is widely reported to possess immunomodulatory activity through inhibition of various inflammatory pathways. Considering the broad spectrum of anti-inflammatory activity, protective effect of doxycycline was evaluated in clinically relevant murine polymicrobial sepsis model induced by caecal ligation and puncture (CLP). In this model, sepsis is accompanied with infection and therefore ceftriaxone at sub-protective dose was combined to retard the bacterial growth. Three hours after CLP challenge, mice were administered vehicle, ceftriaxone (100 mg/kg subcutaneously) alone and in combination with immunomodulatory dose of doxycycline (50 mg/kg, intraperitoneal) and survival were monitored for 5 days. Bacterial count in blood and peritoneal fluid along with cytokines [interleukin (IL)-6, IL-1β, tumour necrosis factor (TNF)-α] and myeloperoxidase (MPO) in plasma and lung homogenate were measured at 18 h post-CLP. Plasma glutathione (GSH) was also determined. Doxycycline in presence of ceftriaxone improved survival of septic mice by significantly reducing the plasma and lung pro-inflammatory cytokines and MPO levels. It also increased plasma GSH levels. Doxycycline did not improve antibacterial effect of ceftriaxone in combination, suggesting that the protective effect of doxycycline was due to its immunomodulatory activity. Doxycycline in the presence of ceftriaxone demonstrated improved survival of septic mice by modulating the immune response.

Paclitaxel-loaded expansile nanoparticles improve survival following cytoreductive surgery in pleural mesothelioma xenografts

ObjectiveMalignant pleural mesothelioma is a lethal malignancy with poor survival and high local recurrence rates despite multimodal therapy with cytoreduction and chemoradiation. We evaluated the antitumor efficacy of a paclitaxel-loaded pH-responsive expansile nanoparticle (PTX-eNP) in 2 clinically relevant murine xenograft models of malignant pleural mesothelioma. MethodsLuciferase-transfected MSTO-211H human mesothelioma cells were injected into the thoracic cavity of immunodeficient Nu/J mice. Tumor burden was monitored by bioluminescent imaging. Animals were randomized into 2 models of disease treatment chemotherapy with PTX-eNPs alone delivered locally for early limited disease or cytoreductive surgery plus local PTX-eNP chemotherapy for advanced disease. Within each disease model, anti-tumor efficacy of PTX-eNP was compared against standard formulation paclitaxel and drug-empty nanoparticles. Influence on survival was calculated. Fluorescently labeled PTX-eNPs and immunohistochemistry evaluated in vivo drug localization to tumor. ResultsIntrathoracic injection of MSTO-211H resulted in large tumor deposits distributed within the pleural space of the murine thoracic cavity. Local multidose treatment with PTX-eNPs alone in limited stage disease more than doubled survival compared with drug-empty nanoparticles (P ≤ .0001) and standard formulation paclitaxel (P = .0004). In the model of advanced disease, local multidose treatment with PTX-eNPs following cytoreductive surgery also prolonged survival by 126% and 69.4% compared with drug-empty nanoparticles (P = .0018) and standard formulation paclitaxel (P = .03457), respectively. Immunohistology demonstrated PTX-eNP accumulation within tumor cells in vitro and in vivo. ConclusionsLocal delivery of paclitaxel via eNPs confers prolonged survival in a murine model of malignant pleural mesothelioma as single modality treatment for limited disease and in combination with cytoreductive surgery for advanced disease.

CCR2 inhibition reduces tumor myeloid cells and unmasks a checkpoint inhibitor effect to slow progression of resistant murine gliomas

Immunotherapy directed at the PD-L1/PD-1 axis has produced treatment advances in various human cancers. Unfortunately, progress has not extended to glioblastoma (GBM), with phase III clinical trials assessing anti-PD-1 monotherapy failing to show efficacy in newly diagnosed and recurrent tumors. Myeloid-derived suppressor cells (MDSCs), a subset of immunosuppressive myeloid derived cells, are known to infiltrate the tumor microenvironment of GBM. Growing evidence suggests the CCL2-CCR2 axis is important for this process. This study evaluated the combination of PD-1 blockade and CCR2 inhibition in anti-PD-1-resistant gliomas. CCR2 deficiency unmasked an anti-PD-1 survival benefit in KR158 glioma-bearing mice. CD11b+/Ly6Chi/PD-L1+ MDSCs within established gliomas decreased with a concomitant increase in overall CCR2+ cells and MDSCs within bone marrow of CCR2-deficient mice. The CCR2 antagonist CCX872 increased median survival as a monotherapy in KR158 glioma-bearing animals and further increased median and overall survival when combined with anti-PD-1. Additionally, combination of CCX872 and anti-PD-1 prolonged median survival time in 005 GSC GBM-bearing mice. In both models, CCX872 decreased tumor associated MDSCs and increased these cells within the bone marrow. Examination of tumor-infiltrating lymphocytes revealed an elevated population, increased IFNγ expression, indicating enhanced cytolytic activity, as well as decreased expression of exhaustion markers in CD4+ and CD8+ T cells following combination treatment. These data establish that combining CCR2 and PD-1 blockade extends survival in clinically relevant murine glioma models and provides the basis on which to advance this combinatorial treatment toward early-phase human trials.

IMMU-05. DISRUPTION OF THE CCR2 CHEMOKINE RECEPTOR PATHWAY OVERCOMES THERAPEUTIC RESISTANCE TO PD-1 BLOCKADE IN MALIGNANT GLIOMA

Abstract INTRODUCTION Large scale clinical trials have failed to determine efficacy of adjuvant PD-1 blockade in recurrent glioblastoma (GBM). However, recent phase 2 clinical trial results demonstrating the potential of neo-adjuvant anti-PD-1 treatment have renewed enthusiasm for the use of immune checkpoint inhibitors (ICIs) in GBM. Additional benefit of ICIs in GBM will likely derive from development of novel therapies directed against immunosuppressive resistance mechanisms. Chemokine receptor 2 (CCR2) expressing tumor infiltrative immune suppressive myeloid cells represent a targetable axis within ICI resistant gliomas. We have established that CCR2 disruption unmasks an effect of anti-PD-1 therapy in ICI resistant murine glioma models. However, the mechanism(s) by which combined inhibition of PD-1 and CCR2 achieves efficacy is unknown. OBJECTIVE Determine the impact of combination anti-PD-1 and CCR2 antagonism on immune cells within anti-PD-1 resistant gliomas. METHODS Immune cell characteristics were determined in CCR2 antagonist (CCX872)/anti-PD-1 treated KR158 or 005 GSC glioma-bearing wild type or CCR2+/rfp/CX3CR1+/gfp reporter mice. RESULTS CCX872 increased median survival (32 vs. 50 days, P=.002) in KR158 tumor-bearing animals and in combination with anti-PD-1 significantly increased durable survival (P=.0005). In 005 GSC glioma-bearing mice, combination therapy also enhanced median survival (30 vs. 49 days, P=.005). CCX872 decreased CD45+/CD11bhi/Ly6Chi MDSCs (40%, P=.038) within gliomas, and increased these cells within bone marrow in both models (74%, P=.020). Three distinct intra-tumoral populations of CCR2+ and CX3CR1+ cells were identified in both glioma models including CCR2+/CX3CR1-/CD45hi/CD11blo, CCR2+/CX3CR1+/CD45hi/CD11bhi/Ly6Chi, and CX3CR1+/CD45lo/CD11bhi/F4/80+/MHCII+, with both CCR2+ populations being reduced by CCX872 treatment. Examination of tumor infiltrating lymphocytes revealed both increased presence and decreased exhaustion (PD-1+/Tim3+) of CD4+ (P=.029) and CD8+ (P=.011) T-cells following CCX872/anti-PD-1 treatment. CONCLUSION CCX872/anti-PD-1 combination therapy is effective in clinically relevant murine glioma models, providing a basis to progress this novel combinatorial treatment to human clinical trials.