Giri, Shailendra

Abstract Introduction Ovarian cancer (OvCa) remains the most lethal gynecologic malignancy, with older women experiencing the poorest outcomes. Aging profoundly remodels the ovarian tumor immune microenvironment (TME), promoting regulatory T cell (Treg) mediated immune evasion; however, the underlying metabolic mechanisms remain unclear. Methods Using young (3-month) and aged (22-month) female mice injected intraperitoneally with ID8p53⁻/⁻ OvCa cells, we investigated how age associated metabolic reprogramming influences tumor immunity Results Aged OvCa bearing mice exhibited accelerated tumor progression, reduced survival (median 54 vs. 72 days), decreased CD4+ and CD8+ T cells, and an expanded Treg population. Metabolomic profiling revealed increased succinate accumulation in ascites, tumor tissue, and serum of aged mice and elderly OvCa patients. Exogenous succinate enhanced CCR4+FOXP3+ Treg frequency and upregulated IL-10 and TGF-β secretion. Elevated levels of CCL22, a CCR4 ligand, were also detected in ascites from aged mice and patients, suggesting a chemokine-driven reinforcement of Treg recruitment. PBMCs from aged patients displayed an OXPHOS-reprogrammed CCR4+FOXP3+ Treg subset with heightened suppressive capacity, markedly inhibiting CD4+ and CD8+ effector cytotoxicity. Treatment with AA6, an α-ketoglutarate dehydrogenase inhibitor that blocks succinate synthesis, reduced CCR4+FOXP3+ Treg accumulation, restored effector T-cell function, and significantly prolonged survival in aged OvCa-bearing mice. Conclusion Together, these findings reveal a novel succinate—CCR4 metabolic—chemokine axis that drives Treg mediated immunosuppression in aged OvCa. Targeting this pathway offers a promising strategy to restore anti-tumor immunity and overcome age-related immunotherapy resistance in ovarian cancer. Funding Source N/A Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

ABSTRACT Epithelial ovarian cancer (EOC) incidence and mortality increase with age, driven in part by chronic inflammation, diminished T cell output, and heightened regulatory T cell (Treg) mediated immunosuppression. In aged EOC‐bearing mice, we observed reduced survival, accompanied by impaired CD4 + and CD8 + T cell responses and a marked expansion of FOXP3 + Tregs exhibiting elevated IL‐10 and TGFβ expression. Metabolic profiling revealed enhanced oxidative phosphorylation in Tregs from aged mice, along with a fivefold increase in intracellular succinate levels. This accumulation of succinate within the aged tumor microenvironment was found to potentiate Treg suppressive function. Notably, pharmacologic inhibition of α‐ketoglutarate dehydrogenase reversed this effect, restoring effector T cell activity. These findings highlight succinate‐driven metabolic reprogramming as a central mechanism of age‐related Treg dysfunction in EOC and suggest that targeting succinate metabolism may offer a promising strategy to rejuvenate antitumor immunity in elderly patients.

Abstract Among all the genetic factors linked with multiple sclerosis (MS), MHC class-II molecules show the strongest association. Generation of HLA class-II transgenic mice has helped to elucidate the role of HLA class-II gene in MS. We have shown that the human HLA-DR3 (DRβ1*0301) gene predisposes to proteolipid protein (PLP)-induced EAE, whereas HLA-DQβ1*0601 (DQ6) was resistant. We also showed that the DQ6 molecule protects from EAE in DR3DQ6 double transgenic mice by producing anti-inflammatory interferon gamma (IFNγ). To further confirm the role of IFNγ in protection, we generated DR3DQ8 mice lacking IFNγ (DR3DQ8.IFNγ-/-). Immunization with PLP91-110 peptide caused atypical EAE in DR3DQ8.IFNγ-/- mice characterized by ataxia and spasticity, hallmarks of brain-specific disease. Severe brain specific inflammation and demyelination in DR3DQ8.IFNγ-/- mice with minimal spinal cord pathology further confirmed brain-specific pathology. Severe disease in DR3DQ8.IFNγ-/- mice was due to increased encephalitogenicity of CD4 T cells and its ability to produce higher levels of IL-17 and GM-CSF compared to DR3DQ8 mice. Further, areas with demyelination showed increased presence of CD68+ inflammatory cells suggesting an important role for monocytes/microglia in causing brain pathology. Thus, our study supports a protective role for IFNγ in inflammatory and demyelinating disease through down regulation of IL-17 and induction of neuro-protective factors in the brain by monocytes/microglial cells.

Abstract Multiple sclerosis is a chronic inflammatory and demyelinating disease of the CNS. Although, MS is well characterized in terms of the role played by immune cells, cytokines and CNS pathology, nothing is known about the metabolic alterations that occur during the disease process in circulation. Here, we profiled the plasma metabolites of B6 with EAE, a known model of chronic MS using untargeted global metabolomics. Using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry, a total of 326 metabolites were identified, with significant changes observed in 105 metabolites (13 up- and 92 down-regulated), that mapped to lipid and amino acid pathways, followed by the peptide, xenobiotic, carbohydrate, nucleotide and energy pathways (p<0.05). To understand the functional role of these alterations, the KEGG metabolic library was analyzed using Metaboanalyst. The top nine most significant pathways in terms of GlobeTest p-value and impact are 1) fatty acid biosynthesis, 2) lysine degradation, 3) histidine metabolism, 4) beta-alanine metabolism, 5) pentose phosphate pathway, 6) arachidonic acid metabolism, 7) linoleic acid, 8) D-arginine and ornithine. Overall, these metabolic changes could be exploited as biomarkers for EAE/MS disease and to design new treatment paradigms where metabolic interventions could be combined with present and experimental therapeutics to achieve better treatment.

Abstract In search of metabolites signature as biomarkers during EAE disease, we profiled urine from B6 EAE using global untargeted metabolomics. Evaluation of metabolomic profiling of urine from EAE and healthy B6 group by using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry, we found that 132 out of 322 (41%) metabolites were differentially altered (P<0.05) indicating robust alteration in the urine metabolomics profile during disease. Among the perturbed metabolites, 11 were up regulated in EAE urine whereas 121 were down regulated. We conducted pathway analysis of the biochemical pathways of the KEGG and considered both concerted changes in metabolite intensity within the pathway (GlobalTest) and alterations of high impact, and found that a number of pathways were significantly altered including glyoxylate and dicarboxylate, phenylanine metabolism, porphyrin & chlorophyll metabolism, primary bile acid biosynthesis, cysteine & methione metabolism, taurine and hypotaurine metabolism, glycine, serine & threonine metabolism, and beta-alanine metabolism. Alteration in these pathways during EAE disease suggesting that perturbation of certain central metabolites could have impact on multiple metabolic pathways. While some of these metabolite changes could easily be developed as biomarkers, the key to translating metabolomics into therapeutics would require figuring out the central altered metabolic pathway(s), once studied in detail.

Abstract AMPK is an energy-sensing metabolic switch implicating in various metabolic disorders. Here we report its new role in controlling the immunopathology of experimental autoimmune encephalitis by polarizing pro-inflammatory phenotype of Macrophage. AMPK alpha1 knockout (alpha1KO) developed severe EAE due to higher expression of IL17 with reduced IFNy and IL4. AMPK alpha1KO also exhibited increased number of infiltrated mononuclear cells in CNS due to impaired blood brain barrier integrity. Macrophage from alpha1KO mice exhibited M1 type phenotype (IL10lowIL12/23high) with stronger capacity to induce allogenic and antigen-specific T-cell response and also heightened the encephalitogenic property of MOG35-55-primed CD4 T cells in B6 mice. Collectively, our data shows AMPK controls the clinical outcome of disease by regulating M1 phenotype-Th17 axis in animal model of MS.

Abstract AMPK is an energy sensing metabolic switch in mammalian cells and regulates diverse metabolic pathway in cells. Recently, we observed that AMPK is lost in all immune cells of Experimental autoimmune encephalomyelitis (EAE), widely used animal model for MS. AMPK is lost at protein level in spleen macrophages, total T cells and their subsets (CD4, CD8 and regulatory T cells) isolated from EAE afflicted animals compared to control. To examine the role of AMPK, EAE was induced in Wt (+/+) and AMPKalpha1 null mice (-/-) and found that AMPKalpha1 (-/-) mice exhibited severe EAE disease. AMPKalpha2 is not involved in enhancing the severity of the disease. Moreover, AMPKalpha1 null mice significantly exhibited severe inflammation and demyelination in CNS. Loss of AMPKalpha1 leads to higher production of IL17 cytokine and reduced production of IFNgamma and IL4 under recall response with MOG35-55 as antigen. AMPKalpha1 KO mice exhibited higher number of infiltrated mononuclear cells in CNS and produced higher amount of IL17 significantly compared to Wt mice. Moreover, adoptive transfer of MOG35-55 specific T cells from AMPKalpha1 KO in Wt mice (KO to Wt) induced significantly higher degree of clinical disease compared to adoptive transfer of MOG specific T cells from Wt to Wt mice suggesting loss of AMPK negatively regulate clinical disease of EAE and identify AMPK as a novel therapeutic target for MS.

Abstract MS is an autoimmune disease of the CNS and among all the genetic factors; MHC class-II molecules show the strongest association. To better understand the role of MHC class-II (HLA-DR/DQ) genes in susceptibility and resistance to MS, we utilized transgenic (Tg) mice expressing HLA class-II gene lacking endogenous mouse class II genes. HLA-DR3 Tg mice are susceptible to PLP91-110 induced experimental autoimmune encephalomyelitis (EAE), whereas DQ8 (DQB1*0302) transgenic mice are resistant. To identify the gene expression changes and pathways required for disease susceptibility vs resistance to EAE, we performed mRNA expression profiling of CD4+ T cells from HLA-DR3 (susceptible) and HLA-DQ8 (resistant) Tg mice. Whole genome expression analysis identified 2945 genes in HLA-DR3 Tg mice (PLP91-110 immunized vs. naïve) and 1053 genes in HLA-DQ8 Tg mice (PLP91-110 immunized vs. naïve). Comparison of modulated genes between DR3 and DQ8 identified 772 genes common to both strains, 2173 genes unique to DR3 whereas 281 genes were unique to DQ8 mice. Ingenuity pathway analysis showed up regulation of pathways involved in T cell signaling in inflammation, NF-kB signaling and those responsible for communication between innate and adoptive immunity. Majority of these pathways have IL1β as a major upstream regulator in disease susceptible HLA-DR3 Tg mice. Thus microarray data indicates an important role of IL1β induced encephalitogenic T helper cell differentiation in disease susceptibility.

Abstract The AMP-activated protein kinase, AMPK, is an energy-sensing, metabolic switch implicated in various metabolic disorders; however, its role in inflammation is not well defined. We have previously shown that loss of AMPK exacerbates experimental autoimmune encephalomyelitis (EAE) disease severity. In this study, we investigated the mechanism through which AMPK modulates inflammatory disease like EAE. AMPKα1 knockout (α1KO) mice with EAE showed severe demyelination and inflammation in the brain and spinal cord compared with wild-type due to higher expression of proinflammatory Th17 cytokines, including IL-17, IL-23, and IL-1β, impaired blood–brain barrier integrity, and increased infiltration of inflammatory cells in the CNS. Infiltrated CD4 cells in the brains and spinal cords of α1KO with EAE were significantly higher compared with wild-type EAE and were characterized as IL-17 (IL-17 and GM-CSF double-positive) CD4 cells. Increased inflammatory response in α1KO mice was due to polarization of macrophages (Mϕ) to proinflammatory M1 type phenotype (IL-10lowIL-23/IL-1β/IL-6high), and these M1 Mϕ showed stronger capacity to induce allogenic as well as Ag-specific (myelin oligodendrocyte glycoprotein [MOG]35–55) T cell response. Mϕ from α1KO mice also enhanced the encephalitogenic property of MOG35–55–primed CD4 T cells in B6 mice. The increased encephalitogenic MOG-restricted CD4+ T cells were due to an autocrine effect of IL-1β/IL-23–mediated induction of IL-6 production in α1KO Mϕ, which in turn induce IL-17 and GM-CSF production in CD4 cells. Collectively, our data indicate that AMPK controls the inflammatory disease by regulating the M1 phenotype–Th17 axis in an animal model of multiple sclerosis.

Abstract Background: The heparan sulfate mimetic PG545 has been demonstrated to inhibit the function of vascular endothelial growth factor (VEGF) and heparanase (HPSE) in vitro whilst downregulating HPSE expression in tumor and metastatic tissue in vivo. Although there are reports of consistent drug-induced increases in plasma VEGF with VEGF inhibitors such as bevacizumab, similar data are lacking for HS mimetics. Given PG545's mechanism of action which involves inhibition of both angiogenesis and metastasis, the evaluation of plasma VEGF and HPSE may represent pharmacodynamic (PD) biomarkers for each of these key processes. Materials and Methods: The levels of plasma VEGF and HPSE were measured using ELISA in samples from preclinical models of ovarian cancer (syngeneic ID8 and xenogeneic A2780 models) and a small cohort of patients (dosed with PG545 by the subcutaneous route) with advanced solid tumors from a Phase I clinical study. In tumor models, PG545 was administered once weekly by intravenous or intraperitoneal routes and efficacy was determined by quantifying tumor burden using the following methods: measurement of palpable solid tumors, abdominal circumference, ascites volume and/or by tracking cells using bioluminescent reporter imaging (depending on the model). In the A2780 model and the clinical samples, the pharmacokinetics of PG545 was also evaluated using LC-MS/MS, which ensured multiple samples to facilitate the measurement of VEGF and HPSE in plasma over the duration of the dosing interval following treatment with PG545. Results: PG545 demonstrated potent antitumor and antimetastatic activity when administered once weekly via intravenous or intraperitoneal routes in preclinical studies. These studies also indicated that PG545 treatment led to increases in plasma VEGF and HPSE. Importantly, plasma samples from advanced cancer patients treated with PG545 also revealed increases in these PD markers with initial peaks often correlating with Cmax of PG545 but longer-term gradual increases also noted in some instances. We consider that these increases in plasma levels result from PG545 binding to, thereby inhibiting, VEGF and HPSE in the tumor microenvironment. Conclusion: Given the questionable utility of single time point measures of plasma PD markers (including VEGF), these preliminary data suggest that kinetic profiling can reveal the previously undetermined dynamics of circulating VEGF and HPSE following treatment with PG545. Thus, the measurement of VEGF and HPSE appears to represent potential markers of pharmacodynamic activity. A new Phase I trial with intravenously-administered PG545 has recently commenced and should further clarify the potential utility of these basic PD biomarker responses in advanced cancer patients. Citation Format: Keith Dredge, Edward Hammond, Boris Winterhoff, Shailendra Giri, Attila Teoman, Darryn Bampton, Michael Millward, Viji Shridhar. The utility of plasma vascular endothelial growth factor and heparanase as pharmacodynamic markers following treatment with PG545, a heparan sulfate mimetic: preliminary evidence from preclinical and clinical settings. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3695. doi:10.1158/1538-7445.AM2014-3695