Improving combination therapies: targeting A2B-adenosine receptor to modulate metabolic tumor microenvironment and immunosuppression.

BACKGROUND: We investigated the role of A2B-adenosine receptor in regulating immunosuppressive metabolic stress in the tumor microenvironment. Novel A2B-adenosine receptor antagonist PBF-1129 was tested for antitumor activity in mice and evaluated for safety and immunologic efficacy in a phase I clinical trial of patients with non-small cell lung cancer. METHODS: The antitumor efficacy of A2B-adenosine receptor antagonists and their impact on the metabolic and immune tumor microenvironment were evaluated in lung, melanoma, colon, breast, and epidermal growth factor receptor-inducible transgenic cancer models. Employing electron paramagnetic resonance, we assessed changes in tumor microenvironment metabolic parameters, including pO2, pH, and inorganic phosphate, during tumor growth and evaluated the immunologic effects of PBF-1129, including its pharmacokinetics, safety, and toxicity, in patients with non-small cell lung cancer. RESULTS: Levels of metabolic stress correlated with tumor growth, metastasis, and immunosuppression. Tumor interstitial inorganic phosphate emerged as a correlative and cumulative measure of tumor microenvironment stress and immunosuppression. A2B-adenosine receptor inhibition alleviated metabolic stress, downregulated expression of adenosine-generating ectonucleotidases, increased expression of adenosine deaminase, decreased tumor growth and metastasis, increased interferon γ production, and enhanced the efficacy of antitumor therapies following combination regimens in animal models (anti-programmed cell death 1 protein vs anti-programmed cell death 1 protein plus PBF-1129 treatment hazard ratio = 11.74 [95% confidence interval = 3.35 to 41.13], n = 10, P < .001, 2-sided F test). In patients with non-small cell lung cancer, PBF-1129 was well tolerated, with no dose-limiting toxicities; demonstrated pharmacologic efficacy; modulated the adenosine generation system; and improved antitumor immunity. CONCLUSIONS: Data identify A2B-adenosine receptor as a valuable therapeutic target to modify metabolic and immune tumor microenvironment to reduce immunosuppression, enhance the efficacy of immunotherapies, and support clinical application of PBF-1129 in combination therapies.

Correction to: Determinant roles of dendritic cell-expressed Notch Delta-like and Jagged ligands on anti-tumor T-cell immunity.

Following publication of the original article [1], the author reported the wrong version of Figs. 5 and 7 have been published. The correct version of the figures can be found below.

Determinant roles of dendritic cell-expressed Notch Delta-like and Jagged ligands on anti-tumor T cell immunity.

BACKGROUND: Notch intercellular communication instructs tissue-specific T-cell development and function. In this study, we explored the roles of dendritic cell (DC)-expressed Notch ligands in the regulation of T-cell effector function. METHODS: We generated mice with CD11c lineage-specific deletion of Notch Delta-like ligand (Dll)1 and Jagged (Jag)2. Using these genetically-ablated mice and engineered pharmacological Notch ligand constructs, the roles of various Delta-like and Jagged ligands in the regulation of T-cell-mediated immunity were investigated. We assessed tumor growth, mouse survival, cytokine production, immunophenotyping of myeloid and lymphoid populations infiltrating the tumors, expression of checkpoint molecules and T-cell function in the experimental settings of murine lung and pancreatic tumors and cardiac allograft rejection. Correlative studies were also performed for the expression of NOTCH ligands, NOTCH receptors and PD-1 on various subsets of myeloid and lymphoid cells in tumor-infiltrating immune cells analyzed from primary human lung cancers. RESULTS: Mice with CD11c lineage-specific deletion of Notch ligand gene Dll1, but not Jag2, exhibited accelerated growth of lung and pancreatic tumors concomitant with decreased antigen-specific CD8+T-cell functions and effector-memory (Tem) differentiation. Increased IL-4 but decreased IFN-γ production and elevated populations of T-regulatory and myeloid-derived suppressor cells were observed in Dll1-ablated mice. Multivalent clustered DLL1-triggered Notch signaling overcame DC Dll1 deficiency and improved anti-tumor T-cell responses, whereas the pharmacological interference by monomeric soluble DLL1 construct suppressed the rejection of mouse tumors and cardiac allograft. Moreover, monomeric soluble JAG1 treatment reduced T-regulatory cells and improved anti-tumor immune responses by decreasing the expression of PD-1 on CD8+Tem cells. A significant correlation was observed between DC-expressed Jagged and Delta-like ligands with Tem-expressed PD-1 and Notch receptors, respectively, in human lung tumor-infiltrates. CONCLUSION: Our data show the importance of specific expression of Notch ligands on DCs in the regulation of T-cell effector function. Thus, strategies incorporating selectively engineered Notch ligands could provide a novel approach of therapeutics for modulating immunity in various immunosuppressive conditions including cancer.

Notch3-dependent ß-catenin signaling mediates EGFR TKI drug persistence in EGFR mutant NSCLC.

EGFR tyrosine kinase inhibitors cause dramatic responses in EGFR-mutant lung cancer, but resistance universally develops. The involvement of ß-catenin in EGFR TKI resistance has been previously reported, however, the precise mechanism by which ß-catenin activation contributes to EGFR TKI resistance is not clear. Here, we show that EGFR inhibition results in the activation of ß-catenin signaling in a Notch3-dependent manner, which facilitates the survival of a subset of cells that we call "adaptive persisters". We previously reported that EGFR-TKI treatment rapidly activates Notch3, and here we describe the physical association of Notch3 with ß-catenin, leading to increased stability and activation of ß-catenin. We demonstrate that the combination of EGFR-TKI and a ß-catenin inhibitor inhibits the development of these adaptive persisters, decreases tumor burden, improves recurrence free survival, and overall survival in xenograft models. These results supports combined EGFR-TKI and ß-catenin inhibition in patients with EGFR mutant lung cancer.

Interstitial Inorganic Phosphate as a Tumor Microenvironment Marker for Tumor Progression.

Noninvasive in vivo assessment of chemical tumor microenvironment (TME) parameters such as oxygen (pO2), extracellular acidosis (pHe), and concentration of interstitial inorganic phosphate (Pi) may provide unique insights into biological processes in solid tumors. In this work, we employ a recently developed multifunctional trityl paramagnetic probe and electron paramagnetic resonance (EPR) technique for in vivo concurrent assessment of these TME parameters in various mouse models of cancer. While the data support the existence of hypoxic and acidic regions in TME, the most dramatic differences, about 2-fold higher concentrations in tumors vs. normal tissues, were observed for interstitial Pi - the only parameter that also allowed for discrimination between non-metastatic and highly metastatic tumors. Correlation analysis between [Pi], pO2, pHe and tumor volumes reveal an association of high [Pi] with changes in tumor metabolism and supports different mechanisms of protons and Pi accumulation in TME. Our data identifies interstitial inorganic phosphate as a new TME marker for tumor progression. Pi association with tumor metabolism, buffer-mediated proton transport, and a requirement of high phosphorus content for the rapid growth in the "growth rate hypothesis" may underline its potential role in tumorigenesis and tumor progression.

Aquaporin 11 variant associates with kidney disease in type 2 diabetic patients.

Kidney disease, a common complication of diabetes, associates with poor prognosis. Our previous animal model studies linked aquaporin (AQP)11 to acute kidney injury, hyperglycemia-induced renal impairment, and kidney disease in diabetes. Here, we report the AQP11 rs2276415 variant as a genetic factor placing type 2 diabetic patients at greater risk for the development of kidney disease. We performed two independent retrospective case-control studies in 1,075 diabetic and 1,619 nondiabetic individuals who were identified in the Synthetic Derivative Database with DNA samples in the BioVU DNA repository at Vanderbilt University (Nashville, TN). A χ(2)-test and multivariable logistic regression analysis with adjustments for age, sex, baseline serum creatinine, and underlying comorbid disease covariates showed a significant association between rs2276415 and the prevalence of any event of acute kidney injury and chronic kidney disease (CKD) in diabetic patients but not in patients without diabetes. This result was replicated in the second independent study. Diabetic CKD patients over 55 yrs old with the minor AQP11 allele had a significantly faster progression of estimated glomerular filtration rate decline than patients with the wild-type genotype. Three-dimensional structural analysis suggested a functional impairment of AQP11 with rs2276415, which could place diabetic patients at a higher risk for kidney disease. These studies identified rs2276415 as a candidate genetic factor predisposing patients with type 2 diabetes to CKD.

Multivalent Forms of the Notch Ligand DLL-1 Enhance Antitumor T-cell Immunity in Lung Cancer and Improve Efficacy of EGFR-Targeted Therapy.

Activation of Notch signaling in hematopoietic cells by tumors contributes to immune escape. T-cell defects in tumors can be reversed by treating tumor-bearing mice with multivalent forms of the Notch receptor ligand DLL-1, but the immunologic correlates of this effect have not been elucidated. Here, we report mechanistic insights along with the efficacy of combinational treatments of multivalent DLL-1 with oncoprotein targeting drugs in preclinical mouse models of lung cancer. Systemic DLL-1 administration increased T-cell infiltration into tumors and elevated numbers of CD44(+)CD62L(+)CD8(+) memory T cells while decreasing the number of regulatory T cells and limiting tumor vascularization. This treatment was associated with upregulation of Notch and its ligands in tumor-infiltrating T cells enhanced expression of T-bet and phosphorylation of Stat1/2. Adoptive transfer of T cells from DLL1-treated tumor-bearing immunocompetent hosts into tumor-bearing SCID-NOD immunocompromised mice attenuated tumor growth and extended tumor-free survival in the recipients. When combined with the EGFR-targeted drug erlotinib, DLL-1 significantly improved progression-free survival by inducing robust tumor-specific T-cell immunity. In tissue culture, DLL1 induced proliferation of human peripheral T cells, but lacked proliferative or clonogenic effects on lung cancer cells. Our findings offer preclinical mechanistic support for the development of multivalent DLL1 to stimulate antitumor immunity.

Aquaporin 11 insufficiency modulates kidney susceptibility to oxidative stress.

Aquaporin 11 (AQP11) is a newly described member of the protein family of transport channels. AQP11 associates with the endoplasmic reticulum (ER) and is highly expressed in proximal tubular epithelial cells in the kidney. Previously, we identified and characterized a recessive mutation of the highly conserved Cys227 to Ser227 in mouse AQP11 that caused proximal tubule (PT) injury and kidney failure in mutant mice. The current study revealed induction of ER stress, unfolded protein response, and apoptosis as molecular mechanisms of this PT injury. Cys227Ser mutation interfered with maintenance of AQP11 oligomeric structure. AQP11 is abundantly expressed in the S1 PT segment, a site of major renal glucose flux, and Aqp11 mutant mice developed PT-specific mitochondrial injury. Glucose increased AQP11 protein expression in wild-type kidney and upregulation of AQP11 expression by glucose in vitro was prevented by phlorizin, an inhibitor of sodium-dependent glucose transport across PT. Total AQP11 levels in heterozygotes were higher than in wild-type mice but were not further increased in response to glucose. In Aqp11 insufficient PT cells, glucose potentiated increases in reactive oxygen species (ROS) production. ROS production was also elevated in Aqp11 mutation carriers. Phenotypically normal mice heterozygous for the Aqp11 mutation repeatedly treated with glucose showed increased blood urea nitrogen levels that were prevented by the antioxidant sulforaphane or by phlorizin. Our results indicate an important role for AQP11 to prevent glucose-induced oxidative stress in proximal tubules.

Single amino acid substitution in aquaporin 11 causes renal failure.

A screen of recessive mutations generated by the chemical mutagen n-ethyl-n-nitrosourea (ENU) mapped a new mutant locus (5772SB) termed sudden juvenile death syndrome (sjds) to chromosome 7 in mice. These mutant mice, which exhibit severe proximal tubule injury and formation of giant vacuoles in the renal cortex, die from renal failure, a phenotype that resembles aquaporin 11 (Aqp11) knockout mice. In this report, the ENU-induced single-nucleotide variant (sjds mutation) is identified. To determine whether this variant, which causes an amino acid substitution (Cys227Ser) in the predicted E-loop region of aquaporin 11, is responsible for the sjds lethal renal phenotype, Aqp11-/sjds compound heterozygous mice were generated from Aqp11 +/sjds and Aqp11 +/- intercrosses. The compound heterozygous Aqp11 -/sjds offspring exhibited a lethal renal phenotype (renal failure by 2 wk), similar to the Aqp11 sjds/sjds and Aqp11-/- phenotypes. These results demonstrate that the identified mutation causes renal failure in Aqp11 sjds/sjds mutant mice, providing a model for better understanding of the structure and function of aquaporin 11 in renal physiology.

Markers of glycemic control in the mouse: comparisons of 6-h- and overnight-fasted blood glucoses to Hb A1c.

The present studies examined the relationship between fasting blood glucose and Hb A(1c) in C57BL/6J, DBA/2J, and KK/HlJ mice with and without diabetes mellitus. Daily averaged blood glucose levels based on continuous glucose monitoring and effects of 6-h vs. overnight fasting on blood glucose were determined. Daily averaged blood glucose levels were highly correlated with Hb A(1c), as determined with a hand-held automated device using an immunodetection method. R(2) values were 0.90, 0.95, and 0.99 in KK/HIJ, C57BL/6J, and DBA/2J, respectively. Six-hour fasting blood glucose correlated more closely with the level of daily averaged blood glucose and with Hb A(1c) than did blood glucose following an overnight fast. To validate the immunoassay-determined Hb A(1c), we also measured total glycosylated hemoglobin using boronate HPLC. Hb A(1c) values correlated well with total glycosylated hemoglobin in all three strains but were relatively lower than total glycosylated hemoglobin in diabetic DBA/2J mice. These results show that 6-h fasting glucose provides a superior index of glycemic control and correlates more closely with Hb A(1c) than overnight-fasted blood glucose in these strains of mice.

Insight into the genetics of diabetic nephropathy through the study of mice.

PURPOSE OF REVIEW: To discuss mouse models of diabetic nephropathy and their use in discovering genetic risk factors predisposing to diabetic nephropathy. RECENT FINDINGS: Despite occurring in only 10-40% of diabetic patients, diabetic nephropathy is the largest single cause of end stage renal disease in the USA. Accumulated evidence points to critical genetic factors that predispose a subset of diabetic patients to nephropathy. Defining the genes that confer risk for nephropathy in human populations has proven challenging. The use of robust genetic reagents available in the laboratory mouse provides a complementary approach to defining genes that predispose to diabetic nephropathy in mice and humans. These findings support the existence of dominant mutations predisposing to diabetic nephropathy in mice as well as substantiating an important role for eNOS in forestalling the development of diabetic nephropathy. SUMMARY: When studied for a sufficient duration of diabetic hyperglycemia, some strains of mice exhibit changes similar to those of human diabetic nephropathy. The unique genetic reagents in mice should help accelerate the identification of genes predisposing to diabetic nephropathy.

A sensitized screen of N-ethyl-N-nitrosourea-mutagenized mice identifies dominant mutants predisposed to diabetic nephropathy.

Diabetic nephropathy (DN) is a late diabetic complication that comprises progressively increasing albuminuria, declining GFR, and increased cardiovascular risk. Only a minority of patients with diabetes (25 to 40%) develop nephropathy, and there is evidence that heritable genetic factors predispose these "at-risk" individuals to DN. Comparing variability among inbred mouse strains with respect to a specific phenotype can model interhuman variability, and each strain represents a genetically homogeneous system with a defined risk for nephropathy. C57BL/6 mice, which are relatively resistant to DN, were mutagenized using N-ethyl-N-nitrosourea and screened for mutants that developed excess albuminuria on a sensitizing type 1 diabetic background contributed by the dominant Akita mutation in insulin-2 gene (Ins2(Akita)). Two of 375 diabetic G1 founders were found to exhibit albumin excretion rates persistently 10-fold greater than albumin excretion rates in nonmutagenized Ins2(Akita) controls. This albuminuria trait was heritable and transmitted to approximately 50% of Ins2(Akita) G2 and G3 progeny, consistent with a simple, dominantly inherited trait, but was never observed in nondiabetic offspring. During the course of 1 yr, albuminuric Ins2(Akita) G2 and G3 progeny developed reduced inulin clearance with elevated blood urea nitrogen and plasma creatinine. Glomerular histology revealed mesangial expansion, and glomerular basement membrane thickening as determined by electron microscopy was enhanced in diabetic mutant kidneys. Hereditary albuminuric N-ethyl-N-nitrosourea-induced mutants were redesignated as Nphrp1 (nephropathy1) and Nphrp2 (nephropathy2) mice for two generated lines. These novel mutants provide new, robust mouse models of DN and should help to elucidate the underlying genetic basis of predisposition to DN.

Differential roles of vascular endothelial growth factor receptors 1 and 2 in dendritic cell differentiation.

Impaired Ag-presenting function in dendritic cells (DCs) due to abnormal differentiation is an important mechanism of tumor escape from immune control. A major role for vascular endothelial growth factor (VEGF) and its receptors, VEGFR1/Flt-1 and VEGFR2/KDR/Flk-1, has been documented in hemopoietic development. To study the roles of each of these receptors in DC differentiation, we used an in vitro system of myeloid DC differentiation from murine embryonic stem cells. Exposure of wild-type, VEGFR1(-/-), or VEGFR2(-/-) embryonic stem cells to exogenous VEGF or the VEGFR1-specific ligand, placental growth factor, revealed distinct roles of VEGF receptors. VEGFR1 is the primary mediator of the VEGF inhibition of DC maturation, whereas VEGFR2 tyrosine kinase signaling is essential for early hemopoietic differentiation, but only marginally affects final DC maturation. SU5416, a VEGF receptor tyrosine kinase inhibitor, only partially rescued the mature DC phenotype in the presence of VEGF, suggesting the involvement of both tyrosine kinase-dependent and independent inhibitory mechanisms. VEGFR1 signaling was sufficient for blocking NF-kappaB activation in bone marrow hemopoietic progenitor cells. VEGF and placental growth factor affect the early stages of myeloid/DC differentiation. The data suggest that therapeutic strategies attempting to reverse the immunosuppressive effects of VEGF in cancer patients might be more effective if they specifically targeted VEGFR1.