Aging is the main risk factor for many chronic degenerative diseases and cancer. Increased senescent cell burden in various tissues is a major contributor to aging and age-related diseases. Recently, a new class of drugs termed senolytics were demonstrated to extending healthspan, reducing frailty and improving stem cell function in multiple murine models of aging. To identify novel and more optimal senotherapeutic drugs and combinations, we established a senescence associated β-galactosidase assay as a screening platform to rapidly identify drugs that specifically affect senescent cells. We used primary Ercc1 −/− murine embryonic fibroblasts with reduced DNA repair capacity, which senesce rapidly if grown at atmospheric oxygen. This platform was used to screen a small library of compounds that regulate autophagy, identifying two inhibitors of the HSP90 chaperone family as having significant senolytic activity in mouse and human cells. Treatment of Ercc1 −/∆ mice, a mouse model of a human progeroid syndrome, with the HSP90 inhibitor 17-DMAG extended healthspan, delayed the onset of several age-related symptoms and reduced p16INK4a expression. These results demonstrate the utility of our screening platform to identify senotherapeutic agents as well as identified HSP90 inhibitors as a promising new class of senolytic drugs.

Background Calcium plays an essential role in nearly all cellular processes. As such, cellular and systemic calcium concentrations are tightly regulated. During sepsis derangements in such tight regulation frequently occur, and treating hypocalcemia with parenteral calcium administration remains the current practice guideline. Objective We investigated whether calcium administration worsens mortality and organ dysfunction using an experimental murine model of sepsis and explored the mechanistic role of the family of calcium/calmodulin-dependent protein kinases in mediating these physiologic effects. To highlight the biological relevance of these observations, we conducted a translational study of the association between calcium administration, organ dysfunction and mortality among a cohort of critically ill septic ICU patients Design Prospective, randomized controlled experimental murine study. Observational clinical cohort analysis. Setting University research laboratory. Eight ICUs at a tertiary care center. Patients 870 septic ICU patients. Subjects C57BL/6 and CaMKK−/− mice. Interventions Mice underwent cecal ligation and puncture polymicrobial sepsis and were administered calcium chloride (0.25 or 0.25 mg/kg) or normal saline. Measurements and Main Results Administering calcium chloride to septic C57BL/6 mice heightened systemic inflammation and vascular leak, exacerbated hepatic and renal dysfunction, and increased mortality. These events were significantly attenuated in CaMKK−/− mice. In a risk–adjusted analysis of septic patients, calcium administration was associated with an increased risk of death, OR 1.92 (95% CI 1.00–3.68, p=0.049), a significant increase in the risk of renal dysfunction, OR 4.74 (95% CI 2.48–9.08, p<0.001), and a significant reduction in ventilator free days, mean decrease 3.29 days (0.50–6.08 days, p=0.02). Conclusions Derangements in calcium homeostasis occur during sepsis that are sensitive to calcium administration. This altered calcium signaling, transduced by the CaMKK cascade, mediates heightened inflammation and vascular leak that culminates in elevated organ dysfunction and mortality. In the clinical management of septic patients calcium supplementation provides no benefit and may impose harm.

Background Immunotherapy with CAR T-cells is actively being explored for pediatric brain tumors in preclinical models and early phase clinical studies. At present it is unclear which CAR target antigens are consistently expressed across different pediatric brain tumor types. In addition, the extent of HLA class-I expression is unknown, which is critical for tumor recognition by conventional αβTCR T-cells. Methods We profiled 49 low- and high-grade pediatric brain tumor patient-derived orthotopic xenografts (PDOX) by flow analysis for the expression of five CAR targets (B7-H3, GD2, IL13Rα2, EphA2, HER2), and HLA class-I. In addition, we generated B7-H3-CAR T-cells and evaluated their antitumor activity in vitro and in vivo. Results We established an expression hierarchy for the analyzed antigens (B7-H3 = GD2 &gt;&gt; IL13Rα2 &gt; HER2 = EphA2) and demonstrated that antigen expression is heterogenous. All high-grade gliomas expressed HLA class-I, but only 57.1% of other tumor subtypes had detectable expression. We then selected B7-H3 as a target for CAR T-cell therapy. B7-H3-CAR T-cells recognized tumor cells in an antigen-dependent fashion. Local or systemic administration of B7-H3-CAR T-cells induced tumor regression in PDOX and immunocompetent murine glioma models resulting in a significant survival advantage. Conclusions Our study highlights the importance of studying target antigen and HLA class-I expression in PDOX samples for the future design of immunotherapies. In addition, our results support active preclinical and clinical exploration of B7-H3-targeted CAR T-cell therapies for a broad spectrum of pediatric brain tumors.

Dysregulated Ca(2+) handling is prevalent during sepsis and postulated to perpetuate the aberrant inflammation underlying subsequent organ dysfunction and death. The signal transduction cascades mediating these processes are unknown. Here, we identify that CaMKIα mediates the Mϕ response to LPS in vitro and the inflammation and organ dysfunction of sepsis in vivo. We show that LPS induced active pThr(177)-CaMKIα in RAW 264.7 cells and murine peritoneal Mϕ, which if inhibited biochemically with STO609 (CaMKK inhibitor) or by RNAi, reduces LPS-induced production of IL-10. Transfection of constitutively active CaMKIα (CaMKI293), but not a kinase-deficient mutant (CaMKI293(K49A)), induces IL-10 release. This production of IL-10 is mediated by CaMKIα-dependent regulation of p38 MAPK activation. CaMKIα activity also mediates the cellular release of HMGB1 by colocalizing with and regulating the packaging of HMGB1 into secretory lysosomes. During endotoxemia, mice receiving in vivo CaMKIα(RNAi) display reduced systemic concentrations of IL-10 and HMGB1 in comparison with mice receiving NT(RNAi). These data support the biological relevance of CaMKIα-dependent IL-10 production and HMGB1 secretion. In a CLP model of sepsis, CaMKIα(RNAi) mice display reduced systemic concentrations of IL-10, IL-6, TNF-α, and HMGB1 in comparison with NT(RNAi) mice, which correlate with reductions in the development of renal dysfunction. These data support that CaMKIα signaling is integral to the Mϕ responding to LPS and may also be operant in vivo in regulating the inflammation and organ dysfunction consequent to sepsis.

Autophagy is an evolutionarily conserved cytoplasmic process regulated by the energy rheostats mTOR and AMPK that recycles damaged or unused proteins and organelles. It has been described as an important effector arm of immune cells. We have shown that the cytoplasmically oriented calcium/calmodulin-dependent protein kinase I α (CaMKIα) regulates the inflammatory phenotype of the macrophage (Mφ). Here, we hypothesize that CaMKIα mediates Mφ autophagy. LPS induced autophagy in RAW 264.7 cells and murine peritoneal Mφ that was attenuated with biochemical CaMK inhibition or CaMKIα siRNA. Inhibition of CaMKIα reduced LPS-induced p-Thr172AMPK and TORC1 activity, and expression of a constitutively active CaMKIα but not a kinase-deficient mutant induced p-Thr172AMPK and autophagy that was attenuated by the AMPK inhibitor Compound C. Co-immunoprecipitation and in vitro kinase assays demonstrated that CaMKIα activates AMPK, thereby inducing ATG7, which also localizes to this CaMKIα-AMPK complex. During LPS-induced lung inflammation, C57Bl/6 mice receiving CaMKIαsiRNA displayed reduced lung and bronchoalveolar immune cell autophagy that correlated with reduced neutrophil recruitment, myeloperoxidase activity, and air space cytokine concentration. Independently inhibiting autophagy, using siRNA targeting the PI3 kinase VPS34, yielded similar reductions in lung autophagy and neutrophil recruitment. Thus, a novel CaMKIα-AMPK pathway is rapidly activated in Mφ exposed to LPS and regulates an early autophagic response, independent of TORC1 inhibition. These mechanisms appear to be operant in vivo in orchestrating LPS-induced lung neutrophil recruitment and inflammation.