Heparan Sulfate Induces Necroptosis in Murine Cardiomyocytes: A Medical-In silico Approach Combining In vitro Experiments and Machine Learning.

Life-threatening cardiomyopathy is a severe, but common, complication associated with severe trauma or sepsis. Several signaling pathways involved in apoptosis and necroptosis are linked to trauma- or sepsis-associated cardiomyopathy. However, the underling causative factors are still debatable. Heparan sulfate (HS) fragments belong to the class of danger/damage-associated molecular patterns liberated from endothelial-bound proteoglycans by heparanase during tissue injury associated with trauma or sepsis. We hypothesized that HS induces apoptosis or necroptosis in murine cardiomyocytes. By using a novel Medical-In silico approach that combines conventional cell culture experiments with machine learning algorithms, we aimed to reduce a significant part of the expensive and time-consuming cell culture experiments and data generation by using computational intelligence (refinement and replacement). Cardiomyocytes exposed to HS showed an activation of the intrinsic apoptosis signal pathway via cytochrome C and the activation of caspase 3 (both p < 0.001). Notably, the exposure of HS resulted in the induction of necroptosis by tumor necrosis factor α and receptor interaction protein 3 (p < 0.05; p < 0.01) and, hence, an increased level of necrotic cardiomyocytes. In conclusion, using this novel Medical-In silico approach, our data suggest (i) that HS induces necroptosis in cardiomyocytes by phosphorylation (activation) of receptor-interacting protein 3, (ii) that HS is a therapeutic target in trauma- or sepsis-associated cardiomyopathy, and (iii) indicate that this proof-of-concept is a first step toward simulating the extent of activated components in the pro-apoptotic pathway induced by HS with only a small data set gained from the in vitro experiments by using machine learning algorithms.This work was supported by an intramural grant to LM (START 46/16) and EZ (START 113/17). LM has received a grant by the Deutsche Forschungsgemeinschaft (DFG, MA 7082/1–1). We thank Dr Claycomb and his coworkers for providing the HL-1 cells and a detailed documentation. The Immunohistochemistry and Confocal Microscopy Unit, a core facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen, within the Faculty of Medicine at the RWTH Aachen University, supported this work

STING agonism overcomes STAT3-mediated immunosuppression and adaptive resistance to PARP inhibition in ovarian cancer

BackgroundPoly (ADP-ribose) polymerase (PARP) inhibition (PARPi) has demonstrated potent therapeutic efficacy in patients with BRCA-mutant ovarian cancer. However, acquired resistance to PARPi remains a major challenge in the clinic.MethodsPARPi-resistant ovarian cancer mouse models were generated by long-term treatment of olaparib in syngeneic Brca1-deficient ovarian tumors. Signal transducer and activator of transcription 3 (STAT3)-mediated immunosuppression was investigated in vitro by co-culture experiments and in vivo by analysis of immune cells in the tumor microenvironment (TME) of human and mouse PARPi-resistant tumors. Whole genome transcriptome analysis was performed to assess the antitumor immunomodulatory effect of STING (stimulator of interferon genes) agonists on myeloid cells in the TME of PARPi-resistant ovarian tumors. A STING agonist was used to overcome STAT3-mediated immunosuppression and acquired PARPi resistance in syngeneic and patient-derived xenografts models of ovarian cancer.ResultsIn this study, we uncover an adaptive resistance mechanism to PARP inhibition mediated by tumor-associated macrophages (TAMs) in the TME. Markedly increased populations of protumor macrophages are found in BRCA-deficient ovarian tumors that rendered resistance to PARPi in both murine models and patients. Mechanistically, PARP inhibition elevates the STAT3 signaling pathway in tumor cells, which in turn promotes protumor polarization of TAMs. STAT3 ablation in tumor cells mitigates polarization of protumor macrophages and increases tumor-infiltrating T cells on PARP inhibition. These findings are corroborated in patient-derived, PARPi-resistant BRCA1-mutant ovarian tumors. Importantly, STING agonists reshape the immunosuppressive TME by reprogramming myeloid cells and overcome the TME-dependent adaptive resistance to PARPi in ovarian cancer. This effect is further enhanced by addition of the programmed cell death protein-1 blockade.ConclusionsWe elucidate an adaptive immunosuppression mechanism rendering resistance to PARPi in BRCA1-mutant ovarian tumors. This is mediated by enrichment of protumor TAMs propelled by PARPi-induced STAT3 activation in tumor cells. We also provide a new strategy to reshape the immunosuppressive TME with STING agonists and overcome PARPi resistance in ovarian cancer.Peer reviewe

Role of STAT3 N-terminal domain and GAS-site recognition in signaling and crosstalk with STAT1 and NF-κB

Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous transcription factor involved in many biological processes, including hematopoiesis, development and immune response. Dysfunctional STAT3 signalling has been reported in many pathophysiological conditions such cancer, chronic inflammation and fibrosis. In the first part of this work, we investigated the functions of the N-terminal domain and GAS-site recognition during IL-6-induced STAT3 signaling. Our results demonstrate the nonessential role of GAS-element recognition for both cytokine-induced and basal nuclear import of STAT3. In turn, deletion of the NTD markedly decreased nuclear accumulation upon IL-6 treatment resulting in a prolonged accumulation of phosphorylated dimers in the cytoplasm, at the same time preserving specific DNA recognition ability of the truncation mutant. Observed defect in nuclear localization could not be explained by flawed importin-α binding. Furthermore, our data indicated mechanistic differences between active and latent nuclear trafficking of STAT3, as well as between STAT1 and STAT3 active nuclear import.In the second part of this thesis, we analyzed the excessive STAT1 activation in STAT3-deficient cells upon IL-6 treatment. Our findings show that STAT3-mediated regulation of IL-6-induced STAT1 signaling depends on STAT3 target gene expression, but not on isolated STAT3 NTD functions. In the third part, we demonstrated that NF-κB and STAT3 have no direct influence on each other canonical signaling pathways. Instead, the expression of NF-κB subunit p65 correlates with total levels of STAT1 and STAT3.In the final part of this work, we characterized a STAT3-YFP knock-in murine model as a potentially powerful tool for the visualization of STAT3 in vivo. Our data show that STAT3-YFP Knock-In mice have been successfully generated and that the YFP fluorescence can be detected by common microscopy techniques. The STAT3-YFP knock-in mice will be a valuable tool for deciphering the function of STAT3 in vivo

Dissecting functions of the N-terminal domain and GAS-site recognition in STAT3

Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous transcription factor involved in many biological processes, including hematopoiesis, inflammation and cancer progression. Cytokine-induced gene transcription greatly depends on tyrosine phosphorylation of STAT3 on a single tyrosine residue with subsequent nuclear accumulation and specific DNA sequence (GAS) recognition. In this study, we analyzed the roles of the conserved STAT3 N-terminal domain (NTD) and GAS-element binding ability of STAT3 in nucleocytoplasmic trafficking. Our results demonstrate the nonessential role of GAS-element recognition for both cytokine-induced and basal nuclear import of STAT3. Substitution of five key amino acids within the DNA-binding domain rendered STAT3 unable to bind to GAS-elements while still maintaining the ability for nuclear localization. In turn, deletion of the NTD markedly decreased nuclear accumulation upon IL-6 treatment resulting in a prolonged accumulation of phosphorylated dimers in the cytoplasm, at the same time preserving specific DNA recognition ability of the truncation mutant. Observed defect in nuclear localization could not be explained by flawed importin-α binding, since both wild-type and NTD deletion mutant of STAT3 could precipitate both full-length and autoinhibitory domain (∆ IBB) deletion mutants of importin-α5, as well as ∆ IBB-α3 and ∆ IBB-α7 isoforms independently of IL-6 stimulation. Despite its inability to translocate to the nucleus upon IL-6 stimulation, the NTD lacking mutant still showed nuclear accumulation in resting cells similar to wild-type upon inhibition of nuclear export by leptomycin B. At the same time, blocking the nuclear export pathway could not rescue cytoplasmic trapping of phosphorylated STAT3 molecules without NTD. Moreover, STAT3 mutant with dysfunctional SH2 domain (R609Q) also localized in the nucleus of unstimulated cells after nuclear export blocking, while upon cytokine treatment the subcellular localization of this mutant had not changed. Our findings support the concept that basal nucleocytoplasmic shuttling of STAT3 is different from active cytokine-induced nuclear import and does not require conserved N- or SH2-terminal domains, preformed dimer formation and GAS-element-specific DNA recognition

Angptl4 is upregulated under inflammatory conditions in the bone marrow of mice, expands myeloid progenitors, and accelerates reconstitution of platelets after myelosuppressive therapy

BACKGROUND: Upon inflammation, myeloid cell generation in the bone marrow (BM) is broadly enhanced by the action of induced cytokines which are produced locally and at multiple sites throughout the body. METHODS: Using microarray studies, we found that Angptl4 is upregulated in the BM during systemic inflammation. RESULTS: Recombinant murine Angptl4 (rmAngptl4) stimulated the proliferation of myeloid colony-forming units (CFUs) in vitro. Upon repeated in vivo injections, rmAngptl4 increased BM progenitor cell frequency and this was paralleled by a relative increase in phenotypically defined granulocyte-macrophage progenitors (GMPs). Furthermore, in vivo treatment with rmAngptl4 resulted in elevated platelet counts in steady-state mice while allowing a significant acceleration of reconstitution of platelets after myelosuppressive therapy. The administration of rmAngptl4 increased the number of CD61(+)CD41(low)-expressing megakaryocytes (MK) in the BM of steady-state and in the spleen of transplanted mice. Furthermore, rmAngptl4 improved the in vitro differentiation of immature MKs from hematopoietic stem and progenitor cells. Mechanistically, using a signal transducer and activator of transcription 3 (STAT3) reporter knockin model, we show that rmAngptl4 induces de novo STAT3 expression in immature MK which could be important for the effective expansion of MKs after myelosuppressive therapy. CONCLUSION: Whereas the definitive role of Angptl4 in mediating the effects of lipopolysaccharide (LPS) on the BM has to be demonstrated by further studies involving multiple cytokine knockouts, our data suggest that Angptl4 plays a critical role during hematopoietic, especially megakaryopoietic, reconstitution following stem cell transplantation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13045-015-0152-2) contains supplementary material, which is available to authorized users