Biology subjects

Horejs-Hoeck, J.

Publications and source records attributed to Horejs-Hoeck, J..

6 recordsLinked to original sources

bioRxiv2024

ADAM17 regulates hepatic DNA damage repair and tumour formation

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths worldwide. Still, therapy options for this tumour entity are limited and novel therapeutic options are highly sought after. Genomic instability of hepatocytes promotes oncogenic transformation and underlies the regulation of micro-environmental signalling cues. The membrane-bound a disintegrin and metalloprotease (ADAM) 17 is a major regulator of micro-environmental signals through the proteolytic release of paracrine factors. However, its role in hepatic DNA damage repair and its contribution to hepatic tumourigenesis is still unclear. MethodsWe investigated the effect of ADAM17 on diethylnitrosamine (DEN)-induced acute DNA damage and subsequent DNA damage repair by utilizing mice with ubiquitous or myeloid-specific genetic deficiency in ADAM17. DNA double strand breaks and inflammation were investigated by immunofluorescence of liver tissue sections. tumourigenesis in mice with myeloid-specific ADAM17-deficiency was investigated in a chemically induced hepatocarcinogenesis model. ResultsADAM17 on myeloid cells, in particular Kupffer cells is essentially involved in the non-cell autonomous regulation of DNA damage repair in hepatocytes. Parenchymal ADAM17 regulates hepatocyte fate and recruitment of infiltrating myeloid cells. Furthermore, myeloid ADAM17 promotes hepatic tumour initiation and correlates with poor prognosis in human HCC. ConclusionsWe identified ADAM17, in particular on myeloid cells as an essential driver of hepatic tumourigenesis and as a potential novel drug target for the treatment of hepatic malignancies.

cancer biology↗

bioRxiv2024

Hedgehog-Hippo pathway interactions promote T cell exclusion from the tumor microenvironment in basal cell carcinoma

Basal cell carcinoma (BCC) is the most common non-melanoma skin cancer driven primarily by genetic activation of the Hedgehog/GLI (HH/GLI) signaling pathway. BCC also displays a high mutational burden, with frequent co-occurrence of mutations in the TP53, NOTCH, N- MYC, and Hippo/YAP pathways, though the impact of these mutations on HH/GLI-driven tumorigenesis remains poorly understood. This study examines the interaction between the HH/GLI and Hippo/YAP signaling pathways in the pathogenesis of BCC and the establishment of an immunosuppressive tumor microenvironment (TME). The study used 3D human organotypic skin models and humanized mouse models to demonstrate that co-activation of HH/GLI and Hippo/YAP in epidermal cells suppresses T cell chemotaxis. Transcriptome analysis revealed a significant downregulation of chemotactic and inflammatory genes, particularly under combined GLI-YAP activation. Analysis of primary human BCC biopsies confirmed these findings and demonstrated a marked suppression of T cell chemo-attractants, including CCL22 and CCL27. Spatial profiling of immune cells in BCC tissues revealed preferential exclusion of CD8+ T cells from the TME, which correlated with high HH/GLI and Hippo/YAP activity. Further, functional assays in humanized mice showed that the GLI-YAP interaction controls immune cell distribution by inhibiting T cell recruitment and migration into the skin. These findings suggest that cooperation between oncogenic HH/GLI and Hippo/YAP signaling contributes to the development of an immunosuppressive TME in BCC by disrupting local chemokine signaling. Understanding these mechanisms provides a basis for the development of targeted combination therapies to enhance the efficacy of existing treatments, such as Smoothened (SMO) inhibitors and immune checkpoint blockers, by promoting T cell infiltration and activation within the TME.

cancer biology↗

bioRxiv2021

The NLRP3/eIF2 axis drives cell cycle progression in acute myeloid leukemia

Aberrant activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome mediates numerous inflammatory diseases. Oncogenes can activate the NLRP3 inflammasome and thereby promote myeloproliferative neoplasia, suggesting a crucial role of NLRP3 in the malignant transformation of hematopoietic cells. Here, we show that bone marrow-derived mononuclear cells of AML patients display enhanced expression of NLRP3, IL-1{beta} and IL-18 and that high-level expression of NLRP3 is linked to poor survival of AML patients. Pharmacological and genetic inhibition of NLRP3 inflammasome activation attenuated cell proliferation of MOLM-13 AML cells in vitro. In vivo, genetic inhibition of NLRP3 in MOLM-13 AML cells resulted in reduced engraftment potential in xenografts, along with reduced splenomegaly and organ infiltration. Differential proteomic analysis revealed the eIF2 pathway as potential target of NLRP3 in AML, with a significant increase of eIF2 phosphorylation upon NLRP3 inhibition. NLRP3 inhibition also caused a strong decrease in cyclin - dependent kinases CDK4 and CDK6, accompanied by an upregulation of the CDK inhibitor p21 (CDKN1A) and a marked arrest of cell cycle progression in the G0/G1 phase, consistent with the role of eIF2 phosphorylation as negative cell cycle regulator. Taken together, we show that inhibition of the NLRP3 inflammasome reduces AML cell proliferation by promoting eIF2 phosphorylation, which in turn enhances the expression of cell cycle arrest genes such as p21. Thus, the study uncovers the NLRP3/eIF2 axis as new driver of AML proliferation and proposes a novel therapeutic treatment of AML by targeted inhibition of NLRP3 activation.

immunology↗

bioRxiv2020

Oncogenic GLI1 and STAT1/3 activation drives immunosuppressive tryptophan/kynurenine metabolism via synergistic induction of IDO1 in skin cancer cells

Pharmacological targeting of Hedgehog (HH)/GLI has proven effective for certain blood, brain and skin cancers including basal cell carcinoma (BCC). However, limited response rates and the development of drug resistance call for improved anti-HH therapies that take into account synergistic crosstalk mechanisms and immune evasion strategies. In previous work, we demonstrated that crosstalk of HH/GLI with pro-inflammatory Interleukin-6 (IL6) signaling drives BCC by promoting tumor cell proliferation [1]. In the present study, we screened for possible mechanisms of cancer immune evasion regulated by synergistic HH-IL6 signaling and identified the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1) as a novel transcriptional target co-regulated by HH-IL6 signaling. Analysis of the cis-regulatory region of IDO1 by chromatin-immunoprecipitation revealed co-occupancy of this region by HH- IL6 induced GLI1 and STAT3 transcription factors along with active chromatin marks at the histone level. Elevated expression of IDO1 in human BCC with high-level HH and IL6 signatures supports the clinical relevance of our mechanistic data. Genetic inhibition of GLI1 expression prevented the induction of IDO1 expression in response to IL6/STAT3 and IFN{gamma}/STAT1 signaling in human melanoma cells. Pharmacological targeting of HH signaling at the level of GLI proteins interfered with IDO1 expression and consequently prevented the production of the immunosuppressive metabolite kynurenine generated by active IDO1 from tryptophan. Further, inhibition of GLI1 enhanced the efficacy of the selective IDO1 inhibitor epacadostat. Of note, inhibition of HH/GLI signaling in melanoma cells not only reduced IDO1 expression but also interfered with the repression of T cell activation by attenuating IDO1/kynurenine-mediated immunosuppression. These data identify the immunosuppressive IDO1-kynurenine pathway as a novel pro-tumorigenic effector of oncogenic HH-IL6 and GLI-STAT cooperation. Our data suggest simultaneous pharmacological targeting of the HH/GLI, JAK/STAT and IDO1- kynurenine axis as rational combination therapy in skin cancers.

cancer biology↗

bioRxiv2020

In silico design of Phl p 6 variants with altered folding stability significantly impacts antigen processing, immunogenicity and immune polarization

AbstactO_ST_ABSIntroductionC_ST_ABSProtein fold stability has been proposed to represent an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Using in silico prediction, we introduced point mutations in proteins that either increase or decrease their fold-stability without altering immunodominant epitopes or changing the overall structure of the protein. Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization. MethodsUsing the MAESTRO software tool, stabilizing or destabilizing mutations were selected and verified by molecular dynamics simulations. The mutants were expressed in E. coli, purified tag-free, and analyzed for thermal stability and resistance to endolysosomal proteases. The resulting peptides were analysed by degradome assay and mass spectrometry. The structure of the most stable mutant protein was obtained by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation in vitro, as well as antibody responses and T cell polarization in vivo in an adjuvant-free BALB/c mouse model. ResultsFour stabilizing and two destabilizing mutations were identified by MAESTRO. Experimentally determined changes in thermal stability compared to the wild type protein ranged from -5 to +14 {degrees}C. Destabilization led to faster proteolytic processing in vitro, whereas highly stabilized mutants were degraded very slowly. However, the overall pattern of identified peptides remained very similar. This was confirmed in bone marrow derived dendritic cells that processed and presented the immune dominant epitope from a destabilized mutant more efficiently. In vivo, stabilization resulted in a shift in immune polarization as indicated by higher levels of IgG2a and increased secretion of TH1/TH17 cytokines. ConclusionMAESTRO was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with susceptibility to protease resistance and presentation of pMHCII on the surface of dendritic cells in vitro. This change in processing kinetics significantly influenced the polarization of T cell responses in vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.

immunology↗

bioRxiv2020

Laser-facilitated epicutaneous immunotherapy with hypoallergenic beta-glucan neoglycoconjugates suppresses lung inflammation and avoids local side effects in a mouse model of allergic asthma

BackgroundAllergen-specific immunotherapy via the skin targets an area rich in antigen presenting cells, but can be associated with local and systemic side effect. Allergen-polysaccharide neoglycogonjugates can increase immunization efficacy by targeting and activating dendritic cells via C-type lectin receptors and reduce side effects. ObjectiveWe investigated the immunogenicity, allergenicity and therapeutic efficacy of laminarin-ovalbumin neoglycoconjugates (LamOVA). MethodsThe biological activity of LamOVA was characterized in vitro using bone marrow derived dendritic cells. Immunogenicity and therapeutic efficacy was analyzed in BALB/c mice. Epicutaneous immunotherapy (EPIT) was performed using fractional infrared laser ablation to generate micropores in the skin and the effects of LamOVA on blocking IgG, IgE, cellular composition of BAL, lung, and spleen, lung function, and T cell polarization was assessed. ResultsConjugation of laminarin to ovalbumin reduced its IgE binding capacity 5-fold and increased its immunogenitiy 3-fold in terms of IgG generation. EPIT with LamOVA induced significantly higher IgG levels than OVA, matching the levels induced by s.c. injection of OVA/alum (SCIT). EPIT was equally effective as SCIT in terms of blocking IgG induction and suppression of lung inflammation and airway hyperresponsiveness, but SCIT was associated with a higher level of therapy induced IgE and TH2 cytokines. EPIT with LamOVA induced significantly lower local skin reactions during therapy compared to unconjugated OVA. ConclusionConjugation of the allergen to laminarin increased its immunogenicity while at the same time reducing local side effects. LamOVA EPIT via laser generated micropores is safe and equally effective to SCIT with alum, without the need for adjuvant.

immunology↗