The emerging and diverse roles of the SLy/SASH1-protein family in health and disease-Overview of three multifunctional proteins.

Intracellular adaptor proteins are indispensable for the transduction of receptor-derived signals, as they recruit and connect essential downstream effectors. The SLy/SASH1-adaptor family comprises three highly homologous proteins, all of them sharing conserved structural motifs. The initial characterization of the first member SLy1/SASH3 (SH3 protein expressed in lymphocytes 1) in 2001 was rapidly followed by identification of SLy2/HACS1 (hematopoietic adaptor containing SH3 and SAM domains 1) and SASH1/SLy3 (SAM and SH3 domain containing 1). Based on their pronounced sequence similarity, they were subsequently classified as one family of intracellular scaffold proteins. Despite their obvious homology, the three SLy/SASH1-members fundamentally differ with regard to their expression and function in intracellular signaling. On the contrary, growing evidence clearly demonstrates an important role of all three proteins in human health and disease. In this review, we systematically summarize what is known about the SLy/SASH1-adaptors in the field of molecular cell biology and immunology. To this end, we recapitulate current research about SLy1/SASH3, SLy2/HACS1, and SASH1/SLy3, with an emphasis on their similarities and differences.

Use of plasma ctDNA as a potential biomarker for longitudinal monitoring of a patient with metastatic high-risk upper tract urothelial carcinoma receiving pembrolizumab and personalized neoepitope-derived multipeptide vaccinations: a case report.

Upper tract urothelial carcinoma (UTUC) is often diagnosed late and exhibits poor prognosis. Only limited data are available concerning therapeutic regimes and potential biomarkers for disease monitoring. Standard therapies often provide only insufficient treatment options. Hence, immunotherapies and complementary approaches, such as personalized neoepitope-derived multipeptide vaccine (PNMV), come into focus. In this context, genetic analysis of tumor tissue by whole exome sequencing represents an essential diagnostic step in order to calculate tumor mutational burden (TMB) and to reveal tumor-specific neoantigens. Furthermore, disease progression is essential to be monitored. Longitudinal screening of individually known mutations in plasma circulating tumor DNA (ctDNA) by the use of next-generation sequencing and digital droplet PCR (ddPCR) might be a promising method to fill this gap.Here, we present the case of a 55-year-old man who was diagnosed with high-risk metastatic UTUC in 2015. After initial surgery and palliative chemotherapy, he developed recurrence of the tumor. Genetic analysis revealed a high TMB of 41.2 mutations per megabase suggesting a potential success of immunotherapy. Therefore, in 2016, off-label treatment with the checkpoint-inhibitor pembrolizumab was started leading to strong regression of the disease. This therapy was then discontinued due to side effects and treatment with a previously produced PNMV was started that induced strong T cell responses. During both treatments, plasma Liquid Biopsies (pLBs) were performed to measure the number of mutated molecules per mL plasma (MM/mL) of a known tumor-specific variant in the MLH1 gene by ddPCR for longitudinal monitoring. Under treatment, MM/mL was constantly zero. A few months after all therapies had been discontinued, an increase of MM/mL was detected that persisted in the following pLBs. When MRI scans proved tumor recurrence, treatment with pembrolizumab was started again leading to a rapid decrease of MM/mL in the pLB to again zero. Treatment response was then also confirmed by MRI.This case shows that use of immunotherapy and PNMV might be a promising treatment option for patients with high-risk metastatic UTUC. Furthermore, measurement of individually known tumor mutations in plasma ctDNA by the use of pLB could be a very sensitive biomarker to longitudinally monitor disease.

Deficiency of PI3-Kinase catalytic isoforms p110γ and p110δ in mice enhances the IL-17/G-CSF axis and induces neutrophilia.

BACKGROUND: Phosphoinositide 3-kinase γ (PI3Kγ) and PI3Kδ are second messenger-generating enzymes with key roles in proliferation, differentiation, survival, and function of leukocytes. Deficiency of the catalytic subunits p110γ and p110δ of PI3Kγ and PI3Kδ in p110γ/δ-/- mice leads to defective B- and T-cell homeostasis. Here we examined the role of p110γ and p110δ in the homeostasis of neutrophils by analyzing p110γ-/-, p110δ-/- and p110γ/δ-/- mice. METHODS: Neutrophils and T cells in leukocyte suspensions from the bone marrow (BM), blood, spleen and lung were analyzed by flow cytometry. Serum concentrations of IL-17, of the neutrophilic growth factor G-CSF, and of the neutrophil mobilizing CXC chemokines CXCL1/KC and CXCL2/MIP-2 were measured by Bio-Plex assay. Production of G-CSF and CXCL1/KC by IL-17-stimulated primary lung tissue cells were determined by ELISA, whereas IL-17-dependent signaling in lung tissue cells was analyzed by measuring Akt phosphorylation using immunoblot. RESULTS: We found that in contrast to single knock-out mice, p110γ/δ-/- mice exhibited significantly elevated neutrophil counts in blood, spleen, and lung. Increased granulocytic differentiation stages in the bone marrow of p110γ/δ-/- mice were paralleled by increased serum concentrations of G-CSF, CXCL1/KC, and CXCL2/MIP-2. As IL-17 induces neutrophilia via the induction of G-CSF and CXC chemokines, we measured IL-17 and IL-17-producing T cells. IL-17 serum concentrations and frequencies of IL-17+ splenic T cells were significantly increased in p110γ/δ-/- mice. Moreover, IFN-γ+, IL-4+, and IL-5+ T cell subsets were drastically increased in p110γ/δ-/- mice, suggesting that IL-17+ T cells were up-regulated in the context of a general percentage increase of other cytokine producing T cell subsets. CONCLUSIONS: We found that p110γ/δ deficiency in mice induces complex immunological changes, which might in concert contribute to neutrophilia. These findings emphasize a crucial but indirect role of both p110γ and p110δ in the regulation of neutrophil homeostasis.