Somatic USP8 alteration affects the immune landscape of corticotroph pituitary adenomas- a pilot study.

INTRODUCTION: Somatic mutations in ubiquitin-specific protease-8 (USP8), encoding a deubiquinating protein, are found in approximately 30% of corticotroph-derived pituitary adenomas (CPAs). Stratifin, a protein encoded by SFN, inhibits USP8 catalytic activity. USP8 has immunomodulating properties that have been demonstrated in non-tumoral diseases. METHODS: We assessed the influence of USP8 on the immune landscape of CPA and validated this effect and its dependency on stratifin in large cohorts of non-pituitary tumors. We analyzed data of CPA samples (n = 20) and additional non-pituitary tumors from the TCGA database, using transcriptome signature-recognition algorithms. Immune tumor microenvironment (iTME) was compared both by USP8 and SFN expression levels (n = 843) and by USP8 mutation status and SFN expression (n = 12,389). RESULTS: CPA with activating USP8 mutations was associated with "cold" iTME compared with wild-type USP8 CPA, as reflected by lower fractions of immune cells, including B cells, CD4, regulatory and gamma/delta T cells, natural killer cells, M0 and M1 macrophages, dendritic cells, and eosinophils (p < 0.05 for all comparisons). Pathways altered by the presence of USP8 mutation, based on the most differentially expressed genes (3061 genes), included microglia pathogen phagocytosis and multiple toll-like receptor signaling pathways (p < 0.0001). In a validation analysis based on large cohorts of non-pituitary tumors, high expression of USP8 was associated with a suppressed iTME effect that was augmented by a low SFN expression. CONCLUSIONS: Our data demonstrate for the first time, to our knowledge, a distinct immune landscape of tumors based on USP8 status and expression and the dependency of this immunological effect on SFN expression.

Visceral Adipose Tissue E2F1-miRNA206/210 Pathway Associates with Type 2 Diabetes in Humans with Extreme Obesity.

Objective: Up-regulated expression of transcription-factor E2F1 in human visceral adipose tissue (VAT) characterizes a dysmetabolic obesity sub-phenotype. An E2F1-miRNA network has been described in multiple cancers. Here we investigated whether elevated VAT-E2F1 in obesity is associated with VAT-miRNA alterations similar to, or distinct from, those described in cancer. Furthermore, we assessed if E2F1-associated miRNA changes may contribute to the link between high- VAT-E2F1 and a dysmetabolic obesity phenotype. Methods: We assembled a cohort of patients with obesity and high-VAT-E2F1, matched by age, sex, ±BMI to patients with low-VAT-E2F1, with and without obesity (8 patients/groupX3 groups). We performed Nanostring©-based miRNA profiling of VAT samples from all 24 patients. Candidate E2F1-related miRNAs were validated by qPCR in an independent cohort of patients with extreme obesity, with or without type-2-diabetes (T2DM) (n = 20). Bioinformatic tools and manipulation of E2F1 expression in cells were used to establish the plausibility of the functional VAT-E2F1-miRNA network in obesity. Results: Among n = 798 identified miRNAs, 17 were differentially expressed in relation to E2F1 and not to obesity itself. No evidence for the cancer-related E2F1-miRNA network was identified in human VAT in obesity. In HEK293-cells, overexpression/downregulation of E2F1 correspondingly altered the expression of miRNA-206 and miRNA-210-5p, two miRNAs with reported metabolic functions consistent with those of E2F1. In VAT from both cohorts, the expression of both miRNA-206 and 210-5p intercorrelated, and correlated with the expression of E2F1. In cohort 1 we did not detect significant associations with biochemical parameters. In cohort 2 of patients with extreme obesity, all those with high VAT-E2F1 showed a diabetes-complicated obesity phenotype and higher expression of miRNA-206 and miRNA-210-5p, which also correlated with fasting glucose levels (both miRNAs) and fasting insulin (miRNA-210-5p). Conclusions: Whilst the previously described cancer-related E2F1-miRNA network does not appear to operate in VAT in obesity, miRNAs-206 and 210-5p may link high-E2F1 expression in VAT with diabetes-complicated extreme obesity phenotype.

Real-world evidence for immunotherapy in the first line setting in small cell lung cancer.

OBJECTIVE: Despite major progress over the past decade in the field of lung cancer care, only mild advances have occurred in Small Cell Lung Cancer (SCLC), with prognosis remaining poor. Based on two randomized clinical trials (RCTs), two checkpoint-inhibitors have recently been approved in extensive-SCLC with moderate improvements in median overall survival (OS). However, only limited data exist regarding the impact of immunotherapy in real-world SCLC patients. This study reports the efficacy of immunotherapy in the first-line treatment of extensive-stage SCLC patients in a real-world setting. METHODS: a retrospective cohort study of all patients treated for extensive-SCLC with chemotherapy with or without immunotherapy, at a single center in Israel between October-2017 and July-2021. Patient characteristics, adverse-events and survival analyses were conducted. RESULTS: Of 102 patients identified, 54 patients (53%) received immunotherapy in addition to chemotherapy. 34.7% of patients had a performance status (PS) of 2-4. Patients that received only chemotherapy were older, had more liver metastasis and a poorer PS. In the whole cohort, patients receiving immunotherapy had a significantly longer median OS (353 days vs 194 days, HR = 0.40p < 0.0001). After stratification by PS groups, survival analysis remained significantly longer in the PS 0-1 group (HR 0.43, p = 0.0036), with a trend for better survival in the PS 2-3 group. Multivariate analysis validated an OS advantage with immunotherapy (HR = 0.46, p = 0.004). CONCLUSION: We present evidence for the efficacy of immunotherapy in SCLC in a real-world setting. Although treatment groups differ in their baseline characteristics, it appears that even some patients not included in RCTs, such as poor PS, may benefit from the addition of immunotherapy to their treatment protocol.

A TRAIL-TL1A Paracrine Network Involving Adipocytes, Macrophages, and Lymphocytes Induces Adipose Tissue Dysfunction Downstream of E2F1 in Human Obesity.

Elevated expression of E2F1 in adipocyte fraction of human visceral adipose tissue (hVAT) associates with a poor cardiometabolic profile. We hypothesized that beyond directly activating autophagy and MAP3K5 (ASK)-MAP kinase signaling, E2F1 governs a distinct transcriptome that contributes to adipose tissue and metabolic dysfunction in obesity. We performed RNA sequencing of hVAT samples from age-, sex-, and BMI-matched patients, all obese, whose visceral E2F1 protein expression was either high (E2F1high) or low (E2F1low). Tumor necrosis factor superfamily (TNFSF) members, including TRAIL (TNFSF10), TL1A (TNFSF15), and their receptors, were enriched in E2F1high While TRAIL was equally expressed in adipocytes and stromal vascular fraction (SVF), TL1A was mainly expressed in SVF, and TRAIL-induced TL1A was attributed to CD4+ and CD8+ subclasses of hVAT T cells. In human adipocytes, TL1A enhanced basal and impaired insulin-inhibitable lipolysis and altered adipokine secretion, and in human macrophages it induced foam cell biogenesis and M1 polarization. Two independent human cohorts confirmed associations between TL1A and TRAIL expression in hVAT and higher leptin and IL6 serum concentrations, diabetes status, and hVAT-macrophage lipid content. Jointly, we propose an intra-adipose tissue E2F1-associated TNFSF paracrine loop engaging lymphocytes, macrophages, and adipocytes, ultimately contributing to adipose tissue dysfunction in obesity.

Leptin stimulates autophagy/lysosome-related degradation of long-lived proteins in adipocytes.

Obesity, a condition most commonly associated with hyper-leptinemia, is also characterized by increased expression of autophagy genes and likely autophagic activity in human adipose tissue (AT). Indeed, circulating leptin levels were previously shown to positively associate with the expression levels of autophagy genes such as Autophagy related gene-5 (ATG5). Here we hypothesized that leptin acts in an autocrine-paracrine manner to increase autophagy in two major AT cell populations, adipocytes and macrophages. We followed the dynamics of autophagosomes following acute leptin administration with or without a leptin receptor antagonist (SMLA) using high-throughput live-cell imaging in murine epididymal adipocyte and macrophage (RAW264.7) cell-lines. In macrophages leptin exerted only a mild effect on autophagy dynamics, tending to attenuate autophagosomes growth rate. In contrast, leptin-treated adipocytes exhibited a moderate, ~20% increase in the rate of autophagosome growth, an effect that was blocked by SMLA. This finding corresponded to mild increases in mRNA and protein expression of key autophagy genes. Interestingly, a long-lived proteins degradation assay uncovered a robust, >2-fold leptin-mediated stimulation of the autophagy/lysosome-related (bafilomycin-inhibited) activity, which was entirely blocked by SMLA. Collectively, leptin regulates autophagy in a cell-type specific manner. In adipocytes, autophagosome dynamics is moderately enhanced, but even more pronounced stimulation is seen in autophagy-related long-lived protein degradation. These findings suggest a causal link between obesity-associated hyperleptinemia and elevated adipocyte and AT autophagy-related processes.