Kinome and phosphoproteome reprogramming underlies the aberrant immune responses in critically ill COVID-19 patients.

SARS-CoV-2 infection triggers extensive host immune reactions, leading to severe diseases in certain individuals. However, the molecular basis underlying the excessive yet non-productive immune responses in severe COVID-19 remains incompletely understood. In this study, we conducted a comprehensive analysis of the peripheral blood mononuclear cell (PBMC) proteome and phosphoproteome in sepsis patients positive or negative for SARS-CoV-2 infection, as well as healthy subjects, using quantitative mass spectrometry. Our findings demonstrate dynamic changes in the COVID-19 PBMC proteome and phosphoproteome during disease progression, with distinctive protein or phosphoprotein signatures capable of distinguishing longitudinal disease states. Furthermore, SARS-CoV-2 infection induces a global reprogramming of the kinome and phosphoproteome, resulting in defective adaptive immune response mediated by the B and T lymphocytes, compromised innate immune responses involving the SIGLEC and SLAM family of immunoreceptors, and excessive cytokine-JAK-STAT signaling. In addition to uncovering host proteome and phosphoproteome aberrations caused by SARS-CoV-2, our work recapitulates several reported therapeutic targets for COVID-19 and identified numerous new candidates, including the kinases PKG1, CK2, ROCK1/2, GRK2, SYK, JAK2/3, TYK2, DNA-PK, PKCδ, and the cytokine IL-12.

Global pannexin 1 deletion increases tumor-infiltrating lymphocytes in the BRAF/Pten mouse melanoma model.

Immunotherapies for malignant melanoma seek to boost the anti-tumoral response of CD8+ T cells, but have a limited patient response rate, in part due to limited tumoral immune cell infiltration. Genetic or pharmacological inhibition of the pannexin 1 (PANX1) channel-forming protein is known to decrease melanoma cell tumorigenic properties in vitro and ex vivo. Here, we crossed Panx1 knockout (Panx1-/-) mice with the inducible melanoma model BrafCA, PtenloxP, Tyr::CreERT2 (BPC). We found that deleting the Panx1 gene in mice does not reduce BRAF(V600E)/Pten-driven primary tumor formation or improve survival. However, tumors in BPC-Panx1-/- mice exhibited a significant increase in the infiltration of CD8+ T lymphocytes, with no changes in the expression of early T-cell activation marker CD69, lymphocyte activation gene 3 protein (LAG-3) checkpoint receptor, or programmed cell death ligand-1 (PD-L1) in tumors when compared to the BPC-Panx1+/+ genotype. Our results suggest that, although Panx1 deletion does not overturn the aggressive BRAF/Pten-driven melanoma progression in vivo, it does increase the infiltration of effector immune T-cell populations in the tumor microenvironment. We propose that PANX1-targeted therapy could be explored as a strategy to increase tumor-infiltrating lymphocytes to boost anti-tumor immunity.

Expression of pannexin1 in lung cancer brain metastasis and immune microenvironment.

Brain metastases are the most common central nervous system malignancy, and the leading cause of cancer-related deaths. Non-small cell lung carcinomas (NSCLC) comprise the most common cell of origin. Immunotherapy, particularly checkpoint inhibitors, has emerged as the standard of care for many patients with advanced lung cancer. Pannexin1 (PANX1) is a transmembrane glycoprotein that forms large-pore channels and has been reported to promote cancer metastasis. However, the roles of PANX1 in lung cancer brain metastases and tumor immune microenvironment have not been characterized. 42 patient-matched formalin-fixed paraffin-embedded tissue samples from lung carcinomas and the subsequent brain metastases were constructed into three tissue microarrays (TMAs). PANX1 and markers of tumor-infiltrating immune cells (CD3, CD4, CD8, CD68, and TMEM119) were assessed using immunohistochemistry and digital image analysis. The expression of PANX1 was significantly higher in brain metastases than in their paired primary lung carcinoma. The high levels of PANX1 in lung carcinoma cells in the brain inversely correlated with infiltration of peripheral blood-derived macrophages. Our findings highlight the role of PANX1 in the progression of metastatic NSCLC, and the potential therapeutic approach of targeting PANX1 enhances the efficacy of immune checkpoint inhibitors in brain metastasis.

The spatial transcriptomic landscape of non-small cell lung cancer brain metastasis.

Brain metastases (BrMs) are a common occurrence in lung cancer with a dismal outcome. To understand the mechanism of metastasis to inform prognosis and treatment, here we analyze primary and metastasized tumor specimens from 44 non-small cell lung cancer patients by spatial RNA sequencing, affording a whole transcriptome map of metastasis resolved with morphological markers for the tumor core, tumor immune microenvironment (TIME), and tumor brain microenvironment (TBME). Our data indicate that the tumor microenvironment (TME) in the brain, including the TIME and TBME, undergoes extensive remodeling to create an immunosuppressive and fibrogenic niche for the BrMs. Specifically, the brain TME is characterized with reduced antigen presentation and B/T cell function, increased neutrophils and M2-type macrophages, immature microglia, and reactive astrocytes. Differential gene expression and network analysis identify fibrosis and immune regulation as the major functional modules disrupted in both the lung and brain TME. Besides providing systems-level insights into the mechanism of lung cancer brain metastasis, our study uncovers potential prognostic biomarkers and suggests that therapeutic strategies should be tailored to the immune and fibrosis status of the BrMs.

Proteome and phosphoproteome signatures of recurrence for HPV+ head and neck squamous cell carcinoma.

Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and the human papillomavirus (HPV+)-driven subtype is the fastest rising cancer in North America. Although most cases of HPV+ HNSCC respond favorably to the treatment via surgery followed by radiochemotherapy, up to 20% recur with a poor prognosis. The molecular and cellular mechanisms of recurrence are not fully understood. Methods: To gain insights into the mechanisms of recurrence and to inform patient stratification and personalized treatment, we compared the proteome and phosphoproteome of recurrent and non-recurrent tumors by quantitative mass spectrometry. Results: We observe significant differences between the recurrent and non-recurrent tumors in cellular composition, function, and signaling. The recurrent tumors are characterized by a pro-fibrotic and immunosuppressive tumor microenvironment (TME) featuring markedly more abundant cancer-associated fibroblasts, extracellular matrix (ECM), neutrophils, and suppressive myeloid cells. Defective T cell function and increased epithelial-mesenchymal transition potential are also associated with recurrence. These cellular changes in the TME are accompanied by reprogramming of the kinome and the signaling networks that regulate the ECM, cytoskeletal reorganization, cell adhesion, neutrophil function, and coagulation. Conclusions: In addition to providing systems-level insights into the molecular basis of recurrence, our work identifies numerous mechanism-based, candidate biomarkers and therapeutic targets that may aid future endeavors to develop prognostic biomarkers and precision-targeted treatment for recurrent HPV+ HNSCC.

Head and neck cancer can be caused by the human papillomavirus. While this type of cancer responds well to chemotherapy given simultaneously with radiation, a significant proportion of cases recur within a few years, leading to illness and sometimes death in these patients. It is therefore important to understand the mechanisms of recurrence in order to develop better treatments. By comparing the levels of proteins and protein phosphorylation­a type of modification that affects how proteins work­between tumors from patients with or without recurrence, we found that the cells surrounding recurrent tumors show signs of fibrosis­the development of fibrous connective tissue­and suppression of the body's immune responses. This suggests that therapies directed towards the regulators of fibrosis and immune suppression may help to overcome recurrent head and neck cancer.

Astrocyte Role in Temporal Lobe Epilepsy and Development of Mossy Fiber Sprouting.

Epilepsy affects approximately 50 million people worldwide, with 60% of adult epilepsies presenting an onset of focal origin. The most common focal epilepsy is temporal lobe epilepsy (TLE). The role of astrocytes in the presentation and development of TLE has been increasingly studied and discussed within the literature. The most common histopathological diagnosis of TLE is hippocampal sclerosis. Hippocampal sclerosis is characterized by neuronal cell loss within the Cornu ammonis and reactive astrogliosis. In some cases, mossy fiber sprouting may be observed. Mossy fiber sprouting has been controversial in its contribution to epileptogenesis in TLE patients, and the mechanisms surrounding the phenomenon have yet to be elucidated. Several studies have reported that mossy fiber sprouting has an almost certain co-existence with reactive astrogliosis within the hippocampus under epileptic conditions. Astrocytes are known to play an important role in the survival and axonal outgrowth of central and peripheral nervous system neurons, pointing to a potential role of astrocytes in TLE and associated cellular alterations. Herein, we review the recent developments surrounding the role of astrocytes in the pathogenic process of TLE and mossy fiber sprouting, with a focus on proposed signaling pathways and cellular mechanisms, histological observations, and clinical correlations in human patients.