Extracellular vesicles are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles, which originate from the endosomal system or which are shed from the plasma membrane, respectively They are present in biological fluids and are involved in multiple physiological and pathological processes Extracellular vesicles are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material Knowledge of the cellular processes that govern extracellular vesicle biology is essential to shed light on the physiological and pathological functions of these vesicles as well as on clinical applications involving their use and/or analysis However, in this expanding field, much remains unknown regarding the origin, biogenesis, secretion, targeting and fate of these vesicles

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Shedding light on the cell biology of extracellular vesicles.

Extracellular vesicles (EVs) have become the focus of rising interest because of their numerous functions in physiology and pathology. Cells release heterogeneous vesicles of different sizes and intracellular origins, including small EVs formed inside endosomal compartments (i.e., exosomes) and EVs of various sizes budding from the plasma membrane. Specific markers for the analysis and isolation of different EV populations are missing, imposing important limitations to understanding EV functions. Here, EVs from human dendritic cells were first separated by their sedimentation speed, and then either by their behavior upon upward floatation into iodixanol gradients or by immuno-isolation. Extensive quantitative proteomic analysis allowing comparison of the isolated populations showed that several classically used exosome markers, like major histocompatibility complex, flotillin, and heat-shock 70-kDa proteins, are similarly present in all EVs. We identified proteins specifically enriched in small EVs, and define a set of five protein categories displaying different relative abundance in distinct EV populations. We demonstrate the presence of exosomal and nonexosomal subpopulations within small EVs, and propose their differential separation by immuno-isolation using either CD63, CD81, or CD9. Our work thus provides guidelines to define subtypes of EVs for future functional studies.

https://www.pnas.org/content/pnas/113/8/E968.full.pdf

Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.

The ability of exosomes to transfer cargo from donor to acceptor cells, thereby triggering phenotypic changes in the latter, has generated substantial interest in the scientific community. However, the extent to which exosomes differ from other extracellular vesicles in terms of their biogenesis and functions remains ill-defined. Here, we discuss the current knowledge on the specificities of exosomes and other types of extracellular vesicles, and their roles as important agents of cell-to-cell communication.

Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication.

The mechanistic target of rapamycin (mTOR) coordinates eukaryotic cell growth and metabolism with environmental inputs, including nutrients and growth factors. Extensive research over the past two decades has established a central role for mTOR in regulating many fundamental cell processes, from protein synthesis to autophagy, and deregulated mTOR signaling is implicated in the progression of cancer and diabetes, as well as the aging process. Here, we review recent advances in our understanding of mTOR function, regulation, and importance in mammalian physiology. We also highlight how the mTOR signaling network contributes to human disease and discuss the current and future prospects for therapeutically targeting mTOR in the clinic.

mTOR Signaling in Growth, Metabolism, and Disease

Release of membrane vesicles, a process conserved in both prokaryotes and eukaryotes, represents an evolutionary link, and suggests essential functions of a dynamic extracellular vesicular compartment (including exosomes, microparticles or microvesicles and apoptotic bodies). Compelling evidence supports the significance of this compartment in a broad range of physiological and pathological processes. However, classification of membrane vesicles, protocols of their isolation and detection, molecular details of vesicular release, clearance and biological functions are still under intense investigation. Here, we give a comprehensive overview of extracellular vesicles. After discussing the technical pitfalls and potential artifacts of the rapidly emerging field, we compare results from meta-analyses of published proteomic studies on membrane vesicles. We also summarize clinical implications of membrane vesicles. Lessons from this compartment challenge current paradigms concerning the mechanisms of intercellular communication and immune regulation. Furthermore, its clinical implementation may open new perspectives in translational medicine both in diagnostics and therapy.

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Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles

Depletion of creatine phosphagen energetics with a covalent creatine kinase inhibitor


 Emerging classifiers that leverage recent advances in the characterization of mutational signatures have identified a broader range of cancer patients with DNA repair deficiencies, many of whom do not harbor mutations or epigenetic silencing of canonical DNA repair genes. Investigating the various mechanisms underlying dysfunctional DNA damage response (DDR) and heterogeneity across patients will be crucial to understand disparate responses to therapy, including to PARP1 inhibitors and to immunotherapy. In this study, we integrate genomic, transcriptomic, proteomic, and post-translational modification (PTM) data across 11 cancer types to elucidate the diverse functional mechanisms of DNA repair deficiencies and their therapeutic implications through an in-depth proteogenomic analysis. We first characterized the landscape of mutational signatures in 1110 patients by applying SignatureAnalyzer, a tool that uses a Bayesian non-negative matrix factorization method to discover patterns of somatic mutations. Our analysis identified 144 homologous recombination-deficient (HRD) and 60 mismatch repair-deficient (MMRD) patients. Multi-omic (mRNA, protein, PTMs) signature based hierarchical clustering revealed two distinct clusters of HRD patients defined by either (i) acute or (ii) chronic hypoxic states. The acute hypoxia cluster exhibited typical HRD phenotypes, including a preference for non-homologous end joining (NHEJ) and enrichment in PARP1 activity, as well as phosphorylation patterns driven by DNA-PKcs, ATM, and ATR. In contrast, tumors under chronic hypoxia exhibited a halting of replication and silencing of DDR, suggesting that patients with these tumors may respond poorly to common HRD treatments that exploit alternative error-prone repair and PARP1 activation. Notably, these tumors were enriched in signatures representative of highly active kinases involved in cellular stress responses, potentially providing additional avenues for therapy. Additional analysis of both HRD and MMRD tumors revealed groups of highly immunogenic DNA repair-deficient tumors that may respond well to some types of immunotherapy given their increased mutagenesis. Moreover, differences in inflammation-related kinase activities offer further insight into the interaction between faulty DDR and the innate immune response (e.g., alterations in the cGAS-STING DNA sensing pathway). Overall, our multi-omic signature analysis provides insights into the mechanisms underlying DDR and its crosstalk with other cancer-related pathways that remain unexplained by evaluating genomic and transcriptomic data alone. Our characterization of the heterogeneity within the population of patients with DNA repair deficiency shows promise for improved prediction of treatment response, as well as identifying additional potential therapeutic targets.
 Citation Format: Yo Akiyama, Yifat Geffen, Shankara Anand, Tomer Yaron, Jared L. Johnson, Emily Huntsman, David Heiman, Chet Birger, Karl Clauser, Özgün Babur, Mendy Miller, Karsten Krug, D R Mani, Lewis C. Cantley, Steven A. Carr, PanCancer Oncogenic Drivers and Pathways Group, Clinical Proteomic Tumor Analysis Consortium (CPTAC), Francois Aguet, Li Ding, Gad Getz. Pan-cancer proteogenomic analysis reveals functional mechanisms underlying DNA repair deficiencies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 794.

Abstract 794: Pan-cancer proteogenomic analysis reveals functional mechanisms underlying DNA repair deficiencies


 Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that responds to cellular and oxidative stress by promoting the transcription of antioxidant response element-containing genes. In an unstressed cellular environment, NRF2 is bound by Kelch-like ECH-associated protein 1 (KEAP1) and tagged for proteasomal degradation. In many cancers, the interaction between NRF2 and KEAP1 is interrupted due to mutations in either protein. Previous work has suggested that NRF2 can promote DNA damage repair, and our preliminary data demonstrates that NRF2 is phosphorylated at Serine-40 by the DNA damage response kinase, Ataxia Telangiectasia Mutated (ATM) in response to DNA damage. To create a system to better study the relationship between NRF2 Serine-40 phosphorylation and DNA damage, we modified the mouse Nfe2l2 locus to generate a mouse model in which Nrf2 Serine-40 is mutated to Alanine on a C57BL/6 background. Utilizing this model, we administered 7.5 Gy of total body irradiation and found that Nrf2S40A/S40A mice had significantly shortened survival compared to Nrf2WT mice. Immunohistochemical analysis of radiation-sensitive tissues including bone marrow and spleen revealed a prolonged persistence of the DNA damage marker γH2AX following irradiation in the Nrf2S40A/S40A group compared to Nrf2WT. We also observed a significantly higher level of cell death in the Nrf2S40A/S40A splenic tissue compared with Nrf2WT as indicated by the marker of apoptotic cell death, Cleaved Caspase-3. Our findings indicate that phosphorylation of Nrf2 at Serine-40 in response to DNA damage promotes DNA damage repair. Our ongoing work is focused on examining these phenotypes in cellular systems to determine the specific mechanisms by which NRF2 promotes DNA damage repair. Future work will include incorporating the systems generated thus far into non-small cell lung cancer (NSCLC) and liver cancer mouse models to further investigate the impact of this phosphorylation site on tumorigenesis and radio-resistance.
 Citation Format: Cheyenne A. Schneider, Tin-Yu Lin, Aimee Falzone, Samantha Caldwell, Jared L. Johnson, Gina M. DeNicola. ATM phosphorylation of NRF2 promotes DNA damage repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 309.

Abstract 309: ATM phosphorylation of NRF2 promotes DNA damage repair

The phosphoinositide 3-kinase, p110α, is an essential mediator of insulin signaling and glucose homeostasis. We systematically interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate and completely inhibit its activity. This inhibitory state corresponds to a conformational change of a membrane binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases, MST1/2, using forskolin or epinephrine is associated with phosphorylation and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules. Our study reveals a novel inhibitory pathway of PI3K signaling and a previously unappreciated link between epinephrine and insulin signaling.

Epinephrine inhibits PI3K alpha via the Hippo kinases

Previously, we showed that coagulation factors directly cleave SARS-CoV-2 spike and promote viral entry (Kastenhuber et al., 2022). Here, we show that substitutions in the S1/S2 cleavage site observed in SARS-CoV-2 variants of concern (VOCs) exhibit divergent interactions with host proteases, including factor Xa and furin. Nafamostat remains effective to block coagulation factor-mediated cleavage of variant spike sequences. Furthermore, host protease usage has likely been a selection pressure throughout coronavirus evolution, and we observe convergence of distantly related coronaviruses to attain common host protease interactions, including coagulation factors. Interpretation of genomic surveillance of emerging SARS-CoV-2 variants and future zoonotic spillover is supported by functional characterization of recurrent emerging features.

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Jared L. Johnson

Papers

Depletion of creatine phosphagen energetics with a covalent creatine kinase inhibitor

Abstract 794: Pan-cancer proteogenomic analysis reveals functional mechanisms underlying DNA repair deficiencies

Abstract 309: ATM phosphorylation of NRF2 promotes DNA damage repair

Epinephrine inhibits PI3K alpha via the Hippo kinases

Evolution of host protease interactions among SARS-CoV-2 variants of concern and related coronaviruses