Modulation of cellular transcriptome and proteome composition by azidohomoalanine-implications on click chemistry-based secretome analysis.

The analysis of the secretome provides important information on proteins defining intercellular communication and the recruitment and behavior of cells in specific tissues. Especially in the context of tumors, secretome data can support decisions for diagnosis and therapy. The mass spectrometry-based analysis of cell-conditioned media is widely used for the unbiased characterization of cancer secretomes in vitro. Metabolic labeling using azide-containing amino acid analogs in combination with click chemistry facilitates this type of analysis in the presence of serum, preventing serum starvation-induced effects. The modified amino acid analogs, however, are less efficiently incorporated into newly synthesized proteins and may perturb protein folding. Combining transcriptome and proteome analysis, we elucidate in detail the effects of metabolic labeling with the methionine analog azidohomoalanine (AHA) on gene and protein expression. Our data reveal that 15-39% of the proteins detected in the secretome displayed changes in transcript and protein expression induced by AHA labeling. Gene Ontology (GO) analyses indicate that metabolic labeling using AHA leads to induction of cellular stress and apoptosis-related pathways and provide first insights on how this affects the composition of the secretome on a global scale. KEY MESSAGES: Azide-containing amino acid analogs affect gene expression profiles. Azide-containing amino acid analogs influence cellular proteome. Azidohomoalanine labeling induces cellular stress and apoptotic pathways. Secretome consists of proteins with dysregulated expression profiles.

ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization.

The actin cytoskeleton is tightly controlled by RhoGTPases, actin binding-proteins and nucleation-promoting factors to perform fundamental cellular functions. We have previously shown that ERK3, an atypical MAPK, controls IL-8 production and chemotaxis (Bogueka et al., 2020). Here, we show in human cells that ERK3 directly acts as a guanine nucleotide exchange factor for CDC42 and phosphorylates the ARP3 subunit of the ARP2/3 complex at S418 to promote filopodia formation and actin polymerization, respectively. Consistently, depletion of ERK3 prevented both basal and EGF-dependent RAC1 and CDC42 activation, maintenance of F-actin content, filopodia formation, and epithelial cell migration. Further, ERK3 protein bound directly to the purified ARP2/3 complex and augmented polymerization of actin in vitro. ERK3 kinase activity was required for the formation of actin-rich protrusions in mammalian cells. These findings unveil a fundamentally unique pathway employed by cells to control actin-dependent cellular functions.

Multiomic analysis of papillary thyroid cancers identifies BAIAP2L1-BRAF fusion and requirement of TRIM25, PDE5A and PKCδ for tumorigenesis.

BACKGROUND: Papillary thyroid carcinoma (PTC) is one of the most common forms of thyroid cancer with a cure rate of over 90% after surgery. However, aggressive forms may still occur, and personalized therapeutic strategies are increasingly required. METHODS: We performed integrated genomic and proteomic analysis of PTC tumor samples from patients who did not harbor BRAF or RAS mutations. We validate the analysis and present in-depth molecular analysis of the identified genetic rearrangement by employing biochemical and cell biological assays. Finally, we employ 3D spheroid models, loss of function studies and chemical inhibitors to target the hitherto upregulated factors. The data are analysed with appropriate statistical tests which are mentioned in the legends section. RESULTS: In a 23-year-old patient with thyroiditis, we identified a novel rearrangement leading to a BAIAP2L1-BRAF fusion that transforms immortalized human thyroid cells in a kinase and CC-domain dependent manner. Moreover, quantitative proteomic analysis of the same patient samples revealed the upregulation of several proteins including the Ubiquitin E3 ligase TRIM25, PDE5A, and PKCδ. Further, in a cohort of PTC patients, we observed higher expression of TRIM25 and PKCδ in the tumor and metastatic lesions, when compared to the matched normal tissue. Inhibition of TRIM25, PDE5A and PKCδ with small molecules or RNA interference affected not only viability and proliferation of BAIAP2L1-BRAF transformed cells, but also the viability, growth and invasion of corresponding 3D spheroid cultures. CONCLUSIONS: Apart from unveiling a novel oncogenic BRAF fusion in PTCs, our data may open a novel avenue of therapeutic targeting in human PTCs.

Recent advances in targeting protein kinases and pseudokinases in cancer biology.

Kinases still remain the most favorable members of the druggable genome, and there are an increasing number of kinase inhibitors approved by the FDA to treat a variety of cancers. Here, we summarize recent developments in targeting kinases and pseudokinases with some examples. Targeting the cell cycle machinery garnered significant clinical success, however, a large section of the kinome remains understudied. We also review recent developments in the understanding of pseudokinases and discuss approaches on how to effectively target in cancer.

Development of fluorizoline analogues as prohibitin ligands that modulate C-RAF signaling, p21 expression and melanogenesis.

Fluorizoline is a cytotoxic trifluorothiazoline that targets the scaffold proteins prohibitins-1 and -2 (PHB1/2) to inhibit the kinase C-RAF and promote the expression of the cyclin-dependent kinase inhibitor p21 to induce cancer cell death. In melanocytes, fluorizoline also induces the synthesis of melanin. Herein we report the first structural requirement of fluorizoline analogues for these activities. We identified in particular some compounds that display enhanced anti-C-RAF and anti-MEK activities, and a higher cytotoxicity in HeLa cells compared to fluorizoline. These results provide a foundation for further optimization of PHB ligands for the treatment of cancers. We also discovered an analogue of fluorizoline that displays pharmacological effects opposed to those of fluorizoline and that can be used as a chemical tool to explore PHB signaling in cancers and other diseases.

The caspase-2 substrate p54nrb exhibits a multifaceted role in tumor cell death susceptibility via gene regulatory functions.

Caspase-2 represents an evolutionary conserved caspase, which plays a role in genotoxic stress-induced apoptosis, ageing-related metabolic changes, and in deleting aneuploid cells in tumors. Genetic deletion of caspase-2 leads to increased tumor susceptibility in vivo. The exact downstream signaling mechanism by which caspase-2 accomplishes its specific tumor suppressor functions is not clear. Caspase-2, uniquely among caspases, resides in the nucleus and other cellular compartments. In this study, we identify a nuclear caspase-2 specific substrate, p54nrb, which is selectively cleaved by caspase-2 at D422, leading to disruption of the C-terminal site, the putative DNA binding region of the protein. P54nrb is an RNA and DNA binding protein, which plays a role in RNA editing, transport, and transcriptional regulation of genes. Overexpression of p54nrb is observed in several human tumor types, such as cervix adenocarcinoma, melanoma, and colon carcinoma. In contrast, the loss of p54nrb in tumor cell lines leads to increased cell death susceptibility and striking decrease in tumorigenic potential. By employing high resolution quantitative proteomics, we demonstrate that the loss/cleavage of p54nrb results in altered expression of oncogenic genes, among which the downregulation of the tumorigenic protease cathepsin-Z and the anti-apoptotic gelsolin can be detected universally across three tumor cell types, including adenocarcinoma, melanoma and colon carcinoma. Finally, we demonstrate that p54nrb interacts with cathepsin-Z and gelsolin DNA, but not RNA. Taken together, this study uncovers a so far not understood mechanism of caspase-2 tumor suppressor function in human tumor cells.

Enhanced pro-apoptosis gene signature following the activation of TAp63α in oocytes upon γ irradiation.

Specialized surveillance mechanisms are essential to maintain the genetic integrity of germ cells, which are not only the source of all somatic cells but also of the germ cells of the next generation. DNA damage and chromosomal aberrations are, therefore, not only detrimental for the individual but affect the entire species. In oocytes, the surveillance of the structural integrity of the DNA is maintained by the p53 family member TAp63α. The TAp63α protein is highly expressed in a closed and inactive state and gets activated to the open conformation upon the detection of DNA damage, in particular DNA double-strand breaks. To understand the cellular response to DNA damage that leads to the TAp63α triggered oocyte death we have investigated the RNA transcriptome of oocytes following irradiation at different time points. The analysis shows enhanced expression of pro-apoptotic and typical p53 target genes such as CDKn1a or Mdm2, concomitant with the activation of TAp63α. While DNA repair genes are not upregulated, inflammation-related genes become transcribed when apoptosis is initiated by activation of STAT transcription factors. Furthermore, comparison with the transcriptional profile of the ΔNp63α isoform from other studies shows only a minimal overlap, suggesting distinct regulatory programs of different p63 isoforms.

ARAF suppresses ERBB3 expression and metastasis in a subset of lung cancers.

RAF kinases are highly conserved serine/threonine kinases, and among the three RAF isoforms (ARAF, BRAF, and CRAF), the pathophysiological relevance of ARAF is not well defined. Here, we show that patients with lung cancer exhibit low expression of ARAF, which is associated with lymph node metastasis and poor patient survival. We uncover that depletion of ARAF promotes anchorage-independent growth and metastasis through activation of AKT signaling in a subset of lung cancer cells. We identified that loss of ARAF was associated with an increase in ERBB3 expression in a kinase-independent manner. ARAF suppressed the promoter activity of ERBB3, and reconstitution of ARAF in ARAF-depleted cells led to the reversal of enhanced ERBB3-AKT signaling. Furthermore, ARAF inhibited neuregulin 1 (hNRG1)-mediated AKT activation through controlling ERBB3 expression via the transcription factor KLF5. Our results disclose a critical dual role for ARAF kinase in the negative regulation of ERBB3-AKT signaling, thereby suppressing tumor metastasis.

Systemically Administered TLR7/8 Agonist and Antigen-Conjugated Nanogels Govern Immune Responses against Tumors.

The generation of specific humoral and cellular immune responses plays a pivotal role in the development of effective vaccines against tumors. Especially the presence of antigen-specific, cytotoxic T cells influences the outcome of therapeutic cancer vaccinations. Different strategies, ranging from delivering antigen-encoding mRNAs to peptides or full antigens, are accessible but often suffer from insufficient immunogenicity and require immune-boosting adjuvants as well as carrier platforms to ensure stability and adequate retention. Here, we introduce a pH-responsive nanogel platform as a two-component antitumor vaccine that is safe for intravenous application and elicits robust immune responses in vitro and in vivo. The underlying chemical design allows for straightforward covalent attachment of a model antigen (ovalbumin) and an immune adjuvant (imidazoquinoline-type TLR7/8 agonist) onto the same nanocarrier system. In addition to eliciting antigen-specific T and B cell responses that outperform mixtures of individual components, our two-component nanovaccine leads in prophylactic and therapeutic studies to an antigen-specific growth reduction of different tumors expressing ovalbumin intracellularly or on their surface. Regarding the versatile opportunities for functionalization, our nanogels are promising for the development of highly customized and potent nanovaccines.

Patterning of Oncogenic Ras Clustering in Live Cells Using Vertically Aligned Nanostructure Arrays.

As a dominant oncogenic protein, Ras is well-known to segregate into clusters on the plasma membrane for activating downstream signaling. However, current technologies for direct measurements of Ras clustering are limited to sophisticated high-resolution techniques like electron microscopy and fluorescence lifetime imaging. To further promote fundamental investigations and the related drug development, we hereby introduce a nanobar-based platform which effectively guides Ras clusters into quantifiable patterns in live cells that is resolvable under conventional microscopy. Major Ras isoforms, K-Ras, H-Ras, and N-Ras, were differentiated, as well as their highly prevalent oncogenic mutants G12V and G13D. Moreover, the isoform specificity and the sensitivity of a Ras inhibitor were successfully characterized on nanobars. We envision that this nanobar-based platform will serve as an effective tool to read Ras clustering on the plasma membrane, enabling a novel avenue both to decipher Ras regulations and to facilitate anti-Ras drug development.

SAMHD1 in cancer: curse or cure?

Human sterile α motif and HD domain-containing protein 1 (SAMHD1), originally described as the major cellular deoxyribonucleoside triphosphate triphosphohydrolase (dNTPase) balancing the intracellular deoxynucleotide (dNTP) pool, has come recently into focus of cancer research. As outlined in this review, SAMHD1 has been reported to be mutated in a variety of cancer types and the expression of SAMHD1 is dysregulated in many cancers. Therefore, SAMHD1 is regarded as a tumor suppressor in certain tumors. Moreover, it has been proposed that SAMHD1 might fulfill the requirements of a driver gene in tumor development or might promote a so-called mutator phenotype. Besides its role as a dNTPase, several novel cellular functions of SAMHD1 have come to light only recently, including a role as negative regulator of innate immune responses and as facilitator of DNA end resection during DNA replication and repair. Therefore, SAMHD1 can be placed at the crossroads of various cellular processes. The present review summarizes the negative role of SAMHD1 in chemotherapy sensitivity, highlights reported SAMHD1 mutations found in various cancer types, and aims to discuss functional consequences as well as underlying mechanisms of SAMHD1 dysregulation potentially involved in cancer development.

ERK5 modulates IL-6 secretion and contributes to tumor-induced immune suppression.

Tumors exhibit a variety of strategies to dampen antitumor immune responses. With an aim to identify factors that are secreted from tumor cells, we performed an unbiased mass spectrometry-based secretome analysis in lung cancer cells. Interleukin-6 (IL-6) has been identified as a prominent factor secreted by tumor cells and cancer-associated fibroblasts isolated from cancer patients. Incubation of dendritic cell (DC) cultures with tumor cell supernatants inhibited the production of IL-12p70 in DCs but not the surface expression of other activation markers which is reversed by treatment with IL-6 antibody. Defects in IL-12p70 production in the DCs inhibited the differentiation of Th1 but not Th2 and Th17 cells from naïve CD4+ T cells. We also demonstrate that the classical mitogen-activated protein kinase, ERK5/MAPK7, is required for IL-6 production in tumor cells. Inhibition of ERK5 activity or depletion of ERK5 prevented IL-6 production in tumor cells, which could be exploited for enhancing antitumor immune responses.

Cilium induction triggers differentiation of glioma stem cells.

Glioblastoma multiforme (GBM) possesses glioma stem cells (GSCs) that promote self-renewal, tumor propagation, and relapse. Understanding the mechanisms of GSCs self-renewal can offer targeted therapeutic interventions. However, insufficient knowledge of GSCs' fundamental biology is a significant bottleneck hindering these efforts. Here, we show that patient-derived GSCs recruit elevated levels of proteins that ensure the temporal cilium disassembly, leading to suppressed ciliogenesis. Depleting the cilia disassembly complex components is sufficient to induce ciliogenesis in a subset of GSCs via relocating platelet-derived growth factor receptor-alpha (PDGFR-α) to a newly induced cilium. Importantly, restoring ciliogenesis enabled GSCs to switch from self-renewal to differentiation. Finally, using an organoid-based glioma invasion assay and brain xenografts in mice, we establish that ciliogenesis-induced differentiation can prevent the infiltration of GSCs into the brain. Our findings illustrate a role for cilium as a molecular switch in determining GSCs' fate and suggest cilium induction as an attractive strategy to intervene in GSCs proliferation.

Famotidine inhibits toll-like receptor 3-mediated inflammatory signaling in SARS-CoV-2 infection.

Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.

SIK2 orchestrates actin-dependent host response upon Salmonella infection.

Salmonella is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in Salmonella infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon Salmonella infection. SIK2 depletion favors the escape of bacteria from the Salmonella-containing vacuole (SCV) and impairs Xenophagy, resulting in a hyperproliferative phenotype. Mechanistically, SIK2 associates with actin filaments under basal conditions; however, during bacterial infection, SIK2 is recruited to the SCV together with the elements of the actin polymerization machinery (Arp2/3 complex and Formins). Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. We propose that SIK2 controls the formation of a protective SCV actin shield shortly after invasion and orchestrates the actin cytoskeleton architecture in its entirety to control an acute Salmonella infection after bacterial invasion.

Druggable genome and precision medicine in cancer: current challenges.

The past decades have seen tremendous developments with respect to "specific" therapeutics that target key signaling molecules to conquer cancer. The key advancements with multiomics technologies, especially genomics, have allowed physicians and molecular oncologists to design "tailor-made" solutions to the specific oncogenes that are deregulated in individual patients, a strategy which has turned out to be successful though the patients quickly develop resistance. The swift integration of multidisciplinary approaches has led to the development of "next generation" therapeutics and, with synergistic therapeutic regimes combined with immune checkpoint inhibitors to reactivate the dampened immune response, has provided the much-needed promise for cancer patients. Despite these advances, a large portion of the druggable genome remains understudied, and the role of druggable genome in the immune system needs further attention. Establishment of patient-derived organoid models has fastened the preclinical validation of novel therapeutics for swift clinical translation. We summarized the current advances and challenges and also stress the importance of biobanking and collection of longitudinal data sets with structured clinical information, as well as the critical role these "high content data sets" will play in designing new therapeutic regimes in a tailor-made fashion.

ERK3/MAPK6 is required for KRAS-mediated NSCLC tumorigenesis.

KRAS is one of the most frequently mutated oncogenes, especially in lung cancers. Targeting of KRAS directly or the downstream effector signaling machinery is of prime interest in treating lung cancers. Here, we uncover that ERK3, a ubiquitously expressed atypical MAPK, is required for KRAS-mediated NSCLC tumors. ERK3 is highly expressed in lung cancers, and oncogenic KRAS led to the activation and stabilization of the ERK3 protein. In particular, phosphorylation of serine 189 in the activation motif of ERK3 is significantly increased in lung adenocarcinomas in comparison to adjacent normal controls in patients. Loss of ERK3 prevents the anchorage-independent growth of KRAS G12C-transformed human bronchial epithelial cells. We further find that loss of ERK3 reduces the oncogenic growth of KRAS G12C-driven NSCLC tumors in vivo and that the kinase activity of ERK3 is required for KRAS-driven oncogenesis in vitro. Our results demonstrate an obligatory role for ERK3 in NSCLC tumor progression and suggest that ERK3 kinase inhibitors can be pursued for treating KRAS G12C-driven tumors.

The non-linearity of RAF-MEK signaling in dendritic cells.

Kinases form the major part of the druggable genome and their selective inhibition in human cancers has had reasonable clinical success. In contrast to tumorigenesis, the role of kinases in mediating immune responses is poorly understood. However, synergistic therapeutic regimens combining targeted therapy and immune therapy have been found to increase the median survival of tumor patients. In this context, we uncovered that RAF and MEK1/2 kinases, which are the integral parts of the classical MAPK cascade, have unique roles in driving DC differentiation and activation. RAF kinases are stabilized in their protein levels during DC differentiation and are obligatory for normal functioning of DCs. But, the targeting of MEK1/2 kinases with specific inhibitors did not phenocopy the effects observed with RAF inhibitors suggesting that RAF and MEK1/2 kinases may have specific and unique roles in driving immune responses, which deserves further studies to successfully administer these inhibitors in clinics.

Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity.

The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses3-5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.