Signaling proteins in HSC fate determination are unequally segregated during asymmetric cell division.

Hematopoietic stem cells (HSCs) continuously replenish mature blood cells with limited lifespans. To maintain the HSC compartment while ensuring output of differentiated cells, HSCs undergo asymmetric cell division (ACD), generating two daughter cells with different fates: one will proliferate and give rise to the differentiated cells' progeny, and one will return to quiescence to maintain the HSC compartment. A balance between MEK/ERK and mTORC1 pathways is needed to ensure HSC homeostasis. Here, we show that activation of these pathways is spatially segregated in premitotic HSCs and unequally inherited during ACD. A combination of genetic and chemical perturbations shows that an ERK-dependent mechanism determines the balance between pathways affecting polarity, proliferation, and metabolism, and thus determines the frequency of asymmetrically dividing HSCs. Our data identify druggable targets that modulate HSC fate determination at the level of asymmetric division.

Sequestration of translation initiation factors in p62 condensates.

Selective autophagy mediates the removal of harmful material from the cytoplasm. This cargo material is selected by cargo receptors, which orchestrate its sequestration within double-membrane autophagosomes and subsequent lysosomal degradation. The cargo receptor p62/SQSTM1 is present in cytoplasmic condensates, and a fraction of them are constantly delivered into lysosomes. However, the molecular composition of the p62 condensates is incompletely understood. To obtain insights into their composition, we develop a method to isolate these condensates and find that p62 condensates are enriched in components of the translation machinery. Furthermore, p62 interacts with translation initiation factors, and eukaryotic initiation factor 2α (eIF2α) and eIF4E are degraded by autophagy in a p62-dependent manner. Thus, p62-mediated autophagy may in part be linked to down-regulation of translation initiation. The p62 condensate isolation protocol developed here may facilitate the study of their contribution to cellular quality control and their roles in health and disease.

RAF1 contributes to cell proliferation and STAT3 activation in colorectal cancer independently of microsatellite and KRAS status.

More than 30% of all human cancers are driven by RAS mutations and activating KRAS mutations are present in 40% of colorectal cancer (CRC) in the two main CRC subgroups, MSS (Microsatellite Stable) and MSI (Microsatellite Instable). Studies in RAS-driven tumors have shown essential roles of the RAS effectors RAF and specifically of RAF1, which can be dependent or independent of RAF's ability to activate the MEK/ERK module. In this study, we demonstrate that RAF1, but not its kinase activity, plays a crucial role in the proliferation of both MSI and MSS CRC cell line-derived spheroids and patient-derived organoids, and independently of KRAS mutation status. Moreover, we could define a RAF1 transcriptomic signature which includes genes that contribute to STAT3 activation, and could demonstrate that RAF1 ablation decreases STAT3 phosphorylation in all CRC spheroids tested. The genes involved in STAT3 activation as well as STAT3 targets promoting angiogenesis were also downregulated in human primary tumors expressing low levels of RAF1. These results indicate that RAF1 could be an attractive therapeutic target in both MSI and MSS CRC regardless of their KRAS status and support the development of selective RAF1 degraders rather than RAF1 inhibitors for clinical use in combination therapies.

Impaired degradation of YAP1 and IL6ST by chaperone-mediated autophagy promotes proliferation and migration of normal and hepatocellular carcinoma cells.

Impaired degradation of the transcriptional coactivator YAP1 and IL6ST (interleukin 6 cytokine family signal transducer), two proteins deregulated in liver cancer, has been shown to promote tumor growth. Here, we demonstrate that YAP1 and IL6ST are novel substrates of chaperone-mediated autophagy (CMA) in human hepatocellular carcinoma (HCC) and hepatocyte cell lines. Knockdown of the lysosomal CMA receptor LAMP2A increases protein levels of YAP1 and IL6ST, without changes in mRNA expression. Additionally, both proteins show KFERQ-dependent binding to the CMA chaperone HSPA8 and accumulate into isolated lysosomes after stimulation of CMA by prolonged starvation. We further show that LAMP2A downregulation promotes the proliferation and migration in HCC cells and a human hepatocyte cell line, and that it does so in a YAP1- and IL6ST-dependent manner. Finally, LAMP2A expression is downregulated, and YAP1 and IL6ST expression is upregulated, in human HCC biopsies. Taken together, our work reveals a novel mechanism that controls the turnover of two cancer-relevant proteins and suggests a tumor suppressor function of CMA in the liver, advocating for the exploitation of CMA activity for diagnostic and therapeutic purposes.Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; ATG7: autophagy related 7; CMA: chaperone-mediated autophagy; eMI: endosomal microautophagy; HCC: hepatocellular carcinoma; HSPA8: heat shock protein family A (Hsp70) member 8; IL6ST: interleukin 6 cytokine family signal transducer; JAK: Janus kinase; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; MAPK8: mitogen-activated protein kinase 8; P6: pyridine 6; SQSTM1: sequestosome 1; TUBA: tubulin alpha; VDAC1: voltage dependent anion channel 1; VP: verteporfin; YAP1: Yes1 associated transcriptional regulator.

Editor Profile: Manuela Baccarini.

In this special interview series, we profile members of The FEBS Journal editorial board to highlight their research focus, perspectives on the journal and future directions in their field. Manuela Baccarini is Professor of Cell Signaling at the University of Vienna, Coordinator of the International PhD Program 'Signaling Mechanisms in Cellular Homeostasis' and Director of the Vienna BioCenter PhD Program, a graduate school of the University and Medical University of Vienna in collaboration with the Institute of Molecular Pathology and the Austrian Academy of Sciences, Institute for Medical Biotechnology and Gregor Mendel Institute, as well as EMBO member and corresponding member of the Austrian Academy of Sciences. She has served as an editorial board member of The FEBS Journal since 2016.

amica: an interactive and user-friendly web-platform for the analysis of proteomics data.

BACKGROUND: Quantitative proteomics has become an increasingly prominent tool in the study of life sciences. A substantial hurdle for many biologists are, however, the intricacies involved in the associated high throughput data analysis. RESULTS: In order to facilitate this task for users with limited background knowledge, we have developed amica, a freely available open-source web-based software that accepts proteomic input files from different sources. amica provides quality control, differential expression, biological network and over-representation analysis on the basis of minimal user input. Scientists can use amica's query interface interactively to compare multiple conditions and rapidly identify enriched or depleted proteins. They can visualize their results using customized output graphics, and ultimately export the results in a tab-separated format that can be shared with collaborators. The code for the application, input data and documentation can be accessed online at https://github.com/tbaccata/amica and is also incorporated in the web application. CONCLUSIONS: The strong emphasis on dynamic user interactions, the integration of various databases and the option to download processed data, facilitate the analysis of complex proteomic data for both first-time users and experienced bioinformaticians. A freely available version of amica is available at https://bioapps.maxperutzlabs.ac.at/app/amica .

BRAF increases endothelial cell stiffness through reorganization of the actin cytoskeleton.

The dynamics of the actin cytoskeleton and its connection to endothelial cell-cell junctions determine the barrier function of endothelial cells. The proper regulation of barrier opening/closing is necessary for the normal function of vessels, and its dysregulation can result in chronic and acute inflammation leading to edema formation. By using atomic force microscopy, we show here that thrombin-induced permeability of human umbilical vein endothelial cells, associated with actin stress fiber formation, stiffens the cell center. The depletion of the MEK/ERK kinase BRAF reduces thrombin-induced permeability prevents stress fiber formation and cell stiffening. The peripheral actin ring becomes stabilized by phosphorylated myosin light chain, while cofilin is excluded from the cell periphery. All these changes can be reverted by the inhibition of ROCK, but not of the MEK/ERK module. We propose that the balance between the binding of cofilin and myosin to F-actin in the cell periphery, which is regulated by the activity of ROCK, determines the local dynamics of actin reorganization, ultimately driving or preventing stress fiber formation.

Listeria monocytogenes infection rewires host metabolism with regulatory input from type I interferons.

Listeria monocytogenes (L. monocytogenes) is a food-borne bacterial pathogen. Innate immunity to L. monocytogenes is profoundly affected by type I interferons (IFN-I). Here we investigated host metabolism in L. monocytogenes-infected mice and its potential control by IFN-I. Accordingly, we used animals lacking either the IFN-I receptor (IFNAR) or IRF9, a subunit of ISGF3, the master regulator of IFN-I-induced genes. Transcriptomes and metabolite profiles showed that L. monocytogenes infection induces metabolic rewiring of the liver. This affects various metabolic pathways including fatty acid (FA) metabolism and oxidative phosphorylation and is partially dependent on IFN-I signaling. Livers and macrophages from Ifnar1-/- mice employ increased glutaminolysis in an IRF9-independent manner, possibly to readjust TCA metabolite levels due to reduced FA oxidation. Moreover, FA oxidation inhibition provides protection from L. monocytogenes infection, explaining part of the protection of Irf9-/- and Ifnar1-/- mice. Our findings define a role of IFN-I in metabolic regulation during L. monocytogenes infection. Metabolic differences between Irf9-/- and Ifnar1-/- mice may underlie the different susceptibility of these mice against lethal infection with L. monocytogenes.

ERK phosphorylation is RAF independent in naïve and activated B cells but RAF dependent in plasma cell differentiation.

Members of the RAF family of serine-threonine kinases are intermediates in the mitogen-activated protein kinase and extracellular signal-regulated kinase (MAPK-ERK) signaling pathway, which controls key differentiation processes in B cells. By analyzing mice with B cell-specific deletion of Raf1, Braf, or both, we showed that Raf-1 and B-Raf acted together in mediating the positive selection of pre-B and transitional B cells as well as in initiating plasma cell differentiation. However, genetic or chemical inactivation of RAFs led to increased ERK phosphorylation in mature B cells. ERK activation in the absence of Raf-1 and B-Raf was mediated by multiple RAF-independent pathways, with phosphoinositide 3-kinase (PI3K) playing an important role. Furthermore, we found that ERK phosphorylation strongly increased during the transition from activated B cells to pre-plasmablasts. This increase in ERK phosphorylation did not occur in B cells lacking both Raf-1 and B-Raf, which most likely explains the partial block of plasma cell differentiation in mice lacking both RAFs. Collectively, our data indicate that B-Raf and Raf-1 are not necessary to mediate ERK phosphorylation in naïve or activated B cells but are essential for mediating the marked increase in ERK phosphorylation during the transition from activated B cells to pre-plasmablasts.

Combination of Hypoglycemia and Metformin Impairs Tumor Metabolic Plasticity and Growth by Modulating the PP2A-GSK3ß-MCL-1 Axis.

Tumor cells may adapt to metabolic challenges by alternating between glycolysis and oxidative phosphorylation (OXPHOS). To target this metabolic plasticity, we combined intermittent fasting, a clinically feasible approach to reduce glucose availability, with the OXPHOS inhibitor metformin. In mice exposed to 24-h feeding/fasting cycles, metformin impaired tumor growth only when administered during fasting-induced hypoglycemia. Synergistic anti-neoplastic effects of the metformin/hypoglycemia combination were mediated by glycogen synthase kinase 3ß (GSK3ß) activation downstream of PP2A, leading to a decline in the pro-survival protein MCL-1, and cell death. Mechanistically, specific activation of the PP2A-GSK3ß axis was the sum of metformin-induced inhibition of CIP2A, a PP2A suppressor, and of upregulation of the PP2A regulatory subunit B56δ by low glucose, leading to an active PP2A-B56δ complex with high affinity toward GSK3ß.

RAF dimers control vascular permeability and cytoskeletal rearrangements at endothelial cell-cell junctions.

The endothelium functions as a semipermeable barrier regulating fluid homeostasis, nutrient, and gas supply to the tissue. Endothelial permeability is increased in several pathological conditions including inflammation and tumors; despite its clinical relevance, however, there are no specific therapies preventing vascular leakage. Here, we show that endothelial cell-restricted ablation of BRAF, a kinase frequently activated in cancer, prevents vascular leaking as well metastatic spread. BRAF regulates endothelial permeability by promoting the cytoskeletal rearrangements necessary for the remodeling of VE-Cadherin-containing endothelial cell-cell junctions and the formation of intercellular gaps. BRAF kinase activity and the ability to form complexes with RAS/RAP1 and dimers with its paralog RAF1 are required for proper permeability control, achieved mechanistically by modulating the interaction between RAF1 and the RHO effector ROKα. Thus, RAF dimerization impinges on RHO pathways to regulate cytoskeletal rearrangements, junctional plasticity, and endothelial permeability. The data advocate the development of RAF dimerization inhibitors, which would combine tumor cell autonomous effect with stabilization of the vasculature and antimetastatic spread.

Activating mutations in MEK1 enhance homodimerization and promote tumorigenesis.

RAS-RAF-MEK-ERK signaling has a well-defined role in cancer biology. Although aberrant pathway activation occurs mostly upstream of the kinase MEK, mutations in MEK are prevalent in some cancer subsets. Here, we found that cancer-related, activating mutations in MEK can be classified into two groups: those that relieve inhibitory interactions with the helix A region and those that are in-frame deletions of the ß3-αC loop, which enhance MEK1 homodimerization. The former, helix A-associated mutants, are inhibited by traditional MEK inhibitors. However, we found that the increased homodimerization associated with the loop-deletion mutants promoted intradimer cross-phosphorylation of the activation loop and conferred differential resistance to MEK inhibitors both in vitro and in vivo. MEK1 dimerization was required both for its activation by the kinase RAF and for its catalytic activity toward the kinase ERK. Our findings not only identify a previously unknown group of MEK mutants and provide insight into some key steps in RAF-MEK-ERK activation but also have implications for the design of therapies targeting RAS-ERK signaling in cancers.

The dimer-dependent catalytic activity of RAF family kinases is revealed through characterizing their oncogenic mutants.

Although extensively studied for three decades, the molecular mechanisms that regulate the RAF/MEK/ERK kinase cascade remain ambiguous. Recent studies identified the dimerization of RAF as a key event in the activation of this cascade. Here, we show that in-frame deletions in the ß3-αC loop activate ARAF as well as BRAF and other oncogenic kinases by enforcing homodimerization. By characterizing these RAF mutants, we find that ARAF has less allosteric and catalytic activity than the other two RAF isoforms, which arises from its non-canonical APE motif. Further, these RAF mutants exhibit a strong oncogenic potential, and a differential inhibitor resistance that correlates with their dimer affinity. Using these unique mutants, we demonstrate that active RAFs, including the BRAF(V600E) mutant, phosphorylate MEK in a dimer-dependent manner. This study characterizes a special category of oncogenic kinase mutations, and elucidates the molecular basis that underlies the differential ability of RAF isoforms to stimulate MEK-ERK pathway. Further, this study reveals a unique catalytic feature of RAF family kinases that can be exploited to control their activities for cancer therapies.

An ERK-Dependent Feedback Mechanism Prevents Hematopoietic Stem Cell Exhaustion.

Hematopoietic stem cells (HSCs) sustain hematopoiesis throughout life. HSCs exit dormancy to restore hemostasis in response to stressful events, such as acute blood loss, and must return to a quiescent state to prevent their exhaustion and resulting bone marrow failure. HSC activation is driven in part through the phosphatidylinositol 3-kinase (PI3K)/AKT/mTORC1 signaling pathway, but less is known about the cell-intrinsic pathways that control HSC dormancy. Here, we delineate an ERK-dependent, rate-limiting feedback mechanism that controls HSC fitness and their re-entry into quiescence. We show that the MEK/ERK and PI3K pathways are synchronously activated in HSCs during emergency hematopoiesis and that feedback phosphorylation of MEK1 by activated ERK counterbalances AKT/mTORC1 activation. Genetic or chemical ablation of this feedback loop tilts the balance between HSC dormancy and activation, increasing differentiated cell output and accelerating HSC exhaustion. These results suggest that MEK inhibitors developed for cancer therapy may find additional utility in controlling HSC activation.

RAF proteins exert both specific and compensatory functions during tumour progression of NRAS-driven melanoma.

NRAS and its effector BRAF are frequently mutated in melanoma. Paradoxically, CRAF but not BRAF was shown to be critical for various RAS-driven cancers, raising the question of the role of RAF proteins in NRAS-induced melanoma. Here, using conditional ablation of Raf genes in NRAS-induced mouse melanoma models, we investigate their contribution in tumour progression, from the onset of benign tumours to malignant tumour maintenance. We show that BRAF expression is required for ERK activation and nevi development, demonstrating a critical role in the early stages of NRAS-driven melanoma. After melanoma formation, single Braf or Craf ablation is not sufficient to block tumour growth, showing redundant functions for RAF kinases. Finally, proliferation of resistant cells emerging in the absence of BRAF and CRAF remains dependent on ARAF-mediated ERK activation. These results reveal specific and compensatory functions for BRAF and CRAF and highlight an addiction to RAF signalling in NRAS-driven melanoma.

RAF1/BRAF dimerization integrates the signal from RAS to ERK and ROKα.

Downstream of growth factor receptors and of the guanine triphosphatase (GTPase) RAS, heterodimers of the serine/threonine kinases BRAF and RAF1 are critical upstream kinases and activators of the mitogen-activated protein kinase (MAPK) module containing the mitogen-activated and extracellular signal-regulated kinase kinase (MEK) and their targets, the extracellular signal-regulated kinase (ERK) family. Either direct or scaffold protein-mediated interactions among the components of the ERK module (the MAPKKKs BRAF and RAF1, MEK, and ERK) facilitate signal transmission. RAF1 also has essential functions in the control of tumorigenesis and migration that are mediated through its interaction with the kinase ROKα, an effector of the GTPase RHO and regulator of cytoskeletal rearrangements. We combined mutational and kinetic analysis with mathematical modeling to show that the interaction of RAF1 with ROKα is coordinated with the role of RAF1 in the ERK pathway. We found that the phosphorylated form of RAF1 that interacted with and inhibited ROKα was generated during the interaction of RAF1 with the ERK module. This mechanism adds plasticity to the ERK pathway, enabling signal diversification at the level of both ERK and RAF. Furthermore, by connecting ERK activation with the regulation of ROKα and cytoskeletal rearrangements by RAF1, this mechanism has the potential to precisely coordinate the proper timing of proliferation with changes in cell shape, adhesion, or motility.

Deciphering the RAS/ERK pathway in vivo.

The RAS/ERK pathway has been intensely studied for about three decades, not least because of its role in human pathologies. ERK activation is observed in the majority of human cancers; in about one-third of them, it is driven by mutational activation of pathway components. The pathway is arguably one of the best targets for molecule-based pharmacological intervention, and several small-molecule inhibitors are in clinical use. Genetically engineered mouse models have greatly contributed to our understanding of signaling pathways in development, tissue homeostasis, and disease. In the specific case of the RAS/ERK pathway, they have revealed unique biological roles of structurally and functionally similar proteins, new kinase-independent effectors, and unsuspected relationships with other cascades. This short review summarizes the contribution of mouse models to our current understanding of the pathway.

Raf Kinases Are Essential for Phosphate Induction of ERK1/2 Phosphorylation in Hypertrophic Chondrocytes and Normal Endochondral Bone Development.

Hypophosphatemia causes rickets by impairing hypertrophic chondrocyte apoptosis. Phosphate induction of MEK1/2-ERK1/2 phosphorylation in hypertrophic chondrocytes is required for phosphate-mediated apoptosis and growth plate maturation. MEK1/2 can be activated by numerous molecules including Raf isoforms. A- and B-Raf ablation in chondrocytes does not alter skeletal development, whereas ablation of C-Raf decreases hypertrophic chondrocyte apoptosis and impairs vascularization of the growth plate. However, ablation of C-Raf does not impair phosphate-induced ERK1/2 phosphorylation in vitro, but leads to rickets by decreasing VEGF protein stability. To determine whether Raf isoforms are required for phosphate-induced hypertrophic chondrocyte apoptosis, mice lacking all three Raf isoforms in chondrocytes were generated. Raf deletion caused neonatal death and a significant expansion of the hypertrophic chondrocyte layer of the growth plate, accompanied by decreased cleaved caspase-9. This was associated with decreased phospho-ERK1/2 immunoreactivity in the hypertrophic chondrocyte layer and impaired vascular invasion. These data further demonstrated that Raf kinases are required for phosphate-induced ERK1/2 phosphorylation in cultured hypertrophic chondrocytes and perform essential, but partially redundant roles in growth plate maturation.

A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths, but its molecular heterogeneity hampers the design of targeted therapies. Currently, the only therapeutic option for advanced HCC is Sorafenib, an inhibitor whose targets include RAF. Unexpectedly, RAF1 expression is reduced in human HCC samples. Modelling RAF1 downregulation by RNAi increases the proliferation of human HCC lines in xenografts and in culture; furthermore, RAF1 ablation promotes chemical hepatocarcinogenesis and the proliferation of cultured (pre)malignant mouse hepatocytes. The phenotypes depend on increased YAP1 expression and STAT3 activation, observed in cultured RAF1-deficient cells, in HCC xenografts, and in autochthonous liver tumours. Thus RAF1, although essential for the development of skin and lung tumours, is a negative regulator of hepatocarcinogenesis. This unexpected finding highlights the contribution of the cellular/tissue environment in determining the function of a protein, and underscores the importance of understanding the molecular context of a disease to inform therapy design.

MEK1 is required for the development of NRAS-driven leukemia.

The dual-specificity kinases MEK1 and MEK2 act downstream of RAS/RAF to induce ERK activation, which is generally considered protumorigenic. Activating MEK mutations have not been discovered in leukemia, in which pathway activation is caused by mutations in upstream components such as RAS or Flt3. The anti-leukemic potential of MEK inhibitors is being tested in clinical trials; however, downregulation of MEK1 promotes Eµ-Myc-driven lymphomagenesis and MEK1 ablation induces myeloproliferative disease in mice, raising the concern that MEK inhibitors may be inefficient or counterproductive in this context. We investigated the role of MEK1 in the proliferation of human leukemic cell lines and in retroviral models of leukemia. Our data show that MEK1 suppression via RNA interference and genomic engineering does not affect the proliferation of human leukemic cell lines in culture; similarly, MEK1 ablation does not impact the development of MYC-driven leukemia in vivo. In contrast, MEK1 ablation significantly reduces tumorigenesis driven by Nras alone or in combination with Myc. Thus, while MEK1 restricts proliferation and tumorigenesis in some cellular and genetic contexts, it cannot be considered a tumor suppressor in the context of leukemogenesis. On the contrary, its role in NRAS-driven leukemogenesis advocates the use of MEK inhibitors, particularly in combination with PI3K/AKT inhibitors, in hematopoietic malignancies involving RAS activation.