Lung adenocarcinoma (LUAD) is one of the most prevalent cancer types worldwide and has one of the poorest survival rates. Understanding its developpment is crucial for improving diagnosis, prognosis, and treatment. A key factor in LUAD is the frequent loss-of-function mutations in LKB1/STK11, a kinase that regulates metabolism. These mutations are linked to increased metastasis and worse clinical outcomes. In this study, we analyzed gene expression data from LUAD patients to explore how LKB1 mutations affect cancer behavior. We found that LKB1 mutations in KRAS-driven LUAD lead to widespread gene downregulation. By integrating avalaible protein interaction data, mass spectrometry analysis of LKB1 nuclear partners, and co-immunoprecipitations experiments, we identified BRG1, a chromatin activator and subunit of the BAF complex, as a nuclear partner of LKB1. Further analysis suggested that LKB1 mutations may impair BRG1 activity, disrupting chromatin regulation and gene expression. Notably, LUAD patients with mutated LKB1 showed gene expression patterns indicative of oxidative stress, defective neuronal-glial and neuroinflammation programs, and altered amino acid homeostasis. These changes resemble the roles LKB1 plays in neural crest stem cells, suggesting that LKB1 may reduce tumor aggressiveness in LUAD by maintaining a developmental gene expression program.

The enteric nervous system (ENS) comprises ganglia of neurons and glial cells derived from migratory multipotent neural crest cells. While the molecular mechanisms of ENS development are well-studied, the involvement of metabolic processes has received less attention. We previously showed that the tumor suppressor kinase LKB1 is essential for the trophic maintenance of postnatal ENS. Here we examined LKB1’s role in ENS formation using a genetically engineered mouse model that conditionally inactivates Lkb1 in neural crest progenitors during gut invasion. We conducted a comprehensive phenotyping of the ENS through histology and 3D imaging of cleared tissue, combining lightsheet microscopy with adaptive optics confocal microscopy. We found that Lkb1 loss impairs early neuronal differentiation, followed by glial degeneration, leading to hypoganglionosis and compromised digestive tissue integrity. Metabolite profiling of digestive tracts revealed an increase of oxidative stress upon Lkb1 ablation. In vitro, Lkb1 knockdown induced oxidative stress in neural crest progenitors and their glial derivatives, causing DNA damage and p53 activation. Ablation of p53 rescued glial specification under these conditions. In vivo , hyperphosphorylation of p53 was also observed; however, deletion of p53 alleles in Lkb1 mutants did not restore enteric neurons number. Instead, it improved axonal fiber organization and partially rescued digestive tissue integrity. These findings establish LKB1 as a key metabolic regulator on both the development and maintenance of the ENS, suggesting that aberrant LKB1 signaling may contribute to human enteric glioneuropathies. ### Competing Interest Statement The authors have declared no competing interest. Ligue Regionale Contre le Cancer, comité Isere Fondation ARC French National Research Agency, ANR-15-IDEX-02, ANR-17-EURE-0003, ANR-10-INBS-04 Centre National de la Recherche Scientifique Institut National de la Santé et de la Recherche Médicale University Grenoble Alpes

Advances in cancer treatment have led to a steady increase in the rate of disease remission. However, while many treatment-related adverse effects gradually resolve after therapy, chemotherapy-induced peripheral neuropathy (CIPN) often persists, with no means of prevention or direct treatment available. Herein, we present Carba1, a novel bi-functional carbazole that mitigates neuropathy through two distinct mechanisms. First, by interacting with tubulin, Carba1 reduces the required dose of taxanes, widely used chemotherapy drugs notorious for their toxic side effects, including CIPN. Second, Carba1 activates nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD salvage pathway, triggering a metabolic rewiring that enhances the resilience of neurons and Schwann cells against chemotherapy-induced toxicity. We demonstrate the neuroprotective efficacy of Carba1 both in vitro, against neurotoxicity induced by paclitaxel (PTX), cisplatin, and bortezomib, and in vivo in a rat model of PTX-induced neuropathy. Importantly, we establish that Carba1 does not compromise the therapeutic efficacy of PTX nor promotes tumor growth. Comparative analyses of Carba1 derivatives further suggest the potential of designing compounds with either dual synergistic and neuroprotective activity or exclusive neuroprotective properties. Altogether, our findings position Carba1 as a promising therapeutic candidate for preventing CIPN, with the potential, if successfully translated to clinical settings, to improve both the quality of life and treatment outcome for cancer patients.

Neural crest cells (NCCs) are highly motile, multipotent, embryonic cells that delaminate from the dorsal edges of the neural tube. NCCs follow stereotypical long-range migratory pathways to reach target organs during development, where they give rise to multiple derivatives. The identification of reservoirs of neural crest stem cells that persist to adulthood has recently aroused renewed interest in the biology of NCCs. In this context, several recent studies have demonstrated the essential role of the metabolic kinase LKB1 in NCC establishment. This review surveys how LKB1 governs the formation and maintenance of several neural crest derivatives, including facial bones, melanocytes, Schwann cells, and the enteric nervous system. We also detail the underlying molecular mechanisms that involve downstream effectors of LKB1, in particular the contribution of the AMPK-mTOR signaling pathway to both polarity and metabolic processes. Collectively, these recent discoveries open promising perspectives for new therapeutic applications for the treatment of neural crest disorders.

Metabolic processes underlying the development of the neural crest, an embryonic population of multipotent migratory cells, are poorly understood. Here, we report that conditional ablation of the Lkb1 tumor suppressor kinase in mouse neural crest stem cells led to intestinal pseudo-obstruction and hind limb paralysis. This phenotype originated from a postnatal degeneration of the enteric nervous ganglia and from a defective differentiation of Schwann cells. Metabolomic profiling revealed that pyruvate-alanine conversion is enhanced in the absence of Lkb1. Mechanistically, inhibition of alanine transaminases restored glial differentiation in an mTOR-dependent manner, while increased alanine level directly inhibited the glial commitment of neural crest cells. Treatment with the metabolic modulator AICAR suppressed mTOR signaling and prevented Schwann cell and enteric defects of Lkb1 mutant mice. These data uncover a link between pyruvate-alanine cycling and the specification of glial cell fate with potential implications in the understanding of the molecular pathogenesis of neural crest diseases.

Head development in vertebrates proceeds through a series of elaborate patterning mechanisms and cell-cell interactions involving cephalic neural crest cells (CNCC). These cells undergo extensive migration along stereotypical paths after their separation from the dorsal margins of the neural tube and they give rise to most of the craniofacial skeleton. Here, we report that the silencing of the LKB1 tumor suppressor affects the delamination of pre-migratory CNCC from the neural primordium as well as their polarization and survival, thus resulting in severe facial and brain defects. We further show that LKB1-mediated effects on the development of CNCC involve the sequential activation of the AMP-activated protein kinase (AMPK), the Rho-dependent kinase (ROCK) and the actin-based motor protein myosin II. Collectively, these results establish that the complex morphogenetic processes governing head formation critically depends on the activation of the LKB1 signaling network in CNCC.

The cytoplasmic protein Alix/AIP1 (ALG-2 interacting protein X) is involved in cell death through mechanisms which remain unclear but require its binding partner ALG-2 (apoptosis-linked gene-2). The latter was defined as a regulator of calcium-induced apoptosis following endoplasmic reticulum (ER) stress. We show here that Alix is also a critical component of caspase 9 activation and apoptosis triggered by calcium. Indeed, expression of Alix dominant-negative mutants or downregulation of Alix afford significant protection against cytosolic calcium elevation following thapsigargin (Tg) treatment. The function of Alix in this paradigm requires its interaction with ALG-2. In addition, we demonstrate that caspase 9 activation is necessary for apoptosis induced by Tg and that this activation is impaired by knocking down Alix. Altogether, our findings identify, for the first time, Alix as a crucial mediator of Ca2+ induced caspase 9 activation.

Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] is a ubiquitinous adaptor protein first described for its capacity to bind to the calcium-binding protein, ALG-2. Alix regulates neuronal death in ways involving interactions with ALG-2 and with proteins of the ESCRT (endosomal sorting complex required for transport). Even though all Alix interactors characterized to date are involved in endosomal trafficking, the genuine function of the protein in this process remains unclear. we have demonstrated recently that Alix and ALG-2 form in the presence of calcium, a complex with apical caspases and with the endocytosed death receptor TNFR1 (tumour necrosis factor a receptor 1), thus suggesting a molecular coupling between endosomes and the cell death machinery.

Alix/AIP1 regulates cell death in a way involving interactions with the calcium-binding protein ALG-2 and with proteins of ESCRT (endosomal sorting complex required for transport). Using mass spectrometry we identified caspase-8 among proteins co-immunoprecipitating with Alix in dying neurons. We next demonstrated that Alix and ALG-2 interact with pro-caspase-8 and that Alix forms a complex with the TNFα receptor-1 (TNF-R1), depending on its capacity to bind ESCRT proteins. Thus, Alix and ALG-2 may allow the recruitment of pro-caspase-8 onto endosomes containing TNF-R1, a step thought to be necessary for activation of the apical caspase. In line with this, expression of Alix deleted of its ALG-2-binding site (AlixΔALG-2) significantly reduced TNF-R1-induced cell death, without affecting endocytosis of the receptor. In a more physiological setting, we found that programmed cell death of motoneurons, which can be inhibited by AlixΔALG-2, is regulated by TNF-R1. Taken together, these results highlight Alix and ALG-2 as new actors of the TNF-R1 pathway.

Alix/AIP1 is a cytosolic protein that regulates cell death through mechanisms that remain unclear. Alix binds to two protein members of the so-called Endosomal Sorting Complex Required for Transport (ESCRT), which facilitates membrane fission events during multivesicular endosome formation, enveloped virus budding and cytokinesis. Alix itself has been suggested to participate in these cellular events and is thus often considered to function in the ESCRT pathway. ESCRT proteins were recently implicated in autophagy, a process involved in bulk degradation of cytoplasmic constituents in lysosomes, which can also participate in cell death. In this study, we shown that, unlike ESCRT proteins, Alix is not involved in autophagy. These results strongly suggest that the capacity of several mutants of Alix to block both caspase-dependent and independent cell death does not relate to their capacity to modulate autophagy. Furthermore, they reinforce the conclusion of other studies demonstrating that the role of Alix is different from that of classical ESCRT proteins.

ALIX AND ALG-2 ARE INVOLVED IN TNF-R1 INDUCED CELL DEATH Anne-Laure Mahul-Mellier , Flavie Strappazzon, Anne Petiot, Christine Chatellard-Causse, Sakina Torch, Béatrice Blot, Kimberley Freeman , Loriane Kuhn , Jérome Garin , Jean-Marc Verna, Sandrine Fraboulet, Rémy Sadoul INSERM, U836, Equipe 2, Neurodégénérescence et Plasticité, Grenoble, F-38042, France. CEA, DSV, iRTSV, Laboratoire d’Etude de la Dynamique des Protéomes, Grenoble, F-38054, France. INSERM, U880, Grenoble, F-38054, France. Université Joseph Fourier, Grenoble Institut des Neurosciences, Grenoble, F-38042, France Present addresses : Imperial College London, Experimental Medicine & Toxicology, London W120NN. European Center for Brain Research, Santa Lucia Foundation, Molecular Neuroembryology Unit, 00143, Rome, Italy. MRC Functional Genetic Unit, University of Oxford, Department of Physiology Anatomy and Genetics, OX1 3QX Oxford, UK Address correspondance to: Rémy Sadoul, Grenoble Institute of Neuroscience, Chemin Fortuné Ferrini, BP 170, F38042 Grenoble, France. tel: ++33 456 52 05 44, email: remy.sadoul@ujf-grenoble.fr and Sandrine Fraboulet, Le Gros Clark Building University of Oxford South Parks Road Oxford OX1 3QX,UK. tel: +44 1865 282 273, email: sandrine.fraboulet@dpag.ox.ac.uk

Numerous extracellular stimuli trigger trans-autophosphorylation at Tyr402 of Pyk2, inducing its activation. Pyk2 is a key mediator of several signaling pathways and has been implicated in apoptosis induced by specific stress signals. We investigated whether Pyk2 participates in cerebellar granule neuron (CGN) apoptosis induced by the suppression of membrane depolarization. We demonstrate that shifting CGN cultures from 25 mM to 5 mM KCl-containing medium induces an early, transient 70% increase in phosphorylated Tyr402 and Tyr580 Pyk2 levels that is triggered by Ca(2+) released from intracellular stores and mediated by calmodulin (CaM). Overexpression of Pyk2 increases CGN survival after 24 h by 70% compared to the control, thus suggesting that Pyk2 is involved in an anti-apoptotic response to K+ lowering. Furthermore, we show that CGN grown in K25 medium exhibit detectable CaM-dependent Pyk2 activity. When silencing Pyk2 activity by expressing a dominant-negative form, only 40% of the transfected neurons were alive 24 h after transfection when compared to the control. Overall, the present findings demonstrate for the first time that Pyk2 is a critical mediator of CGN survival.

Chronic intoxication by 3-nitropropionic acid in the Lewis rat reproduces many features reminiscent of Huntington's disease including behavioural alterations and cortico-striatal degeneration. In particular, in this model, striatal degeneration is accompanied by calpain activation as found in the human disease. The present study was undertaken to determine whether the expression of Alix (apoptosis linked gene-2 interacting protein), a widespread protein involved in neuronal death, would be modified in the striatum and cortex of 3NP-treated rats. The results clearly show that Alix immunoreactivity is increased in the neuronal cell bodies of the lateral striatum, where degeneration is massive. The medial striatum and the cortex that lack neurodegeneration remain only weakly labelled. This is further evidence suggesting an involvement of Alix in the events driving neuronal death.

Alix/AIP1 is a cytoplasmic protein, which was first characterized as an interactor of ALG-2, a calcium-binding protein necessary for cell death. Alix has also recently been defined as a regulator of the endo-lysosomal system. Here we have used post-mitotic cerebellar neurons to test Alix function in caspase-dependent and - independent cell death. Indeed, these neurons survived when cultured in 25 mM potassium-containing medium but underwent apoptosis soon after the extracellular potassium was lowered to 5 mM. In agreement with other studies, we show that caspases are activated after K+ deprivation, but that inhibition of these proteases, using the pancaspase inhibitor boc-aspartyl(OMe)-fluoromethylketone, has no effect on cell survival. Transfection experiments demonstrated that Alix overexpression is sufficient to induce caspase activation, whereas overexpression of its C-terminal half, Alix-CT, blocks caspase activation and cell death after K+ deprivation. We also define a 12-amino acid PXY repeat of the C-terminal proline-rich domain necessary for binding ALG-2. Deletion of this domain in Alix or in Alix-CT abolished the effects of the overexpressed proteins on neuronal survival, demonstrating that the ALG-2-binding region is crucial for the death-modulating function of Alix. Overall, these findings define the Alix/ALG-2 complex as a regulator of cell death controlling both caspase-dependent and - independent pathways. They also suggest a molecular link between the endo-lysosomal system and the effectors of the cell death machinery.

ALG-2-interacting proteinX (Alix), also known as AIP1, is a cytoplasmic protein ubiquitously expressed and concentrated in phagosomes and exosomes. Alix may regulate apoptosis since it bindsapoptosis-linked gene 2 (ALG-2), a Ca2+-binding protein necessary for cell death, and also overexpression of its C-terminal half (Alix-CT) blocks death induced by several stimuli. This part of Alix contains a long proline-rich domain containing several potential SH3-binding sites. Using Alix as bait in a yeast two-hybrid system to screen a mouse brain library, we have found that SH3p4, SH3p8, and SH3p13, collectively known as endophilins, bind to Alix. Co-immunoprecipitations and overlay experiments allowed us to demonstrate that endophilins bind to Alix-CT through an SH3/proline-rich domain interaction. We have narrowed the region of Alix interacting with endophilins down to 14 amino acids containing a PXRPPPP consensus sequence, also present in synaptojanin and germinal center kinase-like kinase, allowing their interaction to endophilins. We further show that overexpression of Alix-CT, which blocks cell death, leads to cytoplasmic vacuolization into tubulo-vesicular structures delineated by Alix-CT. This vacuolization phenomenon is greatly enhanced upon co-expression with endophilins and may be part of the protecting mechanism afforded by Alix-CT.

A better knowledge of molecular pathways involved in the neuronal cell death occurring in Parkinson's disease is essential to achieve therapeutics able to block the degenerative process. These pathways have been partially elucidated using experimental models created by either 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) which are able to mimick the histological (death of dopaminergic nigral neurons) and biochemical (oxidative stress and mitochondriol inhibition) hallmarks of Parkinson's disease. Cell death induced by these neurotoxins is thought to be apoptotic. At the very end-stage, this process depends on activated caspase-3. At earlier stages, in these models, cell death is regulated by p53 and Bcl-2 family proteins but also by the transcriptional factor NF-kappaB and MAP-kinases. Modulating such regulatory processes may have potential therapeutic benefit for Parkinson's disease.

La caractérisation des mécanismes moléculaires impliqués dans la mort neuronale survenant au cours de la maladie de Parkinson idiopa-thique est indispensable pour l’élaboration de traitements capables d’arrêter l’évolution de cette maladie. Ces mécanismes ont essentiellement été décryptés grâce à deux modèles expérimentaux utilisant des neurotoxiques capables de reproduire les caractéristiques anatomo-pathologiques (dégénérescence des neurones dopaminergiques de la substance noire) et biochimiques (stress oxydatif et inhibition mito-chondriale) de la maladie de Parkinson idiopa-thique. La mort cellulaire induite dans ces modèles est de type apoptotique. La phase finale de ce processus met en jeu l’activation de la caspase effectrice de type 3. Les phases plus précoces semblent réglées par la protéine p53 et les protéines pro- et anti-apoptotiques de la famille Bcl-2 mais également modulées par le facteur transcriptionnel NFκB et les MAP-kinases. Toutes ces étapes constituent autant de cibles thérapeutiques potentielles.

The involvement of nuclear Factor-kappaB (NF-κB) transcription factor in PC12 cell death triggered by the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) was investigated. Results show that oxidative stress generated by 6-OHDA activates NF-κB. When the NF-κB activation was inhibited by parthenolide, PC12 cell death induced by 6-OHDA was significantly increased, thus suggesting an involvement of this transcription factor in a protective mechanism against 6-OHDA toxicity. To further assess this hypothesis, we studied the involvement of NF-κB in the protective effect of two anti-apoptotic genes, bcl-2 and bfl-1. Although Bcl-2 and Bfl-1 expression normally protects PC12 cells from 6-OHDA, parthenolide strongly decreased the beneficial effects afforded by transgene expression. These results suggest: (1) that the transcription factor NF-κB is likely associated with the protection of catecholaminergic PC12 cells and (2) that the protective effects afforded by bcl-2 and bfl-1 expression may be dependent on NF-κ activation.