The ERK5/NF-κB signaling pathway targets endometrial cancer proliferation and survival.

Endometrial cancer (EC) is the most common type of gynecologic cancer in women of developed countries. Despite surgery combined with chemo-/radiotherapy regimens, overall survival of patients with high-risk EC tumors is poor, indicating a need for novel therapies. The MEK5-ERK5 pathway is activated in response to growth factors and to different stressors, including oxidative stress and cytokines. Previous evidence supports a role for the MEK5-ERK5 pathway in the pathology of several cancers. We investigated the role of ERK5 in EC. In silico analysis of the PanCancer Atlas dataset showed alterations in components of the MEK5-ERK5 pathway in 48% of EC patients. Here, we show that ERK5 inhibition or silencing decreased EGF-induced EC cell proliferation, and that genetic deletion of MEK5 resulted in EC impaired proliferation and reduced tumor growth capacity in nude mice. Pharmacologic inhibition or ERK5 silencing impaired NF-kB pathway in EC cells and xenografts. Furthermore, we found a positive correlation between ERK5 and p65/RELA protein levels in human EC tumor samples. Mechanistically, genetic or pharmacologic impairment of ERK5 resulted in downregulation of NEMO/IKKγ expression, leading to impaired p65/RELA activity and to apoptosis in EC cells and xenografts, which was rescued by NEMO/IKKγ overexpression. Notably, ERK5 inhibition, MEK5 deletion or NF-kB inhibition sensitized EC cells to standard EC chemotherapy (paclitaxel/carboplatin) toxicity, whereas ERK5 inhibition synergized with paclitaxel to reduce tumor xenograft growth in mice. Together, our results suggest that the ERK5-NEMO-NF-κB pathway mediates EC cell proliferation and survival. We propose the ERK5/NF-κB axis as new target for EC treatment.

Discovery of a selective inhibitor of doublecortin like kinase 1.

Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We used chemoproteomic profiling and structure-based design to develop a selective, in vivo-compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA-sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer.

An ERK5-KLF2 signalling module regulates early embryonic gene expression and telomere rejuvenation in stem cells.

The ERK5 MAP kinase signalling pathway drives transcription of naïve pluripotency genes in mouse Embryonic Stem Cells (mESCs). However, how ERK5 impacts on other aspects of mESC biology has not been investigated. Here, we employ quantitative proteomic profiling to identify proteins whose expression is regulated by the ERK5 pathway in mESCs. This reveals a function for ERK5 signalling in regulating dynamically expressed early embryonic 2-cell stage (2C) genes including the mESC rejuvenation factor ZSCAN4. ERK5 signalling and ZSCAN4 induction in mESCs increases telomere length, a key rejuvenative process required for prolonged culture. Mechanistically, ERK5 promotes ZSCAN4 and 2C gene expression via transcription of the KLF2 pluripotency transcription factor. Surprisingly, ERK5 also directly phosphorylates KLF2 to drive ubiquitin-dependent degradation, encoding negative feedback regulation of 2C gene expression. In summary, our data identify a regulatory module whereby ERK5 kinase and transcriptional activities bi-directionally control KLF2 levels to pattern 2C gene transcription and a key mESC rejuvenation process.

Methuosis Contributes to Jaspine-B-Induced Cell Death.

Methuosis is a type of programmed cell death in which the cytoplasm is occupied by fluid-filled vacuoles that originate from macropinosomes (cytoplasmic vacuolation). A few molecules have been reported to behave as methuosis inducers in cancer cell lines. Jaspine B (JB) is a natural anhydrous sphingolipid (SL) derivative reported to induce cytoplasmic vacuolation and cytotoxicity in several cancer cell lines. Here, we have investigated the mechanism and signalling pathways involved in the cytotoxicity induced by the natural sphingolipid Jaspine B (JB) in lung adenocarcinoma A549 cells, which harbor the G12S K-Ras mutant. The effect of JB on inducing cytoplasmic vacuolation and modifying cell viability was determined in A549 cells, as well as in mouse embryonic fibroblasts (MEF) lacking either the autophagy-related gene ATG5 or BAX/BAK genes. Apoptosis was analyzed by flow cytometry after annexin V/propidium iodide staining, in the presence and absence of z-VAD. Autophagy was monitored by LC3-II/GFP-LC3-II analysis, and autophagic flux experiments using protease inhibitors. Phase contrast, confocal, and transmission electron microscopy were used to monitor cytoplasmic vacuolation and the uptake of Lucifer yellow to assess macropinocyosis. We present evidence that cytoplasmic vacuolation and methuosis are involved in Jaspine B cytotoxicity over A549 cells and that activation of 5' AMP-activated protein kinase (AMPK) could be involved in Jaspine-B-induced vacuolation, independently of the phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin complex 1 (PI3K/Akt/mTORC1) axis.

The novel proautophagy anticancer drug ABTL0812 potentiates chemotherapy in adenocarcinoma and squamous nonsmall cell lung cancer.

Around 40% of newly diagnosed lung cancer patients are Stage IV, where the improvement of survival and reduction of disease-related adverse events is the main goal for oncologists. In this scenario, we present preclinical evidence supporting the use of ABTL0812 in combination with chemotherapy for treating advanced and metastatic Nonsmall cell lung adenocarcinomas (NSCLC) and squamous carcinomas. ABTL0812 is a new chemical entity, currently in Phase 1b/2a clinical trial for advanced squamous NSCLC in combination with paclitaxel and carboplatin (P/C), after successfully completing the first-in-human trial where it showed an excellent safety profile and signs of efficacy. We show here that ABTL0812 inhibits Akt/mTOR axis by inducing the overexpression of TRIB3 and activating autophagy in lung squamous carcinoma cell lines. Furthermore, treatment with ABTL0812 also induces AMPK activation and ROS accumulation. Moreover, combination of ABTL0812 with chemotherapy markedly increases the therapeutic effect of chemotherapy without increasing toxicity. We further show that combination of ABTL0812 and chemotherapy induces nonapoptotic cell death mediated by TRIB3 activation and autophagy induction. We also present preliminary clinical data indicating that TRIB3 could serve as a potential novel pharmacodynamic biomarker to monitor ABTL0812 activity administered alone or in combination with chemotherapy in squamous NSCLC patients. The safety profile of ABTL0812 and its good synergy with chemotherapy potentiate the therapeutic potential of current lines of treatment based on chemotherapy regimens, arising as a promising option for improving these patients therapeutic expectancy.

SUMOylation Is Required for ERK5 Nuclear Translocation and ERK5-Mediated Cancer Cell Proliferation.

The MAP kinase ERK5 contains an N-terminal kinase domain and a unique C-terminal tail including a nuclear localization signal and a transcriptional activation domain. ERK5 is activated in response to growth factors and stresses and regulates transcription at the nucleus by either phosphorylation or interaction with transcription factors. MEK5-ERK5 pathway plays an important role regulating cancer cell proliferation and survival. Therefore, it is important to define the precise molecular mechanisms implicated in ERK5 nucleo-cytoplasmic shuttling. We previously described that the molecular chaperone Hsp90 stabilizes and anchors ERK5 at the cytosol and that ERK5 nuclear shuttling requires Hsp90 dissociation. Here, we show that MEK5 or overexpression of Cdc37-mechanisms that increase nuclear ERK5-induced ERK5 Small Ubiquitin-related Modifier (SUMO)-2 modification at residues Lys6/Lys22 in cancer cells. Furthermore, mutation of these SUMO sites abolished the ability of ERK5 to translocate to the nucleus and to promote prostatic cancer PC-3 cell proliferation. We also show that overexpression of the SUMO protease SENP2 completely abolished endogenous ERK5 nuclear localization in response to epidermal growth factor (EGF) stimulation. These results allow us to propose a more precise mechanism: in response to MEK5 activation, ERK5 SUMOylation favors the dissociation of Hsp90 from the complex, allowing ERK5 nuclear shuttling and activation of the transcription.

TRIB3 silencing promotes the downregulation of Akt pathway and PAX3-FOXO1 in high-risk rhabdomyosarcoma.

Rhabdomyosarcoma (RMS), such as other childhood tumors, has witnessed treatment advancements in recent years. However, high-risk patients continue to face poor survival rates, often attributed to the presence of the PAX3/7-FOXO1 fusion proteins, which has been associated with metastasis and treatment resistance. Despite efforts to directly target these chimeric proteins, clinical success remains elusive. In this study, the main aim was to address this challenge by investigating regulators of FOXO1. Specifically, we focused on TRIB3, a potential regulator of the fusion protein in RMS. Our findings revealed a prominent TRIB3 expression in RMS tumors, highlighting its correlation with the presence of fusion protein. By conducting TRIB3 genetic inhibition experiments, we observed an impairment on cell proliferation. Notably, the knockdown of TRIB3 led to a decrease in PAX3-FOXO1 and its target genes at protein level, accompanied by a reduction in the activity of the Akt signaling pathway. Additionally, inducible silencing of TRIB3 significantly delayed tumor growth and improved overall survival in vivo. Based on our analysis, we propose that TRIB3 holds therapeutic potential for treating the most aggressive subtype of RMS. The findings herein reported contribute to our understanding of the underlying molecular mechanisms driving RMS progression and provide novel insights into the potential use of TRIB3 as a therapeutic intervention for high-risk RMS patients.

Brain specific kinase-1 BRSK1/SAD-B associates with lipid rafts: modulation of kinase activity by lipid environment.

Brain specific kinases 1 and 2 (BRSK1/2, also named SAD kinases) are serine-threonine kinases specifically expressed in the brain, and activated by LKB1-mediated phosphorylation of a threonine residue at their T-loop (Thr189/174 in human BRSK1/2). BRSKs are crucial for establishing neuronal polarity, and BRSK1 has also been shown to regulate neurotransmitter release presynaptically. How BRSK1 exerts this latter function is unknown, since its substrates at the synaptic terminal and the mechanisms modulating its activity remain to be described. Key regulators of neurotransmitter release, such as SNARE complex proteins, are located at membrane rafts. Therefore we initially undertook this work to check whether BRSK1 also locates at these membrane microdomains. Here we show that brain BRSK1, but not BRSK2, is palmitoylated, and provide biochemical and pharmacological evidences demonstrating that a pool of BRSK1, but not BRSK2 or LKB1, localizes at membrane lipid rafts. We also show that raft-associated BRSK1 has higher activity than BRSK1 from non-raft environment, based on a higher T-loop phosphorylation at Thr-189. Further, recombinant BRSK1 activity increased 3-fold when assayed with small multilamellar vesicles (SMV) generated with lipids extracted from synaptosomal raft fractions. A similar BRSK1-activating effect was obtained with synthetic SMV made with phosphatidylcholine, cholesterol and sphingomyelin, mixed in the same molar ratio at which these three major lipids are present in rafts. Importantly, SMV also enhanced the activity of a constitutively active BRSK1 (T189E), underpinning that interaction with lipid rafts represents a new mechanism of BRSK1 activity modulation, additional to T-loop phosphorylation.

ERK5 Inhibition Induces Autophagy-Mediated Cancer Cell Death by Activating ER Stress.

Autophagy is a highly conserved intracellular process that preserves cellular homeostasis by mediating the lysosomal degradation of virtually any component of the cytoplasm. Autophagy is a key instrument of cellular response to several stresses, including endoplasmic reticulum (ER) stress. Cancer cells have developed high dependency on autophagy to overcome the hostile tumor microenvironment. Thus, pharmacological activation or inhibition of autophagy is emerging as a novel antitumor strategy. ERK5 is a novel member of the MAP kinase family that is activated in response to growth factors and different forms of stress. Recent work has pointed ERK5 as a major player controlling cancer cell proliferation and survival. Therefore small-molecule inhibitors of ERK5 have shown promising therapeutic potential in different cancer models. Here, we report for the first time ERK5 as a negative regulator of autophagy. Thus, ERK5 inhibition or silencing induced autophagy in a panel of human cancer cell lines with different mutation patterns. As reported previously, ERK5 inhibitors (ERK5i) induced apoptotic cancer cell death. Importantly, we found that autophagy mediates the cytotoxic effect of ERK5i, since ATG5-/- autophagy-deficient cells viability was not affected by these compounds. Mechanistically, ERK5i stimulated autophagic flux independently of the canonical regulators AMPK or mTORC1. Moreover, ERK5 inhibition resulted in ER stress and activation of the Unfolded Protein Response (UPR) pathways. Specifically, ERK5i induced expression of the ER luminal chaperone BiP (a hallmark of ER stress), the UPR markers CHOP and ATF4, and the spliced form of XBP1. Pharmacological inhibition of UPR with chemical chaperone TUDC, or ATF4 silencing, resulted in impaired ERK5i-mediated UPR, autophagy and cytotoxicity. Overall, our results suggest that ERK5 inhibition induces autophagy-mediated cancer cell death by activating ER stress. Since ERK5 inhibition sensitizes cancer cells and tumors to chemotherapy, future work will determine the relevance of UPR and autophagy in the combined use of chemotherapy and ERK5i to tackle Cancer.

The anti-cancer drug ABTL0812 induces ER stress-mediated cytotoxic autophagy by increasing dihydroceramide levels in cancer cells.

ABTL0812 is a first-in-class small molecule with anti-cancer activity, which is currently in clinical evaluation in a phase 2 trial in patients with advanced endometrial and squamous non-small cell lung carcinoma (NCT03366480). Previously, we showed that ABTL0812 induces TRIB3 pseudokinase expression, resulting in the inhibition of the AKT-MTORC1 axis and macroautophagy/autophagy-mediated cancer cell death. However, the precise molecular determinants involved in the cytotoxic autophagy caused by ABTL0812 remained unclear. Using a wide range of biochemical and lipidomic analyses, we demonstrated that ABTL0812 increases cellular long-chain dihydroceramides by impairing DEGS1 (delta 4-desaturase, sphingolipid 1) activity, which resulted in sustained ER stress and activated unfolded protein response (UPR) via ATF4-DDIT3-TRIB3 that ultimately promotes cytotoxic autophagy in cancer cells. Accordingly, pharmacological manipulation to increase cellular dihydroceramides or incubation with exogenous dihydroceramides resulted in ER stress, UPR and autophagy-mediated cancer cell death. Importantly, we have optimized a method to quantify mRNAs in blood samples from patients enrolled in the ongoing clinical trial, who showed significant increased DDIT3 and TRIB3 mRNAs. This is the first time that UPR markers are reported to change in human blood in response to any drug treatment, supporting their use as pharmacodynamic biomarkers for compounds that activate ER stress in humans. Finally, we found that MTORC1 inhibition and dihydroceramide accumulation synergized to induce autophagy and cytotoxicity, phenocopying the effect of ABTL0812. Given the fact that ABTL0812 is under clinical development, our findings support the hypothesis that manipulation of dihydroceramide levels might represents a new therapeutic strategy to target cancer.Abbreviations: 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATG: autophagy related; ATF4: activating transcription factor 4; Cer: ceramide; DDIT3: DNA damage inducible transcript 3; DEGS1: delta 4-desaturase, sphingolipid 1; dhCer: dihydroceramide; EIF2A: eukaryotic translation initiation factor 2 alpha; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; NSCLC: non-small cell lung cancer; THC: Δ9-tetrahydrocannabinol; TRIB3: tribbles pseudokinase 3; XBP1: X-box binding protein 1; UPR: unfolded protein response.

The antitumour drug ABTL0812 impairs neuroblastoma growth through endoplasmic reticulum stress-mediated autophagy and apoptosis.

Neuroblastoma is the leading cause of cancer death in children aged 1 to 4 years. Particularly, five-year overall survival for high-risk neuroblastoma is below 50% with no curative options when refractory or relapsed. Most of current therapies target cell division and proliferation, thereby inducing DNA damage and programmed cell death. However, aggressive tumours often present alterations of these processes and are resistant to therapy. Therefore, exploring alternative pathways to induce tumour cell death will provide new therapeutic opportunities for these patients. In this study we aimed at testing the therapeutic potential of ABTL0812, a novel anticancer drug that induces cytotoxic autophagy to eliminate cancer cells, which is currently in phase II clinical trials of adult tumours. Here, we show that ABTL0812 impaired the viability of clinical representative neuroblastoma cell lines regardless of genetic alterations associated to bad prognosis and resistance to therapy. Oral administration of ABTL0812 to mice bearing neuroblastoma xenografts impaired tumour growth. Furthermore, our findings revealed that, in neuroblastoma, ABTL0812 induced cancer cell death via induction of endoplasmic reticulum stress, activation of the unfolded protein response, autophagy and apoptosis. Remarkably, ABTL0812 potentiated the antitumour activity of chemotherapies and differentiating agents such as irinotecan and 13-cis-retinoic acid. In conclusion, ABTL0812 distinctive mechanism of action makes it standout to be used alone or in combination in high-risk neuroblastoma patients.

STK11 (LKB1) missense somatic mutant isoforms promote tumor growth, motility and inflammation.

Elucidating the contribution of somatic mutations to cancer is essential for personalized medicine. STK11 (LKB1) appears to be inactivated in human cancer. However, somatic missense mutations also occur, and the role/s of these alterations to this disease remain unknown. Here, we investigated the contribution of four missense LKB1 somatic mutations in tumor biology. Three out of the four mutants lost their tumor suppressor capabilities and showed deficient kinase activity. The remaining mutant retained the enzymatic activity of wild type LKB1, but induced increased cell motility. Mechanistically, LKB1 mutants resulted in differential gene expression of genes encoding vesicle trafficking regulating molecules, adhesion molecules and cytokines. The differentially regulated genes correlated with protein networks identified through comparative secretome analysis. Notably, three mutant isoforms promoted tumor growth, and one induced inflammation-like features together with dysregulated levels of cytokines. These findings uncover oncogenic roles of LKB1 somatic mutations, and will aid in further understanding their contributions to cancer development and progression.

Genetic manipulation of LKB1 elicits lethal metastatic prostate cancer.

Gene dosage is a key defining factor to understand cancer pathogenesis and progression, which requires the development of experimental models that aid better deconstruction of the disease. Here, we model an aggressive form of prostate cancer and show the unconventional association of LKB1 dosage to prostate tumorigenesis. Whereas loss of Lkb1 alone in the murine prostate epithelium was inconsequential for tumorigenesis, its combination with an oncogenic insult, illustrated by Pten heterozygosity, elicited lethal metastatic prostate cancer. Despite the low frequency of LKB1 deletion in patients, this event was significantly enriched in lung metastasis. Modeling the role of LKB1 in cellular systems revealed that the residual activity retained in a reported kinase-dead form, LKB1K78I, was sufficient to hamper tumor aggressiveness and metastatic dissemination. Our data suggest that prostate cells can function normally with low activity of LKB1, whereas its complete absence influences prostate cancer pathogenesis and dissemination.

Neuronal Growth Factor regulates Brain Specific Kinase 1 expression by inhibiting promoter methylation and promoting Sp1 recruitment.

Brain specific kinases (BRSKs) are serine/threonine kinases, preferentially expressed in the brain after Embryonic Day 12. Although BRSKs are crucial neuronal development factors and regulation of their enzymatic activity has been widely explored, little is known of their transcriptional regulation. In this work, we show that Neuronal Growth Factor (NGF) increased the expression of Brsk1 in PC12 cells. Furthermore, during neuronal differentiation, Brsk1 mRNA increased through a MAPK-dependent Sp1 activation. To gain further insight into this regulation, we analyzed the transcriptional activity of the Brsk1 promoter in PC12 cells treated with NGF. Initially, we defined the minimal promoter region (-342 to +125 bp) responsive to NGF treatment. This region had multiple Sp1 binding sites, one of which was within a CpG island. In vitro binding assays showed that NGF-induced differentiation increased Sp1 binding to this site and that DNA methylation inhibited Sp1 binding. In vitro methylation of the Brsk1 promoter reduced its transcriptional activity and impaired the NGF effect. To evaluate the participation of DNA methyltransferases in Brsk1 gene regulation, the 5'Aza-dC inhibitor was used. 5'Aza-dC acted synergistically with NGF to promote Brsk1 promoter activity. Accordingly, DNMT3B overexpression abolished the response of the Brsk1 promoter to NGF. Surprisingly, we found Dnmt3b to be a direct target of NGF regulation, via the MAPK pathway. In conclusion, our results provide evidence of a novel mechanism of Brsk1 transcriptional regulation changing the promoter's methylation status, which was incited by the NGF-induced neuronal differentiation process.

Structural and Atropisomeric Factors Governing the Selectivity of Pyrimido-benzodiazipinones as Inhibitors of Kinases and Bromodomains.

Bromodomains have been pursued intensively over the past several years as emerging targets for the development of anticancer and anti-inflammatory agents. It has recently been shown that some kinase inhibitors are able to potently inhibit the bromodomains of BRD4. The clinical activities of PLK inhibitor BI-2536 and JAK2-FLT3 inhibitor TG101348 have been attributed to this unexpected polypharmacology, indicating that dual-kinase/bromodomain activity may be advantageous in a therapeutic context. However, for target validation and biological investigation, a more selective target profile is desired. Here, we report that benzo[e]pyrimido-[5,4- b]diazepine-6(11H)-ones, versatile ATP-site directed kinase pharmacophores utilized in the development of inhibitors of multiple kinases, including several previously reported kinase chemical probes, are also capable of exhibiting potent BRD4-dependent pharmacology. Using a dual kinase-bromodomain inhibitor of the kinase domains of ERK5 and LRRK2, and the bromodomain of BRD4 as a case study, we define the structure-activity relationships required to achieve dual kinase/BRD4 activity, as well as how to direct selectivity toward inhibition of either ERK5 or BRD4. This effort resulted in identification of one of the first reported kinase-selective chemical probes for ERK5 (JWG-071), a BET selective inhibitor with 1 µM BRD4 IC50 (JWG-115), and additional inhibitors with rationally designed polypharmacology (JWG-047, JWG-069). Co-crystallography of seven representative inhibitors with the first bromodomain of BRD4 demonstrate that distinct atropisomeric conformers recognize the kinase ATP-site and the BRD4 acetyl lysine binding site, conformational preferences supported by rigid docking studies.

ERK5 and Cell Proliferation: Nuclear Localization Is What Matters.

ERK5, the last MAP kinase family member discovered, is activated by the upstream kinase MEK5 in response to growth factors and stress stimulation. MEK5-ERK5 pathway has been associated to different cellular processes, playing a crucial role in cell proliferation in normal and cancer cells by mechanisms that are both dependent and independent of its kinase activity. Thus, nuclear ERK5 activates transcription factors by either direct phosphorylation or acting as co-activator thanks to a unique transcriptional activation TAD domain located at its C-terminal tail. Consequently, ERK5 has been proposed as an interesting target to tackle different cancers, and either inhibitors of ERK5 activity or silencing the protein have shown antiproliferative activity in cancer cells and to block tumor growth in animal models. Here, we review the different mechanisms involved in ERK5 nuclear translocation and their consequences. Inactive ERK5 resides in the cytosol, forming a complex with Hsp90-Cdc37 superchaperone. In a canonical mechanism, MEK5-dependent activation results in ERK5 C-terminal autophosphorylation, Hsp90 dissociation, and nuclear translocation. This mechanism integrates signals such as growth factors and stresses that activate the MEK5-ERK5 pathway. Importantly, two other mechanisms, MEK5-independent, have been recently described. These mechanisms allow nuclear shuttling of kinase-inactive forms of ERK5. Although lacking kinase activity, these forms activate transcription by interacting with transcription factors through the TAD domain. Both mechanisms also require Hsp90 dissociation previous to nuclear translocation. One mechanism involves phosphorylation of the C-terminal tail of ERK5 by kinases that are activated during mitosis, such as Cyclin-dependent kinase-1. The second mechanism involves overexpression of chaperone Cdc37, an oncogene that is overexpressed in cancers such as prostate adenocarcinoma, where it collaborates with ERK5 to promote cell proliferation. Although some ERK5 kinase inhibitors have shown antiproliferative activity it is likely that those tumors expressing kinase-inactive nuclear ERK5 will not respond to these inhibitors.

Mutation of the 3-Phosphoinositide-Dependent Protein Kinase 1 (PDK1) Substrate-Docking Site in the Developing Brain Causes Microcephaly with Abnormal Brain Morphogenesis Independently of Akt, Leading to Impaired Cognition and Disruptive Behaviors.

The phosphoinositide (PI) 3-kinase/Akt signaling pathway plays essential roles during neuronal development. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) coordinates the PI 3-kinase signals by activating 23 kinases of the AGC family, including Akt. Phosphorylation of a conserved docking site in the substrate is a requisite for PDK1 to recognize, phosphorylate, and activate most of these kinases, with the exception of Akt. We exploited this differential mechanism of regulation by generating neuron-specific conditional knock-in mice expressing a mutant form of PDK1, L155E, in which the substrate-docking site binding motif, termed the PIF pocket, was disrupted. As a consequence, activation of all the PDK1 substrates tested except Akt was abolished. The mice exhibited microcephaly, altered cortical layering, and reduced circuitry, leading to cognitive deficits and exacerbated disruptive behavior combined with diminished motivation. The abnormal patterning of the adult brain arises from the reduced ability of the embryonic neurons to polarize and extend their axons, highlighting the essential roles that the PDK1 signaling beyond Akt plays in mediating the neuronal responses that regulate brain development.

The New Antitumor Drug ABTL0812 Inhibits the Akt/mTORC1 Axis by Upregulating Tribbles-3 Pseudokinase.

PURPOSE: ABTL0812 is a novel first-in-class, small molecule which showed antiproliferative effect on tumor cells in phenotypic assays. Here we describe the mechanism of action of this antitumor drug, which is currently in clinical development. EXPERIMENTAL DESIGN: We investigated the effect of ABTL0812 on cancer cell death, proliferation, and modulation of intracellular signaling pathways, using human lung (A549) and pancreatic (MiaPaCa-2) cancer cells and tumor xenografts. To identify cellular targets, we performed in silico high-throughput screening comparing ABTL0812 chemical structure against ChEMBL15 database. RESULTS: ABTL0812 inhibited Akt/mTORC1 axis, resulting in impaired cancer cell proliferation and autophagy-mediated cell death. In silico screening led us to identify PPARs, PPARα and PPARγ as the cellular targets of ABTL0812. We showed that ABTL0812 activates both PPAR receptors, resulting in upregulation of Tribbles-3 pseudokinase (TRIB3) gene expression. Upregulated TRIB3 binds cellular Akt, preventing its activation by upstream kinases, resulting in Akt inhibition and suppression of the Akt/mTORC1 axis. Pharmacologic inhibition of PPARα/γ or TRIB3 silencing prevented ABTL0812-induced cell death. ABTL0812 treatment induced Akt inhibition in cancer cells, tumor xenografts, and peripheral blood mononuclear cells from patients enrolled in phase I/Ib first-in-human clinical trial. CONCLUSIONS: ABTL0812 has a unique and novel mechanism of action, that defines a new and drugable cellular route that links PPARs to Akt/mTORC1 axis, where TRIB3 pseudokinase plays a central role. Activation of this route (PPARα/γ-TRIB3-Akt-mTORC1) leads to autophagy-mediated cancer cell death. Given the low toxicity and high tolerability of ABTL0812, our results support further development of ABTL0812 as a promising anticancer therapy. Clin Cancer Res; 22(10); 2508-19. ©2015 AACR.

Identification of the sucrose non-fermenting related kinase SNRK, as a novel LKB1 substrate.

Recent work has shown that the LKB1 tumour suppressor protein kinase phosphorylates and activates protein kinases belonging to the AMP activated kinase (AMPK) subfamily. In this study, we identify the sucrose non-fermenting protein (SNF1)-related kinase (SNRK), a largely unstudied AMPK subfamily member, as a novel substrate for LKB1. We demonstrate that LKB1 activates SNRK by phosphorylating the T-loop residue (Thr173), and that the LKB1 regulatory subunits STRAD and MO25 are required for LKB1 to activate SNRK. We find that SNRK is not active when expressed in HeLa cells that lack expression of LKB1, and its activity is restored by expression of wild type LKB1, but not catalytically deficient LKB1. We also present evidence that two other AMPK-related kinases more distantly related to AMPK than SNRK, namely NIM1 and testis-specific serine/threonine kinase-1 (TSSK1) are not substrates for LKB1. Tissue distribution analysis indicates that SNRK protein is mainly expressed in testis, similar to TSSK isoforms, whereas NIM1 is more widely expressed. These results provide evidence that SNRK could mediate some of the physiological effects of LKB1.

Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B.

An important mechanism by which insulin regulates cell growth and protein synthesis is through activation of the p70 ribosomal S6 protein kinase (S6K). In mammalian cells, insulin-induced PI3K (phosphoinositide 3-kinase) activation, generates the lipid second messenger PtdIns(3,4,5) P (3), which is thought to play a key role in triggering the activation of S6K. Although the major components of the insulin-signalling pathway are conserved in Drosophila, recent studies suggested that S6K activation does not require PI3K in this system. To investigate further the role of dPI3K (Drosophila PI3K) in dS6K (Drosophila S6K) activation, we examined the effect of two structurally distinct PI3K inhibitors on insulin-induced dS6K activation in Kc167 and S2 Drosophila cell lines. We found that both inhibitors prevented insulin-stimulated phosphorylation and activation of dS6K. To investigate further the role of the dPI3K pathway in regulating dS6K activation, we also used dsRNAi (double-stranded RNA-mediated interference) to decrease expression of dPI3K and the PtdIns(3,4,5) P (3) phosphatase dPTEN ( Drosophila phosphatase and tensin homologue deleted on chromosome 10) in Kc167 and S2 cells. Knock-down of dPI3K prevented dS6K activation, whereas knock-down of dPTEN, which would be expected to increase PtdIns(3,4,5) P (3) levels, stimulated dS6K activity. Moreover, when the expression of the dPI3K target, dPKB (Drosophila protein kinase B), was decreased to undetectable levels, we found that insulin could no longer trigger dS6K activation. This observation provides the first direct demonstration that dPKB is required for insulin-stimulated dS6K activation. We also present evidence that the amino-acid-induced activation of dS6K in the absence of insulin, thought to be mediated by dTOR (Drosophila target of rapamycin), which is unaffected by the inhibition of dPI3K by wortmannin. The results of the present study support the view that, in Drosophila cells, dPI3K and dPKB, as well dTOR, are required for the activation of dS6K by insulin.