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CDK12 is a transcriptional cyclin-dependent kinase (CDK) that interacts with cyclin K to regulate different aspects of gene expression. The CDK12-cyclin K complex phosphorylates several substrates, including RNA Pol II, and thereby regulates transcription elongation, RNA splicing, as well as cleavage and polyadenylation. Because of its implication in cancer, multiple pharmacological inhibitors of CDK12 have been identified to date, including THZ531 and SR-4835. While both CDK12 inhibitors affect Poll II phosphorylation, we found that SR-4835 uniquely promotes cyclin K degradation via the proteasome. Using loss-of-function genetic screening, we found that SR-4835 cytotoxicity depends on a functional CUL4-RBX1-DDB1 ubiquitin ligase complex. Consistent with this, we show that DDB1 is required for cyclin K degradation, and that SR-4835 promotes DDB1 interaction with the CDK12-cyclin K complex. Docking studies and structure-activity relationship analyses of SR-4835 revealed the importance of the benzimidazole side-chain in molecular glue activity. Together, our results indicate that SR-4835 acts as a molecular glue that recruits the CDK12-cyclin K complex to the CUL4-RBX1-DDB1 ubiquitin ligase complex to target cyclin K for degradation.

How the genetic landscape governs a tumor’s response to immunotherapy remains poorly understood. To assess the immune-modulatory capabilities of 573 genes associated with altered cytotoxicity in human cancers, here we perform CRISPR/Cas9 screens directly in mouse lung cancer models. We recover the known immune evasion factors Stat1 and Serpinb9 and identify the cancer testis antigen Adam2 as an immune modulator, whose expression is induced by KrasG12D and further elevated by immunotherapy. Using loss- and gain-of-function experiments, we show that ADAM2 functions as an oncogene by restraining interferon and TNF cytokine signaling causing reduced presentation of tumor-associated antigens. ADAM2 also restricts expression of the immune checkpoint inhibitors PDL1, LAG3, TIGIT and TIM3 in the tumor microenvironment, which might explain why ex vivo expanded and adoptively transferred cytotoxic T-cells show enhanced cytotoxic efficacy in ADAM2 overexpressing tumors. Together, direct in vivo CRISPR/Cas9 screens can uncover genetic alterations that control responses to immunotherapies.

RSK Inhibition in Melanoma Cells do not Affect Glycolytic Enzyme mRNA.

Metabolic reprogramming is a hallmark of cancer that includes increased glucose uptake and accelerated aerobic glycolysis. This phenotype is required to fulfill anabolic demands associated with aberrant cell proliferation and is often mediated by oncogenic drivers such as activated BRAF. In this study, we show that the MAPK-activated p90 ribosomal S6 kinase (RSK) is necessary to maintain glycolytic metabolism in BRAF-mutated melanoma cells. RSK directly phosphorylated the regulatory domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2), an enzyme that catalyzes the synthesis of fructose-2,6-bisphosphate during glycolysis. Inhibition of RSK reduced PFKFB2 activity and glycolytic flux in melanoma cells, suggesting an important role for RSK in BRAF-mediated metabolic rewiring. Consistent with this, expression of a phosphorylation-deficient mutant of PFKFB2 decreased aerobic glycolysis and reduced the growth of melanoma in mice. Together, these results indicate that RSK-mediated phosphorylation of PFKFB2 plays a key role in the metabolism and growth of BRAF-mutated melanomas.Significance: RSK promotes glycolytic metabolism and the growth of BRAF-mutated melanoma by driving phosphorylation of an important glycolytic enzyme. Cancer Res; 78(9); 2191–204. ©2018 AACR.

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PDF file - 186K, Generation of CK1 isoform knockdown cells (S1); Effect of CK1epsilon inhibitor on cell death (S2); CK1epsilon expression in cancer tissue (S3); CK1epsilon interacts with 4E-BP1 (S4); CK1epsilon phosphorylates 4E-BP1 at T41/T50 (S5).

Abolition of the LH-induced ERK1/2 pathway leads to dramatic changes in gene expression in granulosa cells, subsequently abrogating ovulation. Here we explored whether sustained ERK1/2 signaling beyond immediate-early hours of the LH surge is important for ovulation in mice. First, we examined the effect of inhibition of ERK1/2 activity at 4 h after hCG stimulation on ovulation in superovulated immature mice. Treatment with the ERK1/2 pathway inhibitor PD0325901 at 4 h post-hCG disrupted follicular rupture without altering cumulus expansion, oocyte meiotic maturation and luteinization. Profiling the expression pattern of genes of the RSK family of ERK1/2 signal mediators revealed that RSK3, but not other isoforms, was induced by hCG treatment. Further, RSK3-knockout mice were sub-fertile with reduced ovulation rate and smaller litter size compared to WT mice. Given that PD0325901 inhibits all mediators of ERK1/2 signaling, we chose to evaluate the gene expression underlying deficient follicular rupture in ERK1/2 inhibited mice. We found that inhibition of ERK1/2 signaling at 4 h post-hCG resulted in an imbalance in the expression of genes involved in extracellular matrix degradation and leukocyte infiltration necessary for follicular rupture. In conclusion, our data demonstrate that sustained ERK1/2 signaling during ovulation is not required for cumulus expansion, oocyte meiotic maturation and luteinization, but is required for follicular rupture.

How the genetic landscape of a tumor governs the tumor’s response to immunotherapy remains largely elusive. Here, we established a direct in vivo CRISPR/Cas9 gene editing methodology to assess the immune-modulatory capabilities of 573 putative cancer genes associated with altered cytotoxic activity in human cancers. Using KrasG12D- and BrafV600E-driven mouse lung cancer models, we identify Serpinb9 and Adam2 as our top immune suppressive and immune enhancing genes, respectively. Mechanistically, we show that Serpinb9 ablation in KrasG12D- and BrafV600E-mutant lung tumor cells greatly enhances the efficacy of cytotoxic T-cells in vitro and in vivo. ADAM2 is a cancer testis antigen broadly expressed in human cancers such as lung adenocarcinoma (13.9%), renal (74.7%), prostate (72.4%), uterine (28.6%) and invasive breast (9.5%) cancer. In our mouse models, we show that Adam2 expression is induced in KrasG12D- but not BrafV600E-driven murine lung tumors and that its expression is further enhanced by immunotherapy. We show that loss of Adam2 significantly decreases KrasG12D-lung tumor burden but blocks the efficacy of cytotoxic T-cells. Consistently, Adam2 overexpression dramatically increases tumor growth and enhances immunotherapy efficacy. Mechanistically, we find that Adam2’s oncogenic function depends on modulating the tumor immune microenvironment by restraining productive type I and type II interferon responses as well as cytokine signaling, reducing the presentation of tumor-associated antigen, and modulating surface expression of several immunoregulatory receptors within Kras-driven lung tumors. Adam2 expression also leads to reduced levels of immune checkpoint inhibitors such as Pd-l1, Lag3, Tigit and Tim3. This reduced exhaustion within the tumor microenvironment may explain why ex vivo expanded and adoptively transferred cytotoxic T-cells show enhanced cytotoxic efficacy against Adam2 overexpressing lung tumors. Together, our study highlights the power of integrating cancer genomic with in vivo CRISPR/Cas9 screens to uncover how cancer-associated genetic alterations control responses to immunotherapies.

Despite its importance in human cancers, including colorectal cancers (CRC), oncogenic KRAS has been extremely challenging to target therapeutically. To identify potential vulnerabilities in KRAS-mutated CRC, we characterize the impact of oncogenic KRAS on the cell surface of intestinal epithelial cells. Here we show that oncogenic KRAS alters the expression of a myriad of cell-surface proteins implicated in diverse biological functions, and identify many potential surface-accessible therapeutic targets. Cell surface-based loss-of-function screens reveal that ATP7A, a copper-exporter upregulated by mutant KRAS, is essential for neoplastic growth. ATP7A is upregulated at the surface of KRAS-mutated CRC, and protects cells from excess copper-ion toxicity. We find that KRAS-mutated cells acquire copper via a non-canonical mechanism involving macropinocytosis, which appears to be required to support their growth. Together, these results indicate that copper bioavailability is a KRAS-selective vulnerability that could be exploited for the treatment of KRAS-mutated neoplasms., The oncogene KRAS is frequently mutated in cancer, including colorectal cancer. Here, using a cell-surface proteomics approach, KRAS-mutated colorectal cancer cells are shown to express high levels of the copper transporter ATP7A, which has an essential roles in cancer cell survival and proliferation.

Rationale: Vascular remodeling, including smooth muscle cell hypertrophy and proliferation, is the key pathological feature of pulmonary arterial hypertension (PAH). Prostaglandin I2 analogs (beraprost, iloprost, and treprostinil) are effective in the treatment of PAH. Of note, the clinically favorable effects of treprostinil in severe PAH may be attributable to concomitant activation of DP1 (D prostanoid receptor subtype 1).Objectives: To study the role of DP1 in the progression of PAH and its underlying mechanism.Methods: DP1 levels were examined in pulmonary arteries of patients and animals with PAH. Multiple genetic and pharmacologic approaches were used to investigate DP1-mediated signaling in PAH.Measurements and Main Results: DP1 expression was downregulated in hypoxia-treated pulmonary artery smooth muscle cells and in pulmonary arteries from rodent PAH models and patients with idiopathic PAH. DP1 deletion exacerbated pulmonary artery remodeling in hypoxia-induced PAH, whereas pharmacological activation or forced expression of the DP1 receptor had the opposite effect in different rodent models. DP1 deficiency promoted pulmonary artery smooth muscle cell hypertrophy and proliferation in response to hypoxia via induction of mTORC1 (mammalian target of rapamycin complex 1) activity. Rapamycin, an inhibitor of mTORC1, alleviated the hypoxia-induced exacerbation of PAH in DP1-knockout mice. DP1 activation facilitated raptor dissociation from mTORC1 and suppressed mTORC1 activity through PKA (protein kinase A)-dependent phosphorylation of raptor at Ser791. Moreover, treprostinil treatment blocked the progression of hypoxia-induced PAH in mice in part by targeting the DP1 receptor.Conclusions: DP1 activation attenuates hypoxia-induced pulmonary artery remodeling and PAH through PKA-mediated dissociation of raptor from mTORC1. These results suggest that the DP1 receptor may serve as a therapeutic target for the management of PAH.

The RNA-binding protein LARP1 has generated interest in recent years for its role in the mTOR signalling cascade and its regulation of terminal oligopyrimidine (TOP) mRNA translation. Paradoxically, some scientists have shown that LARP1 represses TOP translation while others that LARP1 activates it. Here, we present opinions from four leading scientists in the field to discuss these and other contradictory findings.

Key Points CEACAM1 is a novel cell surface marker of ex vivo expanded LT-HSCs. Sorting umbilical cord blood cells based on CEACAM1 and known HSC marker expression allows purification of LT-HSCs with improved purity.

Ribosome biogenesis lies at the nexus of various signaling pathways coordinating protein synthesis with cell growth and proliferation. This process is regulated by well-described transcriptional mechanisms, but a growing body of evidence indicates that other levels of regulation exist. Here we show that the Ras/mitogen-activated protein kinase (MAPK) pathway stimulates post-transcriptional stages of human ribosome synthesis. We identify RIOK2, a pre-40S particle assembly factor, as a new target of the MAPK-activated kinase RSK. RIOK2 phosphorylation by RSK stimulates cytoplasmic maturation of late pre-40S particles, which is required for optimal protein synthesis and cell proliferation. RIOK2 phosphorylation facilitates its release from pre-40S particles and its nuclear re-import, prior to completion of small ribosomal subunits. Our results bring a detailed mechanistic link between the Ras/MAPK pathway and the maturation of human pre-40S particles, which opens a hitherto poorly explored area of ribosome biogenesis., Author summary Ribosomes are universal molecular machines composed of ribosomal RNAs (rRNAs) and ribosomal proteins (RPs) responsible for the synthesis of cellular proteins. In eukaryotic cells, ribosome biogenesis relies on the assembly of large precursor particles (pre-ribosomes) containing, in addition to rRNAs and RPs, more than 200 accessory proteins named assembly and maturation factors (AMFs). Ribosome biogenesis is a highly energy demanding process which is tightly regulated to accommodate protein synthesis with cell growth requirements. In humans, the Ras/MAPK regulatory pathway is one of the main systems controlling cell growth and proliferation. Under favorable growth conditions, the Ras/MAPK pathway activates several aspects of ribosome production, such as synthesis of rRNAs and RPs. Our study highlights that the MAPK pathway also directly stimulates the assembly and maturation of pre-ribosomes. We identify the kinase RIOK2, an AMF involved in the synthesis of the small (40S) ribosomal subunit, as a new target of the MAPK-activated kinase RSK. We describe molecular events promoted by the MAPK pathway to finely tune the activity of RIOK2 during pre-40S particle maturation. This work is particularly important because of the current lack of mechanistic connections between growth-promoting signaling pathways and the assembly and maturation of pre-ribosomes in human cells.

Inactivation of the protein complex ‘mechanistic target of rapamycin complex 1’ (mTORC1) can increase the nuclear content of transcriptional regulators of metabolism and apoptosis. Previous studies established that nuclear import of signal transducer and activator of transcription-1 (STAT1) requires the mTORC1-associated adaptor karyopherin-α1 (KPNA1) when mTORC1 activity is reduced. However, the role of other mTORC1-interacting proteins in the complex, including ‘protein kinase C delta’ (PKCδ), have not been well characterized. In this study, we demonstrate that PKCδ, a STAT1 kinase, contains a functional ‘target of rapamycin signaling’ (TOS) motif that directs its interaction with mTORC1. Depletion of KPNA1 by RNAi prevented the nuclear import of PKCδ in cells exposed to the mTORC1 inhibitor rapamycin or amino acid restriction. Mutation of the TOS motif in PKCδ led to its loss of regulation by mTORC1 or karyopherin-α1, resulting in increased constitutive nuclear content. In cells expressing wild-type PKCδ, STAT1 activity and apoptosis were increased by rapamycin or interferon-β. Those expressing the PKCδ TOS mutant exhibited increased STAT1 activity and apoptosis; further enhancement by rapamycin or interferon-β, however, was lost. Therefore, the TOS motif in PKCδ is a novel structural mechanism by which mTORC1 prevents PKCδ and STAT1 nuclear import, and apoptosis.

Acute megakaryoblastic leukemia (AMKL) represents ∼10% of pediatric acute myeloid leukemia cases and typically affects young children (

Collective cell migration is involved in development, wound healing and metastasis. In the Drosophila ovary, border cells (BC) form a small cluster that migrates collectively through the egg chamber. To achieve directed motility, the BC cluster coordinates the formation of protrusions in its leader cell and contractility at the rear. Restricting protrusions to leader cells requires the actin and plasma membrane linker Moesin. Herein, we show that the Ste20-like kinase Misshapen phosphorylates Moesin in vitro and in BC. Depletion of Misshapen disrupts protrusion restriction, thereby allowing other cells within the cluster to protrude. In addition, we show that Misshapen is critical to generate contractile forces both at the rear of the cluster and at the base of protrusions. Together, our results indicate that Misshapen is a key regulator of BC migration as it coordinates two independent pathways that restrict protrusion formation to the leader cells and induces contractile forces., Directed motility of cell clusters requires coordination of protrusion formation at the front of leader cells and contractility at the rear. Here the authors show that the kinase Misshapen restricts protrusions to the leader cell and promotes contractile forces at the rear of the cluster.

KRAS-driven cancers acquire profound metabolic dependencies that are intimately linked to tumor growth. Our work revealed that colorectal cancers that harbor KRAS mutations are addicted to copper metabolism. This adaptation renders tumor cells critically dependent on the copper transporter ATP7A, which reveals copper metabolism as a promising therapeutic target for KRAS-driven colorectal cancers.

Lung cancer causes more deaths annually than any other malignancy. A subset of non-small cell lung cancer (NSCLC) is driven by amplification and overexpression or activating mutation of the receptor tyrosine kinase (RTK) ERBB2. In some contexts, notably breast cancer, alternative splicing of ERBB2 causes skipping of exon 16, leading to the expression of an oncogenic ERBB2 isoform (ERBB2ΔEx16) that forms constitutively active homodimers. However, the broader implications of ERBB2 alternative splicing in human cancers have not been explored. Here, we have used genomic and transcriptomic analysis to identify elevated ERBB2ΔEx16 expression in a subset of NSCLC cases, as well as splicing site mutations facilitating exon 16 skipping and deletions of exon 16 in a subset of these lung tumors and in a number of other carcinomas. Supporting the potential of ERBB2ΔEx16 as a lung cancer driver, its expression transformed immortalized lung epithelial cells while a transgenic model featuring inducible ERBB2ΔEx16 specifically in the lung epithelium rapidly developed lung adenocarcinomas following transgene induction. Collectively, these observations indicate that ERBB2ΔEx16 is a lung cancer oncogene with potential clinical importance for a proportion of patients.

Inactivation of the protein complex 'mechanistic target of rapamycin complex 1' (mTORC1) can increase the nuclear content of transcriptional regulators of metabolism and apoptosis. Previous studies established that nuclear import of signal transducer and activator of transcription-1 (STAT1) requires the mTORC1-associated adaptor karyopherin-α1 (KPNA1) when mTORC1 activity is reduced. However, the role of other mTORC1-interacting proteins in the complex, including 'protein kinase C delta' (PKCδ), have not been well characterized. In this study, we demonstrate that PKCδ, a STAT1 kinase, contains a functional 'target of rapamycin signaling' (TOS) motif that directs its interaction with mTORC1. Depletion of KPNA1 by RNAi prevented the nuclear import of PKCδ in cells exposed to the mTORC1 inhibitor rapamycin or amino acid restriction. Mutation of the TOS motif in PKCδ led to its loss of regulation by mTORC1 or karyopherin-α1, resulting in increased constitutive nuclear content. In cells expressing wild-type PKCδ, STAT1 activity and apoptosis were increased by rapamycin or interferon-β. Those expressing the PKCδ TOS mutant exhibited increased STAT1 activity and apoptosis; further enhancement by rapamycin or interferon-β, however, was lost. Therefore, the TOS motif in PKCδ is a novel structural mechanism by which mTORC1 prevents PKCδ and STAT1 nuclear import, and apoptosis.

Summary Most metazoan cells entering mitosis undergo characteristic rounding, which is important for accurate spindle positioning and chromosome separation. Rounding is driven by contractile tension generated by myosin motors in the sub-membranous actin cortex. Recent studies highlight that alongside myosin activity, cortical actin organization is a key regulator of cortex tension. Yet, how mitotic actin organization is controlled remains poorly understood. To address this, we characterized the F-actin interactome in spread interphase and round mitotic cells. Using super-resolution microscopy, we then screened for regulators of cortex architecture and identified the intermediate filament vimentin and the actin-vimentin linker plectin as unexpected candidates. We found that vimentin is recruited to the mitotic cortex in a plectin-dependent manner. We then showed that cortical vimentin controls actin network organization and mechanics in mitosis and is required for successful cell division in confinement. Together, our study highlights crucial interactions between cytoskeletal networks during cell division., Highlights • Comparison of the F-actin interactome in spread interphase and round mitotic cells • Proteomics identifies vimentin and plectin as key regulators of the mitotic cortex • Vimentin intermediate filaments localize under the actin cortex in mitosis • Sub-cortical vimentin regulates actin cortex organization and mechanics in mitosis, Mitotic rounding, which is essential for accurate spindle positioning, is controlled by the sub-membranous cortical actin network. Using proteomics, Serres et al. identify the intermediate filament vimentin as a key regulator of the mitotic cortex. Vimentin forms a sub-cortical layer in mitosis and controls actin cortex organization and mechanics.

Metabolic reprogramming is a hallmark of cancer that includes increased glucose uptake and accelerated aerobic glycolysis. This phenotype is required to fulfill anabolic demands associated with aberrant cell proliferation and is often mediated by oncogenic drivers such as activated BRAF. In this study, we show that the MAPK-activated p90 ribosomal S6 kinase (RSK) is necessary to maintain glycolytic metabolism in BRAF-mutated melanoma cells. RSK directly phosphorylated the regulatory domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2), an enzyme that catalyzes the synthesis of fructose-2,6-bisphosphate during glycolysis. Inhibition of RSK reduced PFKFB2 activity and glycolytic flux in melanoma cells, suggesting an important role for RSK in BRAF-mediated metabolic rewiring. Consistent with this, expression of a phosphorylation-deficient mutant of PFKFB2 decreased aerobic glycolysis and reduced the growth of melanoma in mice. Together, these results indicate that RSK-mediated phosphorylation of PFKFB2 plays a key role in the metabolism and growth of BRAF-mutated melanomas. Significance: RSK promotes glycolytic metabolism and the growth of BRAF-mutated melanoma by driving phosphorylation of an important glycolytic enzyme. Cancer Res; 78(9); 2191–204. ©2018 AACR.

The 90kDa ribosomal S6 kinase (RSK) family is a group of Ser/Thr protein kinases (RSK1-4) that function downstream of the Ras/mitogen-activated protein kinase (MAPK) signalling pathway. RSK regulates many substrates involved in cell survival, growth, and proliferation, and as such, deregulated RSK activity has been associated with multiple cancer types. RSK expression and activity are dysregulated in several malignancies, including breast, prostate, and lung cancer, and available evidence suggests that RSK may be a promising cancer therapeutic target. Current limitations include the lack of RSK inhibitors with suitable pharmacokinetics and selectivity toward particular isoforms. This review briefly describes the current knowledge on RSK activation and function, with a particular emphasis on RSK-dependent mechanisms associated with tumorigenesis and pharmacological inhibition.

Compared to solid tumors, antibody-based immunotherapy is underexploited in acute myeloid leukemia (AML), likely due to the limited availability of targetable surface antigens specifically expressed on AML cells. While targeted immunotherapies hold great promise, their development inexorably depends on the identification of cell surface antigens selectively expressed by cancer cells. The identification of such antigens has been challenging due to the characteristic hydrophobicity of plasma membrane proteins. Another level of complexity is brought by the fact that AML is a heterogeneous disease comprised of different subgroups with associated prognosis ranging from favorable to adverse. In order to identify novel AML antigens to be targeted using immunotherapeutic approaches, we have analysed the surface proteome of a collection of 100 primary human AML specimens, enriched in poor prognosis AML samples. These analyses revealed that AML subgroups have distinct surfaceome signatures. We also confirmed the expression of known AML markers at the surface of specific AML subgroups. Our search for novel AML antigens focused on four poor prognosis AML subgroups: MLL-rearranged, complex karyotype, RUNX1-mutated and normal karyotype with NPM1, DNMT3A and FLT3 (ITD) mutations. We identified proteins selectively expressed at the surface of AML cells from these subgroups. Analysis of the whole cohort of specimens also identified known and novel pan AML antigens. Single cell RNA-sequencing analysis of aforementioned subgroup-specific and pan AML antigens revealed that many of these are expressed by the vast majority of blast populations within primary AML specimens, including most primitive ones. Overall, these results suggest that studying the surface proteome of AML cells has the potential to reveal promising AML antigens to be targeted using immunotherapeutic approaches.

Cell and tissue morphogenesis are fundamental during development. The authors previously characterized Slik kinase and its effector, moesin, as regulators of mitotic morphogenesis and epithelial integrity. Here, they identify dSTRIPAK as a new regulator of Slik localization to promote moesin activation and functions., Proteins of the ezrin, radixin, and moesin (ERM) family control cell and tissue morphogenesis. We previously reported that moesin, the only ERM in Drosophila, controls mitotic morphogenesis and epithelial integrity. We also found that the Pp1-87B phosphatase dephosphorylates moesin, counteracting its activation by the Ste20-like kinase Slik. To understand how this signaling pathway is itself regulated, we conducted a genome-wide RNAi screen, looking for new regulators of moesin activity. We identified that Slik is a new member of the striatin-interacting phosphatase and kinase complex (STRIPAK). We discovered that the phosphatase activity of STRIPAK reduces Slik phosphorylation to promote its cortical association and proper activation of moesin. Consistent with this finding, inhibition of STRIPAK phosphatase activity causes cell morphology defects in mitosis and impairs epithelial tissue integrity. Our results implicate the Slik–STRIPAK complex in the control of multiple morphogenetic processes.

The RAS/mitogen-activated protein kinase (MAPK) signaling pathway regulates various biological functions, including cell survival, proliferation and migration. This pathway is frequently deregulated in cancer, including melanoma, which is the most aggressive form of skin cancer. RSK (p90 ribosomal S6 kinase) is a MAPK-activated protein kinase required for melanoma growth and proliferation, but relatively little is known about its function and the nature of its cellular partners. In this study, we used a proximity-based labeling approach to identify RSK proximity partners in cells. We identified many potential RSK-interacting proteins, including p120ctn (p120-catenin), which is an essential component of adherens junction (AJ). We found that RSK phosphorylates p120ctn on Ser320, which appears to be constitutively phosphorylated in melanoma cells. We also found that RSK inhibition increases melanoma cell-cell adhesion, suggesting that constitutive RAS/MAPK signaling negatively regulates AJ integrity. Together, our results indicate that RSK plays an important role in the regulation of melanoma cell-cell adhesion.

Summary: In human cells, the expression of ∼1,000 genes is modulated throughout the cell cycle. Although some of these genes are controlled by specific transcriptional programs, very little is known about their post-transcriptional regulation. Here, we analyze the expression signature associated with all 687 RNA-binding proteins (RBPs) and identify 39 that significantly correlate with cell cycle mRNAs. We find that NF45 and NF90 play essential roles in mitosis, and transcriptome analysis reveals that they are necessary for the expression of a subset of mitotic mRNAs. Using proteomics, we identify protein clusters associated with the NF45-NF90 complex, including components of Staufen-mediated mRNA decay (SMD). We show that depletion of SMD components increases the binding of mitotic mRNAs to the NF45-NF90 complex and rescues cells from mitotic defects. Together, our results indicate that the NF45-NF90 complex plays essential roles in mitosis by competing with the SMD machinery for a common set of mRNAs.

High hyperdiploidy (HD) is the most common cytogenetic subtype of childhood acute lymphoblastic leukemia (ALL), and a higher incidence of HD has been reported in ALL patients with congenital cancer syndromes. We assessed the frequency of predisposing germline mutations in 57 HD-ALL patients from the California Childhood Leukemia Study via targeted sequencing of cancer-relevant genes. Three out of 57 patients (5.3%) harbored confirmed germline mutations that were likely causal, in NBN, ETV6, and FLT3, with an additional six patients (10.5%) harboring putative predisposing mutations that were rare in unselected individuals (

International audience; Cell shape is controlled by the submembranous cortex, an actomyosin network mainly generated by two actin nucleators: the Arp2/3 complex and the formin mDia1. Changes in relative nucleator activity may alter cortical organization, mechanics and cell shape. Here we investigate how nucleation-promoting factors mediate interactions between nucleators. In vitro, the nucleation-promoting factor SPIN90 promotes formation of unbranched filaments by Arp2/3, a process thought to provide the initial filament for generation of dendritic networks. Paradoxically, in cells, SPIN90 appears to favour a formin-dominated cortex. Our in vitro experiments reveal that this feature stems mainly from two mechanisms: efficient recruitment of mDia1 to SPIN90–Arp2/3 nucleated filaments and formation of a ternary SPIN90–Arp2/3–mDia1 complex that greatly enhances filament nucleation. Both mechanisms yield rapidly elongating filaments with mDia1 at their barbed ends and SPIN90–Arp2/3 at their pointed ends. Thus, in networks, SPIN90 lowers branching densities and increases the proportion of long filaments elongated by mDia1.

ERK is a key coordinator of the epithelial-to-mesenchymal transition (EMT) in that a variety of EMT-inducing factors activate signaling pathways that converge on ERK to regulate EMT transcription programs. However, the mechanisms by which ERK controls the EMT program are not well understood. Through an analysis of the global changes of gene expression mediated by ERK2, we identified the transcription factor FoxO1 as a potential mediator of ERK2-induced EMT, and thus we investigated the mechanism by which ERK2 regulates FoxO1. Additionally, our analysis revealed that ERK2 induced the expression of Dock10, a Rac1/Cdc42 GEF, during EMT. We demonstrate that the activation of the Rac1/JNK signaling axis downstream of Dock10 leads to an increase in FoxO1 expression and EMT. Taken together, our study uncovers mechanisms by which epithelial cells acquire less proliferative but more migratory mesenchymal properties and reveals potential therapeutic targets for cancers evolving into a metastatic disease state.

Inhibition of oxidative phosphorylation (OXPHOS) is a promising therapeutic strategy in Acute Myeloid Leukemia (AML), but patients respond heterogeneously. Through chemically interrogation of 200 sequenced specimens, we identified Mubritinib as a strongin vitroandin vivoanti-leukemic compound, acting through ubiquinone-dependent inhibition of Electron Transport Chain complex I (ETC1). ETC1 targeting showed selective toxicity against a subgroup of chemotherapy-resistant leukemias exhibiting OXPHOS hyperactivity, high expression of mitochondrial activity-related genes, and mutations affectingNPM1, FLT3andDNMT3A. Altogether, our work thus identifies a novel ETC1 inhibitor with high clinical potential and reveals the landscape of OXPHOS dependency in AML.

Translation is a key step in the regulation of gene expression and one of the most energy-consuming processes in the cell. In response to various stimuli, multiple signaling pathways converge on the translational machinery to regulate its function. To date, the roles of phosphoinositide 3-kinase (PI3K)/AKT and the mitogen-activated protein kinase (MAPK) pathways in the regulation of translation are among the best understood. Both pathways engage the mechanistic target of rapamycin (mTOR) to regulate a variety of components of the translational machinery. While these pathways regulate protein synthesis in homeostasis, their dysregulation results in aberrant translation leading to human diseases, including diabetes, neurological disorders, and cancer. Here we review the roles of the PI3K/AKT and MAPK pathways in the regulation of mRNA translation. We also highlight additional signaling mechanisms that have recently emerged as regulators of the translational apparatus.

Messenger RNA (mRNA) translation is highly regulated in cells and plays an integral role in the overall process of gene expression. The initiation phase of translation is considered to be the most rate-limiting and is often targeted by oncogenic signaling pathways to promote global protein synthesis and the selective translation of tumor-promoting mRNAs. Translational control is a crucial component of cancer development as it allows cancer cells to adapt to the altered metabolism that is generally associated with the tumor state. The phosphoinositide 3-kinase (PI3K)/Akt and Ras/mitogen-activated protein kinase (MAPK) pathways are strongly implicated in cancer etiology, and they exert their biological effects by modulating both global and specific mRNA translation. In addition to having respective translational targets, these pathways also impinge on the mechanistic/mammalian target of rapamycin (mTOR), which acts as a critical signaling node linking nutrient sensing to the coordinated regulation of cellular metabolism. mTOR is best known as a central regulator of protein synthesis and has been implicated in an increasing number of pathological conditions, including cancer. In this article, we describe the current knowledge on the roles and regulation of mRNA translation by various oncogenic signaling pathways, as well as the relevance of these molecular mechanisms to human malignancies. This article is part of a Special Issue entitled: Translation and cancer.

The mechanistic/mammalian target of rapamycin (mTOR) is a conserved protein kinase that controls several anabolic processes required for cell growth and proliferation. As such, mTOR has been implicated in an increasing number of pathological conditions, including cancer, obesity, type 2 diabetes and neurodegeneration. As part of the mTOR complex 1 (mTORC1), mTOR regulates cell growth by promoting the biosynthesis of proteins, lipids and nucleic acids. Several mTORC1 substrates have been shown to regulate protein synthesis, including the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) and the ribosomal S6 kinases (S6Ks) 1 and 2. In this work, we focus on the signalling pathways that lie both upstream and downstream of mTORC1, as well as their relevance to human pathologies. We further discuss pharmacological approaches that target mTOR and their applications for the treatment of cancer.

Summary: AXL is activated by its ligand GAS6 and is expressed in triple-negative breast cancer cells. In the current study, we report AXL expression in HER2-positive (HER2+) breast cancers where it correlates with poor patient survival. Using murine models of HER2+ breast cancer, Axl, but not its ligand Gas6, was found to be essential for metastasis. We determined that AXL is required for intravasation, extravasation, and growth at the metastatic site. We found that AXL is expressed in HER2+ cancers displaying epithelial-to-mesenchymal transition (EMT) signatures where it contributes to sustain EMT. Interfering with AXL in a patient-derived xenograft (PDX) impaired transforming growth factor β (TGF-β)-induced cell invasion. Last, pharmacological inhibition of AXL specifically decreased the metastatic burden of mice developing HER2+ breast cancer. Our data identify AXL as a potential anti-metastatic co-therapeutic target for the treatment of HER2+ breast cancers. : Metastasis is responsible for the majority of breast cancer deaths. Goyette et al. report that AXL is expressed in HER2+ human tumors that acquire aggressive features. Blockade of AXL in mice decreases metastasis. These results suggest that co-targeting AXL and HER2 may limit the metastatic progression of HER2+ breast cancer. Keywords: AXL, HER2, breast cancer, metastasis, EMT, PDX, AXL inhibitor

// Ali Ben Djoudi Ouadda 1, 2 , Yi He 1, 2 , Viviane Calabrese 3 , Hidetaka Ishii 1, 2 , Rony Chidiac 4 , Jean-Philippe Gratton 4 , Philippe P. Roux 3 and Nathalie Lamarche-Vane 1, 2 1 Cancer Research Program, Research Institute of the MUHC, Montreal, Quebec, H4A 3J1, Canada 2 McGill University, Department of Anatomy and Cell Biology, Montreal, Quebec, H3A 2B2, Canada 3 Institute for Research in Immunology and Cancer (IRIC), Montreal, Quebec, H3T 1J4, Canada 4 Department of Pharmacology, Faculty of Medicine, Universite de Montreal, Department of pharmacology, Montreal, Quebec, H3T 1J4, Canada Correspondence to: Nathalie Lamarche-Vane, email: nathalie.lamarche@mcgill.ca Keywords: RhoGAPs; RSK; 14-3-3; phosphorylation; cytoskeleton Received: June 29, 2017 Accepted: December 03, 2017 Published: January 10, 2018 ABSTRACT Cdc42 GTPase-activating protein (CdGAP, also named ARHGAP31) is a negative regulator of the GTPases Rac1 and Cdc42. Associated with the rare developmental disorder Adams-Oliver Syndrome (AOS), CdGAP is critical for embryonic vascular development and VEGF-mediated angiogenesis. Moreover, CdGAP is an essential component in the synergistic interaction between TGFβ and ErbB-2 signaling pathways during breast cancer cell migration and invasion, and is a novel E-cadherin transcriptional co-repressor with Zeb2 in breast cancer. CdGAP is highly phosphorylated on serine and threonine residues in response to growth factors and is a substrate of ERK1/2 and GSK-3. Here, we identified Ser1093 and Ser1163 in the C-terminal region of CdGAP, which are phosphorylated by RSK in response to phorbol ester. These phospho-residues create docking sites for binding to 14-3-3 adaptor proteins. The interaction between CdGAP and 14-3-3 proteins inhibits the GAP activity of CdGAP and sequesters CdGAP into the cytoplasm. Consequently, the nucleocytoplasmic shuttling of CdGAP is inhibited and CdGAP-induced cell rounding is abolished. In addition, 14-3-3β inhibits the ability of CdGAP to repress the E-cadherin promoter and to induce cell migration. Finally, we show that 14-3-3β is unable to regulate the activity and subcellular localization of the AOS-related mutant proteins lacking these phospho-residues. Altogether, we provide a novel mechanism of regulation of CdGAP activity and localization, which impacts directly on a better understanding of the role of CdGAP as a promoter of breast cancer and in the molecular causes of AOS.

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways., Beta-arrestins play central roles in the mechanisms regulating GPCR signalling and trafficking. Here the authors identify a selective inhibitor of the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP-2, which they use to dissect the role of the β-arrestin/β2-adaptin interaction in GPCR signalling.

The scaffolding adapter protein Gab2 (Grb2-associated binder) promotes cell proliferation, survival, and motility by engaging several signaling pathways downstream of growth factor and cytokine receptors. In particular, Gab2 plays essential roles in mast cells, as it is required for phosphoinositide 3-kinase (PI3K) activation in response to Kit and the high-affinity IgE receptor. While the positive role of Gab2 in PI3K signaling is well documented, very little is known about the mechanisms that attenuate its function. Here we show that Gab2 becomes phosphorylated on multiple proline-directed sites upon stimulation of the Ras/extracellular signal-regulated kinase (ERK) signaling pathway. We demonstrate that ERK1 and ERK2 interact with Gab2 via a novel docking motif, which is required for subsequent Gab2 phosphorylation in response to ERK1/2 activation. We identified four ERK1/2-dependent phosphorylation sites in Gab2 that prevent the recruitment of the p85 regulatory subunit of PI3K. Using bone marrow-derived mast cells to study Gab2-dependent signaling, we found that the inhibition of ERK1/2 activity promotes Akt signaling in response to Kit and the high-affinity IgE receptor. Together, our results indicate that ERK1/2 participates in a negative-feedback loop that attenuates PI3K/Akt signaling in response to various agonists.

Aberrant signaling by the mammalian target of rapamycin (mTOR) contributes to the devastating features of cancer cells. Thus, mTOR is a critical therapeutic target and catalytic inhibitors are being investigated as anti-cancer drugs. Although mTOR inhibitors initially block cell proliferation, cell viability and migration in some cancer cells are quickly restored. Despite sustained inhibition of mTORC1/2 signaling, Akt, a kinase regulating cell survival and migration, regains phosphorylation at its regulatory sites. Mechanistically, mTORC1/2 inhibition promotes reorganization of integrin/focal adhesion kinase-mediated adhesomes, induction of IGFR/IR-dependent PI3K activation, and Akt phosphorylation via an integrin/FAK/IGFR-dependent process. This resistance mechanism contributes to xenograft tumor cell growth, which is prevented with mTOR plus IGFR inhibitors, supporting this combination as a therapeutic approach for cancers.

The 14-3-3 protein family orchestrates a complex network of molecular interactions that regulates various biological processes. Owing to their role in regulating the cell cycle and protein trafficking, 14-3-3 proteins are prevalent in human diseases such as cancer, diabetes, and neurodegeneration. 14-3-3 proteins are expressed in all eukaryotic cells, suggesting that they mediate their biological functions through evolutionarily conserved protein interactions. To identify these core 14-3-3 client proteins, we used an affinity-based proteomics approach to characterize and compare the human and Drosophila 14-3-3 interactomes. Using this approach, we identified a group of Rab11 effector proteins, termed class I Rab11 family interacting proteins (Rab11-FIPs), or Rip11 in Drosophila. We found that 14-3-3 binds to Rip11 in a phospho-dependent manner to ensure its proper subcellular distribution during cell division. Our results indicate that Rip11 plays an essential role in the regulation of cytokinesis and that this function requires its association with 14-3-3 but not with Rab11. Together, our results suggest an evolutionarily conserved role for 14-3-3 in controlling Rip11-dependent protein transport during cytokinesis.

SUMMARY A complex network of endocrine/paracrine signals regulates granulosa-cell function inovarianfollicles.Mechanistictargetofrapamycin(MTOR)hasrecentlyemergedas a master intracellular integrator of extracellular signals and nutrient availability. The objectives of the present study were to characterize the expression pattern and kinase activity of MTOR during follicular and corpus luteum development, and to examine how inhibition of MTOR kinase activity affects preovulatory maturation of ovarian follicles. MTOR expression was constitutive throughout follicular and corpus luteum development. Gonadotropins induced MTOR kinase activity in the ovary, which was inhibited by rapamycin treatment (10mg/g body weight, intraperitoneal injection). Inhibition of human chorionic gonadotropin (hCG)-induced MTOR activity during preovulatory follicle maturation did not change key events of ovulation. Granulosa cells of rapamycin-treated mice showed reduced MTOR kinase activity at 1 and 4hr post-hCG and overexpression of hCG-induced ovulation genes at 4hr post-hCG. Overexpression of these ovulatory genes was associated with hyperactivation of extracellular signal-regulated kinase 1/2 (ERK1/2), which occurred in response to inhibition of MTOR with rapamycin and suggested that MTOR may function as a negative regulator of the mitogen-activated protein kinase (MAPK) pathway. Indeed, simultaneous inhibition of MTOR and ERK1/2 activities during preovulatory follicle maturation caused anovulation. Inhibition of hCG-induced ERK1/2 activity alone suppressed MTOR kinase activity, indicating that MAPK pathway is upstream of MTOR. Thus, normal ovulation appears to be a result of complex interactions between MTOR and MAPK signaling pathways in granulosa cells of ovulating follicles in mice.

The mammalian target of rapamycin (mTOR) promotes cell growth and proliferation by promoting mRNA translation and increasing the protein synthetic capacity of the cell. Although mTOR globally promotes translation by regulating the mRNA 5′ cap-binding protein eIF4E (eukaryotic initiation factor 4E), it also preferentially regulates the translation of certain classes of mRNA via unclear mechanisms. To help fill this gap in knowledge, we performed a quantitative proteomic screen to identify proteins that associate with the mRNA 5′ cap in an mTOR-dependent manner. Using this approach, we identified many potential regulatory factors, including the putative RNA-binding protein LARP1 (La-related protein 1). Our results indicate that LARP1 associates with actively translating ribosomes via PABP and that LARP1 stimulates the translation of mRNAs containing a 5′ terminal oligopyrimidine (TOP) motif, encoding for components of the translational machinery. We found that LARP1 associates with the mTOR complex 1 (mTORC1) and is required for global protein synthesis as well as cell growth and proliferation. Together, these data reveal important molecular mechanisms involved in TOP mRNA translation and implicate LARP1 as an important regulator of cell growth and proliferation.