Trans-activating mutations of the pseudokinase ERBB3.

Genetic changes in the ERBB family of receptor tyrosine kinases serve as oncogenic driver events and predictive biomarkers for ERBB inhibitor drugs. ERBB3 is a pseudokinase member of the family that, although lacking a fully active kinase domain, is well known for its potent signaling activity as a heterodimeric complex with ERBB2. Previous studies have identified few transforming ERBB3 mutations while the great majority of the hundreds of different somatic ERBB3 variants observed in different cancer types remain of unknown significance. Here, we describe an unbiased functional genetics screen of the transforming potential of thousands of ERBB3 mutations in parallel. The screen based on a previously described iSCREAM (in vitro screen of activating mutations) platform, and addressing ERBB3 pseudokinase signaling in a context of ERBB3/ERBB2 heterodimers, identified 18 hit mutations. Validation experiments in Ba/F3, NIH 3T3, and MCF10A cell backgrounds demonstrated the presence of both previously known and unknown transforming ERBB3 missense mutations functioning either as single variants or in cis as a pairwise combination. Drug sensitivity assays with trastuzumab, pertuzumab and neratinib indicated actionability of the transforming ERBB3 variants.

De Novo multi-omics pathway analysis (DMPA) designed for prior data independent inference of cell signaling pathways.

New tools for cell signaling pathway inference from multi-omics data that are independent of previous knowledge are needed. Here we propose a new de novo method, the de novo multi-omics pathway analysis (DMPA), to model and combine omics data into network modules and pathways. DMPA was validated with published omics data and was found accurate in discovering reported molecular associations in transcriptome, interactome, phosphoproteome, methylome, and metabolomics data and signaling pathways in multi-omics data. DMPA was benchmarked against module discovery and multi-omics integration methods and outperformed previous methods in module and pathway discovery especially when applied to datasets with relatively low sample sizes. Transcription factor, kinase, subcellular location and function prediction algorithms were devised for transcriptome, phosphoproteome and interactome modules and pathways, respectively. To apply DMPA in a biologically relevant context, interactome, phosphoproteome, transcriptome and proteome data were collected from analyses carried out using melanoma cells to address gamma-secretase cleavage-dependent signaling characteristics of the receptor tyrosine kinase TYRO3. The pathways modeled with DMPA reflected the predicted function and its direction in validation experiments.

STAT5b is a key effector of NRG-1/ERBB4-mediated myocardial growth.

The growth factor Neuregulin-1 (NRG-1) regulates myocardial growth and is currently under clinical investigation as a treatment for heart failure. Here, we demonstrate in several in vitro and in vivo models that STAT5b mediates NRG-1/EBBB4-stimulated cardiomyocyte growth. Genetic and chemical disruption of the NRG-1/ERBB4 pathway reduces STAT5b activation and transcription of STAT5b target genes Igf1, Myc, and Cdkn1a in murine cardiomyocytes. Loss of Stat5b also ablates NRG-1-induced cardiomyocyte hypertrophy. Dynamin-2 is shown to control the cell surface localization of ERBB4 and chemical inhibition of Dynamin-2 downregulates STAT5b activation and cardiomyocyte hypertrophy. In zebrafish embryos, Stat5 is activated during NRG-1-induced hyperplastic myocardial growth, and chemical inhibition of the Nrg-1/Erbb4 pathway or Dynamin-2 leads to loss of myocardial growth and Stat5 activation. Moreover, CRISPR/Cas9-mediated knockdown of stat5b results in reduced myocardial growth and cardiac function. Finally, the NRG-1/ERBB4/STAT5b signaling pathway is differentially regulated at mRNA and protein levels in the myocardium of patients with pathological cardiac hypertrophy as compared to control human subjects, consistent with a role of the NRG-1/ERBB4/STAT5b pathway in myocardial growth.

An extracellular receptor tyrosine kinase motif orchestrating intracellular STAT activation.

The ErbB4 receptor isoforms JM-a and JM-b differ within their extracellular juxtamembrane (eJM) domains. Here, ErbB4 isoforms are used as a model to address the effect of structural variation in the eJM domain of receptor tyrosine kinases (RTK) on downstream signaling. A specific JM-a-like sequence motif is discovered, and its presence or absence (in JM-b-like RTKs) in the eJM domains of several RTKs is demonstrated to dictate selective STAT activation. STAT5a activation by RTKs including the JM-a like motif is shown to involve interaction with oligosaccharides of N-glycosylated cell surface proteins such as ß1 integrin, whereas STAT5b activation by JM-b is dependent on TYK2. ErbB4 JM-a- and JM-b-like RTKs are shown to associate with specific signaling complexes at different cell surface compartments using analyses of RTK interactomes and super-resolution imaging. These findings provide evidence for a conserved mechanism linking a ubiquitous extracellular motif in RTKs with selective intracellular STAT signaling.

An Unbiased Functional Genetics Screen Identifies Rare Activating ERBB4 Mutations.

Despite the relatively high frequency of somatic ERBB4 mutations in various cancer types, only a few activating ERBB4 mutations have been characterized, primarily due to lack of mutational hotspots in the ERBB4 gene. Here, we utilized our previously published pipeline, an in vitro screen for activating mutations, to perform an unbiased functional screen to identify potential activating ERBB4 mutations from a randomly mutated ERBB4 expression library. Ten potentially activating ERBB4 mutations were identified and subjected to validation by functional and structural analyses. Two of the 10 ERBB4 mutants, E715K and R687K, demonstrated hyperactivity in all tested cell models and promoted cellular growth under two-dimensional and three-dimensional culture conditions. ERBB4 E715K also promoted tumor growth in in vivo Ba/F3 cell mouse allografts. Importantly, all tested ERBB4 mutants were sensitive to the pan-ERBB tyrosine kinase inhibitors afatinib, neratinib, and dacomitinib. Our data indicate that rare ERBB4 mutations are potential candidates for ERBB4-targeted therapy with pan-ERBB inhibitors. Statement of Significance: ERBB4 is a member of the ERBB family of oncogenes that is frequently mutated in different cancer types but the functional impact of its somatic mutations remains unknown. Here, we have analyzed the function of over 8,000 randomly mutated ERBB4 variants in an unbiased functional genetics screen. The data indicate the presence of rare activating ERBB4 mutations in cancer, with potential to be targeted with clinically approved pan-ERBB inhibitors.

Therapeutic Potential of Targeting the SUMO Pathway in Cancer.

SUMOylation is a dynamic and reversible post-translational modification, characterized more than 20 years ago, that regulates protein function at multiple levels. Key oncoproteins and tumor suppressors are SUMO substrates. In addition to alterations in SUMO pathway activity due to conditions typically present in cancer, such as hypoxia, the SUMO machinery components are deregulated at the genomic level in cancer. The delicate balance between SUMOylation and deSUMOylation is regulated by SENP enzymes possessing SUMO-deconjugation activity. Dysregulation of SUMO machinery components can disrupt the balance of SUMOylation, contributing to the tumorigenesis and drug resistance of various cancers in a context-dependent manner. Many molecular mechanisms relevant to the pathogenesis of specific cancers involve SUMO, highlighting the potential relevance of SUMO machinery components as therapeutic targets. Recent advances in the development of inhibitors targeting SUMOylation and deSUMOylation permit evaluation of the therapeutic potential of targeting the SUMO pathway in cancer. Finally, the first drug inhibiting SUMO pathway, TAK-981, is currently also being evaluated in clinical trials in cancer patients. Intriguingly, the inhibition of SUMOylation may also have the potential to activate the anti-tumor immune response. Here, we comprehensively and systematically review the recent developments in understanding the role of SUMOylation in cancer and specifically focus on elaborating the scientific rationale of targeting the SUMO pathway in different cancers.

Combined genetic and chemical screens indicate protective potential for EGFR inhibition to cardiomyocytes under hypoxia.

The return of blood flow to ischemic heart after myocardial infarction causes ischemia-reperfusion injury. There is a clinical need for novel therapeutic targets to treat myocardial ischemia-reperfusion injury. Here we screened for targets for the treatment of ischemia-reperfusion injury using a combination of shRNA and drug library analyses in HL-1 mouse cardiomyocytes subjected to hypoxia and reoxygenation. The shRNA library included lentiviral constructs targeting 4625 genes and the drug library 689 chemical compounds approved by the Food and Drug Administration (FDA). Data were analyzed using protein-protein interaction and pathway analyses. EGFR inhibition was identified as a cardioprotective mechanism in both approaches. Inhibition of EGFR kinase activity with gefitinib improved cardiomyocyte viability in vitro. In addition, gefitinib preserved cardiac contractility in zebrafish embryos exposed to hypoxia-reoxygenation in vivo. These findings indicate that the EGFR inhibitor gefitinib is a potential candidate for further studies of repurposing the drug for the treatment of myocardial infarction.

Structural Basis for the Functional Changes by EGFR Exon 20 Insertion Mutations.

Activating somatic mutations of the epidermal growth factor receptor (EGFR) are frequently implicated in non-small cell lung cancer (NSCLC). While L858R and exon 19 deletion mutations are most prevalent, exon 20 insertions are often observed in NSCLC. Here, we investigated the structural implications of two common EGFR exon 20 insertions in NSCLC, V769insASV and D770insNPG. The active and inactive conformations of wild-type, D770insNPG and V769insASV EGFRs were probed with molecular dynamics simulations to identify local and global alterations that the mutations exert on the EGFR kinase domain, highlighting mechanisms for increased enzymatic activity. In the active conformation, the mutations increase interactions that stabilize the αC helix that is essential for EGFR activity. Moreover, the key Lys745-Glu762 salt bridge was more conserved in the insertion mutations. The mutants also preserved the state of the structurally critical aspartate-phenylalanine-glycine (DFG)-motif and regulatory spine (R-spine), which were altered in wild-type EGFR. The insertions altered the structure near the ATP-binding pocket, e.g., the P-loop, which may be a factor for the clinically observed tyrosine kinase inhibitor (TKI) insensitivity by the insertion mutants. The inactive state simulations also showed that the insertions disrupt the Ala767-Arg776 interaction that is key for maintaining the "αC-out" inactive conformation, which could consequently fuel the transition from the inactive towards the active EGFR state.

Identification of Predictive ERBB Mutations by Leveraging Publicly Available Cell Line Databases.

Although targeted therapies can be effective for a subgroup of patients, identification of individuals who benefit from the treatments is challenging. At the same time, the predictive significance of the majority of the thousands of mutations observed in the cancer tissues remains unknown. Here, we describe the identification of novel predictive biomarkers for ERBB-targeted tyrosine kinase inhibitors (TKIs) by leveraging the genetic and drug screening data available in the public cell line databases: Cancer Cell Line Encyclopedia, Genomics of Drug Sensitivity in Cancer, and Cancer Therapeutics Response Portal. We assessed the potential of 412 ERBB mutations in 296 cell lines to predict responses to 10 different ERBB-targeted TKIs. Seventy-six ERBB mutations were identified that were associated with ERBB TKI sensitivity comparable with non-small cell lung cancer cell lines harboring the well-established predictive EGFR L858R mutation or exon 19 deletions. Fourteen (18.4%) of these mutations were classified as oncogenic by the cBioPortal database, whereas 62 (81.6%) were regarded as novel potentially predictive mutations. Of the nine functionally validated novel mutations, EGFR Y1069C and ERBB2 E936K were transforming in Ba/F3 cells and demonstrated enhanced signaling activity. Mechanistically, the EGFR Y1069C mutation disrupted the binding of the ubiquitin ligase c-CBL to EGFR, whereas the ERBB2 E936K mutation selectively enhanced the activity of ERBB heterodimers. These findings indicate that integrating data from publicly available cell line databases can be used to identify novel, predictive nonhotspot mutations, potentially expanding the patient population benefiting from existing cancer therapies.

Erbb4 regulates the oocyte microenvironment during folliculogenesis.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders leading to infertility in women affecting reproductive, endocrine and metabolic systems. Recent genomewide association studies on PCOS cohorts revealed a single nucleotide polymorphism (SNP) in the ERBB4 receptor tyrosine kinase 4 gene, but its role in ovary development or during folliculogenesis remains poorly understood. Since no genetic animal models mimicking all PCOS reproductive features are available, we conditionally deleted Erbb4 in murine granulosa cells (GCs) under the control of Amh promoter. While we have demonstrated that Erbb4 deletion displayed aberrant ovarian function by affecting the reproductive function (asynchronous oestrous cycle leading to few ovulations and subfertility) and metabolic function (obesity), their ovaries also present severe structural and functional abnormalities (impaired oocyte development). Hormone analysis revealed an up-regulation of serum luteinizing hormone, hyperandrogenism, increased production of ovarian and circulating anti-Müllerian hormone. Our data implicate that Erbb4 deletion in GCs leads to defective intercellular junctions between the GCs and oocytes, causing changes in the expression of genes regulating the local microenvironment of the follicles. In vitro culture assays reducing the level of Erbb4 via shRNAs confirm that Erbb4 is essential for regulating Amh level. In conclusion, our results indicate a functional role for Erbb4 in the ovary, especially during folliculogenesis and its reduced expression plays an important role in reproductive pathophysiology, such as PCOS development.

Deciphering the Structural Effects of Activating EGFR Somatic Mutations with Molecular Dynamics Simulation.

Numerous somatic mutations occurring in the epidermal growth factor receptor (EGFR) family (ErbB) of receptor tyrosine kinases (RTK) have been reported from cancer patients, although relatively few have been tested and shown to cause functional changes in ErbBs. The ErbB receptors are dimerized and activated upon ligand binding, and dynamic conformational changes of the receptors are inherent for induction of downstream signaling. For two mutations shown experimentally to alter EGFR function, A702V and the Δ746ELREA750 deletion mutation, we illustrate in the following protocol how molecular dynamics (MD) simulations can probe the (1) conformational stability of the mutant tyrosine kinase structure in comparison with wild-type EGFR; (2) structural consequences and conformational transitions and their relationship to observed functional changes; (3) effects of mutations on the strength of binding ATP as well as for binding between the kinase domains in the activated asymmetric dimer; and (4) effects of the mutations on key interactions within the EGFR binding site associated with the activated enzyme. The protocol provides a detailed step-by-step procedure as well as guidance that can be more generally useful for investigation of protein structures using MD simulations as a means to probe structural dynamics and the relationship to biological function.

The guanine nucleotide exchange factor VAV3 participates in ERBB4-mediated cancer cell migration.

ERBB4 is a member of the epidermal growth factor receptor (EGFR)/ERBB subfamily of receptor tyrosine kinases that regulates cellular processes including proliferation, migration, and survival. ERBB4 signaling is involved in embryogenesis and homeostasis of healthy adult tissues, but also in human pathologies such as cancer, neurological disorders, and cardiovascular diseases. Here, an MS-based analysis revealed the Vav guanine nucleotide exchange factor 3 (VAV3), an activator of Rho family GTPases, as a critical ERBB4-interacting protein in breast cancer cells. We confirmed the ERBB4-VAV3 interaction by targeted MS and coimmunoprecipitation experiments and further defined it by demonstrating that kinase activity and Tyr-1022 and Tyr-1162 of ERBB4, as well as the intact phosphotyrosine-interacting SH2 domain of VAV3, are necessary for this interaction. We found that ERBB4 stimulates tyrosine phosphorylation of the VAV3 activation domain, known to be required for guanine nucleotide exchange factor (GEF) activity of VAV proteins. In addition to VAV3, the other members of the VAV family, VAV1 and VAV2, also coprecipitated with ERBB4. Analyses of the effects of overexpression of dominant-negative VAV3 constructs or shRNA-mediated down-regulation of VAV3 expression in breast cancer cells indicated that active VAV3 is involved in ERBB4-stimulated cell migration. These results define the VAV GEFs as effectors of ERBB4 activity in a signaling pathway relevant for cancer cell migration.

Genetic and functional implications of an exonic TRIM55 variant in heart failure.

BACKGROUND: To tackle the missing heritability of sporadic heart failure, we screened for novel heart failure-associated genetic variants in the Finnish population and functionally characterized a novel variant in vitro and in vivo. METHODS AND RESULTS: Heart failure-associated variants were screened in genotyping array data of the FINRISK study, consisting of 994 cases and 20,118 controls. Based on logistic regression analysis, a potentially damaging variant in TRIM55 (rs138811034), encoding an E140K variant, was selected for validations. In HL-1 cardiomyocytes, we used CRISPR/Cas9 technology to introduce the variant in the endogenous locus, and additionally TRIM55 wildtype or E140K was overexpressed from plasmid. Functional responses were profiled using whole-genome RNA sequencing, RT-PCR and Western analyses, cell viability and cell cycle assays and cell surface area measurements. In zebrafish embryos, cardiac contractility was measured using videomicroscopy after CRISPR-mediated knockout of trim55a or plasmid overexpression of TRIM55 WT or E140K. Genes related to muscle contraction and cardiac stress were highly regulated in Trim55 E140K/- cardiomyocytes. When compared to the WT/WT cells, the variant cells demonstrated reduced viability, significant hypertrophic response to isoproterenol, p21 protein overexpression and impaired cell cycle progression. In zebrafish embryos, the deletion of trim55a or overexpression of TRIM55 E140K reduced cardiac contractility as compared to embryos with wildtype genotype or overexpression of WT TRIM55, respectively. CONCLUSIONS: A previously uncharacterized TRIM55 E140K variant demonstrated a number of functional implications for cardiomyocyte functions in vitro and in vivo. These findings suggest a novel role for TRIM55 polymorphism in predisposing to heart failure.

Structural characterization of EGFR exon 19 deletion mutation using molecular dynamics simulation.

Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor important in diverse biological processes including cell proliferation and survival. Upregulation of EGFR activity due to over-expression or mutation is widely implicated in cancer. Activating somatic mutations of the EGFR kinase are postulated to affect the conformation and/or stability of the protein, shifting the EGFR inactive-active state equilibrium towards the activated state. Here, we examined a common EGFR deletion mutation, Δ746ELREA750, which is frequently observed in non-small cell lung cancer patients. By using molecular dynamics simulation, we investigated the structural effects of the mutation that lead to the experimentally reported increases in kinase activity. Simulations of the active form wild-type and ΔELREA EGFRs revealed the deletion stabilizes the αC helix of the kinase domain, which is located adjacent to the deletion site, by rigidifying the flexible ß3-αC loop that accommodates the ELREA sequence. Consequently, the αC helix is stabilized in the "αC-in" active conformation that would prolong the time of the activated state. Moreover, in the mutant kinase, a salt bridge between E762 and K745, which is key for EGFR activity, was also stabilized during the simulation. Additionally, the interaction between EGFR and ATP was favored by ΔELREA EGFR over wild-type EGFR, as reflected by the number of hydrogen bonds formed and the free energy of binding. Simulation of inactive EGFR suggested the deletion would promote a shift from the inactive conformation towards active EGFR, which is supported by the inward movement of the αC helix. The MDS results also align with the effects of tyrosine kinase inhibitors on ΔELREA and wild-type EGFR lung cancer cell lines, where more pronounced inhibition was observed against ΔELREA than for wild-type EGFR by inhibitors recognizing the active kinase conformation.

An unbiased in vitro screen for activating epidermal growth factor receptor mutations.

Cancer tissues harbor thousands of mutations, and a given oncogene may be mutated at hundreds of sites, yet only a few of these mutations have been functionally tested. Here, we describe an unbiased platform for the functional characterization of thousands of variants of a single receptor tyrosine kinase (RTK) gene in a single assay. Our in vitroscreen for activating mutations (iSCREAM) platform enabled rapid analysis of mutations conferring gain-of-function RTK activity promoting clonal growth. The screening strategy included a somatic model of cancer evolution and utilized a library of 7,216 randomly mutated epidermal growth factor receptor (EGFR) single-nucleotide variants that were tested in murine lymphoid Ba/F3 cells. These cells depend on exogenous interleukin-3 (IL-3) for growth, but this dependence can be compensated by ectopic EGFR overexpression, enabling selection for gain-of-function EGFR mutants. Analysis of the enriched mutants revealed EGFR A702V, a novel activating variant that structurally stabilized the EGFR kinase dimer interface and conferred sensitivity to kinase inhibition by afatinib. As proof of concept for our approach, we recapitulated clinical observations and identified the EGFR L858R as the major enriched EGFR variant. Altogether, iSCREAM enabled robust enrichment of 21 variants from a total of 7,216 EGFR mutations. These findings indicate the power of this screening platform for unbiased identification of activating RTK variants that are enriched under selection pressure in a model of cancer heterogeneity and evolution.

Decorin Expression in Human Vulva Carcinoma: Oncosuppressive Effect of Decorin cDNA Transduction on Carcinoma Cells.

The extracellular matrix proteoglycan decorin is well-known for its oncosuppressive activity. Here, decorin expression was examined in human vulva carcinoma tissue samples and in primary and commercial cell lines representing this malignant disease. Furthermore, the effect of adenovirus-mediated decorin cDNA (Ad-DCN) transduction on the viability, proliferation, and the expression and activity of the epidermal growth factor receptor (ErbB/HER) family members of the cell lines were investigated. Using in situ hybridization and immunohistochemistry for decorin, it was demonstrated that malignant cells in human vulva carcinoma tissues lack decorin expression. This result was true independently on tumor stage, grade or human papillomavirus status. RT-qPCR analyses showed that the human vulva carcinoma cell lines used in this study were also negative for decorin expression. Transduction of the cell lines with Ad-DCN caused a marked reduction in cell viability, while the proliferation of the cells was not affected. Experiments examining potential mechanisms behind the oncosuppressive effect of Ad-DCN transduction revealed that ErbB2/HER2 expression and activity in carcinoma cells were markedly downregulated. In conclusion, the results of this study showed that human vulva carcinoma cells lack decorin expression, and that Ad-DCN transduction of these cells induces oncosuppressive activity in part via downregulation of ErbB2/HER2.

Endothelial Cells Regulate Physiological Cardiomyocyte Growth via VEGFR2-Mediated Paracrine Signaling.

BACKGROUND: Heart failure, which is a major global health problem, is often preceded by pathological cardiac hypertrophy. The expansion of the cardiac vasculature, to maintain adequate supply of oxygen and nutrients, is a key determinant of whether the heart grows in a physiological compensated manner or a pathological decompensated manner. Bidirectional endothelial cell (EC)-cardiomyocyte (CMC) cross talk via cardiokine and angiocrine signaling plays an essential role in the regulation of cardiac growth and homeostasis. Currently, the mechanisms involved in the EC-CMC interaction are not fully understood, and very little is known about the EC-derived signals involved. Understanding how an excess of angiogenesis induces cardiac hypertrophy and how ECs regulate CMC homeostasis could provide novel therapeutic targets for heart failure. METHODS: Genetic mouse models were used to delete vascular endothelial growth factor (VEGF) receptors, adeno-associated viral vectors to transduce the myocardium, and pharmacological inhibitors to block VEGF and ErbB signaling in vivo. Cell culture experiments were used for mechanistic studies, and quantitative polymerase chain reaction, microarrays, ELISA, and immunohistochemistry were used to analyze the cardiac phenotypes. RESULTS: Both EC deletion of VEGF receptor (VEGFR)-1 and adeno-associated viral vector-mediated delivery of the VEGFR1-specific ligands VEGF-B or placental growth factor into the myocardium increased the coronary vasculature and induced CMC hypertrophy in adult mice. The resulting cardiac hypertrophy was physiological, as indicated by preserved cardiac function and exercise capacity and lack of pathological gene activation. These changes were mediated by increased VEGF signaling via endothelial VEGFR2, because the effects of VEGF-B and placental growth factor on both angiogenesis and CMC growth were fully inhibited by treatment with antibodies blocking VEGFR2 or by endothelial deletion of VEGFR2. To identify activated pathways downstream of VEGFR2, whole-genome transcriptomics and secretome analyses were performed, and the Notch and ErbB pathways were shown to be involved in transducing signals for EC-CMC cross talk in response to angiogenesis. Pharmacological or genetic blocking of ErbB signaling also inhibited part of the VEGF-B-induced effects in the heart. CONCLUSIONS: This study reveals that cross talk between the EC VEGFR2 and CMC ErbB signaling pathways coordinates CMC hypertrophy with angiogenesis, contributing to physiological cardiac growth.

Gamma-secretase-dependent signaling of receptor tyrosine kinases.

Human genome harbors 55 receptor tyrosine kinases (RTK). At least half of the RTKs have been reported to be cleaved by gamma-secretase-mediated regulated intramembrane proteolysis. The two-step process involves releasing the RTK ectodomain to the extracellular space by proteolytic cleavage called shedding, followed by cleavage in the RTK transmembrane domain by the gamma-secretase complex resulting in release of a soluble RTK intracellular domain. This intracellular domain, including the tyrosine kinase domain, can in turn translocate to various cellular compartments, such as the nucleus or proteasome. The soluble intracellular domain may interact with transcriptional regulators and other proteins to induce specific effects on cell survival, proliferation, and differentiation, establishing an additional signaling mode for the cleavable RTKs. On the other hand, the same process can facilitate RTK turnover and proteasomal degradation. In this review we focus on the regulation of RTK shedding and gamma-secretase cleavage, as well as signaling promoted by the soluble RTK ICDs. In addition, therapeutic implications of increased knowledge on RTK cleavage on cancer drug development are discussed.

ErbB4 tyrosine kinase inhibition impairs neuromuscular development in zebrafish embryos.

Tyrosine kinase inhibitors are widely used in the clinic, but limited information is available about their toxicity in developing organisms. Here, we tested the effect of tyrosine kinase inhibitors targeting the ErbB receptors for their effects on developing zebrafish ( Danio rerio) embryos. Embryos treated with wide-spectrum pan-ErbB inhibitors or erbb4a-targeting antisense oligonucleotides demonstrated reduced locomotion, reduced diameter of skeletal muscle fibers, and reduced expression of muscle-specific genes, as well as reduced motoneuron length. The phenotypes in the skeletal muscle, as well as the defect in motility, were rescued both by microinjection of human ERBB4 mRNA and by transposon-mediated muscle-specific ERBB4 overexpression. The role of ErbB4 in regulating motility was further controlled by targeted mutation of the endogenous erbb4a locus in the zebrafish genome by CRISPR/Cas9. These observations demonstrate a potential for the ErbB tyrosine kinase inhibitors to induce neuromuscular toxicity in a developing organism via a mechanism involving inhibition of ErbB4 function.

Different responses of colorectal cancer cells to alternative sequences of cetuximab and oxaliplatin.

Therapeutic protocols including EGFR antibodies in the context of oxaliplatin-based regimens have variable clinical effect in colorectal cancer. Here, we tested the effect of the EGFR antibody cetuximab in different sequential combinations with oxaliplatin on the growth of colorectal cancer cells in vitro and in vivo. Cetuximab reduced the efficacy of oxaliplatin when administered before oxaliplatin but provided additive effect when administered after oxaliplatin regardless of the KRAS or BRAF mutation status of the cells. Systemic gene expression and protein phosphorylation screens revealed alternatively activated pathways regulating apoptosis, cell cycle and DNA damage response. Functional assays indicated that cetuximab-induced arrest of the cells into the G1 phase of the cell cycle was associated with reduced responsiveness of the cells to subsequent treatment with oxaliplatin. In contrast, oxaliplatin-enhanced responsiveness to subsequent treatment with cetuximab was associated with increased apoptosis, inhibition of STAT3 activity and increased EGFR down-regulation. This preclinical study indicates that optimizing the sequence of administration may enhance the antitumor effect of combination therapy with EGFR antibodies and oxaliplatin.