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

Simple SummaryNon-small cell lung cancer (NSCLC) is the most common type of lung cancer that claims the lives of many worldwide. Activating mutations occurring on the epidermal growth factor receptor (EGFR) protein have been associated with the pathogenesis of NSCLC, among which exon 20 insertion mutations play a significant role. The objective of this study is to examine the dynamic structural changes occurring on the EGFR protein as a result of two common EGFR exon 20 insertion mutations, V769insASV and D770insNPG. The study further aims to uncover the mechanisms by which the insertion mutations increase kinase activity. Our results suggest that the insertion mutations stabilize structural elements key to maintaining the active EGFR conformation. Furthermore, the insertions disrupt an interaction essential in stabilizing the inactive conformation, which could drive the kinase from an inactive to an active EGFR state. AbstractActivating 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 alpha C 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 "alpha C-out" inactive conformation, which could consequently fuel the transition from the inactive towards the active EGFR state.</div

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 Delta(746)ELREA(750) 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

An extracellular receptor tyrosine kinase motif orchestrating intracellular STAT activation

Specificity in signaling activated by receptor tyrosine kinases is typically attributed to characteristics of their intracellular domains. Here, the authors demonstrate that an extracellular receptor sequence motif controls intracellular signaling as a result of extracellular glycan interactions.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 beta 1 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.Peer reviewe

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

While 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 vast 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 (TKI) by leveraging the genetic and drug screening data available in the public cell line databases: Cancer Cell Line Encyclopedia (CCLE), Genomics of Drug Sensitivity in Cancer (GDSC), and Cancer Therapeutics Response Portal (CTRP). 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 to 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. Out of 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 non-hotspot mutations, potentially expanding the patient population benefiting from existing cancer therapies

Immunogenic SARS-CoV-2 Epitopes: In Silico Study Towards Better Understanding of COVID-19 Disease-Paving the Way for Vaccine Development

The emergence of the COVID-19 outbreak at the end of 2019, caused by the novel coronavirus SARS-CoV-2, has, to date, led to over 13.6 million infections and nearly 600,000 deaths. Consequently, there is an urgent need to better understand the molecular factors triggering immune defense against the virus and to develop countermeasures to hinder its spread. Using in silico analyses, we showed that human major histocompatibility complex (MHC) class I cell-surface molecules vary in their capacity for binding different SARS-CoV-2-derived epitopes, i.e., short sequences of 8-11 amino acids, and pinpointed five specific SARS-CoV-2 epitopes that are likely to be presented to cytotoxic T-cells and hence activate immune responses. The identified epitopes, each one of nine amino acids, have high sequence similarity to the equivalent epitopes of SARS-CoV virus, which are known to elicit an effective T cell response in vitro. Moreover, we give a structural explanation for the binding of SARS-CoV-2-epitopes to MHC molecules. Our data can help us to better understand the differences in outcomes of COVID-19 patients and may aid the development of vaccines against SARS-CoV-2 and possible future outbreaks of novel coronaviruses

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 β1 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.</p

DUX4 is a multifunctional factor priming human embryonic genome activation

Double homeobox 4 (DUX4) is expressed at the early pre-implantation stage in human embryos. Here we show that induced human DUX4 expression substantially alters the chromatin accessibility of non-coding DNA and activates thousands of newly identified transcribed enhance-like regions, preferentially located within ERVL-MaLR repeat elements. CRISPR activation of transcribed enhancers by C-terminal DUX4 motifs results in the increased expression of target embryonic genome activation (EGA) genes ZSCAN4 and KHDC1P1. We show that DUX4 is markedly enriched in human zygotes, followed by intense nuclear DUX4 localization preceding and coinciding Kith minor EGA. DUX4 knockdown in human zygotes led to changes in the EGA transcriptome but did not terminate the embryos. We also show that the DUX4 protein interacts with the Mediator complex via the C-terminal KIX binding motif. Our findings contribute to the understanding of DUX4 as a regulator of the non-coding genome.Peer reviewe

The Dynamic Structural Effects of Activating ERBB Kinase Somatic Mutations

The ERBB family of receptor tyrosine kinases, epidermal growth factor receptor (EGFR, ERBB1), ERBB2, ERBB3 and ERBB4, are transmembrane signaling proteins that regulate cellular processes such as cell survival, mobility, proliferation and differentiation. Normal activity of the ERBBs is essential for tissue growth and organ development. An array of somatic mutations of the ERBB genes have been linked to human malignancies. As a result, the ERBBs are important treatment targets, with multiple ERBB-based drugs currently in effective clinical use against e.g. lung, breast and colorectal cancers. A comprehensive characterization of the ERBB somatic mutations identified in cancer samples is essential to unveil the molecular level impacts of the genetic alterations that could play a role in tumorigenesis. In this thesis, the structural consequences of four cancer-associated ERBB kinase mutations that aberrantly activate EGFR and ERBB2 proteins were explored. The mutations include three EGFR alterations, 746ΔELREA750 (ΔELREA), V769insASV and D770insNPG, and an ERBB2 missense mutation: E936K. The ERBB receptors and proteins in general are dynamic molecules. Hence, the possible structural changes exerted by the above activating mutations were examined by employing molecular dynamics simulations, which allow the assessment of time-dependent structural motions. The simulations revealed that the EGFR ΔELREA deletion mutation stabilizes the active state EGFR conformation by conserving the states of key structural units, such as the αC helix and the Lys745 Glu762 salt bridge, which were disrupted in the wild-type EGFR. Furthermore, the deletion resulted in a structural change on the inactive EGFR state, an inward movement of the αC helix, which could drive a conformational change from the inactive towards the active EGFR state. The V769insASV and D770insNPG EGFR insertion mutations also led to the better stability of the active EGFR conformation relative to the wild-type EGFR. Moreover, the insertions obstructed the formation of an autoinhibitory interaction between Ala767 and Arg776 in the inactive EGFR conformation, which would predispose EGFR to transition to the catalytically active EGFR state. The ERBB2 E936K mutation affected the nature of interactions taking place at the ERBB2 homodimer and heterodimer interface, with a new inter-monomer ionic interaction being formed that strengthened the monomer-monomer binding. Consequently, the duration of the activated ERBB2 dimer would be extended, which fuels the phosphorylation of ERBB2. Taken together, this thesis demonstrated that a series of structural changes are at play that collectively elicit the experimentally reported functional changes by these activating ERBB mutations. A thorough examination of the mutation-induced structural alterations furthers our knowledge on the role the mutations play in cancer progression and the results are essential knowledge when using structure-based, rational design of ligands that could inhibit ERBB kinase activity and subsequent receptor signaling. Such ligands have potential for further development towards a therapeutic agent in efforts to tackle cancers.---------- ERBB-familjen av tyrosinkinasreceptorer, epidermala tillväxtfaktorreceptorer (EGFR, ERBB1), ERBB2, ERBB3 och ERBB4 är transmembrana signalproteiner som reglerar cellulära processer så som överlevnad, mobilitet, proliferation och differentiering. Normal aktivitet hos ERBB är nödvändigt för vävnadstillväxt och organutveckling. En mängd somatiska mutationer i ERBB generna har kopplats till cancer. På grund av detta är ERBB-proteinerna viktiga mål för behandling och flera ERBB-baserade läkemedel är för tillfället i klinisk användning, t.ex. vid lung-, bröst- och kolorektalcancer. En omfattande karakterisering av de somatiska mutationerna i ERBB, som identifierats i cancerprover, är nödvändig för att påvisa vilken effekt de genetiska förändringarna på en molekylärnivå kan ha för tumörutveckling. I den här avhandlingen utforskades de strukturella konsekvenserna av fyra cancer-associerade ERBB kinas-mutationer som felaktigt aktiverar EGFR och ERBB2 proteiner. Mutationerna inkluderar tre EGFR förändringar, 746ΔELREA750 (ΔELREA), V769insASV och D770insNPG, och en ERBB2 missense-mutation: E936K. ERBB receptorer och proteiner är dynamiska molekyler och därför användes molekyldynamiska simulationer för att studera de möjliga strukturella förändringar som de ovan nämnda aktiverande mutationerna orsakar, vilket ger möjlighet att utvärdera tidsberoende strukturella rörelser. Simulationerna visade att EGFR ΔELREA deletion-mutationen stabiliserar det aktiva stadies konformation i EGFR genom att bevara stadierna för viktiga strukturella enheter, så som helix αC och saltbryggan mellan Lys745 och Glu762, som båda avbryts i wild-type EGFR. Deletionmutationen resulterade dessutom i en strukturell förändring i det inaktiva EGFR stadiet, en inåtgående rörelse i helix αC som kunde orsaka en konformationsförändring från det inaktiva till det aktiva stadiet i EGFR. V769insASV och D770insNPG EGFR insertion-mutationerna ledde också till bättre stabilitet för den aktiva konfirmationen i EGFR i förhållande till wild-type EGFR. Insertion-mutationerna hindrade också bildningen av en autoinhiberande interaktion mellan Ala767 och Arg776 i den inaktiva konformationen för EGFR, vilket skulle göra det mera troligt att EGFR övergår till det katalytiskt aktiva stadiet. E936K-mutationen i ERBB2 påverkade typen av interaktioner som sker vid ERBB2:s homodimera- och heterodimera gränsytor, där en ny jonisk interaktion formas mellan monomererna och stärkte monomer-monomer bindningen. Följaktligen förlängs varaktigheten hos den aktiverade ERBB2-dimeren, vilket ökar fosforyleringen av ERBB2. Sammanfattningsvis visar den här avhandlingen att en serie av strukturella förändringar spelar en roll i att kollektivt orsaka de experimentellt rapporterade funktionella förändringarna genom de vi aktiverande ERBB-mutationerna. En grundlig genomgång av de mutationsinducerade förändringarna ökar vår kunskap om vilken roll mutationer spelar i cancerprogression, och resultaten är nödvändig kunskap när man använder sig av strukturbaserad, rationell design av ligander som kunde inhibera ERBB kinas-aktiviteten och påföljande receptorsignalering. Dylika ligander kan potentiellt vidareutvecklas till en ny behandlingsmetod för cancer

Probing MST1 Kinase Sequence and Structure for Rational Drug Discovery (Targeting diabetes by blocking protein-protein interactions)

Apoptotic beta cell death is an underlying cause majorly for type I and to a lesser extent for type II diabetes. Recently, MST1 kinase was identified as a key apoptotic agent in diabetic condition. In this study, I have examined MST1 and closely related kinases namely, MST2, MST3 and MST4, aiming to tackle diabetes by exploring ways to selectively block MST1 kinase activity. The first investigation was directed towards evaluating possibilities of selectively blocking the ATP binding site of MST1 kinase that is essential for the activity of the enzymes. Structure and sequence analyses of this site however revealed a near absolute conservation between the MSTs and very few changes with other kinases. The observed residue variations also displayed similar physicochemical properties making it hard for selective inhibition of the enzyme. Second, possibilities for allosteric inhibition of the enzyme were evaluated. Analysis of the recognized allosteric site also posed the same problem as the MSTs shared almost all of the same residues. The third analysis was made on the SARAH domain, which is required for the dimerization and activation of MST1 and MST2 kinases. MST3 and MST4 lack this domain, hence selectivity against these two kinases can be achieved. Other proteins with SARAH domains such as the RASSF proteins were also examined. Their interaction with the MST1 SARAH domain were evaluated to mimic their binding pattern and design a peptide inhibitor that interferes with MST1 SARAH dimerization. In molecular simulations the RASSF5 SARAH domain was shown to strongly interact with the MST1 SARAH domain and possibly preventing MST1 SARAH dimerization. Based on this, the peptidic inhibitor was suggested to be based on the sequence of RASSF5 SARAH domain. Since the MST2 kinase also interacts with RASSF5 SARAH domain, absolute selectivity might not be achieved.Siirretty Doriast