Novel tools for identification of oncogenic driver mutations

Genetic alterations contribute to the development and pathogenesis of several human cancers. These mutations accumulate in a cancer tissue over the course of time due to the instability of the cancer genome. Large-scale sequencing efforts have enabled identification of an abundance of these somatic mutations, and the amount of data is constantly increasing due to the improved accessibility of next-generation sequencing technologies. From this multitude of cancer-associated somatic mutations, a large majority are predicted to be inconsequential “passenger” mutations, (i.e., mutations which do not confer a selective growth advantage to the cancer cells); and only a handful have been validated as “driver” mutations (i.e., mutations playing a critical role in the development or maintenance of cancer). These driver mutations also function as predictive markers for survival, therapeutic efficacy, and often make the cancer cells susceptible to therapeutic intervention. Identification of driver mutations is an integral part of biomarker discovery in cancer research, and my thesis aimed to address this by developing a screening platform and a database. The in vitro Screen for Activating Mutations (iSCREAM) is a high-throughput screening workflow which was established with Epidermal Growth Factor Receptor (EGFR) as a model. The screen was validated by detection of known activating mutations like EGFR L858R. A previously known EGFR variant of unknown significance (VUS), EGFR A702V, was discovered in the screen and was functionally characterized to be an activating mutation. The iSCREAM screening methodology was further used to systematically study ERBB4, another gene in the EGFR family of receptor tyrosine kinases. We detected ERBB4 VUS R687K, and E715K in the screen and identify them as activating mutations. The ERBB4 mutations were characterized for their effect on ERBB4 phosphorylation, their sensitivity to various tyrosine kinase inhibitors, and their tumorigenicity was evaluated with in vivo allografts. The Database Of Recurrent Mutations (DORM), was prepared by analyzing a public registry of somatic mutations and preparing a catalog of the mutations identified from genome-wide studies to recapitulate the “real-world” frequency of all the recurrent (n > 1) somatic mutations. DORM allows limiting the scope of search to 38 tissue types and supports advanced queries using regular expressions. The easy-to-use database and its backend are written to be very responsive and fast in comparison to contemporary public cancer databases. Taken together, the findings and resources presented in this thesis establish grounds for further studies with other tyrosine kinases and potentially enable diversification into new niches.Uusia työkaluja syöpää aiheuttavien mutaatioiden tunnistamiseksi Geneettiset muutokset vaikuttavat useiden ihmisen syöpien syntyyn ja kehittymiseen. Syöpäkudokseen geenimutaatioita kertyy yhä enemmän ajan kuluessa syövän genomisen instabiliteetin vuoksi. Laajamittaisten sekvensointihankkeiden avulla on pystytty tunnistamaan paljon erilaisia somaattisia eli hankinnallisia mutaatioita ja sekvensointitulosten määrä kasvaa jatkuvasti uuden sukupolven sekvensointitekniikoiden (engl. next generation sequencing, NGS) paremman saatavuuden ansiosta. Näistä lukuisista syöpään liittyvistä somaattisista mutaatioista suurin osa on potilaan ennusteen kannalta merkityksettömiä "matkustajamutaatioita" (engl. passenger mutation) eli mutaatioita, jotka eivät anna valikoivaa kasvuetua syöpäsoluille. Vain muutamia somaattisia mutaatioita on validoitu "ajajamutaatioiksi" (engl. driver mutation) eli mutaatioiksi, joilla on kriittinen rooli syövän kehittymisessä tai ylläpitämisessä. Nämä ajajamutaatiot toimivat usein eloonjäämisen sekä hoidon tehon ennusteellisina markkereina ja usein myös herkistävät syöpäsoluja hoidoille. Ajajamutaatioiden tunnistaminen on olennainen osa syövän biomarkkereiden tutkimusta. Väitöskirjatyöni tavoitteena oli kehittää ajajamutaatioiden seulonta-alusta ja tietokanta. Aktivoivien mutaatioiden in vitro -seulonta (engl. in vitro Screen for Activating Mutations, iSCREAM) on tehoseulontamenetelmä, jonka kehittämistyössä käytettiin mallina epidermaalista kasvutekijäreseptoria (EGFR) koodaavaa geeniä iSCREAM-seulonnalla tunnistettiin jo tunnettuja aktivoivia EGFR-mutaatioita, kuten L858R, mikä validoi menetelmän toimivuuden. Seulontamenetelmällä tunnistettiin ja karakterisoitiin myös uusi EGFR-geenin aktivoiva mutaatio, A702V, jonka oletettu toimintamekanismi selvitettiin. iSCREAM-seulontamenetelmää hyödynnettiin tässä työssä myös EGFR-reseptorityrosiinikinaasiperheen toisen geenin, ERBB4-geenin, systemaattiseen tutkimiseen, jonka avulla löydettiin uusina aktivoivina mutaatioina ERBB4 R687K ja E715K. Näiden ERBB4-mutaatioiden vaikutusta ERBB4:n fosforylaatioon ja lääkeherkkyyteen erilaisille tyrosiinikinaasiestäjille karakterisoitiin, ja niiden tuumorigeenisyys validoitiin in vivo -allografteissa. Toistuvien mutaatioiden tietokanta (engl. Database Of Recurrent Mutations, DORM) luotiin analysoimalla somaattisten mutaatioiden julkista rekisteriä ja laatimalla luettelo genominlaajuisissa tutkimuksissa tunnistetuista mutaatioista, jotta kaikkien toistuvien (n > 1) somaattisten mutaatioiden "todellinen" esiintymistiheys voitaisiin laskea. DORM mahdollistaa haun rajoittamisen 38:aan kudostyyppiin ja tukee edistyneempiä kyselyjä säännöllisten lausekkeiden (engl. regular expression) avulla. Helppokäyttöinen tietokanta ja sen taustajärjestelmä kehitettiin hyvin reagoivaksi ja nopeaksi nykyisiin julkisiin syöpätietokantoihin verrattuna. Tässä työssä esitetyt havainnot ja resurssit luovat yhdessä perustan jatkotutkimuksille muilla tyrosiinikinaaseilla ja ovat mahdollisesti laajennettavissa muillekin tutkimusalueille

Gene expression analysis in cancer microarray datasets, investigating the role of an Embryonic Stem Cell Factor in prognosis

Cancer is a condition that is demanding more research with new cases being reported each year. In this thesis the aim was to investigate the behaviour of a embryonic stem cell factor and its partners in various types of cancers. The embryonic stem cell factor under study in this thesis is responsible for the maintenance of pleuripotency in stem cells and its interaction partners maintain the self-renewal ability of the embryonic stem cells. With the discovery of cancer stem cells and detections of stem cell like gene signatures from cancers, it becomes important to address the issue to identify the responsible genes. The embryonic stem cell factor of our interest when knocked down in cell line studies showed downregula- tion of stem cell pleuripotency factors therefore, we believe it may be playing a key role in cancer tissues where it is expressed.We use gene expression analysis of microarray data of cancer patient samples along with the available survival information to test whether the gene and its partners have any ef- fect on survival. We use correlation measures to establish that partners of the embryonic stem cell factor of our interest might be co-expressed in patient samples. In particular, we were able to identify colon cancer and seminoma samples that express our gene of interest at high levels. We used T-test and ROTS (Reproducibility Optimized Test Statistic) on these datasets to detect which genes are differentially expressed.The project also presents a different approach to microarray data analysis where the focus is not on the disease or condition but a set of genes are central theme of the study, and the research is done to find the cancer or datasets where the gene set is perturbed. This is de- sired under situations similar to the premise of this project, that if an embryonic stem cell factor is expressed in adult tissues it is a sign of problems.The project suggests that the embryonic stem cell factor under question alone cannot be held responsible for poor survival of the cancer patients, instead it seems that it is a pro- survival factor after all. But further analyses are being done in this area to uncover more in- formation and also to find factors that can explain the poor survival of the samples for the cancer datasets under study.</p

Receptor tyrosine kinase profiling of ischemic heart identifies ROR1 as a potential therapeutic target

BackgroundReceptor tyrosine kinases (RTK) are potential targets for the treatment of ischemic heart disease. The human RTK family consists of 55 members, most of which have not yet been characterized for expression or activity in the ischemic heart.MethodsRTK gene expression was analyzed from human heart samples representing healthy tissue, acute myocardial infarction or ischemic cardiomyopathy. As an experimental model, pig heart with ischemia-reperfusion injury, caused by cardiopulmonary bypass,was used, from which phosphorylation status of RTKs was assessed with a phospho-RTK array. Expression and function of one RTK, ROR1, was further validated in pig tissue samples, and in HL-1 cardiomyocytes and H9c2 cardiomyoblasts, exposed to hypoxia and reoxygenation. ROR1 protein level was analyzed by Western blotting. Cell viability after ROR1 siRNA knockdown or activation with Wnt-5a ligand was assessed by MTT assays.ResultsIn addition to previously characterized RTKs, a group of novel active and regulated RTKs was detected in the ischemic heart. ROR1 was the most significantly upregulated RTK in human ischemic cardiomyopathy. However, ROR1 phosphorylation was suppressed in the pig model of ischemia-reperfusion and ROR1 phosphorylation and expression were down-regulated in HL-1 cardiomyocytes subjected to short-term hypoxia in vitro. ROR1 expression in the pig heart was confirmed on protein and mRNA level. Functionally, ROR1 activity was associated with reduced viability of HL-1 cardiomyocytes in both normoxia and during hypoxia-reoxygenation.ConclusionsSeveral novel RTKs were found to be regulated in expression or activity in ischemic heart. ROR1 was one of the most significantly regulated RTKs. The in vitro findings suggest a role for ROR1 as a potential target for the treatment of ischemic heart injury.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

Receptor tyrosine kinase profiling of ischemic heart identifies ROR1 as a potential therapeutic target

BackgroundReceptor tyrosine kinases (RTK) are potential targets for the treatment of ischemic heart disease. The human RTK family consists of 55 members, most of which have not yet been characterized for expression or activity in the ischemic heart.MethodsRTK gene expression was analyzed from human heart samples representing healthy tissue, acute myocardial infarction or ischemic cardiomyopathy. As an experimental model, pig heart with ischemia-reperfusion injury, caused by cardiopulmonary bypass,was used, from which phosphorylation status of RTKs was assessed with a phospho-RTK array. Expression and function of one RTK, ROR1, was further validated in pig tissue samples, and in HL-1 cardiomyocytes and H9c2 cardiomyoblasts, exposed to hypoxia and reoxygenation. ROR1 protein level was analyzed by Western blotting. Cell viability after ROR1 siRNA knockdown or activation with Wnt-5a ligand was assessed by MTT assays.ResultsIn addition to previously characterized RTKs, a group of novel active and regulated RTKs was detected in the ischemic heart. ROR1 was the most significantly upregulated RTK in human ischemic cardiomyopathy. However, ROR1 phosphorylation was suppressed in the pig model of ischemia-reperfusion and ROR1 phosphorylation and expression were down-regulated in HL-1 cardiomyocytes subjected to short-term hypoxia in vitro. ROR1 expression in the pig heart was confirmed on protein and mRNA level. Functionally, ROR1 activity was associated with reduced viability of HL-1 cardiomyocytes in both normoxia and during hypoxia-reoxygenation.ConclusionsSeveral novel RTKs were found to be regulated in expression or activity in ischemic heart. ROR1 was one of the most significantly regulated RTKs. The in vitro findings suggest a role for ROR1 as a potential target for the treatment of ischemic heart injury

The L1TD1 Protein Interactome Reveals the Importance of Post-transcriptional Regulation in Human Pluripotency

Peer reviewe

L1TD1-a prognostic marker for colon cancer

BackgroundPrognostic markers specific to a particular cancer type can assist in the evaluation of survival probability of patients and help clinicians to assess the available treatment modalities.MethodsGene expression data was analyzed from three independent colon cancer microarray gene expression data sets (N=1052). Survival analysis was performed for the three data sets, stratified by the expression level of the LINE-1 type transposase domain containing 1 (L1TD1). Correlation analysis was performed to investigate the role of the interactome of L1TD1 in colon cancer patients.ResultsWe found L1TD1 as a novel positive prognostic marker for colon cancer. Increased expression of L1TD1 associated with longer disease-free survival in all the three data sets. Our results were in contrast to a previous study on medulloblastoma, where high expression of L1TD1 was linked with poor prognosis. Notably, in medulloblastoma L1TD1 was co-expressed with its interaction partners, whereas our analysis revealed lack of co-expression of L1TD1 with its interaction partners in colon cancer.ConclusionsOur results identify increased expression of L1TD1 as a prognostic marker predicting longer disease-free survival in colon cancer patients.Peer reviewe

L1TD1 - a prognostic marker for colon cancer

BackgroundPrognostic markers specific to a particular cancer type can assist in the evaluation of survival probability of patients and help clinicians to assess the available treatment modalities.MethodsGene expression data was analyzed from three independent colon cancer microarray gene expression data sets (N = 1052). Survival analysis was performed for the three data sets, stratified by the expression level of the LINE-1 type transposase domain containing 1 (L1TD1). Correlation analysis was performed to investigate the role of the interactome of L1TD1 in colon cancer patients.ResultsWe found L1TD1 as a novel positive prognostic marker for colon cancer. Increased expression of L1TD1 associated with longer disease-free survival in all the three data sets. Our results were in contrast to a previous study on medulloblastoma, where high expression of L1TD1 was linked with poor prognosis. Notably, in medulloblastoma L1TD1 was co-expressed with its interaction partners, whereas our analysis revealed lack of co-expression of L1TD1 with its interaction partners in colon cancer.ConclusionsOur results identify increased expression of L1TD1 as a prognostic marker predicting longer disease-free survival in colon cancer patients.</div

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

Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells

Regulatory T (Treg) cells are critical in regulating the immune response. In vitro induced Treg (iTreg) cells have significant potential in clinical medicine. However, applying iTreg cells as therapeutics is complicated by the poor stability of human iTreg cells and their variable suppressive activity. Therefore, it is important to understand the molecular mechanisms of human iTreg cell specification. We identified hypermethylated in cancer 1 (HIC1) as a transcription factor upregulated early during the differentiation of human iTreg cells. Although FOXP3 expression was unaffected, HIC1 deficiency led to a considerable loss of suppression by iTreg cells with a concomitant increase in the expression of effector T cell associated genes. SNPs linked to several immune-mediated disorders were enriched around HIC1 binding sites, and in vitro binding assays indicated that these SNPs may alter the binding of HIC1. Our results suggest that HIC1 is an important contributor to iTreg cell development and function