Studying the functional relevance of lung cancer genetic drivers in their physiological niche

Lung cancer is the leading cause of cancer related deaths worldwide. It is characterised with a high level of intra- and intertumour heterogeneity. Large lung cancer sequencing efforts have identified clear histopathology-specific genetic alteration patterns, which in the cases of lung adenocarcinomas are applied in clinics to direct treatment. Furthermore, lung cancer immunotherapy approaches have recently shown promising results in clinical trials. However, a deeper understanding of the functional importance of novel lung cancer genes as well as the lung cancer-related niche and cell type specific propensities leading to molecular and microenvironmental tumour heterogeneity is needed to better translate the growing amount of information to patient stratified treatments. The first part of this thesis work concentrated on the functional in vitro and in vivo investigation of putative tumour suppressive characteristics of the EPH receptor A3 (EPHA3), a gene commonly mutated in human lung cancers. Our in vitro findings supported the tumour suppressive characteristics of EPHA3 and indicated that EPHA3-mediated tumour suppression was specifically dependent on its kinase activity. However, our in vivo investigation demonstrated that loss of EphA3 does not co-operate with two known genetic alterations of human lung cancer in murine lung tumourigenesis nor it effects lung morphogenesis. Hence, we conclude that our study demonstrates how functional validation of putative cancer genes can be challenged by biological complexity, which may result in acquired compensation or different functional roles in human and mice. The results from the second part of this thesis work showed that cells in the airways of mouse lungs had a higher propensity to develop faster growing and progressing lung tumours than the cells in the distal alveolar space when exposed to known lung cancer genetic alterations, namely expression of oncogenic Kras and loss of Lkb1 (KL). The lung tumours originated from the airways were predominantly classified as adenosquamous carcinomas (ASCs). ASCs showed elevated levels of genes associated with immunosuppression and a notable immune cell infiltration with an increase in the amount of possible myeloid-derived suppressor cells (MDSCs). The KL ASC model may thus represent a relevant preclinical model for the study of anti-MDSC immune therapy as a treatment for ASCs, which in humans represent a rare but aggressive type of lung cancer. Thus, the findings in this thesis work highlight the importance of the functional niche in the progression of lung cancer and, therefore, possibly affecting a response to treatment. Niche-specific investigation of lung cancer genetic alterations thus leads to a more accurate stratification of the preclinical in vivo models, simultaneously revealing relevant molecular mechanisms underlying lung cancer heterogeneity.Huolimatta uusimmista keuhkosyövän hoitomuodoista, joilla pyritään vaikuttamaan kohdennetusti tiettyjen geenimutaatioiden aiheuttamaan pahalaatuisen syöpäkudoksen kasvuun, ei riittävän hyviä potilaan parantumiseen johtavia hoitoja ole vielä keksitty. Tämän ajatellaan johtuvan muun muassa yksittäisen syöpäkudoksen sisäisestä tai syöpäkasvainten välillä olevasta heterogeenisyydestä eli siitä, että syöpäsolut poikkeavat toisistaan. Laajat keuhkosyövän DNA-sekvensointitutkimukset ovat tunnistaneet valtavan määrän uusia mutaatiota, joiden syvempi toiminnallinen ymmärtäminen on tärkeää, jotta voimme paremmin käyttää hyväksi tätä suurta määrää uutta tietoa kehittäessämme uusia hoitomuotoja syöpiin. Lisäksi potilaan oman immuunipuolustuksen valjastaminen syöpäkasvainta vastaan on kasvava tutkimuskohde. Tämän väitöskirjatyön tavoitteena oli selvittää useissa keuhkosyövissä mutatoituneen EPH-reseptori A3- (EPHA3) geenin osuutta keuhkosyövän synnyssä ja etenemisessä, sekä tutkia kyseisen geenin toimintaa keuhkon kehityksessä. Soluviljelmillä saadut tutkimustuloksemme tukivat EPHA3-geenin syövän kasvua estävää eli tuumorisuppressivista tehtävää soluissa. Tästä huolimatta havaitsimme, että hiiren EphA3-geenin poisto ei vaikuta keuhkosyövän etenemiseen eikä keuhkojen kehitykseen. Näiden hiirimallista saatujen tulosten perusteella teimme johtopäätöksen, että hiiren EphA3- geenin poisto voi esimerkiksi johtaa saman geeniperheen geenien ilmentymisen aikaansaamaan kompensaatioon ja/tai EPHA3-geenin poistolla voi olla eri merkitys hiirelle kuin ihmiselle. Toinen tämän väitöskirjatyön tavoite oli tutkia eri keuhkosolutyyppien alttiutta kehittyä ei-pienisoluisen keuhkosyövän eri histopatologiatyypeiksi. Tutkimustuloksemme keuhkosyöpähiirimallilla, jossa tunnettujen keuhkosyöpägeenien muutokset aikaansaavat ei-pienisoluisen keuhkosyövän synnyn, osoittivat, että keuhkojen kahdella eri solutyypillä on erilainen alttius kehittyä adenoskvamoosi karsinoomaksi. Lisäksi tutkimustuloksemme osoittivat, että kyseisen keuhkosyöpähiirimallin adenoskvamooseissa karsinoomissa on immuunipuolustuksen vaimentamiseen viittaava immuunisoluympäristö. Ihmisellä adenoskvamoosi karsinooma on harvinainen, mutta aggressiivinen keuhkosyöpätyyppi. Tämä väitöskirjatyö on tuottanut merkittävää uutta tietoa EPHA3-geenin toiminnallisesta tehtävästä syövässä, jonka valossa kyseiseen geeniin liittyvää syöpätutkimusta pystytään ohjaamaan tulevaisuudessa oikeaan suuntaan. Lisäksi tämä väitöskirjatyö avasi uusia näkökulmia keuhkosyövän heterogeenisyyteen johtavista tekijöistä sekä osoitti aggressiivisen keuhkosyöpätyypin mahdollisesti omaavan erityisen kasvaimen immuunisoluympäristön, jota voidaan tulevaisuudessa käyttää hyväksi uusien hoitomuotojen kehityksessä

Exploring Transcriptomic Landscapes in Red Blood Cells, in Their Extracellular Vesicles and on A Single-Cell Level

Being enucleated, RBCs lack typical transcriptomes, but are known to contain small amounts of diverse long transcripts and microRNAs. However, the exact role and importance of these RNAs are lacking. Shedding of extracellular vesicles (EVs) from the plasma membrane constitutes an integral mechanism of RBC homeostasis, by which RBCs remove unnecessary cytoplasmic content and cell membrane. To study this further, we explored the transcriptomes of RBCs and extracellular vesicles (EVs) of RBCs using next-generation sequencing. Furthermore, we performed single-cell RNA sequencing on RBCs, which revealed that approximately 10% of the cells contained detectable levels of mRNA and cells formed three subpopulations based on their transcriptomes. A decrease in the mRNA quantity was observed across the populations. Qualitative changes included the differences in the globin transcripts and changes in the expression of ribosomal genes. A specific splice form of a long non-coding RNA, Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), was the most enriched marker in one subpopulation of RBCs, co-expressing with ribosomal structural transcripts. MALAT1 expression was confirmed by qPCR in CD71-enriched reticulocytes, which were also characterized with imaging flow cytometry at the single cell level. Analysis of the RBC transcriptome shows enrichment of pathways and functional categories required for the maturation of reticulocytes and erythrocyte functions. The RBC transcriptome was detected in their EVs, making these transcripts available for intercellular communication in blood

The putative tumor suppressor gene EphA3 fails to demonstrate a crucial role in murine lung tumorigenesis or morphogenesis

Treatment of non-small cell lung cancer (NSCLC) is based on histological analysis and molecular profiling of targetable driver oncogenes. Therapeutic responses are further defined by the landscape of passenger mutations, or loss of tumor suppressor genes. We report here a thorough study to address the physiological role of the putative lung cancer tumor suppressor EPH receptor A3 (EPHA3), a gene that is frequently mutated in human lung adenocarcinomas. Our data shows that homozygous or heterozygous loss of EphA3 does not alter the progression of murine adenocarcinomas that result from Kras mutation or loss of Trp53, and we detected negligible postnatal expression of EphA3 in adult wildtype lungs. Yet, EphA3 was expressed in the distal mesenchyme of developing mouse lungs, neighboring the epithelial expression of its Efna1 ligand; this is consistent with the known roles of EPH receptors in embryonic development. However, the partial loss of EphA3 leads only to subtle changes in epithelial Nkx2-1, endothelial Cd31 and mesenchymal Fgf10 RNA expression levels, and no macroscopic phenotypic effects on lung epithelial branching, mesenchymal cell proliferation, or abundance and localization of CD31-positive endothelia. The lack of a discernible lung phenotype in EphA3-null mice might indicate lack of an overt role for EPHA3 in the murine lung, or imply functional redundancy between EPHA receptors. Our study shows how biological complexity can challenge in vivo functional validation of mutations identified in sequencing efforts, and provides an incentive for the design of knock-in or conditional models to assign the role of EPHA3 mutation during lung tumorigenesis

The putative tumor suppressor gene EphA3 fails to demonstrate a crucial role in murine lung tumorigenesis or morphogenesis

Treatment of non-small cell lung cancer (NSCLC) is based on histological analysis and molecular profiling of targetable driver oncogenes. Therapeutic responses are further defined by the landscape of passenger mutations, or loss of tumor suppressor genes. We report here a thorough study to address the physiological role of the putative lung cancer tumor suppressor EPH receptor A3 (EPHA3), a gene that is frequently mutated in human lung adenocarcinomas. Our data shows that homozygous or heterozygous loss of EphA3 does not alter the progression of murine adenocarcinomas that result from Kras mutation or loss of Trp53, and we detected negligible postnatal expression of EphA3 in adult wildtype lungs. Yet, EphA3 was expressed in the distal mesenchyme of developing mouse lungs, neighboring the epithelial expression of its Efna1 ligand; this is consistent with the known roles of EPH receptors in embryonic development. However, the partial loss of EphA3 leads only to subtle changes in epithelial Nkx2-1, endothelial Cd31 and mesenchymal Fgf10 RNA expression levels, and no macroscopic phenotypic effects on lung epithelial branching, mesenchymal cell proliferation, or abundance and localization of CD31-positive endothelia. The lack of a discernible lung phenotype in EphA3-null mice might indicate lack of an overt role for EPHA3 in the murine lung, or imply functional redundancy between EPHA receptors. Our study shows how biological complexity can challenge in vivo functional validation of mutations identified in sequencing efforts, and provides an incentive for the design of knock-in or conditional models to assign the role of EPHA3 mutation during lung tumorigenesis.Peer reviewe

Absence of NEFL in patient-specific neurons in early-onset Charcot-Marie-Tooth neuropathy

Objective: We used patient-specific neuronal cultures to characterize the molecular genetic mechanism of recessive nonsense mutations in neurofilament light (NEFL) underlying early-onset Charcot-Marie-Tooth (CMT) disease. Methods: Motor neurons were differentiated from induced pluripotent stem cells of a patient with early-onset CMT carrying a novel homozygous nonsense mutation in NEFL. Quantitative PCR, protein analytics, immunocytochemistry, electron microscopy, and single-cell transcriptomics were used to investigate patient and control neurons. Results: We show that the recessive nonsense mutation causes a nearly total loss of NEFL messenger RNA (mRNA), leading to the complete absence of NEFL protein in patient's cultured neurons. Yet the cultured neurons were able to differentiate and form neuronal networks and neurofilaments. Single-neuron gene expression fingerprinting pinpointed NEFL as the most downregulated gene in the patient neurons and provided data of intermediate filament transcript abundancy and dynamics in cultured neurons. Blocking of nonsense-mediated decay partially rescued the loss of NEFL mRNA. Conclusions: The strict neuronal specificity of neurofilament has hindered the mechanistic studies of recessive NEFL nonsense mutations. Here, we show that such mutation leads to the absence of NEFL, causing childhood-onset neuropathy through a loss-of-function mechanism. We propose that the neurofilament accumulation, a common feature of many neurodegenerative diseases, mimics the absence of NEFL seen in recessive CMT if aggregation prevents the proper localization of wild-type NEFL in neurons. Our results suggest that the removal of NEFL as a proposed treatment option is harmful in humans.Peer reviewe

Cell of Origin Links Histotype Spectrum to Immune Microenvironment Diversity in Non-small-Cell Lung Cancer Driven by Mutant Kras and Loss of Lkb1

Lung cancers exhibit pronounced functional heterogeneity, confounding precision medicine. We studied how the cell of origin contributes to phenotypic heterogeneity following conditional expression of Kras(G12D) and loss of Lkb1 (Kras; Lkb1). Using progenitor cell-type-restricted adenoviral Cre to target cells expressing surfactant protein C (SPC) or club cell antigen 10 (CC10), we show that Ad5-CC10-Cre-infected mice exhibit a shorter latency compared with Ad5-SPC-Cre cohorts. We further demonstrate that CC10(+) cells are the predominant progenitors of adenosquamous carcinoma (ASC) tumors and give rise to a wider spectrum of histotypes that includes mucinous and acinar adenocarcinomas. Transcriptome analysis shows ASC histotype-specific upregulation of pro-inflammatory and immunomodulatory genes. This is accompanied by an ASC-specific immunosuppressive environment, consisting of downregulated MHC genes, recruitment of CD11b(+) Gr-1(+) tumor-associated neutrophils (TANs), and decreased T cell numbers. We conclude that progenitor cell-specific etiology influences the Kras; Lkb1-driven tumor histopathology spectrum and histotype-specific immune microenvironment.Peer reviewe

Patient-tailored design for selective co-inhibition of leukemic cell subpopulations

The extensive drug resistance requires rational approaches to design personalized combinatorial treatments that exploit patient-specific therapeutic vulnerabilities to selectively target disease-driving cell subpopulations. To solve the combinatorial explosion challenge, we implemented an effective machine learning approach that prioritizes patient-customized drug combinations with a desired synergy-efficacy-toxicity balance by combining single-cell RNA sequencing with ex vivo single-agent testing in scarce patient-derived primary cells. When applied to two diagnostic and two refractory acute myeloid leukemia (AML) patient cases, each with a different genetic background, we accurately predicted patient-specific combinations that not only resulted in synergistic cancer cell co-inhibition but also were capable of targeting specific AML cell subpopulations that emerge in differing stages of disease pathogenesis or treatment regimens. Our functional precision oncology approach provides an unbiased means for systematic identification of personalized combinatorial regimens that selectively co-inhibit leukemic cells while avoiding inhibition of nonmalignant cells, thereby increasing their likelihood for clinical translation.Peer reviewe