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Molecularly targeted cancer therapy has improved outcomes for patients with cancer with targetable oncoproteins, such as mutant EGFR in lung cancer. Yet, the long-term survival of these patients remains limited, because treatment responses are typically incomplete. One potential explanation for the lack of complete and durable responses is that oncogene-driven cancers with activating mutations of EGFR often harbor additional co-occurring genetic alterations. This hypothesis remains untested for most genetic alterations that co-occur with mutant EGFR. Here, we report the functional impact of inactivating genetic alterations of the mRNA splicing factor RNA-binding motif 10 (RBM10) that co-occur with mutant EGFR. RBM10 deficiency decreased EGFR inhibitor efficacy in patient-derived EGFR-mutant tumor models. RBM10 modulated mRNA alternative splicing of the mitochondrial apoptotic regulator Bcl-x to regulate tumor cell apoptosis during treatment. Genetic inactivation of RBM10 diminished EGFR inhibitor-mediated apoptosis by decreasing the ratio of (proapoptotic) Bcl-xS to (antiapoptotic) Bcl-xL isoforms of Bcl-x. RBM10 deficiency was a biomarker of poor response to EGFR inhibitor treatment in clinical samples. Coinhibition of Bcl-xL and mutant EGFR overcame the resistance induced by RBM10 deficiency. This study sheds light on the role of co-occurring genetic alterations and on the effect of splicing factor deficiency on the modulation of sensitivity to targeted kinase inhibitor cancer therapy.

Molecularly targeted cancer therapy has improved outcomes for patients with cancer with targetable oncoproteins, such as mutant EGFR in lung cancer. Yet, the long-term survival of these patients remains limited, because treatment responses are typically incomplete. One potential explanation for the lack of complete and durable responses is that oncogene-driven cancers with activating mutations of EGFR often harbor additional co-occurring genetic alterations. This hypothesis remains untested for most genetic alterations that co-occur with mutant EGFR. Here, we report the functional impact of inactivating genetic alterations of the mRNA splicing factor RNA-binding motif 10 (RBM10) that co-occur with mutant EGFR. RBM10 deficiency decreased EGFR inhibitor efficacy in patient-derived EGFR-mutant tumor models. RBM10 modulated mRNA alternative splicing of the mitochondrial apoptotic regulator Bcl-x to regulate tumor cell apoptosis during treatment. Genetic inactivation of RBM10 diminished EGFR inhibitor-mediated apoptosis by decreasing the ratio of (proapoptotic) Bcl-xS to (antiapoptotic) Bcl-xL isoforms of Bcl-x. RBM10 deficiency was a biomarker of poor response to EGFR inhibitor treatment in clinical samples. Coinhibition of Bcl-xL and mutant EGFR overcame the resistance induced by RBM10 deficiency. This study sheds light on the role of co-occurring genetic alterations and on the effect of splicing factor deficiency on the modulation of sensitivity to targeted kinase inhibitor cancer therapy.

Purpose: Despite the established role of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutated NSCLC, drug resistance inevitably ensues, with a paucity of treatment options especially in EGFRT790M-negative resistance. Experimental Design: We performed whole-exome and transcriptome analysis of 59 patients with first- and second-generation EGFR TKI-resistant metastatic EGFR-mutated NSCLC to characterize and compare molecular alterations mediating resistance in T790M-positive (T790M(+)) and -negative (T790M(-)) disease. Results: Transcriptomic analysis revealed ubiquitous loss of adenocarcinoma lineage gene expression in T790M(-) tumors, orthogonally validated using multiplex IHC. There was enrichment of genomic features such as TP53 alterations, 3q chromosomal amplifications, whole-genome doubling and nonaging mutational signatures in T790M(-) tumors. Almost half of resistant tumors were further classified as immune(hot), with clinical outcomes conditional on immune cell-infiltration state and T790M status. Finally, using a Bayesian statistical approach, we explored how T790M(-) and T790M(+) disease might be predicted using comprehensive genomic and transcriptomic profiles of treatment-naive patients. Conclusions: Our results illustrate the interplay between genetic alterations, cell lineage plasticity, and immune microenvironment in shaping divergent TKI resistance and outcome trajectories in EGFR-mutated NSCLC. Genomic and transcriptomic profiling may facilitate the design of bespoke therapeutic approaches tailored to a tumor's adaptive potential.

Purpose: Despite the established role of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutated NSCLC, drug resistance inevitably ensues, with a paucity of treatment options especially in EGFRT790M-negative resistance. Experimental Design: We performed whole-exome and transcriptome analysis of 59 patients with first- and second-generation EGFR TKI-resistant metastatic EGFR-mutated NSCLC to characterize and compare molecular alterations mediating resistance in T790M-positive (T790M(+)) and -negative (T790M(-)) disease. Results: Transcriptomic analysis revealed ubiquitous loss of adenocarcinoma lineage gene expression in T790M(-) tumors, orthogonally validated using multiplex IHC. There was enrichment of genomic features such as TP53 alterations, 3q chromosomal amplifications, whole-genome doubling and nonaging mutational signatures in T790M(-) tumors. Almost half of resistant tumors were further classified as immune(hot), with clinical outcomes conditional on immune cell-infiltration state and T790M status. Finally, using a Bayesian statistical approach, we explored how T790M(-) and T790M(+) disease might be predicted using comprehensive genomic and transcriptomic profiles of treatment-naive patients. Conclusions: Our results illustrate the interplay between genetic alterations, cell lineage plasticity, and immune microenvironment in shaping divergent TKI resistance and outcome trajectories in EGFR-mutated NSCLC. Genomic and transcriptomic profiling may facilitate the design of bespoke therapeutic approaches tailored to a tumor's adaptive potential.

Oncogene-induced senescence provides a barrier against malignant transformation. However, it can also promote cancer through the secretion of a plethora of factors released by senescent cells, called the senescence associated secretory phenotype (SASP). We have previously shown that in proliferating cells, nuclear lncRNA MIR31HG inhibits p16/CDKN2A expression through interaction with polycomb repressor complexes and that during BRAF-induced senescence, MIR31HG is overexpressed and translocates to the cytoplasm. Here, we show that MIR31HG regulates the expression and secretion of a subset of SASP components during BRAF-induced senescence. The SASP secreted from senescent cells depleted for MIR31HG fails to induce paracrine invasion without affecting the growth inhibitory effect. Mechanistically, MIR31HG interacts with YBX1 facilitating its phosphorylation at serine 102 (p-YBX1S102) by the kinase RSK. p-YBX1S102 induces IL1A translation which activates the transcription of the other SASP mRNAs. Our results suggest a dual role for MIR31HG in senescence depending on its localization and points to the lncRNA as a potential therapeutic target in the treatment of senescence-related pathologies.

Oncogene-induced senescence provides a barrier against malignant transformation. However, it can also promote cancer through the secretion of a plethora of factors released by senescent cells, called the senescence associated secretory phenotype (SASP). We have previously shown that in proliferating cells, nuclear lncRNA MIR31HG inhibits p16/CDKN2A expression through interaction with polycomb repressor complexes and that during BRAF-induced senescence, MIR31HG is overexpressed and translocates to the cytoplasm. Here, we show that MIR31HG regulates the expression and secretion of a subset of SASP components during BRAF-induced senescence. The SASP secreted from senescent cells depleted for MIR31HG fails to induce paracrine invasion without affecting the growth inhibitory effect. Mechanistically, MIR31HG interacts with YBX1 facilitating its phosphorylation at serine 102 (p-YBX1S102) by the kinase RSK. p-YBX1S102 induces IL1A translation which activates the transcription of the other SASP mRNAs. Our results suggest a dual role for MIR31HG in senescence depending on its localization and points to the lncRNA as a potential therapeutic target in the treatment of senescence-related pathologies.

Lung cancer is the world's leading cause of cancer death and shows strong ancestry disparities. By sequencing and assembling a large genomic and transcriptomic dataset of lung adenocarcinoma (LUAD) in individuals of East Asian ancestry (EAS; n = 305), we found that East Asian LUADs had more stable genomes characterized by fewer mutations and fewer copy number alterations than LUADs from individuals of European ancestry. This difference is much stronger in smokers as compared to nonsmokers. Transcriptomic clustering identified a new EAS-specific LUAD subgroup with a less complex genomic profile and upregulated immune-related genes, allowing the possibility of immunotherapy-based approaches. Integrative analysis across clinical and molecular features showed the importance of molecular phenotypes in patient prognostic stratification. EAS LUADs had better prediction accuracy than those of European ancestry, potentially due to their less complex genomic architecture. This study elucidated a comprehensive genomic landscape of EAS LUADs and highlighted important ancestry differences between the two cohorts. Genomic and transcriptomic analysis of lung adenocarcinoma (LUAD) in Asia indicates that Asian LUADs have fewer mutations, lower driver prevalence and fewer copy number alterations than European LUADs.

Lung cancer is the world's leading cause of cancer death and shows strong ancestry disparities. By sequencing and assembling a large genomic and transcriptomic dataset of lung adenocarcinoma (LUAD) in individuals of East Asian ancestry (EAS; n = 305), we found that East Asian LUADs had more stable genomes characterized by fewer mutations and fewer copy number alterations than LUADs from individuals of European ancestry. This difference is much stronger in smokers as compared to nonsmokers. Transcriptomic clustering identified a new EAS-specific LUAD subgroup with a less complex genomic profile and upregulated immune-related genes, allowing the possibility of immunotherapy-based approaches. Integrative analysis across clinical and molecular features showed the importance of molecular phenotypes in patient prognostic stratification. EAS LUADs had better prediction accuracy than those of European ancestry, potentially due to their less complex genomic architecture. This study elucidated a comprehensive genomic landscape of EAS LUADs and highlighted important ancestry differences between the two cohorts. Genomic and transcriptomic analysis of lung adenocarcinoma (LUAD) in Asia indicates that Asian LUADs have fewer mutations, lower driver prevalence and fewer copy number alterations than European LUADs.

There is an interest in bioprospecting organisms from the aquatic environment to find novel bioactive compounds with health promoting or other functional properties. The aim of this study was to evaluate extracts from untreated and heat-treated salmon tissues for their radical scavenging activities and for their ability to inhibit activity of the proteases angiotensin I-converting enzyme (ACE) and dipeptidyl peptidase 4 (DPP-4). In vitro assays were used to detect these activities and the corresponding candidate bioactive compounds were characterized by LC-MS/MS.Radical scavenging activity was detected in <10kDa extracts of gills, belly flap muscle and skin with EC50 values of 39, 82 and 100 µg/mL, respectively. No ACE or DPP-4 inhibiting activity could be detected. LC-MS/MS analysis of dominating compounds in active fractions from size exclusion chromatography showed that families of related compounds were found in several fractions from different tissues but most pronounced in gills. One family was defined according to content of a specific amino acid sequence (PW). Three families were defined by the m/z value of the smallest compound reported in each family (219, 434 and 403). The three latter families did not contain standard unmodified amino acids, indicating peptides with modified amino acids or other kinds of molecules.Industrial relevance. Bioprospecting in fish tissue traditionally regarded as waste can lead to detection of novel natural bioactive compounds including peptides, which could have nutritional, pharmaceutical or other functional value and be used in health and functional foods, thus increasing the value adding of secondary marine products. A number of naturally occurring antimicrobial peptides have been characterized from fish skin and gills, such as piscidins, but these and other fish tissues may contain numerous other compounds with bioactive properties. Such compounds could be extracted by the subsection of the fish industry that processes

Der er en stigende interesse i bioaktive peptider fra marine sekundære produkter, idet de er i besiddelse af et stort potentiale for at blive inkorporeret til funktionelle fødevarer og til medicinale formål. Bioaktive peptider fra det marine område er observeret værende i besiddelse af et bredt spekter af fysiologiske funktioner bl.a. antioxidative, blodtrykssænkende, antimikrobielle, anticancer samt effekt mod diabetes 2. Størstedelen af forskningen har dog fokuseret på peptider genereret fra hydrolyse med kommercielle industrielle enzymer og anvendelsen af disse hydrolysater. Det kunne være interessant at finde ud af om nedbrydelse af sekundære produkter fra fisk med fordøjelsesproteaser genererer peptider, som også er i besiddelse af disse sundhedsfremmende egenskaber, enten set i relation til fordøjelse eller som et alternativ til anvendelsen/brugen af industrielle proteaser. Generelt må fisk være udstyret med et særligt bioaktivt forsvar mod den store bakteriemængede i vandet, og må derfor forventes at være i besiddelse af bio-molekyler såsom små peptider. Det kunne derfor være relevant at undersøge om disse naturligt forekommende peptider indeholder andre funktionelle og sundhedsfremmende bioaktive peptider. På denne baggrund har dette PhD projekt haft til formål at finde og karakterisere hidtil ukendte bioaktive peptider fra marine sekundære produkter. Projektet var inddelt i to mere specifikke formål. Del I var at undersøge naturligt forekommende peptider for bioaktiviteter som radikal scavenging aktivitet, Angiotensin I-converting enzym (ACE) og dipeptidyl peptidase (DPP-IV) inbiberende egenskaber og protease inhiberende aktivitet i væv fra sekundære produkter som gæller, buglap muskel og skind fra laks (Salmo salar). Dette blev udført i ekstrakter fra ubehandlet og varmebehandlet væv ved hjælp af in vitro assays. Hvis bioaktivitets molekyler blev påvist, var det desuden et mål at karakterisere de korresponderende bioaktivitets kandidater. I del II var form, There is an increasing interest in bioactive peptides from marine secondary products, as they offer a great potential for incorporation into functional food and for medical purposes. Bioactive peptides from marine sources have been found to display a wide range of physiological functions including antioxidative, antihypertensive, antimicrobial, immunomodulatory, anticancer and diabetes 2 effects among others. However, majority of the research has been focusing on the peptides derived from hydrolysis with commercial industrial enzymes and the usefulness of these hydrolysates.It could be interesting whether digestion of fish secondary tissue with gastrointestinal proteases generates peptides, which also have these health promoting properties either in relation to gastrointestinal digestion or as an alternative to the use of industrial proteases. Furthermore, as a bioactive defense system against the bacterial load in the water, fish is expected to possess bio-components as small peptides. It could therefore be relevant whether these naturally occurring peptides exhibit other functional and health promoting bioactive properties.On this background the overall goal of the present PhD research was to discover and characterize novel bioactive peptides from marine secondary products. The research was divided into two more specific objectives in different parts. Part I was to investigate naturally occurring peptides for bioactivities as radical scavenging activity, Angiotensin I-converting enzyme (ACE) and intestinal dipeptidyl peptidase (DPP-IV) inhibiting properties and protease inhibiting activity in tissue of secondary products such as gills, belly flap muscle and skin from salmon (Salmo salar). This was conducted in extracts from untreated and heattreated tissue by using in vitro assays. Furthermore, if any detected, an aim was to characterize the corresponding candidate bioactive molecules. Part II was to investigate peptides in hydrolysates from salmon (Salmo salar) b

Proteins from fish tissue could be a promising source of peptides with a nutritional and pharmaceutical value, e.g. as treatment of type 2 diabetes with dipeptidyl peptidase IV (DPP-IV) inhibiting peptides, and could be used in health and functional foods and thereby increasing the value of secondary marine products. The approach in this study is to hydrolyse skin and belly flap tissue from Salmon with the use of mammalian digestive proteases from pancreas and intestinal mucosa and test hydrolysates for antioxidative capacity, intestinal DPP-IV and angiotensin converting enzyme (ACE) inhibiting properties. 10kDa dialysis bags containing 10ml water were added to homogenized fish tissues, which were subsequently hydrolysed for 24 hours at 37˚C and pH 8 with intestinal mucosa extract and/or pancreatin solution from pig. Dialysis bags were then removed and content were analyzed for free amino groups, antioxidative capacity by ABTS (2,2-azinobis(3-ethylbenzothiazoline-6-sulfonicacid)), DPP-IV and ACE inhibiting activity. Degree of hydrolysis (DH) of hydrolysates was approximately 13% and 10% for belly flap and skin respectively. No clear difference was observed in DH between pancreatin and pancreatin + mucosa hydrolysates. No DH was obtained for tissues hydrolysed with only intestinal mucosa extract. Preliminary results showed antioxidant activity and intestinal DPP-IV and ACE inhibiting activity in 10 kDa fraction from both belly flap and skin hydrolysates but with a higher antioxidative capacity in belly flap hydrolysates. No difference between hydrolysates with pancreatin and pancreatin+mucosa was observed. Hydrolysates will be further fractionated by gelfiltration. Fractions will be analyzed for the three bioactivities and also presented.

Proteins from fish tissue could be a promising source of peptides with a nutritional and pharmaceutical value, e.g. as treatment of type 2 diabetes with dipeptidyl peptidase IV (DPP-IV) inhibiting peptides, and could be used in health and functional foods and thereby increasing the value of secondary marine products. The approach in this study is to hydrolyse skin and belly flap tissue from Salmon with the use of mammalian digestive proteases from pancreas and intestinal mucosa and test hydrolysates for antioxidative capacity, intestinal DPP-IV and angiotensin converting enzyme (ACE) inhibiting properties. 10kDa dialysis bags containing 10ml water were added to homogenized fish tissues, which were subsequently hydrolysed for 24 hours at 37˚C and pH 8 with intestinal mucosa extract and/or pancreatin solution from pig. Dialysis bags were then removed and content were analyzed for free amino groups, antioxidative capacity by ABTS (2,2-azinobis(3-ethylbenzothiazoline-6-sulfonicacid)), DPP-IV and ACE inhibiting activity. Degree of hydrolysis (DH) of hydrolysates was approximately 13% and 10% for belly flap and skin respectively. No clear difference was observed in DH between pancreatin and pancreatin + mucosa hydrolysates. No DH was obtained for tissues hydrolysed with only intestinal mucosa extract. Preliminary results showed antioxidant activity and intestinal DPP-IV and ACE inhibiting activity in 10 kDa fraction from both belly flap and skin hydrolysates but with a higher antioxidative capacity in belly flap hydrolysates. No difference between hydrolysates with pancreatin and pancreatin+mucosa was observed. Hydrolysates will be further fractionated by gelfiltration. Fractions will be analyzed for the three bioactivities and also presented.

Proteins from fish tissue could be a promising source of peptides with a nutritional and pharmaceutical value, e.g. as treatment of type 2 diabetes with dipeptidyl peptidase IV (DPP-IV) inhibiting peptides, and could be used in health and functional foods and thereby increasing the value of secondary marine products. The approach in this study is to hydrolyse skin and belly flap tissue from Salmon with the use of mammalian digestive proteases from pancreas and intestinal mucosa and test hydrolysates for antioxidative capacity, intestinal DPP-IV and angiotensin converting enzyme (ACE) inhibiting properties. 10kDa dialysis bags containing 10ml water were added to homogenized fish tissues, which were subsequently hydrolysed for 24 hours at 37˚C and pH 8 with intestinal mucosa extract and/or pancreatin solution from pig. Dialysis bags were then removed and content were analyzed for free amino groups, antioxidative capacity by ABTS (2,2-azinobis(3-ethylbenzothiazoline-6-sulfonicacid)), DPP-IV and ACE inhibiting activity. Degree of hydrolysis (DH) of hydrolysates was approximately 13% and 10% for belly flap and skin respectively. No clear difference was observed in DH between pancreatin and pancreatin + mucosa hydrolysates. No DH was obtained for tissues hydrolysed with only intestinal mucosa extract. Preliminary results showed antioxidant activity and intestinal DPP-IV and ACE inhibiting activity in 10 kDa fraction from both belly flap and skin hydrolysates but with a higher antioxidative capacity in belly flap hydrolysates. No difference between hydrolysates with pancreatin and pancreatin+mucosa was observed. Hydrolysates will be further fractionated by gelfiltration. Fractions will be analyzed for the three bioactivities and also presented.

The aquatic ecosystem represents a large number of organisms adapted to living conditions remarkably different from the land-living ones. For instance fish generally possess an eminent bioactive defense to protect them from the high bacterial load in water, and must be expected to harbor a large number of bio-components such as bioactive peptides for this purpose. Tissue and proteins from e.g. fish gills, skin and viscera could be a new source of peptides that could have a nutritional and pharmaceutical value, and be used in health and functional foods and thereby increasing the value adding of secondary marine products. Only few naturally occurring bioactive peptides have been characterized such as the antimicrobial polypeptide piscidines from gills. It is therefore hypothesized, that fish tissue also contains numerous other peptides with other bioactive properties. The approach in this project is therefore to extract and identify naturally occurring bioactive peptides from different tissues from salmon. A number of aqueous extracts were made from gills, skin and belly flap. In order to preserve the bioactivity of the peptides mild extraction procedures as acidic, basic and aqueous solutions were used. Combination of different extraction conditions such as with/without boiling, with/without inhibitor and variation of pH resulted in a total of 36 extracts. The activity of the extracts was analyzed in vitro for ACE (angiotensin-converting enzyme) inhibiting activity, and anti-oxidative activity (Free Radical Scavenging assay). A number of extracts showed high ACE inhibiting and anti-oxidative activity. The extracts were then size fractionated by ultrafiltration using a 10 kDa filter, and relevant fractions below 10 kDa from gills, skin and belly flap were further fractionated by gel-filtration on a Superdex peptide HR 10/30 column. Peptide fractions were collected and freeze-dried and will be further characterized with LCMS and MS/MS.