The DLBCL microenvironment: A proteomics approach to identify novel players involved in the tumor cell – microenvironment interactions.
Diffuse large B-cell lymphoma (DLBCL) can present de novo or transform from a less aggressive lymphoma. The pathogenesis of DLBCL is a multistep process that involves genetic alterations, immune status of the patient and tumour microenvironment. The Human Leukocyte Antigen (HLA) system regulates the immune response and loss of HLA expression in the tumour cells leads to an altered host immunity and impairs effective anti-tumour responses. Our aim is to investigate the proteome of tumor cells to identify proteins that play a role in the cross talk between tumor cells and the microenvironment in DLBCL. The ultimate goal is to explain why patients react differently to therapy and to provide a tailored treatment plan in order to avoid over- and undertreatment.

In this thesis, we hypothesized that microRNAs (miRNAs) with deregulated expression in lung fibroblasts, are crucial players in the impaired lung tissue repair and remodelling as observed in chronic obstructive pulmonary disease (COPD). To explore this, we focused on miRNA expression changes in the lung, and in particular in fibroblasts in relation to the effects of TGF-β, and current smoking and associations with COPD and ageing. We identified 106 TGF-β-regulated miRNAs in control and/or in COPD lung fibroblasts. Of these, three miRNAs responded differently to TGF-β in COPD compared to control lung fibroblasts. Only one miRNA was higher expressed in COPD compared to control lung fibroblasts. Furthermore, we identified one miRNA that was lower expressed in lung fibroblasts from current compared to ex-smokers. We identified >960 genes that are actively regulated by miRNAs in lung fibroblasts, which were used to identify fibroblast-specific targets of the differentially expressed miRNAs. Our studies indicate that the identified miRNAs may affect the function of lung fibroblasts through these genes, and affect tissue repair and remodelling, and thus are implicated in COPD pathogenesis. In bronchial biopsies of healthy control subjects, 285 age-related genes and 27 age-related miRNAs were identified. Genes with lower expression with increasing age included several hallmarks of ageing whereas genes with higher expression with increasing age were amongst others involved in synapse-related processes. These studies provide a good stepping stone for further studies aiming to clarify the complex role of these miRNAs in relation to abnormal tissue repair in COPD and ageing.

In this thesis, we studied the role of microRNAs (miRNAs) in the pathogenesis of Hodgkin lymphoma (HL). Small RNA sequencing revealed 84 significantly differentially expressed miRNAs between HL cell lines and normal germinal center B cells. Inhibition of the in HL significantly overexpressed miR-24-3p resulted in decreased growth which was at least in part caused by an increase in apoptotic cells. MiRNA target gene identification using Ago2-IP in HL cell lines revealed 1,142 miRNA target genes of which 52 were predicted to be targeted by miR-24-3p. Western blotting analysis confirmed increased CDKN1B/P27kip1 upon miR-24-3p inhibition, possibly explaining the effect on cell growth. We next set up a next generation sequencing based high throughput loss- and gain-of-function screening approach to identify miRNAs that influence HL cell growth. The overexpression screen revealed that miR-19b-1 may enhance while miR-141 may repress HL cell growth. The inhibition screen revealed that inhibition of miR-449a-5p, miR-625-5p, let-7f-2-3p and miR-21-5p has a negative effect on HL cell growth. The highly abundant miR-21-5p showed significantly higher expression levels in HL. We confirmed the negative effects of miR-21-5p inhibition on cell growth and observed a concomitant significant increase in apoptotic cells. Among the in HL Ago2-IP enriched target genes, we identified 36 predicted miR-21-5p targets. We confirmed targeting of BTG2 and PELI1 by miR-21-5p using reporter assays and Western blot. Overall, we set up the technology for functional miRNA studies in HL and identified two miRNAs and their target genes relevant for the pathogenesis of HL.

While RNA was long thought to act as a mere intermediary between DNA and protein, we now know RNA transcripts can have diverse cellular functions themselves. The major class of long noncoding (lncRNAs) thus represent a missing piece in the puzzle of human cell biology. A variety of transcriptional as well as posttranscriptional regulatory mechanisms have been described for lncRNAs. This thesis addresses the relevance of lncRNAs in normal B cell development and B cell malignancies. Specifically, we study lncRNA expression changes in normal B cells before, during and after the maturation process using naive, germinal center [GC] and memory B cells, respectively. Vast lncRNA expression changes are observed in the highly proliferative GC B cells, indicating significant lncRNA involvement in the B cell maturation process. Furthermore we identify multiple lncRNAs overexpressed in Hodgkin lymphoma cell lines compared to their cell-of-origin, which show cancer cell specific expression in primary patient tissue. We defined lncRNAs regulated by the oncogenic transcription factor Myc and putatively involved in its proliferation-supportive effects. We observe deregulation of hundreds of lncRNAs in response to Myc, thus identifying lncRNAs as a major component of the Myc transcriptional network. Myc-induced lncRNA KTN1-AS1 is studied in more detail and shown to affect Burkitt lymphoma cell growth by reinforcing high Myc expression. In summary, our studies provide novel insights into the role of lncRNAs in normal B cells and B cell malignancies. The individual lncRNAs we identified can potentially serve as biomarkers or therapeutic targets for lymphoma detection and treatment.

In this thesis we have focused on resistance mechanisms in patients treated with afatinib in presence of an EGFR mutation and for crizotinib in patients with an ALK break. Reviewing the literature about known resistance mechanism revealed an extra mutation in EGFR (V834I), MET and FGFR1 amplification, upregulation of IL6R/JAK1/STAT3, Src and autophagy, and changes of glycolysis in treatment with afatinib. Under crizotinib treatment ALK ‘gatekeeper’ mutations, upregulation of EGFR, mutations in KRAS, autophagy en epithelial-mesenchymal transition (EMT) were found as resistance mechanisms. These findings raised questions whether we could find those mechanisms in treated patients as well.
Using whole exome sequencing of biopsies obtained before and after afatinib we found resistance specific mutations in Wnt and PI3K-AKT pathways.
In crizotinib treated patients we performed whole exome sequencing and RNA sequencing to identify resistance associated mechanisms. This revealed treatment induced mutations in genes associated with pathways related to EMT.
The clinically most relevant finding in crizotinib treated patients, was that absence of ALK protein expression was strongly associated with absence of any response to crizotinib treatment. Therefore, we concluded that patients with a FISH based ALK break, without ALK protein expression, should not be treated with crizotinib. Response prediction based on presence of ALK protein expression out performed response based on FISH based ALK break. So, performing FISH-ALK does not add anything. We therefore should proceed ALK break testing with ALK-IHC only.