Exploiting Vulnerabilities in the RNA Splicing Machinery: Novel Therapeutic Targets in Lung Cancer

In this thesis, I propose novel therapeutic targets for the treatment of lung cancer. Although the prognosis for this disease improved slightly over the past years, it is still the most lethal form of cancer worldwide with poor survival rate. Reinstatement of p53 activity might be a therapeutic strategy in treating this disease. Therefore in chapter 2, we screened functional proteins in lung cancer cells for putative p53 inhibitors. Amongst other proteins, members of the RNA splicing machinery were enriched in the candidate list of p53 inhibitors. Surprisingly, silencing several of these RNA splice factors induced cell death only in lung cancer cells, whereas healthy cells remained viable. This might serve as a therapeutic opportunity in which side effects are mitigated. In chapter 3, we describe the structure and function of the spliceosome and its implications in lung cancer. Over the recent years, it has become apparent that RNA splicing is often deregulated in multiple forms of cancer, due to the mutation or up- or downregulation of the expression of splice factors. In the context of what was already known from literature, our finding in chapter 2 that silencing specific RNA splice factors induces lung cancerselective cell death, we were interested in further investigating the spliceosome as therapeutic target. We next screened all 364 splice factors in a panel of cancer cell lines compared to healthy cells to identify the most potent therapeutic targets (chapter 4). From this, we found that the so-called Sm proteins are of particular interest. The Sm proteins were completely enriched in our cancer selective lethality screen hit list. To investigate the potential mechanism behind the selective lethality, we analyzed the transcriptome of lung cancer cells upon silencing of one of the Sm proteins (SNRPD3) compared to a splice factor that we found to be essential for both lung cancer and nonmalignant cells (Splicing Factor 3b Subunit 1; SF3B1) in chapter 5. From this analysis, we found an alternative splicing event in a proteasomal protein (Proteasome 20S Subunit Beta 3; PSMB3) unique to silencing SNRPD3. This alternative splicing event resulted in a mRNA variant of PSMB3 that is predicted to be targeted for nonsensemediated decay, and therefore a decrease in PSMB3 protein expression. Moreover, this splicing switch was associated with decreased proteasomal function and was more pronounced in lung cancer cells compared to healthy cells. It was already established by others that proteasome inhibition is toxic to cells. Therefore, the observed cytotoxic splicing switch in PSMB3 in lung cancer cells upon silencing SNRPD3 might explain the associated cancer-selective lethality. From our finding in this thesis, we propose that targeting Sm proteins is of therapeutic value in the treatment of lung cancer. To date, there is no small molecule inhibitor that targets these proteins. Therefore, in chapter 6, we set out to perform virtual screening to identify compounds that might inhibit Sm proteins. Although we were not able to identify compounds that were eligible for further analysis of their biological activity, we gained insight in Sm-Sm PPIs and protein residues that are important in establishing this interaction. The in silico screening can be further optimized focusing on these residues to find promising compounds capable of targeting the Sm protein ring. As it might be expected that the process of RNA splicing is vital to both lung cancer cells and healthy cells, it was surprising that we identified core spliceosome components such as the Sm proteins as therapeutic targets in lung cancer. Although we identified an interesting cytotoxic splicing event, it is of interest to further elucidate the mechanism through which silencing the Sm proteins elicits cancer-selective lethality.