Exploring alternative splicing in cancer: from insights in drug resistance to new therapies

Genomic characterization of cancer subtypes became a major research goal worldwide after the release of the complete human genome sequence in 2001. International consortia (e.g. The Cancer Genome Atlas - TCGA) have profiled and analyzed large numbers of human tumors matched to non-malignant tissues and created databases of molecular aberrations at the DNA, RNA, protein and epigenetic level. Research efforts aimed at understanding the molecular determinants of cancer prompted the rapid development of molecularly targeted chemotherapeutics which significantly improved clinical outcomes. Unfortunately, most cancer patients experience disease relapse manifested as recurrence of residual malignant cells which apparently did not respond to treatment regimes. Such cells either already carried or acquired genetic alterations (e.g. mutation, gene amplification, gene deletion or chromosomal translocation) that provide a clonal advantage to survive under selective therapeutic pressure. DNA aberrations have largely been documented to activate and drive oncogenic transformation as well as modulate response to therapy. Recent evidence highlighted the connection between alternative pre-mRNA splicing, cancer development and response to therapy. Alternative splicing is the essential process in eukaryotic gene expression by which non-coding intron sequences are removed from the pre-mRNA transcripts and specific exons are included or excluded from mature mRNAs. The aim of this thesis is to unravel the role of altered splicing in drug-resistant pediatric leukemia and evaluate the efficacy of small molecule splicing modulators in rare and aggressive tumors which pose major therapeutic challenges in the adult population. Chapter 2 is an extensive overview of the latest findings in the field of alternative splicing regulation in cancer. We describe the molecular mechanisms by which cancer cells harness alternative splicing to drive oncogenesis and evade anticancer drug treatment. Moreover, we discuss current challenges in the domain of bioinformatic analyses arising from genome-wide detection of aberrant splicing, which form the basis for the research presented in chapters 4 and 5. Finally, we identify splicing-based vulnerabilities that can provide novel treatment opportunities, as illustrated in chapters 6, 7 and 8. In Chapter 3 we studied the association of splicing alterations affecting the gene folylpolyglutamate synthetase (FPGS) with ex vivo methotrexate (MTX) resistance as well as clinical response in pediatric acute lymphoblastic leukemia (ALL) patients. In Chapter 4 we present an experimental protocol for genome-wide detection and validation of alternative splicing associated with in vitro drug resistance in solid tumors and hematological malignancies. We analyzed the transcriptomic profiles of several drug resistant in vitro models through RNA-seq and bioinformatic tools and established a qRT-PCR based method to validate the detected splice variants of candidate genes. This method is applied in chapter 5 to ex vivo leukemic specimens. In Chapter 5 we characterize whole-genome splicing profiles associated with glucocorticoid (GC) resistance in pediatric ALL samples by using RNA-seq-based differential splicing analysis, as established in chapter 3. Our analyses revealed markedly distinct splicing landscapes in ALL samples of B-cell precursor and T-cell lineages associated with GC-resistant phenotype. To determine whether splicing modulation could serve as a novel therapeutic option for GC-resistant patients, we tested the efficacy of splicing modulator Pladienolide-B in GC-resistant ALL cell lines and primary leukemic specimens as single agent or in combination with GCs. Chapter 6 and 7 illustrate the rationale of targeting spliceosome in malignant peritoneal mesothelioma and pancreatic ductal adenocarcinoma. Here we tested the antitumor effects of splicing modulators in relevant in vitro and in vivo disease models. Finally, Chapter 8 provides a novel splicing-based strategy to specifically target non-small cell lung cancer by silencing core spliceosomal Sm proteins as an alternative to SF3b modulation. In Chapter 9 we discuss the relevance of the findings presented in this thesis