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

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

The role of alternative splicing in cancer: From oncogenesis to drug resistance

Alternative splicing is a tightly regulated process whereby non-coding sequences of pre-mRNA are removed and protein-coding segments are assembled in diverse combinations, ultimately giving rise to proteins with distinct or even opposing functions. In the past decade, whole genome/transcriptome sequencing studies revealed the high complexity of splicing regulation, which occurs co-transcriptionally and is influenced by chromatin status and mRNA modifications. Consequently, splicing profiles of both healthy and malignant cells display high diversity and alternative splicing was shown to be widely deregulated in multiple cancer types. In particular, mutations in pre-mRNA regulatory sequences, splicing regulators and chromatin modifiers, as well as differential expression of splicing factors are important contributors to cancer pathogenesis. It has become clear that these aberrations contribute to many facets of cancer, including oncogenic transformation, cancer progression, response to anticancer drug treatment as well as resistance to therapy. In this respect, alternative splicing was shown to perturb the expression a broad spectrum of relevant genes involved in drug uptake/metabolism (i.e. SLC29A1, dCK, FPGS, and TP), activation of nuclear receptor pathways (i.e. GR, AR), regulation of apoptosis (i.e. MCL1, BCL-X, and FAS) and modulation of response to immunotherapy (CD19). Furthermore, aberrant splicing constitutes an important source of novel cancer biomarkers and the spliceosome machinery represents an attractive target for a novel and rapidly expanding class of therapeutic agents. Small molecule inhibitors targeting SF3B1 or splice factor kinases were highly cytotoxic against a wide range of cancer models, including drug-resistant cells. Importantly, these effects are enhanced in specific cancer subsets, such as splicing factor-mutated and c-MYC-driven tumors. Furthermore, pre-clinical studies report synergistic effects of spliceosome modulators in combination with conventional antitumor agents. These strategies based on the use of low dose splicing modulators could shift the therapeutic window towards decreased toxicity in healthy tissues. Here we provide an extensive overview of the latest findings in the field of regulation of splicing in cancer, including molecular mechanisms by which cancer cells harness alternative splicing to drive oncogenesis and evade anticancer drug treatment as well as splicing-based vulnerabilities that can provide novel treatment opportunities. Furthermore, we discuss current challenges arising from genome-wide detection and prediction methods of aberrant splicing, as well as unravelling functional relevance of the plethora of cancer-related splicing alterations

Silencing core spliceosome sm gene expression induces a cytotoxic splicing switch in the proteasome subunit beta 3 mRNA in non-small cell lung cancer cells

The core spliceosomal Sm proteins were recently proposed as cancer-selective lethal targets in non-small cell lung cancer (NSCLC). In contrast, the loss of the commonly mutated cancer target SF3B1 appeared to be toxic to non-malignant cells as well. In the current study, the transcriptomes of A549 NSCLC cells, in which SF3B1 or SNRPD3 was silenced, were compared using RNA sequencing. The skipping of exon 4 of the proteasomal subunit beta type-3 (PSMB3) mRNA, resulting in a shorter PSMB3-S variant, occurred only after silencing SNRPD3. This bservation was extended to the other six Sm genes. Remarkably, the alternative splicing of PSMB3 mRNA upon Sm gene silencing was not observed in non-malignant IMR-90 lung fibroblasts. Furthermore, PSMB3 was found to be overexpressed in NSCLC clinical samples and PSMB3 expression correlated with Sm gene expression. Moreover, a high PSMB3 expression corresponds to worse survival in patients with lung adenocarcinomas. Finally, silencing the canonical full-length PSMB3-L, but not the shorter PSMB3-S variant, was cytotoxic and was accompanied by a decrease in proteasomal activity. Together, silencing Sm genes, but not SF3B1, causes a cytotoxic alternative splicing switch in the PSMB3 mRNA in NSCLC cells only

Using RNA-sequencing to detect novel splice variants related to drug resistance in in vitro cancer models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabineresistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure

Abstract 3082: Targeting hypoxic pancreatic cancer cells with glucose conjugated lactate dehydrogenase inhibitor NHI-Glc-2

Pancreatic ductal adenocarcinoma (PDAC) is an abysmal disease with a 5-year survival rate of merely 8%. The tumor microenvironment of PDAC is one of the factors contributing to drug resistance. More specifically, the hypoxic tumor core and the metabolic switch to aerobic glycolysis (the Warburg effect), contribute to the lack of drug response. Therefore, we investigated the effect of several novel lactate dehydrogenase (LDH-A) inhibitors (N-Hydroxyindole-based LDH-A inhibitors, NHI-1 and NHI-2, and the glucose conjugate NHI-Glc-2) in PDAC cells in vitro and in vivo, in combination with the standard drug gemcitabine. For this purpose we used our primary PDAC cancer cell cultures, tested growth inhibition with the SRB chemosensitivity assay, used 3D cultures and established an in vivo orthotopic bioluminescent model. Additionally, LDH-A enzyme activity inhibition by NHI-Glc-2 was assessed by spectrophotometry. LDH-A is overexpressed in PDAC and its expression is correlated with the prognosis of metastatic PDAC. The glucose transporter 1 (GLUT-1) is also overexpressed in PDAC, which would enable an increased uptake of NHI-Glc-2 by the tumor cells. LDH-A mRNA expression and enzyme activity were about 2-fold higher under hypoxic conditions. NHI-1, NHI-2 and NHI-Glc-2 were 4-15-fold more effective under hypoxic conditions compared to normoxia, but gemcitabine was 10-20-fold less active under hypoxia. NHI-1 showed a synergistic effect with gemcitabine in hypoxic PANC-1 and LPC006 cells (combination index 0.14 ± 0.06 and 0.29 ± 0.53, respectively). NHI-Glc-2 inhibited PDAC cell growth in micromolar range under hypoxic conditions and also showed a synergistic effect with gemcitabine. In a 3D spheroid culture (with a hypoxic core), NHI-Glc-2 disrupted the spheroid integrity. Moreover, in an orthotopic PDAC model NHI-Glc-2 showed a more pronounced inhibition (almost complete) of tumor growth compared to gemcitabine. NHI-Glc-2 also showed a favorable pharmacokinetics with a peak plasma concentration of 26 µM at 4 hr, which is higher than the IC50. In conclusion, LDH-A is a viable target in PDAC, and novel LDH-A inhibitors offer an innovative therapeutic tool. Remarkably, the LDH-A inhibitors NHI-1 and NHI-2 increased the effect of gemcitabine under hypoxic conditions, while the glucose conjugated NHI-Glc-2 showed an improved uptake possibly because of the increased GLUT-1 expression, leading to a pronounced in vivo effect

NF-κB drives acquired resistance to a novel mutant-selective EGFR inhibitor

The clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) harbouring activating EGFR mutations is limited by the emergence of acquired resistance, mostly ascribed to the secondary EGFR-T790M mutation. Selective EGFR-T790M inhibitors have been proposed as a new, extremely relevant therapeutic approach. Here, we demonstrate that the novel irreversible EGFR-TKI CNX-2006, a structural analog of CO-1686, currently tested in a phase-1/2 trial, is active against in vitro and in vivo NSCLC models expressing mutant EGFR, with minimal effect on the wild-type receptor. By integration of genetic and functional analyses in isogenic cell pairs we provide evidence of the crucial role played by NF-κB1 in driving CNX-2006 acquired resistance and show that NF-κB activation may replace the oncogenic EGFR signaling in NSCLC when effective and persistent inhibition of the target is achieved in the presence of the T790M mutation. In this context, we demonstrate that the sole, either genetic or pharmacologic, inhibition of NF-κB is sufficient to reduce the viability of cells that adapted to EGFR-TKIs. Overall, our findings support the rational inhibition of members of the NF-κB pathway as a promising therapeutic option for patients who progress after treatment with novel mutant-selective EGFR-TKIs

Enhanced efficacy of AKT and FAK kinase combined inhibition in squamous cell lung carcinomas with stable reduction in PTEN

Squamous cell lung carcinoma (SCC) accounts for 30% of patients with NSCLC and to date, no molecular targeted agents are approved for this type of tumor. However, recent studies have revealed several oncogenic mutations in SCC patients, including an alteration of the PI3K/AKT pathway, i.e. PI3K point mutations and amplification, AKT mutations and loss or reduced PTEN expression. Prompted by our observation of a correlation between PTEN loss and FAK phosphorylation in a cohort of patients with stage IV SCC, we evaluated the relevance of PTEN loss in cancer progression as well as the efficacy of a new combined treatment with the pan PI3K inhibitor buparlisip and the FAK inhibitor defactinib. An increase in AKT and FAK phosphorylation, associated with increased proliferation and invasiveness, paralleled by the acquisition of mesenchymal markers, and overexpression of the oncomir miR-21 were observed in SKMES-1-derived cell clones with a stable reduction of PTEN. Notably, the combined treatment induced a synergistic inhibition of cell proliferation, and a significant reduction in cell migration and invasion only in cells with reduced PTEN. The molecular mechanisms underlying these findings were unraveled using a specific RTK array that showed a reduction in phosphorylation of key kinases such as JNK, GSK-3 α/β, and AMPK-α2, due to the concomitant decrease in AKT and FAK activation. In conclusion, the combination of buparlisib and defactinib was effective against cells with reduced PTEN and warrants further studies as a novel therapeutic strategy for stage IV SCC patients with loss of PTEN expression