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Objectives An efficient pharmacological response to MTX treatment in RA patients relies on the retention and accumulation of intracellular MTX-polyglutamates catalysed by the enzyme folylpolyglutamate synthetase (FPGS). We recently identified a partial retention of FPGS intron 8 (8PR) as a prominent splice variant conferring FPGS dysfunction and decreased MTX polyglutamylation in acute lymphoblastic leukaemia. Here, we explored the association between FPGS 8PR levels and lack of MTX responsiveness in RA patients. Methods Thirty-six patients undergoing MTX treatment were enrolled from the Combinatie behandeling Reumatoide Artritis (COBRA)-light trial. RNA was isolated from blood samples at baseline, 13 weeks and 26 weeks of therapy, from patients in either COBRA-light (n = 21) or COBRA (n = 15) treatment arms. RT-qPCR analysis was used to assess RNA levels of FPGS 8PR over wild-type FPGS (8WT). Results In the COBRA-light treatment arm, higher baseline ratios of 8PR/8WT were significantly associated with higher 44-joint disease activity score (DAS44) at 13 and 26 weeks. Higher baseline ratios of 8PR/8WT also trended towards not obtaining low disease activity (DAS Conclusion This study is the first to associate alterations in FPGS pre-mRNA splicing levels with reduced responsiveness to MTX treatment in RA patients. Trial registration ISRCTN55552928.

Background Computational tools analyzing RNA-sequencing data have boosted alternative splicing research by identifying and assessing differentially spliced genes. However, common alternative splicing analysis tools differ substantially in their statistical analyses and general performance. This report compares the computational performance (CPU utilization and RAM usage) of three event-level splicing tools; rMATS, MISO, and SUPPA2. Additionally, concordance between tool outputs was investigated. Results Log-linear relations were found between job times and dataset size in all splicing tools and all virtual machine (VM) configurations. MISO had the highest job times for all analyses, irrespective of VM size, while MISO analyses also exceeded maximum CPU utilization on all VM sizes. rMATS and SUPPA2 load averages were relatively low in both size and replicate comparisons, not nearing maximum CPU utilization in the VM simulating the lowest computational power (D2 VM). RAM usage in rMATS and SUPPA2 did not exceed 20% of maximum RAM in both size and replicate comparisons while MISO reached maximum RAM usage in D2 VM analyses for input size. Correlation coefficients of differential splicing analyses showed high correlation (β > 80%) between different tool outputs with the exception of comparisons of retained intron (RI) events between rMATS/MISO and rMATS/SUPPA2 (β Conclusions Prior to RNA-seq analyses, users should consider job time, amount of replicates and splice event type of interest to determine the optimal alternative splicing tool. In general, rMATS is superior to both MISO and SUPPA2 in computational performance. Analysis outputs show high concordance between tools, with the exception of RI events.

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.

The antifolate methotrexate (MTX) is one of the pillars of acute lymphoblastic leukemia (ALL) treatment. However, the efficacy of MTX is frequently hampered by drug resistance, which can contribute to relapse. Although a multitude of molecular mechanisms underlie drug resistance in model cell lines, their clinical relevance often remains elusive. The activity of folylpolyglutamate synthetase (FPGS) enzyme is essential for intracellular retention and cytotoxic activity of MTX. Hence, alterations in the FPGS gene are a plausible contributor to impaired FPGS activity in MTX-resistant leukemia cells. Here we studied the association of FPGS splicing alterations with ex vivo MTX resistance as well as clinical response in 91 pediatric ALL patients. Our findings reveal that one FPGS splicing alteration- intron 8 partial retention (8 PR) exhibited a significant association with overall survival (HR=5.55, P=0.003) and event-free survival (HR=4.24, P=0.024) in a subset of patients displaying impaired accumulation of long-chain MTX polyglutamates. Moreover, high levels of intron 8 PR were indicative of resistance to several other chemotherapeutics including glucocorticoids, suggesting that the presence of intron 8 PR reflects a broader splicing defect resulting in multidrug resistance. These findings have important implications for personalized treatment and the circumvention of drug resistance in ALL patients.

Introduction: Therapeutic options for diffuse malignant peritoneal mesothelioma (DMPM) are limited to surgery and locoregional chemotherapy. Despite improvements in survival rates, patients eventually succumb to disease progression. We investigated splicing deregulation both as molecular prognostic factor and potential novel target in DMPM, while we tested modulators of SF3b complex for antitumor activity. Methods: Tissue-microarrays of 64 DMPM specimens were subjected to immunohistochemical assessment of SF3B1 expression and correlation to clinical outcome. Two primary cell cultures were used for gene expression profiling and in vitro screening of SF3b modulators. Drug-induced splicing alterations affecting downstream cellular pathways were detected through RNA sequencing. Ultimately, we established bioluminescent orthotopic mouse models to test the efficacy of splicing modulation in vivo. Results: Spliceosomal genes are differentially upregulated in DMPM cells compared to normal tissues and high expression of SF3B1 correlated with poor clinical outcome in univariate and multivariate analysis. SF3b modulators (Pladienolide-B, E7107, Meayamycin-B) showed potent cytotoxic activity in vitro with IC50 values in the low nanomolar range. Differential splicing analysis of Pladienolide-B-treated cells revealed abundant alterations of transcripts involved in cell cycle, apoptosis and other oncogenic pathways. This was validated by RT-PCR and functional assays. E7107 demonstrated remarkable in vivo antitumor efficacy, with significant improvement of survival rates compared to vehicle-treated controls. Conclusions: SF3B1 emerged as a novel potential prognostic factor in DMPM. Splicing modulators markedly impair cancer cell viability, resulting also in potent antitumor activity in vivo. Our data designate splicing as a promising therapeutic target in DMPM. Keywords: Mesothelioma, Therapy, SF3b modulation, Splicing, RNA-sequencing

Methotrexate (MTX), a folate antagonist which blocks de novo nucleotide biosynthesis and DNA replication, is an anchor drug in acute lymphoblastic leukemia (ALL) treatment. However, drug resistance is a primary hindrance to curative chemotherapy in leukemia and its molecular mechanisms remain poorly understood. We have recently shown that impaired folylpolyglutamate synthetase (FPGS) splicing possibly contributes to the loss of FPGS activity in MTX-resistant leukemia cell line models and adult leukemia patients. However, no information is available on the possible splicing alterations in FPGS in pediatric ALL. Here, using a comprehensive PCR-based screen we discovered and characterized a spectrum of FPGS splicing alterations including exon skipping and intron retention, all of which proved to frequently emerge in both pediatric and adult leukemia patient specimens. Furthermore, an FPGS activity assay revealed that these splicing alterations resulted in loss of FPGS function. Strikingly, pulse-exposure of leukemia cells to antifolates and other chemotherapeutics markedly enhanced the prevalence of several FPGS splicing alterations in antifolate-resistant cells, but not in their parental antifolate-sensitive counterparts. These novel findings suggest that an assortment of deleterious FPGS splicing alterations may constitute a mechanism of antifolate resistance in childhood ALL. Our findings have important implications for the rational overcoming of drug resistance in individual leukemia patients.

Aberrant pre-mRNA splicing in cancer is emerging as an important determinant of oncogenesis, response to treatment and anticancer drug resistance. At the same time, the spliceosome has become a target for a novel class of pre-clinical chemotherapeutics with a potential future application in cancer treatment. Taken together, these findings offer novel opportunities for the enhancement of the efficacy of cancer therapy.This review presents a comprehensive overview of the molecular mechanisms involved in splicing and current developments regarding splicing aberrations in relation to several aspects of cancer formation and therapy. Identified mutations in the various components of the spliceosome and their implications for cancer prognosis are delineated. Moreover, the contribution of abnormal splicing patterns as well as deregulated splicing factors to chemoresistance is discussed, along with novel splicing-based therapeutic approaches.Significant progress has been made in deciphering the role of splicing factors in cancer including carcinogenesis and drug resistance. Splicing-based prognostic tools as well as therapeutic options hold great potential towards improvements in cancer therapy. However, gaining more in-depth molecular insight into the consequences of mutations in various components of the splicing machinery as well as of cellular effects of spliceosome inhibition is a prerequisite to establish the role of splicing in tumor progression and treatment options, respectively.

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 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Background: Diffuse malignant peritoneal mesothelioma (DMPM) is an aggressive tumor of the lining of the abdomen, characterized by late clinical symptoms and poor prognosis. Systemic chemotherapy together with cytoreductive surgery and intraperitoneal hyperthermic therapy has been introduced as the best treatment option, resulting in an overall 5-year survival rate of approximately 50%. To further increase survival, novel drugs targeting key molecular factors in DMPM are warranted. Analogous to pleural mesothelioma, such a factor may be alternative splicing which can be modulated by inhibiting the SF3B subunit of the spliceosome. This strategy is an emerging therapeutic opportunity for a number of solid tumors and hematological malignancies. In particular, the spliceosome inhibitor Pladienolide B (PB) has low nanomolar IC50 values against a range of cancer cell lines and leukemic cells, but no data are available regarding its antitumor efficacy in DMPM. Aims: This study investigates (1) the activity of PB (alone or in combination with standard chemotherapeutics) in primary peritoneal mesothelioma cells through in vitro and in vivo assays, and (2) the molecular mechanisms of splicing inhibition through whole-genome RNA-seq and PCR of selected genes involved in apoptosis and invasion. Methods: The antiproliferative effect of PB was investigated using the SRB assay on two primary mesothelioma cell cultures (MESOII and STO), obtained from resected tumors with well-annotated clinical characteristics. Further in vitro studies were performed to evaluate the pro-apoptotic and anti-invasive activities, while splicing profiles of treated and untreated cells were determined with RNA-seq. Results: PB impaired DMPM cell growth in a dose-dependent manner, with IC50 values of 1.57 ± 0.30 nM in MESOII and 1.18 ± 0.16 nM in STO (n = 3, mean ± standard deviation). The specific activity on the spliceosome was demonstrated by PB induced time- and dose-dependent alterations of splicing patterns for several apoptotic genes, such as Mcl-1, Bcl-X, Fas, and for the pro-metastatic tyrosine kinase receptor RON, which was shifted to its un-spliced and non-functional variant. In addition, RNA-seq showed several differentially expressed alternatively spliced genes in PB treated samples. The DMPM cells have also been genetically engineered to express Firefly- and Gaussia- luciferases, enabling monitoring of tumor growth inhibition by PB in in vivo orthotopic models. Conclusions: These data provide evidence that PB has a strong antitumor activity against relevant models of DPMP, associated with modulation of splicing, induction of apoptosis and inhibition of invasion. RNA-seq represents a powerful tool for the identification of alternatively spliced genes that could serve as useful diagnostic markers as well as potential therapeutic targets for DMPM. Citation Format: Rocco Sciarrillo, Valentina E. Gomez, Marzia Pennati, Anna Wojtuszkiewicz, Gert-Jan L. Kaspers, Carla Molthoff, Nadia Zaffaroni, Godefridus J. Peters, Gerrit Jansen, Elisa Giovannetti. Spliceosome inhibition as novel strategy against diffuse malignant peritoneal mesothelioma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 332.

Methotrexate (MTX) is one of the key components of current ALL treatment protocols. This antifolate inhibits de novo nucleotide biosynthesis, hindering DNA replication, hence leading to cell death. However, despite the overall good cure rates in ALL, drug resistance continues to hamper the efficacy of MTX, contributing to relapse of leukemia. Multiple molecular mechanisms underlying drug resistance have been characterized, however, their clinical relevance often remains unclear. We recently identified impaired FPGS splicing, in particular intron 8 partial retention (intron 8 PR), as a possible contributor to decreased FPGS activity in both MTX-resistant leukemia cell lines and primary ALL specimens. In the current study we explored the association between FPGS splicing alterations and MTX resistance as well as the clinical outcome of 91 pediatric ALL patients. With respect to MTX resistance, we determined the levels of MTX polyglutamates in leukemic cells, as well as FPGS activity along with the expression of several enzymes and transporters involved in (anti)folate metabolism and transport, including FPGS, FPGH, RFC, DHFR and TS. The sensitivity of leukemic cells to MTX was determined using the thymidylate synthase inhibition assay (TSIA). Moreover, the sensitivity to other chemotherapeutics as measured in the MTT assay was determined for these patient specimens. The levels of FPGS splicing alterations were semi-quantified using PCR and fragment analysis. We found that intron 8 PR plays an important role in MTX resistance in the studied patient cohort. In a subset of patients with relatively low levels of MTX polyglutamates, high levels of intron 8 PR were associated with inferior EFS (HR = 4.2, P = 0.006) and OS (HR = 5.6, P = 0.003). The relation between intron 8 PR and OS remained significant in multivariate analysis, which included known prognostic factors, including WBC and age. Interestingly, in this subgroup of patients, intron 8 PR correlated with higher MTX resistance as determined by the TSIA assay (p = 0.015) and lower accumulation of long chain MTX polyglutamates (p = 0.027). Moreover, high levels of intron 8 PR were associated with resistance to the glucocorticoids dexamethasone and prednisone. Remarkably, we found that a MTX-resistant FPGS-deficient subline of CCRF-CEM - CEM/FPGS-, harbors a high level of intron 8 PR and displays high level of dexamethasone cross-resistance (over 350-fold). Our findings suggest that intron 8 PR is not only relevant for MTX resistance but may also reflect a broader splicing defect affecting multiple genes, thereby resulting in multidrug resistance. These findings may have important implications for personalized treatment as well as circumvention of drug resistance in ALL patients. Financial support by Kika (Children cancer-free) Citation Format: Anna Wojtuszkiewicz, Yehuda G. Assaraf, Mirthe Hoekstra, Gerrit Jansen, Godefridus J. Peters, Edwin Sonneveld, Gertjan L. Kaspers, Jacqueline Cloos. The relevance of aberrant FPGS splicing for ex vivo MTX resistance and clinical outcome in childhood acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4437. doi:10.1158/1538-7445.AM2015-4437

Introduction The process of pre-mRNA splicing is gaining attention as a contributor to anticancer drug resistance and as a promising novel therapeutic target. Splicing of many genes involved in regulation of apoptosis was found to be altered in tumor cells leading to chemoresistance. Similarly, aberrant splicing of genes engaged in drug metabolism was reported to mediate resistance to several anchor drugs of chemotherapeutic protocols in the treatment of leukemia including daunorubicin, cytarabine and methotrexate. Therefore, targeting the spliceosome holds potential to directly activate apoptosis by inducing pro-apoptotic splicing profiles as well as to sensitize cells displaying drug resistance related to altered splicing of genes involved in drug metabolism. In this respect, meayamycin B (MAMB) is a novel compound, which potently inhibits the SF3B1 subunit, which is one of the core components of the spliceosome complex. MAMB was previously shown to induce shifts in splicing of one of the essential apoptosis regulators Mcl-1 in non-small cell lung cancer cell lines A549 and H1299, hence promoting expression of its pro-apoptotic isoform Mcl-1S. The resulting dominance of Mcl-1S was able to sensitize these tumor cells to Bcl-XL inhibitor leading to induction of cell death. Here we evaluated the in vitro impact of MAMB as a single drug in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Methods To achieve this goal, we first assessed the impact of MAMB in short-term exposure on splicing of selected apoptosis-related genes, in particular Mcl-1, and concomitant induction of apoptosis in 3 human ALL and 3 AML cell lines. In addition, MAMB sensitivity was assessed using a 72h MTT assay in a panel of ALL and AML cell lines including sublines displaying resistance to several conventional chemotherapeutics including methotrexate, bortezomib or imatinib. Finally, we assessed MAMB sensitivity in primary ALL and AML samples, as compared to healthy bone marrow specimens. Results As previously shown in solid tumors, MAMB (0.5-1nM) was able to shift splicing of Mcl-1 but not Bcl-X in leukemic cell lines upon 24h exposure. The observed changes in splicing coincided with enhanced apoptosis as determined by flow cytometry. The fraction of apoptotic cells reached approximately 40% in the parental CCRF-CEM cell line (T-ALL) and 10% in the methotrexate-resistant subline which has lost folylpolyglutamate synthetase (FPGS) activity. Intriguingly, we previously found impaired splicing of FPGS in this methotrexate-resistant cell line as compared to intact splicing in parental cells. It should be emphasized that intact FPGS splicing is a key component of intracellular retention and activity of MTX. Induction of apoptosis in AML cell lines ranged between 17% in OCI-AML3 and 53% in KG1a. When MAMB sensitivity was assessed in the 72h MTT assay, the growth of both ALL and AML cell lines was efficiently inhibited with remarkable IC50 values varying between 0.07 and 0.16 nM. Intriguingly, even tumor cell lines which are resistant to conventional chemotherapeutics with different mechanisms of action, such as methotrexate, imatinib and bortezomib, were highly sensitive to MAMB. In line with our results with leukemic cell lines, both ALL and AML primary samples showed a remarkable sensitivity to MAMB (mean LC50 value 0.42 and 0.43 nM, respectively, Figure 1). The normal bone marrow cells obtained from healthy children showed a slightly higher resistance (mean LC50 value 0.57, p=0.03, Figure). Conclusions Our results show that MAMB itself may constitute a therapeutic option for patients displaying drug resistance to conventional chemotherapy, especially in AML, which in general tends to be more resistant to current treatment options compared to ALL. In addition, as a splicing modulating agent it may also be used to reverse aberrant splicing profiles of genes involved in drug metabolism, thereby restoring sensitivity to standard chemotherapeutics. This novel paradigm warrants further exploration using drug combination studies. Further investigation in a larger cohort of leukemia patient samples is warranted. Moreover, mice studies will be performed to reveal possible toxicities and in vivo efficacy. Figure 1 MAMB sensitivity in primary pediatric ALL and AML samples. Figure 1. MAMB sensitivity in primary pediatric ALL and AML samples. Disclosures No relevant conflicts of interest to declare.

The antifolate methotrexate (MTX) is used in acute lymphoblastic leukemia (ALL) treatment. Cellular retention and consequent efficacy of MTX is enhanced by polyglutamylation, a reaction catalyzed by folylpolyglutamate synthetase (FPGS). It has been shown that loss of FPGS activity can confer antifolate resistance. However, the mechanism underlying this phenomenon remains elusive. We have recently shown that alterations in FPGS mRNA splicing may be a plausible determinant of MTX resistance but a comprehensive screen of the spectrum of splicing aberrations throughout the entire FPGS gene is lacking. The aim of the current study was to evaluate the pattern of FPGS mRNA splicing alterations as a predictor of MTX resistance and treatment failure in ALL. A PCR-based assay was devised that allowed for a screen of the entire FPGS mRNA sequence for splicing alterations. This method was subsequently used to screen antifolate-resistant human leukemia cell lines and ALL patient samples. Our results identified multiple FPGS mRNA splicing alterations (Table 1) in MTX resistant cell lines with marked loss of FPGS activity. Moreover, the level of partial retention of intron 8 was markedly increased upon a 24 hour exposure to MTX in the antifolate-resistant leukemia cell lines. Moreover, most of the detected FPGS splicing alterations either introduced a frame-shift or a premature stop codon, hence resulting in either transcripts targeted for the nonsense-mediated decay or truncated proteins devoid of FPGS activity. These in vitro FPGS splicing alterations where corroborated in vivo as aberrations detected in leukemic cell lines were also identified in a panel of 30 ALL patient specimens at diagnosis. In summary, impaired FPGS mRNA splicing could be responsible for loss of FPGS activity and might offer an opportunity for the design of reliable molecular tools allowing for identification of antifolate resistance in leukemia cells. Table 1. FPGS mRNA splicing alterations in cell lines and ALL patient samples. Alteration Frame shift (yes/no) Early stop codon position (nucleotide) Frequency in primary ALL samples (%) Exon 2 skipping − − 71 Exon 5 skipping + 477 50 Exon 6 skipping − − 93 Exon 12 skipping + 1173 57 Exon 14 skipping + 1356 50 Intron 5 retention + 792 43 Intron 6 retention + 660 35 Intron 5 + Intron 6 retention − 879 50 Intron 8 partial retention − 870 93 Intron 6 retention + Intron 8 partial retention + 660 50 Citation Format: Anna Wojtuszkiewicz, Yehuda Assaraf, Shachar Raz, Michal Stark, Edwin Sonneveld, Godefridus Peters, Gerrit Jansen, Gertjan Kaspers, Jacqueline Cloos. Alterations in FPGS splicing as a plausible underlying mechanism of MTX resistance in ALL. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4628. doi:10.1158/1538-7445.AM2013-4628