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Splicing factor (SF) mutations are important contributors to the pathogenesis of hematological malignancies; however, their relevance in risk classification of acute myeloid leukemia (AML) warrants further investigation. To gain more insight into the characteristics of patients with AML carrying SF mutations, we studied their association with clinical features, cytogenetic and molecular abnormalities, and clinical outcome in a large cohort of 1447 patients with AML and high-risk myelodysplastic syndrome. SF mutations were identified in 22% of patients and were associated with multiple unfavorable clinical features, such as older age, antecedent myeloid disorders, and adverse risk factors (mutations in RUNX1 and ASXL1). Furthermore, they had significantly shorter event-free and overall survival. Notably, in European LeukemiaNet (ELN) 2017 favorable- and intermediate-risk groups, SF3B1 mutations were indicative of relatively poor prognosis. In addition, patients carrying concomitant SF mutations and RUNX1 mutations had a particularly adverse prognosis. In patients without any of the 4 most common SF mutations, RUNX1 mutations were associated with relatively good outcome, which was comparable to that of intermediate-risk patients. In this study, we propose that SF mutations be considered for incorporation into prognostic classification systems. First, SF3B1 mutations could be considered an intermediate prognostic factor when co-occurring with favorable risk features and as an adverse prognostic factor for patients currently categorized as having intermediate risk, according to the ELN 2017 classification. Second, the prognostic value of the current adverse factor RUNX1 mutations seems to be limited to its co-occurrence with SF mutations.

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.

Simple Summary In hematological malignancies, genome-wide sequencing studies found the process of splicing to be surprisingly frequently disrupted. While recent studies characterized altered splicing in relation to splicing factor mutations in AML, this study explored differential splicing profiles associated with two most common aberrations in AML: FLT3-ITD and NPM1 mutations. We identified the differential splicing of FAB-type specific gene sets in FLT3-ITD+/NPM1+ specimens as compared to FLT3-ITD−/NPM1− samples. The primary functions perturbed by differential splicing in all three FAB types included cell cycle control and DNA damage response. Interestingly, differential expression mainly affected genes involved in hematopoietic differentiation. Our findings increase our understanding of how genetic mutations translate to phenotypic features of AML cells to further improve response predictions and to find innovative therapeutic approaches. Altogether, to the best of our knowledge, this is the first study to report differential splicing profiles associated with FLT3-ITD with a concomitant NPM1 mutation in AML. Abstract Despite substantial progress achieved in unraveling the genetics of AML in the past decade, its treatment outcome has not substantially improved. Therefore, it is important to better understand how genetic mutations translate to phenotypic features of AML cells to further improve response predictions and to find innovative therapeutic approaches. In this respect, aberrant splicing is a crucial contributor to the pathogenesis of hematological malignancies. Thus far, altered splicing is well characterized in relation to splicing factor mutations in AML. However, splicing profiles associated with mutations in other genes remain largely unexplored. In this study, we explored differential splicing profiles associated with two of the most common aberrations in AML: FLT3-ITD and NPM1 mutations. Using RNA-sequencing data of a total of 382 primary AML samples, we found that the co-occurrence of FLT3-ITD and mutated NPM1 is associated with differential splicing of FAB-type specific gene sets. Despite the FAB-type specificity of particular gene sets, the primary functions perturbed by differential splicing in all three FAB types include cell cycle control and DNA damage response. Interestingly, we observed functional divergence between alternatively spliced and differentially expressed genes in FLT3-ITD+/NPM1+ samples in all analyzed FAB types, with differential expression affecting genes involved in hematopoietic differentiation. Altogether, these observations indicate that concomitant FLT3-ITD and mutated NPM1 are associated with the maturation state-specific differential splicing of genes with potential oncogenic relevance.

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.

To answer the increasing need for detecting and validating protein biomarkers in clinical specimens, proteomic techniques are required that support the fast, reproducible and quantitative analysis of large clinical sample cohorts. Targeted mass spectrometry techniques, specifically SRM, PRM and the massively parallel SWATH/DIA technique have emerged as a powerful method for biomarker research. For optimal performance, they require prior knowledge about the fragment ion spectra of targeted peptides. In this report, we describe a mass spectrometric (MS) pipeline and spectral resource to support data-independent acquisition (DIA) and parallel reaction monitoring (PRM) based biomarker studies. To build the spectral resource we integrated common open-source MS computational tools to assemble an open source computational workflow based on Docker. It was then applied to generate a comprehensive DIA pan-human library (DPHL) from 1,096 data dependent acquisition (DDA) MS raw files, and it comprises 242,476 unique peptide sequences from 14,782 protein groups and 10,943 SwissProt-annotated proteins expressed in 16 types of cancer samples. In particular, tissue specimens from patients with prostate cancer, cervical cancer, colorectal cancer, hepatocellular carcinoma, gastric cancer, lung adenocarcinoma, squamous cell lung carcinoma, diseased thyroid, glioblastoma multiforme, sarcoma and diffuse large B-cell lymphoma (DLBCL), as well as plasma samples from a range of hematologic malignancies were collected from multiple clinics in China, the Netherlands and Singapore and included in the resource. This extensive spectral resource was then applied to a prostate cancer cohort of 17 patients, consisting of 8 patients with prostate cancer (PCa) and 9 with benign prostate hyperplasia (BPH), respectively. Data analysis of DIA data from these samples identified differential expressions of FASN, TPP1 and SPON2 in prostate tumors. Thereafter, PRM validation was applied to a larger PCa cohort of 57 patients and the differential expressions of FASN, TPP1 and SPON2 in prostate tumors were validated. As a second application, the DPHL spectral resource was applied to a patient cohort consisting of samples from 19 DLBCL patients and 18 healthy individuals. Differential expressions of CRP, CD44 and SAA1 between DLBCL cases and healthy controls were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supported that DIA-PRM MS pipeline enables robust protein biomarker discoveries.

Despite carefully proofreading our submission, we missed a mistake in the affiliation of Wolfgang Kern, Claudia Haferlach and Torsten Haferlach. Instead of (2) H3 Biomedicine, their affiliation should be (3) MLL Munich Leukemia Laboratory, Munich, Germany.

Expression of apoptosis-regulating proteins (B-cell CLL/lymphoma 2 - BCL-2, Myeloid Cell Leukemia 1 - MCL-1, BCL-2 like 1 - BCL-X and BCL-2-associated X protein - BAX) in acute myeloid leukemia (AML) blasts at diagnosis is associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. Herein, we further explored this aspect of dynamic apoptosis regulation in AML. First, we showed that the intraindividual ex vivo apoptosis-related profiles of normal lymphocytes and AML blasts within the bone marrow of AML patients were highly correlated. The expression values of apoptosis-regulating proteins were far beyond healthy control lymphocytes, which implicates the influence of microenvironmental factors. Second, we demonstrated that apoptosis-resistant primary AML blasts, as opposed to apoptosis-sensitive cells, were able to up-regulate BCL-2 expression in sensitive AML blasts in contact cultures (p = 0.0067 and p = 1.0, respectively). Using secretome proteomics, we identified novel proteins possibly engaged in apoptosis regulation. Intriguingly, this analysis revealed that major functional protein clusters engaged in global gene regulation, including mRNA splicing, protein translation, and chromatin remodeling, were more abundant (p = 4.01E-06) in secretomes of apoptosis-resistant AML. These findings were confirmed by subsequent extracellular vesicle proteomics. Finally, confocal-microscopy-based colocalization studies show that splicing factors-containing vesicles secreted by high AAI cells are taken up by low AAI cells. The current results constitute the first comprehensive analysis of proteins released by apoptosis-resistant and sensitive primary AML cells. Together, the data point to vesicle-mediated release of global gene regulatory protein clusters as a plausible novel mechanism of induction of apoptosis resistance. Deciphering the modes of communication between apoptosis-resistant blasts may in perspective lead to the discovery of prognostic tools and development of novel therapeutic interventions, aimed at limiting or overcoming therapy resistance.

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.

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.

Background Methotrexate (MTX) eradicates leukemic cells by disrupting de novo nucleotide biosynthesis and DNA replication, resulting in cell death. Since its introduction in 1947, MTX-containing chemotherapeutic regimens have proven instrumental in achieving curative effects in acute lymphoblastic leukemia (ALL). However, drug resistance phenomena pose major obstacles to efficacious ALL chemotherapy. Moreover, clinically relevant molecular mechanisms underlying chemoresistance remain largely obscure. Several alterations in MTX metabolism, leading to impaired accumulation of this cytotoxic agent in tumor cells, have been classified as determinants of MTX resistance. However, the relation between MTX resistance and long-term clinical outcome of ALL has not been shown previously. Methods We have collected clinical data for 235 childhood ALL patients, for whom samples taken at the time of diagnosis were also broadly characterized with respect to MTX resistance. This included measurement of concentrations of MTX polyglutamates in leukemic cells, mRNA expression of enzymes involved in MTX metabolism (FPGS, FPGH, RFC, DHFR, and TS), MTX sensitivity as determined by the TS inhibition assay, and FPGS activity. Results Herein we demonstrate that higher accumulation of long-chain polyglutamates of MTX is strongly associated with better overall (10-year OS: 90.6 vs 64.1 %, P = 0.008) and event-free survival (10-year EFS: 81.2 vs 57.6 %, P = 0.029) of ALL patients. In addition, we assessed both the association of several MTX resistance-related parameters determined in vitro with treatment outcome as well as clinical characteristics of pediatric ALL patients treated with MTX-containing combination chemotherapy. High MTX sensitivity was associated with DNA hyperdiploid ALL (P < 0.001), which was linked with increased MTX accumulation (P = 0.03) and elevated reduced folate carrier (RFC) expression (P = 0.049) in this subset of ALL patients. TEL-AML1 fusion was associated with increased MTX resistance (P = 0.023). Moreover, a low accumulation of MTX polyglutamates was observed in MLL-rearranged and TEL-AML1 rearranged ALL (P < 0.05). Conclusions These findings emphasize the central role of MTX in ALL treatment thereby expanding our understanding of the molecular basis of clinical differences in treatment response between ALL individuals. In particular, the identification of patients that are potentially resistant to MTX at diagnosis may allow for tailoring novel treatment strategies to individual leukemia patients. Electronic supplementary material The online version of this article (doi:10.1186/s13045-015-0158-9) contains supplementary material, which is available to authorized users.

Defects in apoptosis regulation are known to impact chemotherapy resistance and consequently refractoriness and relapse of acute myeloid leukemia (AML). We previously showed that apoptosis-resistant protein profile of AML blasts at diagnosis is associated with shorter disease-free survival. Specifically, by flow cytometry, we measured the expression of Bcl-2, Bcl-xL, Mcl-1 and Bax in leukemic cells and combined these parameters to define their anti-apoptosis index (AAI). Interestingly, the AAI of normal lymphocytes in the AML patients corresponded to the AAI of AML blasts obtained from the same patient, reaching values far outside the normal AAI range of lymphocytes. In addition, the AAI in both cell types displayed parallel changes during the course of therapy. This points to a role of microenvironment in regulation of apoptosis in bone marrow cells of AML patients. Therefore, the aim of the current study was to assess if apoptosis-resistant AML cells are able to regulate the AAI of apoptosis-sensitive cells by influencing the microenvironment, as well as to perform molecular dissection of microenvironment, to identify novel proteins that regulate apoptosis. First, we showed that apoptosis-resistant AML blasts (high AAI) release factors that modulate sensitive AML blasts (low AAI) to upregulate Bcl-2 and become apoptosis-resistant. In the majority of cases (10 out of 14), Bcl-2 expression was significantly increased in apoptosis-sensitive AML blasts upon contact culture with apoptosis-resistant AML blasts (1.7-fold; p=0.0067). To characterize the AML microenvironment, conditioned medium (18 hrs) from patient samples displaying either apoptosis-resistant (n=5) or apoptosis-sensitive profile (n=6) were collected. Using mass spectrometry-based proteomics, comparative analysis was performed on these secretomes. Strikingly, we found that the major functional protein clusters upregulated in secretomes of the apoptosis-resistant AML were involved in mRNA splicing, protein translation and chromatin remodeling/chromosome organization. We further compared protein profiles of the soluble secretome and the extracellular vesicle fraction of a high AAI patient to those of a low AAI patient. Proteomic analysis of these fractions of the conditioned medium showed that the functional protein networks found in the whole secretome are well-represented in extracellular vesicles that are enriched for exosome markers. Transfer of functional proteins between cells by extracellular vesicles is a well documented phenomenon. Therefore, it is conceivable that the regulatory protein networks detected in the vesicles excreted by AML blasts are involved in regulation of apoptosis-related proteins in recipient AML blasts and other cells residing in the bone marrow, thereby contributing to therapy resistance. Funded by STR and KiKa - Children cancer-free Citation Format: Anna Wojtuszkiewicz, Jacqueline Cloos, Floortje L. Kessler, Sander Piersma, Jako Knol, Gerrit Jansen, Yehuda G. Assaraf, Gertjan L. Kaspers, Sonja Zweegman, Gerrit J. Schuurhuis, Connie R. Jimenez. Transfer of regulatory protein networks via extracellular vesicles as a candidate mechanism of apoptosis-resistance in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4764. doi:10.1158/1538-7445.AM2014-4764

Item does not contain fulltext Pediatric acute lymphoblastic leukemia (ALL) is treated with combination chemotherapy including mercaptopurine (6MP) as an important component. Upon its uptake, 6MP undergoes a complex metabolism involving many enzymes and active products. The prognostic value of all the factors engaged in this pathway still remains unclear. This study attempted to determine which components of 6MP metabolism in leukemic blasts and red blood cells are important for 6MP's sensitivity and toxicity. In addition, changes in the enzymatic activities and metabolite levels during the treatment were analyzed. In a cohort (N = 236) of pediatric ALL patients enrolled in the Dutch ALL-9 protocol, we studied the enzymes inosine-5'-monophosphate dehydrogenase (IMPDH), thiopurine S-methyltransferase (TPMT), hypoxanthine guanine phosphoribosyl transferase (HGPRT), and purine nucleoside phosphorylase (PNP) as well as thioguanine nucleotides (TGN) and methylthioinosine nucleotides (meTINs). Activities of selected enzymes and levels of 6MP derivatives were measured at various time points during the course of therapy. The data obtained and the toxicity related parameters available for these patients were correlated with each other. We found several interesting relations, including high concentrations of two active forms of 6MP-TGN and meTIN-showing a trend toward association with better in vitro antileukemic effect of 6MP. High concentrations of TGN and elevated activity of HGPRT were found to be significantly associated with grade III/IV leucopenia. However, a lot of data of enzymatic activities and metabolite concentrations as well as clinical toxicity were missing, thereby limiting the number of assessed relations. Therefore, although a complex study of 6MP metabolism in ALL patients is feasible, it warrants more robust and strict data collection in order to be able to draw more reliable conclusions.

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.

Spliceosome targeting is a novel therapeutic strategy, showing promising results in solid tumors and hematological malignancies. Aberrant splicing of genes involved in apoptosis regulation and drug metabolism was shown to confer chemoresistance in various tumor cells, including acute leukemia. Moreover, increased expression of abnormal splice variants cause reduced sensitivity of leukemic cells to crucial components of current treatment protocols, such as glucocorticoids or methotrexate. Therefore, targeting the spliceosome holds potential to modulate drug resistance-related splicing and to eradicate cells, which do not respond to conventional therapy. In this study, we assessed the in vitro sensitivity of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) cells to spliceosome inhibitors, including meayamycin B (MAMB), pladienolide B (PB) and spliceostatin A (SSA). First, the growth inhibitory activity of MAMB was determined using a 72 h MTT assay in a panel of ALL and AML cell lines, including sublines with acquired resistance to conventional chemotherapeutics. Mechanistically, the effect of MAMB, PB and SSA on splicing profiles, cell cycle distribution and apoptosis induction was assessed in time course experiments. Finally, we compared MAMB sensitivity between 10 primary ALL, 10 AML specimens, and 6 healthy bone marrow (BM) specimens. Remarkably, both ALL and the notoriously apoptosis-resistant AML cell lines responded to subnanomolar concentrations of MAMB, with IC50 values (50% growth inhibition in the MTT assay) ranging between 0.07 and 0.16 nM. Moreover, MAMB retained full sensitivity towards leukemic sublines resistant to conventional chemotherapeutics with various modes of action, including methotrexate, dexamethasone, bortezomib and imatinib. MAMB, PB and SSA-induced growth inhibition was associated with time and dose-dependent alterations in splicing profiles of selected apoptosis-related genes (including Mcl-1, Bcl-X, FAS and Casp2), concomitant cell cycle arrest (in G1 and G2/M phases) and apoptosis induction (up to 40% after 24 h exposure to 1nM MAMB). Consistent with cell line observations, both primary ALL and AML specimens showed remarkable response to MAMB (mean LC50 = 0.42 nM, range: 0.26-0.69 nM and 0.43 nM, range: 0.33-0.44 nM, respectively), with a significantly lower MAMB sensitivity of healthy BM samples (mean lethal concentration causing 50% cell kill [LC50] value 0.57, range: 0.39-1.13 nM, p = 0.02). Collectively, this is the first study to demonstrate that spliceosome inhibition constitutes a promising therapeutic option for both ALL and AML patients, including those with acquired resistance to other anti-leukemic drugs. Financial support by KiKa (Children Cancer Free) Citation Format: Anna Wojtuszkiewicz, Rocco Sciarrillo, Gerrit Jansen, Yehuda G. Assaraf, Kazunori Koide, Robert K. Bressin, Upamanyu Basu, Edwin Sonneveld, Godefridus J. Peters, Gertjan J L Kaspers, Jacqueline Cloos. Spliceosome inhibition as a novel therapeutic option in acute leukemia. [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 4336.

Methotrexate (MTX) is an essential component of contemporary ALL treatment regimens. This antifolate eradicates leukemic cells by disrupting de novo biosynthesis of nucleotides, leading to inhibition of DNA replication and consequent cell death. Over the past decades, MTX-containing chemotherapy has proven to be crucial in achieving curation in ALL. However, drug resistance continues to be a major obstacle to curative ALL treatment. A number of alterations in MTX metabolism, resulting in impaired accumulation of this antifolate in cancer cells, have been identified as determinants of MTX resistance. However, it remains unclear which of these molecular mechanisms have clinical relevance. Therefore, the current study aimed at determining the relation between MTX resistance and long-term clinical outcome of ALL. Towards this end, we collected the available clinical data of 235 childhood ALL patients, including clinical data with a follow-up time of 10 years. For these patients, samples obtained at the time of diagnosis were also characterized with respect to MTX resistance. This included determination of MTX polyglutamate levels in leukemic cells, mRNA expression of enzymes involved in (anti)folate metabolism and transport (i.e. FPGS, FPGH, DHFR, TS and RFC); MTX sensitivity was determined by the thymidylate synthase inhibition assay (TSIA) and in vitro FPGS activity assay was performed. High levels of long-chain MTX polyglutamates was strongly associated with favorable long-term event-free survival (EFS, p = 0.029) and overall survival (OS, p = 0.008) of ALL patients (10-year EFS 81% and OS 91%, compared to EFS 58% and OS 64% in patients with low levels of this active metabolite). Similar relations were observed for the total accumulation of MTX polyglutamates and FPGS activity, which were associated with overall survival (p = 0.018 and p = 0.039, respectively). In the multivariate Cox regression model, including clinical variables, the level of long chain MTX polyglutamates showed a trend towards an association with event-free survival (p = 0.073) but not with overall survival (p = 0.465). In addition, we assessed the association between MTX resistance-related variables and the treatment outcome in patients with different cytogenetic alterations. High MTX sensitivity was associated with hyperdiploid ALL (P Financial support by Kika (Children cancer-free) Citation Format: Anna Wojtuszkiewicz, Godefridus J. Peters, Nicole L. van Woerden, Boas Dubbelman, Gabrielle Escherich, Kjeld Schmiegelow, Edwin Sonneveld, Rob Pieters, Peter M. van de Ven, Gerrit Jansen, Yehuda G. Assaraf, Gertjan J L Kaspers, Jacqueline Cloos. Methotrexate resistance in relation to treatment 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 1624. doi:10.1158/1538-7445.AM2015-1624

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