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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.

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.

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.

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.

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

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.

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