Search

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

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