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Alternative splicing in acute leukemia-relevance in treatment response
Wojtuszkiewicz, A.M.
2016
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citation for published version (APA)
Wojtuszkiewicz, A. M. (2016). Alternative splicing in acute leukemia-relevance in treatment response.
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c h a p t e r
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Manuscript in preparation
Anna Wojtuszkiewicz, Rocco Sciarrillo, Yehuda G. Assaraf, Floortje L. Kessler, Sonja Zweegman, Kazunori Koide, Robert K. Bressin, Upamanu Basu, Edwin Sonneveld, Elisa Giovannetti, Godefridus J. Peters, Gertjan J.L. Kaspers,
Gerrit Jansen* and Jacqueline Cloos*
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ABSTRACT
Pre-mRNA splicing is emerging as an important contributor to drug resistance by affecting genes involved in regulation of apoptosis as well as drug metabolism. Therefore, spliceosome targeted therapies appear interesting as novel treatment strategies, modulating splicing profiles to eradicate or sensitize drug resistant cells. In this respect, pladienolide B (PB) and meayamycin B (MAMB) are potent inhibitors of SF3B, which is one of the core members of the spliceosome. In the current study, we assessed the in vitro impact of PB and MAMB on acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) cells, including leukemic sublines resistant to methotrexate, bortezomib and imatinib, in terms of splicing modulation, apoptosis induction and growth inhibitory sensitivity. We show that both inhibitors were able to promote splicing of Mcl-1L and Bcl-XL in all the analyzed leukemic cell lines with a concomitant apoptosis induction varying between 10 - 40%. Moreover, we noted splicing modulation of previously unreported genes involved in regulation of apoptosis and drug metabolism such as FAS, TNFα and FPGS in a selective, sequence position and cell type specific-manner. Ex vivo analysis revealed that primary ALL (n=10) and AML (n=9) cells were highly sensitive
to MAMB (mean LC50: 0.43 ± 0.15 nM and 0.39 ± 0.03 nM, respectively), while non-malignant bone
marrow samples (n=7) displayed significantly lower sensitivity to MAMB (P=0.018, LC50: 0.57 ± 0.29
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INTROdUCTION
Evidence is accumulating that aberrant pre-mRNA splicing is an important mechanism
contributing to anticancer drug resistance and thereby providing a novel therapeutic target.1,2
Altered splicing patterns of genes engaged in apoptosis regulation were implicated in the
emergence of chemoresistance in tumor cells.3,4 In this respect, survival of cancer cells was
postulated to be enhanced due to elevated expression levels of several anti-apoptotic protein
isoforms, in example BCL-XL and Caspase 2S.3,4 Moreover, splicing aberrations affecting genes
involved in drug metabolism were reported to result in resistance to a panel of chemotherapeutics
commonly used in the treatment of cancer, including leukemia.5–9 In this respect, aberrant
splicing of deoxycytidine kinase (dCK) was uncovered in chemoresistant acute myeloid leukemia
(AML) patient specimens.5 In vitro experiments showed, that dCK isoforms resulting from altered
splicing were inactive, hence suggesting their potential contribution to cytarabine resistance
in AML patients.6 Similarly, skipping of dCK exon 2 and 3 were present in cytarabine-resistant
T-cell acute lymphoblastic leukemia (ALL) cell line - CCRF/CEM.10 Several splice variants of
multidrug resistance protein 1 (MRP1) discovered in ovarian cancer were shown to cause in vitro
doxorubicine resistance.7 We have previously shown that impaired splicing of folylpolyglutamate
synthetase (FPGS) gene is likely one of the mediators of methotrexate (MTX) resistance in ALL
cell lines and patient specimens.8,9 Moreover, mutations affecting several components of the
spliceosome machinery, including splicing factor 3B subunit 1 (SF3B1), were reported to occur in hematological malignancies such as CLL, myelodysplastic syndromes and AML, conferring
a potential therapeutic vulnerability in these cells.11–14
Therefore, novel therapies targeted at the spliceosome hold potential to eradicate tumor cells by either inducing pro-apoptotic splicing or by sensitizing drug resistant cancer cells displaying aberrant splicing of genes engaged in drug metabolism. A group of compounds obtained from bacteria, including spliceostatin A (SSA; FR901464 derivative) as well as pladienolide B (PB), showed
targeting of the SF3B1 subunit of U2 snRNP – an essential component of the spliceosome.15–17 Both SSA
and PB were able to inhibit splicing in vitro leading to accumulation of pre-mRNA and subsequent
cell cycle arrest in G1 and G2/M phases.16–18 A novel SSA - related spliceosome inhibitor - meayamycin
B (MAMB) was previously shown to switch splicing of an important apoptosis regulator - MCL-1 towards its pro-apoptotic variant in non-small cell lung cancer as well as in head and neck squamous
cell carcinoma cell lines.19,20 The dominance of the pro-apoptotic MCL-1S isoform was sufficient to
sensitize tumor cells to BCL-XL inhibitor (ABT-737) resulting in induction of cell death.19,20 Similarly,
SSA was shown to switch splicing of MCL-1 which was paralleled by apoptosis induction in chronic
lymphocytic leukemia (CLL) cells.21 SSA combined with either of the 2 inhibitors of BCL-2 family
members - ABT-199 and ABT-263 showed synergistic effect against chronic lymphocytic leukemia
(CLL) cells.21 Interestingly, even a multidrug resistant subline of Chinese hamster lung cancer cells
(DC3F), VCRd5L was sensitive to subnanomolar concentrations of meayamycin.22 Altogether, these
results suggest that spliceosome inhibition might be a valuable supplement and/or alternative for treating cells resistant to conventional chemotherapy.
Antitumor drug resistance remains a major cause of relapse in leukemia, affecting about
20% of childhood ALL and 40% of childhood AML patients.23–25 As relapsed patients have to face
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phenomena as well as alternative therapies are warranted. Therefore the aim of this study was to assess whether spliceosome inhibitors can present a novel therapeutic option for leukemia patients, including individuals displaying drug resistant disease.
MATERIALS ANd METhOdS
Small molecule agents
MAMB was synthesized and provided by the Department of Chemistry, University of Pittsburgh,19,20
while PB was purchased from Cayman Chemical Company, Ann Arbor, MI, USA. Dexamethasone was purchased from Centrafarm (Etten-Leur, The Netherlands) and ZD1694 was a gift of Dr. A. L.
Jackman (Institute of Cancer Research, Sutton, Surrey, United Kingdom).26
Leukemic cells
The human T-ALL cell line CCRF-CEM and the human acute myeloblastic leukemia cell lines THP1, Kasumi-1 and KG1a were obtained from ATCC (Manassas, VA, USA). Drug-resistant cell lines used in this study included cells derived from CCRF-CEM: (a) 2 sublines resistant to an antifolate - methotrexate (MTX) - R30dm (cells with diminished activity of folylpolyglutamate synthetase, FPGS, a key contributor to intracellular retention of MTX) and CEM/MTX/RFC- (cells are characterized by impaired function of reduced folate carrier, RFC, involved in cellular uptake
of MTX)27,26, (b) CEM/BTZ200 - resistant to a proteasome inhibitor bortezomib (BTZ)28, as well as
(c) 2 cell lines resistant to dexamethasone (Dex): CEM-R5 (carrying a hemi or heterozygous L753F mutation in the glucocorticoid receptor - GR) and CEM-R5C3 (characterized by impaired induction
of GR expression upon Dex treatment).29,30 Similarly, 3 drug-resistant AML cell lines were used: a
bortezomib-resistant THP1/BTZ200 subline31 and 2 imatinib (tyrosine kinase inhibitor) resistant
sublines of Kasumi-1: Kasumi-1/R1 and Kasumi-1/R2 with unidentified mechanism of resistance. CEM/MTX-R30dm, selected by intermittent pulse exposure to high dose MTX was kindly provided
by Prof. J.J. McGuire27, while CEM-R5 and CEM-R5C3 were kindly provided by Prof. R. Kofler.29,30
All other cell lines resistant to various chemotherapeutics were obtained by stepwise increasing extracellular concentrations of the indicated drugs. All cell lines were maintained in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) containing 2.3 mM folic acid supplemented with 10% fetal calf serum (Greiner Bio-One, Frickenhausen, Germany) and 100 units/mL penicillin G, and 100 mg/mL streptomycin sulphate (Gibco).
Cryopreserved mononuclear (bone marrow or peripheral blood) cells of pediatric ALL, AML as well as cardiac patients (serving as non-malignant controls) were collected after obtaining a written informed consent from patients treated on the Dutch Childhood Oncology Group (DCOG) protocols, which have been approved by the local medical ethical committees.
exposure to spliceosome inhibitors and isolation of total rNa
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RNA and Moloney Murine Leukemia Virus (M-MLV; Invitrogen) in a reaction buffer containing random hexamer primers (Roche, Basel, Switzerland), dNTPs (Roche), and a ribonuclease inhibitor Rnasin (Promega, Madison, WI, USA).
reverse transcriptase (rt)-pcr analysis of FpGS splicing patterns
Splicing patterns of selected genes, including MCL-1, BCL-X, FAS, TNFα, CASP-2, CASP-9, BCL-2 and FPGS were examined in human tumor cell lines (treated and untreated with MAMB/PB) using primers listed in Supplemental Table S1. PCR was performed with 2x ReddyMix PCR master mix following the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). The PCR products were resolved on 2% agarose gels with ethidium bromide.
Western blot
Five million cells were harvested, snap frozen in liquid nitrogen and subsequently lysed for 45 min at 4°C in 100 μl RIPA lysis buffer (50mM TRIS-HCl, pH 7.4; 150mM NaCl; 1% Nonidet P-40; 0.5% Na-deoxycholate; 0.1% SDS;) containing Complete™ protease inhibitor cocktail (Boehringer Mannheim, Almere, Netherlands). Subsequently, clarified cell lysates were collected after micro-centrifugation (14,000g, 5 min, 4°C) and used to assess protein expression by Western
blot analysis, as described previously.28 The following antibodies were used: anti-MCL-1 (S-19)
antibody (sc-819, Santa Cruz Biotechnology, Dallas, TX, USA), anti-β-actin (clone c4; Boehringer Mannheim, Germany).
apoptosis assay and cell cycle analysis
For the apoptosis assay cells were analyzed using Apoptest™–FITC kit (VPS Diagnostics, Hoeven, The Netherlands) and 7-AAD (BD Via-Probe™, BD Bioscience, San Jose, CA, USA). For cell cycle analysis the cells were first permeabilized in 70% ethanol, followed by 30 min incubation with RNAse A (100μg/ml, Qiagen) and subsequent staining with propidium iodide (Thermo Fisher Scientific). In both assays fluorescence was measured using BD FACS Canto II flow cytometer (BD Bioscience). Analysis was performed using BD FACS Diva software (BD Bioscience).
Mtt growth inhibition assay
Growth inhibitory effects of MAMB in ALL and AML cell lines as well as primary samples were determined after a continuous exposure using the colorimetric MTT dye reduction assay as
described previously.32,33 ALL cell lines were incubated for 72 h, while AML cell lines as well as
pediatric primary ALL and AML samples for 96 h. The results for cell lines are expressed as IC50 values
– concentration of the drug that inhibits cell growth by 50%, while for primary samples as LC50 values
– the drug concentration that kills 50% of the cells as compared to control. Combination experiments were performed using either a fixed constant ratio of the 2 drugs (PB + antifolate ZD1694) or non-fixed ratio whereby a fixed concentration of one drug is combined with a dilution range of the other (PB +
Dex). Fractional effect analysis was performed as described previously.34 CalcuSyn software (Version
1.1.1 1996, Biosoft, Cambridge, UK) was used to calculate the combination indexes (CI) based on the
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RESULTS
targeting SF3B1 potently inhibits growth of leukemic cells
To determine the sensitivity of ALL and AML cells to spliceosome inhibitors we first assessed the response of T-cell ALL cell line - CCRF-CEM and five of its sublines with acquired resistance to various anti-leukemia chemotherapeutics. These included two methotrexate (MTX)-resistant
cell lines (CEM/R30dm and CEM/MTX/RFC-27,26), a bortezomib (BTZ)-resistant subline (CEM/
BTZ20028) and two sublines resistant to dexamethasone (Dex, CEM-R5 and CEM-R5C329,30). All the
ALL cell lines investigated, including CEM/R30dm and CEM/MTX/RFC-, displayed sensitivity to
subnanomolar concentrations of MAMB with IC50 values ranging from 0.06 - 0.09 nM (Figure 1A and
Table 1) irrespective of the differential underlying mechanisms of resistance involved. To determine whether another SF3B1 inhibitor induces a comparable response in ALL cell lines, CCRF-CEM WT and CEM/R30dm cells as well as CEM-R5 and CEM-R5C3 cells were exposed to a range of PB concentrations. Similarly to MAMB, the growth of ALL cell lines regardless of their sensitivity to
other chemotherapeutics (Figure 1B) was inhibited by PB although with higher IC50 values (CCRF-CEM
WT: 3.9 ± 1.7 nM, CEM/R30dm: 4.1 ± 0.9 nM, CEM-R5: 3.8 ± 1.4 nM and CEM/R5C3: 2.9 ± 0.8 nM). To test whether MAMB is also active in notoriously therapy-resistant AML cells, three AML cell lines
0.01 0.1 1 0 50 100 150 CCRF-CEM WT CEM/R30dm CEM/BTZ200 CEM/MTX/RFC-MAMB conc (nM) Gr ow th (% of co nt ro l) 0.01 0.1 1 0 50 100 150 Kasumi-1 WT Kasumi-1R1 Kasumi-1R2 MAMB conc (nM) Gr ow th (% of co nt ro l) 0.01 0.1 1 0 50 100 150 THP1 THP1ps200 MAMB conc (nM) Gr ow th (% of co nt ro l) Figure 1 A B C D 0.01 0.1 1 10 100 0 50 100 150 CEM/R30dm CEM WT CEM-R5 CEM-R5C3 PB conc (nM) Gr ow th (% of co nt ro l)
Figure 1. Sensitivity of ALL and AML cell lines to spliceosome inhibitors. Cells were incubated with a range of MAMB or PB concentrations for 72 h (ALL cell lines) or 96 h (AML cell lines). A - MAMB sensitivity of the parental CCRF-CEM WT as well as its drug resistant sublines: CEM/R30dm - FPGS deficient MTX resistant cells, CEM/BTZ200 - bortezomib resistant cells, CEM/MTX/RFC- - RFC-deficient MTX resistant cells; B - PB sensitivity of CCRF-CEM WT, CEM/R30dm and two Dex resistant sublines: CEM-R5 and CEM-R5C3; C - MAMB sensitivity of Kasumi-1 and its imatinib resistant sublines: Kasumi-1/R1 and R2; D - MAMB sensitivity of THP1 and its bortezomib resistant subline THP1/BTZ200. Mean ± SD of 3 experiments is plotted.
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were examined: KG1a, THP1 and Kasumi-1, as well as three of their drug-resistant counterparts: BTZ
resistant THP1/BTZ200 cells31 and two imatinib resistant sublines: Kasumi-1/R1 and Kasumi-1/R2.
Remarkably, MAMB was able to potently inhibit growth of all wild type and drug-resistant AML cell
lines (Figure 1C and 1D). Interestingly, MAMB IC50 values were in the same range as observed for the
ALL cell lines (Table 1). Due to limited availability of the compounds, we were not able to perform all the experiments with both agents. Therefore, most of the initial experiments were performed with MAMB, while PB treatments were limited to confirm that both compounds have comparable effects.
SF3B1 inhibition induces shifts in splicing, cell cycle arrest and apoptosis in leukemic cells
To further characterize the response of acute leukemia cells to spliceosome inhibitors, we investigated splicing perturbations, cell cycle distribution as well as apoptosis induction upon drug treatment. Selected ALL and AML cell lines were exposed to a range of MAMB (0.1 nM - 1 nM) and PB (5 nM - 100 nM) concentrations for 24 h followed by PCR analysis performed on selected genes.Since, MAMB was previously shown to shift MCL-1 but not BCL-X splicing in solid tumors19,20, we first
assessed whether MAMB induced changes in MCL-1 and BCL-X splicing. Consistent with their growth inhibition profiles, both compounds were able to shift splicing of MCL-1 gene in leukemic cells towards its pro-apoptotic isoform MCL-1S (Figure 2A and 2B), which was paralleled by a decrease
in MCL-1L protein expression (Figure 2C). However, in line with previously reports21,36, no MCL-1S
induction was observed on protein level with an exception of THP1/BTZ cells. CEM/R30dm cells responded to slightly higher doses of spliceosome inhibitors as compared to the other panel of cell lines (only a minor shift was visible upon 1 nM MAMB, Figure 1A). Intriguingly, we previously found altered splicing of FPGS gene in CEM/R30dm cells as the possible underlying mechanism of
MTX-resistance in these cells.9 In contrast to other reports19,21, all ALL cell lines as well as KG1a displayed
a dose-dependent shift in BCL-X splicing towards its pro-apoptotic isoform BCL-XS, which was abolished at 1 nM MAMB/100 nM PB (Figure 2A and 2B). In CCRF-CEM WT cells this shift was most optimal at 0.25 nM MAMB/10nM PB while, in THP1 cell lines the pattern of shifts in BCL-X splicing was analogous to that of MCL-1.
Table 1. Sensitivity of ALL and AML cell lines to MAMB.
Tumor type Cell line IC50 (±Sd)*
ALL CCRF-CEM WT 0.06 (0.01) CEM/R30dm 0.09 (0.01) CEM/MTX/RFC- 0.09 (0.01) CEM/BTZ200 0.08 (0.01) AML THP1 WT 0.11 (0.03) THP1/BTZ200 0.12 (0.03) KG1a 0.06 (0.01) Kasumi-1 WT 0.06 (0.01) Kasumi-1 R1 0.12 (0.04) Kasumi-1 R2 0.08 (0.04)
*The drug incubation time was 72 h for ALL cell lines and 96 h for AML cell lines. Mean IC50 values (±SD) of at least 3 experiments
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Figure 2 A KG1a CCRF-CEM WT R30dm CEM/RFC-Mcl-1L Mcl-1S Mcl-1L Mcl-1S KG1a CCRF-CEM WT R30dm CEM/RFC-Bcl-XL Bcl-XS Bcl-XL Bcl-XS THP1 WT THP1/BTZ200 THP1 WT THP1/BTZ200 0 0.1 0.25 0.5 1 0 0.1 0.25 0.5 1 0 0.1 0.25 0.5 1 0 0.1 0.25 0.5 1 0 0.1 0.25 0.5 1 0 0.1 0.25 0.5 1 MAMB (nM) MAMB (nM) A B - -- -Bcl-XL Bcl-XS Mcl-1L Mcl-1S 0 5 10 25 50 100 0 5 10 25 50 100 PB (nM) CCRF-CEM WT R30dm CCRF-CEM WT CEM/R30dm MCL-1L 0 0.5 1 0 0.5 1 0 0.5 1 MAMB (nM) CEM/MTX/RFC-actin MCL-1L MCL-1S actin KG1a THP1 WT THP1/BTZ200 CFigure 2. Spliceosome inhibition-induced shifts in splicing of MCL-1 and BCL-X genes. Cells were incubated with a range of MAMB (A) or PB (B) concentrations for 24 h, followed by PCR performed on MCL-1 and BCL-X as well as Western blot analysis of MCL-1 protein expression (C). The figure depicts splicing changes investigated in three ALL (CCRF-CEM WT, R30dm and CEM/MTX/RFC-) and three AML (KG1a, THP1 WT and THP1/BTZ200) cell lines. Pro- and anti-apoptotic splice variants/isoforms for both genes are indicated together with the concentrations of MAMB/PB used.
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B
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The observed changes in splicing were accompanied by cell cycle arrest in G1 and G2/M phases
as well as apoptosis induction as determined by flow cytometry (Figure 3 and 4). In all leukemic cell lines, treatment with 1 nM MAMB for 24 hr resulted in about a 2-fold decrease in percentage
of cells in the S-phase with a concomitant increase in cells in the G2/M phase. The cell cycle arrest
was paralleled by apoptosis induction (Figure 4A and 4B). The fraction of apoptotic cells induced by 1 nM MAMB reached approximately 30-40% in the parental CCRF-CEM and CEM/MTX/RFC- cells while R30dm cells, consistently with their decreased response in MCL-1 splicing, showed only about
CCRF-CEM WT CEM/R30dm CEM /MTX/RFC- KG1a THP1 WT THP1/BTZ200 contr ol 0.1 0.25 0.5 1 0 50 100 150 G1 S G2/M MAMB conc (nM) Ph ase of th e ce ll c ycl e (% ) contr ol 0.1 0.25 0.5 1 0 50 100 150 G1 S G2/M MAMB conc (nM) Ph ase of th e ce ll cycl e (% ) Figure 3 contr ol 0.1 0.25 0.5 1 0 50 100 150 G1 S G2/M MAMB conc (nM) Ph ase of th e ce ll c ycl e (% ) contr ol 0.1 0.25 0.5 1 0 50 100 150 G1 S G2/M MAMB conc (nM) Ph ase of th e ce ll c ycl e (% ) contr ol 0.1 0.25 0.5 1 0 50 100 150 G1 S G2/M MAMB conc (nM) Ph ase of th e ce ll cycl e (% ) contr ol 0.1 0.25 0.5 1 0 50 100 150 G1 S G2/M MAMB conc (nM) Ph ase of th e ce ll cycl e (% ) CCRF-CEM WT CEM/R30dm CEM /MTX/RFC- KG1a THP1 WT THP1/BTZ200 contr ol 0.1 0.25 0.5 1 0 50 100 150 G1 S G2/M MAMB conc (nM) Ph ase of th e ce ll c ycl e (% ) contr ol 0.1 0.25 0.5 1 0 50 100 150 G1 S G2/M MAMB conc (nM) Ph ase of th e ce ll cycl e (% ) Figure 3 contr ol 0.1 0.25 0.5 1 0 50 100 150 G1 S G2/M MAMB conc (nM) Ph ase of th e ce ll c ycl e (% ) contr ol 0.1 0.25 0.5 1 0 50 100 150 G1 S G2/M MAMB conc (nM) Ph ase of th e ce ll c ycl e (% ) contr ol 0.1 0.25 0.5 1 0 50 100 150 G1 S G2/M MAMB conc (nM) Ph ase of th e ce ll cycl e (% ) contr ol 0.1 0.25 0.5 1 0 50 100 150 G1 S G2/M MAMB conc (nM) Ph ase of th e ce ll cycl e (% )
Figure 3. Cell cycle distribution upon MAMB treatment. Cells were incubated with a range of MAMB concentrations for 24 h, followed by flow cytometry-based cell cycle analysis. A fraction of cells (%) in each phase of the cell cycle (G1, S and G2/M) is indicated. Mean ± SD of 3 experiments is plotted.
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Figure 4. Apoptosis induction in ALL and AML cell lines upon treatment with spliceosome inhibitors. Cells were incubated with a range of MAMB (A,C) or PB (B) concentrations for 24 h, followed by flow cytometry-based apoptosis assay. A - apoptosis induction in three AML (KG1a, THP1 WT and THP1/BTZ200) cell lines. B, C - apoptosis induction in ALL cell lines (CCRF-CEM WT, CEM/R30dm and CEM/MTX/RFC-); Mean ± SD of 3 experiments is plotted.
10% of apoptosis induction. Interestingly though, in 72 h cytotoxicity assays, CCRF-CEM WT and CEM/R30dm cell lines showed comparable levels of MAMB sensitivity. To examine this apparent discordance, we have assessed apoptosis induction in CCRF-CEM WT and CEM/R30dm cells after 48 h to determine if the difference between the two cell lines dissolves over time. The apoptosis assay showed that indeed, after the 48 h incubation the difference between the two cell lines is narrowing (54.8% apoptotic cells in CCRF-CEM WT and 29.4% in CEM/R30dm) as compared to their profiles determined after 24 h of incubation (40.8% in CCRF-CEM WT and 10.6% in CEM/R30dm). PB-dependent induction of apoptosis did not seem to be solely PB-dependent on shifts in MCL-1 splicing as treatment with 25 nM PB induced almost 50% of apoptosis but no changes in MCL-1 splicing (Figure 2B and 4B). Induction of apoptosis upon treatment with 1 nM MAMB in AML cell lines ranged between 18% in KG1a and 28% in THP1/BTZ200 cells (Figure 4C).
To determine whether the general influence of MAMB on splicing regulation would also affect other genes important for cell survival, we also investigated splicing profiles of additional genes,
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including the Fas cell surface death receptor (FAS), Caspase 2 (CASP2) and 9 (CASP9), tumor necrosis factor α (TNFα) and Bcl-2 (Figure 5). All the analysed cell lines displayed alternative splicing of FAS mRNA towards 2 variants - exon 6 skipping and exon 8 skipping, both of which were reported
to have anti-apoptotic function. Interestingly, an unspliced form of the TNFα accumulated upon
exposure to 0.5 nM and 1 nM MAMB in ALL as opposed to AML cell lines. With respect to CASP9, MAMB exposure slightly favoured splicing towards the anti-apoptotic CASP9S form in CEM/R30dm, CEM/MTX/RFC- and KG1a cells (Figure 5A and B). No substantial changes were observed for CASP2 splicing, except for THP1 WT cells wherein MAMB exposure effected a change in favour of the anti-apoptotic CASP2S form. However, rather than modulation of their splicing, MAMB did cause an overall decrease in mRNA levels of both caspases. In contrast, Bcl-2 splicing was not influenced within the investigated range of MAMB concentrations.
SF3B1 inhibition as a potential tool to re-sensitize drug resistant cells
For several solid tumor types MAMB was able to sensitize cells to a BCL-X inhibitor, which resulted
in enhanced induction of apoptosis.19,20 Since, the CEM/R30dm cell line displayed altered splicing
of FPGS (particularly intron 8 partial retention - PR) as the molecular basis for its MTX resistant
phenotype9, we hypothesized that MAMB could sensitize these cells to MTX. In order to test our
hypothesis, we have assessed the combined effect of MAMB and ZD1694 (an FPGS-dependent antifolate agent) on growth of CEM/R30dm using a fixed constant ratio of both drugs in a 72 h MTT assay. Subsequently, combination indexes (CIs) were calculated based on the median-effect
principle34,35, which characterize the interaction between the studied drugs. CI<1 suggest synergism,
CI=1 point to additive effect, while CI>1 indicate antagonism. We observed no benefit in combining MAMB with ZD1694 with the CI values well above 1 indicating an antagonistic interaction (data not shown). This observation is in line with the analysis of MAMB-induced changes in FPGS splicing upon a 24 h treatment (data not shown), revealing no changes in intron 8 PR of FPGS. Intriguingly, we noted that the effect of MAMB on FPGS splicing was position-specific: in some regions of the gene exon skipping was potently induced (i.e. exon 14 skipping), while in others the induction of exon skipping was minor (i.e. exon 6 skipping). In addition, it appeared that while moderate concentrations of MAMB (0.25 nM in CCRF-CEM and 0.5 nM in CEM/R30dm) were able to induce alternative splicing events, mainly exon skipping, higher concentrations of this drug resulted in inhibition of splicing and accumulation of unspliced mRNA.
CEM/R30dm cells are highly cross-resistant to Dex, which is another crucial chemotherapeutic in ALL treatment. Dex resistance in ALL patients has previously been linked to increased MCL-1
expression.37,38 Therefore, we assessed if spliceosome inhibition could re-sensitize CEM/R30dm as well
as two other Dex-resistant cell lines - CEM-R5 and CEM-R5C3. Due to different profiles of dose response to Dex and PB in all three cell lines, a combination of fixed concentration of PB (either 0.5 nM or 1 nM) with a dose range for Dex was applied. Interactions were evaluated using the CIs as well as the fractional effect analysis, in which the observed effect is considered synergistic if it exceeds the product of the
effects of 2 agents individually (the expected curve).34 Only the effect of the drug resulting in high
fraction affected (proportion of growth inhibition) was taken into account since it is considered more
clinically relevant.34 For both 0.5 nM and 1 nM PB the observed effect of the combinations was greater
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Figure 5 A B KG1a CCRF-CEM WT R30dm CEM/RFC-FAS WT TNFα unspliced Bcl-2 FAS Δexon 8 FAS Δexon 6 TNFα spliced FAS WT TNFα unspliced Bcl-2 FAS Δexon 8 FAS Δexon 6 TNFα spliced THP1 WT THP1/BTZ200 Casp 2L Casp 2S Casp 2L Casp 2S Casp 9L Casp 9S 0 0.1 0.25 0.5 1 0 0.1 0.25 0.5 1 0 0.1 0.25 0.5 1 0 0.1 0.25 0.5 1 0 0.1 0.25 0.5 1 0 0.1 0.25 0.5 1 Casp 9L Casp 9S MAMB (nM) MAMB (nM)--Figure 5. MAMB induced shifts in splicing of selected apoptosis-related genes. Cells were incubated with a range of MAMB concentrations for 24 h, followed by PCR performed on FAS, TNFα, CASP2, CASP9 and Bcl-2. A - splicing changes in three ALL (CCRF-CEM WT, CEM/R30dm and CEM/MTX/RFC-); B - splicing changes in three AML (KG1a, THP1 WT and THP1/BTZ200) cell lines. Pro- and anti-apoptotic splice variants for all the genes are indicated together with the concentrations of MAMB used.
A B Figure 5 A B KG1a CCRF-CEM WT R30dm CEM/RFC-FAS WT TNFα unspliced Bcl-2 FAS Δexon 8 FAS Δexon 6 TNFα spliced FAS WT TNFα unspliced Bcl-2 FAS Δexon 8 FAS Δexon 6 TNFα spliced THP1 WT THP1/BTZ200 Casp 2L Casp 2S Casp 2L Casp 2S Casp 9L Casp 9S 0 0.1 0.25 0.5 1 0 0.1 0.25 0.5 1 0 0.1 0.25 0.5 1 0 0.1 0.25 0.5 1 0 0.1 0.25 0.5 1 0 0.1 0.25 0.5 1 Casp 9L Casp 9S MAMB (nM) MAMB (nM)
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Dex in combination with 0.5 nM PB
A
Dex in combination with 1 nM PB
B 0.000 1 0.001 0.01 0.1 1 10 100 1000 0 50 100 150 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l)
Dex in combination with 0.5 nM PB Dex in combination with 1 nM PB
CEM/R30dm
CEM-R5C3
C
Dex in combination with 0.5 nM PB Dex in combination with 1 nM PB
0.000 1 0.001 0.01 0.1 1 10 100 1000 70 80 90 100 110 120 130 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l) CEM-R5 0.000 1 0.001 0.01 0.1 1 10 100 1000 0 50 100 150 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l) 0.000 1 0.001 0.01 0.1 1 10 100 1000 0 50 100 150 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l) CI 0.229CI 0.223 CI 0.227CI 0.271 CI 0.176 CI 0.347 CI 0.300CI 0.281CI 0.254 CI 0.198 CI 1.429CI 1.613CI 1.982 CI 2.131 0.000 1 0.001 0.01 0.1 1 10 100 1000 0 50 100 150 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l) 0.000 1 0.001 0.01 0.1 1 10 100 1000 60 80 100 120 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l)
Figure 6. The effect of PB and Dex combination in three Dex-resistant cell lines. For each cell lines a fixed concentration of PB (either 0.5 nM or 1 nM) was combined with a dilution range of Dex. Subsequently, the expected effect was calculated (by multiplying the effects of both single agents alone) and plotted together with the dose-response curve to Dex alone and in combination with PB. The arrows indicate points for which the observed dose-response was greater than the expected response. Combination indexes (CI) calculated using Calcusyn are indicated above the arrows. A - dose-response curves obtained for CEM/R30dm; B - dose-response curves obtained for CEM-R5C3; C - dose-response curves obtained for CEM-R5. Mean of 3 experiments is plotted.
A
Dex in combination with 0.5 nM PB
A
Dex in combination with 1 nM PB
B 0.000 1 0.001 0.01 0.1 1 10 100 1000 0 50 100 150 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l)
Dex in combination with 0.5 nM PB Dex in combination with 1 nM PB
CEM/R30dm
CEM-R5C3
C
Dex in combination with 0.5 nM PB Dex in combination with 1 nM PB
0.000 1 0.001 0.01 0.1 1 10 100 1000 70 80 90 100 110 120 130 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l) CEM-R5 0.000 1 0.001 0.01 0.1 1 10 100 1000 0 50 100 150 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l) 0.000 1 0.001 0.01 0.1 1 10 100 1000 0 50 100 150 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l) CI 0.229CI 0.223 CI 0.227CI 0.271 CI 0.176 CI 0.347 CI 0.300CI 0.281CI 0.254 CI 0.198 CI 1.429CI 1.613CI 1.982 CI 2.131 0.000 1 0.001 0.01 0.1 1 10 100 1000 0 50 100 150 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l) 0.000 1 0.001 0.01 0.1 1 10 100 1000 60 80 100 120 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l) B
Dex in combination with 0.5 nM PB
A
Dex in combination with 1 nM PB
B 0.000 1 0.001 0.01 0.1 1 10 100 1000 0 50 100 150 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l)
Dex in combination with 0.5 nM PB Dex in combination with 1 nM PB
CEM/R30dm
CEM-R5C3
C
Dex in combination with 0.5 nM PB Dex in combination with 1 nM PB
0.000 1 0.001 0.01 0.1 1 10 100 1000 70 80 90 100 110 120 130 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l) CEM-R5 0.000 1 0.001 0.01 0.1 1 10 100 1000 0 50 100 150 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l) 0.000 1 0.001 0.01 0.1 1 10 100 1000 0 50 100 150 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l) CI 0.229CI 0.223 CI 0.227CI 0.271 CI 0.176 CI 0.347 CI 0.300CI 0.281CI 0.254 CI 0.198 CI 1.429CI 1.613CI 1.982 CI 2.131 0.000 1 0.001 0.01 0.1 1 10 100 1000 0 50 100 150 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l) 0.000 1 0.001 0.01 0.1 1 10 100 1000 60 80 100 120 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l)
Dex in combination with 0.5 nM PB
A
Dex in combination with 1 nM PB
B 0.000 1 0.001 0.01 0.1 1 10 100 1000 0 50 100 150 Dex only expected observed Dex conc. (µM) Gr ow th (% of co nt ro l)
Dex in combination with 0.5 nM PB Dex in combination with 1 nM PB
CEM/R30dm
CEM-R5C3
C
Dex in combination with 0.5 nM PB Dex in combination with 1 nM PB
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MaMB eradicates primary aLL and aML cells
Finally, we investigated whether MAMB also effectively targeted primary leukemic cells. Using the cytotoxicity (MTT) assay, we tested primary cells of 10 childhood ALL and 10 childhood AML patients as well as 7 non-malignant bone marrow (BM) controls obtained from pediatric cardiac patients for their MAMB sensitivity. In line with our results in leukemic cell lines, primary leukemic samples showed a remarkable sensitivity to MAMB upon a 96 h exposure (Figure 7). Interestingly, there was even no significant difference in MAMB sensitivity between primary ALL and AML samples (P=0.84,
mean LC50 values 0.43 ± 0.15 nM and 0.39 ± 0.03 nM, respectively). Notably, the LC50 values of
non-malignant BM cells were significantly higher (P=0.018, mean LC50 value 0.57 ± 0.26 nM) as compared
to leukemic cells. ALL AML NBM 0.0 0.5 1.0 1.5 MA MB sens itiv ity (L C50 val ue s) P=0.0178 Figure 7
Figure 7. Sensitivity of primary ALL and AML cell lines to MAMB. Cryopreserved leukemic cells were incubated with a range of MAMB concentrations (0.025 - 1.6 nM) for 96 h. MAMB sensitivity is expressed as the LC50 values (in
nM) - the concentration of the drug that kills 50% of the cell as compared to the control. The p-value is based on Mann-Whitney U test.
dISCUSSION
In this study we showed that spliceosome inhibition via targeting SF3B1 constitutes a novel potential therapeutic option for acute leukemia patients. MAMB was previously shown to be effective against
a range of solid tumors in vitro,19,20 including a multidrug resistant Chinese hamster lung cancer
cell line - VCRd5L.22 Also SSA and another group of spliceosome inhibitors, i.e. sudemycins, which
share structural features of both SSA and PB, recently showed antitumor activity against CLL cells.21,36
Extending on these observations, we found that two structurally different spliceosome inhibitors - MAMB and PB were active against ALL and AML, as indicated both by in vitro leukemic cell line models and ex vivo primary patient samples studies. Remarkably, AML cells, which in general
tend to be more intrinsically resistant to currently available chemotherapy as compared to ALL,39
were highly responsive to both MAMB and PB. From this perspective, a recent report revealing
an association of splicing factor mutations with poor outcome in de novo AML11, underscores the
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spliceosome inhibition might be an alternative for leukemia patients with emerging or established drug-resistant disease. Moreover, PB combined with Dex exerted a strong synergistic anti-tumor effect on CEM/R30dm cells. The exact mechanism underlying the synergy between these two agents awaits further investigations but might involve PB-induced changes in splicing of MCL-1 and the resultant decline in MCL-1 protein levels. Increased MCL-1 protein expression was previously
linked to prednisolone resistance in ALL patients.38 Interestingly, in two Dex-resistant cell lines the
interaction between Dex and PB was either slightly (CEM-R5C3) or strongly (CEM-R5) antagonistic. Dex resistance in these cell lines relied on alterations in glucocorticoid receptor (GR) with CEM-R5C3 displaying impaired induction of GR expression upon Dex treatment and CEM/R5 carrying a hemi
or heterozygous mutation in this gene.29,30 In contrast, CEM/R30dm cells were originally selected
for resistance to high dose MTX and were characterised by changes in splicing of FPGS and MCL-1
as well as glucocorticoid cross-resistance.9,27 Therefore, spliceosome inhibition might constitute
a useful therapeutic strategy for patients displaying MCL-1-dependent but not GR-dependent glucocorticoid resistance.
Obviously, therapeutic application of spliceosome inhibitors comes with minimizing potential toxicity to normal cells and tissues. In this respect, both SSA and sudemycins showed increased
cytotoxic effect on cancerous cells as compared to normal cells.21,36,40 In line with these results,
primary acute leukemia cells were significantly more sensitive to MAMB compared to non-malignant bone marrow in cytotoxicity assays, albeit both types of cells responded to subnanomolar concentrations of MAMB. Therefore, conceivably, a therapeutic window will probably be small to minimize toxicity to normal bone marrow cells and requires further assessment. The first clinical trials involving spliceosome inhibitors recently tested the activity of a compound derived from PB
(E7107) against solid tumors.41,42 E7107 was able to inhibit splicing in the treated patients, resulting in
the accumulation of unspliced pre-mRNA species of several genes (i.e. DNAJB1 and EIF4A1), which confirms the proof of principle activity of this spliceosome inhibitor in vivo. Despite the fact that E7101 was generally well-tolerated, two patients displayed unexpected ophthalmologic side effects
of which the mechanism is currently unclear.41,42 This important study shows, that despite the promise
that spliceosome inhibitors show in cancer treatment, improving their efficacy and minimizing toxicity warrants further (pre)clinical investigations. With respect to leukemia treatment, the effect of spliceosome inhibition on leukemic as well as non-malignant cells should be investigated in mouse models, including assessment of broad changes in splicing profiles.
One of the major changes in splicing patterns we observed in leukemic cells so far upon SF3B1 inhibition included dose-dependent shift in MCL-1 splicing towards its pro-apoptotic variant
MCL-1S, which were also observed in solid tumors.19,20 As opposed to MCL-1, BCL-X splicing remained
unaffected upon spliceosome inhibition in solid tumors as well as in CLL.19–21,40 As opposed to these
previous studies, MAMB/PB also modulated splicing of BCL-X in leukemic cells, leading to elevation of its pro-apoptotic variant BCL-XS which decreased again at higher MAMB/PB concentrations used. Our data suggest that MAMB/PB-mediated apoptosis is dependent on changes in both MCL-1 and BCL-X splicing with BCL-XS induction being the earlier event. Alterations in splicing of CASP2
observed in our study confirm those previously reported to be induced by sudemycins.40 In contrast,
CASP9, which was shown to be differentially spliced upon SF3B1 inhibition in solid tumors,40 only
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included induction of anti-apoptotic variants3,4 of FAS in all the analyzed leukemic cell lines as well
as unsplicing of TNFα identified in ALL but not in AML cells. Interestingly, MAMB-induced splicing changes detected in FPGS mRNA in MTX resistant CEM/R30dm cells differed depending on the region of the gene, indicating sequence-specific influence on splicing. This selective regulation of splicing patterns in a sequence position- and cell type specific-manner is supported by previous studies postulating differential use of splice sites as well as altered expression of particular genes instead
of complete splicing inhibition.15,16,43 Moreover, sudemycins were also shown to induce alterations in
the chromatin structure involving methylation of H3K36, which caused reversible splicing changes
together with altered gene expression, resulting in cell cycle arrest and apoptosis.44 Furthermore,
our bell-shaped dose-response in BCL-X splicing is in line with reports that spliceosome inhibitors used in high doses exert different effects on splicing modulation and cell fate as compared to low
doses.45 Conceivably, MAMB could be used in low doses to reverse drug resistance-related aberrant
splicing profiles, thereby inflicting sensitivity to conventional chemotherapy. This novel paradigm requires experimental exploration involving more extensive drug combination studies.
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Supplemental Table S1. Primers used in the RT-PCR based detection of splicing alterations in selected genes
Primer set Primer Primer sequence
MCL-1 Forward 5’-GCCAAGGACACAAAGCCAAT-3’
Reverse 5’-GCTCCTACTCCAGCAACACC-3’
BCL-X Forward 5’-GGGCATTCAGTGACCTGACA-3’
Reverse 5’-GGGAGGGTAGAGTGGATGGT-3’
FAS Forward 5’-ACTCACCAGCAACACCAAGT-3’
Reverse 5’-CCAAGCAGTATTTACAGCCAGC-3’
TNFα* Forward 5’-GATCATCTTCTCGAACCCCG-3’
Reverse 5’-TGGCAGGGGCTCTTGATG-3’
Caspase 2 Forward 5’-ACTGCCCAAGCCTACAGAAC-3’
Reverse 5’-CACAAGCCCGCTCAGAAAAC-3’
Caspase 9 Forward 5’-TGGAGACTCGAGGGAGTCAG-3’
Reverse 5’-AAGTGGAGGCCACCTCAAAC-3’ Bcl-2 Forward 5’-TCCTGGCTGTCTCTGAAGACT-3’ Reverse 5’-CGGTGCTTGGCAATTAGTGG-3’ FPGS_1* EX1-up 5’-CGCGGCATAACGACCCAG-3’ EX3-dw 5’-TTCCCCTTCGTCCCAGTGAC-3’ FPGS_4 EX4_F 5’-GCTCCACCTGTGCCTTCACG-3’ EX7_R 5’-CCGCCAATGCCCACCTCCAC-3’ FPGS_5 EX5_F 5’-CGCCTCTACCACCGGCTGGA-3’ EX9_R 5’-GCTCGGTCCCTCAGCACTGC’-3’ FPGS_6 EX6_2F 5’-CCGCTTCCTGACACTCATGGC-3’ EX9_2R 5’-CTGCTGGGCTCGGTCCCTCA-3’ FPGS_11* EX11-up 5’-CAAAGGCATCCAGGCCAGG-3’ EX15-dw 5’-TGCTCTTCGTCCAGGTGGTT-3’
*- primers previously published8
