Accepted Article Preview: Published ahead of advance online publication The impact of SF3B1 mutations in CLL on the DNA damage response G D te Raa, I A M Derks, V Navrkalova, A Skowronska, P D Moerland, J van Laar, C Oldreive, H Monsuur, M Trbusek, J Malcikova, M Lode´n, C H Geisler, J Hu¨llein, A Jethwa, T Zenz, S Pospisilova, T Stankovic, M H J van Oers, A P Kater, E Eldering
Cite this article as: G D te Raa, I A M Derks, V Navrkalova, A Skowronska, P D Moerland, J van Laar, C Oldreive, H Monsuur, M Trbusek, J Malcikova, M Lode´n, C H Geisler, J Hu¨llein, A Jethwa, T Zenz, S Pospisilova, T Stankovic, M H J van Oers, A P Kater, E Eldering, The impact of SF3B1 mutations in CLL on the DNA damage response, Leukemia accepted article preview 5 November 2014; doi: 10.1038/leu.2014.318. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Received 15 August 2014; revised 3 October 2014; accepted 23 October 2014; Accepted article preview online 5 November 2014
©
2014 Macmillan Publishers Limited. All rights reserved.
The impact of SF3B1 mutations in CLL on the DNA damage response
1,2
2
3
4
G. Doreen te Raa , Ingrid A.M. Derks , Veronika Navrkalova , Anna Skowronska , Perry D. 5
1,2
4
2
3
Moerland , Jacoline van Laar , Ceri Oldreive , Hanneke Monsuur , Martin Trbusek , Jitka Malcikova 6
7
8
8
3
8,9
Martin Lodén , Christian H. Geisler , Jennifer Hüllein , Alexander Jethwa , Thorsten Zenz , Sarka 3
4
Pospisilova , Tatjana Stankovic , Marinus H.J. van Oers
1
1,10
, Arnon P. Kater
1,10
and Eric Eldering
2,10
2
Departments of Hematology and Experimental Immunology, Academic Medical Center, Amsterdam, 3
The Netherlands, Department of Molecular Medicine, Central European Institute of Technology, 4
Masaryk University, Brno, Czech Republic, School of Cancer Sciences, University of Birmingham, Birmingham, United Kingdom,
5
Bioinformatics Laboratory, Department of Clinical Epidemiology, 6
Biostatistics and Bioinformatics, Academic Medical Center, Amsterdam, The Netherlands, MRC7
Holland, Amsterdam, the Netherlands, Department of Hematology, Rigshospitalet, Copenhagen, Denmark,
8
Department of Translational Oncology, National Center for Tumor Diseases (NCT),
German Cancer Research Center (DKFZ) and
9
Department of Medicine V, University Hospital
Heidelberg, Heidelberg, Germany. 10
Lymphoma and Myeloma Center Amsterdam (LYMMCARE)
Running title: CLL SF3B1 mutations associate with a defective DDR Key words: SF3B1, ATM, NOTCH1, chemoresistance, DNA damage response, apoptosis
Abstract: 188 words Main text: 3918 words Number of figures: 6 Number of supplementary tables: 4 Number of supplementary figures: 8 Number of references: 54
Corresponding author: E. Eldering, PhD Department of Experimental Immunology Academic Medical Center University of Amsterdam Meibergdreef 9 1105 AZ, Amsterdam, The Netherlands tel. +31-20-5667018 fax. +31-20-5669756 e-mail: [email protected]
©
2014 Macmillan Publishers Limited. All rights reserved.
1
ABSTRACT Mutations or deletions in TP53 or ATM are well-known determinants of poor prognosis in chronic lymphocytic leukemia (CLL), but only account for approximately 40% of chemo-resistant patients. Genome-wide sequencing has uncovered novel mutations in the splicing factor sf3b1, that were in part associated with ATM aberrations, suggesting functional synergy. We first performed detailed genetic analyses in a CLL cohort (n=110) containing ATM, SF3B1 and TP53 gene defects. Next, we applied a newly developed multiplex assay for p53/ATM target gene induction and measured apoptotic responses to DNA damage. Interestingly, SF3B1 mutated samples without concurrent ATM and TP53 aberrations (sole SF3B1) displayed partially defective ATM/p53 transcriptional and apoptotic responses to various DNA-damaging regimens. In contrast, NOTCH1 or K/N-RAS mutated CLL displayed normal responses in p53/ATM target gene induction and apoptosis. In sole SF3B1 mutated cases, ATM kinase function remained intact, and γH2AX formation, a marker for DNA damage, was increased at baseline and upon irradiation. Our data demonstrate that single mutations in sf3b1 are associated with increased DNA-damage and/or an aberrant response to DNA damage. Together, our observations may offer an explanation for the poor prognosis associated with SF3B1 mutations.
©
2014 Macmillan Publishers Limited. All rights reserved.
2
INTRODUCTION Chronic lymphocytic leukemia (CLL) is characterized by an extremely heterogeneous clinical course, ranging from an indolent disease without progression towards overt disease requiring immediate treatment. The variable course of CLL is driven, at least in part, by molecular heterogeneity. The most important genetic defects thus far associated with adverse prognosis and chemo-resistance 1;2
are aberrations in the pivotal regulators of the DNA damage response pathway, ATM and TP53 . However, in around 60% of the patients, the molecular basis for chemo resistance is not linked to ATM 3
or TP53 defects . In recent years, a number of novel recurrent somatic mutations in various genes, including but not limited to SF3B1, NOTCH1, KRAS and NRAS have been uncovered by genome and exome-wide sequencing studies mutations
4-7;10
4-8
6;7;9
in CLL. Relatively high frequencies for SF3B1
(10-15%) and NOTCH1 11
(4-16%) were found, whereas mutations in K-/N-RAS occur only occasionally in CLL .
Both SF3B1 and NOTCH1 mutations correspond with inferior prognosis
4;6;7;9;10;12
. In addition, SF3B1
and NOTCH1 were frequently mutated in fludarabine-refractory CLL, which might indicate selection 4;9
due to chemotherapy . Notch1 and K- and N-ras are well known (proto-) oncogenes
11;13
, whereas SF3B1 encodes a
splicing factor. Interestingly, mutations in SF3B1 have also been found in other hematological disorders
14-16
17
and in solid tumors . In addition, mutations in other genes encoding for proteins
involved in the splicing machinery were found in hematological malignancies alterations in splicing have been related to tumorigenesis
6;7;15;18;19
. Moreover,
20-22
. This indicates that spliceosomal defects
constitute an important and ubiquitous pathway in malignant transformation. Although it is tempting to speculate that mutations in SF3B1 might lead to aberrant splicing of specific transcripts that affect the pathogenesis of CLL, the underlying mechanism of dysfunction in SF3B1 mutated patients remains elusive thus far. SF3B1 mutations often occur together with 11q23 deletions (52-57%)
7;23
, the locus for the
ATM gene. In addition, patients with combined SF3B1 and ATM mutation without 11q deletion were 7
identified . This suggests that SF3B1 mutations might be functionally synergistic with ATM loss. 9
Furthermore, SF3B1 mutations were initially reported as mutually exclusive with TP53 disruption , which suggested similarity in biological consequences. Therefore, we hypothesized that SF3B1
©
2014 Macmillan Publishers Limited. All rights reserved.
3
defects may impact on the ATM and/or p53 pathways, either in conjunction with or independently of ATM aberrations. The goal of this study was first to characterize the genetic landscape of SF3B1 mutations in relation to ATM and TP53 aberrations (mutations and deletions) in CLL. Secondly, we investigated the functional consequences of SF3B1 mutations in response to DNA damage, in comparison with other recurrent somatic mutations recently found in CLL. Our data indicate that SF3B1 mutations in CLL independently lead to defects in the DNA damage response.
©
2014 Macmillan Publishers Limited. All rights reserved.
4
M ATERIALS AND METHODS
Patient and Samples CLL patients (n=110) from four different cohorts were enrolled: 1) “AMC cohort”; both untreated and treated patients, 2) “H68 cohort”; treatment-naïve patients with high-risk CLL (defined by either 17p deletion, 11q deletion, or trisomy 12, or unmutated IGHV and/or VH3-21) included in the HOVON68 24
clinical trial , 3) “d’Accord cohort”; fludarabine-refractory patients included in D’accord clinical trial
25
and 4) “Brno cohort”, enriched for 11q deletion and/or ATM mutations. The study was conducted in accordance with the Declaration of Helsinki and informed consent was obtained from all patients. Diagnosis of CLL was according to IWCLL-NCI Working Group criteria. Peripheral blood mononuclear 26
cells (PBMCs) were isolated and frozen as earlier described . After thawing, CLL cells were enriched, in case CD19/CD5 purity was below 90%, via negative depletion using α-CD3, α-CD14 and α-CD16 26
(CLB, Amsterdam) as described .
Mutational analyses Sequence analysis was performed on a set of genes (TP53, NOTCH1, SF3B1, NRAS, KRAS, MYD88, BRAF, EZH2, PIK3CA) by next generation sequencing (NGS) on samples from the “H68 cohort” using a 454-based platform (Roche)
27
2
or by Sanger sequencing on the remaining cohorts (Supplemental
Table 1). Samples containing a sole SF3B1 mutation were also sequenced for TP53 (ex2-11), which revealed no additional mutations. ATM (ex4-65) was analyzed by either Sanger sequencing or using 1;28
resequencing microarray and direct sequencing as described previously
.
P53 and ATM target gene induction analyzed by Reverse Transcriptase Multiplex Ligationdependent Probe Amplification (RT-MLPA) CLL cells were treated with or without irradiation (5Gy), fludarabine or doxorubicin (Sigma-Aldrich), in the presence or absence of 10 μM Ku-55933 (ATM-inhibitor, Selleckchem) and cultured for 16 hours at 37°C. For some experiments, CLL cells were co-cultured with CD40L-expressing (3T40) or control 3T3 fibroblasts for 48 hours as previously described
26;29
. RT-MLPA (MRC-Holland), was performed as
26
described before , using a newly designed RT-MLPA probe set (R016-X2, te Raa et al, manuscript in preparation), which included several p53 and ATM target genes (CD95, Bax, Puma, p21, FDXR,
©
2014 Macmillan Publishers Limited. All rights reserved.
5
30
PCNA, NME1, ACSM3,MYC,PYCR1) . Expression of these genes was normalized to a set of housekeeping genes included in the probeset. See also Supplemental Methods. Representative WT and TP53 mutated samples were included as controls in all functional experiments. WT samples were defined as: absence of TP53 aberrations, 11q deletion, trisomy 12 and SF3B1 mutation, and fludarabine sensitive in vitro. TP53 mutated cases harbored both a TP53 mutation and a 17p deletion and were fludarabine resistant in vitro.
Western Blot and CD95 determination by flowcytometry CLL cells were irradiated (5Gy), cultured for 45 minutes or 16 hours and lysed in Laemmli sample 26
buffer and Western blotting was performed using standard conditions as described previously . The following antibodies were used: puma, p53, phosphorylated-p53 (p-p53-ser15), ATM, KAP (TIF1ß), phosphorylated-KAP (phospho-TIF1ß-ser824;Cell Signalling), sf3b1 (Sigma-Aldrich), p21 and β-actin (Santa Cruz). Protein expression was quantified using Licor Odyssey software and corrected for the expression of actin. CD95 expression on CLL cells was determined by flowcytometry using CD5-PE, CD19-APC (eBioscience) and CD95-FITC (BD). Data were normalized for isotype control.
Apoptosis induction by fludarabine, doxorubicine, irradiation (IR) and nutlin-3a 6
Thawed CLL cells were cultured (1.5x10 /ml) in the presence of fludarabine, nutlin-3a (Sigma-Aldrich) or doxorubicin at indicated concentrations or following exposure to IR (5Gy), for 48 hours at 37°C. 26
Apoptosis was measured by flowcytometry as previously described . Specific cell death was calculated as ((%apoptosistreated cells-% apoptosisuntreated cells)/%viableuntreated cells)x100.
γH2AX expression by flowcytometry Thawed CLL cells were incubated for 30 minutes in the presence or absence of 1 µM NU7441 (DNAPK inhibitor; Selleckchem) at 37°C, followed by irradiation (5 Gy). At the indicated times, CLL cells were permeabilized (Foxp3 staining kit; eBioscience) and stained using the following antibodies: CD5PE, CD19-APC (eBioscience) and isotype-AF488 or γH2AX-AF488 (phosphorylated-H2AX-ser-139; Cell Signaling). Expression of γH2AX was measured using flowcytometry. Data were normalized for isotype control.
©
2014 Macmillan Publishers Limited. All rights reserved.
6
Statistical analysis Non-parametric Mann-Whitney-U test was used for comparison of 2 independent groups. For RTMLPA and apoptotic responses, one-way ANOVA with Dunnett’s post hoc test was used for multiple comparison correction. To test the effect of Nu7441 on γH2AX expression, a non-parametric paired test, Wilcoxon signed rank test, was used. Correlations were analyzed by Spearman’s rank correlation test. A p-value
Cite this article as: G D te Raa, I A M Derks, V Navrkalova, A Skowronska, P D Moerland, J van Laar, C Oldreive, H Monsuur, M Trbusek, J Malcikova, M Lode´n, C H Geisler, J Hu¨llein, A Jethwa, T Zenz, S Pospisilova, T Stankovic, M H J van Oers, A P Kater, E Eldering, The impact of SF3B1 mutations in CLL on the DNA damage response, Leukemia accepted article preview 5 November 2014; doi: 10.1038/leu.2014.318. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Received 15 August 2014; revised 3 October 2014; accepted 23 October 2014; Accepted article preview online 5 November 2014
©
2014 Macmillan Publishers Limited. All rights reserved.
The impact of SF3B1 mutations in CLL on the DNA damage response
1,2
2
3
4
G. Doreen te Raa , Ingrid A.M. Derks , Veronika Navrkalova , Anna Skowronska , Perry D. 5
1,2
4
2
3
Moerland , Jacoline van Laar , Ceri Oldreive , Hanneke Monsuur , Martin Trbusek , Jitka Malcikova 6
7
8
8
3
8,9
Martin Lodén , Christian H. Geisler , Jennifer Hüllein , Alexander Jethwa , Thorsten Zenz , Sarka 3
4
Pospisilova , Tatjana Stankovic , Marinus H.J. van Oers
1
1,10
, Arnon P. Kater
1,10
and Eric Eldering
2,10
2
Departments of Hematology and Experimental Immunology, Academic Medical Center, Amsterdam, 3
The Netherlands, Department of Molecular Medicine, Central European Institute of Technology, 4
Masaryk University, Brno, Czech Republic, School of Cancer Sciences, University of Birmingham, Birmingham, United Kingdom,
5
Bioinformatics Laboratory, Department of Clinical Epidemiology, 6
Biostatistics and Bioinformatics, Academic Medical Center, Amsterdam, The Netherlands, MRC7
Holland, Amsterdam, the Netherlands, Department of Hematology, Rigshospitalet, Copenhagen, Denmark,
8
Department of Translational Oncology, National Center for Tumor Diseases (NCT),
German Cancer Research Center (DKFZ) and
9
Department of Medicine V, University Hospital
Heidelberg, Heidelberg, Germany. 10
Lymphoma and Myeloma Center Amsterdam (LYMMCARE)
Running title: CLL SF3B1 mutations associate with a defective DDR Key words: SF3B1, ATM, NOTCH1, chemoresistance, DNA damage response, apoptosis
Abstract: 188 words Main text: 3918 words Number of figures: 6 Number of supplementary tables: 4 Number of supplementary figures: 8 Number of references: 54
Corresponding author: E. Eldering, PhD Department of Experimental Immunology Academic Medical Center University of Amsterdam Meibergdreef 9 1105 AZ, Amsterdam, The Netherlands tel. +31-20-5667018 fax. +31-20-5669756 e-mail: [email protected]
©
2014 Macmillan Publishers Limited. All rights reserved.
1
ABSTRACT Mutations or deletions in TP53 or ATM are well-known determinants of poor prognosis in chronic lymphocytic leukemia (CLL), but only account for approximately 40% of chemo-resistant patients. Genome-wide sequencing has uncovered novel mutations in the splicing factor sf3b1, that were in part associated with ATM aberrations, suggesting functional synergy. We first performed detailed genetic analyses in a CLL cohort (n=110) containing ATM, SF3B1 and TP53 gene defects. Next, we applied a newly developed multiplex assay for p53/ATM target gene induction and measured apoptotic responses to DNA damage. Interestingly, SF3B1 mutated samples without concurrent ATM and TP53 aberrations (sole SF3B1) displayed partially defective ATM/p53 transcriptional and apoptotic responses to various DNA-damaging regimens. In contrast, NOTCH1 or K/N-RAS mutated CLL displayed normal responses in p53/ATM target gene induction and apoptosis. In sole SF3B1 mutated cases, ATM kinase function remained intact, and γH2AX formation, a marker for DNA damage, was increased at baseline and upon irradiation. Our data demonstrate that single mutations in sf3b1 are associated with increased DNA-damage and/or an aberrant response to DNA damage. Together, our observations may offer an explanation for the poor prognosis associated with SF3B1 mutations.
©
2014 Macmillan Publishers Limited. All rights reserved.
2
INTRODUCTION Chronic lymphocytic leukemia (CLL) is characterized by an extremely heterogeneous clinical course, ranging from an indolent disease without progression towards overt disease requiring immediate treatment. The variable course of CLL is driven, at least in part, by molecular heterogeneity. The most important genetic defects thus far associated with adverse prognosis and chemo-resistance 1;2
are aberrations in the pivotal regulators of the DNA damage response pathway, ATM and TP53 . However, in around 60% of the patients, the molecular basis for chemo resistance is not linked to ATM 3
or TP53 defects . In recent years, a number of novel recurrent somatic mutations in various genes, including but not limited to SF3B1, NOTCH1, KRAS and NRAS have been uncovered by genome and exome-wide sequencing studies mutations
4-7;10
4-8
6;7;9
in CLL. Relatively high frequencies for SF3B1
(10-15%) and NOTCH1 11
(4-16%) were found, whereas mutations in K-/N-RAS occur only occasionally in CLL .
Both SF3B1 and NOTCH1 mutations correspond with inferior prognosis
4;6;7;9;10;12
. In addition, SF3B1
and NOTCH1 were frequently mutated in fludarabine-refractory CLL, which might indicate selection 4;9
due to chemotherapy . Notch1 and K- and N-ras are well known (proto-) oncogenes
11;13
, whereas SF3B1 encodes a
splicing factor. Interestingly, mutations in SF3B1 have also been found in other hematological disorders
14-16
17
and in solid tumors . In addition, mutations in other genes encoding for proteins
involved in the splicing machinery were found in hematological malignancies alterations in splicing have been related to tumorigenesis
6;7;15;18;19
. Moreover,
20-22
. This indicates that spliceosomal defects
constitute an important and ubiquitous pathway in malignant transformation. Although it is tempting to speculate that mutations in SF3B1 might lead to aberrant splicing of specific transcripts that affect the pathogenesis of CLL, the underlying mechanism of dysfunction in SF3B1 mutated patients remains elusive thus far. SF3B1 mutations often occur together with 11q23 deletions (52-57%)
7;23
, the locus for the
ATM gene. In addition, patients with combined SF3B1 and ATM mutation without 11q deletion were 7
identified . This suggests that SF3B1 mutations might be functionally synergistic with ATM loss. 9
Furthermore, SF3B1 mutations were initially reported as mutually exclusive with TP53 disruption , which suggested similarity in biological consequences. Therefore, we hypothesized that SF3B1
©
2014 Macmillan Publishers Limited. All rights reserved.
3
defects may impact on the ATM and/or p53 pathways, either in conjunction with or independently of ATM aberrations. The goal of this study was first to characterize the genetic landscape of SF3B1 mutations in relation to ATM and TP53 aberrations (mutations and deletions) in CLL. Secondly, we investigated the functional consequences of SF3B1 mutations in response to DNA damage, in comparison with other recurrent somatic mutations recently found in CLL. Our data indicate that SF3B1 mutations in CLL independently lead to defects in the DNA damage response.
©
2014 Macmillan Publishers Limited. All rights reserved.
4
M ATERIALS AND METHODS
Patient and Samples CLL patients (n=110) from four different cohorts were enrolled: 1) “AMC cohort”; both untreated and treated patients, 2) “H68 cohort”; treatment-naïve patients with high-risk CLL (defined by either 17p deletion, 11q deletion, or trisomy 12, or unmutated IGHV and/or VH3-21) included in the HOVON68 24
clinical trial , 3) “d’Accord cohort”; fludarabine-refractory patients included in D’accord clinical trial
25
and 4) “Brno cohort”, enriched for 11q deletion and/or ATM mutations. The study was conducted in accordance with the Declaration of Helsinki and informed consent was obtained from all patients. Diagnosis of CLL was according to IWCLL-NCI Working Group criteria. Peripheral blood mononuclear 26
cells (PBMCs) were isolated and frozen as earlier described . After thawing, CLL cells were enriched, in case CD19/CD5 purity was below 90%, via negative depletion using α-CD3, α-CD14 and α-CD16 26
(CLB, Amsterdam) as described .
Mutational analyses Sequence analysis was performed on a set of genes (TP53, NOTCH1, SF3B1, NRAS, KRAS, MYD88, BRAF, EZH2, PIK3CA) by next generation sequencing (NGS) on samples from the “H68 cohort” using a 454-based platform (Roche)
27
2
or by Sanger sequencing on the remaining cohorts (Supplemental
Table 1). Samples containing a sole SF3B1 mutation were also sequenced for TP53 (ex2-11), which revealed no additional mutations. ATM (ex4-65) was analyzed by either Sanger sequencing or using 1;28
resequencing microarray and direct sequencing as described previously
.
P53 and ATM target gene induction analyzed by Reverse Transcriptase Multiplex Ligationdependent Probe Amplification (RT-MLPA) CLL cells were treated with or without irradiation (5Gy), fludarabine or doxorubicin (Sigma-Aldrich), in the presence or absence of 10 μM Ku-55933 (ATM-inhibitor, Selleckchem) and cultured for 16 hours at 37°C. For some experiments, CLL cells were co-cultured with CD40L-expressing (3T40) or control 3T3 fibroblasts for 48 hours as previously described
26;29
. RT-MLPA (MRC-Holland), was performed as
26
described before , using a newly designed RT-MLPA probe set (R016-X2, te Raa et al, manuscript in preparation), which included several p53 and ATM target genes (CD95, Bax, Puma, p21, FDXR,
©
2014 Macmillan Publishers Limited. All rights reserved.
5
30
PCNA, NME1, ACSM3,MYC,PYCR1) . Expression of these genes was normalized to a set of housekeeping genes included in the probeset. See also Supplemental Methods. Representative WT and TP53 mutated samples were included as controls in all functional experiments. WT samples were defined as: absence of TP53 aberrations, 11q deletion, trisomy 12 and SF3B1 mutation, and fludarabine sensitive in vitro. TP53 mutated cases harbored both a TP53 mutation and a 17p deletion and were fludarabine resistant in vitro.
Western Blot and CD95 determination by flowcytometry CLL cells were irradiated (5Gy), cultured for 45 minutes or 16 hours and lysed in Laemmli sample 26
buffer and Western blotting was performed using standard conditions as described previously . The following antibodies were used: puma, p53, phosphorylated-p53 (p-p53-ser15), ATM, KAP (TIF1ß), phosphorylated-KAP (phospho-TIF1ß-ser824;Cell Signalling), sf3b1 (Sigma-Aldrich), p21 and β-actin (Santa Cruz). Protein expression was quantified using Licor Odyssey software and corrected for the expression of actin. CD95 expression on CLL cells was determined by flowcytometry using CD5-PE, CD19-APC (eBioscience) and CD95-FITC (BD). Data were normalized for isotype control.
Apoptosis induction by fludarabine, doxorubicine, irradiation (IR) and nutlin-3a 6
Thawed CLL cells were cultured (1.5x10 /ml) in the presence of fludarabine, nutlin-3a (Sigma-Aldrich) or doxorubicin at indicated concentrations or following exposure to IR (5Gy), for 48 hours at 37°C. 26
Apoptosis was measured by flowcytometry as previously described . Specific cell death was calculated as ((%apoptosistreated cells-% apoptosisuntreated cells)/%viableuntreated cells)x100.
γH2AX expression by flowcytometry Thawed CLL cells were incubated for 30 minutes in the presence or absence of 1 µM NU7441 (DNAPK inhibitor; Selleckchem) at 37°C, followed by irradiation (5 Gy). At the indicated times, CLL cells were permeabilized (Foxp3 staining kit; eBioscience) and stained using the following antibodies: CD5PE, CD19-APC (eBioscience) and isotype-AF488 or γH2AX-AF488 (phosphorylated-H2AX-ser-139; Cell Signaling). Expression of γH2AX was measured using flowcytometry. Data were normalized for isotype control.
©
2014 Macmillan Publishers Limited. All rights reserved.
6
Statistical analysis Non-parametric Mann-Whitney-U test was used for comparison of 2 independent groups. For RTMLPA and apoptotic responses, one-way ANOVA with Dunnett’s post hoc test was used for multiple comparison correction. To test the effect of Nu7441 on γH2AX expression, a non-parametric paired test, Wilcoxon signed rank test, was used. Correlations were analyzed by Spearman’s rank correlation test. A p-value
