University of Veterinary Medicine Hannover
MYCN-dependent and -independent mechanisms targeting drug-induced DNA damage response and
chromosomal instability in neuroblastoma cells
Thesis
Submitted in partial fulfillment of the requirements for the degree
- Doctor of Veterinary Medicine - Doctor medicinae veterinariae
( Dr. med. vet. )
by
Sina Gogolin Potsdam
Hannover 2012
University of Veterinary Medicine Hannover 2. Extern:
Univ. Prof. Dr. Manfred Schwab Division of Tumor Genetics,
German Cancer Research Center (DKFZ) Heidelberg PD Dr. Frank Westermann
Division of Tumor Genetics,
German Cancer Research Center (DKFZ) Heidelberg
1. Referee: Univ. Prof. Dr. Ingo Nolte, joint report with
Univ. Prof. Dr. Manfred Schwab and PD Dr. Frank Westermann 2. Referee: Univ. Prof. Dr. Marion Hewicker-Trautwein
Day of the oral examination: 13.11.2012
The project was financially supported by the European Union (EU, FP6): E.E.T.
Pipeline #037260 and EU (FP7): ASSET.
Contents
1 Introduction 7
1.1 Neuroblastoma in humans and animals 7
1.1.1 Origin 7
1.1.2 Clinical presentation 8
1.1.3 Diagnosis 9
1.1.4 Risk stratification and therapy 11
1.1.5 Differential diagnosis 12
1.2 The Myc family of transcription factors 13
1.3 Outline of the study 15
2 Manuscript I 17
2.1 Abstract 19
2.2 Introduction 20
2.3 Results 22
2.3.1 Impaired drug-induced DNA damage response in neuroblastoma cells dependent on amplified MYCN and chromosomal aberrations of the p53 and/or pRB pathway 22 2.3.2 Deregulated MYCN impairs cell cycle arrest after drug-induced
DNA damage 24
2.3.3 High CDK2 and CDK4 activity despite p21 induction in MYCN- amplified neuroblastoma cells after drug-induced DNA
damage 28 2.3.4 CDK4 inhibition partly restores cell cycle arrest, reduces
viability and increases cell death after drug treatment in
MYCN-amplified cells 31
2.3.5 p19-INK4D, but not p16-INK4A, abrogates cell cycle progression in MYCN-amplified neuroblastoma cells and sensitizes for cell death after drug-induced DNA damage 37
2.5 Material and methods 43
2.5.1 Cell culture 43
2.5.2 Plasmids 43
2.5.3 RNA interference 43
2.5.4 Immunoprecipitation 44
2.5.5 In vitro CDK2 or CDK4 kinase assay 44
2.5.6 Protein expression 44
2.5.7 Viability assay 45
2.5.8 Flow cytometry analysis of cell cycle and cell death 45 2.5.9 (multicolor-)Fluorescence in situ hybridization (FISH/mFISH) 45
2.5.10 Pharmacological inhibition 45
2.6 Conflict of interest 45
2.7 Acknowledgement 46
2.8 References 47
3 Manuscript II 51
3.1 Abstract 53
3.2 Introduction 54
3.3 Material and methods 56
3.3.1 Patients 56
3.3.2 Cell culture 56
3.3.3 Ploidy and cell cycle analysis 56
3.3.4 Reverse transfection on cell arrays 57 3.3.5 Image acquisition, image analysis 57
3.3.6 Quantitative analysis 58
3.3.7 RNA interference 58
3.3.8 Protein expression 59
3.3.9 Fluorescence in situ hybridization (FISH) 59 3.3.10 Indirect immunofluorescence microscopy 59
3.3.11 Pharmacological inhibition 60
3.3.12 GO term enrichment/cluster analysis 60
3.3.13 Statistics 61
3.3.14 Gene expression analysis 61
3.4 Results 62
3.4.1 Tumor ploidy cut-points 1.11 and 1.77 separate
neuroblastoma patient subgroups 62
3.4.2 Mitotic regulatory genes are overexpressed in neuroblastomas
with unfavorable biology 64
3.4.3 Loss of p53-p21 function is associated with tetraploidization in
neuroblastoma cells 68
3.4.4 Selective inhibition of mitotic checkpoint genes causes mitotic- linked cell death in MYCN-amplified and TP53-mutated
neuroblastoma cells 70
3.4.5 MAD2L1 repression in the presence of vincristine induces tetraploidization in neuroblastoma cells with functional p53-
p21 73
3.5 Discussion 78
3.6 Acknowledgement 80
3.7 Conflict of interest 80
4.1 Extra centrosomes and anaphase bridge formation in tetraploid
neuroblastoma cells harboring amplified MYCN and mutated TP53 88 4.2 CDK1 inhibition sensitizes for drug-induced G1-S arrest and cell
death and induces mitotic-linked cell death in MYCN-amplified
neuroblastoma cells 92
5 Overall Discussion 95
6 Conclusion and Perspective 103
7 Summary 104
8 Zusammenfassung 106
9 References 108
10 Appendix 126
10.1 Supplementary data manuscript I 126
10.2 Supplementary data manuscript II 131
Additional publications 184
Acknowledgement 186
Introduction 7
1 Introduction
1.1 Neuroblastoma in Humans and Animals
Neuroblastoma is the most common extracranial solid tumor in young children and accounts for 7% to 10% of all childhood cancer. In Germany, approximately 130 new neuroblastoma cases are diagnosed every year, which corresponds to an incidence of 1.3 per 100,000 children per year. About 90% of all cases are diagnosed until the age of five (SCHWAB et al. 2003).
In contrast, there are only few reports concerning neuroblastoma in animals. The first, published in 1931, described neuroblastoma in the lung and muscles of a six- year old cow after slaughter (Baumgärtner, 1931). 30 years later a study was published investigating the incidence and characteristics of adrenal tumors among 26,667 cattle. Tumors were observed in 253 cases, ten of those diagnosed as neuroblastomas (WRIGHT et al. 1968). During the following years, neuroblastoma tumors were sporadically detected in cattle; the age at diagnosis ranged from five- month-old calves to adult animals (HAYNES et al. 1984; STEINBERG et al. 2006;
UCHIDA et al. 1998). Furthermore, as a single event, neuroblastomas have been found in a newborn piglet and a six-week-old goat (DIESSLER et al. 2002; MARTIN DE LAS MULAS et al. 1991). Most neuroblastomas, however, have been described in dogs so far (FORREST et al. 1997; KELLY 1975; LOUDEN et al. 1992;
MARCOTTE et al. 2004; MATSUSHIMA et al. 1998; MICHISHITA et al. 2010;
SCHWARTZ et al. 2011; SIMON et al. 1960; SUZUKI et al. 2003).
1.1.1 Origin
Neuroblastomas derive from embryonal neuroectodermal sympathetic precursor cells of the neural crest. Primary tumors are localized in human to 65% of cases in the abdomen, half of them in the adrenal gland. Furthermore, neuroblastomas can occur anywhere in the sympathetic nervous system, preferentially alongside the sympathetic chain (neck, thoracic cavity, chest, pelvis) (MARIS et al. 2007).
Neuroblastomas in animals have been observed in the adrenal gland but also in the spinal cord, the mandibulopharyngeal area, the abdominal cavity (sympathetic ganglia) and in other regions of the body (FORREST et al. 1997; FOSTER et al.
1988; MARCOTTE et al. 2004; MATSUSHIMA et al. 1998; MICHISHITA et al. 2010;
SCHWARTZ et al. 2011; STEINBERG et al. 2006; SUZUKI et al. 2003; UCHIDA et al. 1998). The etiology of neuroblastoma remains unclear. It occurs sporadically, even though in 1% to 2% of cases familial neuroblastoma with autosomal dominant inheritance was described (WEINSTEIN et al. 2003). Genetic alterations that are involved in the development of hereditary neuroblastoma are ALK and PHOX2B mutations (MOSSE et al. 2004; MOSSE et al. 2008).
1.1.2 Clinical Presentation
In 1986 the “International Neuroblastoma Staging System” (INSS) (LUNDBERG et al.) was developed to improve diagnosis and for choosing the optimal therapy in human (risk stratification) (BRODEUR et al. 1993). Neuroblastoma is classified by the INSS in different stages (stage 1 – 2A/B - 3 - 4 and 4S) depending on the localization of the primary tumor, the occurrence of metastases and the age of the patient at diagnosis (Figure 1).
Figure 1 International Neuroblastoma Staging System.
(Modified from Schwab et al., 1999 and Maris et al., 2007.) Stage 1: Localized tumor with
complete gross excision;
negative ipsilateral lymph nodes (nodes attached to and removed with the primary tumor could be positive).
Stage 2A: Localized tumor with incomplete gross excision and negative ipsilateral non-adherent lymph nodes.
Stage 2B: Localized tumor with or without complete gross excision, with positive ipsilateral non-adherent lymph nodes.
Stage 3: Unresectable unilateral tumor infiltrating across the midline, with/without regional lymph node involvement;
or localized unilateral tumor with contralateral regional lymph node involvement;
or midline tumor with bilateral extension by infiltration (unresectable) or by lymph node involvement.
Stage 4: Tumor with dissemination to distant lymph nodes, bone (marrow), liver, skin etc. (except as defined for stage 4S).
Stage 4S: Localized tumor in infants <1 year with dissemination limited to skin, liver or bone marrow (<10%
malignant cells).
INSS
Introduction 9
Localized tumors are found in about 40% of all patients, whereas about 60% of patients present metastatic disease at diagnosis. Neuroblastomas usually spread to lymph nodes and the liver, but show also high tendency to metastasize to the cortical bone and bone marrow. Metastases can be further found in the skin, bony orbit or rarely in the brain (MARIS et al. 2007). In animals, metastases have been found in liver, lymph nodes and the brain. The clinical presentation depends on tumor localization and disease progression and is therefore highly variable. Patients, human or animal, with localized neuroblastomas are often in a normal “healthy”
status or show only few symptoms like abdominal pain due to the tumor mass. In contrast, the general condition of patients with metastatic disease is very poor, however, symptoms are often still unspecific like fever, diarrhea, anorexia, weight loss, dyspnea, bone pain, limping. Patients may also show neural symptoms - such as opsoclonus, myoclonus and ataxia. In the case of paraspinal tumors, patients might show signs of spinal cord compression, whereas patients with cervical or apical thoracic tumors could show Horner syndrome (miosis, ptosis, enophthalmus and anhidrosis) (HELMAN et al. 1980; MARCOTTE et al. 2004; STEINBERG et al. 2006;
SUZUKI et al. 2003; UCHIDA et al. 1998; WEINSTEIN et al. 2003).
1.1.3 Diagnosis
To diagnose neuroblastoma in human, computed tomography as well as magnetic resonance imaging are commonly used. For detection of bone metastases and occult soft tissue disease metaiodobenzylguanidine (MIBG) scintigraphy is the method of first choice because of its high sensitivity and specificity for neuroblastoma. 131I- or
123I-labeled MIBG binds to norepinephrine transporters on the cell surface and is selectively concentrated in more than 90% of neuroblastomas. The final diagnosis is done either by characteristic histopathologic evaluation of tumor tissue after surgery or by assessment of tumor cells in biopsies and/or bone marrow aspirates (WEINSTEIN et al. 2003). Maturation/Differentiation to ganglioneuroblastoma or to benign ganglioneuroma is one of the characteristic biological features of neuroblastoma. Therefore, in 1984 the SHIMADA classification and later in 1999 the International Neuroblastoma Pathology Classification (INPC) system were developed
to histologically classify the tumors into the three different categories depending on the degree of neuroblast differentiation, Schwannian stroma content and mitosis- karryorhexis index (Figure 2) (SHIMADA et al. 1999).
Figure 2 Histological classification of neuroblastoma.
(A) neuroblastoma, (B) ganglioneuroblastoma, (C) ganglioneuroma (fully mature ganglion cells embedded in Schwannian stroma, see arrows; modified from Shimada et al., 1999).
Serum markers - including ferritin, neuron-specific enolase, disialoganglioside GD2
and serum lactate dehydrogenase (LDH) - can be assessed at least in some patients with advanced neuroblastoma. These markers, however, are not used to predict outcome or to define therapy because significant correlation with tumor progression has not been proven. Specific for neuroblastomas is increased catecholamines production. The derivatives homovanillic acid and vanillylmandelic acid (VMA) can be detected in the urine of about 90% of all patients and are used as a diagnostic marker for neuroblastoma. In Japan, North America and Europe mass screenings had been undertaken in newborns in the hope that early detection of neuroblastomas by these urinary markers could improve the outcome of patients. Unexpectedly, most patients detected with this method presented tumors with favorable features and showed the tendency to spontaneously mature or regress (BRODEUR 2003).
However, every child with the suspected disease neuroblastoma is still examined for the secretion of catecholamines in the urine to confirm the diagnosis.
In animals, the diagnosis of neuroblastoma seems to be difficult due to unspecific clinical presentation. In most cases neuroblastoma has been diagnosed not until slaughter, euthanasia or death of the patient due to the disease. Small animal surgery offers quite more possibilities to diagnose neuroblastoma before the death of
A B C
Introduction 11
the animal. Similar to diagnosis in human, ultrasonography/computertomography and biopsies are used to detect and histologically classify neoplasia masses. As in human, neuroblastoma tumors are characterized by small round neuroblastic cells, perivascular pseudorosettes and Homer and Wright rosettes, although in some cases the Homer and Wright rosettes had been missed (MARCOTTE et al. 2004; SUZUKI et al. 2003; UCHIDA et al. 1998). Besides undifferentiated neuroblastomas, more differentiated ganglioneuroblastomas and benign ganglioneuromas were also sporadically observed in animals and showed the same histological features as in human (HAWKINS et al. 1987; KUWAMURA et al. 2004; NAKAMURA et al. 2004;
REIMER et al. 1999; RILEY et al. 1976; SCHULZ et al. 1994; SPUGNINI et al. 2008;
VAN DEN INGH et al. 1984; YENER et al. 2002).
1.1.4 Risk Stratification and Therapy
To predict patient outcome and to individually adjust therapy, the International Neuroblastoma Risk Group (INRG) classification system was developed. Patients are classified into three groups: i) low-risk disease, ii) intermediate-risk disease and iii) high-risk disease based on different parameters (age at diagnosis, INSS stage, histopathology, MYCN amplification status and DNA index). Characteristics for unfavorable tumors, besides amplified MYCN, are other structural chromosomal alterations, including unbalanced gain of chromosome arm 17q, 11q aberration and/or chromosome arm 1p deletion (BRODEUR 2003; COHN et al. 2009). Low-risk neuroblastoma patients are treated by surgery alone (MARIS et al. 2007). Compared to other cancers, stage 4S-tumors without amplified MYCN show the highest rate of spontaneous regression without or with only minimal treatment (5%-10% of all neuroblastomas clinically detected) (SCHWAB et al. 2003; WESTERMANN et al.
2002). Spontaneous regression has also been reported for localized neuroblastomas with normal MYCN status. The authors of this study hypothesized that localized neuroblastomas may represent stage 4S disease at different phases and, therefore, suggest to expand the observation period for localized tumors without amplified MYCN to avoid surgery and chemotherapy (HERO et al. 2008). The survival rate of low-risk patients with stage 1, 2A/B and 4S is 80%-90% (BRODEUR 2003). Patients
with intermediate-risk disease receive surgery and therapy consisting of different chemotherapeutic agents (e.g. cisplatin, etoposide, vincristine, doxorubicin, cyclophosphamide) and/or irradiation. The survival rate ranges between 10%-70%.
High-risk neuroblastoma patients are characterized by age at diagnosis older than 18 months and stage 4 disease or any stage with amplified MYCN. These patients need high-modality treatment consisting of high-dose chemotherapy, irradiation, autologous stem cell transplantation, MIBG-targeted radiation therapy and biological therapy targeting the unique biology of neuroblastoma (e.g. 13-cis-retinoic acid to induce differentiation). Relapses frequently occur in high-risk neuroblastoma patients despite intensive multimodality treatment, which is mainly due to drug resistance (WEINSTEIN et al. 2003). Therefore, the event-free survival rate of high-risk neuroblastoma patients is less than 30% (MARIS et al. 2007).
To date, prognostic markers for neuroblastoma in animals are poorly investigated. A specific treatment plan has still to be established. Most animal patients showed a very poor general condition at diagnosis that consequently led in many cases to euthanasia. Therefore, first therapy steps should address the stabilization of the patient, and a symptomatic treatment should be initiated (MARCOTTE et al. 2004).
Surgery is the treatment of choice if a localized tumor is diagnosed and no metastases are observed (HERMEYER et al. 2007; NAKAMURA et al. 2004).
Although chemo- or radiotherapy is not described for animal neuroblastoma patients yet, this could be an alternative and/or complementary therapeutic option in future.
1.1.5 Differential Diagnosis
Neuroblastoma belongs to the group of “small blue round cell tumors” (SBRCT) or
“small round cell tumors” (SRCT). Tumors of this group are histologically characterized by small blue round cells that are often arranged in so called Homer and Wright rosettes. Other SBRC tumors are: primitive neuroectodermal tumors (PNET), Ewing sarcoma, rhabdomyosarcoma, desmoplastic small round cell tumor, Wilms tumor, small cell lymphoma and hepatoblastoma (DAS 2004). To distinguish between these tumors, different techniques are used, including fluorescence in situ hybridization (FISH) or reverse transcription PCR (CHEN et al. 2007).
Introduction 13
1.2 The MYC Family of Transcription Factors
The MYC protein family consists of three members, MYC, MYCN and MYCL, which represent transcription factors that affect essential regulators of physiologic cellular processes, including the cell cycle, differentiation, cell growth, cell adhesion and angiogenesis, but are also involved in transformation (MEYER et al. 2008).
Transcriptional activity of MYC family members is mediated by sequence-specific DNA binding through a characteristic basic region and a helix-loop-helix-leucine zipper domain (ADHIKARY et al. 2005) (Figure 3).
Figure 3 MYC transcription factor.
In 1979 the transforming sequence of the MC29 avian tumor virus was discovered that causes myelocytomatosis, a form of leukaemia, but also endotheliomas, sarcomas and liver or kidney carcinomas in chicken. Only three years later, in 1982, the human homologue of v-gag-myc fusion protein, which is encoded by v-myc, was identified, refered to as MYC (formerly designated as c-MYC). Apart from retroviral
MAX MIZ1
I II IV BR HLH-LZ
PP NLS
MYC N C
sequence specific DNA binding dimerization domain
MYC boxes I-IV are highly conserved elements located in the N-terminus of MYC. MYC box I contains MYC regulatory phosphorylation sites. Activation of gene transcription to a heterologous DNA binding-domain is mediated by the transactivation domain (TAD).
Subcellular localization to the nucleus is encoded primarily by the NLS region (primary nuclear localization signal). The C-terminus of MYC contains the basic-region (BR) and helix-loop-helix-leucine zipper (HLH-LZ) domains,which are essential for specific binding of canonical and non-canonical MYC E-boxes to the DNA, together with MAX or MIZ1.
Interactions resulting in transcriptional repression by MYC are shown in red bars.
Interactions mediating both, transcriptional activation and repression, are shown in dashed bars.
TAD
modified from Meyer and Penn, 2008
cellular proto-oncogene activation there are three other mechanisms known that can lead to oncogene activation: mutation, translocation and gene amplification (MEYER et al. 2008). Activation of the proto-oncogene MYCN as a result of gene amplification was first described in neuroblastoma in 1983 (SCHWAB et al. 1983). Amplified MYCN manifests itself in form of intrachromosomal homogeneously staining regions (HSRs) or extrachromosomal double minutes bodies (dmins) with amplification values frequently ranging from 50 to 100 fold (SCHWAB 1992). In neuroblastoma, amplified MYCN is found in about 20% of all cases, shown to be highly correlated with poor outcome, and represents one of the most powerful prognostic factors for neuroblastoma patients to date (SCHWAB et al. 2003). However, amplified or overexpressed MYCN is also found in other pediatric and adult tumors that are rhabdomyosarcoma, medulloblastoma, retinoblastoma, astrocytoma, glioblastoma, small cell lung carcinoma and Wilms tumor (STRIEDER et al. 2002).
Deregulated expression of MYCN protein contributes to tumor formation by promoting angiogenesis, preventing differentiation and by deregulation of cell proliferation, and might also play a role in promoting genomic instability (BRODEUR 2003). In contrast, MYCN also activates apoptosis (FULDA et al. 1999) mainly by up- regulation of the tumor suppressor p53 (WESTERMANN et al. 2008). This contradiction of oncogenic- and tumor suppressor-like functions of MYC transcription factors and their contribution to tumorigenesis has still to be elucidated. The oncogene addiction model addresses this question and proposes that “the function of these proteins is also influenced by their level of activity and the context in which they are expressed“ (WEINSTEIN et al. 2006). Oncogenes may deregulate specific pathways to compensate for additional gene mutations for maintaining a state in which the evolving cancer cell is able to survive (WEINSTEIN et al. 2006). In line with this proposal, the number of genes directly activated or repressed by MYCN increases in MYCN-amplified neuroblastomas with unfavorable biology compared to tumors with normal MYCN status and favorable biology (WESTERMANN et al. 2008).
This indicates that activation and/or repression of distinct genes by MYCN might decide whether the MYCN-mediated oncogenic-like functions outweigh the tumor suppressor-like functions or vice versa.
Outline of the thesis 15
1.3 Outline of the study
Therapy-resistance and chromosomal instability in form of structural chromosomal alterations and near-di/tetraploidy are characteristics of high-risk neuroblastomas. To date, adaptation processes facilitating these tumor characteristics are poorly understood for neuroblastoma. Identifying these mechanisms may not only increase our understanding of why some neuroblastomas tend to spontaneously regress, whereas others are associated with an aggressive and lethal disease, but further help to develop novel therapeutic concepts to improve the outcome of high-risk neuroblastoma patients. It is still under discussion whether chromosomal instability is a cause or consequence of tumorigenesis. Normal diploid human cell cultures also mis-segregate chromosomes about once every hundred cell divisions (CIMINI et al.
1999; THOMPSON et al. 2008), which normally leads to cell death. Genetic inactivation or functional deregulation of certain genes that regulate pathways, which are important to recognize and eliminate these defective cells and drive cell proliferation, represents a crucial selection step and maybe the initiating step to tumorigenesis. Therapy-resistance might represent another selection process or result from deregulation of the same genes, which facilitate the tolerance of chromosomal instability (SWANTON et al. 2009). Several studies suggest that loss of p53 and/or pRB functionality is involved in the origin and adaptation to chromosomal instability and therapy-resistance (THOMPSON et al. 2010). Since high-risk neuroblastomas are characterized by MYCN overexpression resulting from gene amplification, in turn leading to deregulation of several p53 and/or pRB pathway components, this thesis primarily focusses on the relationship between the MYCN status, p53 and pRB signaling, chromosomal instability and therapy-resistance in neuroblastoma cells:
i) First aim of this thesis was to investigate the role of MYCN in the (de-) regulation of drug-induced DNA damage response in neuroblastoma cells (manuscript I).
ii) Second aim was to investigate mechanisms, potentially MYCN-mediated, that contribute to the development of numerical and structural chromosomal alterations and distinguish neuroblastomas with unfavorable biology from tumors with favorable biology (manuscript II).
17 Manuscript I
2 Manuscript I
The following manuscript was submitted to Oncogene, 04.06.2012.
CDK4 inhibition restores G1-S arrest and induces synthetic lethality in MYCN- amplified neuroblastoma cells in the context of drug-induced DNA damage
Sina Gogolin1, Volker Ehemann2, Gabriele Becker3, Lena M. Brueckner1, Daniel Dreidax1, Steffen Bannert1, Ingo Nolte4, Larissa Savelyeva1, Emma Bell1 and Frank Westermann1
1Division of Tumor Genetics, German Cancer Research Center, Heidelberg, Germany
2Department of Pathology, University of Heidelberg, Heidelberg, Germany
3Clinical Cooporation Unit Pediatric Oncology, German Cancer Research Center, Heidelberg, Germany
4Small Animal Clinic, University of Veterinary Medicine Hannover, Hannover, Germany
Funding: BMBF: NGFNPlus #01GS0896 (L. M. Brueckner, L. Savelyeva, F. Westermann), European Union (EU, FP6): E.E.T.-Pipeline (#037260, D. Dreidax, S. Gogolin, F.
Westermann), EU (FP7): ASSET (#259348-2, D. Dreidax, S. Gogolin, F. Westermann), Helmholtz-Russia Joint Research Groups: HRJRG-006 (L. M. Brueckner, L. Savelyeva), EU Marie-Curie-Fellowship (E. Bell)
Corresponding author Dr. Frank Westermann
German Cancer Research Center Im Neuenheimer Feld 280
69120 Heidelberg, Germany E-mail: f.westermann@dkfz.de
Running title: CDK4 inhibition is synthetically lethal with amplified MYCN
Contributions to the study:
- pharmacological treatment, western blotting, immunoprecipitation, kinase activity assays, siRNA interference experiments and viability assays
- flow cytometric analyses together with Gabriele Becker and PD Dr. Volker Ehemann
- fluorescence in situ hybridization together with Lena M. Brueckner - interpretation of results
- generating the theoretical model
- drafting of the manuscript together with PD Dr. Frank Westermann
19 Manuscript I
2.1 Abstract
Drug resistance of relapse tumors is the main cause of death from disease in patients with neuroblastomas harboring amplified MYCN. In line with this, MYCN- amplified neuroblastoma cells fail to arrest in G1 and show reduced cell death induction upon drug-induced DNA damage. Here, we found that besides amplified MYCN additional chromosomal aberrations, such as CDK4/CCND1/MDM2 amplifications or TP53 mutations, were associated with impaired G1-S arrest resulting in mitotic checkpoint activation and resistance to cell death upon doxorubicin treatment. CDK4 and CDK2 competed for p21-binding in MYCN- amplified cells with wild-type p53 resulting in an insufficient amount of p21 to effectively inhibit CDK2, and consequently resulted in both high CDK4 and CDK2 kinase activity after doxorubicin treatment. CDK4 inhibition either by siRNAs, selective small compounds or p19INK4D overexpression partly restored G1-S arrest and delayed S phase progression upon doxorubicin treatment and enhanced cell death. Our results suggest a specific function of p19INK4D, but not p16INK4A, in inducing S phase arrest in MYCN-amplified cells that sensitizes these cells to doxorubicin-induced cell death. S phase arrest and enhanced cell death after drug treatment was not observed in the presence of CDK2-targeting siRNAs. Moreover, CDK2 inhibition reversed the doxorubicin-sensitizing effect of CDK4 inhibition. In summary, the CDK4/cyclin D-pRB axis controls cell cycle progression after drug- induced DNA damage as an additional layer to the p53-p21 axis. CDK4 inhibition in a MYCN-amplified cellular background combined with drug-induced DNA damage induces synthetic lethality in neuroblastoma cells. Selective CDK4 inhibitors potentiate the effect of conventionally used chemotherapeutic drugs in high-risk and relapsed neuroblastomas by capitalizing on synthetic lethal effects.
2.2 Introduction
Neuroblastoma is the most common solid extracranial tumor in early childhood with clinical phenotypes varying from spontaneous regression or differentiation to relentless progression (1-3). The 5-year survival rate of high-risk neuroblastoma patients remains below 35% (4-6), which is mainly due to drug resistance of tumors and metastases after relapse. Amplified MYCN is a powerful prognostic factor in neuroblastoma. It occurs in about 20% of all primary neuroblastomas and is associated with tumor progression or relapse and poor patient’ outcome (7, 8).
Cellular DNA damage response after irradiation or drug exposure involves different biological processes, including cell cycle arrest, apoptosis, differentiation and DNA repair. The p53 and pRB tumor suppressors are both important to prevent replication of cells with damaged DNA, implying that the clinical efficacy of chemotherapeutic drugs or irradiation will be influenced by both p53 and pRB status in the target tumor.
Following drug-induced DNA damage, p53 protein is up-regulated leading to transcriptional activation of a large number of target genes, including BAX and CDKN1A (p21CIP1). p21, in turn, binds to CDK2 to inhibit its function and cause G1 cell cycle arrest. Proapoptotic genes activated by p53, such as BAX, may trigger cell death when cell cycle arrest to allow DNA repair is insufficient. Functional pRB is also essential for G1 arrest following irradiation or drug treatment (9, 10). This is supported by RB1-/- mouse embryo fibroblasts failing to arrest in G1-S despite p53- p21CIP1 activation (11). Loss of pRB does not interfere with either p21 induction or inhibition of CDK2 activity in response to -irradiation, suggesting that pRB acts downstream of p21 during p53-dependent G1 arrest (12). Intriguingly, drug-induced DNA damage causes RB1+/+ and RB1+/- fibroblasts to arrest but induces apoptosis in RB1-/- fibroblasts. From these findings, it has been hypothesized that the susceptibility of tumor cells - as opposed to normal cells - to undergo p53-dependent apoptosis arises from their inability to enforce an pRB-dependent cell cycle arrest.
Consequently, alterations of the p53 pathway, including TP53 mutations, would mark a switch to a chemotherapy-resistant tumor.
Although frequent in other human cancers (13), TP53 mutations occur only in less than 2% of primary neuroblastomas. MDM2 amplification and loss of p14ARF, which
21 Manuscript I
result in inhibition of p53 functions, occasionally occur in relapse tumors and neuroblastoma cell lines established from tumors following chemotherapy (14-16).
MYCN regulates several components of the p53-p21 axis (17-21): MYCN transcriptionally activates both TP53 and the p53 inhibitor, MDM2, and suppresses p21CIP1 transcription. However, p53 remains transcriptionally active and induces p21 after irradiation- or drug-induced DNA damage in MYCN-amplified neuroblastoma cells (22). This suggests that MYCN does not significantly interfere with p21 induction in response to DNA damage in these cells. Surprisingly, MYCN- amplified neuroblastoma cells failed to induce functional G1 arrest despite p53 and p21 induction (22). We have previously established that MYCN upregulates the CDK4/cyclin D1 complex in high-risk neuroblastomas, and particularly MYCN- amplified tumors (18). We hypothesized here that highly abundant CDK4/cyclin D1 may weaken the G1 checkpoint after drug-induced DNA damage in MYCN-amplified neuroblastoma cells, and investigated whether amplified MYCN and/or chromosomal aberrations of pRB pathway members (e. g. CDK4 or CCND1 amplification, p16- INK4A deletion) are associated with an attenuated G1 arrest after drug-induced DNA damage in neuroblastoma cell lines. Because CDK4- and CDK2-containing complexes both bind p21, we tested whether highly abundant CDK4/cyclin D1 complexes compete with CDK2-containing complexes for newly induced p21 after drug-induced DNA damage. To test whether CDK4 inhibition can restore a functional G1 arrest and sensitize cells to drug-induced death, we inhibited CDK2 and CDK4 using small molecule inhibitors, shRNA/siRNA methodology and tetracycline- inducible cell models to modulate p19INK4D and p16INK4A expression.
2.3 Results
2.3.1 Deregulated MYCN impairs cell cycle arrest after drug-induced DNA damage
To define the role of MYCN after drug-induced DNA damage, we used two MYCN regulatable neuroblastoma cell models, one having a MYCN-amplified genetic background and the other, single-copy MYCN. Cell cycle distribution and cell death were analyzed using flow cytometry following no or doxorubicin (doxo) treatment.
MYCN-amplified IMR5/75-C2 stably express a tetracycline-inducible MYCN shRNA that, upon induction, reduced MYCN protein to approximately 35% (23). Untreated IMR5/75-C2 cultures with high endogenous MYCN expression showed higher numbers of cycling cells (S and G2/M) compared to IMR5/75-C2 expressing the MYCN shRNA, indicating that even reducing MYCN protein levels to ~35% has a robust impact on cell cycle distribution (Figure 1a). Doxo treatment further depleted uninduced IMR5/75-C2 cultures of G0/1 phase cells. Reduction of MYCN by inducing the MYCN-targeting shRNA in these cultures restored G1 arrest after doxo treatment and diminished the G2/M fraction by 2.5-fold (Figure 1a). The subG1 fraction, indicative of apoptosis, after doxo treatment was not significantly different between IMR5/75-C2 cultures expressing low or high MYCN (33.8% ± 0.5 and 34.6% ± 0.4, respectively) (Figure 1b).
We compared the findings in IMR5/75-C2 with those in SH-EP-MYCN (TET21N), which stably express a tetracycline-regulatable MYCN transgene allowing MYCN induction by removal of tetracycline from the culture medium (24). Untreated SH-EP- MYCN cultures expressing the MYCN transgene contained higher numbers of cycling cells (S and G2/M) than cultures without MYCN transgene expression. Doxo treatment of MYCN-expressing SH-EP-MYCN cultures further reduced the G0/1 fraction by 7.4% of untreated cultures, whereas doxo treatment did not affect the fraction of cells in G0/1 in SH-EP-MYCN cultures with an inactive MYCN. Doxo treatment reduced the fraction of SH-EP-MYCN cells in S phase and enriched the fraction of SH-EP-MYCN cells in the G2/M phase regardless of whether the MYCN transgene was activated or not (Figure 1a). The subG1 fraction of either untreated or
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doxo-treated SH-EP-MYCN cells overexpressing MYCN was also higher than in cultures without the active MYCN transgene (Figure 1b).
These experiments demonstrate that ectopic MYCN expression in neuroblastoma cells with a single-copy MYCN genetic background does not fully recapitulate the response to doxo in MYCN-amplified cells, and suggest that the higher MYCN dosage together with the cellular genetic background establishes the impaired DNA damage response in MYCN-amplified cells.
Figure 1 Amplified MYCN and additional chromosomal aberrations impair drug- induced DNA damage response in neuroblastoma cells. SH-EP-MYCN cells were treated with tetracycline to suppress MYCN transgene expression. IMR5/75-C2 cells were treated with tetracycline to induce the shRNA targeting MYCN (=MYCN -). Doxo was added to the culture medium 48h later after tetracycline addition. Cell cycle (a) and cell death (b) were analyzed using flow cytometry 48h after doxo addition. Data are presented as mean ±SD of triplicates. (b) also shows a western blot of MYCN
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2.3.2 MYCN amplification and chromosomal aberrations of p53 and/or pRB pathway members impaired drug-induced DNA damage response in neuroblastoma cells
We next tested whether other genetic aberrations besides MYCN amplification, and particularly those affecting the p53 and/or pRB pathways, establish the impaired drug-induced DNA damage response. We analyzed the effect of doxo treatment on the cell cycle and cell death in 13 well-characterized neuroblastoma cell lines and a primary neuroblastoma short-term culture (NB-7) using flow cytometry (Table 1 and Supplementary Figure 1). The percent change in the fraction of cells in the G0/1 and S phases and the fold-change of the G2/M phase cell enrichment were determined after doxo treatment compared to untreated control cultures. Together these values were used to define characteristic neuroblastoma cell responses to DNA damage and separate the cell lines into defined DNA damage response groups (Table 1).
Eight of nine tested MYCN-amplified neuroblastoma cell lines showed prominent reduction of G0/1 and S phase cells and massive enrichment of G2/M phase cells after doxo treatment (response group 1). Only MYCN-amplified LA-N-5 showed S phase cell enrichment associated with reduction of G0/1 phase cells (response group 2). The failure of most MYCN-amplified cell lines to arrest in G1 and/or S phase after doxo treatment was associated with additional chromosomal aberrations of p53 and/or pRB pathway members. For instance, NGP and LS both harbor amplified MYCN, CDK4 and MDM2, and showed the most pronounced G0/1 fraction reduction and G2/M cell enrichment after doxo treatment (Supplementary Figure 1; LS additionally harbor an amplified CCND1 gene, Supplementary Figure 2).
Neuroblastoma cell lines lacking amplified MYCN responded variably to drug-induced DNA damage, and the response was dependent on chromosomal aberrations affecting p53 and/or pRB pathway members. SK-N-AS harbor a TP53 mutation, and showed a prominent reduction of G0/1 and S phase cells and G2/M fraction enrichment (response group 1) after doxo treatment, similar to MYCN-amplified cells.
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LA-N-6, with a p16-INK4A/p14ARF deletion and a CCND1 duplication had a similar DNA damage response phenotype (response group 2) to MYCN-amplified LA-N-5, but lacked S phase cell enrichment (Table 1 and Supplementary Figure 2). SH-EP, a subclone of SK-N-SH that harbors a p16INK4A/p14ARF deletion, showed an intermediate DNA damage response phenotype (response group 3) with no change in the G0/1 fraction and a reduced S phase fraction. This is probably due to deleted p16INK4A/p14ARF, because another subclone of SK-N-SH with normal p16INK4A/p14ARF, SH-SY5Y (response group 4), did not show a prominent change in the S phase fraction after doxo treatment. In summary, these results demonstrate that – besides amplified MYCN - chromosomal aberrations of p53 and/or pRB pathway members impair G1-S cell cycle arrest and lead to G2/M cell enrichment.
We quantified the enhancement of cell death by doxo treatment by calculating the difference between the subG1 fractions of doxo-treated and untreated cultures. The strongest enhancement of cell death occurred in SH-SY5Y (Table 1). Doxo treatment induced less cell death in the other SK-N-SH subclone, SH-EP, compared to SH- SY5Y. As expected, neuroblastoma cell lines harboring chromosomal aberrations of p53 pathway members (either p14ARF deletion, MDM2 amplification or TP53 mutation) exhibited significantly lower enhancement of cell death (p=0.014, Wilcoxon test).
We further investigated whether doxo-induced cytotoxicity is dependent on the capability of neuroblastoma cells to arrest in G1-S. To determine the cell cycle phase of cell death induction, DNA breaks in G0/1, S and G2/M phases were assessed using a double staining with propidium iodide and BrdUTP after doxo treatment.
MYCN-single-copy SH-SY5Y were used as a reference, because doxo treatment induced cell death most strongly in this cell line. BrdUTP-positive SH-SY5Y cells were predominantly observed in the G0/1 and S phases. Contrastingly, only few BrdUTP-positive cells were observed in the G0/1 and S phases in all the MYCN- amplified cell lines that were analyzed (Figure 1c), suggesting that the failure to arrest in G0/1 and/or S phase after doxo treatment is associated with cell death resistance in MYCN-amplified cells. We also observed that the number of DNA breaks differed in the MYCN-amplified cell lines, which was dependent on additional
chromosomal aberrations of p53 and/or pRB pathway members. Thus, SK-N-BE(2)- C, Kelly and LS, harboring either a TP53 mutation or amplification of CDK4 and MDM2, showed less BrdUTP-positive cells compared to IMR5/75 that has any of the investigated additional chromosomal aberrations besides amplified MYCN.
Collectively, this argues in favor for an important role of chromosomal aberrations in p53 and/or pRB pathway members, including CDK4/CCND1 and MDM2 amplifications, p16INK4A/p14ARF deletion or TP53 mutation, in impairing G1-S arrest and cell death after doxo treatment.
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Table 1. Drug-induced DNA damage response of neuroblastoma cell lines. No. DNA damage response group cell lineG0/1 changes after doxo (%) S changes after doxo (%) G2/M enrichment (fold) cell death enhance ment (%)
MYCN/ MYC status
pRB pathway alterationsp53 pathway alterationsother genetic alterations 1 responseNB-7 -7 -95 4.0 20 MYCN amp 12 1p del; 17q gain 2 group 1 TR14 -11 -37 3.7 3 MYCNamp CDK4amp (dmins) MDM2 amp (dmins) 1p del; 17q gain 3 SK-N-BE(2)-C -11 -78 3.9 7 MYCN amp 1 TP53 mut1p del; 17q gain 4 IMR-32-16 -67 3.6 16 MYCN amp 12 1p del; 11q del; 17q gain 5 IMR5/75 -22 -100 4.1 28 MYCN amp 12 1p del; 11q del; 17q gain 6 Kelly -34 -100 11.1 11 MYCN amp 1 TP53 mut1p del; 11q del; 17q gain 7 SK-N-AS -60 -100 5.0 3 high MYC 1 TP53 mut1p del; 11q del; 17q gain 8 NGP-88 -77 10.0 n.d.MYCN amp CDK4 amp (HSR) MDM2 amp (HSR) t(1p); 11q del; 17q gain 9 LS-90 -99 4.7 11 MYCN amp CDK4 amp (HSR);CCND1 amp (HSR) MDM2 amp (HSR) 1q gain; 17q gain 11 response LA-N-5 -14 33 1.5 17 MYCN amp 12 1p del; 17q gain 10 group 2 LA-N-6 -14 -2 4.2 11 high MYC p16-INK4A del; CCDN1 duplp14ARF del1p del; 11p del; 14q gain; 17q gain 12 responseSH-EP4 -92 2.7 25 high MYC p16-INK4Adelp14ARF deldel (14); 17q gain 13 group 3 SJ-NB-12-4 -91 7.4 11 MYC amp p16-INK4A delp14ARF del1p del; 17q gain 14 response group 4 SH-SY5Y -6 -4 1.5 50 high MYC 12 del (14); 17q gain 15 Ewing sarcomaSK-N-MC 26 0 0.04 42 MYC amp n.d. TP53 mutEWS-FLi1gene fusion Measure for G1 or S arrest after drug-induced DNA damage response is given by the change of cells in G0/1 or S phase after doxo treatment compared to the untreated control;G2/M enrichment = ratio G2/M doxo/G2/M untreated; cell death enhancement = subG1 doxo subG1 untreated; all experiments were done in triplicate; copy number status of p16-INK4A, CDK4/6, RB1, CCND1 normal; 2copy number status ofp14ARF, MDM2, TP53normal; amp = amplification, dupl = duplication, del = deletion, t(1p) = reciprocal 1;15 translocation, high = high expression, mut = mutation, dmins = double minutes, HSR = homogeneously staining region, n.d. = not defined
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2.3.3 Competitive binding of p21 to CDK4 and CDK2 complexes results in high CDK4 and CDK2 kinase activities in MYCN-amplified neuroblastoma cells after drug-induced DNA damage
Deregulated MYCN increases the activity of the G1 cell cycle kinases, CDK4 and CDK2, by transcriptionally activating CDK4 (18) and suppressing p21CIP1 (17). We hypothesized that impaired G1 arrest after drug-induced DNA damage could be associated with high G1 cell cycle kinase activity. CDK2 and CDK4 activity was assessed in untreated and doxo-treated cell lines with various MYCN or MYC genetic backgrounds, including single-copy MYCN (SH-SY5Y and SH-EP), amplified MYCN (IMR-32, Kelly and LS) and amplified MYC (SK-N-MC Ewing sarcoma cell line). The SK-N-SH subclone, SH-SY5Y, showing almost unchanged G0/1 and S phase fractions but massive cell death after doxo treatment, exhibited the lowest CDK2 and CDK4 activity in untreated and doxo-treated cultures of all cell lines that we analyzed.
The other SK-N-SH subclone, SH-EP, harboring the p16INK4A/p14ARF deletion, exhibited higher CDK4 and CDK2 activities than SH-SY5Y, which could be due to the lack of p16INK4A and p14ARF expression. CDK4 activity tended to decrease after doxo treatment in all three MYCN-amplified cell lines, but remained higher than in untreated SH-SY5Y. CDK2 activity decreased in only one out of three MYCN- amplified cell lines after doxo treatment, but remained higher than in both SH-SY5Y and SH-EP subclones with single-copy MYCN (Figure 2a). Similar to CDK4, CDK1 activity also remained high in MYCN-amplified neuroblastoma cell lines after doxo treatment (Supplementary Figure 3). These results indicate that high activity of cell cycle kinases is poorly affected by DNA damage-inducing drug treatment of neuroblastoma cells harboring amplified MYCN.
Following drug-induced DNA damage, activity of CDK4 and CDK2 is controlled by activation of the p53-p21 axis but also - independent of p53 - through proteolysis of cyclin D1 releasing p21 from the CDK4/cyclin D1 complex, which in turn inactivates CDK2 (25). We evaluated the relative amounts of p21 in G1 cell cycle kinase complexes before and after doxo treatment in two MYCN-single-copy, four MYCN- amplified neuroblastoma cell lines and the SK-N-MC Ewing sarcoma cell line using co-immunoprecipitation with antibodies against CDK4 and CDK2. All cell lines
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harboring TP53 mutations, which included Kelly, SK-N-BE(2)-C and SK-N-MC, lacked p21 expression independent of doxo treatment (Figure 2b). Generally, p21 levels were low in untreated neuroblastoma cells. Thus, complexes precipitated with antibodies against either CDK4 or CDK2 in most untreated neuroblastoma cell lines contained barely detectable amounts of p21. With such low levels of available p21, only a small pool can be expected to shift from CDK4- to CDK2-containing complexes upon doxo treatment. Doxo treatment induced p21 expression only in neuroblastoma cells harboring wild-type p53. Induction of p21 after doxo treatment was less in all cell lines that also harbored amplified MYCN, namely IMR-32 and LS (Figure 2b). Following doxo treatment, p21 was distributed in both CDK4- and CDK2- containing complexes. Overall CDK2 activity remained high after doxo treatment in MYCN-amplified, TP53-wild-type cells, despite a substantial amount of p21 in CDK2- containing complexes. Sequestration of p21 produced in response to DNA-damaging drugs away from CDK2 complexes into highly abundant CDK4/cyclin D1 complexes may at least, in part, causes unaltered CDK2 activity in the neuroblastoma cell lines harboring amplified MYCN.
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Figure 2 High CDK4 and CDK2 activity after doxorubicin treatment of MYCN- amplified cells. (a) CDK4 and CDK2 activity were analyzed 48h after treatment using RB and histone 1 as substrates, respectively. Luminescence directly correlates to the amount of produced ADP, indicative for kinase activity. Data are presented as mean ±SD of duplicates. (b) Whole cell protein extracts were prepared 48h after doxo treatment, and immunoprecipitated with anti-CDK4, anti-CDK2 or control anti-p38 antibodies. Then, 50 µg of whole cell protein extracts (ext) and 500 µg of the immunoprecipitates (IP) were separated on 12.5% SDS-PAGE. β-actin was used as loading control for whole cell protein extracts.
2.3.4 CDK4 inhibition partially restores cell cycle arrest, reduces viability and increases cell death after drug treatment in MYCN-amplified cells
To further investigate the effects of CDK4 and CDK2 activity on drug-induced cell cycle arrest in MYCN-amplified cells, CDK4 and/or CDK2 were selectively inhibited in combination with doxo treatment, and cell cycle changes were assessed using flow cytometry. Transient transfection of two CDK4 siRNAs achieved knockdown efficiencies of 76% and ~96% in LS and SK-N-BE(2)-C, respectively, at the protein level (Figure 3a). CDK4 knockdown increased the G0/1 fraction and decreased the number of cells in G2/M phase in both untreated and doxo-treated cultures compared to non-transfected or with control siRNAs transiently transfected cells (Figure 3b and Supplementary Figure 4). To validate this effect of CDK4 inhibition, we treated twelve neuroblastoma cell lines harboring single-copy MYCN or amplified MYCN/MYC and/
or chromosomal aberrations of p53 and/or pRB pathway members with a CDK4- specific small molecule inhibitor (RO050124), which has been shown to delay G0/1 in cells with functional pRB (26). Combined RO050124 and doxo treatment increased the G0/1 and S phase fractions in nine and six cell lines, respectively, and reduced the G2/M fraction in eleven cell lines compared to cultures treated only with doxo (Figure 3c, results are shown for SK-N-BE(2)-C, LS, Kelly and IMR-32). This confirmed our results obtained by transient silencing of CDK4 using siRNAs. We next tested whether CDK4 inhibition by RO050124 was capable of enhancing the inhibitory effect of doxo on neuroblastoma cell viability using the alamarBlue assay.
We here focused on MYCN-amplified neuroblastoma cell lines harboring additional aberrations of p53 pathway members, namely TP53-mutant SK-N-BE(2)-C and MDM2-amplified LS, which responded poorly to doxo treatment with only cell death
responses of 7% and 11%, respectively (Table 1). Combined treatment achieved at least an additive effect on viability reduction for both cell lines compared with the doxo or RO05012 treatment alone (Figure 3d). These results show that CDK4 inhibition sensitize MYCN-amplified neuroblastoma cells to doxo treatment.
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