PhD Thesis

Investigating the role of the PIDDosome in tumorigenesis

Submitted by Lukas Peintner, MSc

for the granting of the academic degree Doctor of Philosophy (PhD)

at the

Medical University of Innsbruck Division of Developmental Immunology Biocenter

Under the supervision of Univ.-Prof. Dr. Andreas VILLUNGER

2015

ABSTRACT Caspase-2 functions as a proteolytic enzyme and was shown to act as a tumor suppressor gene in some mouse tumor models. For example, Caspase-2 was reported to have tumor suppressor functions in c-Myc driven B cell tumors, in ATM deficient T cell lymphomas or in MMTV/c-neu-driven breast cancer as well as K-Ras- driven lung cancer. Extensive efforts were put into understanding how Caspase-2 is activated and which of its postulated biological roles actually drives its tumor suppressor function. A high molecular protein complex called the PIDDosome was reported to form right before activated Caspase-2 emerges in stressed cells. The PIDDosome, consisting of Procaspase-2, the p53-induced protein with a death domain, PIDD, and the scaffold protein RAIDD is since believed to be an important activation platform for Caspase-2. However, mice lacking Pidd still show normal activation of Caspase-2 after genotoxic stress, pointing out that alternative modes of activation likely exist. In the work presented here, I report on my investigation on the role of Caspase-2 and its associated proteins Raidd and Pidd in tumorigenesis. I provide evidence that Caspase-2 does not show tumor suppressor function in tumor models driven by low dose radiation or bulky adduct formation but confirm its tumor suppressor role in the Eµ-Myc mouse model. Surprisingly, loss of Raidd does not shorten tumor latency while loss of Pidd delays tumor onset in this tumor model. The delay of tumor formation cannot be explained by defects in proliferation, differentiation, apoptosis or migration of B-cells, but mice lacking Pidd show deregulated levels of cytokines in their serum, which might influence tumor growth. Altogether, I was able to confirm the tumor suppressor function of Caspase-2 in MYC-driven B cell lymphomagenesis. The tumor suppressive effect of Caspase-2 appears to be independent of the two proteins Raidd and Pidd that were shown to interact with Caspase-2 in the PIDDosome.

ii

Zusammenfassung Caspase-2 ist ein proteolytisches Enzym welches eine wichtige Rolle bei der Unterdrückung von Krebs spielt. Es konnte festgestellt werden, dass Caspase-2 als „Anti-Onkogen“ die Ausbildung von MYC induzierten B Zelllymphomen, ATM- defizienten T Zelllymphomen, MMTV/c-neu gesteuerten Brustkrebs und K-Ras getriebenen Lungenkrebs unterdrücken kann. Von großem Interesse war daher in den letzten Jahren, wie Caspase-2 genau aktiviert wird und welche der verschiedenen biologischen Funktionen, die Caspase-2 zugeschrieben wurden, für die Unterdrückung von Tumorzellwachstum verantwortlich sind. Wenn Zellen DNA-schädigenden Behandlungen ausgesetzt werden, kann es zur Bildung von einem Proteinkomplex, der als das „RAIDD-PIDDosom“ bezeichnet wird, kommen. Dieser Komplex, welcher aus den Proteinen Caspase-2, PIDD und dem Gerüstprotein RAIDD besteht, könnte eine wichtige Rolle in der Aktivierung von Caspase-2 und somit bei der Tumorsuppression spielen. Überraschenderweise konnte aber bereits früh gezeigt werden, dass Caspase-2 nach genomschädigendem Stress auch in Abwesenheit von PIDD aktiviert werden kann. Die vorliegende Arbeit beschreibt meine Untersuchungen zur Rolle von Caspase-2 und deren aktivierenden Proteinen bei der Entwicklung von Krebs in unterschiedlichen Tumormodellen in der Maus. Diese Untersuchungen zeigen, dass Caspase-2 und die anderen Komponenten des PIDDosoms keine Hemmer von Tumoren sind, die z.B. durch wiederholte Bestrahlung oder chemische Mutagenese induziert wurden. Andererseits konnte die tumorunterdrückende Funktion von Caspase-2 in dem Eµ-Myc Lymphom Modell bestätigt werden. Dabei wurde ersichtlich, dass das Gerüstprotein Raidd keinen Einfluss auf die Tumorentstehung hat. Der Verlust von Pidd hingegen verzögert die Bildung von B Zell Lymphomen deutlich. Diese Verzögerung konnte aber nicht durch Fehler im Wachstum, der Differenzierung oder anhand des Zelltodes von B Zellen erklärt werden. Eine Deregulation der Zyktokinausschüttung nach onkogenem Stress, welche das Tumorwachstum eventuell indirekt beeinflusst, könnte hierfür ebenfalls von Bedeutung sein. Zusammenfassend bestätigen diese Untersuchungen die unterdrückende Funktion von Caspase-2 im Tumorwachstum. Diese Rolle von Caspase-2 scheint jedoch komplett unabhängig von den Proteinen RAIDD und PIDD zu sein, obwohl beschrieben wurde, dass diese Proteine mit Caspase-2 interagieren können.

iii

Chapters Introduction ...... 1 Proteolytic Enzymes ...... 1 Family of Caspases ...... 2 Caspase-2 ...... 5 Caspase-2 as a tumor suppressor ...... 11 RAIDD ...... 14 PIDD ...... 17 THE PIDDosome ...... 19 Physiological relevance of the PIDDosome ...... 21 Aims of the study ...... 24 Main Results: ...... 25 Discussion ...... 25 Conclusions: ...... 29 Attached Manuscripts ...... 30 1) P53-induced protein with a death domain (PIDD) – Master of Puppets? ...... 30 2) PIDDosome-independent tumor suppression by Caspase-2 ...... 38 3) Death of p53-defective cells triggered by forced mitotic entry in the presence of DNA damage is not uniquely dependent on Caspase-2 or the PIDDosome ...... 50 4) The tumor-modulatory effects of Caspase-2 and Pidd1 do not require the scaffold protein RAIDD ...... 60 5) Loss of PIDD limits NFκB activation and cytokine production but not cell survival or transformation after DNA-damage ...... 95 REFERENCES for Introduction: ...... 108 SUPPLEMENT ...... 120 Abbreviations ...... 120 Curriculum Vitae ...... 122 List of Publications ...... 125 Conference attendances, fellowships and awards ...... 126 Acknowledgements ...... 127

iv

Introduction

Introduction Thermodynamic systems have a common drive to increase entropy. Life, as we know it, uses this drive to generate energy by concentration gradients. The enclosure of biochemical reactions in compartmentalized areas was the successful foundation of life and this is only possible, when the actions within a compartment or the interactions of many components, such as cells, are tightly regulated and controlled. As usual for social systems there are forces that try to escape the generally accepted codes of practise. In biological systems some cells often disrupt the general tissue architecture, proliferate massively and build their own nutrient supply system. By this reckless behaviour they induce massive damage to the tissue and in long term may also kill the organism from which they were derived. To prevent cells of becoming tumorigenic several mechanisms evolved, closely controlling proliferation, programmed cell death, differentiation or migration of each individual cell. Loss of certain proteins may lead to deficits in this supervision. The present work focuses on the function of such a protein, Caspase-2, that has influences on various levels in the biology of a cell. Since it is that versatile its activation and mode of action is tightly controlled by various means.

Proteolytic Enzymes Targeted degradation of proteins is a crucial feature for the proper functioning of a cell. Protein degrading enzymes come in many different flavours to verify the correct targeting of proteins at the required time in development, for metabolism or cell division1, 2. Cysteine proteases, characterized by an active site cysteine residue, reside mostly in the lysosome or in the cytosol and mediate intracellular protein catabolism and selective activation of signalling molecules. Trypsin belongs to the group of serine proteases. These proteases get activated from inactive zymogens and are important for cell growth and differentiation, since they are involved in food digestion, blood coagulation, fibrinolysis but also control of blood pressure in mammals. Specialists for cleavage of proteins in an acidic environment such as the lysosomes are the aspartate proteases. The degradation of extracellular matrix proteins is a feature of the specialized matrix metalloproteases. Beside this direct protein degradation mediated by the enzyme families mentioned above there is the tightly regulated proteasome-mediated protein degradation. 1

Proteolytic Enzymes

Threonine proteases that make up the proteasome can only decompose proteins that are ubiquitin-tagged by specialized ubiquitin ligases. The protein turnover in eukaryotes is mostly mediated via this proteasomal pathway.

Family of Caspases The caspase family of proteases, a subfamily of the cysteine proteases3, cleaves many proteins resulting in major morphological and biochemical changes within a cell and driving it into programmed cell death1. 12 caspases are known in mammals and at least 7 of them have a defined role in apoptosis4. Caspases (Cysteine-driven aspartate-specific proteases) are proteolytic enzymes belonging to the group of endopeptidases4, 5 that cleave proteins at a specific amino acid recognition motif. Caspases reside in cells as inactive zymogens and need to dimerize or get cleaved to become activated6. Activation can happen via autoproteolysis or cleavage by such an activated caspase. According to the length of their prodomain, caspases are grouped in initiator caspases (e.g. Caspase-8, -9) and executioner caspases (e.g. Caspase-3, -6 and - 7). Initiator caspases present in their prodomain a caspase recruitment domain (CARD) or a death domain (DD) that form a so-called “death fold”, made up by six alpha-helical bundles. Via this domain caspases can dimerize in high molecular weight complexes, such as the death inducing signalling complex (DISC) and usually get activated there. Activated initiator caspases then kick-start executioner caspases by inter-domain cleavage between the large and small subunit and they then cleave essential proteins within a cell that eventually drives it into apoptosis. The function of caspases is conserved across species and can also be found in invertebrates such as the fly or the worm. CED-3 is the proteolytic enzyme in Caenorhabditis elegans, crucial for developmental cell death. This protein has the characteristics of both, an initiator and an executioner Caspase. Drosophila melanogaster already has a more diverse set of cysteine proteases, DRONC is an example for an initiator caspase whereas DRICE or DCP-1 are executioner caspases in flies6. The catalytic process mediated by caspases is based on two chemical reactions called acylation and deacylation3, 6. The enzyme grabs the target peptide bond at the

2

Family of Caspases active site. This brings the substrate specificity pocket close to the active cysteine that is in spatial proximity to a histidine. The substrate pocket of the caspase contains an arginine. This arginine is able to hold an aspartate amino acid of the target protein. Now the acylation step takes place. The cysteine is activated by the nearby histidine and it donates a proton to acetylate the target aspartate. In the following deacylation step the arginine donates a water molecule and the target protein is now cut after its aspartate residue.

Figure 1a: The chemical steps backing a caspase cleavage: Intraprotein lysis is mediated by acylation and deacylation steps. b: Recognition targets for activated caspases: The sequence up the P1 asparagine defines about the substrate specificity of the individual caspase. These five positions are filled by 11 amino acids in various sequences and so caspase cleavage in target proteins can be predicted by sequence comparison. (Modified from Green DR, 2011 6).

The selectivity of the various caspases is mediated by the sequence of amino acids in the active centre. The aspartate (in rare cases also glutamate) also designated as the P1 residue and the three to four upstream amino acids, named P2, P3 and P4

3

Family of Caspases can define the caspase to cut only designated proteins. The caspase then cleaves between the P1 aspartate and the subsequent P1´ amino acid. Caspase - inhibitors contain these polypeptide sequences and therefore block the activity of caspases in cell culture experiments7. Some caspases in mammals can be blocked by the cowpox protein CrmA or other proteins such as the IAP family, Survivin but also by the halomethyl ketone benzyloxycarbony-VAD (Z-VAD-FMK)8.

Figure 2: Caspases in Mammalia can be divided into initiator caspases, executioner caspases and pro- inflammatory caspases. Initiator caspases are characterized by their long prodomain which plays an important role in their activation. Executioner caspases lack this prodomain, since they get activated by live initiator caspases (Modified from McIlwain et al., 2013 4).

Depending on the kind of death stimulus encountered by a cell different caspases are involved. In a tissue the cell homeostasis is an essential feature. Cell numbers need to be tightly controlled and excess cells are removed via the activation of one of the two key apoptosis pathways. One is activated via “death receptor” binding leading to Caspase-8 activation at an activating platform consisting of the proteins CD95, FADD and the inactive form of Caspase-8 forming the “death-inducing signalling complex” (DISC). Caspase-8 further activates executioner Caspase-3 and starts the programmed cell death machinery. Beside CD95 other “death receptors” such as TNFR1 or TRAIL-R1 have the ability, beside the activation of Caspase-8, to also up regulate for instance NFκB signalling that can be antiapoptotic. These various death receptors play a crucial role in the homeostasis of the hematopoietic system9. A different approach to programmed cell death is the intrinsic or mitochondrial pathway. Each individual cell tightly controls its functionality. Once major flaws accumulate, such as DNA damage, mutations or trophic factors are lacking cells can activate the mitochondrial apoptosis pathway. The proteins Bax and Bak form a pore 4

Family of Caspases leading to mitochondrial outer membrane permeabilisation (MOMP). This leads to the release of Cytochrome-c from the mitochondrial lumen, which now can reach the cytosol. There it teams up with Apaf-1 to form the apoptosome, a platform also containing Procaspase-9 that binds and subsequently gets activated. Caspase-9 is an initiator caspase and when activated cleaves Caspase-3. The intrinsic apoptosis pathway is of major importance in development of many organ systems such as the hematopoietic or the neuronal system. The dimerization of Bax and Bak is tightly controlled by a vast meshwork of protein interactions, most prominently by the anti- apoptotic Bcl-2 family proteins and proteins belonging to the BH3-only family10. The literature of the recent years also underlined a prominent role of caspases in other basic mechanisms of life beside their proapoptotic functions. Caspases are reported to be involved in inflammation, proliferation, differentiation and cell cycle progression5, 11, 12.

Figure 3: Cell death pathways: The extrinsic pathway gets activated by a death ligand binding to a cell membrane bound receptor. This activates a caspase cascade which primes the cell to death. Cell internal malfunctions lead to release of Cytochrome-c from the mitochondria that forms the Apoptosome with Apaf-1 in the cytosol. On this protein complex an initiator caspase gets activated and starts the caspase pathway (Modified from Strasser et al., 2011 12).

Caspase-2 In humans 12 caspases have been characterized by now and the variety of functions and specializations for each single caspase became apparent over the years. However one caspase, Caspase-2, is still part of a scientific controversy. Although it

5

Caspase-2

was the second caspase discovered in 199413 and is one of the most conserved among the animal kingdom14 a clear association to a biological progress is still missing for this enigmatic enzyme. Caspase-2 was formerly also known as “Ich-1: ICE/CED-3 homolog 1” (Interleukin 1 converting enzyme/C. elegans cysteine protease-3 homolog) or ”Nedd-2” (neural precursor cells-expressed, developmentally down regulated-2)13. The sequence of Nedd-2 had striking similarity to the C. elegans cysteine protease-3 or the mammalian IL-1β converting enzyme (ICE). Over expression of Nedd-2 also had similar effects on cells in culture as CED-3, i.e. apoptosis. By structure, Caspase-2 can be considered as an initiator Caspase because of its long N-terminal prodomain containing a CARD motive5. To activate Caspase-2 it needs to be cleaved twice15. Similar to other initiator caspases a first processing step occurs between the large p19 and the small p12 subunit. These two fragments then heterodimerize to get subsequently cleaved between the p19 subunit and the CARD domain. Finally, fully mature Caspase-2 consists of a heterodimer build up by two p19 and two p12 subunits. Activation by auto-processing of initiator caspases usually takes place at high molecular weight multi-protein complexes. Probably the best-known example for that is the so-called “Apoptosome” which activates Caspase-9 on a complex of Apaf-1 and mitochondrial released Cytochrome-c16. The activation platform for Caspase-2 was reported to contain the proteins RAIDD (RIP associated Ich-1/CED homologous protein with a Death Domain) and PIDD (p53 induced protein with a death domain) and will be discussed later in this introduction and throughout this work. It is a general feature of caspases that they are expressed within the cells as inactive zymogens. For activation of Capsase-2 two inactive proteins need to get in close proximity. While the expression of Caspase-2 is tightly regulated during embryonic development and varies among organs in adult animals, the activation of Caspase-2 was reported after different types of stress, such as heat shock17, DNA damage18, drug induced endoplasmic reticulum stress19, cytoskeleton disruption20, trophic factor deprivation in neurons21, accumulation of reactive oxygen species22, pore forming toxins23 or mitotic catastrophe24. Caspase-2 is on mouse chromosome 625 and comes in two flavours by differential splicing of the mRNA. The gene of Caspase-2 is made up of 10 exons. Alternative

6

Caspase-2

splicing of exon 7 produces the two isoforms25, 26. Caspase-2L is the longer version and contains fully functional p12 and p19 domains. The short Caspase-2 isoform, Caspase-2S, has an inclusion of an additional exon which contains a stop signal in translation leading to a truncated version of Caspase-2. It is believed to be some kind of endogenous inhibitor of Caspase-2L or actively antagonize apoptosis by the activation of a cytoprotective protein27.

Figure 4: Activation of Caspase-2: To get activated Procaspase-2 dimerizes and gets cleaved twice. This dimerization process is mediated by an adapter protein binding to the CARD domain in the Procaspase. (Modified from Baliga et al., 2004 15).

Independent of each other, two Caspase-2 knockout mouse strains have been generated. The first report focuses on the developmental abilities of Caspase-2 deficient tissues28. Different tissues expressed varying ratios of the long splice variant Caspase-2L and the short variant Caspase-2S. In brain tissue this ratio is almost equal and Caspase-2 knockout mice turned out to show a loss of facial motor neurons during development. On the other side germ cells, where mostly Caspase- 2L is abundant, are resistant to developmental and doxorubicin induced apoptosis, when the Caspase-2 signal is lost. However, the second Caspase-2 knockout report only focused on cell death progression in thymocytes and the developing immune system29. Experiments in thymocytes or dorsal root ganglions proved that Caspase-2 gets activated after incubation in cytotoxic agents, but Caspase-2 was dispensable for the execution of apoptosis in these cells. This apoptosis was found to be entirely dependent on Apaf-

7

Caspase-2

1 and Caspase-9, suggesting, that the pro-apoptotic function of Caspase-2 might be overtaken by other caspases or that it plays no critical role. Several complexes have been described, which interact with Caspase-2. Caspase-2 can be recruited to the CD95 (Fas/Apo-1) death inducing signalling complex (DISC)30. CD95 can be activated after etoposide or 5-FU inflicted DNA damage by p53, since its gene has a p53 response element in its promoter31. After DISC formation Caspase-2 and Caspase-8 get activated and cleave Bid. Truncated tBid then activates the mitochondrial pathway as already explained. This cleavage of Bid is also thought to be of major importance for the execution of cell death by Caspase- 2 after a heat shock event32. Interestingly Caspase-2 activated by CD95 stimulation was not able to execute apoptosis alone in Caspase-8 deficient cells30. This hypothesizes a collector function for the DISC death domain of Procaspase-2 and Procaspase-8. But only Caspase-8 gets directly activated at the DISC and then activates Caspase-2 via cleavage of its prodomain31. A possible interactome of Caspase-2, RhoA and ROCK-II suggests that Caspase-2 might play a critical role in Alzheimer’s disease (AD) progression, since a Caspase-2 knockout model shows decreased numbers of dendritic spines during aging33. This proposed complex requires further confirmation but might point out an alternative function of Caspase-2 entirely unrelated to its apoptotic function. When checking for the exact localization of Caspase-2, upon others, one oddity of Caspase-2 is its possibility to translocate into the nucleus. Normally caspases reside only in the cytoplasm. But Caspase-2 contains a nuclear translocation sequence in its prodomain18. It was shown that it can interact with promyelotic leukaemia protein nuclear bodies (PML-NBs) after DNA damage, during DNA repair and apoptosis. This nuclear bodies are macro-molecular complexes that contain up to 30 different proteins such as SUMO-1, eIF4E, Rb or p5318. In the cytosol, Caspase-2 primarily resides close to the Golgi apparatus29, 34. This is again a unique feature of Caspase-2 but also explaining its target Golgin 160. No confirmed report found that Caspase-2 is located at the mitochondria so far35.

8

Caspase-2

Biological functions of Caspase-2 Most reports provide evidence that Caspase-2 acts in the DNA damage response36. Within the intrinsic apoptotic pathway, the key function of Caspase-2 was primarily placed upstream of the mitochondria, but was also found to be positioned downstream of Caspase-3 in a potential positive feedback loop37. Consistent with the former, active Caspase-2 was found to trigger MOMP by cleaving and activating the proapoptotic BH3-only protein Bid to truncated tBid38. In contrast to this model, processed and proteolytic inactive Caspase-2 was also found to act directly on the mitochondria39, 40. An activation of Caspase-2 at the DISC also underlies a possible involvement to link extrinsic and intrinsic apoptosis pathways. First findings identified Caspase-2 as an upstream promoter of mitochondrial Cytochrome-c release in response to etoposide39. Significantly reduced apoptosis was seen in p53 over expressing cells after knockdown of Caspase-2 upon DNA damage induction via daunorubicin41. Interestingly, knockdown of Caspase-2 in p53- deficient cells showed a similar impact on apoptosis, rendering cells resistant towards γ-irradiation induced apoptosis when Chk-1 was inhibited simultaneously in Zebrafish and HeLa cells42 as discussed extensively further below.

Caspase-2 in cell cycle regulation: Caspase-2 turned out to be tightly regulated during cell cycle progression. Beside a interaction of Caspase-2 and the positive cell cycle regulator of the G1/S transition cyclin D343 an even more fascinating regulation is the phosphorylation of Caspase-2 at position S340 by Cdk-1/Cyclin B1 during mitosis44. This phosphorylation blocks the activation of Caspase-2 by preventing inter-domain cleavage. Consistent with a role in cell cycle progression, Ho et al. reported higher rates of proliferation of primary MEF from Caspase-2-/- mice compared to wt MEF45. Furthermore, after γ-irradiation, BrdU staining showed that more Caspase-2-/- cells than wt cells were still in S-phase, indicating impaired cell cycle arrest upon γ- irradiation after the loss of Caspase-245, 46. Furthermore, there are decreased p21 transcript levels in cells upon Caspase-2 knockdown46, 47. Surprisingly, in another report p21 was found to repress Caspase-2 mRNA levels and Caspase-2 protein expression41, indicative for a molecular interaction between both proteins that remains to be understood in full detail.

9

Caspase-2

These results are in accordance with findings of a paper that describes Caspase-2- mediated cleavage of Mdm-2, the ubiquitin ligase targeting p53 for degradation48. Cleavage of Mdm-2 produces a p60 subunit that is devoid of E3 ubiquitin ligase activity but still able to bind p53. Hence, Caspase-2, located on the RAIDD- PIDDosome, generates a positive feedback loop by preventing ubiquitination and subsequent degradation of p53. Maintenance of p53 levels after initial accumulation was proposed to result in increased transcription of p21 enforcing cell cycle arrest. Later a close correlation between the Mdm-2 cleavage mediated p53 stabilization and K-Ras induced lung tumor progression was found49. A few studies also indicate involvement of Caspase-2 in G2/M checkpoint maintenance upon DNA damage such as repeated reports on interaction of PIDD with PCNA50 and the fact that Caspase-2 processing was found to be repressed during mitosis by its phosphorylation at S340, which can be reversed through activation of the protein phosphatase 1 (PP1) upon DNA damage44. Hence, one can speculate that upon reparable DNA damage Caspase-2 may induce cell cycle arrest whereas irreparable damage may lead to apoptosis through Caspase-251. The regulation of some cell cycle proteins by Caspase-2 might influence the reaction of a cell on catastrophic events during mitosis24. Preliminary experiments performed in our laboratory on Caspase-2 deficient cells revealed higher survival rates of cells after mitotic slippage (L. Fava, personal communication), an effect when cells proceed into G1 phase after a failed separation of the chromosomes during M phase. The mechanism by which Caspase-2 acts under these conditions remain unclear at present, but may be better understood upon identification of its substrates.

Caspase-2 targets The hunt for putative Caspase-2 target proteins proved to be very tricky. Although Caspase-2 has a preferred cleavage sequence (VDQQD,52) the only proteins found so far being cleaved directly by Caspase-2 are Mdm-248, Bid53 and Golgin 16034. Of note only the last one carries a VDQQD motive. Confusingly, Bid and Mdm-2 can also be cleaved by other Caspases which makes Golgin 160 the only protein selectively cut by Caspase-2. This cleavage mainly occurs after cellular stress involving the Golgi apparatus.

10

Caspase-2

The cleavage of Bid by Caspase-2 but also by Caspase-8 proves evolutionary importance of this step38. Bid is a BH3-only protein that connects the extrinsic apoptotic signalling pathway and the mitochondrial pathway. Both Caspase-2 and Caspase-8 are activated at the CD95 DISC, usually a complex involved in the activation of the extrinsic apoptosis pathway. Subsequently, the cleaved form of Bid, tBid moves to the mitochondria and is sufficient to induce Bax-dependent Cytochrome-c release53. The latest report on putative targets of Caspase-2 identified four cytoskeletal proteins being degraded after over expression of Caspase-2 in apoptotic cells. But in this unbiased proteomics approach, Caspase-2 did not cleave the proteins directly, it primed its targets for ubiquitin mediated proteasome degradation54. The exact mechanisms of the connection of Caspase-2 to the ubiquitin machinery remain to be explained.

Caspase-2 as a tumor suppressor Several of the above-mentioned functions of Caspase-2 are features that have an impact on processes that are of crucial importance for the behaviour of a cell in a tissue or an organ. Failing Caspase-2 signalling can lead to deregulated apoptosis, proliferation, metabolism and impairment of transcription factors with tumor suppressor activity, all biological functions that are known as the hallmarks of cancer55. Already one year after its initial characterization, Caspase-2 was listed as a possible tumor suppressor gene since its locus is in a branch of chromosome 7 that is often lost or mutated in blood leukaemia56. Later it was shown, that a reduction of Caspase-2 in acute myeloid leukaemia (AML) or acute lymphoblastic leukaemia (ALL) leads to a poor prognosis of disease progression57, 58, 59. So, a clear role for Caspase-2 as a tumor suppressor in human disease was established, beside Caspase-8 and -1060. Somatic mutations of Caspase-2 in gastric or colorectal cancer are very rare, but mutated Caspase-2 correlates with poor prognosis61. Reports also mentioned a down regulation of the Caspase-2 protein level in solid hepatocellular tumors, invasive breast carcinomas, ovarian adenocarcinomas and glioblastoma in human patients62.

11

Caspase-2

These observations of Caspase-2 levels in human tumors were mostly pure case reports and lacked a mechanistically explanation for the tumor suppressive function of Caspase-2. For instance, TRAIL (TNF related apoptosis inducing ligand) resistant oesophageal cancer cell lines revealed a possible role of Caspase-2 in apoptosis induction of tumor cells63. The protein kinase PKCK2 is able to phosphorylate Caspase-2 at serine S157 and blocks Caspase-2 autoactivation. A lacking PKCK2 signal leads to hyperactive Caspase-2 and subsequent pre-processing of Caspase-8 and enhanced TRAIL-induced DISC formation. TRAIL resistant cancer cell lines often express too much of PKCK2 and this may lead to loss of Caspase-2 activity and reduced TRAIL responsiveness. The first report in mice, however, that paved the way for many publications to come, was a story published by Ho et al. about a possible role of Caspase-2 in cytoskeletal integrity20. Primary Caspase-2-/- MEF proved to be highly resistant to the actin destructing drugs cytochalasin D or microtubule stabilizing drugs such as vincristine or paclitaxel. They linked this effect to a decreased Bid cleavage in knockout MEF and a subsequent delay in Bax translocation and Cytochrome-c release. However, this observed insensitivity of Caspase-2 deficient MEF to cytoskeleton damaging agents proved difficult to recapitulate in our own laboratory (own unpublished observations). First evidence of Caspase-2 in tumor development was given by exploiting a well- established B cell tumor mouse model45. After showing that Caspase-2 has an influence on cell transformation by E1A/RAS where Caspase-2 knockout cells clearly outperform their wt counterparts in cell culture and in an athymic nude mouse model, the intercrossing of Caspase-2 knockout mice to the Eµ-Myc mouse model drastically revealed its tumor suppressor function64. Further molecular dissection of the Caspase-2 molecule showed that the tumor suppressor function is dependent on its catalytic site. A mutation in the cysteine 320 in the catalytic site and the serine residue S139 rendered cells behave similar to Caspase-2 knockout cells. SV40 MEF and E1A/Ras transformed cells with point mutations in these residues grow faster, have elevated clonogenic activity, accelerated anchorage independent growth and a vastly enhanced ability to form tumors in athymic nude mice65.

12

Caspase-2

Beside athymic nude mice as an extension of cell culture experiments several other mouse tumor models have been employed to highlight the Caspase-2 dependent tumor suppression effect. Next to the already mentioned Eµ-Myc mouse model Atm-/- and Casp-2-/- double knockout mice also show accelerated lymphomagenesis66. A role of Caspase-2 in epithelial cell cancer was shown in mice harbouring the MMTV/c-neu oncogene67. Mammary carcinogenesis was significantly enhanced in single-parous mice, accompanied by cells with bizarre mitoses and karyomegaly. Tumors lacking Caspase-2 had also more cells in mitosis in comparison to tumors from their wild type littermates pointing again towards a role for Caspase-2 in genomic stability. Also lung tissue proved, beside the lymphatic system, to be sensitive to the loss of Caspase-2. K-Ras driven lung cancer develops slightly faster in a Caspase-2 free environment, caused by enhanced proliferation and progression49. Although these Caspase-2 deficient tumors are highly sensitive to chemotherapy, tumor cells rapidly recover from impact of the DNA-damaging cisplatin treatment. Over expression of the oncogene N-MYC associates with neuroblastoma and also causes neuronal tumors in mice68. Interestingly, in Casp2-/- mice, N-MYC-driven tumors developed later, additionally, tumors were less vascularized and the RAS/MAP kinase signalling pathway, which is believed to be the main driver in neuroblastoma, was not deregulated after loss of Caspase-2. However, when examining the Caspase-2 level in human neuroblastoma samples, one clearly could see a survival benefit, when the levels of Caspase-2 were low. This effect could only be observed, when also the N-MYC levels in the tumor were low. This finding suggests a role of Caspase-2 in tumor development, which is tissue and context specific. This assertion is backed by the fact, that loss of Caspase-2 has no influence on tumor progression after injection of the DNA damaging agent 3- methylcholanthrene (3-MC) into muscle tissue or repeated low dose γ-irradiation on T-cell lymphoma formation69. It is difficult to pinpoint the exact mechanism that engages Caspase-2 as a tumor suppressor. Of course an educated guess would correlate a possible derailed apoptosis caused by loss of Caspase-2. But in depth analysis of tumors arising from Caspase-2 deficient mouse models revealed, that the tumor growth is mainly driven

13

Caspase-2

by deregulated cell growth and proliferation, which leads to a numerical up regulation of cells in mitosis in the tumor mass47, 49, 67, 69, 70. Controversial data also claimed a deregulation of the DNA damage response machinery and therefore possible problems in the maintenance of the genomic stability71. However, this claim was backed by the finding of deregulation of the mitotic checkpoint machinery in Caspase-2 deficient primary MEF and tumor cells47. Malfunction of this checkpoint easily leads to an accumulation of cells, which faced difficulties in separating their chromosomes during mitosis. This leads in an early phase to the presence of single chromosomes or entire groups of chromosomes that are not part of the main nucleus. These micronuclei, easily visible by DAPI staining of the interphase cell, can be rescued to the main nucleus during the next mitosis phase or can be entirely lost to daughter cells after the consecutive cell division. So, over the course of a few rounds of cell cycle, Caspase-2 deficient cells are susceptible to develop aneuploidy in a cell culture or tissue. These effects, summarized as chromosomal and genomic instability, are known to drive tumor progression since the possibility of acquiring or loosing chromosomes with information for oncogenes or tumor suppressors is very high72. Of note, chromosomal instability is known to accelerate tumor formation, but the effect alone is not sufficient to start tumorigenesis73. Usually, oncogene driven proliferation directs cells rapidly into senescence. So possible tumor cells can be recognized by the immune system and withdrawn from the tissue. This senescence failsafe program is ineffective in tumor cells, when Caspase-2 is down regulated74. As briefly mentioned above, Caspase-2 engages in a p53 feedback loop48, 49. Via the cleavage and inhibition of Mdm-2 by Caspase-2 p53 levels can be rapidly up regulated after stressful events. In Caspase-2 deficient tumors this p53 stabilization is absent and cell cycle regulating factors such as p21, Cyclin G1 and Msh-2 are not up regulated appropriately. In a developing tumor all these meanderings of emergency plans probably lead to the dirty face of a rapidly growing tumor.

RAIDD RAIDD was first presented by the work of Duan and Dixit in 1997 as an adaptor protein which links cell death proteases to signalling downstream of death

14

RAIDD

receptors75. They described RAIDD as a protein with an unusual bipartite architecture that can interact on its C-terminal domain by homotypic interaction with RIP1 and on its N-terminal end it was very homologous with the sequence of human ICH-1 or the Caenorhabditis elegans CED-3 protein. Hence the bulky name “RIP associated ICH- 1/CED-3-homologous protein with a death domain” or short RAIDD. Another report dubbed this double-linker protein CRADD, short for “Caspase and RIP adaptor with death domain” because of the striking similarity of the N-terminal domain to the CARD of Caspase-2 and C-terminal domain to the death domain of RIP76. Although this report already correctly anticipated the connection of this protein to Caspase-2 the term RAIDD dominates over CRADD in the literature and will also be used throughout this work. The genomic structure of the RAIDD locus has four exons separated by three introns77. The genomic regions upstream of RAIDD shows four possible promoters. The gene can undergo various splicing steps that can produce four different splicing variants in the 5’UTR, the coding region, however, remains unchanged. The gene for RAIDD resides in mice on chromosome 10. It was recognized that mice showing the high growth symptoms (30 - 50% increase of mature body size due to cell hyperplasia with a moderate cell hypertrophy) actually have a 460 KB deletion on chromosome 10. This region contains the coding sequence for the proteins Raidd, Socs-2 and Plexin C177. This correlation of the loss of this chromosomal region with the high growth phenotype was also reported later in swine and chicken78, 79. Later the group focussing on the high growth phenomenon reported that deregulation of the suppressor of cytokine signalling 2 (Socs-2) is actually responsible for this phenotype. Socs-2 deficiency leads to deregulation of signalling by the growth hormone Igf-180. However, an over expression experiment of Raidd in a pre- adipocyte cell line revealed a block of differentiation after hormonal induction81. As a consequence, no adipocytes were forming and adipocyte markers stayed low in this pre-adipocyte cell line. This result was unexpected since the authors anticipated up regulation of apoptosis as a consequence of Raidd over expression rather than defective differentiation, but this may be an alternative explanation for deregulated growth rates of animals. Beside its possible connection to the adipose tissue, Raidd was also shown to be up regulated during several stages of the development of mid-gestation mouse

15

RAIDD

embryos82. During important morphological changes of developing embryonic organs such as heart, kidney, endothelial cells and embryonic midgut, Raidd was strongly up regulated. Those are organs known to undergo a lot of apoptosis in various developmental steps, yet, Raidd must play another role there, as Raidd-deficient embryos develop normal. A first attempt to generate a Raidd knockout mouse failed to generate homozygous adult mice, when targeting exon 2 and 3 containing the Caspase-2 binding CARD domain82. The authors interpreted their results as an embryonic lethality of Raidd nullizygous mice or as a possible random integration event of the neo cassette into an unknown important locus. The first successful knockout mouse, however, did not show any overt phenotypes83. Raidd-/- mice have normal development and haematopoiesis, thymocytes are susceptible to various cytotoxic agents and MEF are sensitive against TNF induced killing. However, apoptosis mediated by p53 after cellular stress was slightly down regulated in MEF obtained from Raidd deficient mice. RAIDD was always known to be a scaffold protein and bind via its CARD and DD to various proteins. Beside Caspase-2, Rip-1 or PIDD it was also found to interact with Bcl-1084. By doing so, RAIDD can act as a negative regulator of the CARMA-1 signalosome. A complex consisting out of CARMA-1, MALT-1 and Bcl-10, short CBM, is crucial in activating the NFκB signalling pathway after B- and T- cell antigen receptor stimulation85. Phosphorylation and polyubiquitination of the CBM members leads to an activation of NEMO and subsequently to the activation of NFκB transcription factor family members that then move to the nucleus to regulate expression of target genes86. Here, RAIDD was described to act as a negative regulator of the CBM complex84. In the proposed model, RAIDD competes with CARMA-1 for the same interaction site in Bcl-10. Both proteins carry a CARD domain, allowing homotypic interactions with each other, or with RAIDD, acting as a competitive inhibitor in this context. Absence of RAIDD leads to a hyperactivity of the CBM complex, resulting in elevated expression of pro-inflammatory cytokines in murine thymocytes or increased vascular permeability during inflammation in endothelial cells87. Ironically, although founding the name, RAIDD was never reported to have a physiologically important interaction with Rip-130, 88.

16

RAIDD

Expression in tumors: For RAIDD a first report of deregulation in cancer cells came four years after its initial description. A micro-array on mantle cell lymphoma cells in comparison to non- malignant hyperplastic lymph node tissue revealed a down regulation of RAIDD together with its N-terminal interaction partner Caspase-2, next to other deregulated genes58. However, RAIDD as an independent factor in tumor growth was first reported in a breast cancer screen using the dissociable antibody microarray technique89. Among others RAIDD was over expressed in breast cancer cell lines. Another story of RAIDD being important in tumor development came apparent when a humane gene-array revealed, that from 96 apoptosis related genes only RAIDD was down regulated in U2OS cells90. This was not the case in multidrug resistant cell lines. After over expression of RAIDD in this multidrug resistant osteosarcoma lines sensitivity to paxlitacel was restored. A third report of RAIDD being affected independently of Caspase-2 was in a cDNA microarray screen of oral squamous cancer tissue against healthy mouth cavity tissue. RAIDD was detected as significantly down regulated, next to a deregulation of the apoptosis markers FADD, ATM, Apaf1 and p6391. In western blots the decrease of RAIDD levels was confirmed but these findings were never backed up by independent studies.

PIDD P53 induced protein with a death domain (PIDD) is a gene regulated by p53 after genotoxic stress such as ionizing radiation92 and dampens the pro apoptotic signal of p53 when down-regulated. A further molecular pattern, the leucine rich repeat region, prompted another team to name it LRDD for leucine repeat death domain containing protein93. They did not make the connection to a putative p53 target but presumed the interaction of the PIDD death domain with proteins such as FADD and MADD by bioinformatics means. With 910 amino acids PIDD is a rather large protein and comes in various isoforms. The largest isoform 1 contains several leucine rich repeats close to the N-terminal end and two ZU-5 domains in the centre of the amino acid sequence. The name- giving DD is located on the C-terminal end, similar in sequence to the death domains of other proteins such as RAIDD, FADD or CD9594. Isoforms 2 to 4 are variations of

17

PIDD

isoform 1 that have deletions in the N-terminal end or the part between the ZU-5 domains and the death domain. Isoform 5 was only predicted in silico and just consists out of the two ZU-5 domains95. A few years later the role of PIDD in p53-mediated apoptosis was defined. As discussed in the next chapter, PIDD is able to bind Caspase-2 via RAIDD as a scaffold protein and homotypic interaction of their death domains96. But PIDD can also bind to other proteins and is therefore able to activate the pro survival NFκB pathway97 or the DNA repair machinery50. PIDD binds after mild genotoxic stress to RIP1 and NEMO. In this complex NEMO gets heavily posttranslationally modified via sumoylation, phosphorylation and ubiquitination and thus activates IKK and the NFκB pathway. Alternatively, PIDD-C can also interact with PCNA in response to UV radiation50. DNA damage inflicted by UV light leads to the stabilization and subsequent monoubiquitinaton of PCNA by PIDD-C via preventing its inhibition by p21. This leads to an activation of low fidelity translesion synthesis by polymerase eta (polη) repairing the cyclobutane pyrimidine dimers in the DNA created by UV irradiation. Of note, these features are entirely independent of Caspase-2.

Figure 5: Depending on the posttranscriptional modification states, PIDD can interact with various proteins and so activate pro survival or pro death pathways (Modified from Bock et al., 2013 98).

18

PIDD

So, somehow PIDD is able to serve as a switch between pro- and anti- survival signalling pathways. This dual function was explained via autoproteolysis events happening within the protein99. The entire protein PIDD can cleave at Serine S446 and form the leucine rich repeat containing PIDD-N and a bigger PIDD-C cleavage product. This C-terminal cleavage fragment of PIDD was observed to form the RIP-1- NEMO-PIDDosome and is responsible for the activation of NFκB-signalling or interacts with PCNA. After severe genotoxic stress the PIDD-C fragment gets further cleaved at serine S588 and gives rise to the PIDD-CC fragment, correlating with a strong apoptotic response of the cells. This PIDD-CC fragment is able to bind to RAIDD and Procaspase-2, therefore mediating the activation of the Caspase. The switch from PIDD-C to the PIDD-CC is mediated by the binding of the molecular chaperone Hsp90 and its co-chaperone p23 to idle PIDD-C in the cytosol, while active PIDD-C in the nucleus remains unaffected. After stress situations such as heat shock Hsp90 releases the autoprocessed PIDD-CC fragment and enables its binding to RAIDD and the formation of the RAIDD-PIDDosome100.

THE PIDDosome The three proteins discussed above were shown to interact in a high molecular complex in 293T cell and Jurkat T cell lysates when incubated for 1 hour at 37°C while such a complex did not form at 4°C96. The biological relevance of this protein complex was proven by over expressing PIDD and blocking all the activated Caspases with the Caspase-inhibitor biotin-VAD-fmk. A subsequent biotin immunoprecipitation reported Caspase-2 as the only activated Caspase after PIDDosome formation. PIDD over expressing cells exposed to genotoxic stress induced by doxorubicin died significantly faster in comparison to wt cells or mutants lacking the DD domain. It was also shown, that apoptosis caused by Pidd is dependent on the PIDDosome scaffold protein Raidd83. Cell death induced by γ-irradiation inflicted DNA damage led to the activation of Caspase-2 after the accumulation of Pidd and was dependent on Raidd. Raidd deficient cells failed to induce Caspase-2 cleavage in E1A transformed MEF. The interaction of Procaspase-2 and RAIDD via the CARD domain is conceptually very similar to the interaction of the C.elegans proteases CED-2 and CED-4101 and the solution structure of the RAIDD CARD domain is topologically similar to the Apaf-1

19

The PIDDosome

CARD which is the central part of the Caspase-9 activating apoptosome102, 103. A CARD domain consists out of six tightly packed helices. These helices form an acidic and a basic batch on opposite sides. The CARD domains of Procaspase-2 and RAIDD can therefore interact by homotypic interaction, which is an electrostatic interaction between charged surface residues. The C-terminal binding of RAIDD to PIDD is mediated by homophilic interaction of their death domains. An in-depth analysis claims that it needs seven RAIDD death domains and five PIDD death domains to form a stable complex104. Each death domain consists out of seven helices, and six of them form the tightly packed helix bundle similar to the CARD domain105. The complex between RAIDD DD and PIDD DD forms, without any visible symmetry around a central axis and has the approximate size of 150 KD.

Figure 6: Schematic view of the RAIDD-PIDDosome. PIDD-CC forms a complex with Procaspase-2 using RAIDD as a scaffold protein. This binding is enabled via homophilic interaction of death domains and the CARD domain (Modified from Tinel et al., 2004 96).

Initially, the PIDDosome received much attention as the missing Caspase-2 activating platform. A few months after publication, formation of a physiologically active PIDDosome was shown in PC12 neurons21, 106. After trophic factor deprivation this complex spontaneously formed, was visible in perinuclear aggregates and drove the cells into apoptosis. After inhibition of RAIDD via RNA interference, trophic factor deprivation in PC12 neurons was no longer inducing apoptosis. The PIDDosome mediated death of hippocampal CA1 neurons, when the brain of a rat is not sufficiently supplied with nutrients during an ischemic event107. Only five minutes after cardiac arrest the levels of PIDD-CC rise and so does the activation of Caspase- 2. Artificial down regulation of PIDD two days prior the ischemic infarct lowers the level of hippocampal neuron death after reperfusion. Next to reports naming the PIDDosome only in connection to apoptosis induction, the PIDDosome was also studied in a larger interactome analysis. I already discussed the work of Oliver et al.48 that shows a feedback loop between p53 and Caspase-2 activity. 20

The PIDDosome

This model is only valid when the protein PIDD is activated by p53 and then in turn PIDD activates the proteolytic function of Caspase-2. However, the real biological relevance of this mechanism is unclear, since the changes in p53 destabilization after loss of PIDDosome components are very small, questioning its physiological relevance. Also, cisplatin was used in this work as an inducer of DNA damage that may not be a very potent trigger of PIDDosome mediated apoptosis in this context. Hence, this phenomenon awaits independent confirmation. The kinases ATM/ATR can directly induce Caspase-2 after genotoxic stress, a pathway, which is opposed by the checkpoint kinase Chk-142. Checkpoint kinase 1 is activated after replication stress or single- or double-strand breaks of the DNA by ATR/ATM. This active Chk-1 kinase blocks the function of Cdc25c, preventing premature entry in mitosis. A fine-tuning of PIDD activity via the kinase ATM discretely regulates the function of this pathway108. ATM phosphorylates PIDD on Threonine T788 in the death domain and only in this way the death domain can interact with RAIDD. The dephosphorylated version of PIDD only binds to Rip-1 driving the activation of the NFκB pathway108. This is a further level of life-death decision regulation for the protein PIDD and also explains the difficulties of other laboratories to mimic the RAIDD – PIDD interaction in cell free experiments109, 110.

Physiological relevance of the PIDDosome The discovery of the PIDDosome as an activation platform for Caspase-2 led to enthusiastic concepts about the crucial role of Caspase-2 in p53 induced apoptotic signalling and the tight relationship to pro-survival NFκB signalling97, 111. However, experiments were published which questioned the physiological relevance of this large protein complex. For instance the generation of the Raidd knockout mouse showed that Raidd is indeed important for the activation of Caspase-2 and subsequently Caspase-7 and Caspase-3. However, intriguingly the pro death signal exerted by Pidd was entirely dependent on the PIDDosome, since Caspase-2 knockout MEF were still sensitive to Pidd over expression83. Also the p53 - PIDDosome connection came into question, since colon carcinoma cells treated with 5-fluoruracil (5FU) were killed by activated Caspase-2, even in the absence of p53. The formation of the PIDDosome was unaffected by the loss of p53112.

21

The physiological relevance of the PIDDosome

The significance of this high molecular weight complex, claimed to be the PIDDosome, consisting of the proteins Procaspase-2, Raidd and Pidd got severely challenged, when the Pidd knockout mouse model was published113. In this study normal Caspase-2 activation was observed in primary lymphocytes after treatment with various chemotherapeutics. Only in SV40 immortalized MEF Caspase-2 activation was slightly delayed. But overall, the lack of Pidd in cells did neither alter the rate of apoptosis, nor did it affect the localization of Caspase-2 within the cell. Most strikingly, Caspase-2 accumulated in a high molecular complex upon temperature shift. This strongly supported the existence of another previously overlooked protein complex involved in the activation of Caspase-2. Consistently, activation of Caspase-2 and RAIDD but not of PIDD was seen after a Marba-virus infection when blocking the IRE-1α pathway114. So, the endoplasmic reticulum stress, known to engage Caspase-2, is most probably independent of the PIDD interaction to RAIDD. Similar results were shown upon the inhibition of deacetylases in cancer cells115. This cell death was not dependent of p53 or extrinsic apoptosis but rather the complex formation of Caspase-2 and RAIDD, initiating the mitochondrial apoptotic pathway. Although trophic factor deprivation in neuronal cell lines or hippocampal cells were proven to be highly dependent on the function of the entire PIDDosome, neuronal activity and function of Caspase-2 is apparently again only depended on Raidd but not Pidd after NGF deprivation of hippocampal neuron cell lines or treatment with the neurotoxin β-amyloid116. This Caspase-2-Raidd interaction can activate the kinase JNK which in turn phosphorylates and activates the transcription factor c-Jun believed to culminate in the induction of the BH3-only protein Bim117. Reports like this back the assumption, that components of the PIDDosome can act in various combinations as either pro-survival or pro-apoptotic mediators. On the one hand, Caspase-2 interacting with RAIDD and PIDD in the RAIDD-PIDDosome, or, Caspase-2 and RAIDD alone activating JNK that in turn leads to proapoptotic signalling99, 111, 117. On the other hand, Caspase-2 in an interactome with RhoA and ROCK-II proved to play a central role in development of dendritic spines during aging and in the progression of Alzheimer’s Disease (AD)33. Raidd as a designated scaffold protein can further team up with Bcl-10 to prevent CARMA-1 signalling and negatively regulates the release of pro-inflammatory cytokines84. RAIDD binding to RIP-1 has been described but the proposed biological effects of this possible

22

The physiological relevance of the PIDDosome interaction75, 76 were not confirmed in genetic experiments83. P53 induced protein with a death domain (PIDD) selectively interacts with various proteins, depending on its posttranslational modifications. Autoproteolysis of PIDD producing the PIDD-C fragment favours interaction with PCNA to mediate DNA repair or the formation of a PIDD-RIP1-NEMO complex, both activating survival mechanisms50, 97. Only a further processing of PIDD-C into PIDD-CC, facilitated by Hsp90, then activates the pro- apoptotic function of PIDD100.

23

Aims of the study

Aims of the study When this study was initiated, the PIDDosome was considered as the putative activation platform for Caspase-2. This protease was shown to act as a tumor suppressor. However, no clear insight was found in the literature about the role of the interacting proteins Raidd and Pidd in this process. Exploiting several mouse models and in vitro analysis methods I intended to address the functions of these proteins and hence that of the PIDDosome complex in tumor formation by asking the following questions:  Does the loss of individual components of the PIDDosome alter the tumor latency in DNA-damage driven cancer models?  Is the observed tumor suppressive capacity of Caspase-2 upon MYC deregulation dependent on interaction with Raidd?  What is the molecular basis of Caspase-2 dependent tumor suppression?

All this questions were addressed by using mouse models at the Medical University of Innsbruck, Austria, and at the University of South Australia, Adelaide, Australia, complemented by in vitro analysis of derived primary and transformed cells and cell lines from these gene modified mice. My studies were initiated in October 2010 and finalized in December 2014.

24

Main Results

Main Results: The results obtained from my work on the RAIDD-PIDDosome can be summarized as follows:

 Caspase-2 is a tumor suppressor gene in the Eµ-Myc tumor model but not in DNA damage inflicted malignant disease  The tumor suppressive function of Caspase-2 is independent of its interaction with Raidd or PIDDosome formation  The selective pressure on p53 caused by MYC over expression is decreased in tumors lacking Caspase-2  More tumor cells appear to be in M-phase when Caspase-2 is missing pointing towards defects in cell cycle control  Caspase-2 deficiency leads to increase in genomic and chromosomal instability.  RAIDD has no influence on tumor development or disease burden in any of the models tested  Loss of Pidd delays the onset of lymphoma formation in a MYC dependent mouse B cell tumor model  Pidd-deficiency deregulates cytokine release in primary cells via impaired NFκB activation

Discussion The focus of this study was to test the role of Caspase-2 and its interaction partners Raidd and Pidd as tumor suppressors. Meanwhile, Caspase-2 has been established as a tumor suppressor by us and other groups in MYC-driven lymphoma as well as in a number of other tumor models45, 49, 68, 70. However, the contribution of Raidd and Pidd in this context remained largely elusive (reviewed in attached manuscript #1). Over the course of my experiments it became evident, that the three components of the PIDDosome can have different functions when it comes to the formation of malignant diseases. This is very surprising, since the PIDDosome was believed to be the key activation platform for Caspase-296. Initiator caspases containing a long pro- domain usually need to be activated in high molecular complexes by dimerization, as 25

Discussion

it happens for Caspase-9 in the Apoptosome, for Caspase-8 in the death receptor induced signalling complex (DISC) or in the nematode Apoptosome activating CED- 36. Studies carried out in our own laboratory casted already some doubt on the physiological relevance of the PIDDosome for Caspase-2 activation as in SV40 immortalized MEF lacking Pidd or Raidd formation of high molecular weight complexes containing activated forms of Caspase-2 was still observed113. Unfortunately, over the years the composition of this complex was never analysed in detail, but the data suggests that Caspase-2 can be activated in the absence of Pidd or Raidd, as it might be the case in the context of oncogenic stress (discussed below). Our approach to address the role of Caspase-2 in DNA damage-induced mouse tumor models, such as those induced by fractionated low dose γ-irradiation or the injection of 3-MC, a compound forming bulky adducts on DNA, did not reveal a role for Caspase-2 in the genesis of DNA damage-inflicted tumors (see attached manuscript #2). This was surprising, since activation of Caspase-2 is reportedly regulated by p53, downstream of transcriptional activation of Pidd41 and the fact that loss of p53 significantly accelerates onset of disease in these models118, 119. This suggests that p53 acts by other means to suppress disease in these models than by activating Caspase-2. Following the line of p53 and Caspase-2 interaction we also tested a hypothesis of a possible p53-independent activation of Caspase-2, mediated by Chk-1 and ATM after DNA damage42. As shown in attached manuscript #3 we were not able to further sensitize p53-deficient mouse cells to genotoxic stress when either Caspase-2 or proper Chk-1 signalling was missing120. This finding challenges the previous claim of an evolutionary conserved mechanism mediating death of p53- deficient cells operating in zebrafish as well as in humans. As further shown in attached manuscript #2, MYC induced formation of B cell tumors is suppressed by Caspase-2 confirming the tumor suppressor function first noted by Kumar and colleagues45, 64, 69. Our analysis of premalignant and malignant B cells derived from Eµ-Myc mice revealed interesting facts: Tumors lacking Caspase-2 were preferentially more mature as they mostly expressed the IgM+ surface marker. This points towards a possible deregulation of B cell maturation during development in Casp2-/- mice, at least in the context of MYC over expression. However, no defects in B cell development were noted in Casp2-/- mice, asking for additional experimental

26

Discussion

support for this hypothesis. Furthermore, an increase of cells in M-phase was observed in single cell suspensions stained for the mitotic marker phospho-Histone H3 from tumors lacking Caspase-2. Apparently, one or more missing signals lead to impaired function of the G2/M or spindle assembly checkpoints engaged for entry into or exit from mitosis, when Caspase-2 is missing. This finding is in line with the observation that Caspase-2 deficient Eµ-Myc tumors show signs of chromosomal instability, a feature also examined again in attached manuscript #4. Diploid cells are expected to present 20 pairs of chromosomes in mice. A small number of cells in Eµ- Myc/Casp-2-/- derived tumors actually meet this criteria but it was interesting to observe a high intra-tumor variance of chromosome numbers. While chromosome numbers were quite constant in tumors from wt donors, chromosome numbers in individual tumors varied vastly when Caspase-2 is missing. This observation can be explained using three hypotheses: One would be, that the tumors lose and gain chromosomes more frequently due to failures during chromosome segregation, leading to a plethora of cells with different grades of aneuploidy that survive in the absence of Caspase-2. Alternatively, Caspase-2 may be critical for faithful chromosome segregation. The third possibility is that tumors from Casp-2-/- mice may show a higher degree of oligoclonality, often noted in Eµ-Myc mice and exemplified in the arising IgM+/IgM- tumors. Of note, in SV40 immortalized MEF, Caspase-2 deficiency leads to increased micronuclei formation after cytokinesis, demonstrating that this phenomenon is not limited to c-MYC-transgenic B cells (see also attached manuscript #4 and 47).

To our disappointment we were unable to confirm that Raidd is critical for Caspase-2 function in MYC-driven lymphomagenesis. Deletion of Raidd in mouse did not reveal any overt phenotypes so far28. Only an in vitro analysis of p53-induced killing reported a suboptimal activation of programmed cell death when Raidd levels are insufficient83. Although levels of RAIDD were reported to be deregulated in various human cancers58, 89, 90, 91 our analyses failed to provide a critical function of RAIDD in tumor formation. In all mouse models tested, Raidd-deficient mice developed tumors in a comparable manner to wt control littermates (see attached manuscript #4). To rule out the possibility that the lack of Raidd induces deregulated proliferation, differentiation or apoptosis that does however not culminate in tumor suppression,

27

Discussion

premalignant and malignant cells in the Eµ-Myc mouse model were closely monitored for possible abnormalities in these processes. But neither in the young premalignant mice nor in acute tumors effects of missing Raidd signalling were observed. Also in the SV40 immortalized MEF levels of micronuclei were comparable to wt and significantly lower as in Casp-2-/- MEF. It remains possible, however, that Raidd may contribute to Caspase-2 function in another context than c-MYC over expression, or that its major physiological role may be control of the CBM complex84, 87.

Our laboratory generated also a knockout mouse model lacking Pidd113. Analysing primary cells and MEF generated from this mouse revealed a deregulation of NFκB activation and cytokine release after high dose γ-irradiation of mice, rather than defects in DNA-damage mediated cell death (see attached manuscript 5121). Despite the clear phenotype after DNA damage on cytokine production, in mouse tumor models we did not reveal predicted accelerating effects on tumor formation on a Pidd- /- background (shown in attached manuscript #269). Surprisingly, in our oncogene induced Eµ-Myc B cell tumor mouse model, Pidd exerted actually oncogenic potential. Loss of this protein delayed tumorigenesis significantly, some mice never developing a tumor at all. The cause for this phenotype remains mysterious so far. The analysed premalignant and malignant B cells did not reveal overt differences to that from wt control mice. Only the pre-B cell population was minimally reduced in 5 week-old mice. Levels of proliferation, apoptosis and also susceptibility to various anti-cancer drugs mimicked behaviour of wt cells. Hence, it remains possible that the NFκB-related function of Pidd may be more critical in vivo and if this putative “pro- survival arm” is lost, tumors develop later. This would be consistent with the reported survival deficit of Pidd-defective lung cancer cells and cell lines treated with cisplatin122. Alternatively, if the cytokines released are related to an inflammatory tumor promoting phenotype, lack of this signal may delay tumor onset. This would be consistent with our own data showing impaired cytokine release upon DNA damage in Pidd-deficient cells (see manuscript #5). To further address the function of Pidd in cancer and sterile inflammation it might be worth-while to generate models that specifically express Pidd-C or Pidd-CC in order to separately tackle pro- and anti- apoptotic vs. inflammatory functions of this versatile protein.

28

Discussion

Conclusions: Altogether the biology of Caspase-2 is, more than twenty years after its initial description, far from being completely understood. The work during my thesis is certainly of value in terms of narrowing down the biological function of Caspase-2 as tumor suppressor but also raises a series of questions in relation to the role of RAIDD and PIDD in cancer biology and inflammation. Furthermore, my analysis supports the idea that Caspase-2 may be activated by interaction with different proteins or is able to auto-activate itself, without the need for further protein-protein interactions, in particular settings such as oncogenic stress. Further investigations into this matter will hopefully shed some light and possibly also open new opportunities to fight cancer.

29

Attached Manuscripts

Attached Manuscripts

1) P53-induced protein with a death domain (PIDD) – Master of Puppets? This review gives an exact overview about the knowledge of PIDD at that time. Due to its various cleavage products the interactions of PIDD are very complex and it changes binding partners depending on isoforms and processing products generated. My contribution to this review was the summary of selected articles dealing with the formation and function of PIDD isoforms and the generation of the table and figures.

30

Attached Manuscripts

Pages 31 - 37: Bock FJ, Peintner L, Tanzer M, Manzl C, Villunger A. P53-induced protein with a death domain (PIDD): master of puppets. Oncogene 2012, 31(45): 4733-4739. can only be accessed online via: http://www.nature.com/onc/journal/v31/n45/full/onc2011639a.html

31 Attached Manuscripts

2) PIDDosome-independent tumor suppression by Caspase-2 The initial description of the PIDD knockout mouse, generated previously in our laboratory mainly focused on the function of the PIDDosome in primary and immortalized cell culture. Meanwhile, the importance of Caspase-2 as a tumor suppressor became public and so the logical extrapolation of this finding was to test for the contribution of PIDD to tumor development. Using three different tumor models we examined the role of the PIDDosome on the level of DNA damage and deregulated proliferation rates. Strikingly we were able to show, that the tumor suppressor function of Caspase-2 is not only independent on the formation of the PIDDosome, but PIDD even acts as an oncogene. Mice lacking PIDD in a B cell tumor mouse model developed malignancies significantly later than their wild type littermates. The study was initiated by my former mentor and master thesis supervisor, Claudia Manzl. I was in charge for the continuation of the project during her maternity leave and extending into the first two years of my PhD thesis. I contributed essentially to all major experimental aspects of the study by monitoring mouse tumor cohorts (Figure 1, 2 and 3), by performing cell death and proliferation assays (Figures 4, 5 and 6) on isolated tumor cells as well as Western blot analysis (Figure 3).

38

Cell Death and Differentiation (2012), 1–11 & 2012 Macmillan Publishers Limited All rights reserved 1350-9047/12 www.nature.com/cdd PIDDosome-independent tumor suppression by Caspase-2

C Manzl1, L Peintner1, G Krumschnabel1, F Bock1, V Labi1,4, M Drach2, A Newbold3, R Johnstone3 and Andreas Villunger*,1

The PIDDosome, a multiprotein complex constituted of the ‘p53-induced protein with a death domain (PIDD), ‘receptor- interacting protein (RIP)-associated ICH-1/CED-3 homologous protein with a death domain’ (RAIDD) and pro-Caspase-2 has been defined as an activating platform for this apoptosis-related protease. PIDD has been implicated in p53-mediated cell death in response to DNA damage but also in DNA repair and nuclear factor kappa-light-chain enhancer (NF-jB) activation upon genotoxic stress, together with RIP-1 kinase and Nemo/IKKc. As all these cellular responses are critical for tumor suppression and deregulated expression of individual PIDDosome components has been noted in human cancer, we investigated their role in oncogenesis induced by DNA damage or oncogenic stress in gene-ablated mice. We observed that Pidd or Caspase-2 failed to suppress lymphoma formation triggered by c-irradiation or 3-methylcholanthrene-driven fibrosarcoma development. In contrast, Caspase-2 showed tumor suppressive capacity in response to aberrant c-Myc expression, which did not rely on PIDD, the BH3-only protein Bid (BH3 interacting domain death agonist) or the death receptor ligand Trail (TNF-related apoptosis-inducing ligand), but associated with reduced rates of p53 loss and increased extranodal dissemination of tumor cells. In contrast, Pidd deficiency associated with abnormal M-phase progression and delayed disease onset, indicating that both proteins are differentially engaged upon oncogenic stress triggered by c-Myc, leading to opposing effects on tumor-free survival. Cell Death and Differentiation advance online publication, 18 May 2012; doi:10.1038/cdd.2012.54

Most members of the Caspase family of proteases are However, it remains unclear if Caspase-2 requires activation involved in killing damaged, harmful or unwanted cells while in the PIDDosome, or another putative activation platform, others regulate inflammation or differentiation. Recent studies such as the death inducing signaling complex (DISC),6 suggest that one member of this family, Caspase-2, may exert or none of the above, under such conditions. even directly opposing functions such as either killing In addition, evidence for Caspase-2 as a critical effector or saving cells after DNA damage.1 in p53-induced apoptosis, acting upstream of mitochondria in Activation of Caspase-2 is coordinated by complex pre- or response to anticancer agents such as 5-fluorouracil has also post-translational modifications of enzyme expression been provided.1,7 Caspase-2 also appears to account for the or function, respectively. These involve differential splicing post-mitotic death of cancer cells upon G2/M checkpoint or phosphorylation at several serine sites, translocation failure.8 Consistent with a role for the PIDDosome in between subcellular compartments, and/or recruitment into p53-induced cell death, PIDD overexpression facilitated different activation complexes.2,3 Following identification Caspase-2 activation and apoptosis in response to DNA of a putative activation platform for Caspase-2, called the damage in HeLa cells4 and RNA interference or antisense PIDDosome (PIDD/RAIDD/Caspase-2), which supported its oligonucleotides targeting PIDD mRNA delayed cell death assumed role as an initiator caspase of apoptosis,4 evidence induced by overexpression of p53 in H1299 colon cancer9 was provided that it might also be involved in an Ataxia or K562 myelogenous leukemia cells, respectively.10 telangiectasia mutated (ATM)/Ataxia telangiectasia and While all these in-vitro studies provided evidence of an Rad3-related protein (ATR)-dependent cell death pathway apoptotic function of the Caspase-2 containing PIDDosome, controlled by checkpoint kinase-1 (Chk1). There, Caspase-2 targeting PIDD or RAIDD by siRNA in HCT-116 colon killing appears to occur independent from classical apoptosis carcinoma cells,7 as well as analysis of gene-targeted mice regulators such as Caspase-3 or B-cell lymphoma 2 (Bcl-2).5 lacking Pidd11,12 or Raidd,13 failed to provide supportive

1Division of Developmental Immunology, BIOCENTER, Medical University Innsbruck, Innsbruck, Austria; 2Institute of Pathology, Medical University Innsbruck, East Melbourne, Victoria, Australia and 3Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia *Corresponding author: A Villunger, Division of Developmental Immunology, BIOCENTER, Medical University Innsbruck, Innrain 80-82, Innsbruck 6020, Austria. Tel: þ 43 512 9003 70380; Fax: þ 43 512 9003 73960; E-mail: [email protected] 4Current address: Max-Delbru¨ck Center for Molecular Medicine, Berlin-Buch, Germany. Keywords: apoptosis; Caspase-2; PIDDosome; cancer Abbreviations: PIDD, p53-induced protein with a death domain; RAIDD, receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a death domain; NF-kB, nuclear factor kappa-light-chain enhancer; Nemo/IKKg, NF-kB essential modulator/nuclear factor kappa-B kinase subunit gamma; ATM, Ataxia telangiectasia mutated; ATR, Ataxia telangiectasia and Rad3-related protein; Bcl-2, B-cell lymphoma 2; DISC, death inducing signaling complex; MEF, mouse embryonic fibroblast; 3-MC, 3-methylcholanthrene; Bid, BH3 interacting domain death agonist; TRAIL, TNF-related apoptosis-inducing ligand; Mdm2, murine double minute; PI, propidium iodide; BrdU, Bromodeoxyuridine; 53BP-1, p53 binding protein 1; RIP-1, receptor-interacting serine/threonine-protein kinase 1; TNF, tumor necrosis factor; IL, interleukin Received 7.10.11; revised 29.2.12; accepted 15.3.12; Edited by B Zhivotovsky Caspase-2 in tumor suppression C Manzl et al 2

evidence as normal cell death responses in mouse embryonic fibroblast (MEF), lymphocytes and oocytes in response to DNA damage were reported. Furthermore, conflicting obser- vations regarding cell death resistance of neurons were made in two different Caspase-2 knockout strains generated14,15 and combined deletion of Caspase-2 plus Caspase-9 in hematopoietic cells failed to reveal any redundancy between both initiator caspases in apoptosis induction.16 Regardless of a pro-death or pro-survival function of PIDD or Caspase-2, all mechanisms they seem to be involved in are highly relevant for tumorigenesis. Noteworthy, expression of Caspase-2, encoded on Chr. 7q in humans harboring a putative tumor suppressor, has been reported to be reduced in patients suffering from acute myeloid leukemia or acute juvenile lymphoblastic leukemia where it correlates with poor prognosis or treatment resistance, respectively. Reduced protein expression was also reported in mantle cell lymphoma as well as in solid tumors such as gastric cancer and metastasizing tumors of the brain.17 Along that line, a possible tumor suppressor function of PIDD is also supported by a reported correlation between apoptotic index and its protein levels in oral squamous cell carcinoma patient samples.18 In line with a tumor suppressive role of the PIDDosome, E1A/Ras-transformed MEF lacking Caspase-2 showed increased colony formation potential in soft agar and accelerated tumor formation in xeno-transplant studies. Even more striking, when Em-Myc transgenic mice, predisposed for B-cell lymphomagenesis, were crossed with Casp2 / mice, further acceleration of tumor formation was observed.19 The molecular basis for the accelerated tumorigenesis in relation to PIDDosome formation, however, remains unresolved but Figure 1 Loss of Pidd or Caspase-2 has no influence on DNA damage-induced proposed to be accounted for by a more general resistance of tumorigenesis. (a) Tumor-free survival of wt (n ¼ 18), Pidd / (n ¼ 6) and Caspase-2-deficient cells to oncogenic stress or DNA Casp2 / (n ¼ 16) mice after fractionated irradiation (4 1.75 Gy). No difference damage.19 To address the role of PIDDosome formation in in the development of thymic lymphomas between the genotypes was observed. tumor suppression, we have investigated DNA damage- P53 / mice (n ¼ 7) were included as positive controls, and earlier onset of thymic lymphomas was monitored (Po0.0001). (b) Tumor-free survival of wt (n ¼ 11), induced as well as oncogene-triggered tumor formation in / / þ / Pidd-deficient mice and compared it with the effects observed Pidd (n ¼ 10), Casp2 (n ¼ 11) and p53 (n ¼ 7) mice after a single injection of 3-MC or vehicle (sesame oil; n ¼ 7) to induce fibrosarcomas. in the absence of Caspase-2, or, where appropriate, p53. Sarcomas occurred significantly earlier in p53 þ / animals compared with wt mice (Po0.0001) Results

The PIDDosome fails to prevent tumor formation (3-MC), a carcinogen forming bulky adducts with DNA, leading triggered in response to DNA damage. Both PIDD and to the formation of fibrosarcomas, again in a p53-regulated Caspase-2 have been repeatedly implicated in the cellular manner.22 Consistently, loss of one allele of p53 led to an response to DNA damage. Hence, we triggered lymphoma- accelerated onset of fibrosarcomas (Po0.0001), but loss of genesis in Pidd- and Caspase-2-deficient mice by repeated Pidd or Caspase-2 had no such effect (Figure 1b). exposure to low-dose g-irradiation, a well-established in-vivo Together, our results demonstrate that the PIDDosome model of DNA damage-driven tumor formation.20 Tumor- is dispensable for tumor suppression in response to DNA- igenesis in this model is accelerated by loss of p5321 and damage, inflicted by g-irradiation or bulky-adduct formation. hence appeared most suitable to study the tumor suppressor potential of the PIDDosome. Wild-type (wt) mice developed Loss of Pidd or Caspase-2 deregulate c-Myc-induced thymic lymphomas with a median survival of 181 days (d). lymphomagenesis. To investigate if Caspase-2 depends Consistent with published results, loss of p53 accelerated on PIDDosome formation to suppress c-Myc-driven lympho- tumor formation (median survival 107 d, P o0.0001) but magenesis, Casp2 / and Pidd / mice were intercrossed absence of Pidd or its putative downstream apoptosis with mice overexpressing c-Myc under control of the Ig heavy effector, Caspase-2, had no such effect (median survival chain enhancer, limiting expression of the transgene to 202 d and 185 d, respectively) (Figure 1a). the B-cell lineage.23 Cohorts of wt Em-Myc, Em-Myc/Pidd / To expand these observations into a different model system and Em-Myc/Casp2 / mice were followed till development of DNA damage-driven tumorigenesis, we also challenged of disease. While Em-Myc mice (median survival 122 d) mice by intramuscular injection of 3-methylcholanthrene lacking Caspase-2 succumbed to disease significantly earlier

Cell Death and Differentiation Caspase-2 in tumor suppression C Manzl et al 3

(median survival 90 d, P ¼ 0.0044) and showed increased Previous reports suggested that the pro-apoptotic function tumor burden in the spleen (Supplementary Figure S1), of Caspase-2 relies in part in its capacity to process and Em-Myc/Pidd / mice showed delayed onset of disease activate the BH3-only pro-apoptotic Bcl-2 family protein (median survival 250 d, Po0.0005; Figure 2a). Immunophe- BH3 interacting domain death agonist (Bid) into its active notyping of lymphomas revealed that loss of Caspase-2 truncated form (tBid), upstream of mitochondrial outer promoted mainly B-cell tumors of IgM þ or mixed pre-B/IgM þ membrane permeabilization,24 and/or by sensitizing cells phenotype (presented as IgM þ / ) while early hematopoietic to Trail-mediated cell death by facilitating processing of progenitor-derived lymphomas, represented as B220 þ pro-Caspase-8.25 Therefore, we reasoned that impaired CD4 þ tumors, were not observed so far in the absence activation of Bid or ineffective Trail signaling may contribute of Caspase-2 (P ¼ 0.002, w2-test). Kaplan–Meier analysis to the accelerated tumor formation observed in Caspase-2- confirmed that the altered latency in tumor-free survival deficient mice overexpressing c-Myc. Hence, we also observed in the absence of Caspase-2 was due to the faster investigated cohorts of Em-Myc transgenic mice that lack rise of IgM þ tumors (P ¼ 0.0028; Figures 2b–d). Bid or Trail, but neither Bid / nor Trail / mice developed

Figure 2 Opposing effects of individual PIDDosome components on c-Myc-induced lymphomagenesis. (a) Cohorts of Em-Myc (n ¼ 42), Em-Myc/Pidd / (n ¼ 41) and Em-Myc/Casp2 / (n ¼ 25) mice were monitored for the development of B-cell lymphomas over time. Em-Myc mice lacking Caspase-2 showed a significantly earlier onset of lymphomagenesis (P ¼ 0.0044) compared with wt Em-Myc, while Pidd deficiency delayed lymphomagenesis in Em-mice (P ¼ 0.0005). (b) Distribution of pro/pre-B (white), mixed IgM þ / (light gray), IgM þ (dark gray) and B220 þ CD4 þ (black) lymphomas analyzed from mice of the indicated genotypes. The distribution of lymphomas in Em-Myc/ Casp2 / was significantly different (P ¼ 0.004) compared with wt Em-Myc due to the higher amount of mixed and IgM þ tumors. Kaplan–Meier analysis was depicted as (c) pre-B and (d) IgM þ B lymphomas of indicated genotypes. In Em-Myc/Casp2 / mice, IgM þ tumors developed significantly earlier than in Em-Myc animals (P ¼ 0.0028). (e) Kaplan–Meier analysis of Bid / (n ¼ 13), Em-Myc (median survival 97 d, n ¼ 18), Em-Myc/Bid þ / (median survival 137 d, n ¼ 35) and Em-Myc/Bid / (median survival 134 d, n ¼ 25) is shown. (f) Cohorts of Trail / (n ¼ 22), Em-Myc (median survival 113 d, n ¼ 21), Em-Myc/Trail þ / (median survival 125 d, n ¼ 50) and Em-Myc/ Trail / (median survival 164 d, n ¼ 20) mice were monitored for the development of B-cell lymphomas over time

Cell Death and Differentiation Caspase-2 in tumor suppression C Manzl et al 4

Em-Myc-driven B-cell lymphomas earlier than Em-Myc mice p53 in Em-Myc transgenic mice.27 To monitor, if loss of (Figures 2e and f). Caspase-2 also reduced the frequency of p53 inactivation, Together, our findings demonstrate that Caspase-2- we performed western blot analysis for p53 and ARF, that mediated suppression of c-Myc-driven lymphomagenesis can be used as markers for lost or mutated p53,26 on primary does not depend on the formation of the PIDDosome, tumor samples from Em-Myc, Em-Myc/Pidd / and Em-Myc/ processing of its putative substrate Bid, or sensitization of Casp2 / mice (Figure 3a). Our analysis revealed cells to Trail-induced killing. Furthermore, Pidd appears to reduced p53 inactivation in the absence of Caspase-2 be able to exert oncogenic potential, most likely independent (2/21 cases; 9.5%), when compared with wt (9/40; 22.5%) of its proposed Caspase-2 activating function. or Pidd-deficient (4/17; 23.5%) Em-Myc tumors. Moreover, Kaplan–Meier analysis of lymphomagenesis showed a Caspase-2 deficiency reduces the pressure to lose significantly earlier tumor onset in Em-Myc/Casp2 / tumors p53. Initially, the increased proliferative rate caused by (Po0.0001) with presumably normal p53 status (no immu- c-Myc overexpression is balanced by massive apoptosis noreactivity for ARF or p53 in western analysis) compared until counterselected for, for example, by inactivation of the with wt Em-Myc tumors while Em-Myc/Pidd / lymphomas ARF/murine double minute (Mdm2)/p53 signaling network.26 developed significantly later (P ¼ 0.0291) (Figure 3b, upper Consistently, loss of alternative antagonists of c-Myc-driven graph). In contrast, tumors carrying a mutation (immunor- lymphomagenesis, such as the pro-apoptotic BH3-only eactive for both ARF and p53) or a deletion of p53 proteins Bim or Bmf, relieve the pressure to inactivate (immunoreactivity for ARF but not for p53) showed no

Figure 3 Loss of Caspase-2 relieves the pressure to lose p53.(a) Representative western blot analysis of p19ARF and p53 expression in tumors derived from Em-Myc, Em-Myc/Pidd / and Em-Myc/Casp2 / . GAPDH serves as a loading control. In the last lane, a lysate derived from Em-Myc/Casp2 / (B10) or Em-Myc lymphoma (E91) is loaded as positive control. (b) Kaplan–Meier analysis of lymphomagenesis in mice with normal p53 or modified p53 status (mutated or lost, as assessed by western blotting in a). (c) Cohorts of Em-Myc/p53 þ / (n ¼ 5), and Em-Myc/p53 þ / /Casp2 / (n ¼ 5) mice were monitored for the development of B-cell lymphomas over time and data are depicted as tumor-free survival in a Kaplan–Meier analysis

Cell Death and Differentiation Caspase-2 in tumor suppression C Manzl et al 5 difference in latency between genotypes (Figure 3b, lower that the DNA-damage response to oncogenic stress is largely graph). These data suggest that Caspase-2 acts most likely functional in PIDDosome-defective mice. in an alternative pathway, other than the ones activated by p53 upon oncogenic stress, or upstream of p53 itself. Yet, Loss of the PIDDosome associates with impaired when Caspase-2 deficiency was combined with haplo- M-phase progression in tumor cells. As we failed to insufficiency for p53, no further acceleration of disease onset observe relevant changes in PIDDosome-defective prema- was observed, indicating that loss of heterozygosity that lignant mice, we turned our focus back on diseased mice. usually accelerates disease onset in Em-Myc/p53 þ / mice is Interestingly, Pidd-deficient tumors showed an increase in dominant over loss of Caspase-2 in Em-Myc-driven lympho- the percentage of cells in G2/M phase compared with wt magenesis (Figure 3c). Em-Myc lymphomas (mean 23±6.6% versus 12±1.1% in G2/M), an effect also noted, albeit less pronounced, in the Normal cell death and proliferative responses in PIDDo- absence of Caspase-2 (mean 18.3±1.8%; Figure 5a). some-defective premalignant B cells. As apoptosis Staining of the mitotic marker phospho-histone H3 (pH3) defects are most critical in premalignant Em-Myc animals to revealed a significantly higher percentage of lymphoma cells allow c-Myc-driven transformation, we investigated if Pidd or in mitosis in Pidd-deficient tumor samples (P ¼ 0.0004), and Caspase-2 loss may impact on c-Myc-mediated cell death a less pronounced, but still statistically significant elevation and B-cell homeostasis in premalignant mice. Composition (P ¼ 0.028) was also observed in Caspase-2-deficient and distribution of different B-cell subsets in bone marrow, samples (Figure 5b), a result fitting our cell-cycle analysis. spleens and peripheral blood derived from premalignant This suggests that cell-cycle progression is impaired in both Em-Myc/Casp2 / mice showed no gross alterations, when Em-Myc/Pidd / and Em-Myc/Casp2 / tumors and trans- compared with Em-Myc mice but loss of Pidd appeared to formed cells may either enter mitosis in increased numbers reduce the number of pre-B and immature T1 transitional B and/or have problems to exit from mitosis. cells present in the spleen of transgenic animals (P ¼ 0.0584 Previous reports suggested that Caspase-2 can limit and P ¼ 0.0004, respectively; Figure 4a). transformation by silencing expression of the chromosome The reduced B-cell numbers may be due to increased rates passenger protein Survivin, a protein highly expressed of c-Myc-driven cell death or, alternatively, impaired prolifera- in proliferating (tumor) cells, with putative albeit disputed tion in premalignant cells. Hence, pre-B as well as IgM þ IgD anti-apoptotic potential.28 Therefore, we tested a number of naive B cells were sorted from the bone marrow and spleen IgM þ tumor samples derived from Em-Myc, Em-Myc/Pidd / from wt Em-Myc, Em-Myc/Pidd / and Em-Myc/Casp2 / and Em-Myc/Casp2 / mice for their Survivin protein levels mice and put in culture to assess apoptosis over time by flow but failed to observe consistent differences. The level of cytometric analysis. With the exception of increased apopto- Caspase-2 was comparable in tumors derived from Em-Myc sis due to c-Myc overexpression, comparable cell death rates and Em-Myc/Pidd / mice (Figure 5c) and cleaved fragments were observed across genotypes, indicating that the PIDDo- of Caspase-2 indicative for processing or activation were not some does not regulate c-Myc-induced apoptosis, at least not detected in this analysis. in premalignant B cells (Figure 4b). Similarly, immediate To determine a potential contribution of oncogene-induced assessment of Annexin V/propidium iodide (PI)-positive B senescence to delayed tumor onset in Em-Myc/Pidd / mice, cells after organ harvest failed to reveal genotype-dependent we tested lymphoma samples for the presence of lysine-9 differences in in-situ apoptosis rates (data not shown). methylation of histone H3 by western blotting (Figure 5c), as Together, our results exclude a prominent role for impaired well as for the expression of the senescence-associated CDK apoptosis in the deregulated tumor formation observed in inhibitors p16ink4a and p21 by immunofluorescence and/or PIDDosome-deficient mice, pointing toward possible deficits qRT–PCR. Of note, p21 mRNA levels actually appeared in cell-cycle control or senescence triggered by oncogenic c- decreased in tumors lacking Caspase-2. However, none of Myc. Therefore, we investigated the impact of Pidd or the other markers provided convincing evidence for deregu- Caspase-2 deficiency on proliferation in premalignant mice lated senescence capacity in PIDDosome-deficient cells by in-vivo Bromodeoxyuridine (BrdU)-labeling experiments. that may have explained the differences in tumor onset However, we failed to detect significant differences in the (Figure 2a). Finally, we also quantified serum levels of TGFb percentage of cycling premalignant B-cell subsets in the in premalignant and diseased mice, as this cytokine appears absence or presence of the PIDDosome (Figure 4c), indicat- to be a critical non-cell autonomous inducer of senescence, ing that proliferation is not impaired by the loss of Caspase-2 released by macrophages after engulfing apoptotic lymphoma or Pidd in non-transformed B cells. cells.29 Next to the fact that we observed increased levels of As the PIDDosome has been implicated in DNA-repair pro- this cytokine in diseased mice we failed to observe statistically cesses, we monitored changes in markers of this response, significant differences across genotypes (Supplementary that is, gH2AX and p53 binding protein 1 (53BP-1), by employing Figure 2C). However, variation between individual animals immunofluorescence analysis on FACS-sorted premalignant was rather high. mature (CD19 þ IgM þ ) splenic B cells from PIDDosome- Finally, as PIDD was reported to be involved in NF-kB defective Em-Myc transgenic mice. Again, we noted no activation via complex formation together with RIP-1 kinase significant difference regarding the amount of DNA damage, and NEMO/IKKg30 and it was recently published that persist- as indicated by gH2AX foci formation, or repair capacity, ing DNA damage can promote secretion of cytokines like quantified by counting the number of p53BP-1 containing tumor necrosis factor a (TNFa), interleukin-6 (IL-6) or IL-831 repair foci across genotypes (Figure 4d). These data suggest that may contribute to generate a pro-inflammatory tumor

Cell Death and Differentiation Caspase-2 in tumor suppression C Manzl et al 6

Figure 4 Normal cell death and proliferation rates in PIDDosome-defective premalignant B cells. (a) Distribution of B-cell subsets in bone marrow, or spleens of mice of the indicated genotypes. Single-cell suspensions derived from premalignant animals 5 weeks of age were counted, stained for different B-cell markers and analyzed by flow y cytometry. Data are represented as means of n44 animals/genotype ±S.E.M. *Po0.05 compared with Em-Myc controls, Po0.05 compared with Em-Myc/Casp2 / mice. (b) Sorted pre-B cells from the bone marrow (CD19 þ IgM CD43 ), immature B cells (IgM þ IgDlow) from the spleens of mice of the indicated genotypes were cultured for 0, 6, 24 and 48 h cells and cell survival in CD19 þ B cells was monitored by Annexin V/PI staining and flow cytometric analysis. Data points represent mean values±S.E.M. from n43 cell mice/genotypes. *Po0.05 all Em-myc versus all non-transgenic genotypes (ANOVA and Student–Newman–Keuls test). (c) BrdU incorporation was determined in CD19 þ IgM pro/pre-B cells isolated from bone marrow (left) and mature CD19 þ IgM þ B cells isolated from spleen (right) from indicated genotypes (5 weeks old). Data are mean values±S.E.M from n44 mice/genotypes. (d) Mature B cells (CD19 þ IgM þ ), sorted from spleens derived from premalignant mice of the indicated genotypes, were stained for gH2AX or 53BP-1 and foci/cells is depicted as bar graphs of mean values±S.E.M. of n43 preparations from individual animals

promoting environment, we also quantified these cytokines in human IL-8, MIP-2, were not significantly altered in sera of serum samples from premalignant and malignant mice. Em-Myc mice lacking the PIDDosome but this issue deserves However, levels of TNFa, IL-6 and the mouse ortholog of a more detailed follow-up (Supplementary Figure S3).

Cell Death and Differentiation Caspase-2 in tumor suppression C Manzl et al 7

Normal cell death responses in PIDDosome-deficient tumor cells. Tumors derived from wt Em-Myc, Em-Myc/ Pidd / and Em-Myc/Casp2 / mice showed comparable levels of apoptotic cells in situ and tumor cells died equally fast, when put in culture without further treatment (Figure 6 and data not shown). Similarly, treatment of primary lymphoma cells wt for p53 according to our western blot analysis with a number of different DNA-damaging agents, including Etoposide and Doxorubicin, or other anti-neoplastic agents reported in part to act via Caspase-2 failed to reveal significant differences between wt, Pidd- or Caspase-2- deficient Em-Myc tumor cells (Figure 6).

Tumors lacking Caspase-2 display increased dissemina- tion potential. Having excluded severe defects in apoptosis or DNA repair in PIDDosome-defective Em-Myc mice, we performed histological analysis of H&E-stained tumor samples to gain possible insight why Caspase-2 and Pidd-defective mice behaved so differently in this model. This analysis revealed that there was a higher tumor infiltration rate in cancerous Em-Myc mice lacking Caspase-2, presenting with pulmonary congestion, loss of splenic parenchymatic architecture and visible hematopoi- esis, as well as clear perivascular infiltrations in liver (Figures 7a and b and not shown), a phenomenon not seen to such degree in wt or Pidd-deficient Em-Myc transgenic mice. This observation indicates differences in the migratory potential of tumors lacking Caspase-2, or a more permissive tumor microenvironment in Caspase-2-deficient mice allow- ing B-cell tumors lacking this protease to behave more aggressively, contributing to the reduced disease-free survival. Preliminary data on transplantation experiments suggest that tumors derived from Em-Myc mice do not arise faster when transplanted into Caspase-2-deficient recipients, indicating that the higher dissemination rate observed in Em-Myc/Casp2 / mice is most likely not due to a more permissive environment in Caspase-2-deficient soma (Supplementary Figure S4).

Discussion Figure 5 Lack of Pidd or Caspase-2 leads to a deregulation of cell cycle in Em- Myc tumors. (a) Cell cycle-distribution is depicted with G1 phase (white), S phase The PIDDosome has been implicated in two fundamental, but (light gray) and G2/M phase (dark gray) in tumor cells freshly isolated opposing cellular processes, both being of major importance from Em-Myc (n ¼ 23), Em-Myc/Pidd / (n ¼ 8) and Em-Myc/Casp2 / to limit tumorigenesis, that is, apoptosis and DNA repair. (n ¼ 13). (b) Tumor cells of wt Em-Myc (n ¼ 17), Em-Myc/Pidd / (n ¼ 8) and 19 / Based on the study from Ho et al., showing a tumor Em-Myc/Casp2 (n ¼ 13) were co-stained for mitosis-marker pH3 and PI. Bars suppressor function for Caspase-2 and correlative findings in represent percentages of pH3-positive cells ±S.E.M. *Po0.05. (c) Representative western blot analysis quantifying Caspase-2, Survivin or Lys9 pan-methyl H3 (mH3) human cancer, the role of the PIDDosome was investigated levels in IgM þ tumors derived from Em-Myc, Em-Myc/Pidd / and Em-Myc/ in different tumor models in more detail. Surprisingly, the Casp2 / . Detection of p38 MAPK served as a loading control PIDDosome had no impact on development of radiation- induced thymic lymphomas or chemically induced fibrosarco- mas arguing against a critical role of the PIDDsome in Together these data suggest that B-cell lymphomas from suppressing DNA damage-driven apoptosis and related Em-Myc/Pidd / mice develop and manifest with a significant tumorigenesis (Figure 1). In contrast, however, it had a strong delay that is not directly linked to an increased rate of influence on the development of c-Myc-driven B-cell lympho- oncogene-driven cellular senescence or defective inflamma- mas, as loss of Caspase-2 accelerated disease onset, tory responses but may associate with deregulated M-phase as previously described,19 while, surprisingly, loss of Pidd progression, while those lacking Caspase-2 show a trend delayed disease in Em-Myc transgenic mice (Figure 2). toward reduced p21 mRNA levels. Whether any of these As the mechanism behind the tumor suppressor function of alterations is causal for the phenotypes observed remains to Caspase-2 resistance to oncogene-driven apoptosis was be determined. proposed, based mainly on studies using E1A/Ras-

Cell Death and Differentiation Caspase-2 in tumor suppression C Manzl et al 8

Figure 6 The PIDDosome is dispensible for drug-induced apoptosis in Em-Myc lymphomas. Freshly isolated tumor cells from indicated genotypes were cultivated on irradiated Bcl-2 overexpressing NIH-3T3 feeder cells and stimulated with graded doses of different drugs for 24 h. Apoptotic cell death was investigated using Annexin V/7-AAD in combination with anti-CD19 mAb and flow cytometric analysis. Data points represent means of n43 tumor samples/genotype ±S.E.M.

transformed MEF in-vitro and xeno-transplant studies using of Caspase-2 protein in wt and Pidd-deficient tumors and so such MEF. A direct impact of Caspase-2 deficiency on far found no evidence for activation of Caspase-2 under c-Myc-driven cell death or proliferation of B cells was not conditions of aberrant expression of c-Myc. investigated in this study.19 Our analysis of these parameters In line with Ho et al.,19 we also noted a trend to lower mRNA demonstrated comparable cell death rates in c-Myc over- levels of the p53 target p21 in Caspase-2-deficient tumors. expressing premalignant B cells proficient or deficient for However, our initial analysis on Em-Myc mice lacking p21 Caspase-2 or Pidd, arguing against a general resistance of failed to provide evidence that loss of this CDK inhibitor PIDDosome-deficient cells to oncogene-driven cell death. accelerates onset of lymphomagenesis in this disease model This response may be critical only in response to H-Ras (AN and RJ, unpublished), questioning the importance of this hyperactivation. In contrast to Ho et al., we also failed to observation. Also, we were unable to confirm these findings at observe drug-resistance phenotypes in the Caspase-2-deficient the protein level (Supplementary Figure S2 and data not lymphomas studied in vitro (Figure 6), observations in line with shown) and data from transplantation experiments comparing our studies on primary lymphocytes and MEF from PIDDo- the growth of wt and Caspase-2-defective tumors failed some-defective mice.12,13 The reason for this inconsistency is to reveal reproducible differences between genotypes unclear, but differences in the experimental set-up, such as (Supplementary Figure S4). Hence, we conclude that neither the use of tumor cells of defined immunophenotype and/or increased cell death resistance nor increased rates of p53 status as well as feeder cells in our study may contribute. proliferation account for the observed acceleration of B-cell Since mediation of c-Myc-driven apoptosis is apparently not lymphomagenesis in Casp2 / mice. Of note, our histo- the key mechanism behind the tumor suppressor function of pathological data confirmed a comparable mitotic index Caspase-2 one might speculate whether the proteolytic between wt and Caspase-2-defective lymphomas (data not activity of Caspase-2 itself has an important role. Caspase-2 shown) but revealed that loss of Casp2 leads to a more may act simply as a scaffold for other proteins exerting anti- aggressive type of B-cell lymphoma, reflected in a higher oncogenic properties. In our study, we observed similar levels infiltration rate of non-lymphoid organs and higher splenic

Cell Death and Differentiation Caspase-2 in tumor suppression C Manzl et al 9

show increased frequency of Mdm2 overexpression or ARF loss remains to be analyzed. To our surprise and in contrast to Caspase-2-deficient mice, Em-Myc/pidd / mice developed lymphomas at a signifi- cantly later time point, assigning it an oncogenic role in B-cell lymphomagenesis. The increased proliferative rate caused by c-Myc overexpression is initially balanced by massive apoptosis. Of note, knockdown of PIDD has been proposed to sensitize cells to death upon DNA damage triggered by UV33 or doxorubicin.34 In contrast, its overexpression asso- ciated with growth arrest in K-RasG12D transformed murine lung carcinomas and human lung cancer cell lines, an effect proposed to be due to improved p53 stability thereby enforcing p21 expression,34 providing a possible explanation for the delayed tumor formation we observed. However, although we noted a minor reduction in B-cell progenitors in the spleens of Em-Myc mice lacking Pidd, we failed to observe increased spontaneous cell death in premalignant B cells or reduced proliferation, as monitored by BrdU incorporation. Also, level of p21 mRNA in Pidd-defective lymphomas was not different to those in wt tumors, while M-phase progression seemed to be impaired despite this lack of difference. PIDD-deficient lymphomas also responded normally to DNA damaging agents (Figure 6). Together, this indicates that the role of Pidd might differ between premalignant and tumor cells and cell type-specific differences need to be taken into account. Together, our results indicate that Caspase-2 does not limit c-Myc-driven transformation by mediating apoptosis or limit- ing cell-cycle progression in premalignant or malignant cells, but leads to a more aggressive tumor phenotype, indicated by the enhanced dissemination into non-lymphoid organs that associates with the reduced lifespan in these mice. Hence, inve- stigations into a possible role of Caspase-2 in cell migration and anoikis, but also genomic stability, appear warranted. Furthermore, Pidd seems to have no impact on limiting cell-cycle progression or cell death in premalignant cells but affects mitosis regulation Figure 7 Loss of Caspase-2 promotes dissemination of Em-Myc lymphomas. (a) Representative images of H&E-stained sections from lung (left panel) and in c-Myc transformed tumor cells, leading to delayed onset of spleen (right panel) from mice of the indicated genotypes showing tumor tumorigenesis by a yet to be defined molecular mechanism. manifestation ( 400 magnification). (b) Percentage of tumor infiltrated area in indicated organs and genotypes quantified from H&E-stained sections of n46 mice Materials and Methods y per genotype. Data are represented as mean values±S.E.M. *, Indicate significant Mice. All animal experiments were performed in accordance with the Austrian difference with a P-value of o0.05 comparing Em-Myc/Casp2 / with Em-Myc or legislation (BGBl. Nr. 501/1988 i.d.F. 162/2005, # BMWF-66.011/0137-II/10b/ Em-Myc/Pidd / mice, respectively 2009). The generation and genotyping of the Pidd / , Casp2 / , Bid / , Trail / , p53 / and Em-Myc transgenic mice have been described.12,35 All mice used were maintained on an inbred C57BL/6 genetic background. weight (Figure 7; Supplementary Figure S1B). This observa- Tumorigenesis induced by DNA damage. Mice were exposed to tion points toward a possible role for Caspase-2 in limiting cell whole body irradiation to four weekly doses of g-irradiation with 1.75 Gy in a linear accelerator starting at the age of 4 weeks. To induce fibrosarcomas, mice were migration and/or metastasis potential. Noteworthy here, a injected once i.m. with 1 mg of 3-MC (dissolved in 200 ml sesame oil/mouse; recent study describes H-Ras-dependent downregulation of Sigma, Vienna, Austria). As a control, mice were injected with vehicle alone. Caspase-2 and subsequent resistance to anoikis in intestinal epithelial cells as a means that facilitates Ras-induced Cell culture and reagents. FACS-sorted pre-B, immature and mature þ transformation.32 This observation may in part also explain IgM B cells were cultured in DMEM (PAA) supplemented with 10% FCS (PAA; 19 Linz, Austria), 250 mM L-glutamine (Gibco, Life Technologies, Vienna, Austria), in part the findings by Ho et al. made in E1A/Ras- streptomycin/penicillin (Sigma) and 50 mM beta-2-mercaptoethanol (Sigma). transformed MEF and xeno-transplants, as well as our obser- Isolated lymphoma cells were cultured on supporting irradiated NIH-3T3 cells vation on increased organ infiltration, but clearly requires overexpressing Bcl-2. Source/concentrations of reagents are Etoposide experimental evaluation. Finally, the phenotype of Caspase- (0.01–1 mg/ml), Dexamethasone (10 9–10 7), Paclitaxel (0.5–50 mM), Thapsi- 2-defective Em-Myc mice is most likely independent of p53, gargin (0.5–50 ng/ml) and Doxorubicin (4–400 ng/ml) (all from Sigma). since Caspase-2-deficient lymphomas show a decreased rate Histopathology. Tissues from moribund mice were embedded in paraffin and of p53 inactivation, suggesting that it acts in a parallel tumor sections (10 mm) were H&E stained. Images were taken using a NIKON Eclipse suppressor pathway. Whether Caspase-2-deficient tumors E800 bright field microscope (Vienna, Austria).

Cell Death and Differentiation Caspase-2 in tumor suppression C Manzl et al 10

Flow cytometric analysis and cell sorting. The monoclonal antibodies the FP06 RTN ‘ApopTrain’ to AV and FB as well as the Daniel Swarovski Fond used, and their specificities, are as follows: RA3-6B2, anti-B220; R2/60, anti- (DSF) and the Tyrolean Science Fund (TWF) to CM and the Austrian Krebshilfe- CD43; II/41, anti-IgM; 11/26C, anti-IgD; MB19-1, anti-CD19; 53-7.3, anti-CD5; Tirol (CM and GK). RWJ is a Principal Research Fellow of the National Health and GK1.5, anti-CD4; H57-597, anti-TCRb (all from eBioscience, Vienna, Austria); 53- Medical Research Council of Australia (NHMRC) and supported by NHMRC 6.7, anti-CD8; (all from BD Pharmingen, San Diego, CA, USA) and B3B4, anti- Program and Project Grants, the Susan G Komen Breast Cancer Foundation, the CD23; 7E9, anti-CD21 (Biolegend, Fell, Germany). Biotinylated antibodies were Prostate Cancer Foundation of Australia, Cancer Council Victoria, The Leukemia detected using streptavidin-RPE (DAKO, Vienna, Austria) or streptavidin-PE-Cy7 Foundation of Australia, Victorian Breast Cancer Research Consortium, Victorian (BD Phamingen). Pre/pro B cells (B220 þ IgM ) were sorted from the bone Cancer Agency and the Australian Rotary Health Foundation. marrow stained for B220 and IgM, while immature (IgM þ IgDlow) and mature (IgM þ IgDhigh) B cells were sorted from the spleen using a FACSVantage cell sorter (Becton Dickinson, Heidelberg, Germany). 1. Vakifahmetoglu-Norberg H, Zhivotovsky B. The unpredictable caspase-2: what can it do? Trends Cell Biol 2010; 20: 150–159. Immunoblotting. Membranes were probed with rat anti-p19/ARF (5-C3-1) 2. Kurokawa M, Kornbluth S. Caspases and kinases in a death grip. Cell 2009; 138: 838–854. (Santa Cruz Biotechnology, Szabo-Scandic, Vienna, Austria), Caspase-2 (11B6) 3. Krumschnabel G, Sohm B, Bock F, Manzl C, Villunger A. The enigma of caspase-2: the (Alexis, Vienna, Austria), mouse anti-p53 antiserum (1C12) and pan-methyl laymen’s view. Cell Death Diff 2009; 16: 195–207. Histone H3 (Lys9), Survivin (71G4B7) or p38 MAPK (8690) (all from Cell 4. Tinel A, Tschopp J. The PIDDosome, a protein complex implicated in activation Signaling, New England Biolabs, Frankfurt, Germany). Equal loading of proteins of caspase-2 in response to genotoxic stress. Science 2004; 304: 843–846. was confirmed by probing filters with antibodies specific for GAPDH (Sigma). 5. Sidi S, Sanda T, Kennedy RD, Hagen AT, Jette CA, Hoffmans R et al. Chk1 suppresses a Horseradish peroxidase-conjugated sheep anti-rat Ig antibodies (Jackson caspase-2 apoptotic response to DNA damage that bypasses p53, Bcl-2, and caspase-3. Research, Vienna, Austria), goat anti-rabbit or rabbit anti-mouse antibodies Cell 2008; 133: 864–877. (DAKO) served as secondary reagents and the enhanced chemiluminiscence 6. Olsson M, Vakifahmetoglu H, Abruzzo PM, Hogstrand K, Grandien A, Zhivotovsky B. (ECL; Amersham, Freiburg, Germany) system was used for detection. DISC-mediated activation of caspase-2 in DNA damage-induced apoptosis. Oncogene 2009; 28: 1949–1959. 7. Vakifahmetoglu H, Olsson M, Orrenius S, Zhivotovsky B. Functional connection between Cell-cycle analysis. Cell-cycle analysis was performed by ethanol fixation p53 and caspase-2 is essential for apoptosis induced by DNA damage. Oncogene 2006; (70% in PBS) of the lymphoma cells and PI staining (40 mg/ml; Sigma). The 25: 5683–5692. percentage of cells in M phase was evaluated by combined PI and anti-phospho- 8. Castedo M, Perfettini JL, Roumier T, Valent A, Raslova H, Yakushijin K et al. Mitotic H3Ser10 staining (Cell Signaling) of ethanol fixed lymphoma cells. Samples were catastrophe constitutes a special case of apoptosis whose suppression entails aneuploidy. analyzed in a FACScan flow cytometer and data were evaluated using WinMDI2.8 Oncogene 2004; 23: 4362–4370. freeware. 9. Baptiste-Okoh N, Barsotti AM, Prives C. A role for caspase 2 and PIDD in the process of p53-mediated apoptosis. Proc Natl Acad Sci USA 2008; 105: 1937–1942. 10. Lin Y, Ma W, Benchimol S. Pidd, a new death-domain-containing protein, is induced by p53 BrdU incorporation. In-vivo rates of B-cell proliferation was determined by and promotes apoptosis. Nat Genet 2000; 26: 124–127. BrdU incorporation using an APC-labeled anti-BrdU mAb as described by the 11. Kim IR, Murakami K, Chen NJ, Saibil SD, Matysiak-Zablocki E, Elford AR et al. DNA manufacturer (BrdU-APC flow kit; BD Bioscience, Vienna, Austria). Briefly, healthy damage- and stress-induced apoptosis occurs independently of PIDD. Apoptosis 2009; 14: animals at the age of 5 weeks were injected with 1 mg BrdU/mouse (in 200 ml 1039–1049. saline) and killed after 4 h. B cells were stained with fluorochrome-conjugated 12. Manzl C, Krumschnabel G, Bock F, Sohm B, Labi V, Baumgartner F et al. antibodies staining IgM or CD19 and were analyzed by FACScan (Becton Caspase-2 activation in the absence of PIDDosome formation. J Cell Biol 2009; 185: Dickinson). 291–303. 13. Berube C, Boucher LM, Ma W, Wakeham A, Salmena L, Hakem R et al. Apoptosis caused by p53-induced protein with death domain (PIDD) depends on the death adapter protein Cell viability assay. The percentage of viable cells in culture was determined RAIDD. Proc Natl Acad Sci USA 2005; 102: 14314–14320. by staining cell suspensions with 1 mg/ml 7-AAD (Sigma) plus FITC-coupled 14. O’Reilly LA, Ekert P, Harvey N, Marsden V, Cullen L, Vaux DL et al. Caspase-2 is not Annexin V (Becton Dickinson). The samples were analyzed in a FACScan (Becton required for thymocyte or neuronal apoptosis even though cleavage of caspase-2 is Dickinson). dependent on both Apaf-1 and caspase-9. Cell Death Diff 2002; 9: 832–841. 15. Bergeron L, Perez GI, Macdonald G, Shi L, Sun Y, Jurisicova A et al. Defects in regulation Immunofluorescence staining. To monitor 53BP-1 and gH2AX foci, of apoptosis in caspase-2-deficient mice. Genes Dev 1998; 12: 1304–1314. 16. Marsden VS, Ekert PG, Van Delft M, Vaux DL, Adams JM, Strasser A. Bcl-2-regulated sorted immature and mature B cells from Em-Myc, Em-Myc/Pidd / and Em-Myc/ / 36 apoptosis and cytochrome c release can occur independently of both caspase-2 and Casp2 mice were stained as described elsewhere (Viale et al., 2009). caspase-9. J Cell Biol 2004; 165: 775–780. Pictures were taken with a Leica SP5 confocal laser-scanning microscope 17. Kumar S. Caspase 2 in apoptosis, the DNA damage response and tumour suppression: (Vienna, Austria) equipped with a 63 glycerol immersion objective, and images enigma no more? Nat Rev Cancer 2009; 9: 897–903. were analyzed using CellProfiler freeware (Broad Institute, Harvard, MA, USA). 18. Bradley G, Tremblay S, Irish J, MacMillan C, Baker G, Gullane P et al. The expression of p53-induced protein with death domain (PIDD) and apoptosis in oral squamous cell Statistical analysis. Estimation of statistical differences between groups was carcinoma. Br J Cancer 2007; 96: 1425–1432. 19. Ho LH, Taylor R, Dorstyn L, Cakouros D, Bouillet P, Kumar S. A tumor suppressor function carried out using the unpaired Student’s t-test or ANOVA analysis with Student– for caspase-2. Proc Natl Acad Sci USA 2009; 106: 5336–5341. Newman–Keuls as post hoc test, where appropriate. Comparison of tumor onset 2 20. Kaplan HS, Brown MB. A quantitative dose-response study of lymphoid-tumor was performed using a log-rank test and the w -test (Fisher’s exact) was used for development in irradiated C57 black mice. J Natl Cancer Inst 1952; 13: 185–208. comparison of frequency distributions. P-values of o0.05 were considered to 21. Kemp CJ, Wheldon T, Balmain A. p53-deficient mice are extremely susceptible to indicate statistically significant differences. radiation-induced tumorigenesis. Nat Genet 1994; 8: 66–69. 22. Garcia-Cao I, Garcia-Cao M, Martin-Caballero J, Criado LM, Klatt P, Flores JM et al. "Super p53" mice exhibit enhanced DNA damage response, are tumor resistant and age Conflict of Interest normally. EMBO J 2002; 21: 6225–6235. The authors declare no conflict of interest. 23. Adams JM, Harris AW, Pinkert CA, Corcoran LM, Alexander WS, Cory S et al. The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature 1985; 318: 533–538. 24. Guo Y, Srinivasula SM, Druilhe A, Fernandes-Alnemri T, Alnemri ES. Caspase-2 induces Acknowledgements. We thank K Rossi and B Rieder for animal husbandry; apoptosis by releasing proapoptotic proteins from mitochondria. J Biol Chem 2002; 277: C Soratroi, R Pfeilschifter, I Gaggl and I Bro¨sch for excellent technical assistance; 13430–13437. P Lukas and his team (LINAC1-4) for enabling irradiation of mice (Center of Nuclear 25. Shin S, Lee Y, Kim W, Ko H, Choi H, Kim K. Caspase-2 primes cancer cells for Medicine and Radiotherapy, University Medical Center of Innsbruck); G Bo¨ck for cell TRAIL-mediated apoptosis by processing procaspase-8. EMBO J 2005; 24: 3532–3542. sorting; G Baier and N Herman-Kleiter for help with Bioplex analysis; D Vaux for 26. Eischen CM, Weber JD, Roussel MF, Sherr CJ, Cleveland JL. Disruption of the Casp2 / and M Serrano for p53 / mice. This study was funded by the ARF-Mdm2-p53 tumor suppressor pathway in Myc-induced lymphomagenesis. Genes Dev Austrian Science Fund (FWF; Y212-B12; SFB021), AICR project 06-440 to AV and 1999; 13: 2658–2669.

Cell Death and Differentiation Caspase-2 in tumor suppression C Manzl et al 11

27. Frenzel A, Labi V, Chmelewskij W, Ploner C, Geley S, Fiegl H et al. Suppression of B-cell 34. Oliver TG, Meylan E, Chang GP, Xue W, Burke JR, Humpton TJ et al. Caspase-2-mediated lymphomagenesis by the BH3-only proteins Bmf and Bad. Blood 2010; 115: 995–1005. cleavage of Mdm2 creates a p53-induced positive feedback loop. Mol Cell 2011; 43: 28. Guha M, Xia F, Raskett CM, Altieri DC. Caspase 2-mediated tumor suppression involves 57–71. survivin gene silencing. Oncogene 2010; 29: 1280–1292. 35. Harris AW, Pinkert CA, Crawford M, Langdon WY, Brinster RL, Adams JM. The Em-myc 29. Reimann M, Lee S, Loddenkemper C, Dorr JR, Tabor V, Aichele P et al. Tumor stroma- transgenic mouse: a model for high-incidence spontaneous lymphoma and leukemia of derived TGF-beta limits myc-driven lymphomagenesis via Suv39h1-dependent senes- early B cells. J Exp Med 1988; 167: 353–371. cence. Cancer Cell 2010; 17: 262–272. 36. Viale A, De Franco F, Orleth A, Cambiaghi V, Giuliani V, Bossi D et al. Cell-cycle restriction 30. Janssens S, Tinel A, Lippens S, Tschopp J. PIDD mediates NF-kappaB activation in limits DNA damage and maintains self-renewal of leukaemia stem cells. Nature 2009; 457: response to DNA damage. Cell 2005; 123: 1079–1092. 51–56. 31. Biton S, Ashkenazi A. NEMO and RIP1 control cell fate in response to extensive DNA damage via TNF-alpha feedforward signaling. Cell 2011; 145: 92–103. 32. Yoo BH, Wang Y, Erdogan M, Sasazuki T, Shirasawa S, Corcos L et al. Oncogenic ras- induced downregulation of a pro-apoptotic protease caspase-2 is required for malignant This work is licensed under the Creative Commons transformation of intestinal epithelial cells. J Biol Chem 2011; 286: 38894–38903. 33. Logette E, Schuepbach-Mallepell S, Eckert MJ, Leo XH, Jaccard B, Manzl C et al. PIDD Attribution-NonCommercial-No Derivative Works 3.0 orchestrates translesion DNA synthesis in response to UV irradiation. Cell Death Diff 2011; Unported License. To view a copy of this license, visit http:// 18: 1036–1045. creativecommons.org/licenses/by-nc-nd/3.0

Supplementary Information accompanies the paper on Cell Death and Differentiation website (http://www.nature.com/cdd)

Cell Death and Differentiation Attached Manuscripts

3) Death of p53-defective cells triggered by forced mitotic entry in the presence of DNA damage is not uniquely dependent on Caspase-2 or the PIDDosome This publication scrutinizes the findings published by Samuel Sidi and colleagues in 2008 42. They proposed in this paper a Chk-1 controlled, p53 and Bcl-2 independent, apoptosis pathway activated by ATM/ATR engaging the PIDDosome. Utilizing murine Caspase-2 or PIDD-deficient cells we were unable to confirm cross species conservation of this Chk-1 controlled pathway. My contribution to this work was the breeding and analysis of the Eµ-Myc/p53-/- /Caspase-2-/- and p53-/-/Caspase-2-/- double knockout mice from which several cell lines used in this study were also derived. Data using these cells is presented in Figures 1 and 2.

50

Citation: Cell Death and Disease (2013) 4, e942; doi:10.1038/cddis.2013.470 OPEN & 2013 Macmillan Publishers Limited All rights reserved 2041-4889/13 www.nature.com/cddis Death of p53-defective cells triggered by forced mitotic entry in the presence of DNA damage is not uniquely dependent on Caspase-2 or the PIDDosome

C Manzl1,3, LL Fava1, G Krumschnabel1, L Peintner1, MC Tanzer1,4, C Soratroi1, FJ Bock1,5, F Schuler1, B Luef2,6, S Geley2 and A Villunger*,1

Much effort has been put in the discovery of ways to selectively kill p53-deficient tumor cells and targeting cell cycle checkpoint pathways has revealed promising candidates. Studies in zebrafish and human cell lines suggested that the DNA damage response kinase, checkpoint kinase 1 (Chk1), not only regulates onset of mitosis but also cell death in response to DNA damage in the absence of p53. This effect reportedly relies on ataxia telangiectasia mutated (ATM)-dependent and PIDDosome-mediated activation of Caspase-2. However, we show that genetic ablation of PIDDosome components in mice does not affect cell death in response to c-irradiation. Furthermore, Chk1 inhibition largely failed to sensitize normal and malignant cells from p53 / mice toward DNA damaging agents, and p53 status did not affect the death-inducing activity of DNA damage after Chk1 inhibition in human cancer cells. These observations argue against cross-species conservation of a Chk1-controlled cell survival pathway demanding further investigation of the molecular machinery responsible for cell death elicited by forced mitotic entry in the presence of DNA damage in different cell types and model organisms. Cell Death and Disease (2013) 4, e942; doi:10.1038/cddis.2013.470; published online 5 December 2013 Subject Category: Cancer

The vast majority of human cancers lack functional p53, a key death. The cellular consequences are only beginning to be tumor-suppressor protein that regulates various cellular deciphered and are currently summed up by the term ‘mitotic stress responses, most prominently that induced by DNA catastrophe’.6,7 The molecular machinery responsible for cell damage. In many experimental systems, cell survival upon killing under these conditions is, however, still unclear but DNA damage in p53-deficient cells critically depends on intact identification of cell death mediators appears pivotal for cell cycle checkpoint pathways that operate in parallel to p53. improved anticancer-drug design and optimization of current Targeting these pathways, for example, by chemical inhibition therapies. of the checkpoint kinases, Chk1 and Chk2, in p53-deficient On the basis of studies in p53-mutant zebrafish embryos as tumor cells has been shown to be a promising option for well as p53-defective human cervical (HeLa) and isogenic cancer treatment.1 These checkpoint kinases not only have colon cancer cells (HCT116), it was postulated that pharma- important roles in response to exogenous DNA damage but cological inhibition of Chk1 or siRNA-mediated ablation of also in an unperturbed cell division cycle by co-ordinating the protein expression in combination with g-irradiation (IR) onset of mitosis with completion of DNA synthesis.2,3 activates a novel cell death pathway that depends on the In particular, Chk1 seems to be indispensable for normal multiprotein PIDDosome complex preferentially in p53-defec- development and repeated or high-dose application of Chk1 tive cells.8 This complex contains the p53-induced protein inhibitors may lead to undesired side effects in healthy tissues with a death domain (PIDD), the bipartite adapter RAIDD with high mitotic index such as in the gastrointestinal tract4 or (receptor-interacting protein-associated ICH-1/CED-3 homo- in the immune system.5 Inhibition of Chk1 abrogates the G2/M logous protein with a death domain) and pro-Caspase-2, checkpoint and promotes premature entry into mitosis even in a cell-death-associated protease with poorly defined properties.9 the presence of DNA damage, which frequently results in cell Upon DNA damage, ataxia telangiectasia mutated (ATM)

1Division of Developmental Immunology, Biocenter, Innsbruck Medical University, Innsbruck, Austria and 2Division of Molecular Pathophysiology, Biocenter, Innsbruck Medical University, Innsbruck, Austria *Corresponding author: A Villunger, Division of Developmental Immunology, Biocenter, Innsbruck Medical University, Innsbruck A-6020, Austria. Tel: +43 512 9003 70380; Fax: +43 512 9003 73960; E-mail: [email protected] 3Current address: Department of Pathology, Medical University of Innsbruck, Innsbruck, Austria. 4Current address: The Walter and Eliza Hall Institute for Medical Research, Melbourne, Australia. 5Current address: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, USA. 6Current address: Department of Urology, Medical University of Innsbruck, Innsbruck, Austria. Keywords: Caspase-2; PIDDosome; Chk1; p53; DNA damage Abbreviations: PIDD, p53-induced protein with a death domain; RAIDD, receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a death domain; Chk1, checkpoint kinase 1; NF-kB, nuclear factor kappa-light-chain enhancer; Nemo/IKKg, NF-kB essential modulator/nuclear factor kappa-B kinase subunit gamma; ATM, ataxia telangiectasia mutated; Bcl-2, B-cell lymphoma 2; DISC, death-inducing signaling complex; MEF, mouse embryonic fibroblast; Mdm2, murine double minute; RIP-1, receptor-interacting serine/threonine-protein kinase 1; IR, g-irradiation Received 23.10.13; revised 28.10.13; accepted 31.10.13; Edited by G Melino Chk1 inhibition kills PIDDosome defective cells C Manzl et al 2

directly phosphorylates PIDD on Thr788 within the death a failure of Chk1 inhibition to sensitize these cells to IR. domain promoting the interaction with RAIDD that leads to In contrast, stimulation with the pan-kinase inhibitor stauros- Caspase-2 activation. By a poorly understood mechanism porine (STS) induced cell death independently of the PIDDosome assembly and Caspase-2-dependent cell death genotype confirming general cell death susceptibility in the are inhibited by Chk1.10 In addition, the downstream effectors absence of p53 or p53 and Caspase-2 (Figures 1a and c). of this Chk1-suppressed cell death pathway are undefined but Similar findings were made in B-cell blasts, albeit these cells were found to be independent of several well-known were less radio-resistant in the absence of p53 when apoptosis regulators, including Caspases-3, -8 and -9 and compared with their cycling T-cell counterparts (Figure 1d). B-cell lymphoma 2 (Bcl-2).8,10 Of note, cells expressing Similarly, using doxorubicin as an inducer of DNA damage in functional p53 do not seem to engage the Chk1-suppressed thymocytes (Supplementary Figure S1b) or FACS-sorted pathway effectively, opening a window of opportunity in mature T or B cells from the spleen (Supplementary Figures anticancer therapy.8 Together these studies define the S1c and d) yielded the same results, that is, substantial cell PIDDosome as the initiating protein, promoting death of death resistance in the absence of p53 but lack of sensitization p53-deficient cells. Hence, PIDD activators would constitute by Chk1 inhibition. Finally, reducing expression levels of Chk1 to interesting alternatives to Chk1 inhibitors without the obvious half by removing one allele of the Chk1 locus on a p53 / loss of essential cell cycle checkpoint functions and therefore background also failed to increase cell death suscep- might have less severe side effects than Chk1 blockers.11,12 tibility of T- or B-cell blasts after IR (Supplementary Figure S1e). To further explore the general validity of the Chk1- Taken together, these data show that primary mouse suppressed cell death pathway in more detail in mammalian lymphocytes lacking p53 respond poorly to G2/M checkpoint cells, we investigated its contribution to cell death in wild-type inhibition by Go¨6976 are not sensitized to IR killing and that and p53-deficient mice and derived cell lines. Our study did Caspase-2 does not contribute to their IR-driven cell death. not confirm conservation or general validity of this mechanism To extend our findings to non-hemopoietic cells, in different primary, immortalized or malignant cells derived we investigated the Chk1-suppressed cell death pathway in from p53 / or p53 / Casp2 / (DKO) mice. Interestingly, early passage primary and immortalized mouse embryonic although we could confirm additive effects of Chk1-inhibiton fibroblasts (MEF) generated from E14.5 embryos. and IR-damage as well as partial Caspase-2 dependence of As expected, primary MEF derived from p53 / or DKO this type of cell death in HeLa cervical carcinoma cells, mice showed only moderate cell death, even upon high-dose we failed to notice a clear correlation between the p53 status IR of 30 Gy, as these cells preferentially undergo cell cycle and Caspase-2 dependence of cell death in isogenic HCT116 arrest. Treatment of cells with Go¨6976 before irradiation failed cells. Thus, our findings call for a reassessment of the to substantially sensitize these cells to death, whereas a molecular machinery responsible for Chk1 inhibition-depen- different Chk1 inhibitor, SB218078, stimulated some cell dent cell death in mammals in the context of DNA damage and death in p53-deficient cells when combined with IR. However, exclude Caspase-2 or PIDD, and hence the PIDDosome, this cell death was independent of Caspase-2. If anything, as master regulators of this type of cell death. loss of Casp2 rather increased cell death under these conditions (Figure 1e; Supplementary Figure S2a). This Results finding suggested that Go¨6976 might not even target Chk1 in mouse cells. However, it clearly prevented the arrest of Lack of cross-species conservation of the Chk1- primary MEF in G2/M after IR (Supplementary Figure S2b) suppressed cell death pathway. Investigating the Chk1- and both inhibitors prevented the accumulation of inactive suppressed pathway in primary thymocytes isolated from CDK1, phosphorylated on tyrosine 15 (Y15), a Chk1- wt, Casp2 / , p53 / or p53 / Casp2 / (DKO) mice dependent effect after IR, confirming that these compounds revealed high susceptibility to IR-induced cell death in wt and were indeed blocking Chk1 activity also in mouse cells Caspase-2-deficient cells, whereas those lacking p53 or p53 (Supplementary Figure S2c). plus Caspase-2 were equally resistant. Blocking Chk1 by the To sensitize cells to IR-induced apoptosis and blunt addition of 1 mMGo¨6976 had no significant impact on cell p53-responses by other means, we transduced MEF with a death in culture but also failed to significantly sensitize retrovirus encoding SV40 large T (LT) antigen and subjected thymocytes to 1.25, 2.5 (not shown) or 5 Gy of IR (Figure 1a; them to IR in the presence or absence of Go¨6976. As can be Supplementary Figure S1a). Cleavage of Caspase-2 was seen in Figure1f, immortalization sensitized these cells to cell best detectable by immunoblotting in wild-type cells exposed death by IR (when compared with primary MEF), although to Go¨6976, IR or both. Cells lacking p53 also showed some again rather high doses of 30 Gy had to be applied, as 10 Gy of processing of Caspase-2 after IR (Figure 1b), documenting IR proved rather ineffective to induce cell death (not shown). that it is not required for IR killing as these cells are highly However, Chk1 inhibition per se triggered some death in these radiation resistant. cells. Despite the fact that the combined treatment proved As only a subfraction of thymocytes is actively cycling and additive, all genotypes tested, also those lacking Caspase-2, hence may be poorly susceptible to the effects of Chk1 p53 or both, died at similar rates (Figure 1f; Supplementary inhibition, we next generated spleen-derived T-cell and B-cell Figure S2d). blasts by mitogenic stimulation to test the effects of Chk1 So far all cell types tested were either primary or inhibition on cell death in the absence of p53. The results from immortalized. To test whether the Chk1-suppressed pathway T-cell blasts mirrored our findings in thymocytes, demonstrating was only active in transformed cells, we also transduced lack of substantial cell death in the absence of p53 and MEF with retroviruses encoding the oncogenes c-Myc and

Cell Death and Disease Chk1 inhibition kills PIDDosome defective cells C Manzl et al 3

Primary thymocytes [24h IR] Primary thymocytes [24h IR] 100 wt MW -/- 80 Casp2 (kDa) p53-/- 55 Pro- DKO 60 Casp-2 40 Cleaved * * 17 Casp-2 20 * * Active 17 0 Casp-3 Specific cell death [%] 37 GAPDH -20 1 µM Gö 5 Gy IR 1 µM Gö 100 nM control 1 µM GÖ 5 Gy IR 1 µM Gö 5 Gy IR STS 5 Gy IR

T cell blasts [24h IR] B cell blasts [24h IR] 100 100 wt wt -/- Casp2-/- Casp2 80 p53-/- 80 p53-/- DKO DKO 60 60

40 40 * 20 * * * 20 Specific cell death [%] Specific cell death [%] 0 0 1 µM Gö 5 Gy IR 1 µM Gö 100 nM 1 µM Gö 5 Gy IR 1 µM Gö 100 nM 5 Gy IR STS 5 Gy IR STS

Primary MEF [24h IR] SV40 MEF [24h IR] 100 wt 60 p53-/- Casp2-/- * 80 -/- 50 DKO p53 * * * DKO 40 60 * * 30 40 20 % SubG1 cells

% SubG1 cells 20 10

0 0 control 1 µM 1 µM 30 Gy Gö SB control 1 µM GÖ 30 Gy IR 1 µM Gö Gö SB IR + IR + IR 30 Gy IR Figure 1 Chk1 inhibition fails to sensitize p53-deficient lymphocytes or MEF to DNA damage. Primary thymocytes (a), stimulated T- (c) or B-cell blasts (d) were treated with Go¨6976 and/or exposed to IR or both. As a control, cells were treated with staurosporine (STS). Cell viability was assessed after 24 h using AnnexinV/PI staining and flow cytometric analysis. To account for spontaneous cell death of lymphocytes in culture, specific cell death was calculated throughout using the equation (induced apoptosis spontaneous cell death)/(100 spontaneous cell death). In parallel, thymocytes were harvested and protein lysates were separated by SDS-PAGE to monitor cleavage of Caspase-2 and Caspase-3. Membranes were reprobed with an anti-GAPDH antibody to control protein loading (b). Viability of primary (e) and SV40 immortalized MEFs (f) derived from E14.5 embryos of the indicated genotypes were assessed using sub-G1 staining and flow cytometric analysis. Bars represent means±S.E. of Z3 independent experiments per genotype and treatment. *Indicates significant differences between wt or Casp2 / cells and p53 / or DKO cells (Po0.05) after IR or Go¨ þ IR treatment. No significant differences were noted in (a, c and d) between Go¨ and Go¨ þ IR treatment

Ha-RasV12 that leads to full cellular transformation.13 These genotype. Exposure of MEF to 10 Gy of IR prevented colony cells were also radiosensitive and showed some cell death in formation irrespective of the mode of transformation response to Chk1 inhibition but the combined treatment only and absence or presence of p53, Caspase-2 or both marginally increased cell death when compared with cells (Supplementary Figure S4a). In contrast, exposure to 3 Gy exposed to IR alone. The cell death observed, however, of IR allowed clonal survival of cells from all genotypes and was again independent of Caspase-2 (Supplementary SV40 MEF lacking Caspase-2 alone, or Caspase-2 and p53, Figure S3a). SV40 MEF or E1A/Ha-RasV12 transformed seemed to display superior clonal survival, suggesting for the MEF lacking PIDD were also found equally susceptible to IR first time a role of Caspase-2 under these conditions. Such and Chk1 inhibition, as Caspase-2-deficient or wt cells marked differences, however, were not noted in MEF excluding PIDDosome formation as a rate-limiting step in this immortalized with c-Myc plus Ha-RasV12 or in primary MEF cell death paradigm in MEF (Supplementary Figures S3c–e). (Supplementary Figure S4a). Testing independent batches of In all cases analyzed, cell death was clearly associated with MEF, however, failed to confirm increased clonal survival in Caspase-3 activation and Caspase-2 processing (indicated the absence of Caspase-2 after exposure to 3 Gy of IR±Chk1 by decreased levels of its pro-form) but independent of inhibition. Notably, SV40 MEF lacking PIDD also behaved like Caspase-2 function (Supplementary Figures S2e and S3b). wt cells (Supplementary Figure S4b). E1A plus Ha-RasV12- To assess long-term consequences on clonal survival, transformed MEF were highly sensitive to the effects of colony formation assays were performed using early passage Go¨6976 and did not show long-term clonal survival upon Chk1 primary MEF (passage 2–3) or immortalized/transformed inhibition (Supplementary Figure S4c). On the basis of these MEF generated from at least two independent embryos per observations, we conclude that Caspase-2 on its own,

Cell Death and Disease Chk1 inhibition kills PIDDosome defective cells C Manzl et al 4

or recruited in the PIDDosome, is not rate-limiting for the primary and transformed MEF or thymic lymphoma cells clonal survival of MEF exposed to Chk1 inhibition and IR, (Figures 1e and f), but Caspase-2 is not required for cell death neither in the absence nor in the presence of p53. in these settings.

Combined loss of p53 and Caspase-2 fails to allow Caspase-2 contributes to IR-driven cell death of human postnatal development in the absence of Chk1. Chk1- cancer cells upon Chk1 inhibition. The above experi- deficiency leads to early embryonic lethality shortly after ments suggested that the Chk1-suppressed pathway is not implantation before E6.5 post conception. Notably, lack of conserved in mice. To interrogate the reported requirement p53 cannot restore embryonic development of Chk1-deficient of Caspase-2 in human carcinoma cells, we treated HeLa embryos.3 We reasoned that under such conditions, basal cells with 1 mMofGo¨6976, 10 Gy IR or a combination of both levels of DNA damage in the embryo might then trigger and measured cell death 24 or 48 h later. Although the Caspase-2-dependent cell death curtailing development. combination treatment triggered significant cell death already Therefore, we investigated whether combined loss of at 24 h, all treatments induced some cell death after 48 h. Caspase-2 and p53 might allow postnatal development in However, the strongest effect was observed after treating the absence of Chk1. Inter-crossing of p53 / Casp2 þ / cells with the Chk1 inhibitor plus IR (Figures 3a and b). / Chk1 þ male mice with p53 þ / Casp2 / Chk1 þ / females Interestingly, Go¨6976 treatment alone caused the phosphor- (expected frequency of triple mutants 1:16; observed ylation of Chk1 at S345, suggesting ATR activation, most 0/54 offspring) or p53 / Casp2 / Chk1 þ / males with likely due to DNA damage accumulating upon Chk1 inhibition p53 þ / Casp2 / Chk1 þ / females (expected frequency and G2/M checkpoint override. When Go¨6976 was combined 1:8; observed 0/46) failed to give rise to viable animals that with IR treatment, the induction of inactivating Tyr15 lacked all three alleles, as monitored by genotyping PCR phosphorylation of CDK1 was prevented, suggesting that analysis on tail DNA at the time of weaning, 4 weeks after the kinase inhibitor did inhibit Chk1 (Figures 3c and d). birth (not shown). Together, this demonstrates that combined The induction of cell death by Go¨6976 alone or in combination loss of p53 and Caspase-2 is insufficient to restore normal with IR was clearly correlated with Caspase-2 processing, development in the absence of Chk1. as indicated by a quantitative reduction of its pro-form at 24 h, where indeed no activation of Caspase-3 was detectable by Lack of Caspase-2 does not affect the tumor-suppressor western blotting (Figure 3d). This confirms that Go¨6976 function of p53. To investigate the potential role of treatment can activate Capase-2 in HeLa cells, an effect that Caspase-2 in p53-dependent tumor suppression and to test is enhanced by IR, which suggests that under these cell death sensitivity of p53-deficient tumor cells exposed conditions Caspase-2 might be involved in checkpoint to DNA damage under conditions of Chk1 inhibition, signaling and/or acts as an upstream regulator of cell death we followed mice lacking one or two alleles of p53 on a (Figure 3d). Consistent with a role for Caspase-2 in this cell Caspase-2 deficient or proficient background. Lack of death paradigm, conditional ablation of Caspase-2 expres- Caspase-2 did not have an impact on spontaneous tumor sion by short hairpin RNA (shRNA)-mediated knockdown latency (Figure 2a) or tumor type in these mice (not shown). (Figure 3e) significantly reduced cell death upon IR and Similarly, lack of Caspase-2 had no influence on thymic Go¨6976 treatment (Figures 3f and g). Of note, cell death lymphomagenesis caused by DNA damage using the well- induced by staurosporine was not affected by Caspase-2 established fractionated irradiation protocol (Figure 2b). knockdown (Figure 3f). Freshly isolated lymphoma cells derived from these mice To further investigate whether Caspase-2 knockdown can also were highly susceptible to spontaneous death in culture. rescue other cell types from cell death upon IR and Go¨6976, we Hence, additional Chk1 inhibition or exposure to IR did not next turned to isogenic HCT116 cells lacking or expressing p53 further accelerate cell death ex vivo (Figure 2c). Therefore, and engineered those to conditionally express Caspase-2 RNAi. we also generated lymphoma cell lines from these primary HCT116 cells underwent apoptosis upon IR treatment but this tumors. These cell lines responded with increased cell death effect was independent of Caspase-2 and could not be enhanced upon IR in culture, in line with findings reported earlier.14 significantly by additional treatment with Go¨6976 (Figures 4a Notably, loss of Caspase-2 provided some degree of and b). In p53 þ / þ HCT116 cells, IR treatment triggered the protection that even achieved statistical significance at activation of Caspase-3 and this effect was independent of higher doses of IR. However, these lymphoma cell lines Caspase-2 expression. Interestingly, however, Go¨6976 treatment were also highly vulnerable to the effects of 1 mMGo¨6976 in alone caused a significant induction of cell death upon Caspase-2 the absence of DNA damage, reducing cell viability to below knockdown, but this was only noted in p53 þ / þ HCT116 cells 40%. Reducing the concentration of the Chk1 inhibitor to (Figures 4a and b). This phenomenon remains currently more tolerable doses of 0.1 mM, however, failed to signifi- unexplained but is in line with previously proposed cell cycle cantly increase cell death rates caused by IR in both checkpoint functions of this protease,15,16 that when absent may genotypes tested. Regardless, under conditions of Chk1 sensitize cells to apoptosis. Finally, although HCT116 p53 / inhibition followed by IR, loss of Caspase-2 did not provide cells died at similar rates than p53 þ / þ cells under these significant protection from cell death (Figure 2d). conditions, activation of Caspase-3 was no longer detectable In summary, this documents that Chk1 inhibition per se can after Caspase-2 ablation in the absence of p53 (Figure 4c), kill immortalized or transformed mouse cells in a p53- suggesting an involvement of alternative non-apoptotic cell death independent manner and can increase cell death rates modalities upon DNA damage.6 Notably, ‘sub-G1’ cells can also caused by IR in some mouse cells lacking p53, such as accumulate in response to non-apoptotic cell death inducers.17

Cell Death and Disease Chk1 inhibition kills PIDDosome defective cells C Manzl et al 5

100

80

60

40 p53+/- p53-/-

% Tumor-free survival 20 p53+/-/Casp2-/-

DKO p<0.0001 p<0.0001 0 0 100 200 300 400 500 600 700 Time [d]

100

IR 80

60

40 wt -/- 20 Casp2 p53-/- % Lymphoma-free survival DKO 0 p<0.0001 p<0.0001

0 50 100 150 200 250 300 350 Time [d]

Thymic lymphoma cells [24h IR] 100 p53-/- 80 DKO

60

40 Cell viability [%] 20

0 NT1 µM 0.5 1 µM Gö 5 1 µM Gö Gö Gy IR 0.5 Gy IR Gy IR 5 Gy IR

100 Thymic lymphoma cell lines [48h IR]

80 p53-/- 60 DKO

40 * Cell viability [%] 20

0 cntr 0.1 µM 2.5 Gy 5 Gy 0.1 µM Gö 1 µM Gö 1 µM 1 µM Gö 1 µM Gö Gö IR IR 2.5 Gy IR 5 Gy IR Gö +2.5 Gy IR +5 Gy IR Figure 2 Lack of Caspase-2 has no effect on spontaneous or IR-driven thymic lymphomagenesis in p53-null mice. (a) Kaplan–Meier analysis comparing tumor-free survival of p53 þ / (median survival 492 days, n ¼ 16), p53 / (median survival 158 days, n ¼ 28), p53 þ / Casp2 / (median survival 387 days, n ¼ 23) and DKO (median survival 129 days, n ¼ 8). (b) Kaplan–Meier analysis of tumor-free survival of wt (median survival 204 days, n ¼ 14), Casp2 / (median survival 185 days, n ¼ 13), p53 / (median survival 117 days, n ¼ 8) and DKO (median survival 116 days, n ¼ 16) mice after fractionated irradiation. Indicated P-values in (a and b) refer to differences compared with wt mice. (c)Freshly isolated thymic lymphoma cells from p53 / and DKO mice were put in culture and treated with Go¨6976 and/or exposed to IR. Cell survival was assessed after 24 h by AnnexinV/PI staining. (d) Thymic lymphoma cell lines derived from the indicated genotypes were treated with 0.1 mMor1.0mMGo¨6976 alone, or in combination with increasing doses of IR. Bars represent means±S.E. of four independent experiments. *indicates significant differences (Po0.05) compared between p53 / and DKO cells

Cell Death and Disease Chk1 inhibition kills PIDDosome defective cells C Manzl et al 6

30 100 HeLa * HeLa 25 [24h IR] [48h IR] * 80 + /P

20 + 60 15 40 10 % SubG1 cells % AnnexinV 5 20

0 0 cntr 1µM Gö10Gy 1µM Gö cntr 1µM Gö 10Gy 1µM Gö IR 10Gy IR 10Gy

MW 0 Gy 10 Gy 0 Gy 10 Gy 0 Gy 10 Gy (kDa) -+-+-+-+-+-+1 µM Gö 72 MW - + - + 1 µM GÖ 55 Chk1 (kDa) --+ + 10 Gy IR 72 P-CDK1 p-Chk1 28 55 (Y15) 55 Caspase-2 37 GAPDH Active HeLa [24h IR] 17 Caspase-3

GAPDH 37

Experiment 1 Experiment 2 Experiment 3

60 HeLa [48hIR] HeLa TetR Casp2 [48h IR] 50 TetR Casp2 (-Dox) MW 0 Gy10 Gy 0 Gy 10 Gy (kDa) 40 TetR Casp2 (+Dox) - +-+-+-+1 µM Gö

30 * 55 Casp-2 20 % SubG1 cells 37 GAPDH 10 * 0 ----++++ Dox control1 µM Gö 10 Gy 1 µM Gö 100 nM IR 10 Gy IR STS

Control GöIR [10 Gy] Gö + IR

shCasp2 0.7% 14.2% 7.1% 48.7% -Dox

shCasp2 1.7% 3.3% 6.2% 23.1% +Dox

Figure 3 Chk1-suppressed cell death pathway is active in HeLa cells. Cells were treated with Go¨6796 and/or IR or STS. Cell death was assessed in parallel after 24 h and 48 h of treatment by sub-G1 (a) or AnnexinV/PI staining (b). After 24 h, cells were harvested and lysed for western blot analysis to assess Chk1 inhibition (c). Activation of Chk1, Caspase-2 and Caspase-3 was monitored in parallel by western blotting (d). HeLa cells with regulated knockdown of Caspase-2 were put on doxycycline for 24 h. Impact of Caspase-2 knockdown (e) on cell death induced by Chk1 inhibition and/or IR in HeLa cells, monitored by sub-G1 analysis, is depicted in (f). Representative histograms from sub-G1 analysis are shown in (g). Bars represent means±S.E. of Z3 independent experiments. *Indicates significant differences (Po0.05) between Casp2 proficient and deficient cells

Discussion mice showed trends of increased cell death upon Chk1 In this study, we investigated the relevance of the Chk1- inhibition and IR exposure (Figures 1 and 2). However, these suppressed cell death pathway in primary and transformed effects were rather minor and, for example, in the case of p53- mouse cells and human cancer cell lines. Our experiments deficient primary MEF only noted with one of the two different suggest that primary lymphocytes lacking p53 function cannot Chk1 inhibitors used (Figure 1e). More importantly, lack of be sensitized effectively to cell death upon IR-inflicted DNA Caspase-2 did never protect from cell death under conditions damage by Go¨6976-mediated Chk1 inhibition (Figure 1). MEF of Chk1 inhibition and IR exposure (Figures 1 and 2; and thymic lymphoma cell lines derived from p53-deficient Supplementary Figures S1 and S3). Given the vast body of

Cell Death and Disease Chk1 inhibition kills PIDDosome defective cells C Manzl et al 7

control Gö IR [10 Gy] Gö + IR

shCasp2 2% 3% 27% 31% -Dox

shCasp2 11% 34% 26% 25% +Dox

p53-/- shCasp2 1% 3% 36% 37% -Dox

p53-/- shCasp2 5% 6% 30% 28% +Dox

25 60 HCT116 wt (-Dox) wt (+Dox) HCT116 wt (-Dox) wt (+Dox) -/- -/- 50 [48h IR] -/- -/- 20 [24h IR] p53 (-Dox) p53 (+Dox) p53 (-Dox) p53 (+Dox) * 40 15 * 30 10 20 % SubG1 cells % SubG1 cells 5 10 * * 0 0 control 1 µM Gö 10 Gy IR 1 µM Gö control 1 µM Gö 10 Gy IR 1 µM Gö 10 Gy IR 10 Gy IR

MW 0 Gy 0 Gy 10 Gy 10 Gy 0 Gy 0 Gy 10 Gy10 Gy 0 Gy 0 Gy 10 Gy10 Gy 0 Gy 0 Gy 10 Gy 10 Gy (kDa) --- + + - + + --- + + - + + --- + + - + + --- + + - + + 1 µM Gö 55 Casp-2

active 17 * Casp-3

36 GAPDH 28 wt shCasp2 (-Dox) wt shCasp2 (+Dox) p53-/- shCasp2 (-Dox) p53-/- shCasp2 (+Dox)

Figure 4 Cell death caused by Chk1 inhibition does not correlate with p53 status. Isogenic HCT116 cells lacking or expressing functional p53 and an shRNA targeting Caspase-2 were treated with Go¨6796 and/or exposed to IR. Representative histograms from sub-G1 analysis performed after 48 h are shown in (a). Percentage of cell death was assessed by sub-G1-staining and flow cytometric analysis after 24 h and 48 h (b). Bars represent means±S.E. of Z3 independent experiments per genotype and treatment. *Indicates significant differences (Po0.05) between controls and Dox-treated cells. (c) Caspase-2 expression was ablated by the addition of Doxycycline for 24 h prior exposure of cells to Go¨ or Go¨ þ IR. Lysates for western blotting were harvested after additional 24 h. (*) In the image marks non-specific precipitates that leave the impression that IR triggers Caspase-3 activation. Higher magnification of the image will reveal the lack of the p17 fragment of active Caspase-3 evidence from human cancer cell lines, demonstrating (Supplementary Figure S2c), that serves as a read out for increased cell death sensitivity of p53-defective cells upon Chk1 activity.8 It is worth mentioning here that Go¨6976, an G2/M checkpoint override,11,12,18 we believe that these indolocarbazol, has been identified as a kinase inhibitor that observations are rather surprising, as they question the targets most selectively classical Ca þþ-dependent PKC general validity of this concept, strongly pointing toward isoforms (ab)19 and its inhibitory action on Chk1 was only species and/or cell type-specific differences. One simple noted a decade later.20 Hence, Go¨6976 at the concentrations explanation for our findings may be lack of efficient Chk1 used here and in related studies8,10 may cause cell death not inhibition by Go¨6976 in mouse cells. However, p53-deficient only by inhibition of Chk1 and checkpoint override. Of note, MEF clearly failed to arrest in G2 upon Go¨6976 classical PKC isoforms are generally considered as pro- treatment (Supplementary Figure S2b) and the compound survival kinases21 and inhibition of PKC isozymes, activated prevented the accumulation of Tyr15 phosphorylated CDK1 in response to DNA damage, for example, by the

Cell Death and Disease Chk1 inhibition kills PIDDosome defective cells C Manzl et al 8

bisindolymaleimide Go¨6850 sensitizes human fibroblasts to The tumor-suppressor p53 contributes to the strength of the the effects of IR.22 G2/M checkpoint by regulating p21 and 14-3-3sigma and loss To increase confidence in our observations, we also used a of p53 facilitates premature G2/M transition upon Chk1 different Chk1 inhibitor, that is, SB218087, in some of our inhibition in human cancer cells. Our data (Figure 4) and a experiments, yielding essentially identical results as those previous study show, however, that p53 status is not strongly obtained using Go¨6976 (Figure 1e; Supplementary Figure correlated with cell death initiation.25 As neither p53, PIDD, S3b). Both compounds trigger some death on their own and Caspase-2 nor BCL-2 seem to be commonly involved in the this was found most pronounced in combination with IR, but regulation of cell death following G2/M checkpoint override the lack of Caspase-2 failed to provide protection from killing molecular players in ‘mitotic catastrophe’ remain to be defined (Figure 1e; Supplementary Figure S3e). Notably, ablation of on a (tumor) cell type-specific basis. one allele of Chk1 also failed to sensitize primary lympho- In summary, these observations argue against a general cytes to IR (Supplementary Figure S1e), suggesting that cross-species conservation of the Chk1-suppressed cell mouse cells can deal better than human cells with death pathway in vertebrates and cell type-dependent DNA damage in the presence of reduced Chk1 activity. differences also in human cancer cells, demanding reassess- It also remains possible that in mice G2/M checkpoint ment of the molecular machinery responsible for tumor cell fidelity in the absence of p53 may rely more on alternative death elicited upon forced mitotic entry in the presence of DNA control mechanisms, such as the recently reported MAPKAP damage. kinase 2 (MK2)–p38 MAPK network that putatively may engage additional regulators of mitotic entry next to Materials and Methods Cdc25c.23,24 However, the early lethality of Chk1-deficient Mice. All animal experiments were performed in accordance with the Austrian mice and severe checkpoint deficits in derived ES cell legislation (BGBl. Nr. 501/1988 i.d.F. 162/2005). The generation and genotyping / 30 / 31 f/f 5 cultures argues against a significant degree of redundancy of the Casp2 , p53 mice and Chk1 mice have been described. 2,3 Mice harboring a floxed allele were mated with Ubi-Cre deleter strain to generate between these two checkpoint pathways in vivo. Chk1 þ / mice. To induce thymic lymphomas by IR, mice were exposed to Furthermore, in contrast to Chk1 RNAi, knockdown of MK2 whole body irradiation (4 1.75 Gy) at the age of 4 weeks32 in weekly intervals also failed to cause premature G2/M transition in the in a linear accelerator. All mice were maintained on C57BL/6 genetic presence of DNA damage in human U2OS cells engineered background. to lack p53, a finding in support of species and/or cell type-dependent differences.25 Cell lines, tissue culture and cell sorting. Freshly isolated thymocytes, splenocytes or thymic lymphoma cells33 and MEF28 were isolated and cultured as It remains formally possible that mouse lymphocytes simply described. For survival analysis and colony formation assays using MEF, at least lack the capacity to activate cell death signaling emanating two independent batches from individual embryo preparations were tested and the from premature G2/M transition in the presence of DNA FACS data were pooled. Chk1 inhibitors were commonly applied 1 h prior damage, as none of the normal lymphocyte populations irradiation. tested here showed increased cell death rates after Chk1 Generation of Caspase-2-knockdown lines. Caspase-2 targeting inhibition (Figure 1; Supplementary Figure S1). In contrast, shRNA encoding oligonucleotides was cloned into HindIII-BglII digested pENTR- in MEF expressing different oncogenes, Chk1 inhibition per se THT-III, a GATEWAY cloning compatible ENTR vector harboring a tetracycline- caused already some cell death, and combination with IR was regulatable H1-RNA gene promoter. After sequence verification, the shRNA most effective in cell killing (Supplementary Figures S3 and S4), expression cassette was shuttled into a selectable puromycin resistance conferring similar to findings made in HeLa cells where DNA as well as a TetR-GFP-expressing lentiviral vector. Target cells (HeLa and damage and Chk1 inhibition were shown to integrate on the HCT116) were transduced with lentiviral particles generated by transient transfection of lentiviral constructs with packaging and pseudotyping plasmids formation of the PIDDosome and Caspase-2-dependent cell 34 10 as described previously. Transduced target cells were selected using 2.5 mg/ml death. We also found that DNA damage activates Caspase-2 puromycin and induced using 1 mg/ml doxycycline. The selected target site in before any active Caspase-3 becomes detectable by western human Caspase-2 mRNA was: 50-GCCCAAGCCTACAGAACAA-30. blotting, suggesting that it might be an upstream regulator of Quantification of cell death and cell cycle distribution. Lympho- cell death and/or checkpoint control under these conditions, at 5 least in HeLa cells (Figure 3). However, our mouse and cytes and myeloid progenitors were cultured at a density of 5 10 /ml and after 24 and 48 h post-irradiation cells were stained with AnnexinV-FITC (eBioscience, HCT116 data also show that Caspase-2 is not strictly required Vienna, Austria) plus 5 mg/ml propidium iodide (Sigma, Vienna, Austria) in for cell death under these conditions, which is consistent with AnnexinV staining buffer and analysed using FACS. MEF, HeLa or HCT116 cells earlier studies in mice and human cancer cells that failed to were plated in six-well plates (100 000 cell/dish) and stimulated the following support a proapoptotic role of the postulated PIDDosome morning. Cells were then washed once in PBS, spun down at 1500 r.p.m. at 4 1C complex after DNA damage.26–28 Admittedly, none of these and subsequently fixed in pre-chilled 70% ethanol/PBS overnight at 4 1C. For sub- studies tested the consequences of PIDD loss or knockdown G1 analysis, cells were washed twice in PBS and after RNase A (Sigma) digestion (100 mg/ml in PBS, 30 min at 37 1C) cells were stained with propidium iodide in response to Chk1 inhibition plus DNA damage but our (40 mg/ml) and the percentages of sub-G1, G1, S and G2/M cells were monitored studies in HCT116 cells (Figure 4) and previous studies by flow cytometry. suggesting a dominant role for PIDD in DNA damage-induced nuclear factor kappa-light-chain enhancer (NF-kB) activation29 Colony forming assays. For colony formation assays early passage MEF argue against a general Caspase-2- or PIDDosome depen- (P2) or transduced MEF selected for 1 week were seeded at a density of 2000, 4000, 8000 or 16 000 cells in 3.5 cm tissue culture dishes. The next morning cells dence of this type of cell death. Thus, although Go¨6976 can were pretreated with 0.1 mMGo¨6976 for 1 h before IR (3 or 10 Gy). Cells were sensitize HeLa cells to DNA damage-induced killing the exact fixed after 9 or 12 days by removal of media and a wash in PBS by the addition of role of this kinase inhibitor and the molecular wiring of these crystal violet staining solution (0.2% crystal violet in 50% methanol). Experiments events are still unclear. were performed independently at least three times in triplicates.

Cell Death and Disease Chk1 inhibition kills PIDDosome defective cells C Manzl et al 9

Immunoblotting. Cells were lysed in lysis-buffer (Buffer: 10 mM HEPES, 13. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. Oncogenic ras provokes INK4a 1.5 mM MgCl2, 300 mM sucrose, 10 mM KCl, 0.5% NP40, Roche Complete premature cell senescence associated with accumulation of p53 and p16 . Cell 1997; Protease Inhibitor Cocktail (Roche, Vienna, Austria), pH adjusted to 7.0, 5 mM 88: 593–602. DTT added freshly from a 1 M stock) for at least 1 h on ice. Protein was separated 14. Strasser A, Harris AW, Jacks T, Cory S. DNA damage can induce apoptosis in proliferating by SDS/PAGE, transferred to a nitrocellulose membrane and incubated with the lymphoid cells via p53-independent mechanisms inhibitable by Bcl-2. Cell 1994; 79: 329–339. relevant antibodies Caspase-2 (11B4) Alexis (Vienna, Austria); cleaved Caspase-3 15. Sohn D, Budach W, Janicke RU. Caspase-2 is required for DNA damage-induced (Asp175) (5A1), pCDK1 (Y15), Chk1 and p-Chk1 (S345; 133D3) all from Cell expression of the CDK inhibitor p21(WAF1/CIP1). Cell Death Differ 2011; 18: Signalling (New England Biolabs, Frankfurt, Germany); GAPDH (71.1) Sigma. 1664–1674. Immune-reactivity was visualized using an enhanced chemiluminescence 16. Ho LH, Taylor R, Dorstyn L, Cakouros D, Bouillet P, Kumar S. A tumor suppressor function detection system (ECL) (GE Healthcare, Freiburg, Germany). for caspase-2. Proc Natl Acad Sci USA 2009; 106: 5336–5341. 17. Tischner D, Manzl C, Soratroi C, Villunger A, Krumschnabel G. Necrosis-like death can Statistics. Statistical analysis was performed using unpaired Student t-test and engage multiple pro-apoptotic Bcl-2 protein family members. Apoptosis 2012; 17: ANOVA as indicated and for Kaplan–Meier analysis the Logrank (Mantel–Cox) test 1197–1209. 18. Toledo LI, Murga M, Fernandez-Capetillo O. Targeting ATR and Chk1 kinases for cancer was used, applying Stat-view 4.1 software program. P-values of o0.05 were treatment: a new model for new (and old) drugs. Mol Oncol 2011; 5: 368–373. considered to be statistically different. 19. Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G, Hug H et al. Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976. J Biol Chem 1993; 268: 9194–9197. Conflict of Interest 20. Kohn EA, Yoo CJ, Eastman A. The protein kinase C inhibitor Go6976 is a potent inhibitor of The authors declare no conflict of interest. DNA damage-induced S and G2 cell cycle checkpoints. Cancer Res 2003; 63: 31–35. 21. Reyland ME. Protein kinase C isoforms: multi-functional regulators of cell life and death. Front Biosci 2009; 14: 2386–2399. Acknowledgements. We are grateful to K Rossi, V Rauch and I Gaggl for 22. Bluwstein A, Kumar N, Leger K, Traenkle J, Oostrum J, Rehrauer H et al. PKC signaling technical assistance and animal care; P Lukas and his team (LINAC1-4) from the prevents irradiation-induced apoptosis of primary human fibroblasts. Cell Death Dis 2013; Department of Radio-oncology for enabling irradiation experiments; P Jost for help 4: e498. 23. Reinhardt HC, Aslanian AS, Lees JA, Yaffe MB. p53-deficient cells rely on ATM- and with establishing thymic lymphoma cell lines. We thank D Vaux, M Serrano and / / f/f ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after T Mak for Casp2 , p53 and Chk1 mice, respectively. This work was DNA damage. Cancer Cell 2007; 11: 175–189. supported by grants from the Austrian Science Fund–FWF (SFB021 and the 24. Reinhardt HC, Hasskamp P, Schmedding I, Morandell S, van Vugt MA, Wang X et al. DNA Doctoral College, MCBO) to AV and SG; the EU-FP06 Marie Curie Research damage activates a spatially distinct late cytoplasmic cell-cycle checkpoint network Training Network ‘Apoptrain’ to AV & FB; the Austrian Cancer Society Branch Tirol controlled by MK2-mediated RNA stabilization. Mol Cell 2010; 40: 34–49. (Tiroler Krebshilfe) to FB, GK, CM and LF; and the Medical University Innsbruck 25. Zenvirt S, Kravchenko-Balasha N, Levitzki A. Status of p53 in human cancer cells does not intramural funding program MUI-START to CM. LP is recipient of a Doc-Fellowship, predict efficacy of CHK1 kinase inhibitors combined with chemotherapeutic agents. sponsored by the Austrian Academy of Science (O¨ AW). LF is a recipient of an Oncogene 2010; 29: 6149–6159. 26. Vakifahmetoglu H, Olsson M, Orrenius S, Zhivotovsky B. Functional connection between EMBO-LT fellowship. p53 and caspase-2 is essential for apoptosis induced by DNA damage. Oncogene 2006; 25: 5683–5692. 27. Kim IR, Murakami K, Chen NJ, Saibil SD, Matysiak-Zablocki E, Elford AR et al. DNA 1. Reinhardt HC, Yaffe MB. Kinases that control the cell cycle in response to DNA damage: damage- and stress-induced apoptosis occurs independently of PIDD. Apoptosis 2009; 14: Chk1, Chk2, and MK2. Curr Opin Cell Biol 2009; 21: 245–255. 1039–1049. 2. Takai H, Tominaga K, Motoyama N, Minamishima YA, Nagahama H, Tsukiyama T et al. 28. Manzl C, Krumschnabel G, Bock F, Sohm B, Labi V, Baumgartner F et al. Caspase-2 -/- Aberrant cell cycle checkpoint function and early embryonic death in Chk1 mice. Genes activation in the absence of PIDDosome formation. J Cell Biol 2009; 185: 291–303. Dev 2000; 14: 1439–1447. 29. Bock FJ, Krumschnabel G, Manzl C, Peintner L, Tanzer MC, Hermann-Kleiter N et al. Loss 3. Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K et al. Chk1 is an essential of PIDD limits NF-kappaB activation and cytokine production but not cell survival or kinase that is regulated by Atr and required for the G2/M DNA damage checkpoint. Genes transformation after DNA damage. Cell Death Differ 2013; 20: 546–557. Dev 2000; 14: 1448–1459. 30. O’Reilly LA, Ekert P, Harvey N, Marsden V, Cullen L, Vaux DL et al. 4. Greenow KR, Clarke AR, Jones RH. Chk1 deficiency in the mouse small intestine results in Caspase-2 is not required for thymocyte or neuronal apoptosis even though cleavage p53-independent crypt death and subsequent intestinal compensation. Oncogene 2009; of caspase-2 is dependent on both Apaf-1 and caspase-9. Cell Death Differ 2002; 9: 28: 1443–1453. 832–841. 5. Zaugg K, Su YW, Reilly PT, Moolani Y, Cheung CC, Hakem R et al. Cross-talk between 31. Jacks T, Remington L, Williams BO, Schmitt EM, Halachmi S, Bronson RT et al. Tumor Chk1 and Chk2 in double-mutant thymocytes. Proc Natl Acad Sci USA 2007; 104: spectrum analysis in p53-mutant mice. Curr Biol 1994; 4: 1–7. 3805–3810. 32. Kaplan HS, Brown MB. A quantitative dose-response study of lymphoid-tumor 6. Vitale I, Galluzzi L, Castedo M, Kroemer G. Mitotic catastrophe: a mechanism for avoiding development in irradiated C57 black mice. J Natl Cancer Inst 1952; 13: 185–208. genomic instability. Nat Rev Mol Cell Biol 2011; 12: 385–392. 33. Labi V, Erlacher M, Kiessling S, Manzl C, Frenzel A, O’Reilly L et al. Loss of the BH3-only 7. Janssen A, Medema RH. Mitosis as an anti-cancer target. Oncogene 2011; 30: protein Bmf impairs B cell homeostasis and accelerates gamma irradiation-induced thymic 2799–2809. lymphoma development. J Exp Med 2008; 205: 641–655. 8. Sidi S, Sanda T, Kennedy RD, Hagen AT, Jette CA, Hoffmans R et al. Chk1 suppresses a 34. Ploner C, Rainer J, Niederegger H, Eduardoff M, Villunger A, Geley S et al. The BCL2 caspase-2 apoptotic response to DNA damage that bypasses p53, Bcl-2, and caspase-3. rheostat in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia. Leukemia Cell 2008; 133: 864–877. 2008; 22: 370–377. 9. Bock FJ, Peintner L, Tanzer M, Manzl C, Villunger A. P53-induced protein with a death domain (PIDD): master of puppets? Oncogene 2012; 31: 4733–4739. 10. Ando K, Kernan JL, Liu PH, Sanda T, Logette E, Tschopp J et al. PIDD Death-domain phosphorylation by ATM controls prodeath versus prosurvival PIDDosome signaling. Cell Death and Disease is an open-access journal Mol Cell 2012; 47: 681–693. published by Nature Publishing Group. This work is 11. Ma CX, Janetka JW, Piwnica-Worms H. Death by releasing the breaks: CHK1 inhibitors as licensed under a Creative Commons Attribution-NonCommercial- cancer therapeutics. Trends Mol Med 2011; 17: 88–96. 12. Aarts M, Linardopoulos S, Turner NC. Tumour selective targeting of cell cycle kinases for NoDerivs 3.0 Unported License. To view a copy of this license, visit cancer treatment. Curr Opin Pharmacol 2013; 13: 529–535. http://creativecommons.org/licenses/by-nc-nd/3.0/

Supplementary Information accompanies this paper on Cell Death and Disease website (http://www.nature.com/cddis)

Cell Death and Disease Attached Manuscripts

4) The tumor-modulatory effects of Caspase-2 and Pidd1 do not require the scaffold protein RAIDD Based on the findings reported above, I initiated a study exploring the role of RAIDD in Caspase-2 dependent tumor suppression. Our own observations and other published data already generated doubts related to the relevance of the PIDDosome complex, at least in cancer progression directed by Caspase-2. Surprisingly, I could not find evidence for RAIDD dependence in this process. The results presented in this publication clearly indicate, that Caspase-2 acts independent of RAIDD in its tumor suppressor function. All the major experiments were conducted by me and I also was in charge of writing the manuscript.

60

The tumor-modulatory effects of Caspase-2 and Pidd1 do not require the scaffold protein Raidd

L. Peintner1, L. Dorstyn2, S. Kumar2, T. Aneichyk3, A. Villunger 1,5 and C. Manzl1,4,5

1Division of Developmental Immunology, Medical University of Innsbruck, 6020

Innsbruck, Austria; 2Centre for Cancer Biology - An Alliance between SA Pathology and the University of South Australia, Adelaide, SA 5001, Australia, 3Division of

Molecular Pathophysiology, Biocenter, Medical University of Innsbruck, 6020

Innsbruck, Austria; 4Department of General Pathology, Medical University of

Innsbruck, 6020 Innsbruck, Austria

5 to whom correspondence should be addressed:

Dr. Claudia Manzl Medical University of Innsbruck Department of General Pathology Muellerstrasse 44 6020 Innsbruck, Austria Phone: +43-512-9003-71307 Fax: +43-512-9003-73301 [email protected]

Prof. Dr. Andreas Villunger Medical University of Innsbruck Division of Developmental Immunology Biocenter Innrain 80-82 6020 Innsbruck, Austria Ph: +43-512-9003-70380 Fax: +43-512-9003-73960 [email protected]

Keywords: Caspase-2, Raidd, PIDDosome, tumorigenesis 1

Abstract:

The receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD/CRADD) functions as a dual adaptor and is a constituent of different multi-protein complexes implicated in the regulation of inflammation and cell death. Within the PIDDosome complex, RAIDD connects the cell death-related protease, Caspase-2, with the p53-induced protein with a death domain 1 (PIDD1).

As such, RAIDD has been implicated in DNA-damage induced apoptosis as well as in tumorigenesis. As loss of Caspase-2 leads to an acceleration of tumor onset in the

Eµ-Myc mouse lymphoma model while loss of Pidd1 actually delays onset of this disease, we set out to interrogate the role of Raidd in cancer in more detail. Our data obtained analyzing Eµ-Myc/Raidd-/- mice indicate that Raidd is unable to protect from c-Myc-driven lymphomagenesis. Similarly, we failed to observe a modulatory effect of

Raidd-deficiency on DNA-damage driven cancer. The role of Caspase-2 as a tumor suppressor and that of Pidd1 as a tumor promoter can therefore be uncoupled from their ability to interact with the Raidd scaffold, pointing towards the existence of alternative signaling modules engaging these two proteins in this context.

2

Introduction:

A number of mechanisms have evolved to trace and remove potentially dangerous cells. Deregulation of the induction of apoptosis upon oncogenic stress, for example, can facilitate the accumulation of cells prone to undergo malignant transformation.

Cell death by apoptosis depends on the cascade-like activation of proteases of the

Caspase family 1. Amongst these, the evolutionary most conserved protease,

Caspase-2, turns out to be a potent tumor suppressor in mice 2, 3, 4, 5, 6, 7 and correlative expression data supports a conserved role in human cancer 8, 9, 10, 11, 12, 13.

Early studies suggested that Caspase-2 interacts with other proteins for its activation,

(e.g., after genotoxic stress), but the protease seems also able to auto-activate cell death on its own when present in sufficiently high concentration 14, 15, 16. The most prominent Caspase-2-containing protein complex was dubbed the “PIDDosome” and described to contain the p53-induced protein with a death domain (PIDD1) and receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD, also known as CRADD) 17. Although the molecular details of the pro-apoptotic potential of Caspase-2 are still discussed and alternative roles in the DNA-damage response (DDR), cell-cycle arrest or sensor of metabolic stress are mechanistically poorly understood, Caspase-2 clearly limits tumorigenesis in different settings. These include aberrant expression of c-Myc in B cells 3, 4 or deletion of the

DDR regulator, ATM kinase, both driving lymphomagenesis 6 as well as overexpression of the Her2/ErbB2 oncogene in breast 5 or that of mutated KRAS in the lung epithelium, driving carcinoma formation 7. One of these studies, addressing also the role of Pidd1 in c-Myc-driven lymphomagenesis, revealed an unexpected oncogenic role for Pidd1, thereby questioning the physiological relevance of the

PIDDosome complex in Caspase-2-mediated cell death and tumor suppression 4.

3

However, the exact role of the scaffold-protein Raidd within these processes remains unaddressed so far.

Raidd, a bipartite adapter containing a death domain (DD) and a caspase-recruitment domain (CARD) was first described to bind to the DD-containing kinase RIPK1 and the C. elegans caspase Ced-3 18, supporting a role in cell death initiation.

Subsequently, the interaction of Caspase-2 and Raidd was biochemically proven 19 and proposed to be required for Caspase-2 auto-processing preceding its activation

17. More recent studies propose an anti-inflammatory role for Raidd through suppression of NF-κB activation and cytokine production upon T cell receptor stimulation by negatively interfering with the Carma1/Malt1/Bcl-10 signaling complex

20, 21.

First evidence for a potential role of RAIDD in human cancer was discovered in a biochemical screen using mantle cell lymphomas, which detected a downregulation of RAIDD by microarray analysis 10 while others reported on RAIDD-linked multidrug- resistance in osteosarcoma cells 22. Furthermore, tumor cell apoptosis induced by inhibitors of histone de-acetylases (HDACi) in treatment-resistant adult T-cell leukemia (ATLL) lines reportedly required Caspase-2 and Raidd 23. It is also reported that the Caspase-2/Raidd axis is necessary after ER-stress, e.g., in the course of infection with the oncolytic maraba virus 24.

Taken together, these studies support a role for RAIDD in drug-induced cancer cell death as well as tumor suppression, most likely linked to its role as a direct activator of Caspase-2. Alternatively, RAIDD may negatively interfere with PIDD or BCL10- regulated NF-κB signaling 20, 21, 25 and thereby suppress pro-tumorigenic inflammation. To address the role of Raidd in tumorigenesis in more detail we exploited different mouse models where we induced thymic lymphomas by - irradiation, fibrosarcomas by 3-methylcholantrene (3-MC) injection or B cell 4

lymphomas by aberrant expression of the c-Myc proto-oncogene. Our results suggest that Raidd is not a suppressor of tumors in the mouse models tested.

Results:

Loss of Raidd has no impact on tumor formation after DNA damage

Since Raidd was reported to play a role in DNA-damage-induced apoptosis due to its ability to form a complex with Pidd1 and Caspase-2 (14), we used different cancer models to evaluate the impact of Raidd in tumorigenesis induced by DNA lesions.

Induction of thymic lymphomas was triggered in 4 week-old mice by repeated low- dose irradiation (1.75 Gy). As reported before, tumor formation was significantly accelerated in p53+/- mice when compared to wild-type (wt) controls, showing a mean survival of 115 and 207 days (d), respectively. Absence of Raidd, however, had no effect on the onset of tumorigenesis (Fig. 1a) or tumor immunophenotype (data not shown) and animals developed thymic lymphomas with a mean survival of 190 days, similar to their wt littermates.

In another tumor model, we treated adult mice with a single injection of 3- methylcholantrene (3-MC) causing the formation of bulky adducts in DNA leading to fibrosarcoma formation within 6 months. As reported before 26, p53+/- mice developed

3-MC induced sarcomas significantly earlier but loss of Raidd failed to accelerate tumor onset when compared to wt controls (Fig. 1b).

Together with our previously published data 4 this demonstrates that the PIDDosome, and each of its components individually, are unable to prevent DNA-damage induced tumor formation inflicted by -irradiation or bulky DNA-adduct formation.

c-Myc induced lymphomagenesis is limited by Caspase-2 in a Raidd- independent manner 5

Caspase-2 and Pidd1 were reported to modulate lymphomagenesis in Eµ-Myc mice, a model somewhat mimicking human Burkitt lymphoma 4. Hence, we introduced

Raidd-deficiency into Eµ-Myc transgenic animals to explore its impact on tumorigenesis caused by oncogenic stress. Excess proliferation by aberrant c-Myc expression in premalignant mice is usually counterbalanced by increased cell death rates providing a rational why loss of pro-apoptotic proteins, e.g. of the BH3-only group, accelerates disease onset 27, 28, 29.

In order to evaluate the role of Raidd early in disease development the B cell subset composition was assessed in premalignant Eµ-Myc/Raidd-/- mice. In line with published data 4, 29, 30, 31 the numbers of B220+IgM- pro/pre-B cells were increased in bone marrow and spleen while numbers of mature B cells were diminished in the periphery of Eµ-Myc mice when compared to wt controls (Fig. 2a). However, no other gross alterations within the B cell composition and distribution in Eµ-Myc mice specifically lacking Raidd were noted.

To monitor B cell survival upon oncogenic stress in the absence or presence of

Raidd, we next sorted pre-B and immature B cells of premalignant mice and put them in culture. Notably, B cells from non-transgenic Raidd-/- mice appeared to be slightly less sensitive to spontaneous cell death when compared to wt (Fig. 2b). As reported before 27, 28, 29, B cells derived from Eµ-Myc mice died more rapidly when compared to the non-transgenic counterparts but additional lack of Raidd had no relevant impact (Fig. 2b). Similarly, Raidd-deficiency had no impact on homeostatic or c-Myc driven B cell proliferation, as assessed by BrdU-incorporation analysis in immature

CD19+IgM- and CD19+IgM+ B cells from bone marrow or spleen of premalignant mice

(Fig. 2c).

Next, cohorts of Eµ-Myc, Eµ-Myc/Raidd-/- and, for reference, Eµ-Myc/Casp-2-/- mice were monitored for tumor onset. Strikingly, while loss of Caspase-2 lead to 6

accelerated disease onset (mean survival: 101 d), loss of Raidd had no effect, when compared to Raidd-proficient Eµ-Myc mice presenting with a mean survival: 128 d vs. 124 d (Fig. 3a). White blood cell counts and tumor burden were also comparable between genotypes (Supplementary Figs 1a, 1b).

Tumors were subsequently classified by flow cytometry as either pre-B cell (IgM-), immature B cell (IgM+), mixed (IgM+/-) or as early hematopoietic progenitor-derived lymphomas (B220+CD4+) and we could not detect any differences in the distribution of these sub-classes across genotypes (Fig 3b).

Further, in lymphoma cells derived from Eµ-Myc/Raidd-/- mice, cell cycle distribution

(Figs. 4a, 4b), spontaneous (Fig. 4c) as well as drug-induced cell death (Fig. 4d) were monitored and found comparable to their wt counterparts. Notably, Caspase-2 deficient tumors showed again an increase in phospho-Histone H3 staining, suggesting perturbed cell cycle control (Fig. 4b). In line with a lack of impact on tumor latency, loss of Raidd did not release the pressure to inactivate p53, a known secondary hit, usually observed in about 25% of all Eµ-Myc-lymphomas 32.

Inactivation of p53, as assessed by Western blotting for p53 and p19ARF, was seen at similar rates in Eµ-Myc (26%, n=11) and Eµ-Myc/Raidd-/- (27%, n=19) tumors

(Supplemental Figs. 1c, 1d).

Overall, these data demonstrate that the adaptor protein Raidd is not limiting Myc- driven tumorigenesis thereby uncoupling the tumor suppressor function of Caspase-2 from Raidd-dependent auto-activation and the oncogenic potential of Pidd1 from

Raidd-modulated NF-κB signaling.

Loss of Caspase-2 promotes aneuploidy

As Caspase-2 has been implicated in mediating mitotic catastrophe as a response to

DNA-damage after failed cell cycle arrest 33, 34 and that c-Myc overexpression drives 7

proliferation stress leading to DNA-damage 35, we investigated if lack of Caspase-2 would influence genomic stability of normal, pre-malignant or transformed cells.

First, we investigated chromosomal stability in SV40-immortalized MEFs from wt and

Caspase-2 deficient mice (Figs. 5a, b). Numbers of micronuclei, which emerge after errors during mitosis and result in individual chromosomes or fragments outside of the main nucleus, were significantly increased in cells lacking Caspase-2 in comparison to wt cells. Raidd-/- MEFs did not show increased susceptibility to micronuclei formation (Fig. 5b). Chromosomal stability was further examined by counting chromosome numbers in metaphase spreads in B cell lymphomas (Fig 5c- e). Eµ-Myc transgenic B cell tumors deficient for Caspase-2 did show a higher variation of chromosome numbers within individual tumor samples. Taken together these data are consistent with published results, indicating that Caspase-2 acts in maintaining genomic stability 33.

In an attempt to identify potential effectors of Caspase-2 in this process and based on the reported role of Caspase-2 in p53-activation and target gene expression upon

DNA damage 36, we performed qPCR-analysis as well as unbiased genome-wide expression analysis comparing mRNA from premalignant splenic IgM+D- B cells of

Eµ-Myc transgenic animals deficient or proficient for Caspase-2 or Raidd. We started by comparing expression levels of mRNAs of p21, Noxa, and Puma by qRT-PCR, anticipating that c-Myc-driven p53-activation would result in reduced expression of these targets in the absence of Caspase-2 36. This however was not the case, or at least the noted differences did not reach statistical significant differences (suppl. Fig.

2). Comparison of gene-chip data revealed only few candidates, including Fbxw10,

Fgd6, Hist3h2a, Hmha1, Igf1R, Lyrm7, RhoBTB1, Slc25a13 and Zfp39 that were deregulated more than two-fold in the absence of Caspase-2 or RAIDD when compared to wt (Suppl. Fig. 2). Only one of these candidates, i.e. RhoBTB1, was 8

subsequently confirmed by qPCR analysis. However, as RhoBTB1 is unlikely related to genomic stability this was not followed up in detail during the course of this study.

This indicates that there are no significant changes, at least in the pre-malignant B cells analysed that may contribute to the genomic instability observed following loss of Caspase-2.

Discussion:

In this study, we investigated the relevance of Raidd in tumorigenesis sparked by its function as an adaptor protein for Caspase-2 and Pidd1, both being reported to influence tumorigenesis in vivo. Caspase-2 is well characterized as a tumor suppressor gene in various mouse cancer models 3, 4, 5, 6, 7, while loss of Pidd1 leads to a delayed tumor onset upon c-Myc overexpression (4). Our data demonstrate that loss of Raidd has no influence on the development of radiation induced thymic lymphomas or chemically induced fibrosarcomas (Fig. 1). These observations are similar to the published results in Caspase-2- and Pidd1-deficient mice (4) and exclude a role of any of the PIDDosome components in these DNA damage-driven models of cancer.

Strikingly, Raidd-deficiency also had no influence on tumor latency in c-Myc-driven B cell lymphomas (Fig. 3a), contrasting the findings with Caspase-2 or Pidd1 deficient mice, where Caspase-2 acts as a tumor suppressor and Pidd1 as a tumor promoter in this model. Our data show that loss of Raidd is dispensable for the development of

B cell lymphomas in the Eµ-Myc mouse model and that Raidd-deficiency has no impact on development, proliferation and cell death of premalignant and transformed cells (Fig. 2). The results thus indicate that Caspase-2-mediated tumor suppression is independent of both Pidd1 and Raidd. This is consistent with the idea that

9

Caspase-2 autoactivation can occur without the need for additional accessory proteins 14, 15, 16.

Prior work linked loss of Caspase-2 to chromosomal and genomic instability as a possible driver of accelerated transformation 33. Consistently, SV40 MEFs and c-Myc transgenic B cells lacking Caspase-2 showed significantly higher levels of micronuclei formation and numbers of chromosomes in metaphase spreads diverted more strongly from the normal number. However, our analysis of chromosome numbers in of Eµ-Myc B cell tumors did not show a direct increase in the frequency of aneuploidy in cells lacking Caspase-2. Instead, we found that the aneuploid cells present in the Caspase-2 deficient tumors showed a greater variation of chromosome numbers in the same tumor sample. Furthermore, our data show that the level of high-grade aneuploidy (i.e. a loss or gain of more than 10 chromosomes) was greater in Caspase-2-/- tumors. Together these results clearly support a possible role of

Caspase-2 in maintaining genomic integrity while loss of the adaptor protein Raidd is dispensable for genomic stability (not shown).

As it is difficult to connect these observations with known substrates of Caspase-2 and the analysis of representative p53-induced genes failed to support impaired

Mdm2 activity, as reported in cisplatin-treated lung cancer cells 36, we performed whole transcriptome analysis on premalignant Eµ-Myc pre-B cells lacking or expressing Caspase-2 (or Raidd). However, we again failed to identify possible candidates that may be involved in the observed tumor suppressor phenotype and established p53 targets were also not deregulated (Suppl. Fig 2). Hence, it remains possible that the phenomenon of reduced p53 activation in the absence of Caspase-

2 is cell type-specific, e.g., in lung epithelium 36, and/or a particular secondary hit, or a fully transformed cellular state 33. Consistent with the latter scenario, we previously

10

reported on a reduced selective pressure to inactivate p53 in Eµ-Myc lymphomas lacking Caspase-2 (4).

In summary our data suggest that Raidd does not influence the development of tumors after DNA damage stress or the overexpression of oncogenes. This is in strong contrast to the opposing effects of its putative interaction partners, Caspase-2 or Pidd1 3, 4. Hence, the function of Raidd as a ‘direct activator of Caspase-2’ needs to be re-considered, at least in the context of Eµ-Myc driven tumorigenesis and justifies the search for additional activators as well as substrates of Caspase-2 to understand its tumor suppressive function at the molecular level.

Materials and Methods:

Mice

All animal experiments were performed in accordance to the Austrian legislation

(BGBl. Nr. 501/1988 i.d.F. 162/2005, # BMWF-66.011/0137-II/10b/2009). The generation and genotyping of Casp2-/-, Raidd-/-, p53+/- and Eµ-Myc transgenic mice have been described elsewhere 30, 37, 38, 39. All mice used for the experiments were on an inbred C57BL/6 background.

Tumorigenesis induced by DNA damage

In order to induce thymic lymphomas, mice at the age of four weeks irradiated with

1.75 Gy in a linear accelerator once per week for 4 weeks. Muscular sarcoma formation was induced by a single 200 µl intra muscular injection of 1 mg 3-methyl- cholantrene per mouse (Sigma, Vienna, Austria), dissolved in sesame oil. Mice, treated with sesame oil alone (vehicle) served as a control.

Cell culture and reagents 11

Primary tumor cells derived from Eµ-Myc, Eµ-Myc/Raidd-/-, Eµ-Myc/Casp2-/- and Eµ-

Myc/Pidd1-/- mice were cultured in DMEM (PAA), supplemented with 10% FCS (PAA)

Pen/Strep (Sigma), 250 µM L-Glutamine (Gibco) and 50 µM 2-mercaptoethanol

(Sigma) and were kept on irradiated Bcl-2 overexpressing NIH-3T3 feeder cells.

Tested agents and concentrations were: Etoposide (0.01–1 mg/ml), Dexamethasone

(109–107 M), Paclitaxel (0.5–50 mM), Thapsigargin (0.5–50 ng/ml) and Doxorubicin

(4–400 ng/ml) (all from Sigma). FACS-sorted pre B, immature and IgM+ B cells were cultured in DMEM with supplements and spontaneous cell death was monitored over time.

Flow cytometric analysis and cell sorting

Tumors were analysed by flow cytometry using the following cell surface markers for a) B cells: RA3-6B2, anti-B220; R2/60, anti-CD43; II/41, anti-IgM; 11/26C, anti-IgD;

MB19-1, anti-CD19; 53-7.3, anti-CD5 and B3B4, anti-CD23; 7E9, anti-CD21

(Biolegend, Fell, Germany); and b) T cells: GK1.5, anti-CD4; H57-597, anti-TCRb (all from eBioscience, Vienna, Austria) and 53-6.7, anti-CD8; (from BD Pharmingen, San

Diego, CA, USA). Biotinylated antibodies were monitored with streptavidin-RPE

(DAKO, Vienna, Austria) or streptavidin-PE-Cy7 (BD Phamingen). Using a

FACSVantage cell sorter (Becton Dickinson, Heidelberg, Germany) premalign pre/pro B cells (B220+/IgM-) or immature (IgM+/IgDlow) and mature (IgM+/IgDhigh) B cells were isolated and sorted from bone marrow and spleen, respectively. Flow cytometry data were analysed using Cyflogic free ware and FlowJo.

Immunoblotting

Proteins were extracted from tumor cells for 1h on ice in protein lysis-buffer 32.

Insoluble debris was cleared by centrifugation for 5 min at 13000 rpm and 4°C. For 12

evaluation of the p53 status of tumor cells 30 µg protein/lane was separated by SDS-

PAGE, transferred to a nitro-cellulose membrane and probed with rat anti-p19/ARF

(5-C3-1; Santa Cruz Biotechnology, Szabo-Scandic, Vienna, Austria) or mouse anti- p53 antiserum (1C12; Cell Signaling, New England Biolabs, Frankfurt, Germany).

Comparability of protein loading was assessed by re-probing membranes with an antibody recognizing GAPDH (Sigma). Horseradish peroxidase-conjugated sheep anti-rat Ig antibodies (Jackson Research, Vienna, Austria), goat anti-rabbit or rabbit anti-mouse antibodies (DAKO) were used as secondary antigens. Antibody binding was detected using enhanced chemiluminiscence (ECL; Amersham, Freiburg,

Germany) system.

Cell cycle analysis

Cell cycle analysis was based on fixing lymphoma cells in 70% ethanol and staining with PI (propidium iodide at 40 µg/ml; Sigma). In order to analyze the percentage of cells in M-phase, ethanol-fixed lymphoma cells were permeabilized using TritonX

(0.25%, Sigma) for 15 min on ice and co-stained with phosphorylated H3 (Ser10; Cell

Signalling) and PI. Distribution of cell cycle phases was analysed using a FACScan cell cytometer (BD, Heidelberg, Germany).

BrdU incorporation

Cell proliferation of immature (CD19+IgM-) and mature (CD19+IgM+) B cells in premalignant 4 weeks old mice was assessed by injecting 1 mg BrdU/mouse i.p.

Four hours later primary cells derived from bone marrow and spleen were isolated and stained for BrdU incorporation using the BrdU/APC flow kit (BD, Vienna, Austria) according to manufacturer’s recommendation. Samples were analyzed using FACS-

Calibur (BD). 13

Cell viability assay

Tumor cells were costained with Annexin-V-FITC (1:1800 in Annexin-V binding buffer; BD) and 7-AAD (1 µg/ml, Sigma) and spontaneous cell death was analyzed by subsequent flow cytometric analysis

Cytogenetic analysis

Micronuclei were examined by seeding 2x105 MEF on a sterile coverslip treated with cytochalisin B (4 µg/mL) for 16 h before fixation in PTEMF fixative 40 for 10 min and staining with 4´,6´-diamidino-phenylinodole (DAPI). A minimum of 300 cells per condition and genotype were screened for micronuclei under a ZEISS IMAGER Z1

Microscope and Zeiss EC Plan-NEOFLUAR 40x/0,75 Ph2 objective using AxioVision

Release 4.8.2 imaging software.

Chromosome spreads from freshly isolated tumors were generated by incubating

1x106 lymphoma cells in media containing 1 µM nocodazole (Sigma) for 5 h at 37°C in a CO2 incubator. After collecting and washing the cells in PBS, the pellet was resuspended in 5 mL 0.075 M KCl (Merck) and incubated for 5 min at 37°C. Then

1 mL Carnoys fixative (Methanol:Acetic Acid=3:1) was added. Cells were spun down

(1200 g for 3 min) and fixed in 1 mL Carnoys fixative (repeated three times). Cells were added drop-wise (from approx. 50 cm hight) onto a coverslip, air dry and stain with DAPI. A minimum of 50 chromosome spreads per tumor sample was analyzed at 100x magnification.

qRT-PCR analysis

RNA was isolated using TRIzol (Invitrogen) and transcribed into cDNA (Omniscript,

Quiagen) using random hexamer primers after DNAse digestion (Promega). cDNA of 14

interest was amplified using primers listed in supplemental Table 1 and 2x DyNAmo

Color Flash SYBR Green Master Mix (Thermo Scientific) in an Eppendorf

Mastercycler ep realplex2 cycler. Relative expression of target/housekeeper was calculated using the delta-CT method.

Microarray data set generation and analysis

Microarray data for gene expression was obtained using Affymetrix MoGene 1.0 ST v.1 arrays. Sample preparation was performed according to the manufacturer’s protocol. In brief, 250 ng of high quality RNA per sample was processed using the

Ambion Affymetrix GeneChip WT Expression Kit (Part no. 308 4411974, Ambion) and the Affymetrix GeneChip WT Terminal Labeling Kit (Affymetrix). The resulting biotinylated targets were hybridized in an Affymetrix hybridization oven to a total of 14

Affymetrix MoGene 1.0 ST v.1 microarrays, which were then washed and stained in an Affymetrix fluidic station 450. Raw fluorescence signals were recorded in an

Affymetrix scanner 3000 and image analysis was made with the Affymetrix GeneChip

Command Console software (AGCC).

Subsequent analyses have been performed in R (version 3.0.2) using packages from the Bioconductor project (41, version 2.13). The raw microarray data was pre- processed using “generalgcrma” package 42 and our custom transcript-level “CEL definition file” (CDF) as described in 43. Transcripts for all genes have been defined using Ensembl version 71. The CDF contained a total of 63455 probe sets for 22991 genes. Background adjustment, normalization and summarization of the microarray data were performed using the GCRMA method 44. Further, a single probe set per gene was selected as following: I) if there were probe sets with more than 5 probes, only those were considered for further analysis II) if any of considered probe sets represented protein coding transcripts, the list of probe sets was limited to such and 15

III) the probe set which had the highest multiple of average expression and standard deviation across all samples was chosen for expression analysis.

Differential expression analysis was performed using limma package 45. P-values were adjusted for multiple hypotheses testing correction by method of Benjamini and

Hochberg (BH) 46. Genes with adjusted p-value < 0.05 and absolute log2 fold change

>1 were considered to be significantly regulated.

The raw and preprocessed microarray data have been submitted to the Gene

Expression Omnibus (accession number GSE64920).

Statistical analysis

Statistical analysis was performed using unpaired Student’s t-test or ANOVA with

Student-Newman-Keuls as post hoc test. Comparison of Kaplan-Meier survival plots was performed using a log-rank test and for evaluation of statistical difference in frequency distributions the Chi2 (Fisher’s exact) analysis algorithm was used. P- values of <0.05 (*) or p<0.001 (**) indicate significant differences.

Conflict of Interest:

The authors declare no conflict of interest

Acknowledgements:

We thank I. Gaggl and C. Soratroi for excellent technical assistance as well as K.

Rossi and B. Rieder for animal care. We further thank Prof. P. Lukas and his team

(LINAC 1-4), Department of Therapeutic Radiology and Oncology, Medical University

Innsbruck, for enabling irradiation experiments, R. Kofler for help with gene-chip analysis and G. Böck for cell sorting. L. Peintner was supported by a DOC Fellowship of the Austrian Academy of Sciences (ÖAW). This study was funded by the 16

“Österreichische Krebshilfe”, Branch Tyrol as well as the Austrian Science Fund,

FWF, (Project P 26856) and the MCBO postgraduate program (Project: W1101) as well as the National Health and Medical Research Council grants (1002863 and

1043057).

Supplementary Information accompanies this paper on Cell Death and Differentiation website (http://www.nature.com/cdd)

17

References:

1. McIlwain DR, Berger T, Mak TW. Caspase Functions in Cell Death and Disease. Cold Spring Harbor Perspectives in Biology 2013, 5(4) : 1-29.

2. Puccini J, Dorstyn L, Kumar S. Caspase-2 as a tumour suppressor. Cell Death and Differentiation 2013, 20 (9) : 1133-1139.

3. Ho LH, Taylor R, Dorstyn L, Cakouros D, Bouillet P, Kumar S. A tumor suppressor function for caspase-2. Proceedings of the National Academy of Sciences of the United States of America 2009, 106 (13) : 5336-5341.

4. Manzl C, Peintner L, Krumschnabel G, Bock F, Labi V, Drach M , et al. PIDDosome-independent tumor suppression by Caspase-2. Cell Death and Differentiation 2012, 19 (10) : 1722-1732.

5. Parsons MJ, McCormick L, Janke L, Howard A, Bouchier-Hayes L, Green DR. Genetic deletion of caspase-2 accelerates MMTV/c-neu-driven mammary carcinogenesis in mice. Cell Death and Differentiation 2013, 20 (9) : 1174-1182.

6. Puccini J, Shalini S, Voss AK, Gatei M, Wilson CH, Hiwase DK , et al. Loss of caspase-2 augments lymphomagenesis and enhances genomic instability in Atm-deficient mice. Proceedings of the National Academy of Sciences of the United States of America 2013.

7. Terry MR, Arya R, Mukhopadhyay A, Berrett KC, Clair PM, Witt B , et al. Caspase-2 impacts lung tumorigenesis and chemotherapy response in vivo. Cell Death and Differentation 2014.

8. Kumar S, White D, Takai S, Tuczynowicz S, Juttner C, Hughes T. Apoptosis regulatory gene NEDD2 maps to human chromosome segment 7q34-35, a region fequently affected in haematological neoplasms. Human Genetics 1995, 95 (6) : 641-644.

9. Estrov Z, Thall PF, Talpaz M, Estey EH, Kantarjian HM, Andreeff M , et al. Caspase 2 and Caspase 3 Protein Levels as Predictors of Survival in Acute Myelogenous Leukemia. 1998, 92 (9) : 3090-3097.

10. Hofmann WK, de Vos S, Tsukasaki K, Wachsman W, Pinkus G, Said J , et al. Altered apoptosis pathways in mantle cell lymphoma detected by oligonucleotide microarray. Blood 2001, 98 (3) : 787-794.

11. Holleman A, Boer MLd, Kazemier KM, Beverloo HB, von Bergh ARM, Janka- Schaub GE , et al. Decreased PARP and procaspase-2 protein levels are 18

associated with cellular drug resistance in childhood acute lymphoblastic leukemia. Blood 2005, 106 (5) : 1817-1823.

12. Kim MS, Kim HS, Jeong EG, Soung YH, Yoo NJ, Lee SH. Somatic mutations of caspase-2 gene in gastric and colorectal cancers. Pathology - Research and Practice 2011, 207 (10) : 640-644.

13. Ren K, Lu J, Porollo A, Du C. Tumor-suppressing Function of Caspase-2 Requires Catalytic Site Cys-320 and Site Ser-139 in Mice. Journal of Biological Chemistry 2012, 287 (18) : 14792-14802.

14. Colussi PA, Harvey NL, Kumar S. Prodomain-dependent Nuclear Localization of the Caspase-2 (Nedd2) Precursor: A novel Function for a Caspase Prodomain. Journal of Biological Chemistry 1998, 273 (38) : 24535-24542.

15. Butt AJ, Harvey NL, Parasivam G, Kumar S. Dimerization and Autoprocessing of the Nedd2 (Caspase-2) Precursor Requires both the Prodomain and the Carboxyl-terminal Regions. Journal of Biological Chemistry 1998, 273 (12) : 6763-6768.

16. Baliga BC, Read SH, Kumar S. The biochemical mechanism of caspase-2 activation. Cell Death and Differentiation 2004, 11 (11) : 1234-1241.

17. Tinel A, Tschopp J. The PIDDosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress. Science 2004, 304 (5672) : 843-846.

18. Duan H, Dixit V. RAIDD is a new "death" adaptor molecule. Nature 1997, 238: 86-89.

19. Zhou P, Chou J, Olea R, Yuan J, Wagner G. Solution structure of Apaf-1 CARD and its interaction with caspase-9 CARD- a structural basis for specific adaptor-caspase interaction. Proceedings of the National Academy of Sciences of the United States of America 1999, 96 (20) : 11265-11270.

20. Lin Q, Liu Y, Moore DJ, Elizer SK, Veach RA, Hawiger J , et al. Cutting edge: the "death" adaptor CRADD/RAIDD targets BCL10 and suppresses agonist- induced cytokine expression in T lymphocytes. Journal of Immunology 2012, 188 (6) : 2493-2497.

21. Qiao H, Liu Y, Veach RA, Wylezinski L, Hawiger J. The Adaptor CRADD/RAIDD Controls Activation of Endothelial Cells by Proinflammatory Stimuli. Journal of Biological Chemistry 2014, 289 (32) : 21973-21983.

19

22. Yang C, Hornicek FJ, Wood KB, Schwab JH, Mankin H, Duan Z. RAIDD expression is impaired in multidrug resistant osteosarcoma cell lines. Cancer Chemotherapy and Pharmacology 2009, 64 (3) : 607-614.

23. Hasegawa H, Yamada Y, Tsukasaki K, Mori N, Tsuruda K, Sasaki D , et al. LBH589, a deacetylase inhibitor, induces apoptosis in adult T-cell leukemia/lymphoma cells via activation of a novel RAIDD-caspase-2 pathway. Leukemia 2011, 25 (4) : 575-587.

24. Mahoney D, Lefebvre C, Allan K, Brun J, Sanaei C, Baird S , et al. Virus-Tumor Interactome Screen Reveals ER Stress Response Can Reprogram Resistant Cancers for Oncolytic Virus-Triggered Caspase-2 Cell Death. CancerCcell 2011, 20 (4) : 443-456.

25. Janssens S, Tinel A, Lippens S, Tschopp J. PIDD mediates NF-kappaB activation in response to DNA damage. Cell 2005, 123 (6) : 1079-1092.

26. Matheu A, Maraver A, Klatt P, Flores I, Garcia-Cao I, Borras C , et al. Delayed ageing through damage protection by the Arf/p53 pathway. Nature 2007, 448 (7151) : 375-379.

27. Harris AW, Pinkert CA, Crawford M, Langdon WY, Brinster RL, Adams JM. The Eµ-myc transgenic mouse. A model for high-incidence spontaneous lymphoma and leukemia of early B cells. The Journal of Experimental Medicine 1988, 167 (2) : 353-371.

28. Michalak EM, Jansen ES, Happo L, Cragg MS, Tai L, Smyth GK , et al. Puma and to a lesser extent Noxa are suppressors of Myc-induced lymphomagenesis. Cell Death and Differentiation 2009, 16 (5) : 684-696.

29. Frenzel A, Labi V, Chmelewskij W, Ploner C, Geley S, Fiegl H , et al. Suppression of B-cell lymphomagenesis by the BH3-only proteins Bmf and Bad. Blood 2010, 115 (5) : 995-1005.

30. Adams J, Harris A, Pinkert C, Corcoran L, Alexander W, Cory S , et al. The c- myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature 1985, 318: 533-538.

31. Egle A, Harris AW, Bouillet P, Cory S. Bim is a suppressor of Myc-induced mouse B cell leukemia. Proceedings of the National Academy of Sciences of the United States of America 2004, 101 (16) : 6164-6169.

20

32. Eischen CM, Weber JD, Roussel MF, Sherr CJ, Cleveland JL. Disruption of the ARF–Mdm2–p53 tumor suppressor pathway in Myc-induced lymphomagenesis. Genes & Development 1999, 13 (20) : 2658-2669.

33. Dorstyn L, Puccini J, Wilson CH, Shalini S, Nicola M, Moore S , et al. Caspase- 2 deficiency promotes aberrant DNA-damage response and genetic instability. Cell Death and Differentation 2012, 19 (8) : 1288-1298.

34. Fava LL, Haschka MD, Villunger A. Bim vanishes in the light of a mitotic Aurora. Cell Death and Differentiation 2013, 20 (12) : 1597-1598.

35. Bric A, Miething C, Bialucha CU, Scuoppo C, Zender L, Krasnitz A , et al. Functional Identification of Tumor Suppressor Genes Through an in vivo RNA Interference Screen in a Mouse Lymphoma Model. Cancer Cell 2009, 16 (4) : 324-335.

36. Oliver TG, Meylan E, Chang GP, Xue W, Burke JR, Humpton TJ , et al. Caspase-2-mediated cleavage of Mdm2 creates a p53-induced positive feedback loop. Molecular Cell 2011, 43 (1) : 57-71.

37. O'Reilly L, Ekert P, Harvey N, Mardsen V, Cullen L, Vaux D , et al. Caspase-2 is not required for thymocyte or neuronal apoptosis even though cleavage of caspase-2 is dependent on both Apaf-1 and caspase-9. Cell Death and Differentation 2002, 9(8) : 832-841.

38. Berube C, Boucher LM, Ma W, Wakeham A, Salmena L, Hakem R , et al. Apoptosis caused by p53-induced protein with death domain (PIDD) depends on the death adapter protein RAIDD. Proceedings of the National Academy of Sciences of the United States of America 2005, 102 (40) : 14314-14320.

39. Garcia-Cao I, Garcia-Cao M, Martin-Caballero J, Cridao LM, Klatt P, Flores JM, et al . 'Super p53' mice exhibit enhanced DNA damage response, are tumor resistant and age normally. The EMBO Journal 2002, 21 (22) : 6225- 6235.

40. Elowe S, Dulla K, Uldschmid A, Li X, Dou Z, Nigg EA. Uncoupling of the spindle-checkpoint and chromosome-congression functions of BubR1. Journal of Cell Science 2010, 123 (1) : 84-94.

41. Gentleman R, Carey V, Bates D, Bolstad B, Dettling M, Dudoit S , et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biology 2004, 5(10) : R80.

21

42. Rainer J, Lelong J, Bindreither D, Mantinger C, Ploner C, Geley S, et al . Research Resource: Transcriptional Response to Glucocorticoids in Childhood Acute Lymphoblastic Leukemia. Molecular Endocrinology 2012, 26 (1) : 178- 193.

43. Bindreither D, Ecker S, Gschirr B, Kofler A, Kofler R, Rainer J. The synthetic glucocorticoids prednisolone and dexamethasone regulate the same genes in acute lymphoblastic leukemia cells. BMC Genomics 2014, 15 (1) : 662.

44. Wu Z, Irizarry RA, Gentleman R, Martinez-Murillo F, Spencer F. A Model- Based Background Adjustment for Oligonucleotide Expression Arrays. Journal of the American Statistical Association 2004, 99 (468) : 909-917.

45. Smyth GK. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology 2004, 3(3) : 1-25.

46. Benjamini Y, Hochberg Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society Series B (Methodological) 1995, 57 (1) : 289-300.

22

Figure legends

Figure 1: Loss of Raidd does not impact on DNA damage-induced tumor formation

(a) Kaplan Meier plot of tumor-free survival in response to repeated low dose

irradiation (4 x 1.75 Gy) of wt (n=8, mean=207 days), Raidd-/- (n=8, mean=190 days) and p53+/- mice (n=5, mean=115 days). (b) Kaplan Meier analysis of 3- methylcholantrene (3-MC) treated mice. Wild type (n=6, median=132 days); Raidd-/-

(n=10, mean=131 days); p53+/- (n=5, mean=79 days). Wt mice injected with vehicle only (n=3) were used as solvent controls.

Figure 2: B cell subset composition, proliferation and cell death responsiveness in premalignant Raidd-/- mice

(a) The development and the distribution of B cells in bone marrow and spleen was assessed in 4 week old Eµ-Myc transgenic mice lacking or expressing Raidd. Single cell suspensions of the organs were counted and stained with cell surface marker- specific antibodies and analyzed by flow cytometry. Bars represent means (n=3-5 per genotype) ± S.E.M. (b) Sorted premalignant B cells (pre B from bone marrow,

CD19+IgM-CD43- or immature B cells CD19+IgMlow from spleen) were analyzed for spontaneous death after 6, 24 and 48 h using Annexin-V plus 7AAD staining in a flow cytometer. Symbols represent means of n=3-5 per genotype ± S.E.M. (c)

Proliferation rates were quantified 4h after a single injection of BrdU in bone-marrow- derived pro/pre B cells (CD19+IgM-) or (CD19+IgM+) B cells from spleen. Bars represent means (n=3-5) per genotype ± S.E.M.

Figure 3: Suppression of c-Myc induced B cell lymphoma formation by

Caspase-2 does not depend on Raidd 23

(a) Lymphoma-free survival of Eµ-Myc (n=37, mean=128 days), Eµ-Myc/Raidd-/-

(n=43, mean=124 days) and Eµ-Myc/Casp-2-/- mice (n=15, mean=101 days). (b)

Flow cytometric analysis of tumor immune-phenotype. A comparison of the frequency of the different groups did not reveal significant differences between genotypes.

Figure 4: Normal cell cycle distribution and tumor cell apoptosis in Eµ-Myc tumor cells lacking Raidd

(a) Cell cycle phases G1 (black), S (dotted) and G2/M (white) of lymphoma cells were assessed ex vivo by intracellular DNA content analysis in tumor derived from Eµ-Myc

(n=21), Eµ-Myc/Raidd-/- (n=22), Eµ-Myc/Casp-2-/- (n=10) mice. (b) To define the percentage of cells in M-phase, ex vivo tumor cells were stained with an antibody specific for the phosphorylated variant of Histone H3 (pH3) and propidium iodide.

Bars represent mean values of pH3+ cells ± S.E.M. Eµ-Myc (n=17), Eµ-Myc/Raidd-/-

(n=11) Eµ-Myc/Casp-2-/- (n=5). (c) Apoptosis in situ was assessed by Sub-G1 analysis of freshly isolated lymph node tumor masses followed flow cytometric analysis (wt n=24, Raidd-/- n=22, Casp-2-/- n=10) mean values ± S.E.M. (d) Freshly isolated lymphoma cells were cultivated on feeder cells for 24h and were either left untreated (upper left graph) or exposed to increasing doses of the indicated chemo- therapeutics. Cell death was assessed by Annexin-V/7AAD staining combined with anti-CD19 to identify B cells. Symbols represent mean values, n>3-7 for each genotype ± S.E.M.

Figure 5: Caspase-2-loss leads to increased micronuclei formation and aneuploidy

(a) Representative images of micronuclei formation in SV40 MEFs stained with DAPI.

Arrows indicate micronuclei formation in wt MEFs. Scale bars = 5 µm. (b) 24

Quantification of data assessed in (a). Wt vs. Casp-2-/- (p=0.03; Students t-Test) Bars represent mean of n>4-6 for each genotype ± S.E.M. (c) Representative images of chromosome spreads using freshly isolated Eµ-Myc lymphoma cells. Metaphase spreads are showing representative euploid (left panel) or aneuploidy karyogramms ranging from low (middle panel) to high-grade (right panel). Scale bar = 5 µm. (d)

Variation of counted chromosome numbers within single tumors from Eµ-Myc (n=13) vs. Eµ-Myc/Casp-2-/- (n=10) mice (p=0.0029; Students t-Test). (e) Quantification of the variance of chromosome numbers in tumors derived from indicated genotypes. A

Chi2 test showed a significant difference between wt and Casp-2-/- (p<0.0001).

25

Peintner et al., Fig. 1 Peintner et al., Fig. 2 Peintner et al., Fig. 3 Peintner et al., Fig. 4 Peintner et al., Fig. 5 Supplemental Data:

Figure S1: Organ analysis of Eµ-Myc transgenic mice of different genotypes after tumor onset and evaluation of the functionality of the Myc/p19Arf/p53 axis.

Figure S2: Changes in transcription of p53 target genes upon loss of Caspase-2 or

Raidd in c-Myc transgenic premalignant pre-B cells.

Figure S1:

(a) White blood cell count (WBC) in peripheral blood of diseased Eµ-Myc mice.

Peripheral blood was stained with Turks solution (1:200) and counted in a hemocytometer. Data are given as WBC/µl. (b) Spleen weight in grams of mice with palpable Eµ-Myc tumors of the indicated genotypes. Numbers of animals analyzed are indicated. (c and d) Representative Western blot analysis of the tumor suppressor gene p53 and its target p19ARF in IgM+ and IgM- tumor lysates derived from Eµ-Myc (c) and Eµ-Myc/Raidd-/- (d) mice. Reprobing with GAPDH was used as a control for protein loading.

Figure S2:

(a) Relative mRNA levels of p21, Puma and Noxa in premalignant bone marrow- derived B220+CD43+IgM- pre-B cells (n=3). (b) Vulcano plots of an Affymetrix Chip assay performed using mRNA from spleen-derived B220+IgM+IgD- B cells isolated from indicated genotypes (n=3). Hits with adjusted pBH – value <0.05 were considered as significantly changed. Adjustment was performed using Benjamini

Hochberg method. Confirmative qRT-PCR analysis of hits identified in (b) using mRNA from B cells from Eµ-Myc and Eµ-Myc/Casp-2-/- (c) or Eµ-Myc/Raidd-/- (d) mice. Peintner Suppl_Figure 1 Peintner Suppl_Figure 2 Peintner Suppl_Table 1

List of used Primers

Actin fwd 5′- ACT GGG ACG ACA TGG AGAAG -3′ rev 5′- GGG GTG TTG AAG GTC TCA AA -3′ L32 fwd 5´- ATC CTG ATG CCC AAC ATC GG - 3´ rev 5´- CCA GCT GTG CTG CTC TTT CT - 3´ Fbxw10 fwd 5´- AAA GAC TCT CAC TGG GCA CG - 3´ rev 5´- AGC ACC TCC AGA GAC GAG AT - 3´ Fgd6 fwd 5´- TGT AAC CCG GAA GTG TCA GC - 3´ rev 5´- GCC TTT TTA AGC ACC CCT GC - 3´ Hist3h2a fwd 5´- CGT GCT ACT GCC CAA GAA GA - 3´ rev 5´- AAA AGT CGC ACT AAC GAC CG - 3´ Hmha1 fwd 5´- CAG GAG TTG CAG GAC TGA GA - 3´ rev 5´- CTC GCT TCT TCC GAG AGA ACA - 3´ Igf1R fwd 5´- GTG TGT GTC CTG GAT TTG GGA - 3´ rev 5´- GGC AGA AAT GCG GAG TGG AA - 3´ Lyrm7 fwd 5´- CAC GTT GCA AAT GAG CGA CA - 3´ rev 5´- AAG GGA AAG AGA GGA GCG TC - 3´ NOXA fwd 5´- CCC ACT CCT GGG AAA GTA CA - 3´ rev 5´- AAT CCC TTC AGC CCT TGA TT - 3´ p21 fwd 5´- TTG CAC TCT GGT GTC TGA GC - 3´ rev 5´- TCT GCG CTT GGA GTG ATA GA - 3´ Puma fwd 5´- CAA GAA GAG CAG CAT CGA CA - 3´ rev 5´- TAG TTG GGC TCC ATT TCT GG - 3´ RhoBTB1 fwd 5´- AAA GCC AAT GTC CTC CCT CG - 3´ rev 5´- ACG TTG GGT CGT TCG TAG TC - 3´ Slc25a13 fwd 5´- AGC CCA GAA GTT TGG TCA GG - 3´ rev 5´- CTC GGC CAA GTT GAA GGG TA - 3´ Zfp39 fwd 5´- CTG GGT ACG GGC AGA AAC TT - 3´ rev 5´- CGA CTC CTG CAT GTA CCC TC - 3´ Attached Manuscripts

5) Loss of PIDD limits NFκB activation and cytokine production but not cell survival or transformation after DNA-damage The auto-proteolysis of PIDD into the PIDD-C or the PIDD-CC fragment leads to NFκB or Caspase-2 activation via the formation of two different protein complexes. However, the role of these complexes in a living mouse remained unexplored. In this work we present a crucial role of PIDD in the activation of NFκB after genotoxic stress. Levels of cell death remained constant but interestingly the levels of cytokine production such as TNFα or to a lesser extend IL-1β was strongly abrogated after the loss of PIDD when cells face ionizing radiation. My contribution to the body of this work was the mouse work presented in Figure 7 and 8 and the establishment and conduction of the Bio-Plex cytokine profiling analysis presented in Figure 9.

95

Attached Manuscripts

Pages 96 - 107:

Bock FJ, Krumschnabel G, Manzl C, Peintner L, Tanzer MC, Hermann-Kleiter N, et al. Loss of PIDD limits NF-kappaB activation and cytokine production but not cell survival or transformation after DNA damage. Cell Death and Differentiation 2013, 20(4): 546-557. can only be accessed online via: http://www.nature.com/cdd/journal/v20/n4/full/cdd2012152a.html

96 References

REFERENCES for Introduction: 1. Alberts B. Molecular Biology of the Cell . Garland Science: New York, 2008.

2. Rakashanda S, Rana F, Rafiq S, Masood A, Amin S. Role of proteases in cancer: A Review. Biotechnology and Molecular Biology Reviews 2012, 7(4) : 90-101.

3. Berg J, Tymoczko J, Stryer L. Biochemistry . Springer, 2012.

4. McIlwain DR, Berger T, Mak TW. Caspase Functions in Cell Death and Disease. Cold Spring Harbor Perspectives in Biology 2013, 5(4): 1-29.

5. Riedl SJ, Shi Y. Molecular mechanisms of caspase regulation during apoptosis. Nature Reviews Molecular Cell Biology 2004, 5(11) : 897-907.

6. Green DR. Means to an End: Apoptosis and Other Cell Death Mechanisms . Cold Spring Harbor Laboratory Press, 2011.

7. Garcia-Calvo M, Peterson EP, Leiting B, Ruel R, Nicholson DW, Thornberry NA. Inhibition of Human Caspases by Peptide-based and Macromolecular Inhibitors. Journal of Biological Chemistry 1998, 273 (49) : 32608-32613.

8. Ekert P, Silke J, Vaux D. Caspase inhibitors. Cell Death and Differentation 1999, 6(11) : 1081-1086.

9. Wilson NS, Dixit V, Ashkenazi A. Death receptor signal transducers: nodes of coordination in immune signaling networks. Nature Immunology 2009, 10 (4) : 348-355.

10. Giam M, Huang D, Bouillet P. BH3-only proteins and their roles in programmed cell death. Oncogene 2008, 27 (S1) : S128-S136.

11. Degterev A, Yuan J. Expansion and evolution of cell death programmes. Nature Reviews Molecular Cell Biology 2008, 9(5) : 378-390.

12. Strasser A, Cory S, Adams JM. Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases. The EMBO Journal 2011, 30 (18) : 3667-3683.

108

References

13. Kumar S, Kinoshita M, Noda M, Copeland NG, Jenkins NA. Induction of apoptosis by the mouse Nedd2 gene, which encodes a protein similar to the product of the Caenorhabditis elegans cell death gene ced-3 and the mammalian IL-1 beta-converting enzyme. Genes & Development 1994, 8(14) : 1613-1626.

14. Chew SK, Akdemir F, Chen P, Lu W-J, Mills K, Daish T , et al. The Apical Caspase dronc Governs Programmed and Unprogrammed Cell Death in Drosophila. Developmental Cell 2004, 7(6) : 897-907.

15. Baliga BC, Read SH, Kumar S. The biochemical mechanism of caspase-2 activation. Cell Death and Differentiation 2004, 11 (11) : 1234-1241.

16. Jiang X, Wang X. Cytochrome c Promotes Caspase-9 Activation by Inducing Nucleotide Binding to Apaf-1. Journal of Biological Chemistry 2000, 275 (40) : 31199-31203.

17. Tu S, McStay GP, Boucher L-M, Mak T, Beere HM, Green DR. In situ trapping of activated initiator caspases reveals a role for caspase-2 in heat shock- induced apoptosis. Nature Cell Biology 2006, 8(1) : 72-77.

18. Tang J, Xie W, Yang X. Association of caspase-2 with the promyelocytic leukemia protein nuclear bodies. Cancer Biology & Therapy 2005, 4(6) : 645- 649.

19. Cheung H, Lynn K, Liston P, Korneluk R. Involvement of caspase-2 and caspase-9 in endoplasmic reticulum stress-induced apoptosis: A role for the IAPs. Experimental Cell Research 2006, 312 (12) : 2347-2357.

20. Ho LH, Read SH, Dorstyn L, Lambrusco L, Kumar S. Caspase-2 is required for cell death induced by cytoskeletal disruption. Oncogene 2008, 27 (24) : 3393-3404.

21. Wang Q, Maniati M, Jabado O, Pavlaki M, Troy CM, Greene LA , et al. RAIDD is required for apoptosis of PC12 cells and sympathetic neurons induced by trophic factor withdrawal. Cell Death and Differentiation 2006, 13 (1) : 75-83.

22. Shalini S, Dorstyn L, Wilson C, Puccini J, Ho L, Kumar S. Impaired antioxidant defence and accumulation of oxidative stress in caspase-2-deficient mice. Cell Death and Differentiation 2012, 19 (8) : 1370-1380.

109

References

23. Imre G, Heering J, Takeda AN, Husmann M, Thiede B, zu Heringdorf DM , et al. Caspase-2 is an initiator caspase responsible for pore-forming toxin- mediated apoptosis. The EMBO Journal 2012, 31 (11) : 2615-2628.

24. Castedo M, Perfettini J-L, Roumier T, Valent A, Raslova H, Yakushijin K , et al. Mitotic catastrophe constitutes a special case of apoptosis whose suppression entails aneuploidy. Oncogene 2004, 23 (25) : 4362-4370.

25. Kumar S, Kinoshita M, Dorstyn L, Noda M. Origin, expression and possible functions of the two alternatively spliced forms of the mouse Nedd2 mRNA. Cell Death and Differentation 1997, 4(5) : 378-387.

26. Droin N, Beauchemin M, Solary E, Bertrand R. Identification of a caspase-2 isoform that behaves as an endogenous inhibitor of the caspase cascade. Cancer Research 2000, 60: 7039-7047.

27. Droin N, Rebe C, Bichat F, Hammann A, Bertrand R, Solary E. Modulation of apoptosis by procaspase-2 short isoform: selective inhibition of chromatin condensation, externalization. Oncogene 2001, 20 (2) : 260-269.

28. Bergeron L, Perez GI, Macdonald G. Defects in regulation of apoptosis in caspase-2-deficient mice. Genes & Development 1998, 12: 1304-1314.

29. O'Reilly L, Ekert P, Harvey N, Mardsen V, Cullen L, Vaux D , et al. Caspase-2 is not required for thymocyte or neuronal apoptosis even though cleavage of caspase-2 is dependent on both Apaf-1 and caspase-9. Cell Death and Differentation 2002, 9(8) : 832-841.

30. Lavrik IN, Golks A, Baumann S, Krammer PH. Caspase-2 is activated at the CD95 death-inducing signaling complex in the course of CD95-induced apoptosis. Blood 2006, 108 (2) : 559-565.

31. Olsson M, Vakifahmetoglu H, Abruzzo PM, Hogstrand K, Grandien A, Zhivotovsky B. DISC-mediated activation of caspase-2 in DNA damage- induced apoptosis. Oncogene 2009, 28 (18) : 1949-1959.

32. Bonzon C, Bouchier-Hayes L, Pagliari LJ, Green DR, Newmeyer DD. Caspase-2–induced Apoptosis Requires Bid Cleavage: A Physiological Role for Bid in Heat Shock–induced Death. Molecular Biology of the Cell 2006, 17 (5) : 2150-2157.

110

References

33. Pozueta J, Lefort R, Ribe EM, Troy CM, Arancio O, Shelanski M. Caspase-2 is required for dendritic spine and behavioural alterations in J20 APP transgenic mice. Nature Communications 2013, 4: 1939.

34. Mancini M, Machamer CE, Roy S, Nicholson DW, Thornberry NA, Casciola- Rosen LA , et al. Caspase-2 Is Localized at the Golgi Complex and Cleaves Golgin-160 during Apoptosis. The Journal of Cell Biology 2000, 149 (3) : 603- 612.

35. Loo Gv, Saelens X, Matthijssens F, Schotte P, Beyaert R, Declercq W , et al. Caspases are not localized in mitochondria during life or death. Cell Death and Differentation 2002, 9(11) : 1207-1211.

36. Guha M, Xia F, Raskett CM, Altieri DC. Caspase 2-mediated tumor suppression involves survivin gene silencing. Oncogene 2011, 29 (9) : 1280- 1292.

37. Van de Craen M, Declercq W, Van den Brande I, Fiers W, Vandenabeele P. The proteolytic procaspase activation network: an in vitro analysis. Cell Death and Differentation 1999, 6(11) : 1117-1124.

38. Wagner KW, Engels IH, Deveraux QL. Caspase-2 Can Function Upstream of Bid Cleavage in the TRAIL Apoptosis Pathway. Journal of Biological Chemistry 2004, 279 (33) : 35047-35052.

39. Robertson JD, Enoksson M, Suomela M, Zhivotovsky B, Orrenius S. Caspase- 2 Acts Upstream of Mitochondria to Promote Cytochromec Release during Etoposide-induced Apoptosis. Journal of Biological Chemistry 2002, 277 (33) : 29803-29809.

40. Robertson JD, Gogvadze V, Kropotov A, Vakifahmetoglu H, Zhivotovsky B, Orrenius S. Processed caspase ‐2 can induce mitochondria ‐mediated apoptosis independently of its enzymatic activity. EMBO Reports 2004, 5(6) : 643-648.

41. Baptiste-Okoh N, Barsotti A, Prives C. Caspase-2 is both required for p53- mediated apoptosis and downregulated by p53 in a p21 dependent manner. Cell Cycle 2008, 7: 1133-1138.

42. Sidi S, Sanda T, Kennedy RD, Hagen AT, Jette CA, Hoffmans R , et al. Chk1 suppresses a caspase-2 apoptotic response to DNA damage that bypasses p53, Bcl-2, and caspase-3. Cell 2008, 133 (5) : 864-877.

111

References

43. Mendelsohn AR, Hamer JD, Wang ZB, Brent R. Cyclin D3 activates Caspase 2, connecting cell proliferation with cell death. Proceedings of the National Academy of Sciences of the United States of America 2002, 99 (10) : 6871- 6876.

44. Andersen JL, Johnson CE, Freel CD, Parrish AB, Day JL, Buchakjian MR , et al. Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase-2. The EMBO Journal 2009, 28 (20) : 3216-3227.

45. Ho LH, Taylor R, Dorstyn L, Cakouros D, Bouillet P, Kumar S. A tumor suppressor function for caspase-2. Proceedings of the National Academy of Sciences of the United States of America 2009, 106 (13) : 5336-5341.

46. Sohn D, Budach W, Janicke RU. Caspase-2 is required for DNA damage- induced expression of the CDK inhibitor p21(WAF1/CIP1). Cell Death and Differentation 2011, 18 (10) : 1664-1674.

47. Dorstyn L, Puccini J, Wilson CH, Shalini S, Nicola M, Moore S , et al. Caspase- 2 deficiency promotes aberrant DNA-damage response and genetic instability. Cell Death and Differentation 2012, 19 (8) : 1288-1298.

48. Oliver TG, Meylan E, Chang GP, Xue W, Burke JR, Humpton TJ , et al. Caspase-2-mediated cleavage of Mdm2 creates a p53-induced positive feedback loop. Molecular Cell 2011, 43 (1) : 57-71.

49. Terry MR, Arya R, Mukhopadhyay A, Berrett KC, Clair PM, Witt B , et al. Caspase-2 impacts lung tumorigenesis and chemotherapy response in vivo. Cell Death and Differentation 2014.

50. Logette E, Schuepbach-Mallepell S, Eckert MJ, Leo XH, Jaccard B, Manzl C , et al. PIDD orchestrates translesion DNA synthesis in response to UV irradiation. Cell Death and Differentiation 2011, 18 (6) : 1036-1045.

51. Bouchier-Hayes L, Green DR. Caspase-2: the orphan caspase. Cell Death and Differentiation 2012, 19 (1) : 51-57.

52. Luthi AU, Martin SJ. The CASBAH: a searchable database of caspase substrates. Cell Death and Differentiation 2007, 14 (4) : 641-650.

53. Luo X, Budihardjo I, Zou H, Slaughter C, Wang X. Bid, a Bcl2 Interacting Protein, Mediates Cytochrome c Release from Mitochondria in Response to Activation of Cell Surface Death Receptors. Cell 1998, 94 (4) : 481-490.

112

References

54. Vakifahmetoglu-Norberg H, Norberg E, Perdomo AB, Olsson M, Ciccosanti F, Orrenius S , et al. Caspase-2 promotes cytoskeleton protein degradation during apoptotic cell death. Cell Death and Disease 2013, 4: e940.

55. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011, 144 (5) : 646-674.

56. Kumar S, White D, Takai S, Tuczynowicz S, Juttner C, Hughes T. Apoptosis regulatory gene NEDD2 maps to human chromosome segment 7q34-35, a region fequently affected in haematological neoplasms. Human Genetics 1995, 95 (6) : 641-644.

57. Estrov Z, Thall PF, Talpaz M, Estey EH, Kantarjian HM, Andreeff M , et al. Caspase 2 and Caspase 3 Protein Levels as Predictors of Survival in Acute Myelogenous Leukemia. Blood 1998, 92 (9) : 3090-3097.

58. Hofmann WK, de Vos S, Tsukasaki K, Wachsman W, Pinkus G, Said J , et al. Altered apoptosis pathways in mantle cell lymphoma detected by oligonucleotide microarray. Blood 2001, 98 (3) : 787-794.

59. Holleman A, Boer MLd, Kazemier KM, Beverloo HB, von Bergh ARM, Janka- Schaub GE , et al. Decreased PARP and procaspase-2 protein levels are associated with cellular drug resistance in childhood acute lymphoblastic leukemia. Blood 2005, 106 (5) : 1817-1823.

60. Olsson M, Zhivotovsky B. Caspases and cancer. Cell Death and Differentiation 2011, 18 (9) : 1441-1449.

61. Kim MS, Kim HS, Jeong EG, Soung YH, Yoo NJ, Lee SH. Somatic mutations of caspase-2 gene in gastric and colorectal cancers. Pathology - Research and Practice 2011, 207 (10) : 640-644.

62. Ren K, Lu J, Porollo A, Du C. Tumor-suppressing Function of Caspase-2 Requires Catalytic Site Cys-320 and Site Ser-139 in Mice. Journal of Biological Chemistry 2012, 287 (18) : 14792-14802.

63. Shin S, Lee Y, Kim W, Ko H, Choi H, Kim K. Caspase ‐2 primes cancer cells for TRAIL ‐mediated apoptosis by processing procaspase ‐8. The EMBO Journal 2005, 24 (20) : 3532-3542.

113

References

64. Adams J, Harris A, Pinkert C, Corcoran L, Alexander W, Cory S , et al. The c- myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature 1985, 318: 533-538.

65. Ren K, Lu J, Porollo A, Du C. Tumor-suppressing function of caspase-2 requires catalytic site Cys-320 and site Ser-139 in mice. The Journal of Biological Chemistry 2012, 287 (18) : 14792-14802.

66. Puccini J, Dorstyn L, Kumar S. Caspase-2 as a tumour suppressor. Cell Death and Differentiation 2013, 20 (9) : 1133-1139.

67. Parsons MJ, McCormick L, Janke L, Howard A, Bouchier-Hayes L, Green DR. Genetic deletion of caspase-2 accelerates MMTV/c-neu-driven mammary carcinogenesis in mice. Cell Death and Differentiation 2013, 20 (9) : 1174- 1182.

68. Dorstyn L, Puccini J, Nikolic A, Shalini S, Wilson CH, Norris MD , et al. An unexpected role for caspase-2 in neuroblastoma. Cell Death and Disease 2014, 5: e1383.

69. Manzl C, Peintner L, Krumschnabel G, Bock F, Labi V, Drach M , et al. PIDDosome-independent tumor suppression by Caspase-2. Cell Death and Differentiation 2012, 19 (10) : 1722-1732.

70. Puccini J, Shalini S, Voss AK, Gatei M, Wilson CH, Hiwase DK , et al. Loss of caspase-2 augments lymphomagenesis and enhances genomic instability in Atm-deficient mice. Proceedings of the National Academy of Sciences of the United States of America 2013. 110 (49) :19920-19925

71. Shi M, Vivian CJ, Lee KJ, Ge C, Morotomi-Yano K, Manzl C , et al. DNA-PKcs- PIDDosome: a nuclear caspase-2-activating complex with role in G2/M checkpoint maintenance. Cell 2009, 136 (3) : 508-520.

72. Crasta K, Ganem NJ, Dagher R, Lantermann AB, Ivanova EV, Pan Y , et al. DNA breaks and chromosome pulverization from errors in mitosis. Nature 2012, 482 (7383) : 53-58.

73. Foijer F, Xie SZ, Simon JE, Bakker PL, Conte N, Davis SH , et al. Chromosome instability induced by Mps1 and p53 mutation generates aggressive lymphomas exhibiting aneuploidy-induced stress. Proceedings of the National Academy of Sciences 2014, 111 (37) : 13427-13432.

114

References

74. Gitenay D, Lallet-Daher H, Bernard D. Caspase-2 regulates oncogene- induced senescence. Oncotarget 2014, 5(14) : 5845-5847.

75. Duan H, Dixit V. RAIDD is a new "death" adaptor molecule. Nature 1997, 238: 86-89.

76. Ahmad M, Srinivasula SM, Wang L, Talanian RV, Litwack G, Fernandes- Alnemri T , et al. CRADD, a Novel Human Apoptotic Adaptor Molecule for Caspase-2, and FasL/Tumor Necrosis Factor Receptor-interacting Protein RIP. Cancer Research 1997, 57 (4) : 615-619.

77. Won M, Islas-Trejo A, Medrano J. Structural characterization of the mouse high growth deletion and discovery of a novel fusion transcript between suppressor of cytokine signaling-2 (Socs-2) and viral encoded semaphorin receptor (Plexin C1). Gene 2002, 299 (1) : 153-163.

78. Ramos AM, Pita RH, Malek M, Lopes PS, Guimarães SEF, Rothschild MF. Analysis of the mouse high-growth region in pigs. Journal of Animal Breeding and Genetics 2009, 126 (5) : 404-412.

79. Smith J, Paton IR, Horvat S, Medrano JF, Burt DW. Mapping the RAIDD gene of chicken (Gallus gallus): identification of a region homologous to the mouse high-growth region. Mammalian Genome 2000, 11 (8) : 706-709.

80. Horvat S, Medrano JF. Lack of Socs2 Expression Causes the High-Growth Phenotype in Mice. Genomics 2001, 72 (2) : 209-212.

81. Felmer R, Horvat S, Clinton M, Clark A. Overexpression of Raidd cDNA inhibits differentation of mourse preadipocytes. Cell Proliferation 2002, 36: 45- 54.

82. Motlan H, McWhir J, Horvat S. In situ analysis of Raidd-beta-galactosidase fusion gene expression in transgenic mouse midgestation embryos. Transgenic Res 2005, 14 (1) : 27-40.

83. Berube C, Boucher LM, Ma W, Wakeham A, Salmena L, Hakem R , et al. Apoptosis caused by p53-induced protein with death domain (PIDD) depends on the death adapter protein RAIDD. Proceedings of the National Academy of Sciences of the United States of America 2005, 102 (40) : 14314-14320.

84. Lin Q, Liu Y, Moore DJ, Elizer SK, Veach RA, Hawiger J , et al. Cutting edge: the "death" adaptor CRADD/RAIDD targets BCL10 and suppresses agonist-

115

References

induced cytokine expression in T lymphocytes. Journal of Immunology 2012, 188 (6) : 2493-2497.

85. Turvey SE, Durandy A, Fischer A, Fung S-Y, Geha RS, Gewies A , et al. The CARD11-BCL10-MALT1 (CBM) signalosome complex: Stepping into the limelight of human primary immunodeficiency. Journal of Allergy and Clinical Immunology 2014, 134 (2) : 276-284.

86. Blonska M, Lin X. NF-κB signaling pathways regulated by CARMA family of scaffold proteins. Cell Research 2011, 21 (1) : 55-70.

87. Qiao H, Liu Y, Veach RA, Wylezinski L, Hawiger J. The Adaptor CRADD/RAIDD Controls Activation of Endothelial Cells by Proinflammatory Stimuli. Journal of Biological Chemistry 2014, 289 (32) : 21973-21983.

88. Micheau O, Tschopp J. Induction of TNF Receptor I-Mediated Apoptosis via Two Sequential Signaling Complexes. Cell 2003, 114 (2) : 181-190.

89. Song X, Fu G, Yang X, Jiang Z, Wang Y, Zhou G. Protein expression profiling of breast cancer cells by dissociable antibody microarray (DAMA) staining. Molecular and cellular Proteomics 2008, 7(1) : 163-168.

90. Yang C, Hornicek FJ, Wood KB, Schwab JH, Mankin H, Duan Z. RAIDD expression is impaired in multidrug resistant osteosarcoma cell lines. Cancer Chemotherapy and Pharmacology 2009, 64 (3) : 607-614.

91. Lo Muzio L, Sartini D, Santarelli A, Rocchetti R, Morganti S, Pozzi V , et al. Expression and prognostic significance of apoptotic genes in oral squamous cell carcinoma. Molecular Carcinogenesis 2014, 53 (4) : 264-271.

92. Lin Y, Ma W, Benchimol S. Pidd, a new death-domain containing protein, is induced by p53 and promotes apoptosis. Nature Genetics 2000, 26: 124-126.

93. Telliez J-B, Bean KM, Lin L-L. LRDD, a novel leucine rich repeat and death domain containing protein. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 2000, 1478 (2) : 280-288.

94. Pick R, Badura S, Bosser S, Zornig M. Upon intracellular processing, the C- terminal death domain-containing fragment of the p53-inducible PIDD/LRDD protein translocates to the nucleoli and interacts with nucleolin. Biochemical and biophysical research communications 2006, 349 (4) : 1329-1338.

116

References

95. Cuenin S, Tinel A, Janssens S, Tschopp J. p53-induced protein with a death domain (PIDD) isoforms differentially activate nuclear factor-kappaB and caspase-2 in response to genotoxic stress. Oncogene 2008, 27 (3) : 387-396.

96. Tinel A, Tschopp J. The PIDDosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress. Science 2004, 304 (5672) : 843-846.

97. Janssens S, Tinel A, Lippens S, Tschopp J. PIDD mediates NF-kappaB activation in response to DNA damage. Cell 2005, 123 (6) : 1079-1092.

98. Bock FJ, Peintner L, Tanzer M, Manzl C, Villunger A. P53-induced protein with a death domain (PIDD): master of puppets. Oncogene 2012, 31 (45) : 4733- 4739.

99. Tinel A, Janssens S, Lippens S, Cuenin S, Logette E, Jaccard B , et al. Autoproteolysis of PIDD marks the bifurcation between pro ‐death caspase ‐2 and pro ‐survival NF ‐κB pathway. The EMBO Journal 2007, 26 (1) : 197-208.

100. Tinel A, Eckert MJ, Logette E, Lippens S, Janssens S, Jaccard B , et al. Regulation of PIDD auto-proteolysis and activity by the molecular chaperone Hsp90. Cell Death and Differentiation 2011, 18 (3) : 506-515.

101. Irmler M, Hofmann K, Vaux D, Tschopp J. Direct physical interaction between the Caenorhabditis elegans 'death proteins' CED-3 and CED-4. FEBS Letters 1997, 406 (1-2) : 198-190.

102. Zhou P, Chou J, Olea R, Yuan J, Wagner G. Solution structure of Apaf-1 CARD and its interaction with caspase-9 CARD- a structural basis for specific adaptor-caspase interaction. Proceedings of the National Academy of Sciences of the United States of America 1999, 96 (20) : 11265-11270.

103. Chou J, Matuso H, Duan H, Wagner G. Solution structure of the RAIDD CARD and model for CARD-CARD interaction in caspase-2 and caspase-9 recruitment. Cell 1998, 94: 171-180.

104. Park HH, Logette E, Raunser S, Cuenin S, Walz T, Tschopp J , et al. Death domain assembly mechanism revealed by crystal structure of the oligomeric PIDDosome core complex. Cell 2007, 128 (3) : 533-546.

105. Park HH, Wu H. Crystal structure of RAIDD death domain implicates potential mechanism of PIDDosome assembly. Journal of Molecular Biology 2006, 357 (2) : 358-364. 117

References

106. Jabado O, Wang Q, Rideout HJ, Yeasmin M, Guo KX, Vekrellis K , et al. RAIDD aggregation facilitates apoptotic death of PC12 cells and sympathetic neurons. Cell Death and Differentiation 2004, 11 (6) : 618-630.

107. Niizuma K, Endo H, Nito C, Myer DJ, Kim GS, Chan PH. The PIDDosome mediates delayed death of hippocampal CA1 neurons after transient global cerebral ischemia in rats. Proceedings of the National Academy of Sciences of the United States of America 2008, 105 (42) : 16368-16373.

108. Ando K, Kernan JL, Liu PH, Sanda T, Logette E, Tschopp J , et al. PIDD death-domain phosphorylation by ATM controls prodeath versus prosurvival PIDDosome signaling. Molecular Cell 2012, 47 (5) : 681-693.

109. Jang TH, Bae JY, Park OK, Kim JH, Cho KH, Jeon JH , et al. Identification and analysis of dominant negative mutants of RAIDD and PIDD. Biochimica et biophysica acta 2010, 1804 (7) : 1557-1563.

110. Jang TH, Zheng C, Wu H, Jeon JH, Park HH. In vitro reconstitution of the interactions in the PIDDosome. Apoptosis : An international Journal on Programmed Cell Death 2010, 15 (12) : 1444-1452.

111. Tinel A, Janssens S, Lippens S, Cuenin S, Logette E, Jaccard B , et al. Autoproteolysis of PIDD marks the bifurcation between pro-death caspase-2 and pro-survival NF-kB pathway. The EMBO Journal 2007, 26: 197-208.

112. Vakifahmetoglu H, Olsson M, Orrenius S, Zhivotovsky B. Functional connection between p53 and caspase-2 is essential for apoptosis induced by DNA damage. Oncogene 2006, 25 (41) : 5683-5692.

113. Manzl C, Krumschnabel G, Bock F, Sohm B, Labi V, Baumgartner F , et al. Caspase-2 activation in the absence of PIDDosome formation. The Journal of Cell Biology 2009, 185 (2) : 291-303.

114. Mahoney D, Lefebvre C, Allan K, Brun J, Sanaei C, Baird S , et al. Virus-Tumor Interactome Screen Reveals ER Stress Response Can Reprogram Resistant Cancers for Oncolytic Virus-Triggered Caspase-2 Cell Death. Cancer Cell 2011, 20 (4) : 443-456.

115. Hasegawa H, Yamada Y, Tsukasaki K, Mori N, Tsuruda K, Sasaki D , et al. LBH589, a deacetylase inhibitor, induces apoptosis in adult T-cell leukemia/lymphoma cells via activation of a novel RAIDD-caspase-2 pathway. Leukemia 2011, 25 (4) : 575-587.

118

References

116. Ribe EM, Jean YY, Goldstein RL, Manzl C, Stefanis L, Villunger A , et al. Neuronal caspase 2 activity and function requires RAIDD, but not PIDD. Biochemical Journal 2012, 444 (3) : 591-599.

117. Jean YY, Ribe EM, Pero ME, Moskalenko M, Iqbal Z, Marks LJ , et al. Caspase-2 is essential for c-Jun transcriptional activation and Bim induction in neuron death. Biochemical Journal 2013, 455 (1) : 15-25.

118. Matheu A, Maraver A, Klatt P, Flores I, Garcia-Cao I, Borras C , et al. Delayed ageing through damage protection by the Arf/p53 pathway. Nature 2007, 448 (7151) : 375-379.

119. Garcia-Cao I, Garcia-Cao M, Martin-Caballero J, Cridao LM, Klatt P, Flores JM, et al . 'Super p53' mice exhibit enhanced DNA damage response, are tumor resistant and age normally. The EMBO Journal 2002, 21 (22) : 6225- 6235.

120. Manzl C, Fava LL, Krumschnabel G, Peintner L, Tanzer MC, Soratroi C , et al. Death of p53-defective cells triggered by forced mitotic entry in the presence of DNA damage is not uniquely dependent on Caspase-2 or the PIDDosome. Cell Death and Disease 2013, 4: e942.

121. Bock FJ, Krumschnabel G, Manzl C, Peintner L, Tanzer MC, Hermann-Kleiter N, et al. Loss of PIDD limits NF-kappaB activation and cytokine production but not cell survival or transformation after DNA damage. Cell Death and Differentiation 2013, 20 (4) : 546-557.

122. Oliver TG, Mercer KL, Sayles LC, Burke JR, Mendus D, Lovejoy KS , et al. Chronic cisplatin treatment promotes enhanced damage repair and tumor progression in a mouse model of lung cancer. Genes & Development 2010, 24 (8) : 837-852.

119

Supplement

SUPPLEMENT

Abbreviations 293T Human embryonic kidney cell line expressing SV40 large T antigen 3-MC 3-Methylcholanthrene 5-FU 5-Fluoruracil 5‘UTR 5‘ untranslated region AD Alzheimers disease ALL Acute lymphoblastic leukemia AML Acute myeloblastic leukemia AP-1 Activator protein 1 Apaf1 Apoptotic peptidase activating factor 1 Atm Ataxia telangiectasia mutated Atr Ataxia telangiectasia and Rad3-related protein Bak Bcl-2 homologous antagonist/killer Bax Bcl-2-associated X protein Bcl-10 B cell leukemia/lymphoma 10 BH3-only Proteins containing the Bcl-2 homology domain 3 Bim (Bcl2L11) Bcl-2 interacting mediator of cell death (Bcl-2-like 11) CA1 neurons Human pyramidal hippocampus cells CARD Caspase recruitment domain CARMA-1 CARD membrane-associated guanylate kinase 1 CD Cluster of Differentiation Cdc25 Cell division cycle 25 cDNA complimentary DNA Chk Checkpoint kinase cJun Part of the transcription factor AP-1 Cradd Caspase-2 and RIPK1 domain containing adaptor with a Death Domain CrmA Cytokine response modifier A DAPI 4´,6-diamidino-2-phenylindole DD Death domain DISC Death inducing signalling complex DNA Desoxyribunucleid acid Eµ Immunoglobulin heavy chain E1A Adenovirus early region 1A eIF4E Eukaryotic translation initiation factor 4E Fadd Fas-associated protein with a Death Domain Hsp90 Heat shock protein 90 Iap Inhibitor of apoptosis IgD Immunoglobulin D Igf-1 Insulin-like growth factor 1 IgM Immunoglobulin M IKK Inhibitor of NFκB kinase 120

Supplement

Ire-1α Inositol-requiring enzyme 1α Jurkat Immortalized human T lymphocyte cell line Jnk/Mapk8 Mitogen-activated protein kinase 8 Kb Kilobases Madd Map kinase-activating death domain protein Map Microtubule associated protein Max Myc associated factor X Mdm-2 Mouse double minute 2 MEF Mouse embryo fibroblasts MMTV/c neu Mouse mammary tumor virus MOMP Mitochondrial outer membrane permeabilisation Msh-2 Melanocyte stimulating hormone 2 Myc Myelocytomatosis Nedd Neuronal expressed developmentally down regulated Nemo NFκB essential modulator NFκB Nuclear factor kappa B Ngf Nerve growth factor p21/Waf1/Cip1 CDK-inhibitor 1 p63 Transformation related protein 63 PC12 neurons cell line from rat adrenal medulla pheochromocytoma Pcna Proliferating cell nuclear antigen PIDD p53 induced protein with a death domain Pkck-2 Protein kinase casein kinase 2 PML-NBs Promyelocytic leukemia - nuclear bodies PP1 Protein phosphatase 1 RAIDD RIP associated Ich-1/CED homologous protein with a Death Domain Ras Rat sarcoma Rb Retinoblastoma protein RhoA Ras homolog gene family, member A Rip Receptor-interacting protein RNA Ribonucleic acid Rock-2 Rho-associated coiled-coil containing protein kinase 2 Sumo-1 Small ubiquitin-related modifier 1 SV40 Simian Virus 40 Socs-2 Suppressor of cytokine signalling 2 TALL Adult T-Cell leukemia Tp53 Tumor protein 53 TRAIL Tumor necrosis factor related apoptosis inducing ligand U2OS human osteosarcoma cell line ZU-5 Domain named after ZO-1 and Unc-5

121

Supplement

Curriculum Vitae

Personal information Name: Lukas Peintner, MSc Residency: 29-31/68, Anichstraße, 6020, Innsbruck, Austria Mobile: +43 / 664 / 641 45 64 Fax: +43 / 512 / 9003/ 73960 E-mail : [email protected] Nationality: Austria Date of birth: May 8th, 1987 Gender: male

Education and training Since November 2010 PhD studies in the group of Prof. Dr. Andreas Villunger, Division of Developmental Immunology, Medical University of Innsbruck and the group of Prof. Dr. Sharad Kumar, University of South Australia, Adelaide, Australia within the Molecular Cell biology and Oncology (MCBO) graduate school funded by the FWF and an Austrian Academy of Sciences DOC scholarship. Title: Investigating the role of the PIDDosome in tumor suppression

10/2009 – 11/2010 Master Thesis in the group of Prof. Dr. Andreas Villunger, Division of Developmental Immunology, Medical University of Innsbruck. Title: The role of the PIDDosome in tumor development

10/2005 – 10/2008 Studies in Biology, Leopold Franzens University, Innsbruck, Austria, with special focus on molecular biology and microbiology

122

Supplement

Work experience and Internships 08-2013 – 01/2014 Visiting Student in the group of Prof. Dr. Sharad Kumar, University of South Australia, Adelaide, Australia 08/2009 – 09/2009 Internship in the group of Prof. Dr. Georg Dechant, Medical University of Innsbruck, Austria 07/2004 – 08/2004 Internship in the group of Prof. Dr. Georg Kraft, Medical University of Innsbruck, Austria 07/2002 Internship in the research and development facility, Adler Lacke, Schwaz, Austria

Various Social skills: In my graduate studies I worked as a tutor for undergraduate biology students in microbiological and molecular biology lab courses. During graduate and postgraduate education it was my daily routine to train undergraduate students in basic laboratory skills. Since I was dismissed from the compulsory community service in 2006 I am working voluntarily as a paramedic for the Austrian Red Cross. Organizational skills: From 2009 - 2013 I was the head of the youth group at the local Austrian Red Cross base in Imst, Austria. I was responsible for the administration of all courses and organizing first aid exams. Since 2012 I am the elected representative for the students of the MCBO (graduate student program at the Medical University Innsbruck). For the MCBO Science Day 2013 and 2014 I was inviting and hosting Seamus Martin (Trinity College, Dublin), Claudia Bagni (KU Leuven, Belgium) and Bill Earnshaw (University of Edinburgh, Scotland). 2013 I got elected representative for all PhD students at the Medical University Innsbruck and me and my team are organizing regular meetings and poster sessions, including international lecturers (Jürgen Knoblich, IMBA, Vienna and Anne Spang, Biozentrum, Basel). Further information on www.mcbo.at and www.i-med.ac.at/oeh/phd.

123

Supplement

Computer skills: Familiar with Microsoft and Apple operating systems, Microsoft Office 2010 (European Computer Driving License), Statview, Prism, FlowJo, Adobe Photoshop, Adobe Illustrator, FreeCAD and Arduino;

Artistic skills: I play the violin and guitar. In 2006 I joined the University Orchestra Innsbruck playing violin. Concert trips were heading to Italy, Germany, France and Switzerland.

124

Supplement

List of Publications

Research papers

Peintner L, Dorstyn L, Kumar S, Aneichyk T, Villunger A and Manzl C. Caspase-2- dependent tumor suppression does not depend on the scaffold protein RAIDD. (under revision in Cell Death and Disease)

Manzl C, Fava LL, Krumschnabel G, Peintner L, Tanzer MC, Soratroi C, Bock FJ, Schuler F, Luef B, Geley S and Villunger A, 2014. Death of p53-defective cells triggered by forced mitotic entry in the presence of DNA-damage is not uniquely dependent on Caspase-2 or the PIDDosome. Cell Death and Disease, 2013 Dec 5;4:e942. doi: 10.1038/cddis.2013.470.

Lindner S, Wissler M, Gruender A, Aumann K, Ottina E, Peintner L, Borner C, Charvet C, Villunger A, Pahl H, Maurer C, 2013.Increased leukocyte survival and accelerated onset of lymphoma in absence of MCL-1 S159-phosphorylation. Oncogene, 2013 Nov 11. doi: 10.1038/onc.2013.469.

Manzl C, Baumgartner F, Peintner L, Schuler F, Villunger A, 2013. Possible pitfalls investigating cell death responses in genetically engineered mouse models and derived cell lines. METHODS. 2013. doi:pii: S1046-2023(13)00031-5. 10.1016/j.meth.2013.02.012

Bock FJ, Krumschnabel G, Manzl C, Peintner L, Tanzer MC, Hermann-Kleiter N, Baier G, Llancuna L, Yelamos J, Villunger A, 2012. Loss of PIDD limits NFκB activation and cytokine production but not cell survival or transformation after DNA- damage. Cell Death and Differentiation. 2013. doi: 10.1038/cdd.2012.152.

Manzl C, Peintner L, Krumschnabel G, Bock FJ, Labi V, Drach M, Newbold A, Johnstone R, Villunger A, 2012. PIDDosome-independent tumor suppression by Caspase-2. Cell Death and Differentiation. 2012. doi: 10.1038/cdd.2012.54.

Reviews and Commentaries

Bock FJ, Peintner L, Tanzer M, Manzl C, Villunger A, 2012. P53- induced Protein with a Death Domain (PIDD) Master of Puppets? Oncogene. 2012 doi: 10.1038/onc.2011.639.

125

Supplement

Conference attendances, fellowships and awards

Conference attendances:

2nd CMBI meeting, Sept. 24th-25th 2010, Igls, Austria

6th Swiss Apoptosis Meeting, Sept. 30th - Oct. 1st 2010, Bern, Switzerland

1st PhD Meeting, Vienna, Dec. 10th-12th 2010, Vienna, Austria

EMBO Workshop “Cell death and disease”, March 10th-14th 2011, Obergurgl, Austria

7th Tuscany Retreat on Cancer Research, August 6th-13th 2011, Palazzo di Piero Chiusi, Italy (Shorttalk)

FWF - SFB21 Meeting, January 25th-27th 2012, Obergurgl, Austria (Shorttalk)

8th EWCD meeting, June 3rd-8th, 2012, Monetier les Bians, France (Shorttalk)

EMBO Workshop “Cycling to death”, Jan. 23th-27th 2013, Obergurgl, Austria

OEAW Fellowship Workshop, February 22nd-23rd 2013, Vienna, Austria

3rd Adelaide Cell and Developmental Biology Meeting, Nov. 19th 2013, Adelaide, Australia

6th Barossa Meeting “Cell signaling in the omics era”, Nov 20th-23rd, Barossa Valley, Australia

OEAW Fellowship Workshop, March 7th-8th 2014, Vienna, Austria (Shorttalk)

8th Swiss Apoptosis Meeting, Oct. 10th-12th 2014, Bern, Switzerland

Fellowships and awards:

2011, 2012 and 2013: Tyrolean cancer research grant for a total of €15.000

2013-2014: DOC Fellowship of the Austrian Academy of Sciences, €120.000

Poster prize at the ANZSCDB Annual Meeting, Nov. 19th 2013, Adelaide, Australia

Poster prize at the 6th Barossa Meeting “Cell signaling in the omics era”, Nov. 20th- 23rd 2013, Barossa Valley, Australia

126

Supplement

Acknowledgements The completion of this work cumulating in the graduation as a PhD from this university would not have been possible without the help of several people: The most important role was played by my supervisor Andreas Villunger. He was able to teach me with abundance of patience the principles of scientific thinking and working. Next to training me in various wet lab methods he also put a stress on increasing my scientific writing abilities, introduced me into the art of grant writing and taught me tricks when it comes to organize a scientific meeting.

I want to thank my PhD thesis committee Stephan Geley and Jakob Troppmair and Thomas Kaufmann and Wolfgang Doppler for reviewing my PhD thesis.

Work in a laboratory wouldn’t be that fun if there wasn’t a gorgeous team working with me in the Division of Developmental Immunology. Thanks for the great science but also for numerous entertaining off lab activities to Florian Bock, Fabian Schuler, Selma Tuzlak, Manuel Haschka, Luca Fava, Silke Lindner, Sebastian Herzog, Claudia Soratroi, Irene Gaggl, Katrin Rossi, Florian Baumgartner, Laura Llancuna, Eleonora Ottina, Verena Labi, Claudia Wöss, Claudia Manzl, Günter Böck, Gerhard Krumschnabel, Ilona Lengenfelder and all the other people in the CCB who always have been fun to work with and last but not least the MCBO community. The MCBO doctoral college made it possible for me to visit another lab for half a year. I was invited to the Molecular Regulations Laboratory headed by Prof. Sharad Kumar in Adelaide/Australia. Thank you Prof. Sharad Kumar, Loretta Dorstyn, Joey Puccini, Andrei Nikolic and Pranay Goel for the warm welcome and teaching me new methods and approaches to tackle problems in a project.

Thanks to the funding by the Austrian Academy of Science for granting me the DOC fellowship and the opportunity to attend several scientific meetings as well as the Österreichische Krebshilfe Tirol for financial support.

Der größte Dank gebührt aber meiner Familie, welche mich durch all die Jahre des Studiums immer großzügig unterstützt und motiviert hat. Auch meiner liebsten Freundin Christina möchte ich von ganzem Herzen dafür danken, dass sie das entstehen dieser Arbeit mit so viel Geduld gefördert hat. 127