WO2007020244A1 - The mitochondrial rhomboid protease parl as a target for lymphoma - Google Patents

Abstract

The present invention relates to the field of cancer, more particularly to the field of lymphomas and even more particularly to the field of B- and T-cell tumors. The invention describes a transgenic mouse which does not produce the mitochondrial rhomboid like protease PARL. It is revealed that the phenotype of this mouse is characterized by a massive apoptosis in B- and T-cells. The invention thus relates to molecules which can bind to PARL and are capable of inducing apoptosis in B- and T-cells. The invention further relates to the use of these molecules to treat cancers of B- and T-cells, more commonly designated as lymphomas.

Description

THE MITOCHONDRIAL RHOMBOID PROTEASE PARL AS A TARGET FOR LYMPHOMA

Field of the invention

The present invention relates to the field of cancer, more particularly to the field of lymphomas and even more particularly to the field of B- and T-cell tumors. The invention describes a transgenic mouse which does not produce the mitochondrial rhomboid like protease PARL. It is revealed that the phenotype of this mouse is characterized by a massive apoptosis in B- and

T-cells. The invention thus relates to molecules which can bind to PARL and are capable of inducing apoptosis in B- and T-cells. The invention further relates to the use of these molecules to treat cancers of B- and T-cells, more commonly designated as lymphomas.

Background of the invention

Rhomboid proteases constitute probably the most widely conserved polytopic-membrane- protein family identified until now. They are present in almost every archaea, bacterial and eukaryotic genome sequenced (Koonin et al., 2003). Studies in Drosophila melanogaster identified seven rhomboids so far, which act as essential activators of the epidermal growth factor (EGF) signaling pathway (Freeman, 2004). Rhomboids proteolitically cleave the EGF receptor ligands Spitz, Gurken and Keren (Lee et al., 2001 ; Urban et al., 2001 ; Urban et al., 2002). Since all Rhomboids share a conserved serine protease catalytic dyad (Lemberg et al., 2005; Urban et al., 2001), it has been suggested that they are all able to cleave proteins in their transmembrane domain. Therefore, together with the Presenilin aspartyl proteases and the Site 2 metalloproteases, they have been functionally assigned to a previously unidentified class of highly hydrophobic proteases involved in "regulated intramembranous proteolytic cleavage", a novel cell signaling mechanism (Brown et al., 2000). While the function of Drosophila rhomboids has been at least partially elucidated, to date our knowledge of the mammalian rhomboids is extremely scarce. US2003/0165497 describes rhomboid like proteases and the use of antibodies against rhomboid like proteases for the treatment of tumors. Recently, a mitochondrial rhomboid rbd1/pcp1 was identified in Saccharomyces cerevisiae (Esser et al., 2002; Herlan et al., 2003; McQuibban et al., 2003) (Sesaki et al., 2003). Arbdi cells display fragmented mitochondria and impaired growth on fermentable carbon sources. A similar phenotype was observed following deletion of the dynamin related protein mgmip, which turned out to be a substrate for rbdip. The short isoform of mgmip produced by rbdip is required to maintain mitochondrial morphology (Herlan et al., 2003; McQuibban et al., 2003). Thus, rhomboids and intramembrane proteolysis appear to control mitochondrial dynamics and function in yeast. Mitochondria are crucial organelles in intermediate metabolism and energy production (Danial et al., 2003), Ca²⁺ signaling (Rizzuto et al., 2000), and integration and amplification of apoptotic signals (Green and Kroemer, 2004). Such a functional versatility is mirrored by a complex morphological organization, both at the ultrastructural and at the cellular level (Griparic and van der Bliek, 2001). In the cytosol, mitochondria appear as dynamic organelles whose shape is continuously regulated by fusion and fission events (Bereiter-Hahn and Voth, 1994). Electron tomography of mitochondria revealed that cristae constitute a separate, pleomorphic compartment connected to a very thin intermembrane space by a narrow junction (Frey and Mannella, 2000). Mitochondrial morphology is controlled by a growing family of "mitochondria-shaping" proteins, many of which have been initially identified by genetic screens in budding yeast (Dimmer et al., 2002; Shaw and Nunnari, 2002). Among those, several dynamin-related proteins directly regulate mitochondrial fusion and fission. Dynamins are ubiquitous mechano-enzymes that hydrolyze GTP to regulate fusion, fission, tubulation and elongation of cellular membranes (McNiven et al., 2000). In mammalians, mitochondrial fission is controlled by a cytosolic dynamin related protein DRP-1 (Smirnova et al., 2001) that translocates to sites of mitochondrial fragmentation where it binds to hFisi, its adapter in the outer membrane (Yoon et al., 2003) (James et al., 2003). Fusion is controlled by mitofusin-1 (MFN1) and-2 (MFN2), orthologues of S. cerevisiae fzoip (Rapaport et al., 1998) and Drosophila melanogaster FZO (Hales and Fuller, 1997), located in the outer mitochondrial membrane. OPA1, the mammalian homologue of Saccharomyces cerevisiae mgmip, is the only dynamin-related protein identified in the inner membrane so far (Olichon et al., 2002) Loss-of-function or dominant negative mutations of OPA1 are associated with dominant optic atrophy, the most common cause of inherited optic neuropathy, characterized by apoptotic death of the retinal ganglion cells and by ascending optic neuropathy (Alexander et al., 2000; Delettre et al., 2000). OPA1 promotes mitochondrial fusion by cooperating with MFN1 (Cipolat et al., 2004), similarly to mgmip, which fuses mitochondria by participating in a complex with fzoip, the orthologue of MFNs, and the adapter ugoip (Wong et al., 2003). We used a genetic approach to investigate the physiological function of the mammalian homologue of rbdip, mitochondrial rhomboid PARL. We found that PARL is required for normal life span, given its prominent, unexpected role in controlling apoptosis at the mitochondrial level. PARL is positioned upstream of OPA1 in the pathway of cristae remodelling that regulates the amount of cytochrome c mobilized for release through the outer mitochondrial membrane. Furthermore, it was shown that PARL is a target for treating B- and T-cell lymphomas.

Figure Figure 1 : Generation of Part knock out mice

A. Maps of the targeting vector, the wild-type Part allele, the conditional targeted allele (floxed allele), and the disrupted Pan allele. Exons are indicated as black boxes. LoxP and FRT recombination sites are indicated as white and black arrowheads respectively. Arrows indicate the locations of PCR primers. The expected sizes for the indicated restriction enzyme digested fragments detected by 5'(L), 3'(R) flanking or internal probes (H) (PCR fragments, black bars) from targeted and wild-type alleles are indicated below every construct with line diagrams. Positive selection marker is indicated as a grey box. Relevant restriction sites are shown B (BgIII) and E (EcoRI).

B. Transcripts of OPA1 and Part were detected on a commercially available Mouse MTN™ Blot (BD Biosciences - Clontech) membrane showing ubiquitous expression. The β-actin transcript was detected as control.

C. Examples of Southern blot of DNA isolated from one of the selected ES cell lines, digested with EcoRI, and hybridized with the different probes (L, R and internal (H).

D. PCR analysis of DNA extracted from tail clips of mice using primers as indicated in panel A. The fragments detected for the WT or floxed (+ or F) Part allele and the null (-) Part allele are indicated. A pseudogene for Part is detected with the primers located in exon 2 and exon3.

E. Northern blot of whole embryonic bodies of mice, the transcript of Part is detected in WT and heterozygous Part samples. In the Parf^A a weak signal corresponding to an aberrant transcript is detected. The β-actin transcript was detected as control.

F. 100 μg of WT and Part^1' MEFs total cell lysate was resolved by SDS-PAGE and probed with anti-PARL C-terminus antibody (specific band ca. 30 kDa). The unspecific upper band was used as loading control. G. RT-PCR analysis of Part transcripts in WT and Parf^"AMEF cells. A shorter transcript of Part is detected in the Part^1' cells. Sequencing of the aberrant transcript confirmed a reading shift in remaining transcript.

H. Prediction of the aberrant Parl protein. Bold amino acids are identical to the WT Parl protein.

Aims and detailed description of the invention

In the present invention we have investigated the role of PARL, a rhomboid like protease. We have observed that the phenotype of the Part^1' knock-out mouse is similar to that of BcI-Z^1' mice, which complete embryonic development, but display early postnatal mortality with massive apoptotic involution of thymus and spleen (Veis et al., 1993). BCL-2 is regarded as a crucial regulator of intrinsic cell death by sequestering activated BH3-only proapoptotic members of its family to prevent activation of the multidomain proapoptotics, cytochrome c release and mitochondrial dysfunction (Danial and Korsmeyer, 2004). The role of PARL in apoptosis is herein corroborated in several assays demonstrating that Pan deficiency affects the intrinsic, mitochondrial apoptotic pathway. Part^1' fibroblasts are more sensitive than their wild type counterparts to a variety of intrinsic death stimuli, including BID (Wang et al., 1996; Wei et al., 2000), while the extrinsic pathway was not affected in type I cells, like MEFs. Part'^' cells can be entirely rescued by the expression of exogenous Parl, but not by a catalytically inactive form of PARL (Lemberg et al., 2005). This shows that the putative proteolytic function of PARL is essential for protection from apoptosis.

The present invention further shows that PARL is a target for the treatment of lymphoma.

Thus in one embodiment the present invention relates to molecules which comprise a region that can specifically bind to PARL and said molecules are capable of enhancing or stimulating apoptosis of B- and T-cells. With the wording "enhancing or stimulating apoptosis" it is understood that said stimulation of apoptosis (e.g. in a B- or T-cell line) can occur for at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% as compared to a B-cell or T-cell line which has not been contacted with said molecules. More specifically the invention relates to molecules that can be used to neutralize the activity of PARL by interfering with its synthesis, translation or proteolytical activity. By molecules it is meant peptides, tetrameric peptides, proteins, organic molecules, antibodies, ribozymes, siRNAs, anti-sense nucleic acids and locked nucleic acids (LNA's). Also, the invention is directed to antagonists of PARL such as anti- PARL antibodies and functional fragments derived thereof, anti-sense RNA and DNA molecules and ribozymes that function to inhibit the translation of PARL, all capable of stimulating or enhancing the apoptosis. By synthesis it is meant transcription of PARL. Small molecules can bind on the promoter region of PARL and inhibit binding of a transcription factor or said molecules can bind said transcription factor and inhibit binding to the PARL-promoter. By PARL it is meant 'presenilin associated rhomboid like" protein. The nucleotide sequence of human PARL is depicted in SEQ ID NO: 1 and the amino acid sequence of human PARL is depicted in SEQ ID NO: 2. The activity of PARL can be calculated by measuring the % inhibition of apoptosis. Since PARL also possesses a proteolytic activity its activity can also be calculated by measuring the cleavage of its substrates (e.g. OPA1 as herein further described in the examples).

In a specific embodiment the invention provides an antibody against PARL. The term 'antibody' or 'antibodies' relates to an antibody characterized as being specifically directed against PARL or any functional derivative thereof, with said antibodies being preferably monoclonal antibodies; or an antigen-binding fragment thereof, of the F(ab')₂, F(ab) or single chain Fv type, or any type of recombinant antibody derived thereof. These antibodies of the invention, including specific polyclonal antisera prepared against PARL or any functional derivative thereof, have no cross-reactivity to others proteins. The monoclonal antibodies of the invention can for instance be produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly of a mouse or rat immunized against PARL or any functional derivative thereof, and of cells of a myeloma cell line, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing PARL or any functional derivative thereof which have been initially used for the immunization of the animals. The monoclonal antibodies according to this embodiment of the invention may be humanized versions of the mouse monoclonal antibodies made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains. Alternatively the monoclonal antibodies according to this embodiment of the invention may be human monoclonal antibodies. Such human monoclonal antibodies are prepared, for instance, by means of human peripheral blood lymphocytes (PBL) repopulation of severe combined immune deficiency (SCID) mice as described in PCT/EP 99/03605 or by using transgenic non- human animals capable of producing human antibodies as described in US patent 5,545,806. Also fragments derived from these monoclonal antibodies such as Fab, F(ab)'₂ and ssFv ("single chain variable fragment"), providing they have retained the original binding properties, form part of the present invention. Such fragments are commonly generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin, or other proteases. It is well known to the person skilled in the art that monoclonal antibodies, or fragments thereof, can be modified for various uses. The antibodies involved in the invention can be labeled by an appropriate label of the enzymatic, fluorescent, or radioactive type. In a particular embodiment said antibodies against PARL or a functional fragment thereof are derived from camels. Camel antibodies are fully described in WO94/25591 , WO94/04678 and in WO97/49805. Processes are described in the art which make it possible that antibodies can be used to hit intracellular targets. Since PARL is a mitochondrial protease it is an intracellular target, the antibodies or fragments thereof with a specificity for PARL must be delivered into the cells. One such technology uses lipidation of the antibodies. The latter method is fully described in WO 94/01131 which is herein incorporated by reference.

Small molecules, e.g. small organic molecules, and other drug candidates can be obtained, for example, from combinatorial and natural product libraries. To screen for said candidate/test molecules cell lines that express PARL may be used and the apoptosis is monitored as described in detail in the examples. Said monitoring can be measured using standard biochemical techniques. Other responses such as activation or suppression of catalytic activity, phosphorylation or dephosphorylation of other proteins, activation or modulation of second messenger production, measuring the proteolytic activity, changes in cellular ion levels, association, dissociation or translocation of signalling molecules, or transcription or translation of specific genes may also be monitored. These assays may be performed using conventional techniques developed for these purposes in the course of screening. Cellular processes under the control of the PARL signalling pathway may include, but are not limited to, normal cellular functions, proliferation, differentiation, maintenance of cell shape, and adhesion, in addition to abnormal or potentially deleterious processes such as unregulated cell proliferation, loss of contact inhibition, blocking of differentiation or cell death. The qualitative or quantitative observation and measurement of any of the described cellular processes by techniques known in the art may be advantageously used as a means of scoring for signal transduction in the course of screening.

Random peptide libraries, such as tetrameric peptide libraries, consisting of all possible combinations of amino acids attached to a solid phase support may be used to identify peptides that are able to bind to the ligand binding site of a given receptor or other functional domains of a receptor such as kinase domains (Lam KS et al., 1991 , Nature 354, 82). Also within the scope of the invention are oligonucleotide sequences that include anti-sense RNA and DNA molecules and ribozymes that function to inhibit the translation of PARL mRNA. Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA of PARL, followed by an endonucleolytic cleavage. Within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of PARL RNA sequences. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.

Both anti-sense RNA and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize anti-sense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines. Beside the inhibition of translation the anti-sense oligonucleotide sequences can work through the use of RNA inhibition (RNAi) with here in the invention applying anti-sense oligonucleotides that are specifically directed to the sequence that encodes PARL and forms a siRNA duplex. RNAi is based on the degradation of particular target sequences by the design of short interference RNA oligo^'s (siRNA) which recognize the target sequence and subsequently trigger their degradation by a poorly understood pathway. The siRNA duplexes should preferentially be shorter than 30 nucleotides, because longer stretches of dsRNA can activate the PKR pathway in mammalian cells, which results in a global a-specific shut-down of protein synthesis. Target regions should be AA(N19)TT or AA(N21), should be specific for the gene of interest and should have a GC content of appr. 50%. The siRNAs duplexes can for example be transfected in the cells of interest by oligofectamin (Life Technologies) and the transfection efficiency reaches 90-95%.

In a particular embodiment the invention also aims at a method for the treatment of lymphoma by administering to a patient in need of such treatment an effective amount of an LNA-modified antisense oligonucleotide (LNA stands for locked nucleic acid), or a cocktail of different LNA- modified antisense oligonucleotides, or a cocktail of different LNA-modified and unmodified antisense oligonucleotides specific for the PARL gene. An LNA-modified olignonucleotide contains one or more units of an LNA monomer, preferably one or more 2'-O, 4'-C-methylene bridge monomers (oxy-LNA), see WO9914220. Incorporation of LNA monomers containing a 2'-O, 4'-C-methylene bridge into an oligonucleotide sequence leads to an improvement in the hybridisation stability of the modified oligonucleotide. Oligonucleotides comprising the 2'-O, 4'- C-methylene bridge (LNA) monomers and also the corresponding 2'-thio-LNA (thio-LNA), 2'- HN-LNA (amino-LNA), and 2'-N(R)-LNA (amino-R-LNA) analogue, form duplexes with complementary DNA and RNA with thermal stabilities not previously observed for bi- and tricyclic nucleosides modified oligonucleotides. The increase in T_m per modification varies from +3 to +11 degree Celsius, and furthermore, the selectivity is also improved. An LNA-modified oligonucleotide may contain other LNA units in addition to or in place of an oxy-LNA group. In particular, preferred additional LNA units include 2'-thio-LNA (thio-LNA), 2'-HN-LNA (amino- LNA), and 2'-N(R)-LNA (amino-R-LNA)) monomers in either the D-beta or L-alpha configurations or combinations thereof. An LNA-modified oligonucleotide also may have other internucleoside linkages than the native phosphordiester, e.g. phosphoromonothioate, phosphorodithioate, and methylphosphonate linkages. The LNA-modified oligonucleotide can be fully modified with LNA (i.e. each nucleotide is an LNA unit), but it is generally preferred that the LNA-modified oligomers will contain other residues such as native DNA monomers, phosphoromonothioate monomers, methylphosphonate monomers or analogs thereof. In general, an LNA-modified oligonucleotide will contain at least about 5, 10, 15 or 20 percent LNA units, based on total nucleotides of the oligonucleotide, more typically at least about 20, 25, 30, 40, 50, 60, 70, 80 or 90 percent LNA units, based on total bases of the oligonucleotide. An LNA-modified oligonucleotide used in accordance with the invention suitably is at least a 5- mer, 6-mer, 7-mer, 8-mer, 9-mer or 10-mer oligonucleotide, that is, the oligonucleotide is an oligomer containing at least 5, 6, 7, 8, 9, or 10 nucleotide residues, more preferably at least about 11 or 12 nucleotides. The preferred maximum size of the oligonucleotide is about 40, 50 or 60 nucleotides, more preferably up to about 25 or 30 nucleotides, and most preferably about between 12 and 20 nucleotides. While oligonucleotides smaller than 10-mers or 12-mers may be utilized they are more likely to hybridise with non-targeted sequences (due to the statistical possibility of finding exact sequence matches by chance in the human genome of 3.10⁹ bp), and for this reason may be less specific. In addition, a single mismatch may destabilise the hybrid thereby impairing its therapeutic function. While oligonucleotides larger than 40-mers may be utilised, synthesis, and cellular uptake may become somewhat more troublesome. Although specialised vehicles or oligonucleotide carriers will improve cellular uptake of large oligomers. Moreover, partial matching of long sequences may lead to non-specific hybridisation, and non-specific effects. While in principle oligonucleotides having a sequence complementary to any region of the target mRNA of PARL find utility in the present invention, preferred are oligonucleotides capable of forming a stable duplex with a portion of the transcript lying within about 50 nucleotides (preferably within about 40 nucleotides) upstream (the 5' direction), or about 50 (preferably 40) nucleotides downstream (the 3' direction) from the translation initiation codon of the target mRNA. Also preferred are oligonucleotides which are capable of forming a stable duplex with a portion of the target mRNA transcript including the translation initiation codon. LNA-modified oligonucleotides based on the PARL sequence can be used for the treatment of lymphomas. In general, therapeutic methods of the invention for the treatment of lymphoma include administration of a therapeutically effective amount of an LNA-modified oligonucleotide to a mammal, particularly a human. In antisense therapies, administered LNA-modified oligonucleotide contacts (interacts) with the targeted PARL RNA from the gene, whereby expression of PARL is inhibited and apoptosis of the target cell is induced. Such inhibition of PARL expression suitably will be at least a 10% or 20% difference relative to a control, more preferably at least a 30%, 40%, 50%, 60%, 70%, 80%, or 90% difference in expression relative to a control. It will be particularly preferred where interaction or contact with an LNA-modified oligonucleotide results in complete or essentially complete inhibition of expression relative to a control, e.g. at least about a 95%, 97%, 98%, 99% or 100% inhibition of in expression relative to control. A control sample for determination of such modulation can be comparable cells (in vitro or in vivo) that have not been contacted with the LNA-modified oligonucleotide. The monitoring of the % inhibition of PARL expression can be followed by the % induction of apoptosis of B- or T-cell lines since the two inhibition processes are inversely correlated.

In yet another embodiment the invention provides a method of treating lymphomas in a subject comprising administering a pharmaceutical composition comprising means for modulating PARL together with a pharmaceutical excipient. In yet another embodiment said means for modulating PARL is an antibody binding to PARL and capable of inducing apoptosis of B- and/or T-lymphocytes. In yet another embodiment said means for modulating PARL is a small molecule binding to PARL and capable of inducing apoptosis of B- and/or T-lymphocytes. In yet another embodiment said means for modulating PARL is a ribozyme binding to PARL which and capable of inducing apoptosis of B- and/or T-lymphocytes. In yet another embodiment said means for modulating PARL is an anti-sense nucleic acid binding to PARL and capable of inducing apoptosis of B- and/or T-lymphocytes. In yet another embodiment said means for modulating PARL is an RNAi molecule binding to PARL and capable of inducing apoptosis of B- and/or T-lymphocytes. In yet another embodiment said means for modulating PARL is a small molecule binding to PARL and capable of inducing apoptosis of B- and/or T- lymphocytes. In yet another embodiment said means for modulating PARL is a locked nucleic acid (LNA) binding to PARL and capable of inducing apoptosis of B- and/or T-lymphocytes.

In another embodiment of the invention the above-described molecules that are capable of neutralizing the activity of PARL can be used for the manufacturing a medicament to treat lymphomas. The term 'lymphoma' means any of a group of malignant diseases, usually starting in the lymph nodes or in the lymphoid tissues (including the lung, the gut, or the skin). Lymphomas are generally classified into two types, Hodgkin's disease and non-Hodgkin's lymphoma, each category being further subdivided. Hodgkin's disease is characterised by the presence of Reed Sternberg cells and can be further subdivided into 4 subtypes — lymphocyte predominant, nodular sclerosis, mixed cellularity, and lymphocyte depleted. Non-Hodgkin's lymphoma (NHL) is composed of either B or T cells. B-cell neoplasms comprise precursor B-cell neoplasm, precursor B-acute lymphoblastic leukemia/lymphoblastic lymphoma (B-ALL, LBL), peripheral B-cell neoplasms, B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocyte leukemia, lymphoplasmacytic lymphoma/immunocytoma, mantle-cell lymphoma, follicular lymphoma, extranodal marginal zone B-cell lymphoma of MALT type, nodal marginal zone B-cell lymphoma (+/-monocytoid B-cells), splenic marginal zone lymphoma (+/-villous lymphocytes), hairy-cell leukemia, plasmacytoma/plasma-cell myeloma, diffuse large B-cell lymphoma, Burkitt's lymphoma. T-cell and putative NK-cell neoplasms comprise precursor T-cell neoplasm: precursor T-acute lymphoblastic leukemia/lymphoblastic lymphoma (T-ALL, LBL), peripheral T-cell and NK-cell neoplasms, T-cell chronic lymphocytic leukemia/prolymphocytic leukaemia, T-cell granular lymphocytic leukemia, Mycosis fungoides/Sezary's syndrome, peripheral T-cell lymphoma, not otherwise characterised, hepatosplenic gamma/delta T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal T-/NK cell lymphoma, nasal type, enteropathy-type intestinal T-cell lymphoma, adult T-cell lymphoma/leukaemia (HTLV 1 positive), anaplastic large-cell lymphoma, primary systemic type, anaplastic, large-cell lymphoma, primary cutaneous type and aggressive NK-cell leukaemia.

In a specific embodiment it should be clear that the therapeutic method of the present invention against lymphomas can also be used in combination with any other lymphoma therapy known in the art such as irradiation, chemotherapy or surgery. The term 'medicament to treat' relates to a composition comprising molecules as described above and a pharmaceutically acceptable carrier or excipient (both terms can be used interchangeably) to treat lymphomas. Suitable carriers or excipients known to the skilled man are saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives. Other suitable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers. The 'medicament' may be administered by any suitable method within the knowledge of the skilled man. One route of administration is parenterally. In parental administration, the medicament of this invention will be formulated in a unit dosage injectable form such as a solution, suspension or emulsion, in association with the pharmaceutically acceptable excipients as defined above. However, the dosage and mode of administration will depend on the individual. Generally, the medicament is administered so that the molecule that inhibits PARL activity of the present invention is given at a dose between 1 μg/kg and 10 mg/kg, more preferably between 10 μg/kg and 5 mg/kg, most preferably between 0.1 and 2 mg/kg. It can be given as a bolus dose. Continuous infusion may also be used and includes continuous subcutaneous delivery via an osmotic minipump. If so, the medicament may be infused at a dose between 1 and 20 μg/kg/minute. It is clear to the person skilled in the art that the use of a therapeutic composition comprising for example an antibody against PARL or an LNA capable of binding to PARL for the manufacture of a medicament to treat lymphoma can be administered by any suitable means, including but not limited to, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In addition, the therapeutic composition is suitably administered by pulse infusion, particularly with declining doses of the antibody. Preferably the therapeutic composition is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

Another aspect of administration for treatment is the use of gene therapy to deliver the above mentioned anti-sense gene or functional parts of the RARL gene or a ribozyme directed against the PARL mRNA or a functional part thereof. Gene therapy means the treatment by the delivery of therapeutic nucleic acids to patient's B- and T-cells. This is extensively reviewed in Lever and Goodfellow 1995; Br. Med Bull. ,51, 1-242; Culver 1995; Ledley, F.D. 1995. Hum. Gene Ther. 6, 1129. There are two general approaches to achieve gene delivery; these are non-viral delivery and virus-mediated gene delivery.

In yet another embodiment the invention provides a non-human transgenic animal whose genome comprises a disruption in the endogenous PARL gene wherein said disruption results in a decreased expression or a lack of expression of said endogenous PARL gene.

The term "animal" is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and foetal stages. A "transgenic animal" is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by targeted recombination or microinjection or infection with recombinant vector. The term "transgenic animal" is not meant to encompass classical cross- breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by or receive a recombinant DNA molecule as described above. The latter molecule may be specifically targeted to a defined genetic locus, be randomly integrated within a chromosome, or it may be extra-chromosomally replicating DNA. The term "germ cell line transgenic animal" refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the genetic information to offspring. If such offspring in fact, possess some or all of that alteration or genetic information, then they, too, are transgenic animals. The alteration or genetic information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene (e.g. lack of expression in a specific organ or tissue). In another embodiment the invention provides a transgenic, non-human animal characterised by having an endogenous nucleic acid sequence encoding a non-functional PARL expression.

In yet another embodiment the invention provides a transgenic, non-human animal characterised by having an endogenous nucleic acid sequence encoding a non-functional PARL wherein said non-functional PARL expression is in a specific tissue or in a specific organ.

Thus in other words the present invention provides a transgenic non-human animal in which in at least one organ or tissue the PARL gene has been selectively inactivated. In a preferred embodiment the non-functional expression of the PARL gene is in the brain or in a specific region of the brain. Mice comprising conditionally targeted PARL (PARL^flx/flx) can be crossed with mice expressing Cre recombinase under the transcriptional control of for example neurotransmitter transporter promoters that drive selective Cre expression in specific brain regions (Zhuang X et al (2005) J. Neurosci. Methods 143(1 ):27-32. Examples of specific regions in the brain are for example the substantia nigra or the hippocampus. It is envisaged that an animal model with a specific inactivation (also equivalent to the term disruption) of PARL in the substantia nigra is a model for Parkinson's disease. It is also envisaged that an animal model with a specific inactivation of PARL in the hippocampus is a model for Alzheimer's disease. More specifically, the present invention provides a transgenic non-human animal whose genome comprises a disruption in the PARL gene, wherein the transgenic animal exhibits a decreased level or no functional PARL protein relative to wild-type. The non- human animal may be any suitable animal (e.g., cat, cattle, dog, horse, goat, rodent, and sheep), but is preferably a rodent. More preferably, the non-human animal is a rat or a mouse. Unless otherwise indicated, the term "PARL gene" refers herein to a nucleic acid sequence encoding PARL protein, and any allelic variants thereof. Due to the degeneracy of the genetic code, the PARL gene of the present invention includes a multitude of nucleic acid substitutions which will also encode a PARL protein. An "endogenous" PARL gene is one that originates or arises naturally, from within an organism. Additionally, as used herein, "PARL protein" includes both a "PARL protein" and a "PARL protein analogue". A "PARL analogue" is a functional variant of the "PARL protein", having PARL-protein biological activity, that has 60% or greater (preferably, 70% or greater) amino-acid-sequence homology with the PARL protein, as well as a fragment of the PARL protein having PARL-protein biological activity. As further used herein, the term "PARL-protein biological activity" refers to protein activity, which regulates apoptosis or has proteolytic activity. In yet another embodiment the invention provides cell lines derived from the above described transgenic animals, in particular cell lines lacking PARL. In a particular embodiment said cells are primary neurons. As further used herein, the term "transgene" refers to a nucleic acid (e.g., DNA or a gene) that has been introduced into the genome of an animal by experimental manipulation, wherein the introduced gene is not endogenous to the animal, or is a modified or mutated form of a gene that is endogenous to the animal. The modified or mutated form of an endogenous gene may be produced through human intervention (e.g., by introduction of a point mutation, introduction of a frameshift mutation, deletion of a portion or fragment of the endogenous gene, insertion of a selectable marker gene, insertion of a termination codon, insertion of recombination sites, etc.). A transgenic non-human animal may be produced by several methods involving human intervention, including, without limitation, introduction of a transgene into an embryonic stem cell, newly fertilized egg, or early embryo of a non-human animal; integration of a transgene into a chromosome of the somatic and/or germ cells of a non-human animal; and any of the methods described herein. The transgenic animal of the present invention has a genome in which the PARL gene has been selectively inactivated, resulting in a disruption in its endogenous PARL gene in at least one tissue or organ. As used herein, a "disruption" refers to a mutation (i.e., a permanent, transmissible change in genetic material) in the PARL gene that prevents normal expression of functional PARL protein (e.g., it results in expression of a mutant PARL protein; it prevents expression of a normal amount of PARL protein; or it prevents expression of PARL protein). Examples of a disruption include, without limitation, a point mutation, introduction of a frameshift mutation, deletion of a portion or fragment of the endogenous gene, insertion of a selectable marker gene, and insertion of a termination codon. As used herein, the term "mutant" is used herein to refer to a gene (or its gene product), which exhibits at least one modification in its sequence (or its functional properties) as compared with the wild-type gene (or its gene product). In contrast, the term "wild-type" refers to the characteristic genotype (or phenotype) for a particular gene (or its gene product), as found most frequently in its natural source (e.g., in a natural population). A wild-type animal, for example, expresses functional PARL. Selective inactivation of a gene in a transgenic non-human animal may be achieved by a variety of methods, and may result in either a heterozygous disruption (wherein one PARL gene allele is disrupted, such that the resulting transgenic animal is heterozygous for the mutation) or a homozygous disruption (wherein both PARL gene alleles are disrupted, such that the resulting transgenic animal is homozygous for the mutation). In one embodiment of the present invention, the endogenous PARL gene of the transgenic animal is disrupted through homologous recombination with a nucleic acid sequence that encodes a region common to PΛRL gene products. By way of example, the disruption through homologous recombination may generate a knockout mutation in the PARL gene, particularly a knockout mutation wherein at least one deletion has been introduced into at least one exon of the PARL gene. In a preferred embodiment of the present invention, the knockout mutation is generated in a coding exon of the PARL gene. Additionally a disruption in the PARL gene may result from insertion of a heterologous selectable marker gene into the endogenous PARL gene. As used herein, the term "selectable marker gene" refers to a gene encoding an enzyme that confers upon the cell or organism in which it is expressed a resistance to a drug or antibiotic, such that expression or activity of the marker can be selected for (e.g., a positive marker, such as the neo gene) or against (e.g., a negative marker, such as the dt gene). As further used herein, the term "heterologous selectable marker gene" refers to a selectable marker gene that, through experimental manipulation, has been inserted into the genome of an animal in which it would not normally be found. The transgenic non-human animal exhibits decreased expression of functional PARL protein relative to a corresponding wild-type non-human animal of the same species. As used herein, the phrase "exhibits decreased expression of functional PARL protein" refers to a transgenic animal in whom the detected amount of functional PARL is less than that which is detected in a corresponding animal of the same species whose genome contains a wild-type PARL gene. Preferably, the transgenic animal contains at least 90% less functional PARL than the corresponding wild-type animal. More preferably, the transgenic animal contains no detectable, functional PARL as compared with the corresponding wild-type animal. Levels of PARL in an animal, as well as PARL activity, may be for example detected using appropriate antibodies against the PARL protein. Accordingly, where the transgenic animal of the present invention exhibits decreased expression of functional PARL protein relative to wild-type, the level of functional PARL protein in the transgenic animal is lower than that which otherwise would be found in nature. In one embodiment of the present invention, the transgenic animal expresses mutant PARL (regardless of amount). In another embodiment of the present invention, the transgenic animal expresses no PARL (wild-type or mutant). In yet another embodiment of the present invention, the transgenic animal expresses wild-type PARL protein, but at a decreased level of expression relative to a corresponding wild-type animal of the same species. The transgenic, non-human animal of the present invention, or any transgenic, non-human animal exhibiting decreased expression of functional PARL protein relative to wild-type, may be produced by a variety of techniques for genetically engineering transgenic animals. For example, to create a transgenic, non-human animal exhibiting decreased expression of functional PARL protein relative to a corresponding wild-type animal of the same species, a PARL targeting vector is generated first. As used herein, the term "PARL targeting vector" refers to an oligonucleotide sequence that comprises a portion, or all, of the PARL gene, and is sufficient to permit homologous recombination of the targeting vector into at least one allele of the endogenous PARL gene within the recipient cell. In one embodiment of the present invention, the targeting vector further comprises a positive or negative heterologous selectable marker gene (e.g., the positive selection gene, neo). Preferably, the targeting vector may be a replacement vector (i.e., the selectable marker gene replaces an endogenous target gene). Such a disruption is referred to herein as a "null" or "knockout" mutation. By way of example, the PARL targeting vector may be an oligonucleotide sequence comprising at least a portion of a non-human PΛRL gene in which there is at least one deletion in at least one exon. In a particular embodiment the PARL targeting vector comprises recombination sites (e.g. loxP sites or FRT sites) which do not interrupt the coding region of the PARL gene. In the method of the present invention, the PARL targeting vector that has been generated then may be introduced into a recipient cell (comprising a wild-type PARL gene) of a non- human animal, to produce a treated recipient cell. This introduction may be performed under conditions suitable for homologous recombination of the vector into at least one of the wild- type PARL gene in the genome of the recipient cell. The non-human animal may be any suitable animal (e.g., cat, cattle, dog, horse, goat, rodent, and sheep), as described above, but is preferably a rodent. More preferably, the non-human animal is a rat or a mouse. The recipient cell may be, for example, an embryonic stem cell, or a cell of an oocyte or zygote. The PARL targeting vector of the present invention may be introduced into the recipient cell by any in vivo or ex vivo means suitable for gene transfer, including, without limitation, electroporation, DEAE Dextran transfection, calcium phosphate transfection, lipofection, monocationic liposome fusion, polycationic liposome fusion, protoplast fusion, creation of an in vivo electrical field, DNA-coated microprojectile bombardment, injection with recombinant replication-defective viruses, homologous recombination, viral vectors, and naked DNA transfer, or any combination thereof. Recombinant viral vectors suitable for gene transfer include, but are not limited to, vectors derived from the genomes of viruses such as retrovirus, HSV, adenovirus, adeno-associated virus, Semiliki Forest virus, cytomegalovirus, and vaccinia virus.

In accordance with the methods of the present invention, the treated recipient cell then may be introduced into a blastocyst of a non-human animal of the same species (e.g., by injection or microinjection into the blastocoel cavity), to produce a treated blastocyst. Thereafter, the treated blastocyst may be introduced (e.g., by transplantation) into a pseudopregnant non- human animal of the same species, for expression and subsequent germline transmission to progeny. For example, the treated blastocyst may be allowed to develop to term, thereby permitting the pseudopregnant animal to deliver progeny comprising the homologously recombined vector, wherein the progeny may exhibit decreased expression of PARL relative to corresponding wild-type animals of the same species. It then may be possible to identify a transgenic non-human animal whose genome comprises a disruption in its endogenous PARL gene. The identified transgenic animal then may be interbred with other founder transgenic animals, to produce heterozygous or homozygous non-human animals exhibiting decreased expression of functional PARL protein relative to corresponding wild-type animals of the same species.

A type of recipient cell for transgene introduction is the embryonal stem cell (ES). ES cells may be obtained from pre-implantation embryos cultured in vitro. Transgenes can be efficiently introduced into the ES cells by standard techniques such as DNA transfection or by retrovirus- mediated transduction. The resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. As used herein, a "targeted gene" or "knock-out" is a DNA sequence introduced into the germline or a non-human animal by way of human intervention, including but not limited to, the methods described herein. The targeted genes of the invention include DNA sequences which are designed to specifically alter cognate endogenous alleles. In order to produce the gene constructs used in the invention, recombinant DNA and cloning methods, which are well known to those skilled in the art, may be utilized (see Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, NY). In this regard, appropriate PARL coding sequences may be generated from genomic clones using restriction enzyme sites that are conveniently located at the relevant positions within the PARL sequence. Alternatively, or in conjunction with the method above, site directed mutagenesis techniques involving, for example, either the use of vectors such as M13 or phagemids, which are capable of producing single stranded circular DNA molecules, in conjunction with synthetic oligonucleotides and specific strains of Escherichia coli (E. coli) (Kunkel, T. A. et al., 1987, Meth. Enzymol. 154:367-382) or the use of synthetic oligonucleotides and PCR (polymerase chain reaction) (Ho et al., 1989, Gene 77:51-59; Kamman, M. et al., 1989, Nucl. Acids Res. 17:5404) may be utilized to generate the necessary PARL nucleotide coding sequences. Appropriate PARL-sequences may then be isolated, cloned, and used directly to produce transgenic animals. The sequences may also be used to engineer the chimeric gene constructs that utilize regulatory sequences other than the PARL promoter, again using the techniques described here. These chimeric gene constructs can then also be used in the production of transgenic animals.

In a particular embodiment a non-human, transgenic animal comprising a targeting vector which further comprises recombination sites (e.g. Lox sites, FRT sites) can be crossed with a non-human, transgenic animal comprising a recombinase (e.g. Cre recombinase, FLP recombinase) under control of a particular promoter. It has been shown that these site-specific recombination systems, although of microbial origin for the majority, function in higher eukaryotes, such as plants, insects and mice. Among the site-specific recombination systems commonly used, there may be mentioned the Cre/Lox and FLP/FRT systems. The strategy normally used consists in inserting the loxP (or FRT) sites into the chromosomes of ES cells by homologous recombination, or by conventional transgenesis, and then in delivering Cre (or FLP) for the latter to catalyze the recombination reaction. The recombination between the two loxP (or FRT) sites may be obtained in ES cells or in fertilized eggs by transient expression of Cre or using a Cre transgenic mouse. Such a strategy of somatic mutagenesis allows a spatial control of the recombination, because the expression of the recombinase is controlled by a promoter specific for a given tissue or for a given cell. A second strategy consists in controlling the expression of recombinases over time so as to allow temporal control of somatic recombination. To do this, the expression of the recombinases is controlled by inducible promoters such as the interferon-inducible promoter, for example. The coupling of the tetracycline-inducible expression system with the site-specific recombinase system described in WO 94 04672 has made it possible to develop a system for somatic modification of the genome which is controlled spatiotemporally. Such a system is based on the activation or repression, by tetracycline, of the promoter controlling the expression of the recombinase gene. It has been possible to envisage a new strategy following the development of chimeric recombinases selectively activated by the natural ligand for the estrogen receptor. Indeed, the observation that the activity of numerous proteins, including at least two enzymes (the tyrosine kinases c-abl and src) is controlled by estrogens, when the latter is linked to the ligand-binding domain (LBD) of the estrogen receptor alpha has made it possible to develop strategies for spatiotemporally controlled site-specific recombination. The feasibility of the site-specific somatic recombination activated by an antiestrogenic ligand has thus been demonstrated for "reporter" DNA sequences, in mice, and in particular in various transgenic mouse lines which express the fusion protein Cre-ER^T activated by Tamoxifen. The feasibility of the site-specific recombination activated by a ligand for a gene present in its natural chromatin environment has been demonstrated in mice.

Initial screening of the transgenic animals may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include but are not limited to Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of brain may be evaluated immunocytochemically using antibodies specific for PARL. In another embodiment the transgenic, non-human animals with a specific disruption of PARL in a brain-specific region (e.g. the substantia nigra or the hypothalamus) can be used for the testing of compounds for neurodegeneration disorders, and more specifically for the testing of compounds for neurodegeneration disorders. Drug screening assays in general suitable for use with transgenic animals are known. See, for example US patents Nos. 6028245 and 6455757. Thus, the transgenic animals may be used as a model system for human neurodegeneration disorders and/or to generate neuronal cell lines that can be used as cell culture models for these disorders. The transgenic animal model systems for neurodegeneration disorders may be used as a test substrate to identify drugs, pharmaceuticals, therapies and interventions which may be effective in treating such disorders. Therapeutic agents may be administered systemically or locally. Suitable routes may include oral, rectal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, intracerebral, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few. The response of the animals to the treatment may be monitored by assessing the reversal of one or more symptoms associated with neurodegeneration (e.g. Parkinson's disease, Alzheimer's disease). With regard to intervention, any treatments which reverse any aspect of neurodegeneration should be considered as candidates for therapeutic intervention. However, treatments or regimes which reverse the constellation of pathologies associated with any of these disorders may be preferred. Dosages of test agents may be determined by deriving dose-response curves. The transgenic animal model systems for neurodegeneration disorders may also be used as test substrates in identifying environmental factors, drugs, pharmaceuticals, and chemicals which may exacerbate the progression of the neuropathologies that the transgenic animals exhibit. In an alternate embodiment, the transgenic animals of the invention may be used to derive a cell line which may be used as a test substrate in culture, to identify both agents that reduce and agents that enhance the neuropathologies. While primary cultures (e.g. hypocampal neurons) derived from the transgenic animals of the invention may be utilized, continuous cell lines can also be obtained. For examples of techniques which may be used to derive a continuous cell line from the transgenic animals, see Small et al., 1985, MoI. Cell Biol. 5:642- 648.

The following examples more fully illustrate preferred features of the invention, but are not intended to limit the invention in any way. All of the starting materials and reagents disclosed below are known to those skilled in the art, and are available commercially or can be prepared using well-known techniques.

Examples

1. Targeted inactivation of the mouse Parl gene

Pan deficient (Part'^') mice were generated by homologous recombination (Fig. 1A). The Pan gene was targeted conditionally with loxP sequences in intron 2 and intron 3 (Parf") in embryonic stem cells. Correct homologous recombination was confirmed by Southern blotting using internal and external probes (Fig 1C). Two independent mouse strains were generated transferring the targeted allele in a Mendelian fashion to their offspring (Fig 1 D). Conditionally targeted (Parf¹^¹") were healthy and fertile. Mice were crossed with a 'total deletor' mouse strain expressing Cre under the PGK promotor. Cre-mediated excision of the region between the loxP sites generated a Part null allele by introducing a frame shift in the open reading frame of the rest of the Pan gene (deletion from aa 50 on and introduction of a premature stopcodon, Fig. 1 H). The successful recombination was confirmed by Northern blotting (Fig. 1 E) of RNA from total embryos. Some aberrant RNA was still observed in the Part^1' mice but RT-PCR confirmed the absence of exon 2 and thus of functional mRNA (Fig. 1G). This was further corroborated by immunoblotting of fibroblasts derived from Part^1' mice using a Parl specific polyclonal antibody (Fig. 1F).

2. Parl^'A mice prematurely die of progressive cachexia

Part^1' mice were born in a normal Mendelian frequency and developed apparently normally up to 4 weeks after birth. From then on, mice displayed severe growth retardation compared to control littermates. Strikingly, all animals died between 8 and 12 weeks after birth most likely as a consequence of mixed feeding, moving and breathing defects and general cachexia. Major alterations were observed in muscle, thymus, spleen and in the male and female reproductive tracts. Part^1' mice beyond 6 weeks lost muscle mass especially at the level of proximal muscles (erector spinae, transverse and oblique abdominal muscles, and diaphragm) and to a lesser degree in distal (limb and limb girdle) muscles. Mice developed a severe posture defect with hunchback deformity of the lower thoracic region, augmented lumbar lordosis and concave flanks. Microscopically, a reduction in the diameter of individual muscle fibers, but no evidence for muscular cell death or dystrophy was observed. Spinal motoneurons were normal and AChE histochemistry failed to reveal signs of a neurogenic atrophy. Ultrastructurally, no gross alterations of the muscle fibers were observed apart from the occasional presence of megamitochondria in some fibers, where the majority of mitochondria remained however normal. At 8 weeks thymus and spleen were dramatically atrophic, weighing 10% or less compared to controls. Microscopically, the internal organization of both thymus and spleen (cortex/medulla structure of thymus lobuli, white/red pulp structure of spleen) was completely deranged, with massive loss of lymphocytes, leaving only the epithelial and reticular stroma behind. Uteri remained macroscopically prepubertal, while ovaries were histologically normal and follicles reached at least tertiary state. On the other hand males showed cryptorchidism with associated size reduction of testes, epidydimis and accessory glands. Gross mitochondrial morphology is normal in both Leydig cells and germinative epithelium. No obvious abnormalities were observed in the central nervous system, albeit fluoro-Jade or immune staining for activated caspase 3 documented limited neurodegeneration and apoptotic cell death in thalamus and striatum. Of note, no morphological or major functional abnormalities in the visual system were recorded, including the lack of optic neuropathy. Both at 4 and 6 weeks, pinna, cornea and righting reflexes were vigorous in Pan^1' mice, and their cage activity and muscular strength measurements (grip strength and grid hang test) were not significantly different from those of wt littermates. Tail flick latency, a measure of nociception, was not different from controls. Latency of the acoustic startle response (ASR) was increased in Parf^A mice (p<0.001), whereas ASR amplitude was decreased (p<0.001), which obviously correlated with body weight (r=0.71 , p<0.001). Increased ASR latency could relate to defects in neural conduction, striatal dysfunction and/or increased reaction time. Body temperature was significantly lower in Part^1' mice at 6 weeks, but not at 4 weeks. Following cold challenge, body temperature decreased more in knockouts both at 4 and 6 weeks (p<0.01). Most notably, temperature decreased 9.5% in the Part^1' mice, compared to 4.5% in wildtypes (at 6 weeks). This more pronounced temperature decrease in Part^1' mice cannot be reduced entirely to their lower body size or wasting, since it was actually positively correlated with body weight (r=0.55; p=0.002). Overall, Part^1' mice have greatly reduced life span due to progressive cachexia, characterized by severe atrophy of spleen, thymus, muscular tissue and indications of increased apoptosis and disturbed body temperature regulation (DosSantos et al., 2003). Since Parl (presenilin associated rhomboid like) protein was originally identified as a presenilin interacting protein, we were surprised to observe no developmental problems suggestive of Presenilin dysfunction in the Part^1' mice. We therefore investigated γ-secretase processing of the Amyloid Precursor Protein which is a substrate for Presenilin, but found no alterations in Parl^"'^" mice. In total, these data show a critical role for PARL in controlling tissue homeostasis, a task accomplished by the balance between proliferation and apoptosis.

3. Parl is not required for normal mitochondrial function The multisystemic atrophy and the body temperature dysregulation evident in the adult Part^1' mouse prompted us to investigate whether it could be related to primary mitochondrial dysfunction, a frequent cause and consequence of tissue atrophy. To this end, we turned to mitochondria isolated from liver, where PARL is abundantly expressed. We measured basal (state 4) and maximal (uncoupled) respiratory rates (Jo₂) in mouse liver mitochondria (MLM) energized with different substrates. State-4 as well as uncoupled Jo₂ was similar in wt and Pan

'^' MLM, irrespective of the substrates used to feed the electron respiratory chain. Similar results were obtained with mitochondria isolated from wt and Part^1' mouse embryonic fibroblasts (MEFs). Moreover, we measured changes in mitochondrial membrane potential in wt and Part^1' mouse embryonic fibroblasts (MEFs) treated with the F₁F₀ATPaSe inhibitor oligomycin [an assay of latent mitochondrial dysfunction in intact cells (Irwin et al., 2003) and found that lack of PARL did not increase susceptibility to oligomycin. Thus, Part^1' mouse mitochondria do not display intrinsic respiratory defects nor latent mitochondrial dysfunction. This contrasts with yeast where rbdip is required to maintain mitochondrial DNA and respiratory competent mitochondria (Herlan et al., 2003; McQuibban et al., 2003).

4. Parl is dispensable for mitochondrial fusion Since Part^1' mitochondria are respiratory competent, we wished to ascertain whether Part was required for maintenance of mitochondrial shape and fusion like rbdip (Herlan et al., 2004; Herlan et al., 2003; McQuibban et al., 2003; Sesaki et al., 2003). Mitochondria of Part^1' MEFs transfected with a mitochondrial^ targeted yellow fluorescent protein (mtYFP) appeared globular or rod-shaped, their elongation being visually and quantitatively undistinguishable from that of wt cells. Moreover, mitochondrial fusion rates were identical in wt and Parf^MEFs, as measured by a specific polyethylene glycol (PEG) fusion assay. In yeast, rbdip controls mitochondrial morphology by cleaving mgmip, and expression of an rbdip-cleaved mgmip isoform in delta-rbdip cells partially complements mitochondrial shape defects (Herlan et al., 2003; McQuibban et al., 2003). OPA1 , the mammalian orthologue of mgmip, induces MFN1- dependent mitochondrial elongation and fusion when expressed in MEFs (Cipolat et al., 2004) and is a natural candidate substrate to explain the multiple defects of the Part^1' mouse. We therefore tested whether expression of OPA1 promoted mitochondrial elongation and fusion in the absence of Part. Expression of OPA1 caused comparable mitochondrial elongation and fusion in wt and Part^1' MEFs. Thus, Part is not required for the pro-fusion effect of OPA1. We extended our analysis to other mitochondria-shaping proteins that promote fusion, like MFN1 and of MFN2, which promoted equal mitochondrial elongation in wt and Part^1' cells. In total, these data indicate that Parl is not required for maintenance of mitochondrial shape and of mitochondrial fusion.

5. Absence of Parl results in massive apoptosis of T and B lymphocytes

As mitochondrial function and fusion were unaffected by ablation of Part, we sought other mechanistic explanations for the multiple tissue atrophy. We turned to a deeper analysis of thymus and spleen, two of the organs most affected by Part ablation. Thymic cellularity was massively reduced. We therefore analyzed T cell development in mutant animals. The great majority of T cells follow the normal developmental sequence CD4^~CD8^~ (double-negative) towards CD4⁺CD8⁺ (double-positive) towards CD4⁺CD8^" or CD4^"CD8⁺ (single-positive). We found that the percentage of double-negative T cells was increased while the fraction of double-positive cells was decreased in thymi of Part^1' animals, as compared to their wt littermates. When we calculated the absolute numbers of thymocytes in each fraction, we found that in Part^1' mice the total number of double-negative thymocytes was unchanged, while the total number of double-positive cells was reduced over 50-fold compared to wt. These results suggest that the thymic atrophy can be caused by death of the double-positive cells. Part^1' thymic sections displayed marked TUNEL staining further substantiating the loss of lymphocytes. We next addressed the developmental stage of the dying lymphocytes. By counterstaining CD4 and CD8 positive cells with annexin-V we found that many (>40%) double-positive Part^1' cells were apoptotic. Splenic cellularity was similarly greatly reduced, from 41.5±2.5x10⁶ in wt to 10.2±1.2x10⁶ cells in Part^1' spleens (n=8, p<0.0001 in an independent Student's t test). Loss of B220+ B cells was largely responsible for the loss of cellularity, as showed by specific CD4, CD8, B220 triple staining. TUNEL assay revealed accumulation of apoptotic cells in Part^1' spleens. Annexin-V staining indicated that indeed most Part^1' B220+ lymphocytes were apoptotic. When spontaneous death of thymocytes isolated from 7 weeks old mice and cultured in complete media was measured, Parl-I- cells always displayed increased apoptosis. This shows that augmented death of Parl-I- double positive cells does not depend on extrinsic, thymic factors like for example in the KA cytochrome c knockin mouse (Hao et al., 2005). In total, our results show that in the adult Part^1' mouse B220+ as well as double-positive T undergo apoptosis in and ex vivo.

6. Parl regulates mitochondrial apoptosis by controlling OPA1 antiapoptotic function To investigate whether PARL regulates the core apoptotic machinery, we turned to mouse embryonic fibroblasts (MEFs). SV-40 transformed Part^1' MEFs proved more susceptible to a panoply of intrinsic pro-apoptotic stimuli acting via mitochondria, including the "BH3-only" member of the BCL-2 family BID. Conversely, death by the extrinsic stimulus TNF-alfa was comparable in wt and Part^1' MEFs, consistent with the equivalent cleavage of the apical caspase-8, as judged by specific immunoblotting. Of note, apoptosis induced by TNF-alfa in wt and Part^1' MEFs was not prevented by overexpression of BCL-2, indicating that these are "type I" cells where recruitment of death receptors results in direct activation of caspase-3 by caspase-8 (Scaffidi et al., 1998). Primary MEFs from a different clone also displayed increased sensitivity to intrinsic stimuli, confirming that this was not a consequence of transformation or was clonally selective. Reintroduction of active Parl in Part^1' MEFs decreased death to wt levels, irrespectively of the death stimulus employed. Conversely, a catalytically inactive mutant of Parl, 335His to GIy (H335G), was unable to rescue the pro-apoptotic phenotype of knockout cells. Taken together, these data show that catalytically active PARL is a required component to keep in check apoptosis induced by intrinsic, mitochondria utilizing stimuli. We addressed whether PARL exerted its antiapoptotic effect at the mitochondrial level. Following treatment with hydrogen peroxide Part^1' MEFs released cytochrome c more rapidly than their wt counterparts. Mitochondrial dysfunction, which accompanies cytochrome c release, also occurred faster in Part^1' MEFs. This was not related to differences in BAK activation, as demonstrated by its comparable oligomerization. We next turned to a reconstituted, in vitro system in which mitochondria isolated from wt and Part^1' mouse livers were treated with recombinant caspase-8 cleaved BID (p7/p15 BID) and cytochrome c release was quantified by a specific ELISA. Mitochondria lacking PARL released cytochrome c faster than their wt counterparts. This was not due to enhanced BAK oligomerization, as p7/p15 BID induced the appearance of higher order BAK oligomers with the same kinetics in wt and Part^1' mitochondria. We investigated whether Part was required for antiapoptotic function of OPA1. Following transfection of wt and Part^1' MEFs, OPA1 was expressed at similar levels. OPA1 protected wt but not Part^1' MEFs from death by etoposide, staurosporine and H₂O₂. Thus, in the absence of Pan, OPA1 is unable to block apoptosis induced by intrinsic stimuli. Expression of OPA1 in Part^1' MEFs indeed did not reduce cytochrome c release following intrinsic stimuli, nor mitochondrial depolarization. These data show that PARL is positioned upstream of OPA1 in the mitochondrial apoptotic pathway. We therefore addressed whether lack of Part resulted in enhanced cytochrome c mobilization from cristae stores. We measured cytochrome c redistribution following treatment of isolated wt and Part^1' mitochondria with active BID, using a specific assay based on the differential ability of ascorbate and N,N,N',N'-tetramethyl-p- phenylenediamine (TMPD) to reduce free and membrane bound, cristae located cytochrome c, respectively (Scorrano et al., 2002). The ratio of ascorbate-driven over TMPD-driven (ascorbate/TMPD ratio) respiration increases when cytochrome c is mobilized from the cristae (Scorrano et al., 2002). While ascorbate/TMPD ratio of Part^1' mitochondria was comparable to that of wt under resting conditions, it resulted substantially higher following treatment with p7/p15 BID. To investigate the morphological substrate for these differences we acquired transmission electron microscopy (TEM) images of resting and BID treated wt and Part^1' mitochondria, coupled to morphometric analysis (Scorrano et al., 2002). Class Il mitochondria, which account for mobilized cytochrome c, are equally represented in untreated wt and Part^1' samples, but they appear much faster in samples from Part^1' livers. Thus, PARL is required to keep in check cristae remodelling and cytochrome c redistribution following an apoptotic stimulus. We reasoned that PARL can produce an "antiapoptotic" isoform of OPA1 , explaining the interplay between OPA1 and PARL in the pathway of cristae remodelling. Since OPA1 electrophoretic mobility was apparently not affected by lack of Part, we decided to analyze OPA1 submitochondrial localization in wt and Part^1' mitochondria. In wt a small fraction of OPA1 was released from membranes following hypotonic swelling and salt washes, a treatment known to dissociate weakly bound proteins, like cytochrome c, from the inner mitochondrial membrane(Jacobs and Sanadi, 1960). This soluble form of OPA1 was almost entirely lacking in Part^1' mitochondria. Taken together, our data show that PARL is a required component of the pathway controlling cytochrome c mobilization during apoptosis and that it operates upstream of OPA1 , by partially converting it into a protein weakly bound to the inner mitochondrial membrane, facing the intermembrane space. 7. Inactivation of PARL in leukemic B- and T-cells leads to apoptosis of the leukemic cells To determine whether a block of PARL activity results in a cell type specific apoptotic response, we first downregulate PARL mRNA using siRNA technology in in vitro conditions using human cancer cell lines. In a second step, we use in vitro mouse models for leukemia to test the effect of the downregulation of Part on the survival of the leukemic cells. Finally we use an in vivo mouse model with inducible inactivation of Part.

Human T- and B-cell leukemia/lymphoma cell lines (T-cell: jurkat, ALL-SIL, HSB-2, HPB-ALL, B-cell: REH, SU-DHL-6) and myeloid leukemia cell lines (K562, MV4-11, U937, HL-60, EOL-1) are transfected (using AMAXA electroporation) or transduced with siRNA's or retroviral constructs (MSCV hairpin structure construct) to downregulate PARL protein levels. We predict that cell death is induced in those cell lines that are derived from T and B lymphocytes. In addtion, we investigate if downregulation of PARL works synergistically with chemotherapy (cytarabine, daunorubicin, mitoxantrone, etoposide) as well as with small molecule kinase inhibitors (imatinib, SU5614, PP2, K-252a). Similar to the experiments using established human cancer cell lines, we also generate mouse transformed B- and T-cells ex vivo, using expression of activated notchi (T-cell leukemia, as described by Aster J.C. et al., MoI. Cell. Biol. (2000), 20:7505-7515.) or BCR-ABL (B-cell leukemia, as described by Li S. et al., J. Exp. Med. (1999), 189:1399-1412). To generate these cell lines we start from bone marrow cells from control mice, and we downregulate Pan using RNA interference, as described for the human cell lines. In addition, we start from bone marrow isolated from our "floxed" Pan mice (at 4 weeks of age), or from fetal liver cells from these mice. The murine B- and T-cell lines derived in this way are then transduced with a retroviral construct expressing the Cre recombinase in combination with an EGFP marker. The effect of Parl inactivation can then be followed by apoptosis assays and by calculating the percentage of apoptosis in EGFP positive and negative cell populations. Again, the effect of combined inhibition of BCR-ABL (with the ABL inhibitor imatinib) and inactivation of Pan can be studied as well, and it will be determined if there is an additive or synergistic effect.

Finally, based on the results from the in vitro and ex vivo studies, an in vivo mouse study is conducted to determine if downregulation/ablation of Parl activity blocks the development of blood cancer. Transgenic mice harboring the Cre recombinase gene under an IFN-inducible

MX1 promoter (mouse strain B6. Cg-Tg(MxI -cre)1Cgn/J from JAX® Mice stock number

003556) and transgenic mice harboring a Cre/Esr1 fusion protein gene (mouse strain B6.Cg-

Tg(cre/Esr1 )5Amc/J from JAX® Mice stock number 004682) is crossed with mice homozygous and heterozygous for a floxed Pan allele. Bone marrow cells are isolated from those mice and these cells are transduced with viral vectors containing BCR-ABL, NOTCH1-ICN (Aster J.C. et al., MoI. Cell. Biol. (2000), 20:7505-7515; Li et al., J. Exp. Med. (1999), 189:1399-1412). The transduced bone marrow cells are injected into irradiated recipient animals. Once these recipient mice start to develop leukemia/lymphoma, induction of Cre expression leads to generation of heterozygote and full Parl knockout cells. We predict that development of blood cancer will be significantly delayed in animals where the expression of Cre recombinase was induced.

Materials and methods

Generation of Parl deficient mice

A mouse Bac clone containing the 5' end of the Parl gene was isolated from a 129/sVJ ES BAC clone library (Genome Systems). A 9.5 kb BgIII DNA restriction fragment of Parl covering the ATG-start codon, exons 2 and 3 and a part of intron 4 was subcloned into the plasmid vector pUC-18. The hygromycin B resistance gene, driven by the phosphoglycerate kinase (PGK) promoter flanked with two FRT sequences, and one loxP sequence downstream of the hygromycin B resistance gene was inserted in intron 2. A second loxP sequence was inserted into the Smal site in intron 3 (figure 1A). The targeting vector was linearized using Sail and introduced into the ES cell line E14 by electroporation. Hygromycin B resistant (100μg/ml) colonies were screened by Southern blot analysis. Genomic DNA of Parl targeted ES cells was digested with EcoRI and hybridized with a 5' external gDNA probe (400bp Ndel-Bglll fragment), a Hygromycin c DNA probe and a 3' external gDNA probe (800 bp EcoRI-Bglll fragment, figure 1C). Two mutated ES cell lines were microinjected into blastocysts of C57BI/6J mice. Chimeric males were obtained and mated with C57BI/6J females to transmit the modified Parl alleles to the germline. Animals carrying a null allele were obtained after breeding with transgenic females expressing a PGK-driven Cre-recombinase. Determinations of the genotypes of the floxed or knock out mice or yolk sac of embryos were done by Southern blotting or PCR analysis using the probes and primers as indicated in figure 1A.

Morphological procedures and observational assessment

Mice were deeply anaesthetized and fixed by transcardiac perfusion with either modified

Bouin's solution (original Bouin recipe, diluted 1 :3 with 0.1 M PBS, Dulbecco formula) or 10% neutral buffered formaline (NBF). After dissection, tissues of interest were kept in the same fixative overnight on a shaker, rinsed in PBS and after dehydration in graded ethanols embedded in Paraplast Plus (TM), using Clear-Rite as an intermediate. Sections were cut at 7 to 10 μm on a SLEE rotary microtome and mounted on aminosilane - coated glass slides. After deparaffinization in Clear-Rite and rehydration in graded ethanols and antigen retrieval by either limited proteolysis or heating in citrate buffer, sections were blocked consecutively with 3% hydrogen peroxide and 2% bovine serum albumin / 2% normal serum of the secondary antibodies donor species. Bound primary antibodies were detected with HRP - tagged secondaries followed by fluorochrome - labelled tyramide. After counterstaining with bisbenzimide, specimens were photographed with a Zeiss Axioplan 2e microscope. In parallel to detection of cleaved caspase 3, apoptotic cell death in sections was detected by a TUNEL assay (ApopTag ® Peroxidase, Chemicon) according to the instructions of the manufacturer. Briefly, after labeling free 3'OH ends of DNA strand fragments with digoxigenin-tagged - tagged nucleotides, these nucleotides were detected by modified HRP-conjugated anti-dig antibodies, of which the Fc portion has been enzymatically removed to further reduce nonspecific binding, and subsequent incubation with a DAB substrate. PARL^"'^" mice (n=16) and wild-type littermates (n=16) were examined at the age of 4 and 6 weeks. A general clinical and behavorial assessment was performed as recommended (Crawley, 1999). Pinna, cornea and righting reflexes were scored and used as an index of neurological status (O'Donoghue, 1996) Nociception was evaluated by submerging the distal end of the mouse tail in tap water of 50⁰C and measuring time before withdrawal. Acoustic startle was recorded using a startle chamber (Med Associates Inc, Georgia, Vermont) with automatic presentation of 120db white noise pulses and recording of the average amplitude and latency of whole body flinch. Body temperature was measured using a rectal probe before and after a 30' cold challenge at 5°C.

Plasmids Part cDNA was cloned into pSG5 mammalian expression vector. The catalytically inactive Parl H335G mutant cloned into pcDNA3.1 mammalian expression vector was a gift from Dr. Luca Pellegrini (Quebec). pMSCV-OPA1 , pCB6-MFN1 , pCB6-MFN2, mitochondrially targeted dsRED (mtRFP), pEYFP-mito (mtYFP), pEGFP-mito were all described in(Cipolat et al., 2004).

Cell culture and transfection

SV40 transformed as well as primary (passage 3) wt and Part^1' mouse embryonic fibroblast (MEFs) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 50 U/mL Penicillin, 50 μg/mL Streptomycin, 100 μM non essential aminoacids (MEM, Gibco/lnvitrogen) and 2 mM glutamine (Gibco). Transfection was performed using Transfectin Lipid Reagent (Biorad) following manufacturer instructions. In cotransfection experiments, plasmids were always cotransfected in equimolar ratios.

PEG fusion assay For PEG fusion assay, 1x10⁶ MEFs of the indicated genotype were transfected with mtGFP or with mtRFP alone, or cotransfected with mtGFP plus OPA1 or with mtRFP plus OPA1. After 24 hrs cells labelled with different fluorescent proteins were coplated in a 1 :1 ratio onto 13 mm round coverslips. Fusion was induced after 24 hrs by a 60 sec treatment with a 50% (w/V) solution of PEG1500 in PBS (Sigma), followed by extensive washes in DMEM supplemented with 10% FCS. To inhibit de novo synthesis of fluorescent proteins, 30 min before PEG treatment cells were incubated with the protein synthesis inhibitor cycloheximide (20 μg/ml, Sigma), which was subsequently kept in all solutions and tissue culture media until cells were fixed for 30 min with ice-cold 3.7% (V/V) formaldehyde in PBS. Following two washes with PBS, coverslips were mounted on slides using Anti-Fade Reagent (Molecular Probes).

Confocal and epifluorescence imaging For imaging of mitochondrial network, 6x10⁴ cells of the indicated genotype were seeded onto 25 mm-round glass coverslips and transfected as indicated. After 24 hrs cells were incubated in Hank's Balanced Salt Solution (HBSS) supplemented with 10 mM Hepes and coverslips were placed on the stage of a Nikon Eclipse TE300 inverted microscope equipped with a spinning-disk Perkin Elmer Ultraview LCI confocal system, a piezoelectric z-axis motorized stage (Pifoc) and a Orca ER 12-bit CCD camera (Hamamatsu). Cells expressing mtYFP were excited using the 488 nm line of the He-Ne laser (Perkin Elmer) with exposure times of 50 msec using a 6OX 1.4 NA Plan Apo objective (Nikon). Morphometric analysis was performed exactly as described in (Cipolat et al., 2004). For imaging of polykarions, cells were placed on the stage of a Nikon Eclipse E600FN upright microscope equipped with a Biorad Radiance 2100 Confocal Laser Scanning system. Cells were excited using the 457 nm laser line for GFP and the 543 nm line for RFP and emitted light was collected using 515/30 BP and 570 LP filters, respectively.

Analysis of cell death For analysis of cell death, 1x10⁵ MEFs of the indicated genotype were seeded in 12-well plates and after 24 hrs treated as detailed in the text, collected and stained with propidium iodide (0.1 μM) and annexin-V-FLUOS (1mM, Roche Biochemicals). Viability was measured by flow cytometry (FACSCalibur, BD) as the percentage Annexin-V, Pl negative cells. For analysis of apoptosis in transfected cells, 1x10⁵ MEFs of the indicated genotype grown in 12-well plates, were cotransfected with pEGFP and the indicated vector. After 24 hrs cells were treated as detailed in the text, collected at the indicated times and then stained with Annexin-V-Alexa568 (Roche Biochemicals) according to the manufacturer protocol. Apoptosis was measured by flow cytometry (FACSCalibur, BD) as the percentage of annexin-V positive events in the GFP positive population. Isolation, staining and viability assays of lymphocytes

Thymus and spleen suspensions were prepared from animals killed at 8-10 weeks of age, by forcing the organs through a 100 μm mesh screen. Meshes were then washed thoroughly with PBS supplemented with 3% FBS. Erythrocytes from spleen homogenates were lysed by incubation in red blood cells solution (RBC, 178 mM NH₄CI, 3mM NH₄HCO₃) for 20 min at room temperature and lymphocytes were then spun at 20Ox g for 10 min at 4°C. Lymphocytes were resuspended in PBS, 3% FBS, stained with the antibodies detailed in the figure legends for 30 min at 37°C, washed and then analyzed by flow cytometry using with a FACSCalibur cytometer (Becton-Dickinson). For analysis of apoptosis, thymic lymphocytes were triple stained with CD4-FITC, CD8-PECy5 and Annexin-V-Alexa568, while splenic lymphocytes were stained with B220-FITC and Annexin-V-Alexa568 and the percentage of Annexin-V positive events in the gated CD4, CD8 positive or B220 positive population was determined.

Antibodies A Parl carboxyterminal antibody was generated by immunizing rabbits with a synthetic peptide (HEIRTNGPKKGGGSK) coupled to keyhole limpet haemocyanin (KLH). For flow cytometric analysis of lymphocytes the following antibodies (BD Biosciences) were used at a final concentration of 1 μg/ml: FITC-conjugated anti-mouse CD4, PECyδ-conjugated anti-mouse CD8a, FITC-conjugated anti-mouse CD4, PECyδ-conjugated anti-mouse CD8a, and PE- conjugated anti-mouse B220.

For immunoblotting experiments, the following antibodies were employed: Rabbit polyclonal anti-OPA1 antibody [1 :1000 (Misaka et al., 2002); 1 :900 (Cipolat et al., 2004), monoclonal anti- OPA1 (1 :500, BD Biosciences), rabbit polyclonal anti-BAK (1 :1000, Santa Cruz), rabbit polyclonal anti caspase 8 (1 :1000 BD Biosciences). lsotype matched, horseradish peroxidase conjugated secondary antibodies (Sigma) were used followed by detection by chemiluminescence (Amersham).

In vitro mitochondrial assays

Mitochondrial oxygen consumption was measured by using a Clarke-type oxygen electrode (Hansatech Instruments). Mitochondria were incubated in experimental buffer (EB, 125 mM KCI, 10 mM Tris-MOPS, 1 mM KPi, 10 μM EGTA-Tris, pH 7.4, 25°C) supplemented with the substrates detailed in the text. Cytochrome c redistribution and release in response to recombinant p7/p15 BID was determined as described in (Scorrano et al., 2002). For detection of intermembrane space mitochondrial proteins, mitochondria were hypotonically swollen in 10 mM KPi, spun at 10-OOOxg and the pellet was further washed with 100 mM KCI, 1OmM Tris- HCI. The supernatants from the former and the latter washes were pooled and constitute the intermembrane space fraction. Equal amounts of proteins from mitochondrial membranes and intermembrane space fractions were dissolved in gel loading buffer (NuPAGE, Invitrogen) and electrophoresed.

Immunoblottinq Proteins dissolved in reduced gel loading buffer (NuPAGE, Invitrogen) were separated by 4- 12% SDS-PAGE (NuPAGE, Invitrogen) transferred onto PVDF membranes (Millipore) and probed using the antibodies indicated in the text

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Claims

Claims

1. A molecule which: comprises a region specifically binding to PARL or nucleic acids encoding said PARL, and

- induces apoptosis of B- and T-lymphocytes.

2. A molecule according to claim 1 which is chosen from the group comprising: an antibody or any fragment thereof, and which specifically binds to PARL and is capable of inducing apoptosis of B- and T-lymphocytes, a small molecule specifically binding to PARL or nucleic acids encoding said PARL and is capable of inducing apoptosis of B- and T- lymphocytes,

- a ribozyme against nucleic acids encoding PARL and is capable of inducing apoptosis of B- and T- lymphocytes, and - anti-sense nucleic acids hybridising with nucleic acids encoding PARL and is capable of inducing apoptosis of B- and T- lymphocytes, and a locked nucleic acid hybridizing with RNA molecules encoding PARL and is capable of inducing apoptosis of B- and T- lymphocytes.

3. A molecule according to any of claims 1 to 2 for use as a medicament.

4. Use of a molecule according to any of claims 1 to 3 for the preparation of a medicament to treat lymphomas.

5. A transgenic, non-human animal characterised by having an endogenous nucleic acid sequence encoding a non-functional PARL expression.

6. A transgenic, non-human animal according to claim 5 wherein said non-functional PARL expression is in a specific tissue or in a specific organ.