WO2013053765A1 - Modèle animal non humain de lymphome du tissu lymphoïde associé aux muqueuses (malt) - Google Patents

Modèle animal non humain de lymphome du tissu lymphoïde associé aux muqueuses (malt) Download PDF

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WO2013053765A1
WO2013053765A1 PCT/EP2012/070070 EP2012070070W WO2013053765A1 WO 2013053765 A1 WO2013053765 A1 WO 2013053765A1 EP 2012070070 W EP2012070070 W EP 2012070070W WO 2013053765 A1 WO2013053765 A1 WO 2013053765A1
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cell
cells
gene
human
lymphoma
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Cesar José COBALEDA HERNÁNDEZ
Lorena FONTÁN GABÁS
José Ángel MARTÍNEZ-CLIMENT
Isidro SÁNCHEZ-GARCÍA
Carolina VICENTE-DUEÑAS
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Proyecto De Biomedicina Cima, S.L.
Consejo Superior De Investigaciones Científicas
Universidad De Salamanca
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE

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  • the present invention relates to a transgenic non-human animal whose genome comprises a polynucleotide comprising a hematopoietic stem cell-specific transcriptional regulatory sequence operatively linked to a heterologous nucleotide sequence encoding a human MALT1 protein.
  • This animal model is useful for studying human B-cell lymphoma, such as Mucosa- Associated Lymphoid Tissue (MALT) lymphoma or activated B-cell like Diffuse Large B-Cell Lymphoma (ABC-DLBCL).
  • MALT Mucosa- Associated Lymphoid Tissue
  • ABSC-DLBCL activated B-cell like Diffuse Large B-Cell Lymphoma
  • the invention also relates to methods for the screening of compounds with pharmacologically relevant activities using said transgenic non-human animals.
  • Mucosa-associated lymphoid tissue (MALT) lymphoma is a distinct clinico-pathologic entity that accounts for approximately 8% of all non-Hodgkin lymphomas. MALT lymphomas can be distinguished from other lymphomas because they occur in various extranodal locations, primarily in the stomach where they are preceded by Helicobacter pylori infection. In addition, they develop in the ocular adnexa, lungs, salivary glands, intestinal tract, skin, thyroid and genitourinary tract, being associated with chronic microbial infections or autoimmune disorders.
  • MALT lymphomas show a typical histopathological picture composed of a morphologically heterogeneous neoplastic B- cell population that arises from the marginal zone of reactive B-cell follicles, extends to the inter- follicular region and infiltrates the epithelium, forming the characteristic lympho epithelial lesions.
  • the lymphoma cells usually express IgM, CD20, CD79a and the marginal zone cell-associated antigens CD21 and CD35, with low or null IgD expression.
  • most MALT lymphomas typically show prominent plasmacytic differentiation (Farinha and Gascoyne, 2005; Isaacson and Du, 2004).
  • MALT lymphomas are mainly associated with two chromosomal translocations involving the MALT1 gene.
  • the t(l I;18)(q21;q21) generates an API2- MALT1 fusion transcript and occurs in up to 30% of the cases, whereas the t( 14; 18)(q32;q21 ) results in MALT1 gene deregulation through juxtaposition with immunoglobulin (Ig) gene enhancers and is detected in 15-20% of cases.
  • Ig immunoglobulin
  • a third translocation present in ⁇ 1% of MALT lymphomas is the t(l;14)(p22;q32), which forms a BCLIO-IGH fusion causing BCL10 over-expression.
  • MALT1 and BCL10 physically cooperate in activating the NF- ⁇ pathway. Indeed, stimulation of the B-cell antigen receptor (BCR) in lymphocytes activates protein- kinase-C that in turn phosphorylates the scaffolding protein CARD11, which subsequently recruits both BCL10 and MALT1 to form the CARD 11 /BCL 10/MALT 1 "signalosome".
  • BCR B-cell antigen receptor
  • MALT1 functions as the effector protein and mediates activation of the IKK complex, leading to the release of NF- ⁇ which translocates to the nucleus to regulate target genes involved in immune responses to foreign antigens.
  • the caspase-like domain of MALT 1 shows proteolytic activity and can cleave BCL 10 and the NF- ⁇ inhibitor TNFAIP3 (A20).
  • MALT lymphomas generally show an indolent course. While some patients can be cured by eradicating the causative infectious agent, most others require radio- chemo-immunotherapy, and 15-20% of the cases eventually transform to diffuse large B-cell lymphoma (DLBCL) which is frequently refractory to common therapies.
  • DLBCL B-cell lymphoma
  • emerging data implicate the CARD 11 /BCL 10/MALTl complex in the pathogenesis of activated B-cell-like (ABC) DLBCL, a distinct lymphoma subtype that can be distinguished from the germinal center B-cell like (GCB) DLBCL.
  • a hallmark of ABC-DLBCLs is the constitutive activation of the NF- ⁇ pathway, which is caused by various mutations in genes regulating NF- ⁇ , including those that activate CARD 11 or repress TNFAIP3 genes.
  • ABC-DLBCLs display somatic mutations in CD79A and CD79B which induce chronic BCR signaling, and in MyD88 (an adaptor protein that mediates toll and interleukin receptor signaling), which leads to CARD 11 phosphorylation, BCL 10- MALT1 complex formation and NF- ⁇ activation.
  • MyD88 and TNFAIP3 genes have been also found in MALT lymphomas, suggesting a molecular link between these two lymphoma entities.
  • US patent application US2006117399 discloses an animal model for human lymphomas obtained by targeting expression of NF-KB2 in both B and T-lymphocytes.
  • the invention relates to a polynucleotide comprising
  • hematopoietic stem cell-specific transcriptional regulatory sequence (i) a hematopoietic stem cell-specific transcriptional regulatory sequence, and (ii) a nucleotide sequence encoding a MALT1 protein or a functionally equivalent variant thereof,
  • the invention relates also to vectors and cells comprising said polynucleotide as well as to a transgenic non-human animal whose genome comprises said polynucleotide.
  • the invention relates to the use of a transgenic non-human animal of the invention as an animal model for studying a human B-cell lymphoma, to a cell derived from said transgenic non-human animal and to the use of said transgenic non- human animal or a cell derived from said transgenic animal for the identification of inhibitors of MALT 1 protease activity.
  • the invention relates to the use of a transgenic non-human animal according to the invention or to a cell derived from said non-human transgenic animal for
  • the invention in another aspect, relates to a method for producing a transgenic non- human animal, said method comprising chromosomally incorporating into the genome of said non-human animal a polynucleotide encoding MALT1 or a functionally equivalent variant thereof, operatively linked to a tissue-specific promoter which is specific for expression in hematopoietic stem/progenitor cells.
  • FIG. 1 Generation of a mouse model of ectopic expression of MALTl under the control of Seal promoter.
  • A Schematic representation of the genomic structure of the mouse Seal locus and the Seal -MALTl transgenic vector, indicating NotI sites used to excise the transgene fragments and EcoRI sites used to examine Southern blots.
  • B RT-PCR analysis revealed MALTl expression in stem/progenitor (Scal+Lin ) cells from the BM of Seal -MALTl mice but not in normal stem/progenitor cells of WT mice.
  • C MALTl immunofluorescence of cells sorted from Seal- MALTl mice (Scal+Lin- from bone marrow; CD19+IgM+ from spleen).
  • the human MALT1- expressing SSK41 cell line was used as a positive control.(Sanchez-Izquierdo et ah, 2003) MALTl is shown in green and nuclei counterstained with DAPI in blue.
  • D-H Flow cytometry analysis (FACS) of hematopoietic cell compartments in Seal -MALTl mice. Two month-old mice had an expanded hematopoietic stem/progenitor cell compartment (D), as shown by an increase in the proportion of Scal+Kit+Lin- cells. There was selective expansion of pro-B (E) and pre-B lymphoid (F) cells in BM.
  • FIG. 4 Histopathological studies of the human-like MALT lymphomas arising in Seal- MALT1 mice. Representative hematoxylin-eosin stainings of mouse lymphomas. Image magnification is indicated.
  • A Lymphoid infiltrate in the small intestine of Seal - MALT1 mice. Tumor cells infiltrate the lamina intestinal and the epithelium in small groups, giving rise to lymphoepithelial lesions. The destruction of intestinal crypts by tumor cells is accompanied by the presence of surrounding plasma cells.
  • B Lymphoid tumor cells also infiltrate other tissues.
  • C The epithelium of the infiltrated tissues is also damaged, as shown in salivary glands, lung and in the tubuli and glomeruli of the kidney.
  • C In 15% of Scal-MALTl mice, spontaneous transformation of low-grade lymphomas was accompanied by high-grade lymphomas resembling DLBCL.
  • the figure shows a typical intestinal MALT lymphoma (CI) that infiltrates lung (C2) and salivary gland (C3), evolving into a high-grade lymphoma resembling DLBCL, involving spleen (C4), stomach (C5), kidney (C6) and liver (C7).
  • C Histopathological analysis of a Scal-MALTl lymphoma in the kidney showing prominent plasma cells surrounding blood vessels.
  • E-F Representative immunohistochemical analysis of Scal-MALTl lymphomas. To determine the identity of the neoplastic cells, stainings were performed using the B-cell specific markers CD20 and Pax5, as well as IgM, IgD, and the T-cell marker CD3. The presence of neoplastic B-cells was detected in small intestine, kidney, salivary gland, lung and spleen. Magnification is 200X for figures Al- A5, B5-B8, Dl and E1-E5, 400X for figures A6-A9, B1-B4, D2-D3 and all figures in C, and 40X for figures in F.
  • FIG. 5 Gene expression profiling of Scal-MALTl lymphomas and human MALT lymphomas.
  • A Cells from splenic lymphomas from Scal-MALTl transgenic mice or spleens from their WT counterparts were isolated and analyzed using gene expression microarrays, defining the Scal-MALTl lymphoma transcriptional signature (B>0; 291 probes, 246 genes).
  • F Genes related to plasma cell differentiation were upregulated in Scal+Lin— sorted cells from transgenic Scal-MALTl relative to WT mice (including Ig genes, Xbpl and Prdml). The XBP1 target gene signature was then studied using GSEA in all datasets, showing significant enrichment in sorted progenitor cells, mice tumors and human MALT lymphomas.
  • G The human MALT lymphoma transcriptional signature was significantly enriched in the mouse lymphomas (FDR q ⁇ 0.0001).
  • FIG. 6 Scal-MALTl-p53-/- mice develop lymphomas displaying the main histopathological and immunohistochemical features of ABC-DLBCL.
  • A Kaplan- Meier survival plots of Scal-Maltl mice, p53-/- mice, Scal-Maltl , p53-/- mice and control mice. The total number of mice analyzed in each group is indicated. Statistical analysis of differences in survival was performed using the log-rank (Mantel-Cox) test, and the corresponding P value is given.
  • C, D Representative histopathological (HE) staining and immunohistochemical analysis of the lymphomas developed in Scal-Maltl , p53-/- mice. Tumor expressed CD20 and Foxpl, while they were negative for Muml, Gcetl, CD 10 and Bcl6, allowing their classification as a human- like ABC-DLBCL.
  • the image magnification is 20X and 100X for HE staining images, and 40X for all IHC images.
  • FIG. 7 Malt protease activity is present in mouse lymphoma cells.
  • B Total splenic extracts from WT mice or Seal -MALT 1 splenic lymphomas were assayed for MALT1 proteolytic activity over BcllO. Lane 1 (left): BcllO recombinant protein. Lanes 2 and 3: extracts from Scal-MALTl splenic lymphomas without or with BcllO recombinant protein, respectively.
  • Lanes 4 and 5 extracts from WT spleens without or with BcllO recombinant protein, respectively.
  • C Proteolytic activity of MALT1 over BcllO was tested in BaF3 cell extracts. Cell extracts were assayed for MALT1 mediated cleavage and Western blot for BcllO was performed. Lane 1 : protein extract with recombinant BcllO, Lane 2: recombinant BcllO protein, Lane 3: protein extract. ( ⁇ ) cleaved BcllO.
  • the present invention relates to a transgenic non-human animal that can be used as a model of human B-cell lymphomas as well as to the polynucleotide molecules required for obtaining said transgenic non-human animals.
  • lymphoma development has been modelled in mice by targeting oncogene expression to committed B lymphocytes.
  • this strategy has failed to generate experimental models of MALT lymphoma.
  • the inventors have engineered transgenic mice wherein the expression of human MALT1 protein is targeted to the hematopoietic stem/progenitor cell compartment by placing the coding region of MALT1 under the control of the Ly-6E. l promoter. These animals fully recapitulate the features of human MALT lymphoma.
  • mice expression of MALT1 in hematopoietic stem/progenitor cells modulated their normal expression profile by activating NF-KB signaling as well as pro -inflammatory and plasmacytic differentiation pathways, induced the expansion of the Scal+ cells, and promoted selective B-cell differentiation and mature lymphocyte accumulation in extranodal tissues (salivary glands, intestine, orbit, lung, etc) that led to the development of clonal B-cell lymphomas.
  • mouse lymphomas accurately reflected the main clinical, histopathological and molecular features of human MALT lymphomas.
  • the invention relates to a polynucleotide, hereinafter polynucleotide of the invention, comprising
  • hematopoietic stem cell-specific transcriptional regulatory sequence (i) a hematopoietic stem cell-specific transcriptional regulatory sequence, and (ii) a nucleotide sequence encoding a MALT1 protein or a functionally equivalent variant thereof,
  • polynucleotide refers to a polymer composed of a multiplicity of nucleotide units (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural variants on synthetic analogues thereof).
  • polynucleotide includes double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense polynucleotide (although only sense stands are being represented herein).
  • polynucleotide sequences are understood to encompass complementary strands as well as alternative backbones described herein.
  • the polynucleotide comprises a hematopoietic stem cell-specific transcriptional regulatory sequence operatively linked to a nucleotide sequence encoding a MALT1 protein or an equivalent functional variant thereof.
  • a transcriptional regulatory sequence refers to a sequence that controls and regulates transcription and, where appropriate, the translation of the polynucleotide of the invention.
  • a transcriptional regulatory sequence includes promoter sequences, sequences encoding transcriptional regulators, ribosome binding sequences (RBS) and/or transcription terminator sequences.
  • a hematopoietic stem cell-specific transcriptional regulatory sequence relates to the transcriptional regulatory sequence which is capable of activating transcription of an operatively linked gene in hematopoietic stem cells and/or progenitor cells but not in differentiated cells, in particular, in differentiated blood cells.
  • HSCs are multipotent stem cells that give rise to all the blood cell types from the myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells).
  • a hematopoietic stem cell-specific transcriptional regulatory sequence allows expression of the polynucleotides which are operatively coupled to it in hematopoietic stem cells and/or in blood progenitor cells but not in mature blood cells such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and cells of the lymphoid lineages (T-cells, B-cells, NK-cells).
  • Suitable methods for the determination of whether a transcriptional regulatory sequence is hematopoietic stem cell-specific have been described, for instance, in Miles et al.
  • transgenic mice wherein a reporter gene is operatively linked to the hematopoietic stem cell-specific transcriptional regulatory sequence followed by:
  • hematopoietic stem cells include, without limitation, Sca-1, CD341o/-, SCA-1+ , Thyl .
  • mature blood cells such as mature blood lymphocytes (identified e.g. by expressing CD 19 and IgM).
  • Suitable hematopoietic stem cell transcriptional regulatory sequences include, without limitation, the Ly-6E. l gene promoter and any functionally equivalent variant thereof.
  • the Ly-6E. l gene promoter is the sequence which regulates transcription of the gene which encodes Sca-1.
  • the Sca-1 is encoded by the allelic Ly-6E.l and Ly-6A.2 genes which are members of the Ly-6 family consisting of at least 18 highly related genes.
  • the Ly-6E.l and Ly-6A.2 genes contain four exons that encode 876 and 830 base transcripts, respectively, and a 10-12 kDa GPI-linked cell surface glycoprotein.
  • telomere binding motifs and promoters involved in hematopoietic stem cell specific transcription have been described [Onyango et al. (1999) Exp. Hematol. 27, 313-25; Meng et al. (1999) Blood 93, 500-8; Nony et al. (1998) J. Biol. Chem. 273, 32910-9; Ye et al. (1998) Hum. Gene Ther. 9, 2197-205].
  • the pLy-6El promoter is well characterized and contains all the elements necessary for the selective expression in Scal + cells (Miles C. et al, 1997, Development 124:537- 547).
  • the promoter that directs the expression of said activatable gene in Scal + cells is the mouse pLy-6E. l promoter or a functional fragment or equivalent thereof.
  • the pLy-6El promoter region comprises at least nucleotides 3000 to 3501 of the polynucleotide shown in the NCBI database under accession number M37707 (version of June 23, 2010) (SEQ ID NO: l), at least nucleotides 2500 to 3501 of the polynucleotide shown in the NCBI database under accession number M37707 (version of June 23, 2010) (SEQ ID NO:2), at least nucleotides 2000 to 3501 of the polynucleotide shown in the NCBI database under accession number M37707 (version of June 23, 2010) (SEQ ID NO:3), at least nucleotides 1500 to 3501 of the polynucleotide shown in the NCBI database under accession number M37707 (version of June 23, 2010) (SEQ ID NO:4), at least nucleotides 1000 to 3501 of the polynucleotide shown in the NCBI database under accession number M37707 (version of June 23, 2010) (SEQ
  • the invention also contemplates polynucleotides wherein the sequence encoding the MALT1 protein or the functionally equivalent variant thereof is operatively linked to additional regions of the Ly-6E.l gene which may have a role in determining specific expression in the hematopoietic stem/progenitor cell compartment.
  • polynucleotide of the invention further comprises the sequence as defined in SEQ ID NO:7, corresponding to 3' distal region in the Ly-6E.l as defined by Ma et al. (Br. J. Haematol. 2001, 114:724-30) and shown in the NCBI database under accession number L76154 (database version of September, 9, 1996).
  • the polynucleotide of the invention comprises, in the 5' to 3' orientation:
  • a first polynucleotide comprising a sequence selected from the group consisting of: a. a sequence comprising at least nucleotides 3000 to 3501 of the polynucleotide shown in the NCBI database under accession number M37707 (version of June 23, 2010) (SEQ ID NO: l),
  • the polynucleotide of the invention comprises a 98 kb fragment downstream from the transcriptional start site. In another embodiment, the polynucleotide of the invention comprises a 14 kb fragment which comprises the complete Ly-6E. l gene. The 14 kb fragment is flanked by BamHI sites in the mouse genome. In yet another preferred embodiment, the polynucleotide of the invention comprises the complete Ly-6E. l gene. In another embodiment, the polynucleotide of the invention comprises the complete Ly-6E.
  • the polynucleotide encoding the MALT1 protein or the functionally equivalent variant thereof is inserted within the first exon of the Ly-6E. l gene, which corresponds to part of the 5'-UTR region of the Ly-6E. l mR A.
  • the skilled person will appreciate that the transcription regulatory regions derived from the Ly-6E.
  • the invention contemplates polynucleotides wherein the polynucleotide encoding the MALT1 protein or the functionally equivalent variant thereof is under the control of functionally equivalent variants of the transcriptional regulatory regions in the Ly-6E.l gene.
  • Variants of the transcription regulatory regions derived from the Ly-6E. l gene according to the present invention include polynucleotides that are at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98% or 99% similar or identical to the sequence of the transcription regulatory regions derived from the Ly-6E. l gene (either the mouse or the human gene).
  • the "similarity" between two polynucleotides is determined by comparing the nucleic acid sequence and its conserved nucleotide substitutes of one polynucleotide to a sequence of a nucleic acid sequence.
  • the degree of identity between two polynucleotides is determined using computer algorithms and methods that are widely known for the persons skilled in the art.
  • Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms Both the BLASTN and BLASTP software are available on the NCBI anonymous FTP server (ftp://ncbi The BLASTN algorithm version 2
  • the use of the BLAST family of algorithms, including BLASTN and BLASTP, is described at NCBI's website at URL http://www.ncbi.nlm.nih.gov/BLAST/newblast.html and in the publication of Altschul, Stephen F., et al.
  • the computer algorithm FASTA is available on the Internet at the ftp site ftp://ftp.virginia.edu/pub/fasta/. Version 2.0u4, February 1996, set to the default parameters described in the documentation and distributed with the algorithm, is also preferred for use in the determination of variants according to the present invention.
  • the use of the FASTA algorithm is described in Pearson and Lipman, (Proc. Natl. Acad. Sci. USA, 1998, 85:2444-2448) and Pearson (Methods in Enzymo logy, 1990, 183:63-98).
  • the term "functionally equivalent variant" of a transcriptional regulatory region refers to nucleic acids which specifically hybridize to the native transcriptional regulatory region of the Ly.6E.1 gene.
  • stringent conditions means hybridization to filter-bound nucleic acid in 6xSSC at about 45 Degrees C followed by one or more washes in 0. lxSSC/0.2% SDS at about 68°C.
  • Other exemplary stringent conditions may refer, e. g., to washing in 6xSSC/0.05% sodium pyrophosphate at 37°C, 48°C, 55°C, and 60°C as appropriate for the particular probe being used.
  • moderate conditions means hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45 Degrees C followed by one, preferably 3-5 washes in 0.2xSSC/0.1% SDS at about 42-65 Degrees C.
  • SSC sodium chloride/sodium citrate
  • the invention contemplates the use of the native transcription regulatory regions derived from the Ly-6E. l gene in the 5' and 3' region, the use of the native transcription regulatory regions derived from the Ly-6E.
  • MALTl protein refers to the protein encoded by the mucosa associated lymphoid tissue lymphoma translocation gene 1.
  • the MALTl protein refers to the MALT protein from Homo sapiens (hMALTl gene) and, in particular, to any of the two alternatively spliced transcript variants encoding different iso forms of the MALTl protein.
  • the transcript variant 1 (NCBI Reference Sequence: NM 006785.2, GL27886564, PRI 14-AUG-2011) encodes to isoform a of the MALTl protein (NCBI Reference Sequence NP 006776.1, GL5803078, PRI 14- AUG-2011) and the transcript variant 2 (NCBI Reference Sequence: NM_173844.1, GL27886565, PRI 14-AUG-2011) encodes the isoform ⁇ of the MALTl protein (NCBI Reference Sequence NP 776216.1, GL27886566, PRI 14-AUG-2011). Either of the two transcripts or isoforms can be used interchangeably in the context of the present invention.
  • the present invention also includes functionally equivalent variants of MALTl protein.
  • the term "functional equivalent variant”, as used herein relates to any polypeptide, the sequence of which can be obtained from the sequence of MALTl protein as defined above by means of insertion of one or more amino acids in the sequence, the addition of one or more amino acids in any end or inside the sequence, or the deletion of one or more amino acids in any end or inside the sequence, and which substantially preserves the biological activity of native MALTl protein. Suitable methods for determining if a function of the native MALTl protein is substantially maintained include, but no limiting to, in vitro MALTl protease activity assay as shown in the Example of the present patent application.
  • Variants of MALTl protein may be obtained by substituting nucleotides within the polynucleotide accounting for codon preference in the host cell that is to be used to produce the MALTl protein.
  • Such "codon optimization” can be determined via computer algorithms which incorporate codon frequency tables such as "ecohigh. cod” for codon preference of highly expressed bacterial genes as provided by the university of Wisconsin package version 9.0, genetics computer group, madison,wis. other useful codon frequency tables include “celegans_ high.cod”, “celegans low.cod”, drosophila high.cod”, “human_ high.cod”, “maize_ high.cod”, and "yeast_ high.cod”.
  • Variants of MALTl protein may also be generated by making conservative amino acid changes and testing the resulting variant in one of the functional assays described above or another functional assay known in the art.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine
  • a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine
  • a group of amino acids having amide-containing side chains is asparagine and glutamine
  • a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan
  • a group of amino acids having basic side chains is lysine, arginine, and histidine
  • a group of amino acids having sulfur- containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-i
  • variants of the MALTl protein may be: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code; (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups; (iii) one in which the protein is an alternative splice variant of the proteins of the present invention and/or; (iv) fragments of the proteins.
  • the fragments include proteins generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants are deemed to be within the scope of those skilled in the art from the teaching herein.
  • Variants according to the present invention include amino acid sequences that are at least 60%, 70%, 80%, 90%, 95% or 96% similar or identical to the original amino acid sequence of the MALT 1 protein.
  • the "similarity" between two proteins is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one protein to a sequence of a second protein.
  • the degree of identity between two proteins is determined using computer algorithms and methods that are widely known for the persons skilled in the art.
  • the identity between two amino acid sequences is preferably determined by using the BLASTP algorithm [BLASTManual, Altschul, S., et aL, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990)].
  • the proteins can be post-translationally modified.
  • post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc.
  • the proteins may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation.
  • variants or fragments of MALT 1 protein can be generated using conventional techniques, such as mutagenesis, including creating discrete point mutation(s), or by truncation. For instance, mutation can give rise to variants which retain substantially the same, or merely a subset, of the biological activity of a polypeptide from which it was derived.
  • the expression "operatively linked” means that the transcriptional regulatory sequence is covalently bound to the nucleotide sequence encoding the human MALT1 protein (or an equivalent functional variant thereof as defined above) and that both are arranged so that expression of the nucleotide sequence encoding the MALT1 protein occurs under the control of the transcriptional regulatory sequence.
  • Regulating sequences useful for the present invention are hematopoietic stem cell-specific promoting sequences or, alternatively, enhancer sequences and/or other regulating sequences increasing the expression of the heterologous nucleic acid sequence in hematopoietic stem cells.
  • the polynucleotide of the invention can be found isolated as such or forming part of vectors allowing the propagation of said polynucleotides in suitable host cells. Therefore, in another aspect, the invention relates to a vector, hereinafter vector of the invention, comprising the polynucleotide of the invention as described above.
  • Vectors suitable for the insertion of said polynucleotide are vectors derived from expression vectors in prokaryotes such as pUC18, pUC19, Bluescript and the derivatives thereof, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phages and "shuttle" vectors such as pSA3 and pAT28; expression vectors in yeasts such as vectors of the type of 2 micron plasmids, integration plasmids, YEP vectors, centromere plasmids and the like; expression vectors in insect cells such as vectors of the pAC series and of the pVL; expression vectors in plants such as pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series and the like; and expression vectors in eukaryotic cells, including baculovirus suitable
  • the vectors for eukaryotic cells include preferably viral vectors (adenoviruses, viruses associated to adenoviruses such as retroviruses and, particularly, lentiviruses) as well as non-viral vectors such as pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1/hyg, pHMCV/Zeo, pCR3.1, pEFI/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAXl, pZeoSV2, pCI, pSVL and PKSV-10, pBPV-1, pML2d and pTDTl .
  • viral vectors adenoviruses, viruses associated to adenoviruses such as retroviruses and, particularly, lentiviruses
  • non-viral vectors such as p
  • the vectors may also comprise a reporter or marker gene which allows identifying those cells that have been incorporated the vector after having been put in contact with it.
  • reporter genes in the context of the present invention include lacZ, luciferase, thymidine kinase, GFP and on the like.
  • Useful marker genes in the context of this invention include, for example, the neomycin resistance gene, conferring resistance to the aminoglycoside G418; the hygromycin phosphotransferase gene, conferring resistance to hygromycin; the ODC gene, conferring resistance to the inhibitor of the ornithine decarboxylase (2-(difluoromethyl)-DL-ornithine (DFMO); the dihydrofolatereductase gene, conferring resistance to methotrexate; the puromycin-N- acetyl transferase gene, conferring resistance to puromycin; the ble gene, conferring resistance to zeocin; the adenosine deaminase gene, conferring resistance to 9-beta-D- xylofuranose adenine; the cytosine deaminase gene, allowing the cells to grow in the presence of N-(phosphonacetyl)-L-aspartate; th
  • the selection gene is incorporated into a plasmid that can additionally include a promoter suitable for the expression of said gene in eukaryotic cells (for example, the CMV or SV40 promoters), an optimized translation initiation site (for example, a site following the so-called Kozak's rules or an IRES), a polyadenylation site such as, for example, the SV40 polyadenylation or phosphoglycerate kinase site, introns such as, for example, the beta-globulin gene intron.
  • a promoter suitable for the expression of said gene in eukaryotic cells for example, the CMV or SV40 promoters
  • an optimized translation initiation site for example, a site following the so-called Kozak's rules or an IRES
  • a polyadenylation site such as, for example, the SV40 polyadenylation or phosphoglycerate kinase site
  • introns such as, for example, the beta-globulin gene in
  • the choice of the vector will depend on the host cell in which it will subsequently be introduced.
  • the vector in which said polynucleotide is introduced can also be a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or a PI -derived artificial chromosome (PAC).
  • YAC yeast artificial chromosome
  • BAC bacterial artificial chromosome
  • PAC PI -derived artificial chromosome
  • the vector of the invention can be obtained by conventional methods known by persons skilled in the art [Sambrook J. et al, 2000 "Molecular cloning, a Laboratory Manual", 3 rd ed., Cold Spring Harbor Laboratory Press, N.Y. Vol 1-3].
  • the polynucleotide of the invention can be introduced into the host cell in vivo as naked DNA plasmids, but also using vectors by methods known in the art, including but not limited to transfection, electroporation (e.g. transcutaneous electroporation), microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter.
  • electroporation e.g. transcutaneous electroporation
  • microinjection e.g. transcutaneous electroporation
  • Biolistic transformation is commonly accomplished in one of several ways.
  • One common method involves propelling inert or biologically active particles at cells. See Sanford J, et al, US 4,945,050, US 5,036,006, and US 5,100,792.
  • the vector can be introduced in vivo by lipofection.
  • cationic lipids can promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes. See Feigner P, Ringold G, Science 1989; 337:387-388. Particularly useful lipid compounds and compositions for transfer of nucleic acids have been described. See Feigner P, et al, US 5,459,127, Behr J, et al, WO1995018863, and Byk G, WO1996017823.
  • the invention relates to a cell, hereinafter cell of the invention, comprising the polynucleotide of the invention or a vector of the invention.
  • the cells can be obtained by conventional methods known by persons skilled in the art [see e.g. Sambrook et al, cited ad supra] as explained before.
  • a "host cell” includes any cultivatable cell that can be modified by the introduction of heterologous DNA.
  • a host cell is one in which the polynucleotide of the invention can be stably expressed, post-translationally modified, localized to the appropriate subcellular compartment, and made to engage the appropriate transcription machinery. The choice of an appropriate host cell will also be influenced by the choice of detection signal.
  • reporter constructs can provide a selectable or screenable trait upon activation or inhibition of gene transcription in response to a transcriptional regulatory protein; in order to achieve optimal selection or screening, the host cell phenotype will be considered.
  • a host cell of the present invention includes prokaryotic cells and eukaryotic cells.
  • Prokaryotes include gram negative or gram positive organisms, for example, E. coli or Bacilli. It is to be understood that prokaryotic cells will be used, preferably, for the propagation of the transcription control sequence comprising polynucleotides or the vector of the present invention.
  • Suitable prokaryotic host cells for transformation include, for example, E.
  • Eukaryotic cells include, but are not limited to, yeast cells, plant cells, fungal cells, insect cells (e.g., baculovirus), mammalian cells, and the cells of parasitic organisms, e.g., trypanosomes.
  • yeast includes not only yeast in a strict taxonomic sense, i.e., unicellular organisms, but also yeast-like multicellular fungi of filamentous fungi.
  • Exemplary species include Kluyverei lactis, Schizosaccharomyces pombe, and Ustilaqo maydis, with Saccharomyces cerevisiae being preferred.
  • Other yeasts which can be used in practicing the present invention are Neurospora crassa, Aspergillus niger, Aspergillus nidulans, Pichia pastoris, Candida tropicalis, and Hansenula polymorpha.
  • Mammalian host cell culture systems include established cell lines such as COS cells, L cells, 3T3 cells, Chinese hamster ovary (CHO) cells, embryonic stem cells, with BHK, HeK or HeLa cells being preferred.
  • Eukaryotic cells are, preferably, used to for recombinant gene expression by applying the transcription control sequence or the expression vector of the present invention.
  • the described polynucleotides, vectors or cells of the invention can be used to obtain a transgenic non-human animal having, inserted in the genome thereof, a hematopoietic stem cell-specific transcriptional regulatory sequence or a functionally equivalent variant thereof operatively linked to a nucleotide sequence encoding a human MALT1 protein or an equivalent functional variant thereof.
  • the invention relates to a transgenic non-human animal, hereinafter transgenic non-human animal of the invention, comprising the polynucleotide of the invention inserted in its genome.
  • transgenic non-human animal of the invention reliably reproduces the phenomena and characteristics of human B-cell lymphomas, preferably, mucosa-associated lymphoid tissue (MALT) lymphoma or activated B-cell like (ABC) Diffuse Large B-Cell Lymphoma (ABC- DLBCL) occurring in humans.
  • MALT mucosa-associated lymphoid tissue
  • ABSC activated B-cell like
  • ABS- DLBCL Large B-Cell Lymphoma
  • the transgenic non- human animal of the invention may possess inserted in its genome one or more copies of the polynucleotide of the invention, preferably, between 2 and 8 copies, more preferably, between 2 and 4 copies.
  • the genome of the transgenic non-human animal of the invention comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more copies of the polynucleotide of the invention.
  • the transgenic non-human animal is a mammal.
  • non-human mammal animals include, without limitation, those belonging to one of the following: the family Bovidae, the family Canidae, the family Suidae, the order Rodentia, the order Lagomorpha, and the order Primates (excluding humans). More preferably, the non- human mammal is selected from the group consisting of a cow, a dog, a pig, a sheep, a goat, a rat, a mouse, a rabbit, a chimpanzee, and a gorilla.
  • the transgenic non- human animal of the invention is a rodent, preferably a mouse or a rat.
  • the transgenic non- human animal of the invention can have any genetic background of those known in the state of the art by a person skilled in the art, i.e., said transgenic non- human animal can come from a wild-type (wt) animal or from a non-human animal with a genetic background incorporating any molecular marker that is directly or indirectly related to human B-cell lymphomas, preferably, MALT lymphoma or ABC-DLBCL.
  • the transgenic non-human animal of the invention can express, in addition to the gene encoding the MALT1 protein, one or more genes that are directly or indirectly related to the development of human B-cell lymphomas.
  • Virtually any molecular marker that is directly or indirectly related to the development of human B-cell lymphomas can be used.
  • said genes recognized as molecular markers of human B-cell lymphomas include CD 19, CD20, IgM, IgD, CD79a, CD79b, among others.
  • the inventors have discovered that crossing the transgenic non-human animal of the invention with a p53 _/ ⁇ background accelerates tumor onset and caused the transformation of MALT lymphoma to aggressive ABC-DLBCL.
  • the non-human animal carries an inactivating mutation in at least one allele of the p53 gene.
  • inactivating mutation refers to mutations that partially or completely abrogate the activity of the polypeptide encoded by the mutated polypeptide.
  • inactivating mutations are those which result in a partial or total deficiency in its ability to initiate a DNA-repair response.
  • Suitable mutations leading to inactivation of p53 are usually missense mutations such as those known in the art (Michalovitz et al, J. Cell. Biochem., 1991, 45(l):22-9; Vogelstein and Kinzler, Cell, 1992, 70(4):523-6; Donehower and Bradley, Biochim. Biophys. Acta., 1993, 1155(2): 181- 205; Levine, Cell, 1997, 88(3):323-31).
  • the transgenic non- human animal of the invention homozygous for a totally defective p53 gene (p53 "/_ ).
  • the invention relates to a cell derived from the transgenic non-human animal of the invention or to an isolated cell population which comprises, i.e. that is enriched in, a cell derived from the transgenic non- human animal of the invention.
  • cell population refers to a number of cells obtained by isolation directly from a suitable mammalian tissue source and subsequent culturing in vitro.
  • the cell populations may comprise at last 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the cells according to the invention.
  • isolated when referred to the population of cells, as used herein, refers to a population of cells that has been removed and separated from a biological sample, or a mixed or heterogeneous population of cells found in such a sample.
  • the cell of the invention which is derived from the non- human transgenic animal of the invention is selected from the group consisting of a cancer stem cell, a hematopoietic stem cell and a lymphoma cell.
  • the isolated cell population of the invention is enriched in cells which derive from the non-human transgenic animal of the invention wherein said cells are selected from the group consisting of a cancer stem cell, a hematopoietic stem cell and a lymphoma cell.
  • cancer stem cell and “CSC” and “tumor stem cell” are used interchangeably herein and refer to cells from a cancer that: (1) have extensive proliferative capacity; (2) are capable of asymmetric cell division to generate one or more kinds of differentiated progeny with reduced proliferative or developmental potential; and (3) are capable of asymmetric cell divisions for self-renewal or self-maintenance. These properties confer on the cancer stem cells the ability to form or establish a tumor or cancer upon serial transplantation into an immunocompromised host (e.g., a mouse) compared to the majority of tumor cells that fail to form tumors. Cancer stem cells undergo self-renewal versus differentiation in a chaotic manner to form tumors with abnormal cell types that can change over time as mutations occur. "Cancer stem cell” as used herein may comprise lymphoma-initiating cells.
  • cancer stem cells are Scal+Lin-.
  • Scal+ refers to cells which express the gene products of the Ly-6E. l and/or Ly-6A.2 genes.
  • Lin- or Lineage- refers to cells which do not express one or more lineage markers.
  • a set of 'Lin' markers which are either not expressed or expressed at low levels in murine Lin- cells include CD2, CD3, CD4, CD5, CD8, NK1.1, B220, TER-119, Mac-1 and Gr-1 wherein:
  • - CD2 is expressed on thymic and peripheral T-cells, thymocytes, NK-cells, many thymic B-cells, and may be expressed also on mature B-cells.
  • - CD3 is expressed on thymocytes and T-cells.
  • CD4 is expressed in subsets of thymocyte and T-lymphocytes, peripheral blood monocytes, tissue macrophages, granulocytes.
  • CD5 is expressed at thymocytes, T-cells, a small subset of mature B- lymphocytes.
  • CD8 is expressed in subsets of thymocytes and cytotoxic T-cells
  • - NK1.1 is expressed by the majority of NK-cells and a small subpopulation of T- cells.
  • - B220 is expressed, typically at high levels, on all hematopoietic cells.
  • TER-119 (alternative designation: Ly76) TER-119 is expressed on erythroid cells and used as a marker for the erythroid lineage.
  • Mac-1 (alternative designation CD l ib) Mac-1 is expressed mainly on myeloid cells ((granulocytes, monocytes), NK-cells, and subsets of T-cells and B-cells.
  • - Gr-1 (alternative designation: Ly6G)
  • Gr-1 is a myeloid differentiation antigen expressed by myeloid cells in a developmentally regulated manner in the bone marrow. Monocytes only express Gr-1 transiently during their development in the bone marrow. Expression of Gr-1 on bone marrow granulocytes and peripheral neutrophils is a good marker for these cell types.
  • hematopoietic stem cell or "HSC” as used herein refers to adult multipotent stem cells that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK- cells).
  • hematopoietic stem cells can also refer to stem cells or progenitor cells found in bone marrow and peripheral blood that are greater than about 10 ⁇ in diameter and are capable of differentiating into any of the specific types of hematopoietic or blood cells, such as erythrocytes, lymphocytes, macrophages and megakaryocytes.
  • HSCs are reactive with certain monoclonal antibodies which are now recognized as being specific for hematopoietic cells, for example, CD34+/CD45+.
  • the term HSC includes both short term and long term stem cells.
  • lymphoma cell is a cell sample containing lymphoma cells from a subject or a patient that have been diagnosed with a type of B-cell lymphoma.
  • lymphoma refers to a malignant growth of B or T cells in the lymphatic system, presenting as an enlargement of the node (a tumor).
  • Mature T cell and natural killer (NK) cell neoplasms are exemplified by T cell prolymphocyte leukemia, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia/lymphoma, extranodal NK/T cell lymphoma, nasal type, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides / Sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disorders, angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma, unspecified, and anaplastic large cell lymphoma.
  • the lymphoma cell is a MALT lymphoma cell or a ABC-DLBCL cell.
  • the cell derived from the transgenic non- human animal of the invention by conventional methods known by the persons skilled in the art [Sambrook J. et al., cited ad supra], and can be isolated from any tissue from the transgenic non- human animal. Nevertheless, in a particular embodiment, the cell is selected from the group consisting of a hematopoietic stem cell and a lymphoma cell.
  • the cells according to the present invention can be isolated from the transgenic animals of the invention by means of the immunophenotype profile, i.e the presence or absence of a certain set of surface markers. These markers are epitopes that can be identified with specific antibodies, constituting a valuable tool that allows not only to identify the cells, but also to design a strategy for isolation or purification thereof.
  • Monoclonal antibodies against said surface markers can be used to identify the somatic lymphoma cells of the invention.
  • the determination of the profile of surface markers by antibodies may be direct, using a labelled antibody, or indirect, using a second labelled antibody against the primary specific antibody of the cell marker, thus achieving signal amplification.
  • the presence or absence of binding of the antibody may be determined by different methods that include but are not limited to immunofluorescence microscopy and radiography.
  • specific reagent refers to a member of a specific binding pair; members of a specific binding pair, include but are not limited to, binding pairs of antigens and antibodies, pairs comprising MHC antigens and T-cell receptors, complementary nucleotide sequences, as well as pairs of peptide ligands and their receptor.
  • the specific binding pairs include analogues, fragments and derivatives of the specific member of the binding pair.
  • antibodies as reagents with affinity is of particular interest.
  • the production of specific monoclonal antibodies will be evident to any ordinary skilled person in the art.
  • the antibodies are labelled.
  • markers that are used include but are not limited to: magnetic particles, biotin and fluorochromes that will allow identification or separation of the cell type to which the antibody has bound.
  • the analysis of the cell population comprising the somatic lymphoma cell of the invention by flow cytometry allows different antibodies labelled with fluorochromes that emit at different wavelengths to be used in the same sample.
  • the separation of the cells can be carried out by affinity separation techniques, which include magnetic separation (using magnetic particles coated with specific antibodies), affinity chromatography, cytotoxic agents bound to monoclonal antibodies or used along with monoclonal antibodies and panning with the antibody attached to a solid support, as well as by other techniques that are appropriate.
  • affinity separation techniques include magnetic separation (using magnetic particles coated with specific antibodies), affinity chromatography, cytotoxic agents bound to monoclonal antibodies or used along with monoclonal antibodies and panning with the antibody attached to a solid support, as well as by other techniques that are appropriate.
  • a more precise separation would be obtained by flow cytometry, a technique that allows the separation of cell populations according to the intensity of staining, along with other parameters such as cell size and cell complexity.
  • the cell derived from the transgenic non-human cell of the invention is also capable of being expanded ex vivo. That is, after isolation, cell lines can be maintained and allowed to proliferate ex vivo in culture medium.
  • Such medium comprises, for example, Dulbecco's Modified Eagle's Medium (DMEM), antibiotics, and glutamine, and it is usually supplemented with 2-20% fetal bovine serum (FBS). It is within the skill of one in the art to modify or modulate concentrations of media and/or media supplements as necessary for the cells used. Sera often contain cellular factors and components that are necessary for viability and expansion. Examples of sera include FBS, bovine serum (BS), calf serum (CS), fetal calf serum (FCS), newborn calf serum (NCS), goat serum (GS), horse serum (HS), porcine serum, sheep serum, rabbit serum, rat serum (RS), etc.
  • BS bovine serum
  • CS calf serum
  • FCS fetal calf serum
  • NCS newborn calf serum
  • GS horse serum
  • HS horse serum
  • porcine serum sheep serum, rabbit serum, rat serum (RS), etc.
  • Modulation of serum concentrations, withdrawal of serum from the culture medium can also be used to promote survival of the cell line.
  • cell line of the invention will benefit from FBS concentrations of about 2% to about 25%.
  • the cell line of the invention can be expanded in a culture medium of definite composition, in which the serum is replaced by a combination of serum albumin, serum transferrin, selenium, and recombinant proteins including but not limited to: insulin, platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF).
  • PDGF platelet-derived growth factor
  • bFGF basic fibroblast growth factor
  • amino acids include, but are not limited to, L-alanine, L- arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L- glutamic acid, L-glutamine, L-glycine, and the like.
  • Antimicrobial agents are also typically used in cell culture to mitigate bacterial, mycoplasmal, and fungal contamination.
  • antibiotics or anti-mycotic compounds used are mixtures of penicillin/streptomycin, but can also include, but are not limited to amphotericin (Fungizone®), ampicillin, gentamicin, bleomycin, hygromacin, kanamycin, mitomycin, etc.
  • the transgenic non- human animal of the invention e.g., a mouse
  • non- human animals which can be crossed with the non- human animal of the invention include, without limiting to:
  • non-human animals expressing allelic variations, polymorphisms or mutations in genes involved in B-cell lymphomas, or other non-human animals which can be used as B-cell lymphomas models.
  • the invention relates to the use of the transgenic non-human animal of the invention as an animal model for studying human B-cell lymphomas.
  • B-cell lymphomas refers to a type of lymphoma affecting B cells. Lymphomas are "blood cancers" in the lymph nodes. . They develop more frequently in older adults and in immunocompromised individuals (such as those with AIDS). B-cell lymphomas include both Hodgkin's lymphomas and most non-Hodgkins lymphomas. They are often divided into indolent (slow-growing) lymphomas and aggressive lymphomas.
  • B-cell lymphomas include, not limiting to, Diffuse large B cell lymphoma (DLBCL) [including activated B-cell (ABC-DLBCL), germinal center (GCB DLBCL), and primary mediastinal B-cell lymphoma (PMBL)], Follicular lymphoma, Mucosa-Associated Lymphatic Tissue lymphoma (MALT), Small cell lymphocytic lymphoma (overlaps with Chronic lymphocytic leukemia), Mantle cell lymphoma (MCL), Burkitt lymphoma, Waldenstrom macroglobulinemia, Nodal marginal zone B cell lymphoma (NMZL), Splenic marginal zone lymphoma (SMZL), Intravascular large B-cell lymphoma, and Primary effusion lymphoma.
  • DLBCL Diffuse large B cell lymphoma
  • ABSB DLBCL activated B-cell
  • GCB DLBCL germinal center
  • PMBL primary mediastinal B-
  • the B-cell lymphoma is mucosa-associated lymphoid tissue (MALT) lymphoma or activated B-cell like Diffuse Large B-Cell Lymphoma (ABC- DLBCL).
  • MALT lymphomas relates to a form of lymphoma involving the mucosa-associated lymphoid tissue (MALT), frequently of the stomach, but virtually any mucosal site can be afflicted. It is a cancer originating from B cells in the marginal zone of the MALT, and is also called extranodal marginal zone B cell lymphoma.
  • activated B-cell like Diffuse Large B-Cell Lymphoma or "ABC- DLBCL” relates to a subtype of aggressive non-Hodgkin lymphoma with a pattern of genetic expression that is similar to healthy, activated B cells.
  • the cells isolated from the transgenic non- human animal according to the invention can also be used for:
  • the invention relates to the use of the transgenic non-human animal or of the cell of the invention for:
  • the invention relates to a method for the identification of compounds suitable for the treatment and/or prevention of MALT lymphoma which comprises the steps of:
  • suitable for the treatment of MALT lymphoma any activity in directly or indirectly mediating any effect in preventing or inhibiting tumor development of growth which may provide for a beneficial effect to the host. This encompasses, for example, prevention of or inhibition of cancer cell and/or tumor development, inhibition of tumor cell growth, reduction of tumor load, and the like.
  • candidate agents can be screened for anti-cancer activity.
  • “Candidate agents” is meant to include synthetic, naturally occurring, or recombinantly produced molecules (e.g., small molecule; drugs; peptides; antibodies (including antigen-binding antibody fragments, e.g., to provide for passive immunity); endogenous factors present in eukaryotic or prokaryotic cells (e.g., polypeptides, plant extracts, and the like)); etc.). Of particular interest are screening assays for agents that have a low toxicity for human cells.
  • Candidate agents also include agents that may serve as cancer vaccines, e.g., to provide for production of an immune response in a subject that will in turn have anticancer activity.
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among bio molecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Candidate agents can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. In a first step, the method comprises the administration to a administering a transgenic non-human animal according to the invention with a candidate compound.
  • the candidate agent can be administered in any manner desired and/or appropriate for delivery of the agent in order to examine anti-cancer activity.
  • the candidate agent can be administered topically, by injection (e.g., by injection intravenously, intramuscularly, subcutaneously, and the like), orally, or by any other desirable means.
  • the screening method can involve administering varying amounts of the candidate agent (from no agent to an amount of agent that approaches an upper limit of the amount that can be delivered successfully to the animal, e.g., within toxicity limits), and may include delivery of the agent in different formulations and routes.
  • the agents can be administered singly or can be combined in combinations of two or more, especially where administration of a combination of agents may result in a synergistic effect.
  • the method for the identification of compounds according to the invention is carried out with mice showing expression of MALT1 or of a functionally equivalent variant thereof in the hematopoietic stem cell compartment and wherein the animal does not contain any mutation in the p53 locus.
  • the method is particularly useful for the identification of compounds useful for the treatment of MALT lymphoma.
  • the method for the identification of compounds according to the invention is carried out with mice showing expression of MALT 1 or of a functionally equivalent variant thereof in the hematopoietic stem cell compartment and showing an inactivating mutation in at least one allele of p53, preferably being homozygous for a totally defective p53 gene.
  • the method is particularly useful for the identification of compounds useful for the treatment of ABC-DLBCL.
  • the method comprises determining in said animal the appearance of one or more molecular or clinical findings of MALT lymphoma or of ABC-DLBCL
  • Suitable molecular findings of MALT lymphoma that can be used for the screening of potentially useful compounds include, without limitation, constitutive NF- ⁇ activation of the lymphoid- infiltrating cells, MALT1 expression in the lymphoid- infiltrating cells, proteolytic activity of MALT 1 (determined e.g. by its potential to cleave A20 or BcllO proteins), expression of B-cell- specific markers CD20 and Pax5, moderate to strong IgM expression or weak IgD expression in the lymphoid-infiltrating cells, expression of the XBP-1 target gene signature.
  • Suitable clinical findings of MALT lymphoma that can be used for the screening of potentially useful compounds include, without limitation, extranodal lymphomas in different tissues such as small intestine, salivary glands, kidneys, lungs, liver, stomach, ocular adnexa and spleen, presence of extranodal cell infiltration comprising a morphologically heterogeneous lymphoid population, predominantly composed of marginal zone (centrocyte- like) cells mixed up with small lymphocytes, scattered large blasts and plasma cells.
  • ABC-DLBCL include, without limitation, appearance in the lymphoma cells of a pattern of genetic expression similar to healthy activated B cells, constitutive NF- ⁇ activation of the lymphoid-infiltrating cells, expression in the lymphoma cells of CD20 and/or Foxpl, lack of expression of CD 10, Gcetl, Muml and/or Bcl6.
  • Clinical findings of ABC-DLBCL include, without limitation, appearance of tumors in the lymph nodes and in other lymphoid territories such as spleen, tonsils, Peyer ' s patches, as well as in non-nodal territories (liver, skin, lungs, etc) which are usually classified as aggressive B-cell lymphomas characterized by a diffuse infiltrate of large lymphocytes with nuclei with dispersed chromatin, deeply basophilic cytoplasm and multiple mitotic figures, resembling human DLBCL, with moderate expression of CD20 and strong expression of Foxpl, with negative expression for Bcl6, Gcetl, Muml and CD 10, and with few scattered CD3 + T-cells.
  • the ability of a candidate agent to treat a pre-existing lymphoma can be assessed by administering the candidate agent to the transgenic mouse which already shows the disease. Modulation of a cancer phenotype can be assessed by evaluating the present or absence of an effect on, for example, tumor burden, number of tumors, tumor size, metabolic activity of tumor cells, and the like. The ability of a candidate agent to facilitate prevention of cancer can be assessed by administering the candidate agent to the transgenic mice prior to the appearance of the clinical or molecular signs of the disease. "Prevention" of cancer refers to reduction in the incidence and/or severity of tumors in the animal relative to the incidence and/or severity of cancer expected in the absence of intervention (e.g., without administration of an agent having anti-cancer activity).
  • a candidate compound is then considered as being useful for the treatment of MALT lymphoma or for ABC-DLBCL wherein it results in a decrease in the appearance of said one or more molecular or clinical findings of MALT lymphoma or of ABC- DLBCL with respect to a control animal.
  • a control animal is an animal which has been left untreated or which has been treated with the vehicle in which the candidate compound is administered.
  • the invention relates to a method for monitoring the effect of the therapy administered to a subject having a human B-cell lymphoma, said method comprising the steps of
  • the method comprises the administration to a transgenic non-human animal according to the invention with a therapy suitable for the treatment of MALT lymphoma or ABC-DLBCL.
  • Suitable therapies for MALT lymphoma include, without limitation, triple therapy with antibiotics and anti-acids in the case of MALT lymphoma restricted to the stomach, Monoclonal antibodies such as rituximab, surgery, chemotherapy and, in particular, chlorambucil, cyclophosphamide, fludaribine and cladribine, radiotherapy and steroid therapy.
  • Suitable therapies for ABC-DLBCL include, without limitation, the so-called CHOP treatment (a combination of 3 chemotherapy drugs (cyclophosphamide, doxorubicin, vincristine) and one steroid (prednisone)); R-CHOP(a combination of one monoclonal antibody (rituximab), 3 chemotherapy drugs (cyclophosphamide, doxorubicin, vincristine) and one steroid (prednisone)), and the proteosome inhibitor drug bortezomib.
  • CHOP treatment a combination of 3 chemotherapy drugs (cyclophosphamide, doxorubicin, vincristine) and one steroid (prednisone)
  • R-CHOP a combination of one monoclonal antibody (rituximab), 3 chemotherapy drugs (cyclophosphamide, doxorubicin, vincristine) and one steroid (prednisone)
  • the method involves determining in said animal the evolution of one or more molecular or clinical findings of MALT lymphoma or of ABC-DLBCL, wherein a decrease in one or more of said molecular or clinical findings of MALT lymphoma or of ABC-DLBCL with respect to a control animal is indicative that the compound is responding to the treatment of MALT lymphoma or for ABC-DLBCL.
  • the expressions "molecular finding of MALT lymphoma”, “clinical finding of MALT lymphoma”, “molecular finding of ABC-DLBCL”, “clinical finding of ABC-DLBCL” have been described above in detail and apply equally to the present method.
  • the term "decrease" in a molecular or clinical finding refers to a decrease in a parameter which measures said molecular or clinical finding of at least 0.9-fold, 0.75- fold, 0.2-fold, 0.1 -fold, 0.05-fold, 0.025-fold, 0.02-fold, 0.01 -fold, 0.005-fold or even less compared of the initial value of said parameter.
  • control animal refers to an animal, preferably a non-human animal of the same species as the animal being studied which has been left untreated or which has been treated with the vehicle in which the therapeutic agent is administered.
  • control animal is allogeneic (individuals which are genetically dissimilar but are from the same species) or syngeneic (individuals which genetically identical) with respect to the non-human animal to which the therapy has been administered.
  • the invention relates to the use of the transgenic non-human animal or the cell of the invention for the identification of inhibitors of MALT 1 protease activity.
  • the invention relates to a method for the identification of inhibitors of the MALT protease activity which comprises: (i) contacting a candidate compound with a population of cells containing lymphoma cells isolated from a transgenic mice according to the invention and
  • lymphoma cells populations of cells containing lymphoma cells, as used herein, as well as cells isolated from extranodal organs wherein the lymphoma cells accumulate such as small intestine, salivary glands, kidneys, lungs, liver, stomach, ocular adnexa, and spleen.
  • the population of cells are splenocytes.
  • the population of cells containing lymphoma cells is then contacted with a candidate compound or preparation.
  • "putting in contact" a cell population with the candidate compound includes any possible way of taking the candidate compound inside the cells of the population.
  • the candidate compound is a molecule with low molecular weight
  • the candidate compound is a molecule with a high molecular weight (for example, biological polymers such as a nucleic acid or a protein)
  • the candidate molecule is a nucleic acid
  • conventional transfection means can be used such as precipitating DNA with calcium phosphate, DEAE-dextran, polybrene, electroporation, microinjection, liposome-mediated fusion, lipofection, infection by retrovirus and biolistic transfection.
  • the candidate compound is a protein
  • the cell can be put in contact with the protein directly or with the nucleic acid encoding it coupled to elements allowing its transcription / translation once they are in the cell interior. To that end, any of the aforementioned methods can be used to allow its entrance in the cell interior.
  • a variant of the protein to be studied which has been modified with a peptide which can promote the translocation of the protein to the cell interior such as the Tat peptide derived from the HIV-1 TAT protein, the third helix of the Antennapedia homeodomain protein from D.melanogaster, the VP22 protein of the herpes simplex virus and arginine oligomers (Lindgren, A. et al, 2000, Trends Pharmacol. Sci, 21 :99-103, Schwarze, S.R. et al, 2000, Trends Pharmacol. Sci., 21 :45- 48, Lundberg, M et al, 2003, Mol. Therapy 8: 143-150 and Snyder, E.L. and Dowdy, S.F., 2004, Pharm. Res. 21 :389-393).
  • a variant of the protein to be studied which has been modified with a peptide which can promote the translocation of the protein to the cell interior such as the Tat peptide derived from the HIV-1 TAT
  • the MALTl protease activity can be determined by any assay known in the art which allows to specifically determine the protease activity of MALTl without interference from other proteases.
  • the protease activity is assayed using specific MALTl substrates and detecting the cleavage of the substrate by any known assay.
  • the MALTl substrate is BLC10 as described in WO08146259.
  • the inhibitors of MALTl protease activity thus obtained can be used for treating diseases selected from the group consisting of hypertension, diabetic nephropathy, congestive heart failure, sepsis due to infection, preferably fungal infection, IgE- mediated diseases such as anaphylaxis and T-cell and/or B-cell receptor linked diseases.
  • diseases selected from the group consisting of hypertension, diabetic nephropathy, congestive heart failure, sepsis due to infection, preferably fungal infection, IgE- mediated diseases such as anaphylaxis and T-cell and/or B-cell receptor linked diseases.
  • the inhibitors obtained by using the transgenic non-human animal of the invention are used for the preparation of a medicament to treat T-cell and/or B-cell receptor linked diseases.
  • the US patent application US 2011021548 discloses inhibitors of MALTl proteolytic activity and uses thereof.
  • Lymphoma cell treatment with a MALTl protease inhibitor blocks A20 and BCL10 cleavage, reduces NF-KB activity and decreases the expression of the NF- ⁇ targets BCL-XL, IL-6, and IL-10, resulting in ABC-DLBCL cell death and growth retardation.
  • the transgenic non-human animal of the invention can be obtained by any transgenesis method known by the person skilled in the art, including, but not limiting to, microinjection, electroporation, particle bombardment, cell transformation followed by cloning (the nuclei of the successfully transformed cells are transferred to enucleated ova and are implanted in receptor females), gamete transformation (introducing genes in oocytes or spermatocytes and using the transformed gametes for fertilization, generating a complete animal) and/or intracytoplasmic sperm microinjection (ICSI).
  • ICSI intracytoplasmic sperm microinjection
  • the invention relates to a method for producing a transgenic non-human mammalian animal, hereinafter method of the invention, said method comprising chromosomally incorporating in the genome of a non-human mammalian animal a polynucleotide encoding MALT1, operatively linked to a tissue-specific promoter which is specific for expression in hematopoietic stem/progenitor cells.
  • the transgenic animals of the present invention are preferably generated by introduction of the targeting vectors into embryonic stem (ES) cells.
  • ES cells can be obtained by culturing pre-implantation embryos in vitro under appropriate conditions (Evans, et al, 1981, Nature 292: 154-156 ; Bradley, et al, 1984, Nature 309: 255-258 ; Gossler, et al, 1986, Proc. Natl. Acad. Sci. USA 83: 9065-9069 ; and Robertson, et al, 1986, Nature 322: 445-448 ).
  • Transgenes can be efficiently introduced into the ES cells by DNA transfection using a variety of methods known to the art including electroporation, calcium phosphate co -precipitation, protoplast or spheroplast fusion, lipofection and DEAE-dextran-mediated transfection. Transgenes may also be introduced into ES cells by retro virus-mediated transduction or by microinjection. Such transfected ES cells can thereafter colonise an embryo following their introduction into the blastocoel of a blastocyst-stage embryo and contribute to the germ line of the resulting chimeric animal. For review, see Jaenisch (1988, Science 240: 1468-1474).
  • the transfected ES cells Prior to the introduction of transfected ES cells into the blastocoel, the transfected ES cells may be subjected to various selection protocols to enrich for ES cells which have integrated the transgene assuming that the transgene provides a means for such selection.
  • the polymerase chain reaction may be used to screen for ES cells which have integrated the transgene. This technique obviates the need for growth of the transfected ES cells under appropriate selective conditions prior to transfer into the blastocoel.
  • transgenic animals are known to those skilled in the art. For example, embryonic cells at various developmental stages can be used to introduce transgenes for the production of transgenic animals. Different methods are used depending on the stage of development of the embryonic cell.
  • the zygote particularly at the pronucleal stage (i.e. prior to fusion of the male and female pronuclei), is a preferred target for micro-injection.
  • the male pronucleus reaches the size of approximately 20 micrometers in diameter, which allows reproducible injection of 1-2 picoliters (pi) of DNA solution.
  • zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host genome before the first cleavage (Brinster, et al, 1985, Proc. Natl. Acad. Sci. USA 82: 4438-4442). As a consequence, all cells of the transgenic non- human animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbour the transgene. Micro -injection of zygotes is the preferred method for random incorporation of transgenes. U.S. Pat. No. 4,873191 describes a method for the micro -injection of zygotes.
  • Retroviral infection can also be used to introduce transgenes into a non-human animal.
  • the developing non-human embryo can be cultured in vitro to the blastocyst stage.
  • the blastomeres can be targets for retroviral infection (Janenich, 1976, Proc. Natl. Acad. Sci. USA 73: 1260-1264).
  • Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan et al., 1986, in Manipulating the Mouse Embryo, Cold Spring Harbour Laboratory Press, Plainview, N.Y.).
  • the viral vector system used to introduce the transgene is typically a replication- defective retrovirus carrying the transgene (Jahner, D.
  • Retroviral infection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart, et al, 1987, EMBO J. 6: 383-388). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner, D. et al, 1982, Nature 298: 623-628).
  • founders will be mosaic for the transgene since incorporation occurs only in a subset of cells which form the transgenic animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome, which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germline, albeit with low efficiency, by intrauterine retroviral infection of the mid-gestation embryo (Jahner, D. et al, 1982, supra).
  • retroviruses or retroviral vectors to create transgenic animals known to the art involves the micro-injection of retroviral particles or mitomycin C-treated cells producing retrovirus into the perivitelline space of fertilised eggs or early embryos (PCT International Application Publication No. WO 90/08832) and Haskell and Bowen, 1995, Mol. Reprod. Dev. 40: 386).
  • lines of any mammalian species to work this invention they may be selected for criteria such as embryo yield, pronuclear visibility in the embryos, reproductive fitness, colour selection of transgenic offspring or availability of ES cell clones. For example, if transgenic mice are to be produced, lines such as C57/B16 or 129 may be used.
  • mice The age of the mammals that are used to obtain embryos and to serve as surrogate hosts is a function of the species used. When mice are used, for example, pre-puberal females are preferred as they yield more embryos and respond better to hormone injections.
  • a primed female i. e. one that is producing eggs that may fertilised
  • a stud male i. e. one that is producing eggs that may fertilised
  • eggs and sperm may be obtained from suitable females and males and used for in vitro fertilisation to produce an embryo suitable for introduction of the transgene.
  • fertilised embryos are incubated in suitable media until the pronuclei appear. At about this time, the exogenous nucleic acid sequence comprising the transgene of interest is introduced into the male or female pronucleus.
  • the male pronuclease is preferred.
  • Introduction of nucleic acid may be accomplished by any means known in the art such as, for example, microinjection. Following introduction of the nucleic acid into the embryo, the embryo may be incubated in vitro for varied amounts of time prior to reimplantation into the surrogate host. One common method is to incubate the embryos in vitro for 1 to 7 days and then reimplant them into the surrogate host.
  • Transgenic offspring of the surrogate host may be screened for the presence of the transgene by any suitable method. Screening may be accomplished by Southern or northern analysis using a probe that is complimentary to at least a portion of the transgene (and/or a region flanking the transgene) or by PCR using primers complementary to portions of the transgene (and/or a region flanking the transgene). Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening.
  • transgenic mammals may be obtained by mating the transgenic mammal with a suitable partner or by in vitro fertilisation using eggs and/or sperm obtained from the transgenic mammal. Where in vitro fertilisation is used, the fertilised embryo is implanted into a surrogate host or incubated in vitro or both. Where mating is used to produce transgenic progeny, the transgenic mammal may be back-crossed to a parental line, otherwise inbred or cross-bred with mammals possessing other desirable genetic characteristics.
  • suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular markers or enzyme activities and the like.
  • the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
  • the transgenic animals according to the invention are mated or their eggs/sperm are used for in vitro fertilization with an animal of the same species which carries an inactivating mutation in at least one allele of the p53 gene. This will give raise to progeny which may carry the transgene and an inactivating mutation in one or more of the p53 genes.
  • the non-human transgenic animals according to the invention are mated with a non- human animal of the same species which is homozygous for a totally defective p53 gene (p53 _/ ⁇ ).
  • the hematopoietic stem cell-specific transcriptional regulatory sequence is the Ly-6E. lgene promoter.
  • the polynucleotide further comprises a region of the Ly-6E. l-gene located 3' with respect to the end of the nucleotide sequence encoding a human MALT1 protein or a functionally equivalent variant thereof.
  • the polynucleotide comprises the complete Ly-6E. l- gene wherein the nucleotide sequence encoding a human MALT1 protein or a functionally equivalent variant thereof is inserted in the first exon of the Ly-6E.1-gene.
  • the Ly-6E.1-gene comprises at least 9 kb with respect to the transcription initiation site of said gene.
  • the non-human recipient animal carries an inactivating mutation in at least one allele of the p53 gene, preferably, the non-human recipient animal is homozygous for a totally defective p53 gene (p53 _/ ⁇ ).
  • the Scal-MALTl vector was generated by inserting the human MALT I cDNA into the Clal site of the pLy6 vector.
  • the transgene fragment was excised from its vector by restriction digestion with Notl, purified and injected (2 ng/mL) into CBAxC57BL/6J fertilized eggs.
  • Transgenic mice were identified by Southern blot analysis of tail snip DNA after EcoRI digestion, using MALT I cDNA to detect the transgene. Three independent transgenic lines were generated and analyzed. Heterozygous p53+/- mice were bred to Seal -MALT 1 mice to generate compound heterozygotes. Fl animals were crossed to obtain null p53-/- mice hemyzygous for Seal - MALT1.
  • mice Upon signs of disease, mice were sacrificed and subjected to standard necropsy procedures. All major organs were examined under the dissecting microscope. Tissue samples were taken from homogenous portions of the resected organ and fixed immediately after excision. Plasmids, cell culture and transfections.
  • Murine pro-B BAF3 cells were grown in RPMI 1640 medium supplemented with 10% FCS, 10% WEHI-conditioned medium, 2 mM Gin, and penicillin/streptomycin. Cells were transfected using Nucleofector technology (Amaxa, Koln, Germany) and selected using 1 mg/mL G418.
  • Clones were isolated by limiting dilution, and stable transfectants were maintained at 500 ng/mL G418.
  • transfected and exponentially-growing BaF3 cells were washed twice with PBS and incubated in complete medium with or without 10% WEHI-conditioned medium for the indicated times. Cell viability was determined by the trypan blue exclusion method. Results were measured as the percentage of the living cells grown without versus with WEHI-conditioned medium.
  • Paraffin-embedded tumor xenografts, transgenic mice tissues and human lymphoma samples were sectioned, dewaxed, and heated in 10 mmol/1 sodium citrate buffer for 30 min. Slides were incubated with primary antibodies (see Tables 2 and 3). Samples were centrally reviewed by a panel of pathologists and diagnosed using uniform criteria based on clinical, histological, immunophenotypical, and molecular characteristics. For comparative studies, age-matched WT mice were used.
  • Equal amounts of total protein (10-50 ⁇ g) were separated on sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), and electrotransferred onto nitrocellulose membranes. Membranes were incubated with primary antibodies (see Tables 2 and 3), followed by secondary antibodies conjugated to horseradish peroxidase, which were detected by chemiluminescence (Applied Biosystems, and Pierce, respectively).
  • Nucleated cells were obtained from total mouse BM (flushing from the long bones), PB, thymus, liver or spleen. Data were analyzed in a FACSCalibur using the CellQuest program (Becton Dickinson). Specific fluorescence of FITC and PE excited at 488 nm (0.4 W) and 633 nm (30 mW), respectively, as well as known forward and orthogonal light scattering properties of mouse cells were used to establish gates. Nonspecific antibody binding was suppressed by preincubation of cells with CD16/CD32 Fc-block solution (BD Pharmingen). Flow cytometry antibodies used are shown in Table 4. All of them were purchased from from BD Pharmingen.
  • Scal+Lin- cells For cell sorter separation of Scal+Lin- cells, BM cells were incubated with anti- Seal and anti-lineage markers antibodies (CD4, CD8, B220, Grl , and Macl). Scal+Lin- cells were isolated and highly purified from the BM of Seal -MALT 1 mice or control mice by fluorescence-activated cell sorting (FACS) (FACSVANTAGE, Becton Dickinson). Sorted cells were then reanalyzed for purity with the FACS and determined to be over 98% pure. V(D)J Recombination.
  • FACS fluorescence-activated cell sorting
  • Ig gene rearrangements were amplified by PCR using the primers listed in Table 5.
  • CD19 + cells were activated with anti-human IgM 10 ⁇ g/mL (Sigma), anti- human CD40 2 ⁇ g/mL (R&D Systems) and recombinant human IL4 10 ng/mL (R&D Systems) for 24 hours. Informed consent was obtained from the patients in accordance with the Declaration of Helsinki. The study was approved by an Institutional Research Ethics Committee.
  • Samples were processed following Affymetrix recommendations and cDNA was hybridized to the Affymetrix Human Genome U133 Plus 2.0 array (human samples) and to the Affymetrix Mouse Genome 430 2.0 (mouse samples).
  • Raw array microarray data files were submitted to GEO and are available under the accession numbers GSE25636, GSE25637, GSE25638 and GSE25639. Background correction and normalization were done with the RMA (Robust Multichip Average) algorithm. R and Bioconductor were used for preprocessing and statistical analysis.
  • BAF3 cells or splenocytes obtained from WT and Seal- MALTl transgenic mice were lysed in cleavage assay buffer (50 mM Tris-HCl, pH 7.4, 60 mM NaCl, 10 mM KC1, 20 mM MgCb, 100 mM CaCh and 10 mM dithiothreitol) and briefly sonicated.
  • Recombinant human BCLIO-His tagged (1-5 ng) was used as substrate for the Maltl activity and incubated the reactions for 4 h at 30°C. Cleavage of recombinant BCL10 was evaluated by immunoblot analysis with an anti-BCLlO antibody.
  • MALTl but not BCL10 or API2-MALT1, shows oncogenic properties in primitive hematopoietic cells.
  • MALTl, BCL10 or API2-MALT1 could be tumorigenic when expressed in more primitive hematopoietic cells.
  • Human full-length MALTl and BCL10 genes and the API2-MALT1 fusion gene were cloned and stably transfected into BCR- activated murine IL3-dependent hematopoietic BaF3 cells.
  • isolated single- cell clones expressing either MALTl, BCL10 or API2-MALT1 genes exhibited NF-KB activation, only MALTl -expressing cells grew independently of IL3 ( Figures 1A-B).
  • mice in which the human MALTl gene was ectopically expressed in the hematopoietic stem/progenitor cell (HS/PC) compartment was generated.
  • the mouse Ly-6E.l gene promoter was used to drive the expression of human MALTl transgene under the control of the stem cell antigen 1 (Seal) gene regulatory sequences in C57BL/6 x CBA mice ( Figure 2A). Southern blot analysis of the three founders obtained showed transgene copy numbers ranging from approximately 2 to 4, which were used to examine the mouse phenotypes ( Figure 3).
  • MALTl in Scal+ cells induces NF- ⁇ signaling, promotes hematopoietic progenitor cell expansion, and enhances B-cell lymphopoiesis in young mice
  • the different hematopoietic cell compartments were studied by flow cytometry in young Seal- MALTl mice. At 2 months of age, no major abnormalities were detected in myeloid cells or T-cell subpopulations (as determined by Grl, Macl, CD4 or CD8 staining) in BM, peripheral blood (PB), spleen and thymus.
  • BM-sorted Scal+Lin- cells were compared using gene expression microarrays.
  • Linear Models of Microarray Data Analysis identified 110 genes that were differentially expressed between transgenic and WT cells, defining the Scal+Lin- MALTl signature. This included underexpression of genes encoding essential regulators of early B-cell development such as Sox4, E2A (Tcefla), Vprebl, Vpreb2 and Ragl/Rag2 and upregulation of differentiated B-cell markers such as Prdml, Xbpl and Ig genes.
  • Immunohistochemistry (IHC) analysis showed that the neoplastic lymphoid-infiltrating cells expressed the B-cell- specific markers CD20 and Pax5, with moderate to strong IgM expression and weak IgD expression, while CD3 highlighted a few scattered T- cells within the tumor ( Figures 4E-F). Tumor clonality was identified by PCR analysis of VDJ rearrangements of the heavy and light chains of the Ig locus.
  • Scal-MALTl lymphomas frequently spread to the spleen, and a spontaneous transformation into a high-grade lymphoma resembling human DLBCLs of ABC subtype (CD20+, Foxpl+, CD10-, Gcetl-, Muml- and Bcl6 ) was observed in 4 of 26 (15%) of the cases ( Figure 4C).
  • Scal-MALTl mice developed clonal B-cell lymphomas that involved extranodal sites and displayed clinical, histological and immunophenotypic features recapitulating the main characteristics of human MALT lymphoma.
  • Bioinformatic analysis of the gene expression microarray data using the Prediction Analysis for Microarrays (PAM) software identified 132 genes that distinguished MALT lymphoma from other B-cell lymphomas, thereby defining the human MALT lymphoma transcriptional signature.
  • a representative selection of deregulated genes and proteins was studied by quantitative PCR (RT-qPCR) and IHC analyses, respectively, in normal tissue samples and lymphoma cells, validating the microarray data.
  • LIMMA Linear Models of Microarray Data Analysis
  • comparison of the gene expression profiles between splenic Scal-MALTl lymphomas and WT mouse spleens identified 246 differentially expressed genes that composed the Scal-MALTl lymphoma transcriptional signature (Figure 5A).
  • Murine Maltl protein shows proteolytic activity in Scal-MALTl lymphomas
  • the model according to the present invention is a suitable tool for studying inhibitors of MALTl protease activity, whose relevance to the treatment of NF-KB- activated B-cell lymphomas has already been highlighted.

Abstract

La présente invention concerne un animal transgénique non humain dont le génome contient un polynucléotide comprenant une séquence régulatrice transcriptionnelle spécifique des cellules souches hématopoïétiques, fonctionnellement liée à une séquence nucléotidique hétérologue codant pour une protéine humaine MALT1 et qui recouvre la plupart des caractéristiques des lymphomes humains à cellules B, tels que le lymphome du tissu lymphoïde associé aux muqueuses(MALT) ou le lymphome B diffus à grandes cellules de type activé (ABC-DLBCL). En outre, l'invention concerne également des méthodes de criblage de composés présentant des activités ayant un intérêt pharmacologique utilisant lesdits animaux transgéniques non humains.
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