The telomeres are specialized structures at the end of the chromosomes composed of the repeated DNA sequence (TTAGGG)n and specific proteins bound to the DNA. The telomeres protect the chromosomes from degradation and end to end fusions. Due to the end-replication problem, the telomeric DNA shortens every cell division, forcing the cells into senescence at a critical telomere length. This process can be counteracted by activating a specialized enzyme, telomerase, which adds telomeric repeats to the chromosome ends leading to an extended or infinite cellular life span. Telomerase activity is absent in most somatic tissues but is found in germ cells, stem cells, activated lymphocytes and the vast majority of tumor cells and permanent cell lines. Hence, telomerase has been suggested as a target for cancer treatment as malignant cells almost exclusively express the enzyme and in that context telomere length measurements will be of great importance. Telomere length is traditionally measured with a Southern blot based technique. A new method for telomere analysis of cells in suspension, called flow-FISH, was developed based on fluorescence in situ hybridization using a telomeric peptide nucleic acid (PNA) probe, DNA staining with propidium iodide and quantification by flow cytometry. Flow-FISH had high reproducibility and the telomere length measurements showed good correlation with Southern blotting results. The flow-FISH technique also allows studies of cells in specific phases of the cell cycle and the replication timing of telomeric, centromeric and other repetitive sequences were analyzed in a number of cells. Like previous studies, centromeres were shown to replicate late in S phase while the telomere repeats were found to replicate early in S phase or concomitant with the bulk DNA, which is opposite to the patterns described in yeast. In benign immunopurified lymphocytes from tonsils, high telomerase activity was found in germinal center (GC) B cells. This population also had high hTERT mRNA levels and displayed a telomere elongation as shown by flow-FISH and Southern blotting. Combined immunophenotyping and flow-FISH on unpurified tonsil cells confirmed the results. Chronic lymphocytic leukemia (CLL), the most common leukemia in adults, can be divided into pre-GC CLL, characterized by unmutated immunoglobulin VH genes and worse prognosis, and post-GC CLL, with mutated VH genes and better prognosis. In 61 cases of CLL, telomere length was measured with Southern blotting and VH gene mutation status was analyzed. A new association was found between VH mutation status and telomere length, where cases with longer telomeres and mutated VH genes (post-GC CLL) had better prognosis than CLL with short telomeres and unmutated VH genes (pre-GC CLL). A larger study of 112 CLL cases was performed using flow-FISH. The same correlation between telomere length and VH mutation status was found but gender seemed to be of importance as telomere length was a significant prognostic factor for the male CLL patients but not in the female group. Age of the patients and spread of disease seemed to affect the prognostic value of VH gene mutation status.

In chronic lymphocytic leukemia (CLL), the presence of multiple subsets expressing ‘stereotyped’ B-cell receptors (BCRs) has implicated antigen(s) in leukemogenesis. These stereotyped subsets display similar immunoglobulin (IG) gene usage, almost identical complementarity determining region 3’s and may share clinical features. For instance, subsets #1 (IGHV1/5/7/IGKV1-39) and #2 (IGHV3-21/IGLV3-21) have inferior outcome compared to non-subset patients, whereas subset #4 (IGHV4-34/IGKV2-30) display a favourable prognosis. The aim of this thesis was to investigate genomic aberrations, gene expression patterns and methylation profiles in stereotyped subsets and compare epigenetic profiles in CLL and mantle cell lymphoma (MCL). In paper I, we investigated genomic aberrations in subsets #2, #4 and #16 and in non-stereotyped samples (n=101) using high-density 250K SNP arrays. Subset #2 and non-subset #2 IGHV3-21 cases displayed a higher frequency of aberrations than subset #4 cases. The high incidence of del(11q) in both subset #2/non-subset #2 may reflect the adverse survival reported for IGHV3-21 patients. In contrast, the lower frequency of genetic events and lack of poor-prognostic aberrations in subset #4 may partially explain their indolent disease. In paper II, we analysed the global RNA expression in subset #4, #16 and non-subset IGHV4-34 CLL patients (n=25). Subsets #4 and 16 showed distinct gene expression profiles, where genes involved in cell regulatory pathways were significantly lower expressed in subset #4, in line with their low-proliferative disease. In paper III, a genome-wide methylation array was applied to investigate methylation profiles in subsets #1, #2 and #4 (n=39). We identified differential methylation patterns for all subsets and found affected genes to be involved in e.g. apoptosis and therapy resistance. When performing functional annotation, a clear enrichment of genes involved in adaptive immunity was observed. These genes were preferentially methylated in subset #1 when compared to either subset #2 or #4, possibly due to different antigen responses. In paper IV, the genome-wide methylation profiles for 30 CLL and 20 MCL patients were investigated. Distinct methylation profiles were observed, where MCL displayed a more homogeneous profile. Homeobox transcription factor genes showed a higher degree of methylation in MCL, while apoptosis-related genes and proliferation-associated genes were methylated in CLL. In summary, this thesis demonstrates that stereotyped CLL subsets display differences in gene expression profiles, genetic aberrations and methylation patterns, underscoring the functional relevance of subgrouping according to BCR stereotypy. The distinct methylation profiles of CLL and MCL suggests that different epigenetic mechanisms are involved in the pathogenesis of these B-cell malignancies.

<p>B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Although treatment of this disease has advanced considerably over the past decade, CLL remains incurable with current chemotherapeutics. In addition, available drug regimens for CLL are associated with frequent cytopenia-related complications, such as infection and fatigue. Thus, the major challenge in CLL treatment today is the need for alternative therapeutics with decreased toxicity and improved efficacy for disease refractory to currently available drugs.</p><p> </p><p>CLL is characterized by slow accumulation of malignant cells, which are supported in the microenvironment by cell-cell interactions and soluble cytokines such as tumor necrosis factor (TNF). We evaluated the effect of the small molecule TNF inhibitor LMP-420 on primary CLL cells. LMP-420 exhibited cytotoxic activity against these cells in the MTS assay, with similar potency to the front-line CLL drug fludarabine. LMP-420 induced time- and dose-dependent apoptosis in CLL cells, as demonstrated by annexin V staining, caspase activation, and DNA fragmentation. These changes were associated with decreased expression of the anti-apoptotic proteins Mcl-1, Bcl-xL, Bcl-2, and XIAP. CLL cells from patients with poor prognostic indicators exhibited LMP-420 sensitivity equal to that for cells from patients with favorable characteristics. In addition, LMP-420 potentiated the cytotoxic effect of fludarabine and inhibited in vitro proliferation of CLL cells. In contrast to other CLL therapeutics, LMP-420 exhibited minimal effects on normal peripheral blood mononuclear cell viability, mitogen-stimulated B- and T-cell proliferation, and hematopoietic colony formation. Our data suggest that LMP-420 may be a useful treatment for CLL with negligible hematologic toxicities. </p><p> </p><p>The effect profile of this compound in normal immune cells and the microarray studies in CLL cells indicate that the mechanism of action of LMP-420 likely involves modulation of the NF-kB pathway. Our initial studies demonstrate moderate but significant inhibitory activity against p65, a key member of the NF-kB transcription factor family. Research is ongoing to gain a better understanding of the specific cytotoxicity of LMP-420 for CLL cells and to elucidate other components of its mechanism of action. Regardless of the ultimate mechanistic findings with LMP-420, our studies support this molecule as a promising new CLL therapeutic that warrants further preclinical evaluation.</p> / Dissertation

A previous population-based study of survival in Chronic Lymphocytic Leukemia (CLL) patients in the province of Manitoba demonstrated a lower five-year relative survival among CLL patients compared with the age- and gender-adjusted general population. This decreased relative survival was most pronounced among elderly male CLL patients. In this study, we have demonstrated that the reduced five-year relative survival observed in CLL patients compared to the general population of Manitoba may partially be attributed to increased risk of second cancers and non-referral to specialized CLL clinics. The increased risk of second cancers in CLL patients compared to Follicular Lymphoma (FL), a similar indolent B cell malignancy, was only observed after CLL diagnosis indicating that a CLL-specific factor may be responsible for the increased risk of second cancers in these patients. The risk of second cancers is independent of treatment and surveillance bias but is further increased with chemotherapy. A superior outcome in CLL patients who have been referred to the CancerCare Manitoba (CCMB) specialized CLL clinic was observed that was independent of age, gender, treatment and history of previous cancers. This superior outcome was most pronounced in the elderly CLL patients. We propose that CLL patients should be referred to CLL-specific hematologists and, where not possible, that guidelines created by such experts be followed. Appropriate screening for second cancers should be performed during routine follow up of CLL patients.

Uncovering the chromosomal abnormalities associated with human malignancy can provide significant insights into the molecular basis of tumorigenesis, as well as identifying potential targets for therapy. The present study set out to examine the genetic characteristics of t(2;7)(p11-12;q21-22) translocations arising in conjunction with chronic B-cell neoplasia. Using long-range PCR, a t(2;7) was initially mapped in an individual presenting with the preclinical entity CD5- monoclonal B-cell lymphocytosis. This revealed a breakpoint at 2p11.2 localized to the recombination signal of the immunoglobulin kappa (IGK) variable gene IGKV3-15, and a breakpoint at 7q21.2 located 520 bp upstream of cyclin dependent kinase 6 (CDK6). The same approach was subsequently employed to elucidate near-identical t(2;7) breakpoints in 4 additional cases presenting with chronic lymphocytic leukemia or indolent non-Hodgkin lymphomas. The remarkable consistency of these translocations implicates the dysregulation of CDK6 via translocation to IGK as a recurrent pathomechanism during the emergence of B-cell lymphoproliferative disorders.

Uncovering the chromosomal abnormalities associated with human malignancy can provide significant insights into the molecular basis of tumorigenesis, as well as identifying potential targets for therapy. The present study set out to examine the genetic characteristics of t(2;7)(p11-12;q21-22) translocations arising in conjunction with chronic B-cell neoplasia. Using long-range PCR, a t(2;7) was initially mapped in an individual presenting with the preclinical entity CD5- monoclonal B-cell lymphocytosis. This revealed a breakpoint at 2p11.2 localized to the recombination signal of the immunoglobulin kappa (IGK) variable gene IGKV3-15, and a breakpoint at 7q21.2 located 520 bp upstream of cyclin dependent kinase 6 (CDK6). The same approach was subsequently employed to elucidate near-identical t(2;7) breakpoints in 4 additional cases presenting with chronic lymphocytic leukemia or indolent non-Hodgkin lymphomas. The remarkable consistency of these translocations implicates the dysregulation of CDK6 via translocation to IGK as a recurrent pathomechanism during the emergence of B-cell lymphoproliferative disorders.

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) both belong to the group of mature B-cell malignancies. However, MCL is typically clinically aggressive while the clinical course of CLL varies. CLL can be divided into prognostic subgroups based on IGHV mutational status and into multiple subsets based on closely homologous (stereotyped) B-cell receptors. In paper I we investigated 31 MCL cases using high-density 250K single-nucleotide polymorphism arrays and gene expression arrays. Although most copy-number aberrations (CNAs) were previously reported in MCL, a novel deletion was identified at 20q (16%) containing the candidate tumor suppressor gene ZFP64. A high proliferation gene expression signature was associated with poor prognosis, large CNAs, 7p gains and 9q losses. Losses at 1p/8p/13q/17p were associated with increased genomic complexity. In paper II we sequenced exons 4 to 8 of the TP53 gene in 119 MCL cases. 17p copy-number status was known from previous studies or determined by real-time quantitative polymerase chain reaction. TP53 mutations were detected in 14% of cases and were strongly associated with poor survival while 17p deletions were more common (32%) but did not predict survival. In papers III and IV we applied high-resolution genomic 27K methylation arrays to 20 MCL and 39 CLL samples. In paper III MCL displayed a homogenous methylation profile without correlation with the proliferation signature whereas MCL was clearly separated from CLL. Gene ontology analysis revealed enrichment of developmental genes, in particular homeobox transcription factor genes, among targets methylated in MCL. In paper IV we compared three different stereotyped CLL subsets: #1 (IGHV unmutated), #2 (IGHV3-21) and #4 (IGHV mutated). Many genes were differentially methylated between each two subsets and immune response genes (e.g. CD80 and CD86) were enriched among genes methylated in subset #1 but not in subsets #2/#4. In summary, CNAs were frequent and not random in MCL. Specific CNAs correlated with a high proliferation gene expression signature or genomic complexity. TP53 mutations predicted short survival whereas 17p deletions did not. A high proliferation signature was not associated with differential DNA methylation in MCL, which demonstrated a homogeneous methylation pattern. In contrast, genomic methylation patterns differed between MCL and CLL and between stereotyped CLL subsets.

Les neuropathies à anticorps anti-MAG sont secondaires à une gammapathie monoclonale IgM dirigée contre la MAG des gaines de myéline des nerfs périphériques. Le traitement est celui de l’hémopathie sous‐jacente. Même si les thérapeutiques sont de plus en plus efficaces, les hémopathies restent le plus souvent incurables. Le rituximab est couramment utilisé dans le traitement des neuropathies à anticorps anti‐MAG, mais son efficacité n’a pas pu être clairement démontrée dans deux études contrôlées. Le FTY720 ou fingolimod est un sphingolipide, analogue de la sphingosine, qui inhibe les récepteurs de la sphingosine-1-phosphate (S1P). Il est utilisé comme immunosuppresseur dans la Sclérose en Plaques. Des études ont également rapporté un effet cytotoxique du FTY720 dans des hémopathies sans toutefois clairement expliquer son mécanisme d’action. L’objectif de ce travail est d’élucider les mécanismes moléculaires de l’effet cytotoxique du FTY720 dans un modèle d’hémopathie B, la leucémie lymphoïde chronique (LLC). Des cellules leucémiques primaires de LLC et une lignée cellulaire MEC1 ont été utilisées comme modèle expérimental in vitro. Le FTY720, comme la sphingosine, entraîne une cytotoxicité dose‐dépendante dans la LLC. Cet effet, médié par la forme non phosphorylée de FTY720, est indépendant des récepteurs au S1P. Le FTY720 induit l’expression de marqueurs d’apoptose: exposition de la phosphaJdylsérine, clivage de PARP et de caspase 3. Cependant sa toxicité apparaît indépendante des caspases. La lipidation accrue de LC3 et la formation d’autophagolysosomes indiquent que le FTY720 augmente également le flux autophagique. Cependant, des inhibiteurs de l’autophagie ne permettent pas de bloquer la mort cellulaire induite par le FTY720, suggérant que l’autophagie a ici un rôle protecteur vis à vis de la toxicité du FTY720. Plusieurs éléments permettent de conclure que le FTY720 est responsable d’une nécrose cellulaire : aspect morphologique de nécrose en microscopie électronique, perméabilisation membranaire précoce avec relocalisation cytoplasmique de HMGB1, libération extracellulaire de LDH, perméabilisation de la membrane lysosomale associée à une activation des cathepsines. Au niveau moléculaire, l’action du FTY720 n’est pas bloquée par la nécrostatine 1, indiquant que la nécrose induite par le FTY720 est indépendante de RIPK1 (receptor interacJng protein 1), une kinase clef des voies extrinsèques de nécrose cellulaire programmée. Par contre, nos travaux ont établi l’implication de DRP1 (dynamin related protein), une enzyme régulatrice de la fission mitochondriale, dans le processus de nécrose induite par le FTY720. En plus d’une relocalisation précoce de DRP1 à la mitochondrie accompagnée d’une augmentation de sa phosphorylation sur des sites régulateurs de son activité, nos expériences montrent que la suppression de son expression par interférence à ARN dans les cellules leucémiques réduit fortement la mort cellulaire induite par le FTY720. Le FTY720 est donc responsable dans la LLC d’une nécrose cellulaire programmée dépendante de DRP1. Nos résultats illustrent l’implication des sphingolipides dans la régulation de la survie cellulaire et dans les voies de nécrose programmée. Le FTY720 a un mode d’action original différent de l’apoptose induite par les chimiothérapies classiques. Le FTY720 pourrait donc être une alternative thérapeutique dans les néoplasies B résistantes aux chimiothérapies usuelles et dans certaines manifestations auto‐immunes des hémopathies comme les neuropathies à anticorps anti‐MAG. / Fingolimod (FTY720) is an immunosuppressive drug that was recently approved for the treatment of multiple sclerosis and is currently under pre-clinical investigation as a therapy for a number of haematological malignancies. Previous studies have indicated a role for FTY720 in inducing autophagy and caspase-independent cell death in cancer cells through incompletely characterized molecular mechanisms. Our study thus aims at a beeer understanding of the way of action of FTY720. In chronic lymphocytic leukaemia (CLL) cells, FTY720 induced cell death with typical features of apoptosis, including phosphatidylserine exposure and caspase-3 activation, and features of autophagy, including LC3 conversion, autophagolysosome formation and lysosomal cathepsins activation. However, neither caspase nor autophagy blockade prevented the cytotoxic effect of FTY720, suggesting another mechanism of cell death. Using electron and fluorescence microscopy, flow cytometry and biochemical analyses, we found that FTY720 treatment increased a fraction of annexin V-/7-AAD+ cells both in primary and transformed leukemic cells and induced morphological changes representative of necrosis, including oncosis, mitochondrial and plasma membrane alteration. FTY720 treatment resulted in increased plasma membrane permeability as shown by the extracellular translocation of the nuclear high mobility group box 1 (HMGB1) protein and by the release into the culture medium of the cytosolic enzyme lactate dehydrogenase (LDH). Interestingly, cell death induced by FTY720 was not prevented by pharmacological inhibition of RIPK1 and PP2A. In contrast, FTY720--‐induced necrosis was accompanied by an early relocation to the mitochondria of Dynamin Related Protein 1, DRP1. Importantly, FTY720 stimulation led to ma tior changes in the phosphorylation of serine residues associated with the mitochondrial fission activity of DRP1. Finally, siRNA--‐mediated knockdown of DRP1 significantly reduced necrotic cell death induced by FTY720. In this study, we thus demonstrate that in leukemic cells the cytotoxic effect of the immunosuppressive drug Fingolimod involves a DRP1--‐dependent regulated necrosis. These observations are important in line of the future development of Fingolimod as a new therapeutic agent in haematological malignancies.

Les protéines de choc thermiques (HSP) sont des chaperons moléculaires qui stabilisent le pliage et la conformation de protéines normales et oncogéniques, prévenant la formation d'agrégats protéiques. Elles sont impliquées dans la régulation de l'apoptose, de la survie cellulaire et dans la cancérogénèse. HSP90 est la protéine chaperone majeure de stabilisation d'oncogènes impliqués dans les hémopathies malignes. L'objectif de notre travail était de déterminer l'implication de HSP90 dans différents types d'hémopathies malignes, les Leucémies Aiguës Myéloïdes (LAM), les Syndromes Myélodysplasiques (SMD) et les Leucémies Aiguës Lymphoblastiques (LAL), et de tester son inhibition par un inhibiteur spécifique, la tanespimycine (17- AAG). Dans les LAM, nous avons évalué l'implication des différentes isoformes de la protéine dans la résistance aux chimiothérapies et aux inhibiteurs de HSP90. Ce travail met en évidence la valeur péjorative de l'expression de HSP90 dans les différents sous types d'hémopathies, corrélant avec un risque de rechute élevé ou d'évolution vers des formes plus agressives. L'utilisation de la tanespimycine a permis de déclencher l'apoptose dans les cellules immatures impliquées dans ces pathologies. HSP90 constitue donc une protéine majeure de la cellule leucémique, et son ciblage offre des perspectives intéressantes dans le traitement des hémopathies malignes / Heat shock proteins (HSP) are molecular chaperones that stabilize the folding and conformation of normal and oncogenic proteins, preventing the formation of protein aggregates. They are involved in the regulation of apoptosis, cell survival and carcinogenesis. HSP90 is the major chaperone implicated in stabilization of oncogenes involved in hematologic malignancies. The aim of our study was to determine the involvement of HSP90 in various types of malignancies, Acute Myeloid Leukemia (AML), Myelodysplastic Syndromes (MDS) and Acute Lymphoblastic Leukemia (ALL) and to test its inhibition by a specific inhibitor, the tanespimycine (17-AAG). In acute myeloid leukemia, we evaluated the involvement of different isoforms of the protein in resistance to chemotherapy and inhibitors of HSP90. This work highlights the pejorative value of HSP90 expression in different subtypes of malignancies, correlated with a high risk of relapse or progression to more aggressive forms. Use of tanespimycine has triggered apoptosis in immature cells involved in these diseases. HSP90 is therefore a major protein of the leukemic cell and its targeting offers interesting perspectives in the treatment of hematologic malignancies

Cytogenetic analysis of cells of chronic lymphocytic leukemia (CLL) is difficult because of their low proliferative activity. To obtain sufficient number of mitoses for performing chromosomal analysis a suitable stimulation of cell division is needed. Using DSP30/IL2 stimulated cultivation 391 CLL samples were investigated in 5 years' period. The cultivation was showed to have high success rate (96%; 375/391) with also high rate of detection of pathological clones by both karyotype and metaphase FISH analyses (in 84% of samples; 329/391). Almost in half of samples (44%; 171/391) other aberrations than recurrent FISH (i.e. 13q14 deletion, trisomy 12, TP53, ATM genes deletions) were found. Also high frequency of translocations (37%; 144/391), complex karyotypes (28%; 111/391) and clonal evolution, which was detected in one third of all samples (34% of samples with presence of more than two clones; 133/391) and like a new event in disease duration even more frequently (in 39% of samples repeatedly investigated after stimulated cultivation; 21/54), was revealed. The presence of translocations, complex karyotypes and clonal evolution was associated with progressive form of disease (P 0,000003, resp. P 0,0002 and P 0,05/P 0,04). In cases of the recurrent deletions the detailed analysis of metaphase...