Tesis sobre el tema "B-acute lymphoblastic leukemia"

Acute leukemias, such as acute lymphoblastic leukemia (ALL), are aggressive cancers characterized by the rapid proliferation of malignant hematopoietic cells. Throughout the last 60 years, childhood ALL’s long-term survival rates increased from less than 10% to more than 90%. Despite these improvements, certain subtypes remain hard to manage (e.g. mixed lineage leukemia, MLL-r), and even new therapies frequently fail. Therefore, identifying leukemia-cell restricted antigens in these ALL subtypes remains crucial in the quest to develop novel diagnostic tools and specific treatments. Traditionally, ALL research has mostly been focusing on genomics and transcriptomics efforts, mainly due to the limited amounts of patient material, and technical difficulties throughout sample processing. The potential encompassed in the use of other -omics technologies has remained unexplored in the context of ALL. For the work presented in this thesis, I have focused on undertaking the first comprehensive characterisation of glycocalyx alterations that occur in ALL and particularly in MLL-r in primary, patient derived cancer cells. This work supports the concept that the glycocalyx of ALL and MLL-r cells undergoes dramatic alterations that clearly differentiate these cells from healthy precursor B- (pre-B) cells. These findings might open doors towards novel potential diagnostic and therapeutic targets. The studies encompassed in chapters III, IV and V were performed in collaboration with Prof. Eleonora Heisterkamp’s team at the Beckman Research Institute (City of Hope, CA, USA). I have performed the first multi-omics analyses of primary patient MLL-r cells by integrating data from the transcriptome, glycome, and proteome of these cells. These results revealed that MLL-r cells exhibit distinct glycosylation features that differentiate them from healthy pre-B cells, which I was able to correlate with alterations at the transcript level of relevant glycosyltransferases. In depth proteome analyses revealed an overall good correlation between proteomics and transcriptomics findings, but also uncovered numerous examples where significant changes were just found in one but not the other approach. Nevertheless, this integrated approach used for the systematic evaluation of MLL-r allowed to obtain significant data for putative novel diagnostic/therapeutic protein markers and revealed important features of the disease that remained elusive until now. I was also able to apply the developed integrated multi-omics workflow to investigate the protective role of the surrounding microenvironment and its impact in environmentmediated drug resistance (EMDR) of pre-B ALL cells, which remains a major obstacle for the efficacy of chemotherapeutics in patients. To date the relevance of glycoconjugates for the development of EMDR has been largely unexplored. I explored a long-term co-culture system using human pre-B ALL cells and mitotically inactivated supporting murine stromal cells (OP9 cells), where pre-B ALL cells were put under a selective pressure to survive in the presence of vincristine, a widely used chemotherapeutic drug. I have performed a multi-omics analyses to understand the effect vincristine-resistance has on the cells' glycocalyx. These results demonstrated both glycome-wide and glycoprotein site-specific alterations, which could potentially be employed to identify emerging drug-resistance at an earlier stage or possibly serve as treatment targets in pre-B ALL. Throughout these studies, I have observed a significant modulation of the sialylation profile on pre-B ALL cells in patients and during EMDR development. Unsurprisingly, changes in sialylation have frequently been linked with development and progression of many cancer types but remain largely unexplored in the context of pre-B ALL. I investigated the impact of the major sialyltransferase, ST6Gal1, on the glycome of pre-B ALL cells. ST6Gal1 is the transferase known to be largely responsible for attaching sialic acids in an a2-6 linkage onto N-glycans. Surprisingly, these results demonstrated that a ST6GAL1 knockout did not ablate the production of a2-6 sialylated N-glycans, unless these N-glycans carried a core fucose residue. Demonstrating for the first time how core-fucosylation regulates a2-6 sialylation also allowed me to unravel the existence of ST6Gal1 independent, alternative a2-6 sialylation pathways that are specific for non-fucosylated N-glycans. I demonstrated that ST6GalNAc3-6 are capable to produce a2-6 sialylated N-glycans in the absence of core-fucose and that ST6Gal1 is required to introduce a2-6 sialylation on corefucosylated N-glycans. These results challenge long standing dogmas in glycobiology while delivering a novel understanding of hitherto unknown mechanism that regulate protein glycosylation, which will have a significant impact on our understanding of glycosylation changes in health and disease.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Griffith Health
Full Text

Thesis: S.M., Massachusetts Institute of Technology, Department of Biology, 2018.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 18-21).
B-cell acute lymphoblastic leukemia is the most common pediatric cancer, responsible for the most cancer-related deaths in children. Advances in chemotherapy over the past half-century have steadily increased the remission and survival of children with B-cell acute lymphoblastic leukemia to nearly 90%. However, the problems of minimal residual disease and relapsed and refractory disease persist. Personalized, targeted therapies have improved outcomes among the minority of patients for whom chemotherapy is ineffective. Immunotherapy, specifically bispecific T-cell engaging antibody therapy and chimeric antigen receptor T-cell therapy, has proven an effective treatment for relapsed and refractory B-cell acute lymphoblastic leukemia in children. These new modalities, however, have also introduced new adverse side effects to the treatment regimen. Though immunotherapy has increased remission and survival, more work must be done to reduce adverse effects and eliminate relapsed and refractory disease.
by Rana Hany Besada.
S.M.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis. Vita.
Includes bibliographical references.
Mutations in the zinc finger gene PHF6 are seen in approximately 20% of adult T-cell acute lymphoblastic leukemias and 3% of adult acute myeloid leukemias. The notable absence of PHF6 mutations in B-cell lineage malignancies has led to the hypothesis that PHF6 may act as a lineage-specific tumor suppressor gene. Recent work from our group described a role for PHF6 as a positive regulator of growth in B-cell acute lymphoblastic leukemia (B-ALL). To identify the mechanisms by which PHF6 acts to promote B-ALL growth in vivo, we utilized CRISPR-Cas9 to delete Phf6 in murine B-ALL cells. Transplantation of Phf6 knockout cells (Phf6KO) into immunocompetent recipients significantly extended disease latency and survival. Strikingly, these mice developed lymphomas, characterized by significantly enlarged lymph nodes, decreased disease burden in the spleen and increased expression of the canonical T-cell marker CD4, suggesting that Phf6KO B-ALL cells adopt alternate lineage programs in vivo. To dissect the molecular mechanisms by which Phf6 regulates this lineage decision, we carried out a combination of RNA and chromatin immunoprecipitation (ChIP-Seq) sequencing analyses in Phf6WT and Phf6KO cells. RNA sequencing analysis revealed many differentially expressed genes in Phf6KO B-ALL cells. Notably, genes and gene sets that were significantly down-regulated in Phf6KO cells included those involved in pathways important for B-cell development and function. ChIP-Seq analysis of PHF6 and several histone marks revealed that PHF6 and H3K27ac signals co-localize close to the transcription start site and enhancer regions of a significant proportion of differentially expressed genes. Transcription factor binding motif analysis revealed significant enrichment for several well-described transcriptional regulators of B-cell development. Importantly, we demonstrated that the transcription factors TCF12 and NF-kB co-immunoprecipitated with PHF6 in Phf6WT B-ALL cells. These findings discovered a novel role for PHF6 in the maintenance of B-cell identity in B-ALL, by activating genes that are crucial for B-cell lineage maintenance. Collectively, these results indicate that loss-of-function of Phf6 in B-ALL leads to an unstable cell identity state, in which cells need to acquire alternate developmental programs in order to survive. These findings could potentially explain the absence of PHF6 mutations in human B-cell lineage malignancies.
by Yadira M. Soto-Feliciano.
Ph. D.

Les cellules Natural Killer (NK) représentent une population de cellules innées lymphoïdes aux fonctions anti-infectieuses et antitumorales. Les leucémies aiguës lymphoblastiques pré-B (LAL pré-B) constituent le cancer de l’enfant le plus fréquent et ont été décrites comme résistantes à la cytotoxicité médiée par les NK bien que les bases moléculaires demeurent inconnues.L’objectif de ces travaux a été de caractériser cette résistance. En développant un essai de cytotoxicité par cytométrie en flux et en utilisant des cellules effectrices activées in vitro, nous avons établi la sensibilité retardée des LAL pré-B à la cytotoxicité NK : initialement résistantes après 4h d’incubation, elles sont fortement tuées après 25h.Cette cytotoxicité est contact-dépendante mais ni la voie de l’exocytose des granules cytotoxiques ni celle des récepteurs de mort n’y contribuent. La mort cellulaire des cibles est de profil apoptotique mais indépendante des caspases ; la signalisation mitochondriale l’amplifie partiellement. Interférer avec les dérivés de l’oxygène par un antioxydant diminue la cytotoxicité. Nous montrons que les cellules NK de patients atteints de granulomatose septique chronique liée à l’X présentent un défaut de cette nouvelle cytotoxicité. Nous démontrons l’expression par les NK des composants clefs d’une NADPH oxydase distincte du complexe utilisé par les phagocytes. Nos travaux établissent l’existence d’une voie de cytotoxicité non conventionnelle et en définissent les principaux prérequis moléculaires
Natural Killer (NK) cells are innate lymphoid cells with anti-infectious and anti-tumoral activities. Among neoplasia, pre-B acute lymphoblastic leukemias (pre-B ALL) represent the most common form of cancer in childhood and were shown to be resistant to NK cell mediated cytotoxicity although the mechanisms explaining this phenomenon are incompletely understood.In the present work, we investigated the relative immune resistance of pediatric pre-B ALL targets to activated NK cells. We developed a flow cytometry based cytotoxicity assay to assess the NK activity and the involvement of long term cytotoxic pathways. Although pre-B ALL blasts were strongly resistant at 4h, we found a considerable delayed NK killing at 25h.Further investigations revealed that cell contact was mandatory for efficient killing but also that neither the granule exocytosis nor the death receptor pathway were involved. Target cell death was caspase independent but mitochondria signaling amplified it. We then showed that NK cells from patients with X-linked chronic granulomatous disease could not kill efficiently ALL blasts and that NK cells expressed key components of a NADPH oxidase complex that was distinct from the phagocyte type. Our work reveals an uncharacterized effector pathway among cytotoxic lymphocytes and establishes key molecular requirements for this unconventional pathway

Background: The treatment of B-cell acute lymphoblastic leukemia (B-ALL) has been enriched by novel agents targeting surface markers CD19 and CD22. Inotuzumab ozogamicin (INO) is a CD22-calicheamicin conjugated monoclonal antibody approved in the setting of relapse/refractory (R/R) B-ALL able to induce a high rate of deep responses, not durable over time. Aims: This study aims to identify predictive biomarkers to INO treatment in B- ALL by flow cytometric analysis of CD22 expression and gene expression profile. Materials and methods: Firstly, the impact on patient outcome in 30 R/R B-ALL patients of baseline CD22 expression in terms of CD22 blast percentage and CD22 fluorescent intensity (CD22-FI) was explored. Secondly, baseline gene expression profile of 18 R/R B-ALL patient samples was analyzed. For statistical analysis of differentially expressed genes (DEGs) patients were divided in non-responders (NR), defined as either INO-refractory or with duration of response (DoR) < 3 months, and responders (R). Gene expression results were analyzed with Ingenuity pathway analysis (IPA). Results: In our patient set higher CD22-FI, defined as higher quartiles (Q2-Q4), correlated with better patient outcome in terms of CR rate, OS and DoR, compared to lower CD22-FI (Q1). CD22 blast percentage was less able to discriminate patients’ outcome, although a trend for better outcome in patients with CD22 ≥ 90% could be appreciated. Concerning gene expression profile, 32 genes with corrected p value <0.05 and absolute FC ≥2 were differentially expressed in NR as compared to R. IPA upstream regulator and regulator effect analysis individuated the inhibition of tumor suppressor HIPK2 as causal upstream condition of the downregulation of 6 DEGs. Conclusions: CD22-FI integrates CD22-percentage on leukemic blasts for a more comprehensive target pre-treatment evaluation. Moreover, a unique pattern of gene expression signature based on HIPK2 downregulation was identified, providing important insights in mechanisms of resistance to INO.

Acute lymphoblastic leukemia is the most common childhood tumor and about 85% of cases are due to the expansion of a clone of B-cell precursors (BCP-ALL). Steady progress in development of effective treatments has led to an elevated rate of success in treating this disease. To date, even thought about 75% of patients are cured, 25% of cases having a relapse has a survival probability of only 30%. Of note, more than 50% of relapses concern patients not classified in high-risk groups based on assessment of prognostic factors at diagnosis or on measurement of Minimal Residual Disease (MRD), a surrogate parameter of individual response to therapy. The understanding of the molecular pathogenesis of the disease and the biological basis for explaining different clinical response represent the most relevant challenges in the field that could open new perspectives in the identification of either new prognostic factors or new molecules for targeted therapeutic approaches. In this setting we focused our studies on a poor prognosis subtype of BCP-ALL, bearing rearrangements in the Cytokine Receptor Like Factor 2 (CRLF2) gene. Alterations in the CRLF2 gene (CRLF2r) are present in about 10% of childhood BCP-ALL, 50% of Down Syndrome ALL and 50% of Ph-like ALL and are responsible of the overexpression of Thymic Stromal Lymphopoietin Receptor (TSLPR).We first studied the phenotypic expression of TSLPR and its associated molecular and phosphosignaling profile in a large and prospective cohort of patients, demonstrating that the TSLPR screening at diagnosis can be successfully performed by standardized flow cytometry protocols. We further found an activation of both JAK/STAT and PI3K/mTOR pathways in the CRLF2 rearranged patients giving the rationale to test targeted tyrosine kinase inhibitors (TKIs) to treat this subgroup of patients. Starting from these evidences we investigate the TSLPR-driven pathway by using a new high dimensional single cell approach, called mass cytometry (CyTOF), that allow the simultaneous measurement of about 50 parameters per cell. By applying this technology we studied 15 BCP-ALL (9 CRLF2r and 6 CRLF2wt) primary samples treating the cells with 3 different TKIs (Dasatinib, Ruxolitinib and BEZ235) and 2 anti-TSLPR monoclonal antibodies. We demonstrated a strong signaling inhibition with Dasatinib, Ruxolitinib and one anti-TSLPR mAb in CRLF2r BCP-ALL samples associated with a synergic in vitro efficacy of JAK/STAT inhibitors (Ruxolitinib and anti-TSLPR mAb) with Dasatinib and BEZ235 in BaF3 CRLF2/IL7Rα expressing cells. We also studied the MRD cells at Day 8 and Day 15 showing that the MRD cells of CRLF2r patients maintain their TSLPR positivity and responsiveness to both stimulation and drug treatment. Meanwhile, we also tested a histone deacetylase inhibitor, Givinostat, as a new therapeutic tool to use with conventional chemotherapy for childhood CRLF2r BCP-ALL. We demonstrated an in vitro inhibition and cell death induction of BCP-ALL CRLF2r cell lines at very low doses confirmed by a cytotoxic effect also in ex-vivo experiments where Givinostat was able to kill blast cells preserving the normal hematopoietic counterpart as demonstrated by a CyTOF analysis. In vivo Givinostat was able to markedly reduce the engraftment of leukemic blasts in the bone marrow of treated mice causing a transcriptional modulation of genes involved in JAK/STAT pathway leading to the inactivation of the signaling network. In the last part of our project we studied, in collaboration with Dr.Kara Davis at Stanford University, 60 primary diagnostic samples with known outcome in order to investigate possible features correlated with relapse. By using CyTOF we identified 8 predictors that separate patients who will relapse from whose who will not and these features suggested a high basal activation of IL-7 signaling node in late pro-B cells and poor response following pre-BCR engagement in pre-BI cells in patients going to relapse.

An 11 year old, hispanic girl with a history of B-cell acute lymphoblastic leukemia was admitted to the hospital for symptoms compatible with Bartonella henselae infection. The first molecularly diagnosed case of disseminated Bartonella henselae infection was reported in an immunocompromised patient in Lima, Peru. The analysis was confirmed by Polymerase Chain Reaction and automated sequencing of a liver biopsy sample, even though the serologic tests were negative. In conclusion, Bartonella spp. infection should have a particular diagnostic consideration in immunocompromised patients with fever of unknown origin and further investigation regarding the patient's past exposures with cats should also be elicited.

La leucemia linfoblastica acuta B (LLA-B) è la leucemia più comune nei bambini (80%), ma ha anche un picco di incidenza in età adulta. Recentemente, gli approcci di immunoterapia diretti contro la molecola CD19 hanno dimostrato un notevole successo nel trattamento della LLA-B recidiva e refrattaria, che rimane una delle principali necessità cliniche. Svantaggi importanti di tali strategie sono la comparsa di recidive CD19-negative e aplasia delle cellule B come risultato della persistenza delle cellule anti-CD19 CAR. In questo contesto, abbiamo ipotizzato che il recettore per il fattore di attivazione delle cellule B (BAFF-R), una proteina transmembrana fondamentale nella maturazione delle cellule B e nella loro sopravvivenza, potrebbe essere una molecola interessante per strategie di targeting, avendo come vantaggio il fatto che questo recettore non è rilevabile nelle cellule B precursori del midollo osseo. Nel nostro lavoro abbiamo dimostrato che BAFF-R è altamente espresso in tutti i campioni primari di LLA-B sia all'insorgenza che alla ricaduta. Al fine di sviluppare un approccio mediato da un recettore chimerico (CAR) specifico per l’antigene BAFF-R, sono state sviluppate sei tipologie di CAR anti-BAFFR che si differenziano per l'inversione della VH e VL e la lunghezza del dominio cerniera. Cellule killer indotte da citochine (CIK), ingegnerizzate utilizzando un sistema non virale che sfrutta trasposoni Sleeping Beauty (SB), esprimono stabilmente anti-BAFFR.CARs e mantengono il loro caratteristico fenotipo. Tra i CAR generati, il CAR più breve VHVL esercita la massima attività anti-leucemica verso le cellule bersaglio, come NALM-6, con un'attività citotossica in vitro pari al 60%. Abbiamo valutato anche le funzioni effettrici a lungo termine di rilascio di citochine mediante colorazione intracellulare (8,9 ± 2% di cellule producesti IFN-γ e 16,4 ± 5,5% di cellule producesti IL-2). Inoltre, abbiamo anche rilevato una specifica attività citotossica nei confronti di blasti primari di LLA-B (media 65,6 ± 4,5%, n = 9). Combinando le cellule trasdotte Invsh.CAR con le cellule trasdotte CD19.CAR abbiamo rilevato una attività antitumorale superiore nei confronti di tutti le linee target. Infine, utilizzando un campione raccolto da un paziente con recidiva di malattia negativa per l’antigene CD19, abbiamo dimostrato la capacità del INVsh.CAR di lisare blasti CD19-negativi. Concludendo, questi risultati dimostrano come questo recettore sia un bersaglio sicuro e attraente per un trattamento immunoterapeutico di seconda linea per la LLA-B in caso di recidiva dopo terapia con approcci di targeting dell’antigene CD19 o per un approccio di targeting doppio. Essendo BAFF-R ristretto a cellule B mature e assente in precursori e plasmablast, la nostra strategia potrebbe avere una tossicità inferiore, per quanto riguarda l'emergere di aplasia delle cellule B osservata nei pazienti trattati con le cellule T anti-CD19 CAR.
B-cell Acute Lymphoblastic Leukemia (B-ALL) is most common in children (80%), but it has also a peak of incidence in adult age. Recently, immunotherapeutic approaches targeting the CD19 molecule have demonstrated remarkable success in the treatment of relapsed and refractory B-ALL, which remains a major clinical need. Important downsides of these strategies are the emergence of CD19-negative relapses and B-cell aplasia as a result of anti-CD19 CAR T-cell persistence. In this context, we hypothesized that the receptor for B-cell activating factor (BAFF-R), a transmembrane protein fundamental in B-cell maturation and survival, could be an interesting molecule to be targeted, taking the advantage that this receptor is undetectable on bone marrow B-cell precursors. Here we showed that BAFF-R is highly expressed in B-ALL primary samples at the onset and relapse In order to develop a chimeric antigen receptor (CAR) approach targeting BAFF-R molecule, six anti-BAFFR CAR genes that differ for the inversion of the VH and VL and the length of the spacer domain have been generated. Cytokine-induced Killer (CIK) cells, engineered using an improved Sleeping Beauty (SB) transposon system, stably expressed anti-BAFFR.CARs, and maintained their characteristic phenotype. Among the newly constructed CARs, the shortest VHVL CAR exerted the highest anti-leukemic activity towards target cells, such as NALM-6, with an in vitro killing activity of 60%. We also evaluated later effector functions in terms of cytokine release by intracellular staining (8,9±2% of IFN-γ and 16,4±5,5% of IL-2 producing cells). Importantly, we also detected a specific cytotoxic activity towards primary B-ALL blasts (average 65,6±4,5%, n=9). Combining the Invsh.CAR with CD19.CAR we detected a superior antitumor activity towards ALL targets. Furthermore, by using a sample collected from a patient relapsed with CD19 negative disease, we demonstrated the ability of the INVsh.CAR to lysate CD19-negative blasts. Taken together, these findings make this receptor a safe and attractive target for a second line B-ALL immunotherapy in case of relapse after CD19-targeting therapies or for a double targeted approach. Being restricted to mature B cells, but absent in precursors and plasmablasts, our strategy could have an inferior toxicity concerning the emergence of B-cell aplasia observed in patients treated with anti-CD19 CAR-modified T cells.

Treatment of pediatric acute lymphoblastic leukemia (ALL) is increasingly successful, achieving cure rates of over 80%. Early identification of patients with high risk for relapse has led to improved outcome, however, two third of patients encountering relapse were initially stratified into low-intermediate risk groups. The identification of better upfront prognostic factors remains an important challenge in childhood ALL. In this thesis, gene expression profiling (GEP) was applied to different research approaches aiming to dissect subgroups and to find novel therapeutic targets in B cell precursor ALL (BCP ALL). Among BCP ALL, the patients lacking major genomic aberrations (B-others) represent the subgroup that is most in need of in depth investigations in order to indentify new prognostic factors and improve of risk stratification. To advance biological knowledge of B-others we performed an integrated study of gene and non coding RNAs expression and genetic aberrancies. Chapter 1 reports a study on profiling by gene expression arrays of 145 Italian B-others BCP ALL patients and in a representative subcohort of patients microRNAs (miRNAs) expression profiling and genome-wide DNA copy number variation analysis. In this study we found that 25% of Italian B-others patients fits in a group with unique signature and is associated to a favourable outcome. MicroRNAs expression profiling of this group revealed an unique miRNAs signature characterized by over expression of hsa-miR-125b, -125b-2*, -99a, -100, -125a-3p and has-miR-491-5p. Over expression of cluster miR-125b-2 in region 21q21.1 goes along with over expression of genes in the same chromosomal region. Genome-wide analysis excluded copy number alteration of the 21q21.1 region. The frequent involvement of human chromosome 21 (Hsa21) aberrations in ALL (e.g. hyperdiploidy (HD), t(12;21) or iAmp21) and the involvement of the 21q21.1 region suggest a direct and functional contribution of Hsa21 genes to the malignant transformation of hematopoietic cells. There for there is high interest in studying ALL in children with Down Syndrome (DS), where trisomy 21 is constitutional and where the incidence of ALL is approximately 20-fold higher than in the general population. In Chapter 2 is presented a study of genomic analysis of a large group of DS ALLs that characterizes molecular abnormalities specific of this ALL group. Gene expression analysis revealed that DS ALL is a highly heterogeneous disease not definable as a unique ALL subtype with an enrichment of DNA damage and BCL6 responsive genes suggesting B-cell lymphocytic genomic instability. Surprisingly, only a single Hsa21 gene, SON was included in the DS ALL signature and it was only slightly upregulated. Furthermore gene expression data suggested that DS ALL and HD ALL are very different leukemias, reflecting the fundamental differences between constitutional and acquired trisomy such as the developmental stage in which the trisomy occurs and the fact that a constitutional trisomy is present both in the leukemia cells and in their microenvironment. The study further revealed that 62% of the DS ALL samples were characterized by aberrant expression of the type I cytokine receptor CRLF2. Two kind of aberrations involving CRLF2 were identified: a cryptic translocation involving IGH@ and CRLF2 in the pseudoautosomal region (PAR1) of the sex chromosomes and a deletion within PAR1. This aberration resulted in the P2RY8-CRLF2 fusion and leads to overexpression of CRLF2. Furthermore a novel activating somatic mutation, F232C, in CRLF2 was identified. We demonstrated that CRLF2 and mutated JAK2 cooperate in conferring cytokine independent growth to pro-B cells suggesting that the DS ALL children with CRLF2 aberrant expression may benefit from therapy blocking the CRLF2-JAK2 pathway. Since CRLF2 aberrations were found also among non DS patients, we further analyzed the incidence and prognostic impact of this potential new marker in BCP ALL Italian patients enrolled into the AIEOP-BFM ALL2000 study. Chapter 3, presents the study of a representative cohort of 464 non DS BCP ALL patients that was analyzed for the expression levels of CRLF2 and for the occurrence of CRLF2 rearrangements. In this study we found that the P2RY8-CRLF2 rearrangements in association with 20 times over expression of CRLF2 identifies BCP ALL patients with a very poor prognosis and, among them, an important subset of patients currently stratified in the intermediate risk need to be considered for treatment adaptation. Investigating the pathways highlighted by GEP analysis and testing drug effects require a substantial availability of leukemia samples. Primary ALL samples are difficult to culture in vitro and currently available cell lines poorly reflect the heterogeneous nature of the disease. Mouse xenotransplantation models are therefore widely used for in vivo testing and to amplify the number of leukemia cells to be used for various analyses. In Chapter 4 study we assessed the capability of xenografted samples to recapitulate their respective primary leukemia, and we investigated whether the murine microenvironment selects for leukemia initiating cells leading to a bulk tumor markedly different from the diagnostic patient sample. We analysed the gene expression profiles of 7 primary paediatric ALL samples at diagnosis as well as of their respective xenograft leukaemia samples after serial primary, secondary and tertiary passages in the NOD/SCID/huALL transplant model. In this study we demonstrated that the NOD/SCID/huALL transplant model recapitulates the primary human leukaemia, mimics the features of the primary malignancy and retains these characteristics over serial passages without selection for a subclone of the initial leukaemia. Chapter 5 reports on a study that investigated engraftment properties of 50 pediatric ALL samples transplanted into NOD/SCID mice. Time to leukemia (TTL) was determined for each patient sample engrafted as weeks from transplant to overt leukemia. The study shows that short TTL was strongly associated with high risk for early relapse, identifying a new independent prognostic factor. The high risk phenotype is reflected by a gene signature that identified patients with early relapse in an independent patient cohort. Gene expression profiling revealed a set of genes associated with this aggressive phenotype providing a potential strategy to identify these high-risk patients. Most importantly, pathways involving mTOR regulating cell growth were identified, providing targets for alternative therapeutic strategies for these high risk patients. Concluding, ten years after its introduction in oncohematology, GEP constitutes to be a valuable research tool, efficacious in subtype discovery, biomarkers identification and discoveries of deregulated molecular pathways.
La cura della leucemia linfoblastica acuta (ALL) sta migliorando con successo, raggiungendo un tasso di guarigione che va oltre l’80%. L’identificazione precoce dei pazienti con alto rischio di ricaduta ha portato ad un miglioramento generale dell’outcome, tuttavia, due terzi dei pazienti che incorrono nell’evento di ricaduta vengono inizialmente stratificati in gruppi a basso rischio o rischio intermedio. L’identificazione di migliori fattori prognostici rimane un’importante sfida nelle ALL pediatriche. In questa tesi, lo studio del profilo di espressione genica è stato applicato a diversi approcci di ricerca, con lo scopo di individuare sottogruppi e trovare nuovi target terapeutici nelle ALL a cellule precursori B (BCP ALL) Tra le BCP ALL, i pazienti privi delle aberrazioni genomiche più riccorrenti (B-others) rappresentano il sottogruppo che più necessita di studi approfonditi, tesi ad identificare nuovi fattori prognostici e migliorare la loro stratificazione nelle classi di rischio. Per aumentare le conoscenze biologiche riferite al gruppo dei B-others, è stato eseguito uno studio integrato di espressione genica, espressione di non coding RNAs e analisi delle aberrazioni genetiche. Il Capitolo 1 riporta lo studio mediante microarrays di espressione genica di 145 pazienti Italiani affetti da BCP ALL e, in una sotto-coorte rappresentativa, lo studio dell’espressione dei microRNAs (miRNAs) e l’analisi di variazione di DNA copy number estesa all’intero genoma. Da questo studio è emerso che il 25% dei pazienti Italiani di tipo B-others rientrano in un gruppo con una signature specifica e sono associati ad un outcome favorevole. Lo studio del profilo di espressione dei miRNAs rivela in questo gruppo una specifica signature di miRNAs caratterizzata dalla sovra espressione di hsa-miR-125b, -125b-2*, -99a, -100, -125a-3p e has-miR-491-5p. La sovra espressione del cluster miR-125b-2 nella regione 21q21.1 è accompagnata dalla concomitante sovra espressione dei geni nella stessa regione cromosomica. Le analisi sul genoma hanno portato ad escludere la presenza di alterazioni di DNA copy number nella regione 21q21.1. Il frequente coinvolgimento di aberrazioni a carico del cromosoma 21 nelle ALL (come nel caso di iperdiploidia (HD), t(12;21) o iAmp21) e il coinvolgimento della regione 21q21.1, suggeriscono un diretto e funzionale contributo dei geni nel cromosoma 21 alla trasformazione maligna delle cellule ematopoietiche. A questo proposito c’è un grande interesse nello studio delle ALL nei bambini affetti dalla Sindrome di Down (DS), nei quali la trisomia 21 è costituzionale e per i quali l’incidenza di ALL è approssimativamente 20 volte maggiore che nel resto della popolazione. Nel Capitolo 2 viene presentato uno studio di analisi genomica di un grande gruppo di DS ALL che mira a caratterizzare le anomalie molecolari specifiche di questo gruppo di ALL. L’analisi di espressione genica ha rivelato che le DS ALL sono leucemie molto eterogenee, non definibili come un unico sottotipo di ALL, con un arricchimento di geni rispondenti al signalling di BCL6 e di risposta al danno al DNA, che suggerisce un’instabilità genomica dei linfociti B. Sorprendentemente, solamente un gene appartenente al cromosoma 21, SON, è compreso nella signature delle DS ALL e risulta solo debolmente up-regolato. Inoltre, i dati di espressione genica suggeriscono che le DS ALL e le HD ALL sono leucemie molto diverse, riflettendo le differenze fondamentali tra trisomia costituzionale e acquisita, quali lo stadio di sviluppo nel quale la leucemia insorge e il fatto che la trisomia costituzionale è presente sia nelle cellule leucemiche che nel microambiente. Lo studio ha inoltre rilevato che il 62% delle DS ALL sono caratterizzate da un’aberrante espressione del recettore per le citochine di tipo I CRLF2. Due tipi di aberrazioni che coinvolgono CRLF2 sono state identificate: una traslocazione criptica che coinvolge il locus IGH@ e CRLF2 nella regione pseudoautosomale PAR1 dei cromosomi sessuali e una delezione in PAR1. Queste aberrazioni danno luogo alla formazione del trascritto di fusione P2RY8-CRLF2 che determina la sovra espressione di CRLF2. Inoltre una nuova mutazione somatica attivante, F232C, in CRLF2 è stata identificata. E’ stato dimostrato che CRLF2 e JAK2 mutato cooperano nel conferire capacità di crescita indipendente da citochine a cellule pro-B suggerendo che i bambini affetti da DS e ALL con un’espressione aberrante di CRLF2 possono trarre beneficio da terapie mirate a bloccare il pathway di CRLF2-JAK. Dal momento che le aberrazioni a carico di CRLF2 sono state trovate anche tra i pazienti non affetti dalla Sindrome di Down, è stata analizzata l’incidenza e l’impatto prognostico di questo potenziale nuovo marcatore nei pazienti Italiani con BCP ALL arruolati nello studio AIEOP-BFM ALL2000. Il Capitolo 3 presenta lo studio di una coorte rappresentativa di 464 pazienti con BCP ALL non affetti da DS che è stata analizzata per l’espressione di CRLF2 e per la presenza di riarrangiamenti a carico di CRLF2. Da questo studio è emerso che il riarrangiamento P2RY8-CRLF2 in associazione con la sovra espressione di CRLF2 (di almeno 20 volte maggiore che nel resto della coorte), identifica pazienti con una prognosi molto sfavorevole e, tra essi, un inportante sottogruppo di pazienti attualmente stratificati nella classe di rischio intermedia e che necessitano di essere considerati per un adeguamento della terapia. Per investigare i pathways emersi dalle analisi di espressione genica e per testare l’effetto dei farmaci è necessaria una grande disponibilità di cellule leucemiche. Le cellule da leucemia primaria sono difficili da coltivare in vitro e le linee cellulari attualmente disponibili non riescono a riflettere la natura eterogenea della malattia. Per questo motivo i modelli di xenotrapianto in topo sono ampiamente usati sia per lo studio in vivo che per amplificare il numero di cellule leucemiche da usare nelle varie analisi. Nello studio riportato nel Capitolo 4 è stata verificata la capacità delle cellule leucemiche ottenute da xenotrapianto di ricapitolare la loro rispettiva leucemia primaria ed è stata valutata la possibilità di una selezione da parte del microambiente murino per particolari cellule “inizianti” la leucemia che portino ad una massa tumorale marcatamente diversa da quella dei pazienti alla diagnosi. E’stato analizzato il profilo di espressione genica di 7 ALL primarie pediatriche alla diagnosi e le rispettive cellule leucemiche ottenute da xenotrapianto dopo un primo, un secondo ed un terzo passaggio seriale nel modello di trapianto di leucemia umana in topo NOD/SCID/huALL. In questo studio è stato dimostrato che il modello di trapianto NOD/SCID/huALL ricapitola la leucemia primaria umana, mima le caratteristiche del tumore primario e ne trattiene le caratteristiche durante i passaggi seriali senza selezionare per un sottoclone della leucemia primaria iniziale. Il Capitolo 5 riporta uno studio che ha investigato le proprietà di attecchimento di 50 ALL pediatriche trapiantate in topi NOD/SCID. Il tempo di attecchimento (Time To Leukemia – TTL) è stato determinato per ogni campione attecchito in termini di settimane trascorse dal trapianto alla manifestazione della leucemia. Lo studio ha mostrato che un breve TTL è fortemente associato con un alto rischio di ricaduta precoce, costituendo di fatto un nuovo marcatore prognostico indipendente. Il fenotipo di alto rischio è riflesso in una signature in grado di identificare pazienti incorsi precocemente nell’evento di ricaduta in una coorte di pazienti indipendente. Lo studio di espressione genica rivela una serie di geni associati con questo fenotipo aggressivo, mettendo a diposizione una potenziale strategia per identificare i pazienti ad alto rischio. In modo ancora più importante, pathways che regolano la crescita cellulare e che coinvolgono mTOR sono stati identificati, indicando dei target per strategie terapeutiche alternative per i pazienti ad alto rischio di riaduta. Concludendo, dieci anni dopo la sua introduzione in oncoematologia, lo studio del profilo di espressione genica si conferma essere un valido strumento di ricerca, efficace nella scoperta di nuovi sottotipi, nell’individuazione di biomarcatori e nel portare alla luce pathways molecolari deregolati.

Acute lymphoblastic leukaemia (ALL) is the most common cancer diagnosed in children aged 1-14 years. There have been vast improvements in clinical outcomes for children diagnosed with ALL with cure rates of up to 90% achievable for some forms of the disease. Despite these successes, some patients still relapse and the prognosis for these individuals is poor, thus there is still a great deal to be learned about the complex biology underlying ALL. Connective tissue growth factor (CTGF/CCN2) is a novel candidate gene in precursor B-cell (pre-B) ALL, and is aberrantly expressed in a high proportion (around 75%) of cases. While the CTGF protein has no known role in lymphocyte biology or haemopoiesis, CTGF gene expression has been associated with a poor outcome in children and adults, particularly in those patients deemed to have a high-risk of relapse. The primary aims of this study were to characterise CTGF expression in paediatric pre-B ALL tumours and cell lines, and to investigate what mechanisms were responsible for its deregulated expression by examining the contribution of both genetic and epigenetic factors.Analysis of a cohort of 73 primary paediatric pre-B ALL specimens, confirmed CTGF was aberrantly expressed in 75% of patients at heterogeneous levels. CTGF expression was found to be associated with prognosis in this cohort, as there was a trend toward lower 5 year relapse free survival (RFS) in patients with CTGFpos ALL (71% RFS in CTGFpos, 83% RFS in CTGFlow/neg), however this did not reach statistical significance (p=0.39). There was no difference in overall 5 year survival (OS) between patients with CTGFpos and CTGFlow/neg ALL. The association between CTGF expression and clinical features recorded at the time of biopsy was undertaken. These features included; age, gender, percentage blasts in bone marrow, peripheral haemoglobin level, spleen or lymph node involvement, or whether the sample was obtained at diagnosis or relapse. Patients with enlarged lymph nodes displayed a lower mean CTGF expression compared to those patients with no lymph node involvement. A similar pattern was observed with patients exhibiting enlargement of the spleen, however this did not reach statistical significance. No other clinical features were associated with CTGF expression.Analysis of global gene expression patterns in three independent paediatric pre-B ALL cohorts (PMH; n=73, Ross; n=118, Kang; n=207), identified five genes that were highly correlated with CTGF expression; SOCS2, MEF2C, ADD3, GSN and DPYSL2. In silico analysis of the 5’ flanking sequences of these genes, as well as CTGF identified predicted binding sites for the two Ikaros family proteins IKAROS and HELIOS, with at least one HELIOS site present in the 5’ flanking sequence of all correlated genes. This suggested a possible role for Ikaros family proteins in modulating CTGF gene expression. Subsequent analysis of a microarray cohort recently characterised for IKAROS gene deletions revealed that mutations or deletions within the IKAROS coding region were significantly associated with higher mean CTGF gene expression, implying that a loss of IKAROS function may promote activation of the CTGF locus. This is the first evidence implicating the Ikaros family of lymphoid transcriptional regulators in deregulation of the CTGF locus.Northern blotting of RNA from B-lineage ALL cell lines uncovered evidence of alternative splicing of CTGF mRNA. Non-canonical transcripts of approximately 1.3kb and 1.6 kb were hybridised by a CTGF-specific probe in extracts from all CTGFpos cell lines. Sequencing of cDNA fragments as well as 5’ and 3’ RACE products from a cell line with the highest level of CTGF expression revealed a number of CTGF transcripts exhibiting internal deletions of exons 2 and 3, as well as truncation of exons 1 and 4. 3’ RACE also identified a 1.3kb transcript that was devoid of 3’ UTR regulatory elements as a result of premature polyadenylation. These findings represent the first direct evidence of alternative splicing of CTGF pre-mRNA in any tissue type and further investigation is warranted to fully characterise these tumour-associated transcripts and their protein coding potential.Structural changes within the genome are common in leukaemia and deletions affecting the long arm of chromosome 6 occur in around 30% of cases of pre-B ALL. To investigate whether deregulation of CTGF expression has a genetic basis, the CTGF locus at 6q23.1 was investigated for structural alterations or mutations. Southern blotting performed in seven B-lineage ALL cell lines (4 CTGFpos, 3 CTGFneg) confirmed that the CTGF locus was not cytogenetically rearranged. Furthermore, analysis of CTGF copy number by qPCR in primary paediatric pre-B ALL specimens (n=17) and B-lineage ALL cell lines (n=7) confirmed that gene amplification could not account for CTGF overexpression. Sequencing of the CTGF promoter and 3’ UTR in three B-lineage ALL cell lines confirmed that there were no mutations affecting these important regulatory regions, although one cell line harboured the rs6918698 -739 C>G SNP, which is predicted to disrupt SP3-mediated repression of CTGF gene expression.Epigenetic regulation of gene expression is frequently altered in neoplasia. The CTGF locus contains a CpG island, and methylation of this region was found to be inversely correlated with CTGF gene expression in B-lineage ALL cell lines, as assessed by both methylation-specific PCR and by bisulfite sequencing. Bisulfite sequencing of primary tumour specimens revealed that hypomethylation of the CTGF locus was a widespread feature of pre-B ALL. By contrast, analysis of primary T-ALL specimens demonstrated extensive methylation at the CTGF locus, indicating that CTGF may be permissive for expression specifically in pre-B ALL.This study has highlighted several novel aspects of CTGF expression in pre-B ALL, including a potential role for Ikaros family proteins in regulating the CTGF locus, and the existence of CTGF mRNA transcripts generated through alternative pre-mRNA splicing. Investigation of mechanisms promoting CTGF gene expression in pre-B ALL revealed that the rs6918698 C>G SNP was present in one pre-B ALL cell line. Hypomethylation of the CTGF locus was a notable feature of primary pre-B ALL specimens and was in contrast to the hypermethylation observed in 2 T-ALL specimens and CD34pos bone marrow cells. These findings will direct future research to elucidate the complex mechanisms regulating CTGF expression in pre-B ALL. It is anticipated that illuminating the role of CTGF in the pathogenesis of ALL may result in significantly improved patient outcomes through the development of targeted and less toxic therapies, and through improved risk-based stratification of patients to ensure those at a high risk of relapse are directed toward an appropriate level of therapeutic intervention.

I tumori delle cellule B sono un gruppo eterogeneo di patologie per le quali le opzioni terapeutiche includono chemioterapia e immunoterapia. Nonostante il recente sviluppo di nuove strategie terapeutiche, la maggior parte dei pazienti, tuttavia, sviluppa resistenze o non risponde alle terapie. L'obiettivo di questo progetto di dottorato è, pertanto, lo sviluppo preclinico di un nuovo strumento terapeutico per il trattamento delle neoplasie pediatriche a cellule B. In primo luogo sono state caratterizzate le nanobolle di chitosano (NBs) caricate con AntagomiR-17, sui è stato legato un anticorpo anti-CD20 (rituximab). AntagomiR-17 è in grado di legarsi e sconfiggere il miRNA-17, che è una molecola iperespressa in diverse neoplasie delle cellule B, incluso BL, ed è associata allo sviluppo di meccanismi di resistenza ai farmaci. Rituximab è stato impiegato per indirizzare specificamente le NB alle cellule B che esprimono l'antigene CD20 in vivo. Le NB in chitosano risultano efficaci in vitro e in vivo nella riduzione del tumore nei topi portatori di tumore BL ma, la loro carica positiva ha dimostrato alcune limitazioni nella biodistribuzione. Inoltre, il meccanismo di targeting scelto non copre altre patologie pediatriche, come l’ALL che origina da blasti che non possiedono già l'antigene CD20 sulla loro superficie. Per superare queste limitazioni sono state prodotte e caratterizzate nanoparticelle (NPs) PLGA-PVA. In primis, queste NP sono state confrontate con le loro controparti rivestite con HSA, aggiunta per nascondere le NP dall'IS. Una volta dimostrata l'efficacia del rivestimento in vitro e in vivo in uno zebrafish sano, è stato prodotto il meccanismo di targeting anti-CD19. L'efficacia del meccanismo di targeting è stata testata in vitro e, una volta impostato il modello di zebrafish portatore di tumore, anche in vivo. Infine, diversi farmaci sono stati testati sui linfociti B e la doxorubicina è stata scelta come candidata per riempire le NP PLGA-PVA. Le NP caricate con il farmaco sono state nuovamente testate in vitro e in vivo in un modello diffuso di ALL in zebrafish, dimostrando di ridurre significativamente la crescita del tumore e aumentando parallelamente la sopravvivenza degli animali trattati. Tutti questi risultati insieme, evidenziano che le NP PLGA-PVA mirate con anticorpi anti-CD19 e riempite con doxorubicina rappresentano un approccio promettente per il trattamento dei tumori delle cellule B, ma anche per altre patologie. Queste nanostrutture, infatti, possono essere immaginate come una nano-piattaforma in cui i singoli componenti, come il farmaco o il meccanismo di targeting, possono essere sostituiti per raggiungere diversi obiettivi.
B-cell malignancies are a heterogeneous group of diseases, for whom treatment options include chemotherapeutics and immunotherapy. Despite the recent development of new therapeutic strategies, most patients develop resistance or do not respond to therapies. The aim of this PhD project was the preclinical development of a new therapeutic tool for the treatment of pediatric B-cell malignancies. Firstly, chitosan-nanobubbles (NBs) loaded with AntagomiR-17 and joined with an anti-CD20 antibody (rituximab) were characterized. AntagomiR-17 is capable of pairing and defeating miRNA-17, which is a molecule upregulated in several B-cells malignancies, including BL, and it is associated with the development of drug resistance mechanisms. Rituximab was employed to specifically target NBs to B-cells expressing the antigen CD20 in vivo. Chitosan-NBs result effective in vitro and in vivo in the reduction of the tumor burden in BL-tumor-bearing mice, however, their positive charge demonstrated some limitation in the biodistribution. Moreover, the targeting mechanism chosen does not cover other pediatric pathologies, such as ALL that originates from blasts that do not already possess the CD20 antigen on their surface. To overcome these limitations PLGA-PVA Nanoparticles (NPs) were produced and characterized. In primis, these NPs were compared to their counterparts coated with HSA, which was added to conceal NPs from the IS. Once demonstrated the efficacy of the coating in vitro and in vivo in healthy zebrafish, the targeting mechanism anti- CD19 was produced. The efficacy of the targeting mechanism was tested in vitro and, once the tumor-bearing zebrafish model was set, also in vivo. Finally, different drugs were tested on B-cells and the doxorubicin was chosen as a candidate to fill PLGA-PVA NPs. Drug-loaded NPs were tested again in vitro and in vivo in a diffused zebrafish model of ALL, significantly reducing the tumor growth and parallelly augmenting the survival of treated animals. All these results together, already highlighted that PLGA-PVA NPs targeted with anti- CD19 antibodies and filled with doxorubicin represent a promising approach for the treatment of B-cell malignancies, but also for other pathologies. In fact, these nanostructures can be imagined as a nano-platform in which the single components, such as the payload or the targeting mechanism, can be replaced to achieve different goals.

La Leucemia Linfoblastica Acuta (LLA) è il tumore più frequente in età pediatrica. Nonostante l’alto successo terapeutico raggiunto, una percentuale di pazienti non risponde alle terapie convenzionali. Numerosi studi hanno mostrato come il microambiente midollare giochi un importante ruolo nella progressione tumorale. Il nostro scopo è stato quello di identificare i pahways cruciali coinvolti nel cross-talk tra le cellule leucemiche e il microambiente stromale e che possano essere dei potenziali target terapeutici. Abbiamo studiato AttivinaA, molecola della superfamiglia del TGF-β, all’interno della nicchia midollare leucemica. Questa molecola è nota nel contesto dei tumori solidi per la sua capacità di promuovere la progressione tumorale attraverso la regolazione della motilità e invasività cellulare. Abbiamo qui definito per la prima volta AttivinaA come un fattore associato alla leucemia: all’esordio di leucemia, questa molecola è altamente espressa nel midollo leucemico e risulta essere prodotta a più alti livelli dalle cellule stromali mesenchimali (MSC) in seguito a co-coltura con le cellule leucemiche. Anche nel contesto della B-LLA, abbiamo dimostrato che AttivinaA promuove la motilità ed invasività cellulare in presenza o meno del fattore chemiotattico CXCL12, fondamentale nella localizzazione delle cellule staminali ematopoietiche (HSC) e delle cellule leucemiche all’interno della nicchia midollare. In particolare, il meccanismo d’azione di AttivinaA dipende dall’incremento dei livelli di calcio intracellulare e della polimerizzazione dell’actina che sono importanti regolatori della riorganizzazione del citoscheletro e del movimento cellulare. Interessante, AttivinaA modula in modo specifico le proprietà biologiche delle cellule leucemiche in quanto svolge un effetto contrario sulle HSC, favorendo quindi le cellule leucemiche nella competizione per la nicchia. Come già riportato in letteratura, abbiamo trovato livelli ridotti di CXCL12 nel midollo leucemico e abbiamo osservato che AttivinaA è responsabile almeno in parte di questa riduzione a causa di un suo effetto diretto sulla secrezione della chemochina da parte delle MSC. Tuttavia, essendo AttivinaA capace di aumentare la migrazione cellulare anche verso concentrazioni subottimali di CXCL12, questi dati suggeriscono un possibile meccanismo tramite il quale le cellule leucemiche persistono all’interno della nicchia midollare distruggendo l’ematopoiesi sana. I nostri dati in vitro circa il ruolo pro-migratorio e pro-invasivo di AttivinaA sono stati confermati anche in vivo. I processi di regolazione dei flussi di calcio e della riorganizzazione del citoscheletro da parte di AttivinaA sono importanti per stimolare anche la vescicolazione da parte delle cellule. Abbiamo dimostrato che AttivinaA è in grado di aumentare il rilascio di vescicole extracellulari (VE) da parte delle cellule di B-LLA. Tali vescicole trasportano al loro interno l’oncogene t(1;19), tipico delle cellule dalle quali esse originano. Sia AttivinaA sia le VE da essa indotte conferiscono chemioresistenza alle cellule leucemiche, diminuendo in maniera significativa la loro sensibilità al farmaco Asparaginasi, che viene poi ripristinata bloccando la via di segnalazione di AttivinaA. Il meccanismo alla base della chemioprotezione esercitata dalle VE può essere spiegato dalla presenza al loro interno di microRNA differenzialmente espressi in seguito al trattamento con AttivinaA, tra cui il miRNA-491-5p precedentemente associato a chemioresistenza all’Asparaginasi nella leucemia pediatrica. Complessivamente, i nostri dati suggeriscono che AttivinaA è una molecola chiave della nicchia leucemica, che conferisce un vantaggio migratorio alle cellule leucemiche e le protegge dai farmaci convenzionali attraverso la produzione di VE. Il nostro lavoro potrebbe portare dunque allo sviluppo di nuovi farmaci in grado di agire sul cross-talk stroma-leucemia.
Acute Lymphoblastic Leukemia (ALL) is the most common type of cancer in children. About 80% of the cases arises from precursor B cells (BCP-ALL), which abnormally accumulate as a consequence of genetic alterations associated to differentiation inhibition and abnormal expansion. Despite the 85% survival rate, a total of 10-15% of patients retains leukemic stem cells and their progenitors in the bone marrow (BM), thereby relapsing following treatment cessation. The importance of BM microenvironment for cancer progression has been widely recognized in recent years. In this study, we aimed to identify the crucial pathways involved in the bi-directional leukemia-stroma cross-talk that could be an attractive target for future antileukemic therapy. We focused our attention on the characterization of ActivinA, a TGF-β family member, within the BM leukemic niche. Here, we identified ActivinA as a new crucial factor exploited by leukemic cells to create a self-reinforcing niche: indeed, this molecule was highly expressed in the BM plasma of leukemic patients. Furthermore, we reported that BCP-ALL cells, along with the highly pro-inflammatory environment of leukemic BM, induced a strong increase in the molecule secretion by Mesenchymal Stromal Cells (MSCs). In accordance with its protumoral role in solid tumors, ActivinA strongly induced both random and CXCL12-driven migration of cells also in the context of BCP-ALL. We observed that ActivinA selectively stimulated these leukemic cell biological properties with a calcium- and actin polymerization-mediated mechanism as this molecule showed an opposite effect on Hematopoietic stem cells (HSCs). According to the literature, we found reduced CXCL12 levels in the leukemic BM, but ActivinA enhanced cell migration also towards suboptimal CXCL12 concentrations, suggesting a possible mechanism by which leukemic cells could persist in the BM niche, displacing healthy hematopoiesis. Our in vitro data about the pro-migratory and pro-invasive role of ActivinA were confirmed also in vivo. By using a xenograft mouse model of human BCP-ALL, we demonstrated the ability of ActivinA to enhance both BM engraftment and metastatic potential intro extra-medullary sites of leukemic cells. Notably, the regulation of calcium influx and cytoskeleton organization by ActivinA is an important process to stimulate also cell vesiculation. Recent studies have shown that cancer extracellular vesicles (EVs) can mediate cell-cell communication and potentially contribute to tumor progression. Therefore, we investigated whether ActivinA was able to influence vesiculation by leukemic cells. We demonstrated that ActivinA increased the production of both exosomes and MVs by BCP-ALL cells. We found that EVs transport the t(1;19) fusion transcript, typical of cells from which they originate. We then studied the biological effects by which ActivinA-induced leukemia EVs can actively promote BCP-ALL disease, focusing our attention on resistance to therapy. Firstly, we demonstrated that ActivinA significantly decreased the sensitivity of leukemic cells to the anti-leukemic drug Asparaginase (ASNase) which was re-stored by blocking ActivinA signaling. Interestingly, also ActivinA-induced leukemia EVs conferred resistance to leukemic cells. To understand the mechanism underlying EV chemoprotection, we explored their miRNA cargo and identified differentially expressed miRNAs induced by ActivinA treatment. Of these, miR-491-5p has been previously reported to be associated with ASNase chemoresistance in childhood leukemia. The discovery of ActivinA signaling between BCP-ALL cells and MSCs adds significant insights into the mechanisms of communication in the leukemic niche. Moreover, ActivinA-induced leukemia EVs seem to play a crucial role in sustaining leukemic cells, by conferring them drug resistance. Our data provide a new concept to develop alternative therapeutic strategies that include targeting of the leukemic niche in BCP-ALL.

L'équipe s'intéresse aux altérations de facteurs de transcription impliquées dans les leucémies aiguës, dont PAX5 qui est essentiel pour le développement des cellules B. C'est pourquoi le modèle murin transgénique PAX5-ELN a été généré, qui exprime la protéine de fusion oncogénique durant le développement des lymphocytes B, et récapitule le processus multi-étapes des LAL-B (Jamrog L et al., PNAS, 2018). J'ai participé à l'identification des cellules à l'origine de la LAL-B et à la caractérisation de leur propriétés fonctionnelles et moléculaires. Nos travaux indiquent qu'au stade pré-leucémique, PAX5-ELN induit l'émergence d'une population aberrante de progéniteurs B avec une propriété anormale d'auto-renouvellement. Cette population est enrichie en cellules quiescentes résistantes aux agents de chimiothérapie, activent un programme moléculaire de cellule souche, et sont le support de l'initiation leucémique à long terme. Ce travail fait l'objet d'une publication récente que je signe en deuxième auteure (Fregona V, Bayet M et al., J Exp Med, in press). En parallèle, ma thèse s'est tournée vers la modélisation de l'initiation et de la transformation leucémique induite par la mutation PAX5P80R, une altération initiatrice fréquente chez les patients. J'ai donc utilisé des cellules de foie fœtal issus d'embryon de souris Pax5-/- pour sélectionner les progéniteurs lymphoïdes non engagés dans le lignage B. Après transduction avec des rétrovirus CTL, PAX5 Wt ou PAX5P80R, j'ai montré que PAX5P80R ne restaure pas efficacement l'engagement définitif des cellules vers le lignage B. Les études de transplantation ont permis de montrer que PAX5P80R induit un potentiel de prise de greffe aberrant suivi du développement de la LAL-B. Cette transformation leucémique est associée à la sélection de clones porteurs de mutations additionnelles affectant la voie JAK/STAT. Nos analyses ont permis d'identifier Hif2 comme un candidat potentiel dans la leucémogenèse. Enfin, un criblage pharmacologique d'inhibiteurs de Hif révèle l'Acriflavine comme un composé intéressant ciblant les cellules leucémiques. Ainsi, la modélisation de la LAL-B par la mutation PAX5P80R fournit à l'équipe un nouvel outil mimant le processus multi-étapes de la LAL-B, et permet de déchiffrer les mécanismes biologiques par lesquels la mutation mène à la transformation tumorale. Ce travail fait l'objet d'un manuscrit en préparation que je signe en première auteure (Bayet M, Fregona V, et al., in preparation). Les modèles PAX5-ELN et PAX5P80R permettent non seulement d'étudier les différentes étapes de la leucémogenèse B, mais servent aussi de support pour le développement de criblage de petites molécules sur cellules primaires. J'ai donc mis en place un protocole miniaturisé et robuste par FACS pour cribler des composés chimiques ciblant les cellules pré-leucémiques. Notre approche multiparamétrique permet d'évaluer simultanément l'effet des composés sur les cellules pré-leucémiques et les sous-populations B normales. J'ai donc criblé une banque de 1040 composés synthétiques et naturels (chimiothèque essentielle) reflétant la diversité chimique de la chimiothèque nationale française. Ce criblage, associé à des contre-criblages en dose-réponse, m'a permis d'identifier 5 molécules d'intérêt. Dans l'ensemble, mon travail montre la faisabilité d'un criblage de petites molécules sur une population enrichie en cellules initiatrice de la leucémie et prenant en compte la complexité intrinsèque des cellules primaires du lignage B. Enfin, j'ai procédé à la rédaction et la publication d'une revue dans le journal Cancers qui expose les concepts d'hétérogénéité tumorales des cellules leucémiques des patients, l'utilité des modèles de souris transgéniques pour explorer le compartiment des cellules initiatrices de la leucémie, et les efforts actuels visant à découvrir de nouvelles thérapies ciblées (Fregona V*, Bayet M* et al, Cancers (Basel), 2021), que je signe en co-première auteure
The team is interested in alterations in transcription factors involved in acute leukemia, including PAX5, which is essential for B-cell development. This is why the PAX5-ELN transgenic mouse model was generated, which expresses the oncogenic fusion protein during B-cell development, and recapitulates the multi-step process of B-ALL (Jamrog L et al., PNAS, 2018). I was involved in identifying the cells at the origin of-B-ALL and characterizing their functional and molecular properties. Our work indicates that at pre-leukemic stage, PAX5-ELN induces the emergence of an aberrant population of B-progenitors with an abnormal self-renewal property. This population is enriched in quiescent cells resistant to chemotherapeutic agents, activating a molecular stem cell program and supporting long-term leukemic initiation. This work is the subject of a recent publication signed by myself as second author (Fregona V, Bayet M et al., J Exp Med, in press). In parallel, my thesis focused on modeling the initiation and leukemic transformation induced by the PAX5P80R mutation, a frequent initiating alteration in patients. I used fetal liver cells derived from Pax5-/- mouse embryos to select lymphoid progenitors not committed to the B lineage. After transduction with CTL, PAX5 Wt or PAX5P80R retroviruses, I showed that PAX5P80R does not restore efficiently definitive commitment of cells to the B lineage. Transplantation experiments have shown that PAX5P80R induces aberrant engraftment potential followed by the development of B-ALL. This leukemic transformation is associated with the selection of clones carrying additional mutations affecting the JAK/STAT signaling pathway. Our analyses identified Hif2 as a potential candidate for leukemogenesis. Finally, pharmalogical screening of Hif inhibitors revealed Acriflavine as an interesting compound targeting leukemic cells. Thus, the modeling of B-ALL by the PAX5P80R mutation provides the team with a new tool to mimic the multi-step process of B-ALL, and to decipher the biological mechanisms by which the mutation leads to tumor transformation. This work is the subject of a manuscript in preparation which I have signed as first author (Bayet M, Fregona V, et al., in preparation). The PAX5-ELN and PAX5P80R models not only make it possible to study the various stages of B leukemogeneis, but also serve as a basis for the development of small molecule screening on primary cells. I therefore set up a miniaturized and robust protocol by FACS to screen chemical compounds targeting pre-leukemic cells. Our multiparametric approach enables us to simultaneously assess the effect of compounds on pre-leukeic cells and normal B subpopulations. I screened a bank of 1040 synthetic and natural compounds (essential chemical library) reflecting the chemical diversity of the French national chemical library. This screening, combined with dose-response counter-screening, enabled me to identify 5 molecules of interest. Overall, my work demonstrates the feasibility of small-molecule screening on a population enriched in leukemia-initiating cells, taking into account the intrinsic complexity of primary B-cells. Finally, I edited and published a review in the journal Cancers outlining the concepts of tumor heterogeneity in patients' leukemic cells, the utility of transgenic mouse models to explore the leukemia initiating cell compartment, and current efforts to discover new targeted therapies (Fregona V*, Bayet M* et al, Cancers (Basel), 2021), wich I co-authored

Normal karyotype pediatric B-cell precursor ALL patients are heterogeneous with respect to chemotherapy response, relapse rates and prognosis and the reason is unknown. These patients are treated with a standard protocol, and stratified using MRD methodology that shows they have variable responses and predicted outcomes. This study aims to determine the reasons behind such heterogeneity. This study shows that through miRNA profiling, miR-221/222 are differentially expressed in normal karyotype patients and are up regulated in Poor Responder patients. Through proliferation, apoptosis, viability assays and cell cycle analysis, proliferation advantage was identified as the main cause of resistance to glucocorticoid treatment in miR-221/222 over expressing cell lines. SMAD1 and CREBBP were down regulated in miR-221/222 over expressing cell lines implicating the dysregulation of TGFβ and glucocorticoid-receptor pathways, while NLK was identified as a novel target of miR-221/222 through which resistance to chemotherapy is mediated.

O tratamento para a Leucemia Linfoblástica Aguda LLA utiliza, entre outros fármacos, a enzima L-asparaginase (ASNase) proveniente da bactéria Escherichia coli. Reações imunológicas estão entre os problemas do tratamento com ASNase, e a formação de anticorpos contra essa proteína pode impedir o sucesso no tratamento. Duas cisteíno proteases lisossomais estão relacionadas com a degradação de ASNase nos seres humanos, a Catepsina B (CTSB) e Asparagina Endopeptidase (AEP). Em estudos prévios do nosso grupo obteve-se mutantes de ASNase resistentes a degradação por CTSB e/ou AEP in vitro. Nesse trabalho avaliamos essas mutantes quanto a sua citotoxicidade em linhagens celulares de leucemia e conduzimos estudos in vivo, aplicando as proteoformas de ASNases em camundongos Balb C para avaliar a atividade asparaginase sérica das enzimas ao longo do tempo, bem como obter informações sobre a formação de anticorpos contra essas proteoformas. Nos ensaios de citotoxicidade, duas das proteoformas testadas tiveram efeito citotóxico semelhante a forma selvagem, enquanto uma outra proteoforma tem a citotoxicidade sensivelmente reduzida. Já nos ensaios in vivo, uma proteoforma demonstrou meia vida sérica maior da atividade asparaginásica, e duas proteoformas causaram reduzida formação de anticorpos. Juntos, esses resultados colaboram para a obtenção de uma nova geração de ASNases com melhor biodisponibilidade, e efeitos adversos reduzidos, gerando a possibilidade de menores doses e frequência de aplicações.
The Treatment for Acute Lymphoblastic Leukemia (ALL) includes the biopharmaceutical L-asparaginase (ASNase) from Escherichia coli. Immunological reactions are among the problems of treatment using ASNase, and the antibodies formation protein may prevent success in treatment. Lysosomal cysteine proteases are related to ASNase degradation, Cathepsin B (CTSB) and Asparagine Endopeptidase (AEP). In previous studies, ASNase mutants resistant to CTSB and / or AEP degradation in vitro were obtained. In this work, mutants were evaluated in cytotoxicity in ALL cell lines and, in vivo studies, applying doses of the wild and mutant ASNases in Balb C mice to evaluate serum asparaginase activity of the enzymes over time, as well as to obtain information on the formation of antibodies against these proteoforms. Regarding to the cytotoxicity, two proteoforms among the tested had similar cytotoxicity than the wild-type. While another proteoform had the cytotoxicity severely reduced. One proteoform have demonstrated greater serum half-life of asparaginase activity, while two other mutants caused reduced antibody formation. Together, these results collaborate to obtain a new generation of ASNases with increased bioavailability and reduced side effects, generating the possibility of lower doses and frequency of applications.

Les leucémies aiguës lymphoblastiques T (LAL-T) sont caractérisées par la prolifération maligneincontrôlée de précurseurs lymphoïdes T bloqués dans la différenciation. Les stades d’arrêt dematuration observés dans les LAL-T reproduisent fidèlement les différentes étapes de la maturationthymique humaine. Ainsi nous avons montré que le facteur de transcription myéloïde CEBPA, expriméuniquement dans les précurseurs thymiques les plus immatures (ETP), est réprimé par un mécanismed’hyperméthylation dans les LAL-T à l’exception des formes les plus immatures. Il est aujourd’huicommunément admis que les LAL-T constituent une pathologie dite « multi-hits » où les oncogènesde type A affectent la différenciation tandis les oncogènes de type B sont impliqués dans la régulationdu cycle cellulaire, l’auto-renouvellement et/ou l’engagement dans la lignée T. La voie de signalisationde NOTCH, cruciale pour le développement lymphoïde T, est constitutivement activée par la survenuede mutations des gènes NOTCH1 et/ou FBXW7 (N/F) dans environ 60% des LAL-T. La valeurpronostique de ces mutations est controversée. Dans notre travail, nous avons montré que lesmutations de N/F sont plus fréquentes dans les LAL-T arrêtées à un stade de maturation cortical etconfèrent un bon pronostic qui semble toutefois dépendre de la chimiothérapie administrée. Grâce àl’étude de cette large cohorte de LAL-T nous avons pu également établir la fréquence de l’anomalieoncogénique CALM-AF10. Cette dernière est très fréquente dans les LAL-T qui se développent àpartir des ETP dites de mauvais pronostic. Nous avons montré que c’est la présence de l’anomalieCALM-AF10 qui confère le pronostic défavorable à ce sous-type de LAL-T. Contrairement à lalittérature nous n’avons pas retrouvé de valeur pronostique liée à la surexpression des gènes ERG etBAALC. L’étude des anomalies génétiques des LAL-T permet de mieux comprendre l’oncogénèse etd’identifier les anomalies avec une valeur pronostique. L’intérêt de ces travaux est d’apporter une aideaux cliniciens pour une stratification thérapeutique adaptée afin de donner les meilleures chances desurvie aux patients
T-cell acute lymphoblastic leukemia (T-ALL) are lymphoid neoplasms characterized by theproliferation of malignant T lymphoblasts arrested at early stages of maturation. Maturation arrest in TALLmirrors normal lymphopoiesis. Thus we have shown that the myeloid transcription factor CEBPA,expressed only in the most immature thymic precursors (ETP), is commonly repressed byhypermethylation in T-ALL with the exception of the most immature subset. It is now widely acceptedthat T-ALL is a “multi-hits” disease where the type A oncogenes affect the differentiation while type Boncogenes are involved in cell cycle regulation, self-renewal and T-cell commitment. The Notchsignaling pathway, crucial for T cell development, is constitutively activated by the occurrence ofmutations in NOTCH1 and /or FBXW7 (N / F) genes in approximately 60% of T-ALL. The prognosticvalue of these mutations is controversial. In our study, we showed that N/F mutations are morefrequently observed in T-ALL arrested at a cortical stage of maturation and confer a good prognosiswhich seems to be influenced by the therapeutic regimen. In this large cohort of T-ALL we could alsodetermine the frequency of the CALM-AF10 oncogenic abnormality. The latter is very common in TALLdeveloped from ETP wich are of very poor prognosis. We have shown that this is the presence ofCALM-AF10 which confers the poor prognosis in this subtype of T-ALL. Contrary to the litterature wedid not find any prognostic value associated with the overexpression of ERG and BAALC genes. Thestudy of genetic abnormalities in T-ALL provides a better understanding of oncogenesis and identifyabnormalities with prognostic value. The interest of this work is to assist clinicians for an efficienttherapeutic stratification to overcome the poor outcome of T-ALL patients

Le gène PAX5 (Paired boX 5) code un facteur de transcription essentiel pour la différenciation lymphoïde B. Nous avons montré que les deux isoformes PAX5A et PAX5B étaient différentiellement régulées mais pouvaient exercer une fonction équivalente durant l'induction de la différenciation lymphoïde B et pourraient présenter des différences fonctionnelles à la suite de l'activation des lymphocytes B. Le contrôle précis de leur expression peut ainsi refléter un moyen d'ajuster finement le dosage de PAX5 pendant le processus de différenciation des cellules de la lignée lymphoïde B. PAX5 est la cible principale d'une large diversité d'altérations somatiques dans les LAL-B de l'enfant et de l'adulte. Cependant, le rôle des protéines de fusion impliquant PAX5 dans l'initiation et la transformation des LAL-B est encore méconnu. Nous avons précédemment décrit une nouvelle translocation chromosomique récurrente t(7;9)(q11;p13) dans les LAL-B qui juxtapose PAX5 à la séquence codante du gène de l'élastine (ELN). Pour étudier la fonction de la protéine de fusion résultante, PAX5-ELN, au cours du développement leucémique, nous avons créé un modèle murin dans lequel le transgène PAX5-ELN est exprimé spécifiquement dans le compartiment B. Les souris exprimant PAX5-ELN développent un phénotype de LAL-B avec une pénétrance de 80%. Leur transformation leucémique est associée à l'acquisition de mutations secondaires récurrentes des gènes Ptpn11, Kras, Pax5, et Jak3 affectant d'importantes voies de signalisation requises pour la prolifération cellulaire. Nos études fonctionnelles ont démontré que PAX5-ELN altérait in vitro et in vivo le développement lymphoïde B et pouvait induire une expansion aberrante du compartiment progéniteur B (pro-B) au stade préleucémique. Nos approches moléculaires et computationnelles ont identifié des gènes-candidats régulés par PAX5-ELN et pouvant être impliqués dans l'initiation leucémique. Nos données fournissent ainsi un nouveau modèle d'étude in vivo récapitulant la leucémogenèse multi-étapes des LAL-B décrites chez les patients et impliquent fortement les protéines de fusion engageant PAX5 en tant que puissantes oncoprotéines dans le développement leucémique. Par ailleurs, il existe de plus en plus de preuves d'une base génétique héréditaire de prédisposition aux LAL-B pédiatriques. Dans ce contexte, quatre cas de familles non-apparentées affectées par des LAL-B et exprimant des mutations ponctuelles germinales et hétérozygotes de PAX5 ont récemment été rapportés : la mutation PAX5 G183S altérant le domaine octapeptide de PAX5 a été décrite chez trois familles alors que la mutation PAX5 R38H altérant le domaine de liaison à l'ADN de PAX5 a été identifiée chez une autre. Nous avons renforcé l'hypothèse du caractère héréditaire des LAL-B familiales avec la description de trois nouveaux cas de LAL-B au sein d'une même famille exprimant la mutation germinale PAX5 R38H. Pour étudier l'effet intrinsèque de la protéine mutée PAX5 R38H dans le développement lymphoïde B, nous avons effectué des tests fonctionnels in vitro et in vivo combinés à une analyse d'expression génique, basés sur une approche de complémentation rétrovirale. Nos résultats ont indiqué que PAX5 R38H agissait comme un fort variant hypomorphique qui échouait à induire la différenciation lymphoïde B et n'exerçait pas d'effet dominant-négatif sur la forme sauvage de PAX5. Des transplantations syngéniques de cellules exprimant PAX5 R38H ont démontré qu'elles maintenaient une capacité de prise de greffe et menaient au développement leucémique chez la souris. Notre analyse transcriptomique a confirmé la perte de fonction de PAX5 sur ses gènes cibles et a révélé une signature moléculaire spécifique au mutant. Nos données mettent ainsi en évidence l'importance de la dérégulation transcriptionnelle dans la leucémogenèse des LAL-B familiales, en particulier des gènes impliqués dans la différenciation lymphoïde B
The PAX5 (Paired boX 5) gene encodes a key transcription factor crucial for B-cell differentiation. We showed that the two PAX5 isoforms are differentially regulated but have equivalent function during early B-cell differentiation. Indeed, PAX5A and PAX5B isoforms can both induce B-cell program but may have functional differences after B-cell activation. The tight control of their expression may thus reflect a way to finely tune PAX5 dosage during B-cell differentiation process. PAX5 is a well-known haploinsufficient tumor suppressor gene in human B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and is the main target of a wide diversity of somatic alterations in childhood and adult BCP-ALL, occurring in one third of sporadic cases. However, the role of PAX5 fusion proteins in BCP-ALL initiation and transformation is ill-known. We previously reported a new recurrent t(7;9)(q11;p13) chromosomal translocation in human BCP-ALL that juxtaposed PAX5 to the coding sequence of elastin (ELN). To study the function of the resulting PAX5-ELN fusion protein in BCP-ALL development, we generated a mouse model in which the PAX5-ELN transgene is expressed specifically in B cells. PAX5-ELN-expressing mice efficiently developed BCP-ALL phenotype with a penetrance of 80%. Leukemic transformation was associated with clonal Immunoglobulin gene rearrangement and recurrent secondary mutations in Ptpn11, Kras, Pax5, and Jak3 genes affecting key signaling pathways required for cell proliferation. Our functional studies demonstrated that PAX5-ELN impairs B-cell development in vitro and in vivo and induces an aberrant expansion of the pro-B cell compartment at the preleukemic stage. Our molecular and computational approaches identified PAX5-ELN-regulated candidate genes that establish the molecular bases of the preleukemic state to drive BCP-ALL initiation. In conclusion, our study provides a new in vivo model recapitulating the multistep leukemogenesis process of human BCP-ALL and strongly implicates PAX5 fusion proteins as potent oncoproteins in leukemia development. Furthermore, there is increasing evidence for an inherited genetic basis of susceptibility to childhood BCP-ALL. In this context, four unrelated families with childhood BCP-ALL expressing heterozygous PAX5 germline point mutations were recently reported: the recurrent mutation PAX5 G183S affecting the octapeptide domain of PAX5 has been described in three families while PAX5 R38H affecting its DNA-binding paired domain has been identified in another one. We strengthen the hypothesis of inherited character of familial BCP-ALL with the description of three novel familial BCP-ALL cases in related patients that express the germline PAX5 R38H mutation. To uncover the intrinsic effect of PAX5 R38H mutant in B-cell development, we performed in vitro, and in vivo functional assays combined with a gene expression analysis, based on a retroviral complementation approach. Our results indicated that PAX5 R38H mutant acts as a strong hypomorphic variant that fails to drive B-cell differentiation and does not exert a dominant-negative effect on wild-type PAX5. Syngeneic transplantation of PAX5 R38H-expressing cells demonstrated maintenance of engraftment capacity and led to development of BCP-ALL phenotype in mice. Our transcriptomic analysis of these PAX5 R38H-expressing cells showed that PAX5 R38H drastically alters the pattern of expression of PAX5 target genes but also revealed a distinct molecular signature specific to PAX5 R38H. Together with previous unrelated family study, our observations allow to establish the recurrence of the germline PAX5 R38H mutation associated with BCP-ALL. Our data also highlight the importance of transcriptional dysregulation in leukemogenesis of familial BCP-ALL, particularly of genes involved in B-cell differentiation

Chez l’adulte les premières étapes du développement hématopoïétique se déroulent dans la moelle osseuse (MO). La contribution de cellules d’origine mésenchymateuse, appelées niches stromales, a été démontrée dans le cas de la maintenance des cellules souches hématopoïétiques (CSH) et du développement des lymphocytes B (LB). Ainsi la maintenance des CSH dépend de niches périvasculaires sécrétant CXCL12 et SCF. Par ailleurs les LB les plus précoces (preproB) sont en contact de cellules stromales CXCL12+, puis migrent vers des cellules stromales exprimant l’interleukine-7 lors de leur différentiation en cellules proB. L’expression du préBCR, marque ensuite l’entrée dans le stade préB. À ce stade, les cellules sont au contact de cellules stromales galectine-1+.Malgré les progrès obtenus dans la compréhension du rôle des niches stromales, leur hétérogénéité et les mécanismes contrôlant la migration et l’adhésion des cellules hématopoïétiques en différenciation restent à mieux définir. Dans cet objectif, nous avons caractérisé phénotypiquement les cellules stromales de la MO mais aussi démontré l’existence d’une niche multi-spécifique, associée aux sinusoïdes et capable de soutenir les CSH et les LB.La contribution des niches dans le développement et la résistance aux traitements des Leucémies Aigues Lymphoblastiques de type B (LAL-B), équivalents pathologiques des LB en développement, a aussi été démontrée. Au cours de mon travail de thèse nous avons révélé l'influence d'un facteur exprimé par des cellules stromales de la MO sur la prolifération des LAL-B. À terme, ces travaux permettront de développer des traitements ciblant les fonctions protectrices des niches tumorales
In adults, the early stages of hematopoietic development take place in the bone marrow (BM). The contribution of specialized cells of mesenchymal origin, called stromal niches, has been demonstrated in the case of hematopoietic stem cell (HSC) maintenance and B lymphocyte development. Indeed, the maintenance of HSC depends on perivascular niches secreting CXCL12 and SCF. Furthermore progenitor B cells (preproB) are in contact with CXCL12+ stromal cells and migrate towards interleukin 7 expressing stromal cells during their differentiation into proB cells. PreBCR expression then marks the entrance into the preB cell stage. At this point, the cells are in contact with galectin-1+ stromal cells.Although progress have been made in understanding the role of stromal cell niches, their heterogeneity and the mechanisms controlling migration and adhesion of differentiating hematopoietic cells are controversial and remain to be defined. With this objective, we characterized phenotypically BM stromal cells but also demonstrated the existence of a multi-specific niche, associated to sinusoids and able to support both HSC and early B cells.The contribution of BM niches in the development and resistance to treatment of B cell Acute Lymphoblastic Leukemia (B-ALL), pathological equivalent of developing B cells has also been demonstrated. During my PhD, our work revealed the influence of a factor expressed by BM stromal cells on the proliferation of B-ALL. Ultimately, this work will allow the development of treatments targeting the protective functions of tumor niches

Les leucémies aiguës lymphoblastiques T (LAL-T) sont des maladies des progéniteurs T qui surviennent chez l'enfant et le jeune adulte. Des facteurs intrinsèques et extrinsèques à la cellule blastique sous-tendent le développement leucémique. Les LAL-T humaines peuvent être étudiées in vivo après injection dans des souris immunodéficientes ou in vitro dans des systèmes de cocultures avec des cellules stromales MS5 surexprimant Delta-likel. Dans la première partie de cette thèse, nous avons étudié l'hétérogénéité de croissance dans ces deux modèles. Nos résultats montrent que la croissance est parfaitement corrélée in vivo et in vitro. De plus, les LAL-T CD8+, TCRalpha-beta+ ou avec une délétion SIL-TAL1 développent plus rapidement une LAL-T en xénogreffe. Enfin, les LAL-T qui se développent rapidement in vivo présentent une activation de NFKB augmentée. Dans la seconde partie de ce travail, nous avons étudié les facteurs extrinsèques influençant la croissance blastique. La moelle osseuse est composée de moelle rouge - siège de l'hématopoïèse - et de moelle jaune composée d'adipocytes. Chez la souris, les vertèbres thoraciques et caudales contiennent respectivement de la moelle rouge et jaune. Grâce à ce modèle de vertèbres et à des cocultures in vitro, nous avons démontré que les adipocytes ne soutiennent pas la croissance blastique. Au cours du développement leucémique, l'infiltration des vertèbres caudales est un événement tardif associé à une expansion de cellules myéloïdes suppressives. Dans cette niche caudale, les LAL-T présentent des caractéristiques spécifiques, en particulier une progression du cycle cellulaire diminuée et une intensité diminuée des marqueurs T de surface
T-cell acute lymphoblastic leukemia (T-ALL) is T-cell progenitor disease which affects children and young adults. Intrinsic and extrinsic factors underlie leukemic development. Human T-ALL can be studied in vivo after injection into immunodeficient mice or in vitro in coculture with Delta-I ike I expressing MS5 murine stromal cells. In the first part of this PhD, we explored T-ALL heterogeneity growth in these two models. T-ALL growth is highly correlated in the xenograft and coculture models. Furthermore, CD8+, TCRalpha-béta+ and SIL-TAL I deleted T-ALL exhibit an increased ability to grow in immunodeficient mice. T-ALL samples which engraft rapidly into immunodeficient mice display an increased NFKB activation. In the second part of this work, we explored extrinsic factors which may alter T-ALL growth. Red bone marrow contains many hematopoietic cells while yellow marrow is mainly filled with adipocytes. Murine thoracic and tail vertebrae contain red and yellow marrow, respectively. Using this vertebrae model and in vitro cocultures, we could demonstrate that adipocytes do not sustain T-ALL growth. However, T-ALL infiltration of tait vertebrae is a late event in the course of T-ALL development and is associated with the expansion of Myeloid-Derived Suppressor Cells. In this tail niche, T-ALL cells exhibit specific biological characteristics including decreased cell cycle progression and altered cell surface phenotype

Les leucémies aigües lymphobastiques (LAL) représentent un tiers des leucémies et constituent le cancer pédiatrique le plus fréquent chez l’enfant. Les LAL de type T (LAL-T)sont caractérisées par l’expansion anormale de progéniteurs de lymphocytes T. Aujourd’hui,la réponse curative aux traitements est proche de 80% chez l’enfant et 50% chez l’adulte. La rechute reste donc fréquente et souvent de mauvais pronostic. Pour ces raisons,l’identification de nouvelles voies de signalisation en vue de développer de nouvelles stratégies thérapeutiques est cruciale afin d’améliorer le traitement des LAL-T.Les résultats précédents du laboratoire ont révélé l’activation soutenue de la voie calcineurine (Cn)/NFAT dans des échantillons humains de lymphomes et de LAL, ainsi que dans des modèles murins de ces pathologies. Le laboratoire a ensuite montré que Cn est intrinsèquement requise pour la capacité des cellules leucémiques de LAL-T à propager la maladie (activité LIC « Leukemia Initiating Cells ») dans un modèle murin de LAL-T induit parun allèle activé de NOTCH1 (ICN1). Puisque l’inhibition pharmacologique de Cn induit de nombreux effets secondaires, la recherche de cibles thérapeutiques en aval de Cn constitue un axe de recherche important. J’ai participé à une étude du laboratoire montrant que l’expression à la surface cellulaire de CXCR4 est régulée par Cn et requise pour la migration des cellules de LAL-T, mais non suffisante pour rétablir le potentiel de ré-initiation suggérant que d’autres effecteurs doivent être impliqués dans cette activité.Les facteurs de transcription NFAT (NFAT1, NFAT2 et NFAT4) sont des effecteurs importants de Cn en réponse à la signalisation calcique lors du développement des thymocytes, mais également dans les lymphocytes T. L’essentiel de ce travail de thèse a utilisé des LAL-T induites par ICN1 dans lesquelles l’inactivation génique des trois facteurs NFAT par recombinaison homologue. Nous avons ainsi montré que (i) les facteurs NFAT sont requis en aval de Cn pour le potentiel LIC des LAL-T-ICN1 in vivo, (ii) leur inactivation altère la survie, la prolifération et la migration des cellules de LAL-T in vitro, (iii) NFAT1,NFAT2 et NFAT4 ont une fonction largement redondante dans les LAL-T. Nous avons également par une approche transcriptomique identifié deux gènes dont l’expression estsous contrôle des facteurs NFAT et impliqués dans la régulation de la survie et de la prolifération des LAL-T in vitro : CDKN1A et MAFB.Tout comme la voie Cn/NFAT, les CaMKs sont des protéines kinases activées en aval de la signalisation calcique dans les lymphocytes T. Nous avons montré par une approche pharmacologique que l’inhibition des CaMKs dans les LAL-T-ICN1 in vitro altère la survie etla prolifération des cellules leucémiques. L’inhibition spécifique par une approche d’ARN interférence de deux isoenzymes CaMKIIγ et CaMKIIδ suggèrent que ces protéines jouent dans le maintien des cellules leucémiques in vitro
T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of T cell progenitors. Despite initial response to chemotherapy, relapses remain frequent in children and adults. Previous results identify sustained activation of Calcineurin (Cn)/NFAT signaling pathway in human T-ALL and murine T-ALL models. Importantly, they also demonstrated Cn is essential for T-ALL Leukemia Initiating Cells (LIC) activity in a murine model of T-ALL induced by an activated allele of NOTCH1 (ICN1). Since pharmacologic inhibition of Cn induces side effects, we aim to identify downstream effectors involved in T-ALL. NFAT (Nuclear Factor of Activated T cells) factors play crucial roles downstream Cn during development and activation of T cells. To address their role in T-ALL, we generated mouse ICN1-induced T-ALL in which NFAT genes can be inactivated either single or in combination following Cre-mediated gene deletion. We demonstrated that (i) NFAT factors are required downstream Cn for LIC activity in T-ALL in vivo (ii) ex vivo NFAT factors deletion alters survival, proliferation and migration of T-ALL (iii) NFAT1, 2 and 4 have a largely redundant function in T-ALL. Moreover, the NFAT-dependant transcriptome allowed to identify important targets (CDKN1A, MAFB) involved in T-ALL survival and proliferation in vitro. Calmodulin-dependant kinases (CaMK) are kinases activated by calcium signaling in T cells. We showed that pharmacologic inhibition of CaMKs in ICN1-induced T-ALL alters survival and proliferation of T-ALL in vitro. Beside, specific inhibition by RNA interference of CaMKIIg and CaMKIId suggests a putative role of these kinases in T-ALL maintenance

A L-Asparaginase II de Escherichia coli (EcA II) é uma enzima amplamente utilizada no tratamento da Leucemia Linfoblástica Aguda (LLA), atuando na depleção do aminoácido L-asparagina, o qual é fundamental para a multiplicação das células cancerosas. Contudo, o tratamento com a EcA II está associado a altos índices de hipersensibilidade, devido à formação de anticorpos anti-L-asparaginase e à clivagem da enzima pelas proteases sanguíneas asparagina endopeptidase (AEP) e catepsina B (CTSB). Também ocorre neurotoxicidade associada ao efeito L-glutaminase da enzima. O principal objetivo do presente trabalho é a obtenção de mutantes da EcA II (gene ansB) com equivalente eficiência catalítica, maior resistência à clivagem proteolítica e menor atividade glutaminase. Para este propósito, através da reação em cadeia da polimerase propensa a erro (epPCR) do gene ansB, foi construída uma biblioteca de 1128 clones expressos no vetor pET15b em BL21(DE3). Nenhum mutante com atividade asparaginásica equivalente à EcA II selvagem apresentou atividade glutaminásica inferior à esta. Dentre os clones triados obtivemos um mutante (T161I) resistente à clivagem proteolítica pela CTSB e dois mutantes (Q190L e P40S/S206C) resistentes à clivagem proteolítica por ambas AEP e CTSB. Estes três mutantes apresentaram atividade asparaginásica e glutaminásica equivalentes a EcA II selvagem. Nossos resultados mostram promissoras possibilidades de EcA II mutantes com maior estabilidade frente às proteases sanguíneas humanas e possivelmente menos imunogênicas.
Escherichia coli L-asparaginase (EcA II) is an enzyme widely used in the treatment of acute lymphoblastic leukemia (ALL), acting in the depletion of the amino acid L-asparagine, which is essential for cancer cells proliferation. However, treatment with L-asparaginase is associated with a high rate of hypersensitivity, due to formation of anti-L-asparaginase antibody and the enzyme cleavage by the serum proteases asparagine endopeptidase (AEP) and cathepsin B (CTSB). Furthermore, the neurotoxicity is associated with the effect of the enzyme L-glutaminase activity. The main aim of the current work is to obtain variants of EcA II (gene ansB) with an equivalent catalytic efficiency, greater resistance to proteolytic cleavage and a reduced glutaminase activity. For such purpose, through error-prone polymerase chain reaction (epPCR) of gene ansB, a library of 1128 clones was constructed in pET15b vector and expressed in BL21(DE3). None mutant with an asparaginase activity equivalent to EcA II wild type showed a reduced glutaminase activity. Among the screened clones, one mutant (T161I) was resistant to CTSB proteolytic cleavage and two mutants (Q190L e P40S/S206C) were resistant to both CTSB and AEP proteolytic cleavages. These three mutants were EcA II wild type equivalents in asparaginase and glutaminase activities. Our data show promising new possibilities of mutant EcA II presenting higher stability against human serum proteolytic cleavage and maybe lower immunogenicity.

Contrairement aux réponses cliniques favorables chez les patients porteurs de Leucémie Myéloïdes Chronique traités avec des inhibiteurs ciblant l'activité tyrosine kinase de l'oncoprotéine de fusion BCR-ABL, les Leucémie Aigues Lymphoblastiques (LAL-B) BCR-ABL-positives restent de mauvais pronostic et requièrent de nouvelles stratégies thérapeutiques. Cette situation résulte de la présence d'autres altérations génétiques récurrentes, telle que la délétion mono- ou bi-allélique du gène codant le facteur de transcription Ikaros, observée dans plus de 83% des patients. Notre laboratoire a créé un modèle murin permettant l'analyse des conséquences de la perte de fonction d'Ikaros dans les LAL-B BCR-ABL+, et identifié une signature transcriptomique liée à l'absence d'un allèle sauvage d'Ikaros dans ces cellules. Cette signature transcriptomique inclut la surexpression du récepteur tyrosine kinase IGF1R. Mon travail de thèse montre : (i) que l'inhibition pharmacologique de IGF1R sensibilise les LAL-B BCR-ABL+ déficientes pour Ikaros aux effets antiprolifératifs et pro-apoptotiques de Nilotinib, (ii) que la délétion du gène IGF1R inhibe l'expansion de ces cellules in vivo, (iii) que le traitement des souris leucémiques avec NVP-AEW541 (un inhibiteur de IGF1R) en combinaison avec Nilotinib augmente significativement la survie des souris traitées par rapport aux souris contrôles, (iv) que l'augmentation de la survie des souris traitées est accompagnée par une augmentation de l'apoptose et une diminution de la prolifération des cellules leucémiques, et (v) que la voie AKT/mTORC1/S6K est une cible moléculaire de cette combinaison d'inhibiteurs
In recent years several inhibitors have been developed targeting the tyrosine kinase activity of the BCR-ABL fusion oncoprotein in Chronic Myeloid Leukemia (CML). Unlike the favorable clinical response observed in CML cases, BCR¬ABL` B-cell Acute Lymphoblastic Leukemias (B-ALLs) remain of poor prognosis. The likely reason for this aggressive behavior is the presence in these leukemias of additional genetic alterations. The most frequent of these is the mono-or bi-allelic deletion of the gene encoding the Ikaros transcription factor (IKZF1), observed in over 83% of patients. Our laboratory has studied the functional consequences of IKZF1 haploinsufficiency in a BCR-ABL-induced B-ALL mouse model and identified an Ikaros-dependent transcriptomic signature in these leukemic cells. This signature includes the overexpression of IGF1R, a tyrosine kinase receptor for IGF1. Based on these premises my PhD thesis work shows (i) that pharmacological inhibition of IGF1R sensitizes Ikaros-deficient BCR-ABL+ B-ALL to the antiproliferative and pro-apoptotic effects of Nilotinib, (ii) that IGF1R gene deletion impairs in vivo expansion of these leukemias in vivo, (iii) that treatment of leukemic mice with NVP-AEW541 (an IGF1R inhibitor) in combination with Nilotinib significantly increases survival of treated mice as compared to control mice, (iv) that the increased survival of treated mice is accompanied by an increase in apoptosis and a decrease in the proliferation of leukemic cells and (v) that inhibition of the AKT/mTORC1/S6K signalling pathway is a point of convergence of these inhibitor combination

Malgré l'amélioration des traitements, environ 20% des patients atteints de leucémie aigue lymphoblastiques (LAL) rechutent dans la moelle osseuse ou dans des sites extra-médullaires tels que les ovaires et les testicules, ce qui est particulièrement fréquent dans les rechutes tardives de LAL-B présentant un remaniement ETV6/RUNX1. Les travaux réalisés par Virginie Gandemer en 2007, ont montré que l'expression de CD9 permettait de distinguer les leucémies ETV6/RUNX1 des autres types de leucémie. Le gène CD9 code pour une protéine de la famille des tétraspanines dont l'expression a été corrélée avec le risque métastatique et la survie des patients. Par ailleurs il a été démontré que la protéine CD9 était impliquée dans le homing et la prise de greffe des cellules souches hématopoïétiques et leucémiques. Nous avons donc émis l'hypothèse qu'à travers ses propriétés fonctionnelles sur la migration et le homing, CD9 pourrait être un acteur clé des rechutes de LAL-B. Le but de ce travail de thèse était donc premièrement de déterminer le mode de régulation de CD9 dans les LAL-B ETV6/RUNX1 et deuxièmement de déterminer les effets de l'expression de CD9 sur la motilité et la prise de greffe des LAL-B. Les analyses préalablement réalisées au laboratoire avaient suggéré que CD9 pouvait être régulé par des miARNs. Nous avons identifié un cluster de 3 miARNs potentiellement impliqués dans la régulation de CD9 dans les LAL-B ETV6/RUNX1. Ces résultats doivent cependant être complétés par d'autres analyses fonctionnellles afin d'être confirmés. Nous avons étudié le rôle de la protéine CD9 dans la dissémination des cellules de LAL-B. Nous avons démontré que CD9 était un régulateur potentiel de l'adhésion et un nouveau facteur impliqué dans la migration et le homing dépendants de CXCR4 en favorisant l'activation de RAC1 et les réarrangements de l'actine en réponse au CXCL12. Enfin, nous avons décrit pour la première fois l'influence de CD9 sur la migration et le homing dans les testicules via RAC1. Nos résultats montrent donc que CD9 favorise la dissémination des cellules de LAL-B dans les testicules et suggèrent que cette protéine pourrait constituer un acteur majeur des rechutes tardives de LAL-B dont les mécanismes d'apparitions sont peu connus
Despite improvements in survival rates, approximately 20% of children suffering from acute lymphoblastic leukemia (B-ALL) present relapses from bone marrow or from B-extramedullary sites, such as the testes or ovaries, particularly in cases of late relapse of ETV6/RUNX1-ALL. Virgine Gandemer showed in 2007, that the expression of CD9, a protein from the tetraspanin superfamily, can be used to distinguish ETV6/RUNX1 lymphoblastic leukemia from other types of ALL. CD9 expression has been correlated with the risk of metastasis and is associated with a poor clinical outcome in various types of cancer. Moreover CD9 has been implicated in hematopoietic and leukemic stem cell homing. We hypothesized, that CD9 protein, through its functional properties on migration and homing, could be a key actor of B-ALL relapses. The purpose of our study was then to investigate, first the transcriptional regulation of CD9 in ETV6/RUNX1 B-ALL and secondly, the effect of CD9 expression on motility and engrafment of B lymphoblasts. The analysis of CD9 transcriptional regulation previously made in the team, suggested that it could be regulated by miRNAs. We identified a cluster of 3 miRNAs potentially implicated in the regulation of CD9 expression in ETV6/RUNX1 B-ALL. This result has to be confirmd by more functional analysis. We investigated the role of CD9 in the dissemination of B-ALL. We identified CD9 as a potential regulator of B-ALL cell adhesion and a new factor involved in CXCR4-mediated migration and homing, through the promotion of actin rearrangement in response to CXCL12. We also characterized the effect of CD9 protein expression on RAC1 activation, which had an impact on blast migration and engraftment. Finally, we described, for the first time, the influence of CD9, mediated by RAC1 signaling, on B-cell chemotactic migration and homing in the testis. Our work provides evidence for an impact of CD9 on the ability of pre-B leukemic cells to disseminate to testes, through its effects on migration and homing, and suggests that CD9 may be a key player in late relapses of B-ALL, which are currently poorly understood

El 75% de los pacientes con leucemia aguda linfoblástica (LAL) tiene alteraciones cromosómicas recurrentes (numéricas o estructurales). Dichas anomalías (alteraciones primarias) tienen valor pronóstico independiente de otras variables y ello se tiene en cuenta para administrar un tratamiento adaptado al riesgo de cada leucemia. Sin embargo, el impacto pronóstico que pueden tener las alteraciones secundarias (p.ej. mutaciones puntuales o alteraciones en el número de copias) no está tan claro. Es probable que éstas jueguen un papel importante en la respuesta al tratamiento, ya que no todos los pacientes con el mismo cariotipo responden igual a la terapia. Con el fin de identificar nuevos marcadores pronósticos en la LAL de línea B, se ha analizado la frecuencia y significado pronóstico de las alteraciones en el número de copias (CNA) en muestras de pacientes de LAL B madura y LAL de precursores B. En el primer trabajo de esta Tesis Doctoral se identificaron las deleciones de los genes EBF1, IKZF1 y CDKN2A/B como factores pronósticos independientes en una serie de 142 pacientes adolescentes y adultos de LAL de precursores B analizados en el momento del diagnóstico. Concretamente, las deleciones de EBF1 se asociaron con una menor probabilidad de alcanzar la remisión completa, las deleciones de IKZF1 se relacionaron con una mayor probabilidad de sufrir una recaída de la enfermedad, mientras que las deleciones de CDKN2A/B estaban presentes en pacientes con menor probabilidad de supervivencia global. Por tanto, la detección de estas alteraciones genéticas identifica un subgrupo de pacientes con LAL de alto riesgo, los cuales podrían beneficiarse de tratamientos más intensivos, a la espera de tratamientos más específicos contra dichas alteraciones. En el segundo trabajo se evidenció que las LAL de precursores B y las LAL B maduras tienen un perfil genético diferente. En este sentido, la frecuencia de alteraciones de mal pronóstico identificadas en el primer trabajo fue mucho menor en los pacientes con LAL B madura que en los pacientes con LAL de precursores B. Este hecho podría explicar, en parte, el mejor pronóstico que muestran las LAL B maduras en comparación con las de precursores B. Sin embargo, pese a que se detectaron deleciones potencialmente relacionadas con la agresividad y diseminación de las LAL B maduras (CDKN2A/B, RB1 y región 14q32.33), no se consiguió identificar ninguna CNA con valor pronóstico en estos pacientes. Finalmente, en el tercer trabajo se analizaron muestras en recaída de pacientes con LAL de precursores B. Mediante la comparación de muestras pareadas de diagnóstico y recaída de cada paciente se observó un aumento significativo del número de CNA durante la progresión de la leucemia. Además, se evidenció una gran diversidad de rutas metabólicas afectadas por CNA, sin que se detectara una firma genética recurrente en recaída, a excepción de la adquisición de deleciones homocigotas en el gen CDKN2A/B (frecuencia del ~50%). Las rutas metabólicas más afectadas en recaída fueron las que afectan a la diferenciación de los linfocitos B, al control del ciclo celular y a la apoptosis, entre otras. Las alteraciones en genes involucrados en la proliferación celular, la homeostasis de las células madre hematopoyéticas y la resistencia a fármacos fueron las más comunes entre las CNA adquiridas de novo en recaída. A excepción de una paciente, todos los pacientes mostraron cambios genéticos respecto al diagnóstico y la mayoría de recaídas provenían de un clon ancestral al detectado en el momento del diagnóstico. La identificación de CNA específicas de recaída puede contribuir a diseñar nuevos tratamientos que aumenten la probabilidad de supervivencia de los pacientes en esta situación, que actualmente es de tan sólo el 10%.
Approximately, 75% of acute lymphoblastic leukemia (ALL) patients show recurrent chromosomal abnormalities (numerical and structural). These are primary alterations that have been extensively related to patients’ prognosis. The predictive value of these chromosomal aberrations is taken into account for treatment assignment. However, the prognostic value of secondary alterations (i.e., point mutations or copy number alterations) is less clear. These alterations may play an important role in the response to the therapy, as not all patients with the same karyotype show the same response to treatment. With the aim to identify new prognostic markers in patients with B lineage ALL, the frequency and prognostic significance of copy number alterations (CNA) in patients with mature and precursor B ALL have been analyzed. In the first study of this Doctoral Thesis EBF1, IKZF1 and CDKN2A/B deletions at diagnosis were identified as markers with independent prognostic value in a series of 142 adolescent and adult patients with precursor B ALL. Specifically, EBF1 deletions were associated with a lower probability of achieving complete remission, IKZF1 deletions were related to a greater relapse probability while CDKN2A/B deletions were present in patients with lower survival probability. Therefore, the detection of these genetic alterations identifies a subset of high risk patients who may benefit from a more intensive treatment, while new and more specific treatments are developed against these alterations. In the second study, a clear difference in the genetic profiles of mature B and precursor B ALL was observed. In this sense, the frequency of the alterations associated with poor prognosis detected in the first study was much lower in mature B ALL compared to precursor B ALL patients. This fact may explain, in part, the better prognosis of mature B compared to that of precursor B ALL. However, although some deletions potentially related to the aggressiveness and dissemination of mature B ALL (CDKN2A / B, RB1 and 14q32.33 region) were detected, no CNAs with prognostic value could be identified in these patients. Finally, relapse samples of patients with precursor B ALL were analyzed in the third study. Comparison of paired diagnostic and relapse samples from each patient showed a significant increase in the number of CNAs during the progression of leukemia. In addition, a great diversity of metabolic pathways affected by CNA without a recurrent genetic signature at relapse was evidenced, except for the acquisition of homozygous deletions in the CDKN2A/B gene (~50% frequency). The most frequently involved metabolic pathways at relapse were those related with differentiation of B lymphocytes, cell cycle control and apoptosis, among others. Alterations in genes involved in cell proliferation, hematopoietic stem cell homeostasis, and drug resistance were the most common among the newly acquired CNAs at relapse. With the exception of one patient, all patients showed at relapse genetic changes compared to the diagnosis and most relapses came from an ancestral clone to that detected at the time of diagnosis. The identification of relapse-specific CNAs may contribute to the design of new treatments that could increase the probability of survival of relapsed patients, which currently stands at 10%.

碩士
臺灣大學
醫學檢驗暨生物技術學研究所
98
PAX5 (Paired box 5), a member of PAX gene family, is expressed in the central nervous and the hematopoietic systems and is known to play an important role in B cell commitment. Childhood acute lymphoid leukemia (ALL) is a malignant disease resulting from uncontrolled proliferation of lymphoid progenitors. In previous large-scaled study, the PAX5 gene was the most frequent target of somatic mutation, being altered in about 30% in both pediatric and adult patients with B-ALL. However, whether PAX5 mutation plays an important role in B-ALL leukemogenesis remains uncharacterized. In this study, we intend to investigate the molecular or clinical characteristics of PAX5 expression alteration in B-ALL. The RNA and protein expression, DNA status (loss of heterozygosity, LOH) of PAX5 gene, as well as clinical data were investigated in 25 diagnostic bone marrow samples obtained from childhood B-ALL patients. Peripheral blood mature B cells obtained from 3 healthy subjects were used as control. Decreased PAX5 mRNA expression was noted in 14 out of 25 leukemic samples as compared to normal mature B cells (median (25%-75%), ALL: 0.4 (0-0.9), Normal: 0.9 (0.9-1), p=0.1438). Although it was not statistically significant, it revealed that the leukemic cells with decreased level of PAX5 mRNA expression tend to carry more non-B markers. There were more mRNAs with variant sizes in leukemic cells than in normal mature B cells. PAX5 protein level was also investigated in the leukemic cells of 7 B-ALL patients. Four showed decreased PAX5 expression and the other 3 were of comparable PAX5 level as compared to normal mature B cells. We also investigated LOH of PAX5 locus in 6 paired-samples (leukemic bone marrow cells and remission peripheral blood cells as normal reference) from B-ALL patients via short tandem repeat analysis. Three showed LOH and one remained heterozygous. The DNA status was not correlated with mRNA expression level. In summary, the trend of decreased PAX5 protein expression was parallel to that of PAX5 mRNA expression level, but is not correlated with DNA status. The results indicate that: 1. PAX5 mRNA expression is generally decreased in B-ALL patients; 2. There were more PAX5 C-terminal variant forms present in B-ALL leukemic samples, including transcript without exons 7 and 8, which may influence trans-activating ability of PAX5; 3. Significantly decreased protein level in 14 out of 25 B-ALL patients reveals the association in B-ALL and decreased PAX5 expression; 4. Besides the gene dosage, some other factors could regulate PAX5 transcription through mechanisms awaited further studies.

Growing evidence suggests that Notch signaling pathway can modulate drug response in hematological malignancies including T-ALL, B-CLL and AML. In B-ALL we have previously demonstrated that Notch3 and Notch4 support survival of primary B-ALL cells, suggesting a role for Notch signaling in drug response. Here, we used in vitro, in silico, and in vivo approaches to comprehensively the role of Notch pathway in B-ALL pathogenesis in terms of prognosis, proliferation, survival and drug response. B-ALL cell lines were obtained from ATCC, while B-ALL primary cells were isolated from bone marrow or peripheral blood of 51 B-ALL patients. Flow cytometry and western immunoblotting analyses showed that primary leukemia cells from high-risk patients overexpressed Notch3, Notch4, and Jagged2 while displaying a reduction in expression levels of Notch1-4 following chemotherapy, suggesting that Notch signaling may be critical to drug response in B-ALL. We then analyzed in vitro cell survival of B-ALL cells treated with conventional chemotherapeutic agents (Cytarabine, Ara-C; Dexamethasone, Dexa; Doxorubicin, Doxo) alone or in combination with Notch signaling modulators, including anti-Notch blocking antibodies, gamma secretase inhibitors (GSIs), and Notch transcription factor inhibitor (SAHM1). GSIs and anti-Notch4 were all capable of potentiating drug-induced cell death in B-ALL cells, up-regulating intracellular levels of reactive oxygen species (ROS) that were then capable to modulate pro-survival protein levels such as mTor, Akt, NFκ-B and Erk. In vitro observations were successfully translated in mouse-based xenograft models of B-ALL, obtained by injecting the B-ALL line RS4;11 in NOG mice. The in vivo co-administration of Notch inhibitor GSI-XII or anti-Notch4 with the chemotherapeutic agent Ara-C lowered bone marrow leukemic burden, thus prolonging mouse survival, compared with DMSO or Ara-C alone. Overall, our results highlighted the prognostic value of Notch expression in B-ALL as well as its critical role in B-ALL cell survival and response to chemotherapy in vitro and in vivo. We demonstrated that inhibition of Notch signaling enhances the chemosensitivity of B-ALL cells, improving Ara-C-mediated reduction of blast cells in bone marrow, suggesting that Notch signaling is a possible therapeutic strategy to eradicate the minimal residual disease in B-ALL.

La leucémie lymphoblastique aigüe (LLA) est une maladie génétique complexe. Malgré que cette maladie hématologique soit le cancer pédiatrique le plus fréquent, ses causes demeurent inconnues. Des études antérieures ont démontrées que le risque à la LLA chez l’enfant pourrait être influencé par des gènes agissant dans le métabolisme des xénobiotiques, dans le maintient de l’intégrité génomique et dans la réponse au stress oxydatif, ainsi que par des facteurs environnementaux. Au cours de mes études doctorales, j’ai tenté de disséquer davantage les bases génétiques de la LLA de l’enfant en postulant que la susceptibilité à cette maladie serait modulée, au moins en partie, par des variants génétiques agissant dans deux voies biologiques fondamentales : le point de contrôle G1/S du cycle cellulaire et la réparation des cassures double-brin de l’ADN. En utilisant une approche unique reposant sur l’analyse d’une cohorte cas-contrôles jumelée à une cohorte de trios enfants-parents, j’ai effectué une étude d’association de type gènes/voies biologiques candidats. Ainsi, j’ai évaluer le rôle de variants provenant de la séquence promotrice de 12 gènes du cycle cellulaire et de 7 gènes de la voie de réparation de l’ADN, dans la susceptibilité à la LLA. De tels polymorphismes dans la région promotrice (pSNPs) pourraient perturber la liaison de facteurs de transcription et mener à des différences dans les niveaux d’expression des gènes pouvant influencer le risque à la maladie. En combinant différentes méthodes analytiques, j’ai évalué le rôle de différents mécanismes génétiques dans le développement de la LLA chez l’enfant. J’ai tout d’abord étudié les associations avec gènes/variants indépendants, et des essaies fonctionnels ont été effectués afin d’évaluer l’impact des pSNPs sur la liaison de facteurs de transcription et l’activité promotrice allèle-spécifique. Ces analyses ont mené à quatre publications. Il est peu probable que ces gènes de susceptibilité agissent seuls; j’ai donc utilisé une approche intégrative afin d’explorer la possibilité que plusieurs variants d’une même voie biologique ou de voies connexes puissent moduler le risque de la maladie; ces travaux ont été soumis pour publication. En outre, le développement précoce de la LLA, voir même in utero, suggère que les parents, et plus particulièrement la mère, pourraient jouer un rôle important dans le développement de cette maladie chez l’enfant. Dans une étude par simulations, j’ai évalué la performance des méthodes d’analyse existantes de détecter des effets fœto-maternels sous un design hybride trios/cas-contrôles. J’ai également investigué l’impact des effets génétiques agissant via la mère sur la susceptibilité à la LLA. Cette étude, récemment publiée, fût la première à démontrer que le risque de la leucémie chez l’enfant peut être modulé par le génotype de sa mère. En conclusions, mes études doctorales ont permis d’identifier des nouveaux gènes de susceptibilité pour la LLA pédiatrique et de mettre en évidence le rôle du cycle cellulaire et de la voie de la réparation de l’ADN dans la leucémogenèse. À terme, ces travaux permettront de mieux comprendre les bases génétiques de la LLA, et conduiront au développement d’outils cliniques qui amélioreront la détection, le diagnostique et le traitement de la leucémie chez l’enfant.
Childhood acute lymphoblastic leukemia (ALL) is a complex and heterogeneous genetic disease. Although it is the most common pediatric cancer, its etiology remains poorly understood. Previous studies provided evidence that childhood ALL might originate through the collective contribution of different genes controlling the efficiency of carcinogen metabolism, the capacity of maintaining DNA integrity and the response to oxidative stress, as well as environmental factors. In my doctoral research project I attempted to further dissect the genetic intricacies underlying childhood ALL. I postulated that a child’s susceptibility to ALL may be influenced, in part, by functional sequence variation in genes encoding components of two core biologic pathways: G1/S cell cycle control and DNA double-strand break repair. Using a unique two-tiered study design consisting of both unrelated ALL cases and healthy controls, as well as case-parent trios, I performed a pathway-based candidate-gene association study to investigate the role of sequence variants in the promoter regions of 12 candidate cell cycle genes and 7 DNA repair genes, in modulating ALL risk among children. Polymorphisms in promoter regions (pSNPs) could perturb transcription factor binding and lead to differences in gene expression levels that in turn could modify the risk of disease. To better depict the complex genetic architecture of childhood ALL, I used multiple analytical approaches. First, individual genes/variants were tested for association with disease, while functional in vitro validation was performed to evaluate the impact of the pSNPs on differential transcription factor binding and allele-specific promoter activity. These analyses led to four published articles. Given that these genes are not likely to act alone to confer disease risk I used an integrative approach to explore the possibility that combinations of functionally relevant pSNPs among several components of the same or of interconnected pathways, could contribute to modified childhood ALL risk either through pathway-specific or epistatic effects; this work was recently submitted for publication. Finally, childhood ALL is thought to arise in utero suggesting that the parents, and in particular the mother, may play an important role in shaping disease susceptibility in their offspring. Using simulations, I investigated the performance of existing methods to test for maternal genotype associations using a case-parent trio/case-control hybrid design, and then assessed the impact of maternally-mediated genetic effects on ALL susceptibility among children. This published work was the first to show that the mother’s genotype can indeed influence the risk of leukemia in children, further corroborating the importance of considering parentally-mediated effects in the study of early-onset diseases. In conclusion, my doctoral work lead to the identification of novel genetic susceptibility loci for childhood ALL and provided evidence for the implication of the cell cycle control and DNA repair pathways in leukemogenesis. Better elucidation of the genetic mechanisms underlying the pathogenesis of ALL in children could be of great diagnostic value and provide data to help guide risk-directed therapy and improve disease management and outcome. Ultimately, this study brings us one step closer to unraveling the genetic architecture of childhood ALL and provides a stepping-stone towards disease prevention.

Dissertação de mest., Ciências Biomédicas, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010
A leucemia linfoblástica aguda de linfócitos T (LLA-T) é uma patologia hematológica maligna que afecta essencialmente crianças e adultos jovens e é fatal na ausência de um diagnóstico precoce e uma terapêutica apropriada. Sabe-se que a LLA-T tem origem em precursores dos linfócitos T, também designados por timócitos, que sofrem um bloqueio da diferenciação e expansão clonal durante o seu desenvolvimento no timo. As células imaturas transformadas acabam por entar na corrente sanguínea e invadir vários órgãos pelo que esta doença se caracteriza por leucocitose, maior ou menor invasão da medula óssea por células leucémicas e pelo envolvimento de órgãos como o baço, fígado e gânglios linfáticos e, por vezes, do sistema nervoso central. Na origem do desenvolvimento da LLA-T encontram-se sucessivas alterações génicas que afectam os timócitos como, por exemplo, translocações cromossómicas que levam à sobre-expressão de factores de transcrição oncogénicos ou criam genes de fusão aberrantes, delecções, duplicações e mutações específicas em proto-oncogenes ou em genes supressores de tumores. Estas alterações acabam por desregular processos cujo controlo tem uma importância extrema e que acabam por conduzir as células transformadas a sofrer um bloqueio da sua diferenciação e a adquirir capacidades de auto-renovação ilimitada, de subverter os controlos da proliferação e de resistir a sinais pro-apoptóticos. Apesar da grande maioria dos conhecimentos adquiridos até à data sobre o desenvolvimento da LLA-T dizerem respeito a alterações génicas presentes nas células leucémicas, a importância dos factores do microambiente onde as células cancerosas se desenvolvem tornou-se evidente nas últimas décadas. Acredita-se que as células neoplásicas podem interagir com as células que compõem o microambiente de forma a estas modificarem a expressão génica para produzir factores que favoreçam o desenvolvimento das primeiras. Como a LLA-T tem origem no timo e este órgão é caracterizado por possuir um microambiente dinâmico, rico em factores de crescimento, citoquinas e contactos linfo-estromais indispensáveis para o desenvolvimento dos timócitos ou das próprias células do estroma tímico, será razoável acreditar que as células que constituem o estroma do timo poderão intervir de alguma forma no desenvolvimento da doença em questão. Recentemente, num modelo murino de LLA-T, descobriu-se que a proteína RelB expressa em células do estroma tímico, pertencente à família de factores de trancrição NF-κB, é importante para o desenvolvimento desta doença. Esta descoberta apoia a hipótese de que sinais moleculares produzidos pelos timócitos transformados podem, através da activação do factor de transcrição RelB em células do estroma tímico, levar à expressão de factores importantes para o desenvolvimento de leucemia. Contudo, os sinais provenientes do estroma tímico que favorecem o desenvolvimento da LLA-T ainda constituem um enigma. A proteína RelB é activada por membros da superfamília de receptores TNF que activam a via alternativa do NF-κB como, por exemplo, o receptor da linfotoxina beta (LTβR) ou o receptor activador do NF-κB (RANK) e, tal como no caso da proteína RelB, a sua ausência em células do estroma tímico leva a defeitos na microestrutura do timo. Estudos prévios de transcriptómica forneceram dados adicionais que poderão implicar o LTβR no desenvolvimento da leucemia. Estes estudos revelaram níveis de expressão aumentados dos genes que codificam o ligando do LTβR, Lta e Ltb, em células leucémicas TEL-JAK2 quando comparadas com timócitos normais. As proteínas resultantes da expressão dos genes referidos, linfotoxina α e linfotoxina β, formam o heterotrímero LTα1β2 que é expresso na superfície dos timócitos e que, por ligação ao receptor da linfotoxina β expresso na superficie de células do estroma tímico, iniciam a via de sinalização do LTβR. Esta ligação induz a formação de complexos de sinalização citoplasmáticos contendo factores associados a TNFR (TRAFs) que regulam interacções do receptor com vias de sinalização intracelulares a jusante do mesmo. A via de sinalização LTα1β2/LTβR pode activar a via canónica ou a via alternativa do NF-κB conduzindo a diferentes padrões de expressão conforme a via activada. A activação dos heterodímeros p52/RelB (via alternativa) induzida pelo LTβR resulta na expressão de quimiocinas como CCL19, CCL21, CXCL12 ou CXCL13. Destas, as quimiocinas CCL19 e CCL21 e o seu receptor CCR7 que é expresso na superfície de timócitos, foram implicados no desenvolvimento de cancro. Diversos estudos recentes têm sugerido também o envolvimento da via de sinalização LTα1β2/LTβR no desenvolvimento de cancro por activação constitutiva de factores de transcrição da família NF-κB. É portanto possível que a interacção do heterotrímero LTα1β2 produzido pelos timócitos transformados com o receptor da linfotoxina β em células do estroma tímico promova a activação do factor de transcrição RelB e consequente expressão dos seus genes-alvo que podem favorecer o desenvolvimento da leucemia linfoblástica aguda de células T. A realização deste trabalho teve como objectivo compreender o papel do LTβR no desenvolvimento da LLA-T. Para isto, recorreu-se ao ratinho transgénico TEL-JAK2 que desenvolve LLA-T a partir de timócitos que expressam a proteína de fusão e que constituem um modelo relevante para a LLA-T humana pois a proteína de fusão TEL-JAK2 também foi identificada em amostras primárias de doentes. Através da realização de RT-PCR semi-quantitativo e quantitativo, verificámos que as células T leucémicas apresentavam uma expressão mais elevada dos genes que codificam o ligando Ltα1β2, Lta e Ltb, quando comparadas com os timócitos normais. Também verificámos que o gene Ltbr é expresso em tumores do timo de ratinhos TEL-JAK2 o que demonstra que a via de sinalização LTα1β2/LTβR pode ocorrer no timo. De forma a avaliar se o desenvolvimento da leucemia induzida pela proteína de fusão TEL-JAK2 é comprometido na ausência do LTβR, cruzou-se ratinhos trangénicos TEL-JAK2 com ratinhos nos quais a expressão do gene Ltbr foi eliminada, de forma a gerar coortes de ratinhos TEL-JAK2;Ltbr-/- e TEL-JAK2;Ltbr+/-. Estes ratinhos foram monitorizados de forma a verificar se haviam diferenças significativas em termos de tempo de desenvolvimento da leucemia entre os dois grupos. Verificou-se que a inexistência do LTβR atrasou significativamente o desenvolvimento da leucemia em ratinhos TEL-JAK2 apesar da massa tumoral nos órgãos linfóides e o fenótipo da superfície celular das células leucémicas não apresentarem alterações significativas entre os dois grupos estudados. Assim, podemos concluir que o receptor da linfotoxina β deve contribuir para o desenvolvimento da LLA-T. No entanto, será necessário desenvolver mais estudos para se compreender como a sinalização através deste receptor afecta o desenvolvimento da LLA-T, antes que esta via possa constituir um alvo terapêutico a considerar para a doença em questão.

The aim of my PhD research project was to develop an advanced optical technique, based on Raman Spectroscopy (RS) and its integration with other optical (e.g. fluorescence microscopy, flow cytometry, Raman imaging) and biological modalities (e.g. Western Blotting), in order to provide a fast and reliable procedure for identification and classification of single cells (hemogram) from peripheral blood of healthy donors as well as of cancer B-ALL patients. The work can be divided into three main parts: • Deep study of Raman spectroscopy literature with particular attention to its sensitivity, specificity and bio-applications. • Development and characterization of the Raman microscope including the design of optics and development mathematical methods for analysis of the data (Principal Component Analysis). Since the aim of my thesis is to study living cells in vivo, with particular reference to normal and leukemia cells, the main challenges have been to find correct laser wavelength and intensity, alignment and calibration of the Raman system and finally its application to demonstrate its usefulness. • Completion of four experiments that demonstrate the suitability of Raman technique for studies of live normal and leukemia cells. Schematically, my research project concerns: (i) Raman identification and classification of three leukemia cell lines, that closely mimic the different differentiation/maturation stages of B-ALL cells; (ii) Spectroscopic regression assessment of leukemia after low- and not-cytotoxic dose treatments of methotrexate (MTX) and 6-mercaptopurine (6MP), two key drugs currently used in the B-ALL maintenance therapy; (iii) RS analysis of clinical samples collected from peripheral blood of patients with B-ALL; (iv) Raman identification and differentiation of the most important leukocyte populations isolated from peripheral blood of several human volunteers via conventional flow cytometry (granulocytes, monocytes, T cells, B cells and NK cells). The results presented here demonstrate that RS in conjunction with multivariate statistical technique has potential for rapid label-free diagnosis, classification and follow-up after chemotherapy treatment of B-ALL based on the optical evaluation of spectral features of biomolecules.

La leucemia linfoblastica acuta B (B-ALL) è il tipo più comune di leucemia acuta che si sviluppa nel midollo osseo. Mentre esiste molta letteratura sui dati inerenti il ruolo della segnalazione di Notch nella leucemia linfoblastica acuta T in biologia, l'importanza di questo stradino molecolare per lo sviluppo delle cellule di B-ALL nel midollo osseo non era finora noto. In questo studio, abbiamo utilizzato anticorpi neutralizzanti di Notch ed l’ inibitori della gamma-secretasi (GSI XII) per analizzare il ruolo del percorso della segnalazione di Notch nella promozione di B-ALL umane sopravvissute in presenza di cellule stromali sostegno. Il trattamento con combinazioni degli anticorpi neutralizzanti di Notch ha causato la diminuzione della sopravvivenza delle cellule di B-ALL, sia nelle colture univoche sia in co-coltura in presenza di cellule stromali sostegno. E' interessante notare che l'inibizione del Notch-3 e -4 o Jagged-1/-2 e DLL-1 determina un drammatico aumento dell’apoptosi delle cellule di B-ALL in circa 3 giorni, similmente a ciò che è stato ottenuto bloccando tutti le segnalazioni di Notch con la GSI XII. I nostri dati suggeriscono che le cellule stromali hanno un effetto anti apoptotico sulla linea di B-ALL.
B-cell acute lymphoblastic leukemia (B-ALL) is the most common type of acute leukemia developing in the bone marrow. While many literature data are available on the role of Notch signaling in T-cell ALL biology, the importance of this molecular pathway in the development of B-ALL cells in bone marrow microenvironment is unknown so far. In this study, we used anti-Notch molecules neutralizing antibodies and γ-secretase inhibitor (GSI) XII to investigate the role of Notch signaling pathway in the promotion of human B-ALL cell survival in the presence of stromal cell support. The treatment with combinations of anti-Notch molecules neutralizing antibodies resulted in the decrease of B-ALL cell survival, either cultured alone or co-cultured in the presence of stromal cell support. Interestingly, the inhibition of Notch-3 and -4 or Jagged-1/-2 and DLL-1 resulted in a dramatic increase of apoptotic B-ALL cells by 3 days, similar to what is obtained by blocking all Notch signaling with the γ-secretase inhibitor (GSI) XII. Our data suggest that the stromal cell-mediated anti-apoptotic effect on B-lineage ALL cells is mediated by Notch-3 and -4 or Jagged-1/-2 and DLL-1 in a synergistic manner.

Currently, the intensive chemotherapy remains the first line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although these regimens have significantly improved patient outcomes, their use is associated with debilitating morbidities and fatal relapses, highlighting the great need in new agents that target essential survival signals in leukemia. Thus, the overall goal of my project was to gain insights into the signaling abnormalities that regulate aberrant proliferation and survival of B-ALL cells in an effort to identify novel targets in this malignancy. This study demonstrated that pre-B cell receptor (pre-BCR)-independent spleen tyrosine kinase (SYK) activity was required for the survival and proliferation of a p53-/-PrkdcSCID/SCID mouse model of B-ALL. I extended this discovery to human disease, demonstrating that SYK was activated in primary B-ALL, independent of the pre-BCR expression. The small molecule SYK inhibitor fostamatinib (fosta) significantly attenuated proliferation of 79 primary diagnostic B-ALL samples at clinically achievable concentrations. Importantly, fosta treatment reduced dissemination of engrafting B-ALL cells into the spleen, liver, kidney and central nervous system (CNS) in a NOD.Prkdcscid/scidIl2rgtm1Wjl/SzJ xenotransplant model of B-ALL. Analysis of signaling abnormalities using a high-throughput phospho-flow cytometry platform demonstrated that pediatric and adult B-ALL samples exhibit variable basal activation of BCR, iii PI3K/AKT/mTOR, MAPK and JAK/STAT pathways. Importantly, we identified that fosta-mediated inhibition of SYK, PLC2, CRKL and EIF4E phosphorylation in B-ALL was predictive of its anti-leukemic activity, and was distinct from the cellular actions of other small molecule inhibitors of key nodal signaling pathways. Examination of molecular mechanism of fosta action by gene expression profiling revealed transcriptional effects of fosta treatment that included, most notably, potent inhibition of pathways involved in lymphocyte activation and inflammation. In conclusion, this study demonstrates that SYK signaling is crucial for B-ALL survival and provides detailed characterization of cellular and molecular mechanisms of fosta action in B-ALL. These data argue in favor of testing small molecule SYK inhibitors in pediatric and adult B-ALL.

L’ostéonécrose (ON) et les fractures (FR) sont des complications qui prennent de plus en plus place dans le traitement pédiatrique de la leucémie aiguë lymphoblastique (LAL). L’ON peut être causée par différents facteurs, dont principalement l’utilisation de glucocorticoïdes. Les glucocorticoïdes sont administrés lors du traitement de la leucémie dans le but d’initier l’apoptose des cellules malignes tout en ayant un effet anti-inflammatoire. Cependant, l’utilisation de ces corticostéroïdes comprend des effets secondaires sérieux, notamment le développement d’ostéonécrose. Des variantes génétiques peuvent mettre certains patients plus à risque que d’autres. Plusieurs gènes ont déjà été signalés comme régulés par les actions glucocorticoïdes (GC). Les variations génétiques présentes dans les régions régulatrices de ces gènes peuvent affecter leur fonctionnement normal et, en fin de compte, de déterminer un risque accru de développer l’ON associé au traitement contre la leucémie. Pour cette raison, plusieurs polymorphismes ont été identifiés et étudiés dans la cohorte QcALL de Ste-Justine, concernant les gènes suivants : ABCB1, ACP1, BCL2L11, NFKB1, PARP1, et SHMT1. Ces gènes jouent majoritairement un rôle dans les mécanismes d’action des glucocorticoïdes, mais quelques-uns ont plutôt un effet direct sur le développement d’ostéonécrose. Nos recherches ont démontré une corrélation entre ces polymorphismes et l’apparition d’ostéonécrose chez les patients de la cohorte QcALL, traités aux glucocorticoïdes. L'incidence cumulative de l'ostéonécrose a été évaluée rétrospectivement chez 305 enfants atteints de la leucémie qui ont subi un traitement à l’hôpital Ste-Justine selon les protocoles DFCI de Boston (87-01, 91-01, 95-01 et 2000-01). Parmi les huit polymorphismes de BCL2L11 étudiés, les 891T> G (rs2241843) et 29201C> T (rs724710) ont été significativement associés à ON (p = 0.01 et p = 0.03, respectivement). L'association du polymorphisme 891T> G a été modulée par le type de corticostéroïde (CS), l’âge, le sexe et le groupe à risque (p ≤ 0,05). Le polymorphisme 29201C> T était particulièrement apparent chez les patients à haut risque (p = 0,003). La même étude était conduite en parallèle sur des patients de la cohorte DFCI de Boston (N = 192), et montrait des résultats significatifs pour les polymorphismes étudiés. En conclusion, les résultats de cette étude permettront de confirmer l’association de ces polymorphismes au développement d’ON chez les patients de LLA traités aux GC.
Osteonecrosis (ON) and fractures (FR) are complications that take place in the treatment of children acute lymphoblastic leukemia (cALL). They can be caused by various factors, mainly using glucocorticoids. The corticosteroids, dexamethasone (DXM) and prednisone (PDN) are administered during the treatment of leukemia to initiate apoptosis of malignant cells; while having an anti-inflammatory effect. However, the use of these corticosteroids has severe side effects, including the development of osteonecrosis. Moreover, some patients develop resistance to treatment, and are at risk of developing side effects. The genetic variants predispose some patients at higher risk than others. Several genes have been previously reported as up- or down regulated by the GCs actions. The genetic variations present in gene coding or regulatory regions can affect their function and ultimately determine an increased risk of developing ON associated to ALL therapy. Therefore, we investigated the association between several single nucleotide polymorphisms (SNPs) in six candidate genes: BCL2L11, NFKB1, PARP1, ABCB1, ACP1, and SHMT1. These genes play a role in the mechanisms of action of glucocorticoids, but some have more of a direct effect on the development of osteonecrosis. Our research has shown a correlation between these polymorphisms and the occurrence of osteonecrosis in patients in the QCALL cohort, treated with glucocorticoids. Cumulative incidence of osteonecrosis was assessed retrospectively in 305 children with ALL who underwent treatment with DFCI protocols (87-01, 91-01, 95-01 and 2000-01) in childhood ALL cohort from Quebec (QcALL). Among the eight tag BCL2L11 polymorphisms studied the 891T>G (rs2241843) and 29201C>T (rs724710) were significantly associated with ON (p = 0.01 and p = 0.03, respectively). Association of 891T>G polymorphism was modulated by type of corticosteroid (CS), age, sex and risk group (p ≤ 0.05 and that of 29201C>T was particularly apparent among high risk (p = 0.003) patients. These polymorphisms have shown significant ON association in several QcALL risk groups, mainly in corticosteroid groups, age < 10 years, and high risk (HR) group. Furthermore, the same study was conducted in parallel with patients in the replication (DFCI) cohort (N = 192), and we showed significant genetic association results for all studied polymorphisms. In conclusion, this study identifies that some ALL children have a high incidence of ON during the treatment that is highly associated with polymorphisms in different genes regulated by corticosteroids and ALL prognostic factors.

abstract: The Philadelphia chromosome in humans, is on oncogenic translocation between chromosomes 9 and 22 that gives rise to the fusion protein BCR-Abl. This protein is constitutively active resulting in rapid and uncontrolled cell growth in affected cells. The BCR-Abl protein is the hallmark feature of chronic myeloid leukemia (CML) and is seen in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) cases. Currently, the first line of treatment is the Abl specific inhibitor Imatinib. Some patients will, however, develop resistance to Imatinib. Research has shown how transformation of progenitor B cells with v-Abl, an oncogene expressed by the Abelson murine leukemia virus, causes rapid proliferation, prevents further differentiation and produces a potentially malignant transformation. We have used progenitor B cells transformed with a temperature-sensitive form of the v-Abl protein that allows us to inactivate or re-activate v-Abl by shifting the incubation temperature. We are trying to use this line as a model to study both the progression from pre-malignancy to malignancy in CML and Imatinib resistance in Ph+ ALL and CML. These progenitor B cells, once v-Abl is reactivated, in most cases, will not return to their natural cell cycle. In this they resemble Ph+ ALL and CML under Imatinib treatment. With some manipulation these cells can break this prolonged G1 arrested phenotype and become a malignant cell line and resistant to Imatinib treatment. Cellular senescence can be a complicated process requiring inter-play between a variety of players. It serves as an alternate option to apoptosis, in that the cell loses proliferative potential, but does not die. Treatment with some cancer therapeutics will induce senescence in some cancers. Such is the case with Imatinib treatment of CML and Ph+ ALL. By using the S9 cell line we have been able to explore the possible routes for breaking of prolonged G1 arrest in these Ph+ leukemias. We inhibited the DNA damage sensor protein ataxia telangiectasia mutated (ATM) and found that prolonged G1 arrest in our S9 cells was broken. While previous research has suggested that the DNA damage sensor protein ataxia-telangiectasia mutated (ATM) has little impact in CML, our research indicates that ATM may play a role in either senescence induction or release.
Dissertation/Thesis
M.S. Microbiology 2011

Leukemia is the most common malignant disease in children patients. In our laboratory (CLIP) a novel subtype of B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL) with lineage switch during early phase of treatment towards myeloid lineage (swALL) was recently documented. SwALL incidence is almost 4 % of all BCP-ALLs (Slámová et al., 2014). DNA methylation (presence of 5-methylcytosine) is together with post-translational histone modifications and non- coding RNAs an epigenetic mechanism which regulates gene expression without changes of genetic code. DNA methylation is easily detected by bisulphite conversion and subsequent sequencing. The aim of this work was to compare genome-wide DNA methylation patterns between patients with swALL and control BCP-ALLs. The first step in achieving that was revision and improvement of bioinformatic processing protocol for eRRBS data from massive parallel sequencing. To improve the sequence adapter trimming I tested four bioinformatic tools - FAR, cutadapt, Trimmomatic and fastx_clipper. I implemented the fastest and most effective - Trimmomatic into the processing protocol. As a next step I analysed the data with improved protocol and extended the analysis in R programming environment where the comparison of studied groups was performed. The comparison of...

The t(1;19) chromosomal translocation is present in 5% of acute lymphoblastic leukemia (ALL) cases and leads to expression of the oncogenic transcription factor, E2A-PBX1. Although t(1;19) is exclusively associated with pre-B ALL in clinical cases, murine models produce myeloid or T-lymphoid leukemias, which are not representative of the clinical disease. In this work, we have advanced progress towards the development an E2A-PBX1-driven experimental leukemia model. We initially determined that lineage-negative (lin-) hematopoietic progenitors expressing E2A-PBX1 expression fail to repopulate the B-lymphoid lineage when transplanted into irradiated recipient mice. Furthermore, E2A-PBX1 expressing, lin- fetal liver progenitors (FLPs) fail to differentiate into B-lymphocytes ex vivo. The majority of E2A-PBX1-expressing FLPs manifested an immature phenotype and displayed stem cell factor (SCF)-dependency and enhanced self-renewal. Additionally, these cells retained myeloid potential upon transplantation or stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF). DNA binding was required for the differentiation block, suggesting that E2A-PBX1 target genes are incompatible with B-lineage specification. E2A-PBX1 FLPs had a stem cell like gene expression profile, including up-regulation of the leukemic transcription factors, Hoxa9 and Meis1. These findings explain why E2A-PBX1-driven bone marrow transplant models fail to generate B-lymphoid disease and suggest that future efforts in developing a model of E2A-PBX1-driven pre-B ALL leukemia should focus on expressing E2A-PBX1 subsequent to B-lymphoid commitment. In an attempt to override the B-lymphoid differentiation block, we next expressed E2A-PBX1 in primary pre-B cells. E2A-PBX1 induced an apoptotic response in pre-B cells, which was consistent with previous observations. Since pre-B ALL induction requires secondary genetic events, we attempted to abrogate these E2A-PBX1-mediated effects by modulating expression of the Cdkn2a locus. Loss of Cdkn2a through deletion or Bmi1 overexpression failed to ameliorate the apoptotic response, suggesting that E2A-PBX1 mediated apoptosis occurs independently of Cdkn2a in murine pre-B cells. However, in the absence of Cdkn2a, co-expression of constitutively active MerTK or Ras attenuated the E2A-PBX1 mediated apoptosis. Cumulatively, these results support the notion that t(1;19) occurs subsequent to B-lymphoid commitment and requires multiple secondary genetic lesions. Data presented in this thesis represents crucial initiating steps towards the development of a pre-B ALL model mediated by E2A-PBX1.
Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2013-09-03 00:09:29.299

It has been long known that many types of cancers have high metabolic requirements and use reprogrammed metabolism to support cellular activities. The first identified metabolic alteration in cancer cells was elevated glucose uptake, glycolysis activity and lactate production even in the presence of oxygen. This metabolic program, termed aerobic glycolysis or the Warburg effect, provides cells with energy as well as biosynthetic substrates to sustain cell survival and rapid cell proliferation. Cancer metabolism is closely linked to genetic mutations and oncogenic signaling pathways, such as PI3K/Akt, cMyc and HIF pathways. These oncogenic signals can direct metabolic reprogramming while changes in metabolic status can regulate activities of these signaling pathways in turn. In addition to glucose, later studies also found utilization of alternate nutrients in cancer cells, including glutamine and lipids. Glutamine is the second major metabolic fuel and can be converted to various substrates to support cell bioenergetics needs and biosynthetic reactions. Usage of metabolic fuels in cancer cells, however, is variable. While certain cancers display addiction to one type of nutrient, others are capable of using multiple nutrients.

The unique metabolic features of cancer cells raise the possibility of targeting metabolism as a novel therapeutic approach for cancer treatment. Using pharmacological inhibitors, previous research has provided corroborating evidence that metabolic stress can impact survival and growth of proliferative cancer cells by regulating cell apoptotic machinery and cell cycle checkpoints. Due to lack of genetic tools and side effects from these inhibitors, however, mechanistic understanding of cell response to metabolic inhibition was limited in these studies. More importantly, how metabolic stress affects cancer progression in a physiological condition has not yet been well investigated. Lastly, current research has not examined metabolic program in indolent cancers and the metabolic requirements and activities in less proliferative cells also remain to be understood.

This work examines nutrients utilization in B cell derived acute and chronic leukemia (B-ALL and B-CLL). B-ALL is an aggressive form of leukemia. Using cell lines and primary patient samples, we found B-ALL cells primarily used glucose through aerobic glycolysis, similar to other proliferative cancer cells. B-ALL cells were also more sensitive to inhibition of glycolysis than normal B cells. Employing an untargeted metabolomics profiling in combination with isotope labeled glucose tracing approach, we show in a B-ALL model that genetic ablation of glucose transporter Glut1 partially reduced glucose uptake, sufficiently hindered anabolic pathways and promoted catabolic metabolism. This metabolic shift led to sharply curtailed B-ALL proliferation in vitro and reduced leukemic burden in vivo. Furthermore, this partial inhibition of glucose metabolism sensitized B-ALL cells to apoptotic stimuli and non-cytotoxic metabolic inhibition significantly enhanced efficacy of a tyrosine kinase inhibitor to eliminate B-ALL cells in vitro and in vivo. Thus, partial inhibition of glucose metabolism can provide a plausible adjuvant therapy to treat cancers that depend on glycolysis for survival and proliferation.

In contrast to B-ALL, B-CLL is an indolent form of cancer. Most B-CLL cells exhibited low glucose metabolic activities that were comparable with normal B cells at resting stage. Similar to chronically stimulated and anergic B cells, these B-CLL cells also failed to upregulate glucose metabolism in response to IgM stimulation. We also observed an altered amino acid and acyl-carnitine profile and increased glutaminase mRNA in B-CLL relative to normal B cells, suggesting the capability of using alternate nutrients such as glutamine in these cells. Finally, we explored the possibility of suppressing mitochondria metabolism to induce B-CLL cell death through inhibition of the nuclear hormone receptor and metabolic regulator ERRalpha. ERRalpha is known to regulate mitochondrial metabolism and was expressed higher in B-CLL than normal B cells. ERRalpha inhibition decreased viability of oncogene transformed pro-B cells, suggesting ERRalpha as a potential target for B-CLL treatment.

Collectively, this work investigates metabolic phenotype in two forms of leukemia derived from B cells. It reveals different metabolic requirements and activities in aggressive and indolent leukemia and explores different approaches to suppress metabolism in these cancers. Findings of this work shed light on how to potentially design metabolic approach to improve cancer treatment.

Dissertation

T-ALL accounts for 15% of childhood leukemias and approximately 60% of patients overexpress TAL1. TAL1/SCL encodes a transcription factor that regulates hematopoiesis by dimerizing with additional transcription factors including E12, E47, and GATA-1. TAL1 has also been found to repress expression of NF-κB1, potentially promoting formation of an NF-κB p65/c-Rel heterodimer that encourages cell survival by up-regulating IAPs and IκB. However, the correlation between TAL1 and p65/c-Rel expression and their effects on downstream targets like IKK, IκB, and other anti-apoptotic proteins is poorly understood. Jurkat cells, expressing TAL1, were treated with TNFα and/or etoposide to induce apoptosis and experiments were performed to assess the expression of proteins of interest. Caspase-8 activity assays were also performed to help delineate the apoptotic signal present in these cells. Determining if interactions between TAL1, NF-κB, and other downstream targets help promote apoptotic resistance will further research into better, more targeted treatments for T-ALL.
Department of Biology

Tese de mestrado, Oncobiologia, Faculdade de Medicina, Universidade de Lisboa, 2015
Acute lymphoblastic leukemia (ALL) is the most frequent childhood malignancy and it is characterized by the accumulation of immature lymphoid cells within the bone marrow and lymphoid tissues. Approximately 85% of pediatric ALL patients have a B-cell phenotype (B-ALL), and, despite significant improvements in treatment outcome, around 10-20% still relapse. Thus, there is a clear need for new prognostic factors capable of accurately predicting response to therapy. PI3K/Akt/mTOR and JAK/STAT5 pathways are extensively implicated in cancer. Both cell-autonomous factors and microenvironmental cues, such as interleukin 7 (IL-7), contribute to the activation of these pathways in ALL. However, it remains to be determined whether their activation status has a prognostic value in this malignancy. In the current thesis, we proposed to tackle this issue by analyzing the phosphorylation levels of key elements of both pathways in a retrospective cohort (n=58) of pediatric B-ALL cases. Methodologically, we decided to use phospho-flow cytometry, given its potential applicability in clinical diagnostics. Overall, our results show that pediatric B-ALL samples display significant interpatient heterogeneity in the constitutive and IL-7-triggered levels of PI3K/Akt/mTOR and JAK/STAT5 pathway activation. Interestingly, we found that the response to IL-7 does not correlate with the levels of IL-7 receptor α expression. Most importantly, correlation of basal activation levels of both pathways with clinical features with known prognostic value revealed that higher constitutive levels of phosphorylation of S6 on S235/236 and Akt on S473, but not on T308, are associated with higher white blood cell counts. These results suggest the existence of two independent mechanisms leading to Akt activation in ALL, with different biological outcomes. Overall, our preliminary results suggest that there is a positive association of high Akt S473 and S6 S235/236 phosphorylation levels with high risk, which is often associated with a poor prognosis
A leucemia linfoblástica aguda (LLA) é o cancro mais frequente em crianças, apresentando um pico de incidência entre os 2 e os 5 anos de idade. Esta doença caracteriza-se pela expansão clonal descontrolada e consequente acumulação de linfócitos imaturos na medula óssea, com posterior infiltração de outros órgãos. O subtipo mais comum de LLA é a leucemia linfoblástica aguda de células B (LLA-B), constituindo cerca de 85% dos casos pediátricos e 75% dos casos adultos. Os tratamentos actuais apresentam uma elevada eficácia e aproximadamente 80% dos doentes pediátricos apresentam-se livres de doença 5 anos após o início do tratamento. Contudo, cerca de 10-20% dos doentes sofrem recidivas, frequentemente associadas a complicações a longo prazo, resultantes da elevada toxicidade dos tratamentos. Existem vários factores de prognóstico em LLA pediátrica essenciais para definir o tratamento mais adequado dos doentes, incluindo idade, contagem de leucócitos na fase de diagnóstico e presença de anomalias citogenéticas (trissomia 21 ou cromossoma de Filadélfia). Um factor de grande importância para a progressão da doença é a activação de vias de transdução de sinal fundamentais. Sabe-se, por exemplo, que mutações em elementos destas vias podem afectar a resposta dos doentes ao tratamento. No entanto, e apesar da contribuição destas vias para o desenvolvimento de LLA-B, o seu valor prognóstico não é conhecido. Importa salientar que a caracterização da activação das vias de transdução de sinal à data do diagnóstico poderá auxiliar na escolha de terapias mais específicas, com consequente aumento da eficácia e diminuição da toxicidade do tratamento. As vias de sinalização PI3K/Akt/mTOR and JAK/STAT5 têm sido amplamente implicadas em cancro de um modo geral e, em particular, em LLA. A via PI3K/Akt/mTOR encontra-se constitutivamente hiperactivada em doentes pediátricos com leucemia linfoblástica aguda de células T (LLA-T), promovendo a viabilidade das células leucémicas. Foi também demonstrado que esta via é activada pela citocina IL-7 (que se encontra presente no microambiente tumoral), modulando a resistência das células leucémicas face à quimioterapia. A corroborar este facto, diferentes estudos indicam que a citocina IL-7 é capaz de modular, tanto in vitro como in vivo, a resposta das células de LLA-B a inibidores farmacológicos de mTOR (Rapamicina). Existe igualmente evidência a nível genético que apoia um possível valor prognóstico para esta via em LLA. Vários estudos realizados em LLA-T mostram que mutações ou delecções que levam à inactivação do principal regulador negativo da via, o supressor tumoral PTEN, estão associadas a um pior prognóstico. Este regulador pode ainda estar sujeito a inactivação pós-tradução, um processo bastante frequente tanto em LLA-T como em LLA-B. Tal como a via PI3K/Akt/mTOR, a via de sinalização JAK/STAT5 é activada em resposta a estimulação com IL-7, ou quando o receptor desta citocina, IL-7R, se encontra constitutivamente activado devido a mutações. O principal papel desta via no desenvolvimento de LLA-B tem sido maioritariamente demonstrado pela activação constitutiva do factor de transcrição STAT5 a jusante da translocação BCR-ABL. Doentes com esta translocação, conhecidos como Filadélfia-positivos, apresentavam outrora muito mau prognóstico, uma situação resolvida com a inclusão no tratamento de terapias direccionadas especificamente para BCR-ABL, entre as quais o Imatinib foi o primeiro exemplo. Tendo em conta as razões acima descritas, o principal objectivo desta tese é determinar, pela primeira vez, se o estado de activação das vias de sinalização PI3K/Akt/mTOR e JAK/STAT5 tem valor prognóstico em LLA-B pediátrica. Para responder a esta questão, avaliaram-se os níveis de fosforilação de elementos chave de cada uma das vias de transdução de sinal num grupo retrospectivo (n=58) de casos pediátricos de LLA-B provenientes do Departamento de Pediatria do Instituto Português de Oncologia de Lisboa Francisco Gentil (IPOLFG). Para determinar o estado de activação de PI3K/Akt/mTOR analisaram-se os níveis de fosforilação de Akt e de S6, um alvo de mTOR; quanto à segunda via, JAK/STAT5, avaliou-se o nível de fosforilação de STAT5. Estes níveis foram medidos tanto basalmente como após estimulação com IL-7, utilizando citometria de fluxo (phosphoflow cytometry). Posteriormente, e uma vez que dispomos dos dados clínicos de todos os doentes utilizados neste estudo, correlacionaram-se os valores de fosforilação obtidos com os parâmetros clínicos com valor prognóstico, tais como idade, contagem de leucócitos à data do diagnóstico e doença residual mínima após a terapia de indução. Correlacionaramse, também, com o estado de maturação de LLA-B (classificação de EGIL), com o objectivo de melhor compreender a biologia da doença. Adicionalmente a esta análise molecular, procedeu-se a uma análise funcional onde se avaliou a sensibilidade de cada amostra primária à citocina IL-7, medindo parâmetros como a viabilidade e a proliferação das células primárias em resposta à IL-7. Mediram-se, ainda, os níveis de expressão da subunidade α do IL-7R (IL-7Rα) nestas amostras, com o intuito de os correlacionar tanto com os resultados moleculares como com os funcionais. É importante referir que a metodologia phospho-flow cytometry foi seleccionada tendo por base a enorme quantidade de informação passível de ser obtida através da análise de uma única célula, e também por facilmente poder ser introduzida como técnica de diagnóstico em contexto clínico num futuro próximo. Na verdade, a técnica de citometria de fluxo já é actualmente utilizada na clínica para proceder à sub- lassificação dos doentes com LLA com base no imunofenótipo das células. Tendo em conta os nossos resultados, demonstrou-se que as amostras pediátricas de LLA-B são bastante heterogéneas no que diz respeito aos níveis de activação constitutiva das vias de transdução de sinal PI3K/Akt/mTOR e JAK/STAT5. Verificou-se, também, que a estimulação com IL-7 induz um aumento de activação de ambas as vias, embora com grande variabilidade entre as amostras. Quanto à análise funcional, e em concordância com o que já se tinha observado, a maioria das amostras primárias é sensível à estimulação com IL-7, traduzindo-se num aumento de viabilidade e proliferação celular. No que diz respeito aos níveis de expressão do IL-7Rα, também eles bastante variáveis, verificou-se que os mesmos não se correlacionam com os resultados moleculares e/ou funcionais. Isto é, níveis elevados de expressão do receptor não se traduzem necessariamente em maior activação das vias após estimulação com IL-7, nem num maior aumento de viabilidade ou proliferação celular. Para terminar, procedeu-se à correlação dos níveis de activação de ambas as vias de sinalização, tanto basais como após estimulação com IL-7, com as características clínicas anteriormente mencionadas. Não se encontrou nenhuma correlação significativa quando os níveis de activação foram comparados com a idade, o estado de maturação ou a doença residual mínima. Curiosamente, níveis basais elevados de fosforilação de S6 nas serinas 235 e 236 e de Akt na serina 473 (mas não na treonina 308) correlacionam-se com níveis elevados de leucócitos no diagnóstico que, por sua vez, estão associados a um risco elevado. Compararam-se, também, os níveis de expressão do IL-7Rα com os mesmos parâmetros clínicos e, embora não se tenha encontrado nenhuma associação significativa, existe uma tendência para níveis elevados de expressão em crianças com idade igual ou superior a 10 anos, normalmente associada a um pior prognóstico. Concluindo, estes resultados, embora preliminares, parecem sugerir uma possível associação entre níveis elevados de fosforilação de S6 (serinas 235 e 236) e Akt (serina 473) e risco elevado, que se encontra normalmente associado a um mau prognóstico. O facto de esta correlação apenas abranger a fosforilação de Akt na serina 473, e não a na treonina 308, aponta para possível existência de dois mecanismos de activação de Akt em LLA, afectando diferencialmente os dois resíduos com consequências biológicas distintas. É nossa intenção repetir as análises realizadas neste estudo num maior número de amostras primárias, com o objectivo de validar as conclusões apresentadas nesta tese.