Chronic Lymphocytic Leukaemia (CLL) is a disease of B-lymphocytes that account for significant morbidity and mortality in mostly elderly patients (aged ≥ 70 years). The relative survival of patients with CLL has been shown to decrease with patient age, and this age-related reduction in survival was found to correlate with the levels of two inflammatory cytokine levels in the patients’ plasma. The levels of two inflammatory cytokines, interleukin-6 and -8 (IL-6, IL-8) were found to correlate positively with patient age, and increased levels were associated with lower overall survival. Addition of IL-6 or IL-8 to a co-culture system of CLL cells with bone marrow stromal cells increased the CLL-stromal cell adhesion, and co-culturing increased IL-8 secretion. In a search of a treatment regimen that may be effective and readily tolerated by elderly patients, we examined the combination of fludarabine with valproic acid (VPA), an epileptic that was found to inhibit histone deacetylases (HDACs). The combination was synergistic against human leukaemic cells, including primary CLL cells. In a phase II clinical trial where six elderly patients with relapsed, previously treated CLL were enrolled (half of whom were clinically refractory to fludarabine), the VPAfludarabine combination induced reduction in the peripheral and lymph node tumour loads. Mechanistically, the fludarabine treatment induced disruption of the lysosomes, while VPA induced increase in the level and activity of cathepsin B, a lysosomal protease. The VPA-induced increase in cathepsin B levels was observed in in cell lines (in vitro), primary CLL cells (ex vivo) and in patients treated with VPA (in vivo). Chemical inhibition of cathepsin B was sufficient to dampen the VPA-fludarabine cytotoxicity, and the addition activated cathepsin B to leukaemic cell lysates was sufficient to induce caspase cleavage and reduction in anti-apoptotic protein levels. The VPA-fludarabine combination also lowered phospho-Akt levels and ATM activation, which also contributed to the VPA-fludarabine synergy, and VPA treatment lowered ATM levels and phospho-Akt levels in vivo. In summary, there lies a biological explanation for the poor survival observed with elderly patients, and the VPA-fludarabine may be a useful regimen for these patients.

Chronic Lymphocytic Leukaemia (CLL) is a disease of B-lymphocytes that account for significant morbidity and mortality in mostly elderly patients (aged ≥ 70 years). The relative survival of patients with CLL has been shown to decrease with patient age, and this age-related reduction in survival was found to correlate with the levels of two inflammatory cytokine levels in the patients’ plasma. The levels of two inflammatory cytokines, interleukin-6 and -8 (IL-6, IL-8) were found to correlate positively with patient age, and increased levels were associated with lower overall survival. Addition of IL-6 or IL-8 to a co-culture system of CLL cells with bone marrow stromal cells increased the CLL-stromal cell adhesion, and co-culturing increased IL-8 secretion. In a search of a treatment regimen that may be effective and readily tolerated by elderly patients, we examined the combination of fludarabine with valproic acid (VPA), an epileptic that was found to inhibit histone deacetylases (HDACs). The combination was synergistic against human leukaemic cells, including primary CLL cells. In a phase II clinical trial where six elderly patients with relapsed, previously treated CLL were enrolled (half of whom were clinically refractory to fludarabine), the VPAfludarabine combination induced reduction in the peripheral and lymph node tumour loads. Mechanistically, the fludarabine treatment induced disruption of the lysosomes, while VPA induced increase in the level and activity of cathepsin B, a lysosomal protease. The VPA-induced increase in cathepsin B levels was observed in in cell lines (in vitro), primary CLL cells (ex vivo) and in patients treated with VPA (in vivo). Chemical inhibition of cathepsin B was sufficient to dampen the VPA-fludarabine cytotoxicity, and the addition activated cathepsin B to leukaemic cell lysates was sufficient to induce caspase cleavage and reduction in anti-apoptotic protein levels. The VPA-fludarabine combination also lowered phospho-Akt levels and ATM activation, which also contributed to the VPA-fludarabine synergy, and VPA treatment lowered ATM levels and phospho-Akt levels in vivo. In summary, there lies a biological explanation for the poor survival observed with elderly patients, and the VPA-fludarabine may be a useful regimen for these patients.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17820号 / 医博第3818号 / 新制||医||999(附属図書館) / 30635 / 京都大学大学院医学研究科医学専攻 / (主査)教授 富樫 かおり, 教授 伊藤 壽一, 教授 楠見 明弘 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18152号 / 医博第3872号 / 新制||医||1002(附属図書館) / 31010 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 武藤 学, 教授 戸井 雅和 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Human cells possess multiple nucleoside transporters (NTs) that belong to either the human equilibrative or concentrative NT (hENT: hENT1/2/3/4; hCNT: CNT1/2/3) families. In the kidney, coupling of apical hCNT3 activities to basolateral hENT1/2 activities is hypothesized to mediate renal nucleoside proximal tubular absorption while apical ENT1 may have a role in secretion. The overall aim of this research was to increase understanding of the roles of hENTs and hCNTs in renal handling of physiological nucleosides and anti-cancer nucleoside analog drugs. This was achieved by investigating the distribution of hENTs and hCNTs in human kidney tissue and the function of hENTs and hCNTs in cellular uptake and transepithelial fluxes of nucleosides in cultured human renal proximal tubule cells (hRPTCs). Immunolocalization of hCNT3 and hENT1 in human kidney tissue revealed that hENT and hCNT3 were present in apical membranes of proximal tubules. Production and characterization of adherent hRPTC cultures demonstrated endogenous hCNT3, hENT1, and hENT2 activities. These results provided evidence for the involvement of hCNT3, hENT1, and hENT2 in renal handling of nucleosides. Comparison of adherent hRPTC cultures derived from kidneys from different individuals demonstrated that hCNT3 activities varied between cultures. Also, the extent of cellular uptake of fludarabine, an anti-cancer nucleoside drug, and degree of cytotoxicity was reflected in the different hCNT3 activities observed between cultures. These results suggested that hCNT3 plays an important role in fludarabine renal handling and is a determinant of potential renal toxicities. Production of polarized monolayer cultures of hRPTCs on transwell permeable inserts enabled the functional localization of hCNT3 and hENT1 to apical membranes and hENT2 to basolateral membranes. Transepithelial flux studies demonstrated that (i) apical-to-basolateral fluxes of adenosine were mediated by apical hCNT3 and basolateral hENT2, (ii) basolateral-to-apical fluxes of 2′-deoxyadenosine were mediated, in part, by apical hENT1 and basolateral hOATs, and (iii) apical-to-basolateral fluxes of fludarabine, cladribine, and clofarabine were mediated by apical hCNT3. These studies showed that coupling of apical hCNT3 to basolateral hENT2 mediates proximal tubular nucleoside reabsorption, that coupling of basolateral human organic anion transporters (hOATs) to apical hENT1 mediates proximal tubular nucleoside secretion, and that hCNT3 is a key determinant of fludarabine proximal tubular reabsorption and cytoxicity.

Il existe plusieurs défis au développement d’une thérapie visant à stimuler l’immunité cellulaire. Dans la prévention contre certains virus et en immunothérapie du cancer, l’induction de lymphocytes T spécifiques est cependant primordiale. Dans la première partie de l’étude, nous avons porté notre attention sur la compréhension de la présentation croisée par le complexe majeur d’histocompatibilité de classe I (CMH I) médiée par des particules pseudo-virales (VLP) composées de la protéine de surface de potexvirus à laquelle nous avons ajouté un épitope de la protéine M1 du virus de l’influenza ou un épitope de la protéine gp100 du mélanome. Cette VLP se caractérise par sa capacité à stimuler, sans l’aide d’adjuvant, le système immunitaire et de présenter de façon croisée l’épitope inséré dans sa protéine de surface et ce, indépendamment de l’activité du protéasome. Nous avons, tout d’abord, comparé les propriétés de présentation antigénique croisée des VLP formées du virus de la mosaïque de la malva (MaMV) à celles des VLP du virus de la mosaïque de la papaye (PapMV). Les résultats confirment que ces propriétés sont partagées par plusieurs membres de la famille des potexvirus malgré des divergences de séquences (Hanafi et al. Vaccine 2010). De plus, nous avons procédé à des expériences pour préciser le mécanisme menant à la présentation de l’épitope inséré dans les VLP de PapMV. Les résultats nous confirment une voie vacuolaire dépendante de l’activité de la cathepsine S et de l’acidification des lysosomes pour l’apprêtement antigénique. L’induction de l’autophagie par les VLP semble également nécessaire à la présentation croisée par les VLP de PapMV. Nous avons donc établi un nouveau mécanisme de présentation croisée vacuolaire dépendant de l’autophagie (Hanafi et al. soumis Autophagy). En second lieu, en immunothérapie du cancer, il est aussi important de contrôler les mécanismes d’évasion immunitaire mis en branle par la tumeur. Nous avons spécifiquement étudié l’enzyme immunosuppressive indoleamine 2,3-dioxygénase (IDO) (revue de la littérature dans les tumeurs humaines; Hanafi et al. Clin. Can. Res 2011) et son inhibition dans les cellules tumorales. Pour ce faire, nous avons tenté d’inhiber son expression par la fludarabine, agent chimiothérapeutique précédemment étudié pour son activité inhibitrice de l’activation de STAT1 (signal transducers and activators of transcription 1). Étonnamment, nos résultats ont montré l’inhibition d’IDO dans les cellules tumorales par la fludarabine, indépendamment de l’inhibition de la phosphorylation de STAT1. Nous avons démontré que le mécanisme d’action dépendait plutôt de l’induction de la dégradation d’IDO par le protéasome (Hanafi et al. PlosOne 2014). Les travaux présentés dans cette thèse ont donc portés autant sur la compréhension d’une nouvelle plateforme de vaccination pouvant médier l’activation de lymphocytes T CD8+ cytotoxiques et sur le contrôle d’une immunosuppression établie par les cellules tumorales pour évader au système immunitaire. Ces deux grandes stratégies sont à considérer en immunothérapie du cancer et la combinaison avec d’autres thérapies déjà existantes pourra permettre une meilleure réponse clinique. / There are several challenges to the development of therapies aimed at stimulating cellular immunity. In viral infection prevention and in cancer immunotherapy, the induction of specific T lymphocytes is, however, of paramount importance. In the first part of this study, we focused our attention on understanding the major histocompatibility complex class I (MHC I) cross-presentation mediated by virus-like particles (VLP) composed of potexvirus coat protein, in which we had inserted an epitope from the M1 protein of the Influenza virus or an epitope from gp100, a tumour antigen of melanoma. This particular VLP is characterized by its ability to stimulate the immune system with no adjuvant and its cross-presentation of the inserted epitope independently of proteasome activity. First, we compared the antigenic cross-presentation properties of Malva mosaic virus (MaMV) VLPs to that of Papaya mosaic virus (PapMV) VLPs. The results confirm that cross-presentation mechanisms are shared among different members of the potexvirus family despite marked differences in their sequences (Hanafi et al. Vaccine 2010). Furthermore, we have conducted experiments to clarify the mechanism leading to the cross-presentation of the inserted epitope in PapMV VLPs. The results confirm a vacuolar pathway dependent on cathepsin S activity and on lysosomal acidification for antigen presentation. Autophagy induction by VLPs is also important to PapMV VLP antigen cross-presentation. We have herein described a new vacuolar MHC I cross-presentation pathway dependent on autophagy (Hanafi et al. in preparation). Secondly, in cancer immunotherapy, it is crucial to control immune evasion mechanisms that are initiated by the tumour. We have specifically studied the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) (human cancer literature review in Hanafi et al. Clin. Can. Res. 2011), and its inhibition in tumour cells. To this end, we inhibited its expression using fludarabine, a chemotherapeutic agent previously studied for its inhibitory effect on STAT1 (signal transducers and activators of transcription 1) phosphorylation. Surprisingly, our results demonstrate that IDO inhibition in cancer cells by fludarabine was independent of STAT1 phosphorylation. We showed that the mechanism of action was rather dependent on the induction of IDO degradation by the proteasome (Hanafi et al. PlosOne 2014). The work presented in this thesis provides a better understanding of how a new vaccine platform can mediate cytotoxic CD8+ T lymphocytes activation and the control of the problem of immunosuppression by tumour cells for the evading of the immune system. These two main strategies are to key for the consideration of cancer immunotherapy in combination with other existing therapies, as these should allow a better clinical response to cancer treatment.