T-cell prolymphocytic leukemia (T-PLL) is the most common mature T-cell leukemia. It is a typically aggressively growing and chemotherapy-resistant malignancy with a poor prognosis. T-PLL cells resemble activated, post-thymic T-lymphocytes with memorytype effector functions. Constitutive transcriptional activation of genes of the T-cell leukemia 1 (TCL1) family based on genomic inversions/translocations is recognized as a key event in T-PLL’s pathogenesis. TCL1’s multiple effector pathways include the enhancement of T-cell receptor (TCR) signals. New molecular dependencies around responses to DNA damage, including repair and apoptosis regulation, as well as alterations of cytokine and non-TCR activation signaling were identified as perturbed hallmark pathways within the past years. We currently witness these vulnerabilities to be interrogated in first pre-clinical concepts and initial clinical testing in relapsed/refractory TPLL patients. We summarize here the current knowledge on the molecular understanding of T-PLL’s pathobiology and critically assess the true translational progress around this to help appraisal by caregivers and patients. Overall, the contemporary concepts on T-PLL’s pathobiology are condensed in a comprehensive mechanistic disease model and promising interventional strategies derived from it are highlighted.

Throughout society’s continual war against cancer, we have attempted pharmacological intervention only to find that tumors develop modes of resistance. It is well known that genetics play an integral role in cancer. Technological advances have greatly improved our ability to study cancer biochemistry beyond the genome by measuring changes in the expression and activity of RNA, proteins, and lipids in experimental models and human patients. As our techniques and technology to perform cancer research progresses, it is becoming more evident that cancer cells develop stress tolerance mechanisms at multiple levels within the central dogma, including altering mRNA expression, enzyme concentrations, and functional activity of cellular proteins and lipids. In the first chapter, I review previous discoveries demonstrating the importance of metabolic reprogramming in cancer cells and how shifts in metabolic pathways contribute to cancer progression and therapeutic challenges. I discuss how mass spectrometry is a multifunctional research tool that can be used to identify global shifts in gene expression, identify oncogenic roles of specific metabolites and corresponding metabolic pathways, conduct enzyme activity assays, and understand the effects of drugs on cell signaling and metabolic flux through specific pathways. While metabolic reprogramming is a complex and multifaceted concept, the following chapters focus on two specific stress tolerance pathways of lipid and protein metabolism we have shown to significantly promote cancer cell evolution, proliferation, and drug resistance in models of human pancreatic and colon cancer. I describe novel mass spectrometry-based lipidomics and proteomics methods we developed to measure and determine the biological impact of these pathways in each model. I discuss the contributions we have made toward increasing general knowledge of metabolic reprogramming networks in cancer and how they may be targeted in more specific and effective manners to sensitize cancers to therapeutic drugs. Specifically, the second chapter entails our study of a pro-survival lipid metabolic pathway driven by the sphingolipid modifying enzyme sphingosine kinase in a panel of differentially reprogrammed pancreatic cancer subclones. The third chapter describes our novel kinetic proteomics approach to identify how the cellular degradation system autophagy is used to selectively remodel the proteome of colon tumor cells in a xenograft mouse model of colon cancer. Lastly, I discuss how these and other projects completed during my graduate work lay a foundation for ongoing research to further our fundamental understanding of cancer metabolism and treatment development.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20989号 / 医博第4335号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 朝長 啓造, 教授 松田 文彦, 教授 小柳 義夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Clonal evolution is a multistep process characterized by progression of the disease, adverse prognosis and shortening of overall survival. The aim of the dissertation was a detailed characterization of identified changes in patients with myelodysplastic syndromes (MDS) and clonal evolution and evaluation of their prognostic impact. We performed detail cytogenomic analyses in 36/469 (8%) patients with confirmed linear clonal evolution. We described 57 primary abnormalities (32% MDS-specific) at the time of diagnosis, the most frequent was deletion of long arm of chromosome 5. We proved 156 secondary aberrations (21% MDS-specific) during the course of the clonal evolution, the most frequent were trisomies/tetrasomies of chromosome 8. We identified acquired uniparental disomies (aUPD) in 19% of patients. In MDS-specific aUPDs 4q, 11q and 17p, we proved homozygous mutations of TET2, c-CBL and TP53 genes. We found a statistically significant difference in overall survival between the groups of patients divided according to their diagnostic cytogenomic findings. In patients with clonal evolution before treatment 54% of aberrations were gains of whole chromosomes, by contrast 44% of abnormalities identified in patients with clonal evolution after treatment were monosomies or deletions. The study of clonal...

A anemia aplástica severa (AAS) pode ser tratada com sucesso na maioria dos casos com terapia imunossupressora (IS) ou transplante alogenêico de medula óssea (TMO). Os principais fatores que determinam a escolha da modalidade terapêutica são a idade e a disponibilidade de um doador HLA-histocompatível. Em pacientes mais jovens, o TMO de um doador aparentado é preferível, enquanto que em pacientes acima de 40-50 anos, a terapia IS é a modalidade terapêutica de escolha. Resposta hematológica é obtida em 60-75% dos casos com terapia IS na AAS, o que correlaciona com melhor sobrevida. Recidivas ocorrem em aproximadamente um terço dos respondentes e evolução clonal para mielodisplasia em 10-15% ao longo termo. A doença do enxerto-versus-hospedeiro (GVHD) agudo ocorre em 30-40% dos casos sendo a forma crônica presente em 40-50%. Infecções são frequentes e podem complicar o transplante. Portanto, a refratariedade à terapia IS, recidivas e evolução clonal limitam o sucesso da terapia IS na AAS, enquanto rejeição do enxerto, GVHD, e infecções limitam o sucesso do TMO na clínica. Fatores preditivos dessas complicações seriam de grande valor na clínica, uma vez que poder-se-iam realizar decisões terapêuticas com base mais racional, onde pacientes fossem alocados a diferentes tratamentos com base no seu perfil de risco. Ou seja, pacientes com alta probabilidade de resposta e baixo risco de recidiva e evolução clonal se beneficiariam de terapia IS, enquanto àqueles com baixa probabilidade de resposta e alto risco de recidiva e/ou evolução clonal teriam mais benefícios do TMO, por exemplo. Com base nessa premissa, desenvolvemos estudos para investigar fatores que pudessem estar associados ao sucesso da terapia IS na AAS. Os principais achados de 3 análises distintas sobre o tema evidenciou: 1) crianças (< 18 anos) apresentam alta taxa de resposta à terapia IS (em torno de 75%) com uma excelente sobrevida geral em pacientes respondentes; 2) o número absoluto de reticulócitos e de linfócitos pré-tratamento correlaciona com resposta hematológica aos seis meses após terapia IS; e 3) o comprimento telomérico não está associado à resposta hematológica, porém, está associado a probabilidade de recidiva, evolução clonal, e sobrevida geral após terapia IS. Esses parâmetros identificados nesses estudos podem servir de base em algoritmos futuros onde faz-se estratificação de risco de cada paciente, a fim de alocar a modalidade terapêutica mais apropriada com base no perfil individual de risco. No que diz respeito ao comprimento telomérico, é provável que esse marcador biológico não só esteja associado ao processo de evolução clonal na AAS, mas que também participe na biologia da instabilidade genômica de células na medula óssea levando a aberrações cromossômicas e o desenvolvimento de mielodisplasia e leucemias. / Severe aplastic anemia (SAA) can be treated successfully in the majority of cases with immunosuppressive therapy (IST) or allogeneic bone marrow transplantation (BMT). The principal factors that determine the choice of treatment modalities are age and availability of an HLA-histocompatible donor. In younger patients, BMT from a related donor is preferred, while in patients over 40-50 years of age, IST is often employed. Hematologic response is achieved in 60-75% of cases with IST, which correlates with better survival. Relapses occur in approximately one third of responders and clonal evolution to myelodysplasia occurs in 10-15% of cases long-term. Acute graft-versus-host disease (GVHD) occurs in 30-40% of cases and chronic GVHD in 40-50%. Infections are common and complicate transplant outcomes. Therefore, refractoriness, relapses and clonal evolution limit the success of IST in SAA, while graft rejection, GVHD, and infections limited the success of BMT in the clinic. Predictors for these complications would be of great value in the clinic since one could make more rational treatment decisions where patients were allocated to different treatment modalities based on their risk profile. For example, patients with high probability of response and low risk of relapse and clonal evolution would benefit more from IST, while those with low probability of hematologic response and high risk of recurrence and/or clonal evolution most likely to benefit from BMT. Based on this premise, we developed studies to investigate factors that could be associated with the success of IST in SAA. The main findings of three separate analysis on the subject showed: 1) children ( < 18 years) have a high response rate to IST (around 75%) with an excellent long-term survival rate among responders; 2) the absolute number of reticulocytes and lymphocytes pre-treatment correlates with hematologic response at 6 months after IST, and 3) telomere length is not associated with hematologic response, but, associated with the likelihood of relapse, clonal evolution, and overall survival after IST. These parameters may serve as a basis for future algorithms allowing for risk stratification for each individual patient allowing for better treatment allocation. With respect to the telomere length, it is likely that it not only represents a biological marker but that it is involved in the process of clonal evolution contributing to genomic instability in bone marrow cells leading to the development of myelodysplasia and leukemia

A anemia aplástica severa (AAS) pode ser tratada com sucesso na maioria dos casos com terapia imunossupressora (IS) ou transplante alogenêico de medula óssea (TMO). Os principais fatores que determinam a escolha da modalidade terapêutica são a idade e a disponibilidade de um doador HLA-histocompatível. Em pacientes mais jovens, o TMO de um doador aparentado é preferível, enquanto que em pacientes acima de 40-50 anos, a terapia IS é a modalidade terapêutica de escolha. Resposta hematológica é obtida em 60-75% dos casos com terapia IS na AAS, o que correlaciona com melhor sobrevida. Recidivas ocorrem em aproximadamente um terço dos respondentes e evolução clonal para mielodisplasia em 10-15% ao longo termo. A doença do enxerto-versus-hospedeiro (GVHD) agudo ocorre em 30-40% dos casos sendo a forma crônica presente em 40-50%. Infecções são frequentes e podem complicar o transplante. Portanto, a refratariedade à terapia IS, recidivas e evolução clonal limitam o sucesso da terapia IS na AAS, enquanto rejeição do enxerto, GVHD, e infecções limitam o sucesso do TMO na clínica. Fatores preditivos dessas complicações seriam de grande valor na clínica, uma vez que poder-se-iam realizar decisões terapêuticas com base mais racional, onde pacientes fossem alocados a diferentes tratamentos com base no seu perfil de risco. Ou seja, pacientes com alta probabilidade de resposta e baixo risco de recidiva e evolução clonal se beneficiariam de terapia IS, enquanto àqueles com baixa probabilidade de resposta e alto risco de recidiva e/ou evolução clonal teriam mais benefícios do TMO, por exemplo. Com base nessa premissa, desenvolvemos estudos para investigar fatores que pudessem estar associados ao sucesso da terapia IS na AAS. Os principais achados de 3 análises distintas sobre o tema evidenciou: 1) crianças (< 18 anos) apresentam alta taxa de resposta à terapia IS (em torno de 75%) com uma excelente sobrevida geral em pacientes respondentes; 2) o número absoluto de reticulócitos e de linfócitos pré-tratamento correlaciona com resposta hematológica aos seis meses após terapia IS; e 3) o comprimento telomérico não está associado à resposta hematológica, porém, está associado a probabilidade de recidiva, evolução clonal, e sobrevida geral após terapia IS. Esses parâmetros identificados nesses estudos podem servir de base em algoritmos futuros onde faz-se estratificação de risco de cada paciente, a fim de alocar a modalidade terapêutica mais apropriada com base no perfil individual de risco. No que diz respeito ao comprimento telomérico, é provável que esse marcador biológico não só esteja associado ao processo de evolução clonal na AAS, mas que também participe na biologia da instabilidade genômica de células na medula óssea levando a aberrações cromossômicas e o desenvolvimento de mielodisplasia e leucemias. / Severe aplastic anemia (SAA) can be treated successfully in the majority of cases with immunosuppressive therapy (IST) or allogeneic bone marrow transplantation (BMT). The principal factors that determine the choice of treatment modalities are age and availability of an HLA-histocompatible donor. In younger patients, BMT from a related donor is preferred, while in patients over 40-50 years of age, IST is often employed. Hematologic response is achieved in 60-75% of cases with IST, which correlates with better survival. Relapses occur in approximately one third of responders and clonal evolution to myelodysplasia occurs in 10-15% of cases long-term. Acute graft-versus-host disease (GVHD) occurs in 30-40% of cases and chronic GVHD in 40-50%. Infections are common and complicate transplant outcomes. Therefore, refractoriness, relapses and clonal evolution limit the success of IST in SAA, while graft rejection, GVHD, and infections limited the success of BMT in the clinic. Predictors for these complications would be of great value in the clinic since one could make more rational treatment decisions where patients were allocated to different treatment modalities based on their risk profile. For example, patients with high probability of response and low risk of relapse and clonal evolution would benefit more from IST, while those with low probability of hematologic response and high risk of recurrence and/or clonal evolution most likely to benefit from BMT. Based on this premise, we developed studies to investigate factors that could be associated with the success of IST in SAA. The main findings of three separate analysis on the subject showed: 1) children ( < 18 years) have a high response rate to IST (around 75%) with an excellent long-term survival rate among responders; 2) the absolute number of reticulocytes and lymphocytes pre-treatment correlates with hematologic response at 6 months after IST, and 3) telomere length is not associated with hematologic response, but, associated with the likelihood of relapse, clonal evolution, and overall survival after IST. These parameters may serve as a basis for future algorithms allowing for risk stratification for each individual patient allowing for better treatment allocation. With respect to the telomere length, it is likely that it not only represents a biological marker but that it is involved in the process of clonal evolution contributing to genomic instability in bone marrow cells leading to the development of myelodysplasia and leukemia

La protéine p53 (« Gardien du génome ») doit être altérée pour que le cancer puisse se développer. Les nombreuses thérapies anti-cancéreuses disponibles sont très efficaces mais la réponse clinique est souvent transitoire et les cancers disséminés rechutent ou progressent du fait de l'évolution de sous-populations cancéreuses résistantes au traitement, impliquant souvent TP53 qui est le gène le plus muté dans les formes agressives de nombreux cancers. Nous l’avons étudié dans la leucémie lymphoïde chronique (LLC) et les syndromes myélodysplasiques avec délétion 5q (SMD del(5q)). Grâce à l’étude rétrospective longitudinale de 40 patients atteints de SMD del(5q), nous avons généré des données de NGS ciblé et montré que le statut mutationnel de TP53 au diagnostic ne permettait pas de prédire la progression tumorale, contrairement à ce qui avait été publié précédemment (Jädersten et al., JCO 2011). Nous avons montré que c’était l’évolution clonale du gène TP53 qui était l’élément clé de la progression des SMD del(5q). Nous avons observé de nombreuses émergences de clones mutés entre le stade diagnostique et un stade ultérieur de la maladie, toujours après initiation du traitement par lénalidomide.Le lénalidomide a été approuvé comme nouveau traitement spécifique et très efficace contre l’anémie liée aux SMD del(5q), permettant à la plupart des patients d’être indépendants des transfusions sanguines. Le lénalidomide permet souvent d’éradiquer le clone tumoral porteur de l’anomalie génétique del(5q) isolée, induisant une rémission clinique. Malheureusement, cette rémission est courte avec une durée médiane de 2 ans, puis, dans environ 1 cas sur 2, survient une transformation en leucémie aiguë secondaire de pronostic péjoratif.Nous avons étudié un possible lien entre le traitement par lénalidomide et l’évolution clonale de TP53 par annotation clinico-bio-thérapeutiques des résultats de séquençage de TP53 chez les 24 patients dont les échantillons séquentiels avaient été analysés. Dans notre étude, les patients avec progression tumorale (dont 10 évolutions clonales de TP53 et 1 évolution clonale de RUNX1) avaient reçu une dose cumulée de lénalidomide supérieure à celle reçue par les patients dont la tumeur était restée stable (p = 0.036). Nous avons observé chez plusieurs patients que l’éradication de la tumeur n’était pas utile à l’amélioration de la qualité de vie des patients. La non-éradication semblait même permettre un maintien de l’équilibre clonal et une compétition entre les différents sous-clones de la tumeur, résistants ou non au lénalidomide.Nous discutons de l’évolution de l'écologie de la tumeur au cours du traitement, i.e., l’évolution de ses interactions avec son micro-environnement qui se modifie après chaque nouvelle dose de traitement. Un modèle évolutif dit théorie de la thérapie adaptative, développée récemment remet en question les protocoles conventionnels de thérapie anti-cancéreuse qui préconisent souvent d'administrer la dose maximale tolérée par le patient (Gatenby, 2009). Elle suggère que la dose minimale efficace présenterait l’avantage de ne pas éradiquer les cellules cancéreuses sensibles au traitement pour qu'elles restent en compétition avec les cellules cancéreuses résistantes et limiter la progression ou la rechute. Nous suggérons de prendre en compte également la diminution des effets indésirables pour le patient, améliorant ainsi sa qualité de vie, et enfin la diminution des dépenses de santé pour la collectivité. A ce jour, peu d’études cliniques évaluent l’intérêt de l’adoption de tels protocoles de thérapie adaptative.Néanmoins, des modèles in vivo (xénogreffes) et in silico (modèles statistiques) ont permis d’analyser la dynamique évolutive des populations tumorales en fonction du traitement reçu. Ces modèles prédisent que la survie de l’hôte peut être maximisée par la mise en place d’une thérapie adaptative. / P53 protein is named «guardian of the genome » because it must be altered to let cancer grow.TP53 is the most mutated gene in agressive cancers.Numerous systemic therapies are successful for treatment of disseminated cancers. However, clinical response is often transient, and cancer undergo relapse or progression due to emergence of resistant populations. These latter often harbour TP53 mutations. We studied TP53 in chronic lymphoid leukemia (CLL) and lower-risk myelodysplastic syndroms with del(5q), MDS del(5q). We conducted a retrospective longitudinal study in 40 patients suffering from MDS del(5q). We obtained targeted NGS data showing that TP53 mutational status at diagnosis could not predict tumor progression, by contrast with previously published data (Jädersten et al., JCO 2011). We show that TP53 clonal evolution is the key feature of tumor progression in MDS del(5q). We observed numerous mutated sub-clones emerging between diagnosis and follow-up. In our study, this emergence always followed onset of lenalidomide treatment. Lenalidomide was recently approved as a new therapy specifically improving anemia in patients with MDS del(5q). It allows most patients to become red-blood-cells-transfusion independent. Lenalidomide often eradicates the major tumor clone harbouring the isolated genetic abnormality deletion (5q) and allows clinical remission. Unfortunately, this remission is short (median, 2 years) and is followed, in 1 case out of 2, by a secondary acute leukemic transformation with a very poor prognosis.We studied the issue of a possible link between lenalidomide therapy and TP53 clonal evolution by annotating TP53 sequencing results with acute biological, clinical and therapeutic features in the 24 patients with sequential samples analyzed. In our study, patients with tumor progression (10 TP53 clonal evolution and 1 RUNX1 clonal evolution) were given a higher cumulative dose of lenalidomide compared to patients with stable disease (p = 0.036). Similarly to « adaptive therapy theory »(Gatenby 2009), we observed that eradication of the tumor wasn’t useful for improvement of quality of life. Absence of eradication might even allow to maintain a clonal equilibrium and a clonal competition between the distinct tumor sub-clones, resistant to lenalidomide or not, and therefore maintain stable disease.This theory of adaptive therapy questions the classical protocols of treatments against cancer, in which the maximal tolerated dose is preferred to the minimal effective dose. The latter might however slow down cancer progression or cancer relapse, with decreased side effects in patients, and decreased health costs.To date, few clinical trials (if any) questions such protocols of adaptive therapy. However, in vivo experiments (xenografts) and in silico statistical models allowed to study evolutionary dynamics of tumor sub-populations with and without therapy.The models predict that host survival can be maximized if “treatment-for-cure strategy” is replaced by “treatment-for-stability.” Specifically, the models predict that an optimal treatment strategy will modulate therapy to maintain a stable population of chemosensitive cells that can, in turn, suppress the growth of resistant populations under normal tumor conditions, Dr Gatenby said.

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

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