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<p>Radiation is a prominent source of environmental oxidative stress that can have deleterious consequences for health. Despite its well-known negative effects, radiation is commonly employed clinically for disease treatment and diagnosis. Bone marrow transplantation (BMT), used in the treatment of a variety of diseases, is preceded by a myeloablative regimen that usually involves radiation. Mortality associated with BMT is quite high and the aggressive radiation pre-treatment regimen contributes to these high rates of mortality. Interventions that inhibit the negative consequences of irradiation and promote BMT success would have significant implications for public health. Exercise-induced adaptations in numerous body tissues have been associated with amelioration of a variety of pathologies, particularly those associated with oxidative stress, and an overall improvement in health. Whether these adaptations can protect from damage induced by an external source of oxidative stress, such as a high dose of radiation, or promote BMT success is unknown. The purpose of this thesis was to determine if the adaptive response to exercise training could inhibit the negative effects of irradiation in skeletal muscle and bone marrow, and promote BMT success. To apply these adaptations to BMT, we examined the response of hematopoietic stem cells (HSC) and their niche to exercise. We report that muscle from exercise trained mice exhibits an enhanced response to radiation characterized by increased antioxidant and mitochondrial metabolic enzyme activity. Extending these findings to cells in the bone marrow, we demonstrated that exercise training inhibited radiation-induced genotoxicity and cytotoxicity. With respect to BMT, exercise training increased HSC quantity with no effects on HSC function; however, preconditioning BMT recipients with exercise training resulted in improved probability of survival and enhanced hematopoietic regeneration. Collectively, results from the studies presented herein suggest that exercise training may be a successful therapeutic intervention to inhibit the damaging effects of radiation and improve BMT outcomes.</p> / Doctor of Philosophy (PhD)

Mutations and deletions in ribosomal proteins are associated with a group of diseases termed ribosomopathies. Collectively, these diseases are characterized by ineffective hematopoiesis, bone marrow failure, and an increased risk of developing myelodysplastic syndrome (MDS) and subsequently acute myeloid leukemia (AML). This observation highlights the role of dysregulation of this class of proteins in the development and progression of myeloid neoplasms. Analysis of gene expression in CD34+ hematopoietic stem cells (HSC) from 183 MDS patients demonstrated that ribosomal protein L22 (Rpl22) expression exhibited a greater reduction than any other ribosomal protein gene in MDS. Interestingly, we observed that AML patients with lower expression of Rpl22 had a significant reduction in their survival (TCGA cohort, N=200, Log Rank P value&lt;0.05). To assess the mechanism of reduced expression, we developed a FISH probe complementary to the RPL22 locus and assessed for deletion of this locus in an independent set of 104 MDS/AML bone marrow samples. Strikingly, we found that RPL22 deletion was enriched in high-risk MDS and secondary AML cases. We, therefore, sought to investigate whether reduced Rpl22 expression played a causal role in leukemogenesis. Using Rpl22-/- mice, we found that Rpl22-deficiency resulted in a constellation of phenotypes resembling MDS. Indeed, Rpl22-deficiency caused a macrocytic reduction in red blood cells, dysplasia in the bone marrow, and an expansion of the early hematopoietic stem and progenitor compartment (HSPC). Since MDS has been described as a disease originating from the stem cell compartment, we next sought to determine if the hematopoietic defects were cell autonomous and resident in Rpl22-/- HSC. Competitive transplantation revealed that Rpl22-/- HSC exhibited pre-leukemic characteristics including effective engraftment, but a failure to give rise to downstream mature blood cell lineages. Importantly, there was a strong myeloid bias in those downstream progeny derived form Rpl22-/- HSC. To determine how Rpl22-deficiency increased the causes these deficiencies in HSC, we performed whole transcriptome analysis on Rpl22-/- HSC. Interestingly, alterations in genes associated with both ribosomal proteins and mitochondrial components were observed. We found that protein synthesis was unchanged in Rpl22-deficient HSCs, sharply contrasting the reductions in global protein synthesis that usually accompany ribosomal protein insufficiency. Consequently, we shifted our focus to the dysregulated mitochondrial genes, which were linked to the processes of oxidative phosphorylation and fatty acid metabolism. We observed that oxidative phosphorylation was decreased in Rpl22-deficient HSCs while fatty acid oxidation was increased. Increased fatty acid oxidation is associated with maintenance of the hematopoietic stem cells. Interestingly, inhibiting fatty acid oxidation mitigated this attribute in Rpl22 deficient HSCs. Because Rpl22 is an RNA-binding protein, we asked if Rpl22 was regulating fatty acid oxidation by directly binding mRNAs encoding regulators of fatty acid oxidation. We found that Rpl22 is able to directly bind the coding region of an upstream regulator of fatty acid oxidation, Alox12. Thus, we hypothesized that Rpl22-deficiency increased fatty acid oxidation through increased expression of Alox12. Consistent with this hypothesis, knockdown of Alox12 impaired the function of Rpl22 deficient HSC. Because the increased fatty acid oxidation promotes self-renewal of Rpl22-deficient HSC and blocks their differentiation, we also hypothesized that this would predispose them to leukemogenesis. We examined the potential for Rpl22-deficient HSPC to be transformed upon ectopic expression of the MLL-AF9 oncogenic fusion. Indeed, Rpl22 deficiency increased predisposition to transformation both in vitro and in vivo, in MLL-AF9 knockin mice. Furthermore, Rpl22 deficient leukemias were preferentially sensitive to pharmacologic inhibition of fatty acid oxidation or Alox12 knockdown, indicating that leukemia cell survival was also dependent upon fatty acid oxidation. Taken together, these findings indicate that Rpl22-insufficiency predisposes HSPC to leukemic transformation and aggressive growth by regulating mitochondrial function, providing an explanation for the reduced survival observed in Rpl22-low AML patients. We also sought to determine how Rpl22 may be contributing to another subset of AML known as Therapy-related AML. Most commonly, these patients develop AML after previously being treated with an alkylating chemotherapeutic drug. Interestingly, we found that Rpl22-deficient HSPC are resistant to treatment with these agents, despite having evidence of DNA damage. The ultimate consequence of the insensitivity of Rpl22-deficient HSPC to alkylating agents was that mice given serial doses of cyclophosphamide exhibited an increased incidence of leukemic-like changes. This chemo-resistant phenotype in Rpl22-/- cells was related to increased expression of the DNA repair protein MGMT. Inhibition of this protein abrogated the ability of these cells to survive following treatment with cyclophosphamide. Ultimately, this study implicates Rpl22 as a regulator of alkylating DNA damage repair and suggests that both patients with hematologic or solid cancers that express reduced levels of Rpl22 are at increased risk for development of therapy related AML is they are treated with alkylating agents. / Cancer Biology & Genetics

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25196号 / 医博第5082号 / 新制||医||1072(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 金子 新, 教授 滝田 順子, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Diss. (sammanfattning) Stockholm : Karol inst., 2003. / Härtill 5 uppsatser.

Acute myeloid leukemia (AML) develops following oncogenic alterations to hematopoietic stem (HSC) and progenitor cells (HSPCs) in the bone marrow, resulting in dysregulated proliferation of immature myeloid progenitors that interferes with normal hematopoiesis. Understanding the mechanisms of HSPC protection against damage and excessive division, and how these pathways are altered during leukemic progression, is vital for establishing effective therapies. Here, we show that autophagy, a lysosomal degradation pathway, is increased in HSPCs using a novel imaging flow cytometry autophagy assay. Loss of hematopoietic autophagy following deletion of key gene Atg5 resulted in increased HSC proliferation, leading to HSC exhaustion and bone marrow failure. Although erythrocyte and lymphocyte populations were negatively impacted by autophagy loss, myeloid cells showing immature characteristics were expanded. Deletion of Atg5 in an AML model resulted in increased proliferation under metabolic stress, dependent on the glycolytic pathway, and aberrant upstream mTOR signaling. Moreover, modulation of Atg5 altered leukemic response to culture with stromal cells. Finally, primary AML cells displayed multiple markers of decreased autophagy. These data suggest a role for autophagy in preserving HSC function, partially through suppression of HSPC proliferation, and indicate that decreased autophagy may benefit AML cells. We postulate that modulation of autophagy could help maintain stem cell function, for example during transplantation, and aid AML therapy in a setting-specific manner.

The advent of high-throughput sequencing has brought about the creation of an unprecedented amount of research data. Analytical methodology has not been able to keep pace with the plethora of data being produced. Two assays, ImmunoSEQ and the cytokinesisblock micronucleus (CBMN), that both produce count data and have few methods available to analyze them are considered. ImmunoSEQ is a sequencing assay that measures the beta T-cell receptor (TCR) repertoire. The ImmunoSEQ assay was used to describe the TCR repertoires of patients that have undergone hematopoietic stem cell transplantation (HSCT). Several different methods for spectratype analysis were extended to the TCR sequencing setting then applied to these data to demonstrate different ways the data set can be analyzed. The different methods include CDR3 distribution perturbation, Oligoscores, Simpson's diversity, Shannon diversity, Kullback-Liebler divergence, a non-parametric method and a proportion logit transformation method. Herein we also demonstrate adapting compositional data analysis methods to the TCR sequencing setting. The various methods were compared when analyzing a set of 13 subjects who underwent hematopoietic stem cell transplantation. The eight subjects who developed graft versus host disease were compared to the five who did not. There was no little overlap in the results of the different methods showing that researchers must choose the appropriate method for their research question of interest. The CBMN assay measures the rate of micronuclei (MN) formation in a sample of cells and can be paired with gene expression or methylation assays to determine association between MN formation and other genetic markers. Herein we extended the generalized monotone incremental forward stagewise (GMIFS) method to the situation where the response is count data and there are more independent variables than there are samples. Our Poisson GMIFS method was compared to a popular alternative, glmpath, by using simulations and applying both to real data. Simulations showed that both methods perform similarly in accurately choosing truly significant variables. However, glmpath appears to overfit compared to our GMIFS method. Finally, when both methods were applied to two data sets GMIFS appeared to be more stable than glmpath.

La transplantation de cellules souches hématopoïétiques (CSH) est un traitement couramment utilisé pour traiter plusieurs types de maladies hématologiques telles que les leucémies. Par contre, une limite importante de ce type de traitement est la quantité restreinte de CSH disponibles pour la transplantation. Il importe donc de trouver des moyens pour expandre efficacement ces cellules ex vivo tout en préservant leurs propriétés. Le gène HOXB4 est présentement un candidat très prometteur pour atteindre cet objectif. Il a en effet été montré que HOXB4 est capable d’expandre les CSH in vivo et in vitro sans mener au développement de leucémie. Le gène HOXC4, qui appartient au même paralogue est aussi en mesure d’expandre les cellules hématopoïétiques primitives suggérant un rôle commun pour les gènes HOX du paralogue 4 dans l’autorenouvellement des CSH. Le gène HOXA4 est dix fois plus exprimé que le gène HOXB4 dans des CSH du foie fœtal au moment de leur principale expansion. De plus, les CSH mutantes pour Hoxa4, contrairement aux CSH mutantes pour Hoxb4, sont incapables de reconstituer un receveur irradié lorsqu’elles sont transplantées en condition de compétition. HOXA4 pourrait donc jouer un rôle plus important que les autres gènes du paralogue 4 pour l’expansion des CSH au niveau physiologique. Nous avons donc posé l’hypothèse que HOXA4 est capable d’expandre des CSH de façon plus importante que HOXB4. Les résultats obtenues dans le cadre de ce projet de recherche ont montré que la surexpression de HOXA4 était capable d’expandre les CSH et les progéniteurs hématopoïétiques primitifs dans le même ordre que ce qui est connu pour HOXB4. Des cultures et des essais de transplantation en situation de compétition ont confirmé la capacité égale des CSH surexprimant HOXA4 et HOXB4 de proliférer et de reconstituer les receveurs irradiés à long terme. Par contre, nous avons observé une meilleure reconstitution périphérique à court terme par les CSH HOXA4+ par rapport aux CSH HOXB4+, associée à une meilleure reconstitution lymphoïde. Nous avons aussi comparé les niveaux d’expression de gènes cibles potentiels dans des CSH surexprimant HOXA4 ou HOXB4 et observer que plusieurs gènes importants pour la fonction des CSH était régulé positivement suite à leur surexpression, notamment plusieurs gènes impliqués dans les voies de signalisation Notch et Wnt, tels que des récepteurs et ligands. Les gènes HOX du paralogue 4 pourraient donc réguler la communication entre les CSH et leur microenvironnement via ces voies de signalisation majeures et ainsi réguler leur autorenouvellement. La modulation de différents gènes codant pour des facteurs de transcription et des molécules impliquées dans la pluripotence suggère également que HOXA4 et HOXB4 utilisent des mécanismes intrinsèques et extrinsèques pour réguler leur potentiel d’autorenouvellement. Ces connaissances pourront ainsi être utilisées pour optimiser les protocoles d’expansion ex vivo des CSH dans un but thérapeutique. / Transplantation of hematopoietic stem cells (HSC) is a treatment commonly used to treat several types of hematological diseases such as leukemia. However, a major limitation of this type of treatment is the limited number of HSC available for transplantation. It is therefore important to develop ways to expand these cells ex vivo. The HOXB4 gene is a promising candidate for achieving this goal. It has indeed been shown that HOXB4 is able to expand HSC in vivo and in vitro without inducing leukemia. HOXC4, which belongs to the same paralog group, is also able to expand primitive hematopoietic cell suggesting a common role for paralog 4 HOX genes in the self-renewal of HSC. HOXA4 is ten times more expressed in fetal liver HSC during their primary expansion. Furthermore, Hoxa4 mutant HSC, unlike Hoxb4 mutant HSC, are unable to reconstitute an irradiated recipient when transplanted in competition. Therefore, HOXA4 could play a more important role than other paralog 4 genes for HSC expansion at the physiological level and we hypothesized that HOXA4 can expand HSC more efficiently than HOXB4. The results obtained during this research project showed that the overexpression of HOXA4 expand HSC and primitive hematopoietic progenitors in the same order as HOXB4. Direct competitive culture and transplantation assays confirmed the equal capacity of HSC overexpressing HOXA4 and HOXB4 to proliferate and engraft at long-term. However, we observed a better short-term peripheral reconstitution by HOXA4+ HSC compared to HOXB4+ HSC, which was associated with a better lymphoid reconstitution. We also compared the expression levels of potential target genes in HSC overexpressing HOXA4 or HOXB4 and observed that many genes important for HSC function were upregulated following their overexpression, including several genes involved in the Notch and Wnt signaling pathway. These included both receptors as well as ligands, indicating that HOX4 genes might regulate the communication of primitive HSCs with their environment through these major signaling pathways and promote self-renewal. In addition, modulation of genes coding for transcription factors and molecules known for their function in pluripotency suggest that HOXA4 and HOXB4 have both intrinsic and extrinsic potential to control self renewal potential. This knowledge can then be further explored and used to optimize ex vivo HSC expansion protocols for clinical purposes.