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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<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

Acute myeloid leukemia (AML) is an aggressive malignancy of the hematopoietic system, characterized by the accumulation of abnormally differentiated blast cells that is driven by leukemic stem cells (LSCs). In murine AML, Musashi-2 (MSI2), an RNA-binding protein and positive regulator of stemness, has been implicated in the propagation of disease. While its enhanced expression correlated with poor disease outcome for human AML patients, no study has yet examined its actual functional role in human leukemia. In normal human hematopoietic stem cells (HSCs), we have recently reported the inhibitory effects of MSI2 on the pro-differentiative aryl hydrocarbon receptor (AHR) signaling pathway as a mechanism for promoting self-renewal in HSCs. We hypothesized that elevated MSI2 is critical for maintenance of human AML and promotes unrestrained self-renewal of LSCs in part through constitutive repression of AHR signaling. Our work aimed to unravel the relationship between MSI2 and AHR in the human leukemic context and to determine if activation of AHR signaling can promote differentiation. Results confirmed that MSI2 is preferentially expressed in primary patient LSCs and is negatively correlated with the expression of AHR gene targets. Upon lentiviral knockdown of MSI2 in-vitro and in-vivo, leukemic growth was compromised and increased AHR signaling was observed. Circumventing the inhibitory role of MSI2 in AML, activation of AHR with a potent agonist impaired leukemic progenitor activity and proliferation. In-vivo studies employing reconstitution of immunodeficient mice with primary AML samples showed impairment of AML engraftment for a significant proportion of tested samples upon treatment with an AHR agonist. Overall, our findings from this project indicated that MSI2 is required for human AML propagation and that a decrease in MSI2 inhibitory effects on AHR signaling or direct activation of the AHR signaling pathway via a potent agonist can promote AML cell differentiation and loss. / Thesis / Master of Science (MSc) / The human blood system is sustained by a population of blood stem cells that are tightly regulated in their production of stem and differentiated cells. The Musashi-2 (MSI2) protein is a key regulator of blood stem cell identity through its inhibition of the aryl hydrocarbon receptor (AHR) signaling pathway. When there is dysregulation of blood cell homeostasis, blood malignancies such as acute myeloid leukemia (AML) may arise. In this work, the relationship between MSI2 and the AHR signaling pathway was explored within a myeloid leukemic context. It was shown that MSI2 imposes inhibitory effects on AHR to promote disease progression and that its reduction could help alleviate disease burden. Additionally, it was found that activation of the AHR signaling pathway could overcome the MSI2 differentiation block to create a therapeutic effect. Overall, the results of this project shed light on novel therapeutic strategies and targets for the treatment of AML.

This interpretive consumer research study interrogates the idea that people turn to consumption as a means of self-determination. Proceeding from the understanding that the consumer enacts the development of their identity within the marketplace, it takes as its subject those in transition. Its context is a support group community of people brought together by an illness - multiple myeloma. Here, through a phenomenological approach designed to explore the lived experience of illness, the thesis discovers community to be the enabling context for the consumer’s negotiation of both selfhood and the market. Conclusions are drawn about the incremental, complex nature of identity work, and the collective practices that empower it. It is found that the marketplace requires significant mediation, but that the social resources of the community can equip the consumer to navigate its challenges. This transformation is manifested in the newly-diagnosed patient’s journey from dislocation and passivity to the empowered status of ‘skilled consumer’. The importance of the often-overlooked emotional texture of exchange within consumption communities is highlighted. In conclusion, it is offered that this study extends the concept of communities of practice into the field of consumption.

<p>Despite major advances in our understanding of the biology of childhood acute myeloid leukemia (AML) and the development of new cytotoxic drugs, the prognosis of long-term survival is still only 60-65 %.</p><p>In the present research, we studied the pharmacokinetics of drugs used in the induction therapy of childhood AML and performed in vitro drug sensitivity testing of leukemic cells from children with AML.</p><p>The aims of the studies were to correlate the results of the analysis to biological and clinical parameters and to identify subgroups of AML with specific drug sensitivity profiles in order to better understand why treatment fails in some patients and how therapy may be improved.</p><p>Blood samples were analysed to study the pharmacokinetics of doxorubicin (n=41), etoposide (n=45) and 6-thioguanine (n=50). Doxorubicin plasma concentration and total body clearance were correlated to the effect of induction therapy, and doxorubicin plasma concentration was an independent factor for complete remission, both in univariate and multivariate analysis including sex, age, and white blood cell count at diagnosis. For etoposide and 6-thioguanine no correlation was found between pharmacokinetics and clinical effect. Children with Down syndrome (DS) tended to reach higher blood concentrations of etoposide and thioguanine nucleotides, indicating that dose reduction may be reasonable to reach the same drug exposure as in children without DS.</p><p>Leukemic cells from 201 children with newly diagnosed AML, 15 of whom had DS, were successfully analysed for in vitro drug sensitivity by the fluorometric microculture cytotoxicity assay (FMCA). We found that samples from children with DS were highly sensitive to most drugs used in AML treatment. In non-DS children, the t(9;11) samples were significantly more sensitive to cytarabine (p=0.03) and doxorubicin (p=0.035) than other samples. The findings might explain the very favorable outcome reported in children with DS and t(9;11)-positive AML. A specific drug resistance profile was found for several other genetic subgroups as well. A detailed study of MLL-rearranged leukemia showed that cellular drug sensitivity is correlated both to partner genes and cell lineage, findings that support the strategy of contemporary protocols to include high-dose cytarabine in the treatment of patients with MLL-rearrangement, both in AML and acute lymphoblastic leukemia (ALL).</p><p>Our results indicate that drug resistance and pharmacokinetic studies may yield important information regarding drug response in different sub-groups of childhood AML, helping us to optimize future chemotherapy in childhood AML.</p>