Acute myeloid leukemia (AML) is a deadly cancer of the blood and bone marrow defined by the accumulation of immature and non-functional myeloid progenitor cells. While AML is associated with a high success of chemotherapy-induced remission, it is accompanied by high relapse rates with poor response to subsequent therapies. Therefore, relapsed AML patients only have a 10% probability of long-term survival. An effective postinduction therapy is allogeneic hematopoietic stem cell transplantation (HSCT). However, complications associated with HSCT can be more severe than the AML disease itself. To date, no robust methodology is available to prospectively identify and distinguish AML patients that are more likely to benefit from HSCT. Our group has shown that AML patients with high leukemic progenitor cell content (LPC+) have a significantly lower overall survival (OS) when compared to patients with lower LPC content (LPC-). The objective of this study was to determine whether the LPC assay can be used as a functional predictor of post-HSCT survival. We hypothesized that LPC content correlates to post-HSCT survival times. We performed LPC assays on over 100 primary AML patient samples, showing that HSCT significantly improved OS in both LPC+ and LPC- patients, but LPC+ patients benefited more strongly than LPC- patients. This provides an initial basis to suggest that HSCT can offset the negative prognostic impact associated with high LPC content. To understand the biology of LPCs, we employed the Infinium HumanMethylation450 BeadChip assay to determine whether there are any methylation patterns that distinguish LPC+ and LPC- patients. However, we were not able to discover any uniquely methylated regions that separate the two groups, suggesting for further studies with an increased patient cohort, or extending the analyses to the transcript level. Given the rarity of curative approaches to cancers, a prognostic measure that could determine whether any single patient will benefit from HSCT will have an immediate impact. / Thesis / Master of Science (MSc)

Tissue engineering is a promising approach that combines the interactions of biomaterials, cells, and growth factors to stimulate tissue growth and regeneration. As such, selecting a suitable biomaterial is vital to the success of the procedure. Ideally, the material should show similarity to the extracellular matrix in the structure and relative stiffness, and biofunctionality beside others to provide a comfortable environment for the cells. Additionally, the biomaterial properties should allow for the effective diffusion of relevant growth factors and nutrients throughout the material to enable cell growth. Because peptides are composed of amino acids found naturally within the human body, they are considered non-toxic and biocompatible. Ultrashort peptides are peptides with three to seven amino acids that can be self-assembled into helical fibers forming scaffolds of supramolecular structures. These peptide hydrogels formed a highly porous network of nanofibers which can quickly solidify into nanofibrous hydrogels that resemble the extracellular matrix (ECM) and provide a 3D environment for cells with suitable mechanical properties. Furthermore, we can easily tune the stiffness of these peptide hydrogels by just increasing peptide concentration, thus providing a wide range of peptide hydrogels with different stiffness for 3D cell culture applications. Herein we describe the use of ultrashort peptide hydrogels for the maintenance and the differentiation of human mesenchymal stem cells into the osteogenic lineage. Furthermore, we develop a three dimensional (3D) biomimicry acute myeloid leukemia (AML) disease model using biomaterial from a tetramer ultrashort self-assembling peptide. In addition, we evaluate the potential application of peptide hydrogels as a hemostatic agent. The results presented in this study suggest that our biomimetic ultrashort tetrapeptide hydrogels are an excellent candidate for tissue engineering and biomedical applications.

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BCR-ABL1 transforms hematopoietic stem cells into leukemia stem cells (LSCs) to induce chronic myeloid leukemia in chronic phase. Expression of BCR-ABL1 stimulates production of elevated levels of reactive oxygen species (ROS), which induce oxidative DNA damage. CML cells accumulate excessive amounts of ROS-induced DNA damage which can be converted to potentially lethal DNA double strand breaks (DSBs). BCR-ABL1 stimulates enhanced Rad51-mediated DSB repair by the homologous recombination repair (HRR) pathway. In these studies we show BCR-ABL1-transformed cells depend on Rad52-mediated HRR to promote repair of ROS-induced DSBs and that this activity is dependent on Rad52 binding to single-stranded DNA (ssDNA). Our results show in the absence of Rad52, BCR-ABL1-positive hematopoietic cells accumulated elevated numbers of DSBs as detected by enhanced γ--H2AX foci formation compared to cells with wild-type Rad52 which resulted in a decrease in proliferation and expansion of the Rad52-null LSC population. Expression of wild-type Rad52 in Rad52-null cells decreased the accumulation of DSBs and restored expansion of the LSC population. Inhibition of ROS with the antioxidants Vitamin E or N-acetyl cysteine exerted similar effects on the LSC population of Rad52-null cells as restoration of wild-type Rad52. Our studies also show Rad52's ssDNA-binding activity is required for the proliferation of CML cells as evidenced by the accumulation of DSBs and impairment of clonogenic potential in cells in which the Rad52-F79A ssDNA-binding deficient mutant was expressed. Inhibition of Rad52 DNA binding activity by a peptide aptamer targeting Rad52-F79 resulted in a synthetic lethal phenotype in BCR-ABL1-positive cells due to impairment of the Rad52-dependent HRR pathway, as demonstrated by immunofluorescence and HRR repair assays. Altogether we identify Rad52 as a novel target in the treatment of CML, and other BRCA1- and/or BRCA2-deficient cancers, by showing induction of synthetic lethality in proliferating BCR-ABL1-positive cells in which Rad52 ssDNA-binding activity is inhibited. / Biology

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