Inhibition of mitochondrial translation as a therapeutic strategy for acute myeloid leukemia Marko Škrtić Doctor of Philosophy Institute of Medical Science University of Toronto 2012 Abstract Intro: Acute myeloid leukemia (AML) therapies have remained unchanged for 20 years, and thus new therapies are needed. Objective: To identify FDA-approved agents with anti-leukemia stem cell activity, we performed a screen and identified the antimicrobial tigecycline (TIG). Methods: Primary AML mononuclear cells were isolated by Ficoll centrifugation from peripheral blood. Flow cytometry dye; JC-1, Carboxy-H2DCFDA, Mitotracker GreenFM. Leukemia stem cell activity was assayed by human AML engraftment in NOD/SCID mice. Results: TIG induced cell death in primary AML patient samples (LD50, 3-6μM n=14), preferentially over normal hematopoietic cells. Likewise, in colony assays, TIG (5μM) reduced the clonogenic growth of AML samples (n=7) by 93%, demonstrating an effect on leukemia progenitor cells, but not normal hematopoietic cells (34% reduction, n=5). A yeast genome-wide screen identified mitochondrial translation inhibition as the mechanism of tigecycline-mediated cell death in eukaryotic cells. TIG decreased the expression of mitochondrial peptides, enzyme activity and membrane potential preferentially in AML cells over normal hematopoietic cells. ShRNA knockdown of TuFM mitochondrial translation factor in leukemia cells reproduced TIG anti-leukemia target effects previously described. We discovered that primary AML CD34+/CD38- stem cells have greater mitochondrial mass (3-fold, n=5) than normal CD34+ cells (n=4). Higher baseline mitochondrial mass in primary AML samples was predictive for tigecycline sensitivity in vitro (r=-0.71, p<0.05). We assessed the effect of TIG on primary AML stem cells defined by their ability to initiate leukemic engraftment in vivo. NOD/SCID mice treated with TIG had decreased human AML engraftment (n=3 AML patients) compared to control. Conclusions: We identified mitochondrial translation inhibition as a novel therapeutic strategy for AML. Currently, a Phase I clinical trial of tigecycline in hematological malignancies is underway.

The incidence of childhood leukemia is increasing, especially in urbanized areas. It is hypothesised that transplacental exposure to environmental carcinogens, such as benzene, plays a role in the etiology of childhood cancers. The studies in this thesis investigated mechanisms of transplacental benzene tumourigenesis focusing on the role of reactive oxygen species (ROS). Initially, we investigated the effect of maternal benzene exposure on fetal erythroid progenitor cell number and the role of ROS in benzene metabolite-induced dysregulation of erythropoiesis. In the CD-1 mouse, in utero benzene exposure caused significant alterations in female fetal liver erythroid progenitor cell numbers at gestational day 16 and postnatal day 2. Using an in vitro chicken erythroblast cell line capable of erythropoiesis, we found that hydroquinone significantly inhibited erythropoiesis and this effect was prevented by pretreatment with PEG-superoxide dismutase. The second objective investigated the role of ROS in dysregulated fetal hematopoietic progenitor cell growth after maternal benzene exposure in C57Bl/6N mice. In utero exposure to benzene caused changes in fetal hematopoietic progenitor cell numbers, an increase in levels of fetal liver intracellular ROS, and a decrease in IκB-α protein levels, which were all prevented by pretreatment with PEG-catalase. The final objective determined the incidence of cancer in offspring transplacentally exposed to benzene. This study compared two strains of mice (C57Bl/6N and CD-1), as well as male and female offspring. This study also measured levels of benzene and benzene metabolites present in maternal blood and fetal liver tissue after maternal benzene exposure. Transplacental exposure to benzene induced hepatic and hematopoietic tumours in male and female CD-1 mice, respectively. Interestingly, there were no significant changes in tumour incidence in C57Bl/6N mice demonstrating a significant strain difference in susceptibility to transplacental benzene carcinogenesis. Levels of fetal liver benzene metabolites also differed between genders and strains of mice suggesting that the gender and strain differences in tumour formation may be dependent on fetal benzene metabolism capability. In conclusion, this thesis supports the hypothesis that benzene exposure to pregnant women contributes to the etiology of childhood cancers and highlights ROS and fetal benzene metabolism as potential mechanisms. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2009-12-21 13:10:12.747

A new method for the preparation of 1,5-disubstituted-1Z,4Z-pentadienes by the addition of alkenyl cuprates to vinyltriphenylphosphonium bromide followed by an aldehyde is described. This method is used for the syntheses of 6Z,9Z-heneicosadiene and 15,15-(diethyldithio)-13S-t-butyldiphenylsilyloxy-6Z,9Z,11E-pentadecatriene. Syntheses of $(-)$-(5R)-argentilactone and (+)-(5R)-goniothalamin are described. Model studies towards the synthesis of the novel antileukemic agent CI-920 were carried out.

The overall aim of my thesis is to gain insight into the cellular and molecular basis of the hierarchical organization of the human blood system, and how these normal development processes are subverted into leukemogenesis. To date, the major cellular classes that comprise human blood remain ill defined as rigorous clonal analysis required to define the self-renewal and lineage potential of single cells has not yet been performed. Here, identification CD49f as a novel marker of human HSC led to the ability to transplant single human HSC in NOD-scid IL2Rgc-/- mice. Loss of CD49f and Thy1 uniquely demarcated multi-potent progenitors (MPP) from HSC. The classical model of hematopoiesis posits the segregation of lymphoid and myeloid lineages as the earliest fate decision during lineage restriction from HSC. The validity of this model in the mouse has been questioned; however, little is known about the lineage potential of human progenitors. By clonally mapping the developmental potential of seven progenitor classes from neonatal cord blood and adult bone marrow, human multi-lymphoid progenitors (MLP) were identified as a distinct population of Thy1-/loCD45RA+ cells in the CD34+CD38- stem cell compartment that can give rise to all lymphoid cell types, as well as monocytes, macrophages and dendritic cells. This indicates that these myeloid lineages arise in early lymphoid lineage specification. Thus, as in the mouse, human hematopoiesis does not follow a rigid model of myeloid-lymphoid segregation. While non-genetic mechanisms govern cell-fate commitment and lineage specification, hematopoietic malignancies are often initiated by aberrant gene rearrangements that can subvert normal cellular processes. Full transformation requires the accumulation of multiple genetic lesions. Most tumours exhibit dramatic genetic heterogeneity downstream of the initiating oncogenic event and are composed of pockets of genetically distinct clonal subpopulations. However little is known of how diversity evolves or the impact diversity has on functional properties. Here, using xenografting and DNA copy number alteration (CNA) profiling of human BCR-ABL1 lymphoblastic leukaemia, it was demonstrated that genetic diversity occurs in functionally defined leukaemia-initiating cells (L-IC) and that many diagnostic patient samples contain multiple genetically distinct L-IC subclones. Reconstructing the subclonal genetic ancestry of several samples by CNA profiling demonstrated a branching multi-clonal evolution model of leukaemogenesis, rather than linear succession. For some patient samples, the predominant diagnostic clone repopulated xenografts, while in others it was outcompeted by minor subclones. Reconstitution with the predominant diagnosis clone was associated with more aggressive growth properties in xenografts, deletion of CDKN2A/B, and a trend to poor patient outcome. Our findings link clonal diversity with L-IC function and underscore the importance of developing therapies that eradicate all intratumoural subclones.

Hematopoiesis describes the formation and development of blood cells. All blood cells originate from a pluripotent hematopoietic stem cell (HSC) which has the capacity for long and short term self-renewal as well as differentiation into myeloid or lymphoid lineages. The balance between HSC differentiation and proliferation is tightly controlled by both extrinsic and intrinsic factors. During leukemogenesis, this regulation is disrupted as transformation events lead to changes in proliferation, differentiation, and survival. Two distinct experimental strategies were utilized to examine the role of the hematopoietic adaptor protein GADS (GRB2-related adaptor downstream of SHC) in both leukemogenesis and in normal hematopoiesis. Philadelphia chromosome positive (Ph+) leukemias, including chronic myeloid leukemia (CML) and B cell acute lymphoblastic leukemia (B-ALL), are mediated by the oncogenic BCR-ABL fusion protein. Animal modeling experiments utilizing retroviral transduction and subsequent bone marrow transplantation have demonstrated that BCR-ABL generates both myeloid and lymphoid disease in mice when whole bone marrow is used as donor material. Strikingly, we observe that the lymphoid disease is absent in experiments completed with Gads-deficient bone marrow expressing BCR-ABL. Gads-deficient mice were generated previously and display a decrease in mature T cells, indicating that GADS is critical to T cell differentiation. Through immunophenotyping analysis we observed that Gads-deficient mice have increased numbers of lymphoid progenitors and HSCs. The increased number of HSCs in Gads-deficient mice did not induce enhanced HSC function as Gads-deficient bone marrow cells have impaired repopulation potential. Our data suggests that the T cell defect previously observed in Gads-deficient mice may be due to an early block in differentiation and functional defect in the HSC. Taken together, our studies demonstrate that GADS is a critical mediator in the hematopoietic system for both normal and leukemic differentiation and proliferation.

Acute Promyelocytic Leukemia is a subset of Acute Myeloid Leukemias, and is commonly associated with the presence of chromosomal translocations leading to the expression of the PML-RARA fusion protein. Less frequent cases of APL have been identified that express rare variant RARA fusion proteins, such as NPM-RARA. The presence of these fusions results in deregulated RARA signaling and response to the RARA ligand, ATRA. However, studies have indicated that loss of retinoid signaling alone is not sufficient to result in the leukemia. The goals of this thesis were to determine genes and pathways deregulated in variant APL that can serve as cooperating events in APL, through candidate pathway analysis and high-throughput gene expression profiling. Using a cell line model expressing variant fusion proteins associated with APL, we identified the deregulation of the NF-kappaB signaling pathway in APL, and describe the functional analysis of this pathway in our in vitro model. We next assessed whether promotion of survival signals could serve as a contributing factor in the accumulation of leukemic blasts in APL patient bone marrow. Our results indicated that PML-RARA and NPM-RARA expressing cells showed increased survival in the presence of TNFalpha, compared to wild type control cells. These data suggested a greater ability on the part of NPM-RARA cells to survive in the presence of TNFalpha. We also report for the first time the gene expression profiles of, and transcriptional effects of ATRA on, cells expressing the variant fusion proteins. Finally, we determined that the partner protein Nucleophosmin (NPM) has increased cytoplasmic localization in cells expressing the APL fusion proteins, and interacts within complexes comprising of RARA and RXRA. We further determined that the fusion can interfere with NPM function and cellular localization. Taken together, these studies provide evidence for the involvement of secondary hits in APL biology.

Glucocorticoids (GCs) are commonly used in the clinic as a treatment for Chronic Lymphocytic Leukemia (CLL). The exact mechanism of GC action remains unclear and patients eventually develop resistance to this group of agents. Our findings show that GC-cytotoxicity in circulating CLL cells is caused by bioenergetic restriction resulting from the down-regulation of a key glycolytic enzyme, pyruvate kinase, muscle isozyme 2 (PKM2). Conversely, GCs were shown to promote fatty acid oxidation instead by up-regulating the expression of peroxisome proliferator activated receptor α (PPARα). These findings establish PPARα and fatty acid oxidation as novel mediators of GC resistance in CLL. Our findings also demonstrate that GCs enhance the cytotoxic effects of membrane-damaging agents such as ionophores and complement-mediated cytotoxicity. A clinically relevant agent known to intercalate in the cell membrane, Danazol was also found to have activity against CLL and can be combined safely with GCs for enhanced treatment efficacy.

The overall aim of my thesis is to gain insight into the cellular and molecular basis of the hierarchical organization of the human blood system, and how these normal development processes are subverted into leukemogenesis. To date, the major cellular classes that comprise human blood remain ill defined as rigorous clonal analysis required to define the self-renewal and lineage potential of single cells has not yet been performed. Here, identification CD49f as a novel marker of human HSC led to the ability to transplant single human HSC in NOD-scid IL2Rgc-/- mice. Loss of CD49f and Thy1 uniquely demarcated multi-potent progenitors (MPP) from HSC. The classical model of hematopoiesis posits the segregation of lymphoid and myeloid lineages as the earliest fate decision during lineage restriction from HSC. The validity of this model in the mouse has been questioned; however, little is known about the lineage potential of human progenitors. By clonally mapping the developmental potential of seven progenitor classes from neonatal cord blood and adult bone marrow, human multi-lymphoid progenitors (MLP) were identified as a distinct population of Thy1-/loCD45RA+ cells in the CD34+CD38- stem cell compartment that can give rise to all lymphoid cell types, as well as monocytes, macrophages and dendritic cells. This indicates that these myeloid lineages arise in early lymphoid lineage specification. Thus, as in the mouse, human hematopoiesis does not follow a rigid model of myeloid-lymphoid segregation. While non-genetic mechanisms govern cell-fate commitment and lineage specification, hematopoietic malignancies are often initiated by aberrant gene rearrangements that can subvert normal cellular processes. Full transformation requires the accumulation of multiple genetic lesions. Most tumours exhibit dramatic genetic heterogeneity downstream of the initiating oncogenic event and are composed of pockets of genetically distinct clonal subpopulations. However little is known of how diversity evolves or the impact diversity has on functional properties. Here, using xenografting and DNA copy number alteration (CNA) profiling of human BCR-ABL1 lymphoblastic leukaemia, it was demonstrated that genetic diversity occurs in functionally defined leukaemia-initiating cells (L-IC) and that many diagnostic patient samples contain multiple genetically distinct L-IC subclones. Reconstructing the subclonal genetic ancestry of several samples by CNA profiling demonstrated a branching multi-clonal evolution model of leukaemogenesis, rather than linear succession. For some patient samples, the predominant diagnostic clone repopulated xenografts, while in others it was outcompeted by minor subclones. Reconstitution with the predominant diagnosis clone was associated with more aggressive growth properties in xenografts, deletion of CDKN2A/B, and a trend to poor patient outcome. Our findings link clonal diversity with L-IC function and underscore the importance of developing therapies that eradicate all intratumoural subclones.

Hematopoiesis describes the formation and development of blood cells. All blood cells originate from a pluripotent hematopoietic stem cell (HSC) which has the capacity for long and short term self-renewal as well as differentiation into myeloid or lymphoid lineages. The balance between HSC differentiation and proliferation is tightly controlled by both extrinsic and intrinsic factors. During leukemogenesis, this regulation is disrupted as transformation events lead to changes in proliferation, differentiation, and survival. Two distinct experimental strategies were utilized to examine the role of the hematopoietic adaptor protein GADS (GRB2-related adaptor downstream of SHC) in both leukemogenesis and in normal hematopoiesis. Philadelphia chromosome positive (Ph+) leukemias, including chronic myeloid leukemia (CML) and B cell acute lymphoblastic leukemia (B-ALL), are mediated by the oncogenic BCR-ABL fusion protein. Animal modeling experiments utilizing retroviral transduction and subsequent bone marrow transplantation have demonstrated that BCR-ABL generates both myeloid and lymphoid disease in mice when whole bone marrow is used as donor material. Strikingly, we observe that the lymphoid disease is absent in experiments completed with Gads-deficient bone marrow expressing BCR-ABL. Gads-deficient mice were generated previously and display a decrease in mature T cells, indicating that GADS is critical to T cell differentiation. Through immunophenotyping analysis we observed that Gads-deficient mice have increased numbers of lymphoid progenitors and HSCs. The increased number of HSCs in Gads-deficient mice did not induce enhanced HSC function as Gads-deficient bone marrow cells have impaired repopulation potential. Our data suggests that the T cell defect previously observed in Gads-deficient mice may be due to an early block in differentiation and functional defect in the HSC. Taken together, our studies demonstrate that GADS is a critical mediator in the hematopoietic system for both normal and leukemic differentiation and proliferation.

Acute Promyelocytic Leukemia is a subset of Acute Myeloid Leukemias, and is commonly associated with the presence of chromosomal translocations leading to the expression of the PML-RARA fusion protein. Less frequent cases of APL have been identified that express rare variant RARA fusion proteins, such as NPM-RARA. The presence of these fusions results in deregulated RARA signaling and response to the RARA ligand, ATRA. However, studies have indicated that loss of retinoid signaling alone is not sufficient to result in the leukemia. The goals of this thesis were to determine genes and pathways deregulated in variant APL that can serve as cooperating events in APL, through candidate pathway analysis and high-throughput gene expression profiling. Using a cell line model expressing variant fusion proteins associated with APL, we identified the deregulation of the NF-kappaB signaling pathway in APL, and describe the functional analysis of this pathway in our in vitro model. We next assessed whether promotion of survival signals could serve as a contributing factor in the accumulation of leukemic blasts in APL patient bone marrow. Our results indicated that PML-RARA and NPM-RARA expressing cells showed increased survival in the presence of TNFalpha, compared to wild type control cells. These data suggested a greater ability on the part of NPM-RARA cells to survive in the presence of TNFalpha. We also report for the first time the gene expression profiles of, and transcriptional effects of ATRA on, cells expressing the variant fusion proteins. Finally, we determined that the partner protein Nucleophosmin (NPM) has increased cytoplasmic localization in cells expressing the APL fusion proteins, and interacts within complexes comprising of RARA and RXRA. We further determined that the fusion can interfere with NPM function and cellular localization. Taken together, these studies provide evidence for the involvement of secondary hits in APL biology.