Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Chronic lymphocytic leukemia (CLL) is a cancer of B cells and is the most common leukemia in North America. Current therapies are fraught with challenges, and drug resistance and disease relapse remain common occurrences. Therefore, novel therapies and novel therapeutic strategies are needed to improve CLL therapy. Better yet, therapies targeted at specific weaknesses of CLL cells will ensure maximum efficacy and minimum adverse toxicity. To this end, this thesis focuses on targeting the susceptible BCR pathway and lysosome-mediated cell death pathway using gefitinib and lysosomotropic agents, respectively. Firstly, the novel use of the tyrosine kinase inhibitor gefitinib was explored. This drug was most effective in aggressive ZAP-70+ CLL cells and cell lines. A similar inhibitor, erlotinib, had no effect in CLL. Gefitinib inhibited phosphorylation of Syk and ZAP-70, prevented downstream kinase activation, and supressed the pro-survival BCR response. ZAP-70 is implicated in the mechanism of action of gefitinib as introduction of ZAP-70 into a B cell line increased their sensitivity to gefitinib. Secondly, the novel strategy of targeting lysosomes was explored. The lysosomotropic drugs siramesine, nortriptyline, desipramine, mefloquine, and tafenoquine were all found to induce cytotoxicity and lysosome permeabilization. Lysosome permeabilization was accompanied with lipid peroxidation and followed by loss of mitochondrial membrane potential. Compared with healthy B cells, CLL cells were more sensitive to this cell death pathway. This was potentially due to the overexpression of SPP1 and overproduction of sphingosine, which destabilized lysosomes. Lastly, this thesis explored the clinical utility of these targeted therapies. Both gefitinib and siramesine were more effective in CLL cells than patient T cells. Furthermore, they retained efficacy amid protective stromal cells. Clinical correlations revealed that gefitinib and siramesine were effective in CLL cells with poor prognostic features. Siramesine was more effective in male cells and in previously-treated cells. Gefitinib was most effective in young patients. Overall, work presented herein demonstrates the efficacy of the tyrosine kinase inhibitor gefitinib and lysosomotropic agents in primary CLL cells. This work investigates the altered biology of the BCR pathway and lysosomes in CLL cells, and takes advantage of these weaknesses using targeted therapies. / October 2016

Thesis advisor: Thomas N. Seyfried / Gangliosides are glycosphingolipids (GSLs) containing sialic acids that play numerous roles in neuronal maturation, apoptotic signaling, angiogenesis, and cell surface receptor activity. The GM1 and GM2 gangliosidoses are a series of autosomal recessive lysosomal storage disorders (LSDs) characterized by an inability to degrade these lipid molecules. GM1 gangliosidosis is caused by a mutation in the lysosomal hydrolase β-galactosidase, resulting in neuronal storage of ganglioside GM1 and asialo GA1. Tay-Sachs (TS) and Sandhoff Disease (SD) are GM2 gangliosidoses caused by mutations in either the α or β subunits, respectively, of the heterodimeric protein β- hexosaminidase A, resulting in the storage of ganglioside GM2 and asialo GA2. The accumulation of excess ganglioside in the central nervous system leads to abnormal intracellular vacuoles, neuronal loss, demyelination, ataxia, dementia, and premature death. In my studies, I have shown that accumulation of GM1 ganglioside may not coincide with secondary storage of cholesterol, by providing evidence that cholesterol-binding fluorescent molecule filipin reacted to GM1 ganglioside in the absence of cholesterol. In an effort to combat the early-onset gangliosidoses, I have explored the effects of combining Neural Stem Cells (NSCs) with Substrate Reduction Therapy (SRT) in juvenile Sandhoff mice. The analysis showed that SRT was more effective than NSCs in reducing stored GM2 and GA2 in young mice, and no synergy was observed. In adult GM1 gangliosidosis, Tay- Sachs, and Sandhoff mice, Adeno-Associated Viral (AAV) vector gene therapy was used to restore therapeutic levels of wild-type enzyme to the CNS. AAV therapy corrected ganglioside storage and ameliorated myelin-associated lipid loss in all tissues assayed, increasing motor performance and life in effected animals. Lastly, AAV therapy was also successful in a feline model of Sandhoff disease. These results in juvenile and adult model systems point the way towards multiple effective clinical therapies in the near future. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Ubiquitination is a post-translational modification tht mediates sorting of integral membrane proteins to lysosomes for their degradation. ESCRTs (Endosomal Sorting Complex Required For Transport) bind and sequester ubiquitinated membrane proteins and direct them into multivesicular bodies (MVBs). ESCRTs themselves become covalently ubiquitinated, simply by virtue of non-covalently binding Ub. However, it is unclear whether this regulates a critical aspect of ESCRT function. In yeast, many MVB cargo proteins are ubiquitinated by the HECT-type Ub-ligase Rsp5, sometimes via the action of Rsp5 adaptor proteins. While many Rsp5 targets are modified by polyubiquitination, it remains unclear whether polyubiquitination is a necessary signal for their incorporation into MVBs. Despite years of research, these and related questions have been difficult to resolve because it is technically quite challenging to control the level of a given protein's ubiquitination. The aim of this research was to develop a novel technique, which can render proteins resistant to ubiquitination. The technique involved the fusion of the Ub-peptidase to a protein of interest via a flexible linker, essentially creating a "DUb module". The intent of this module would be to cleave any Ub form the target protein, essentially immunizing it from the effects of ubiquitination. This novel method was used in combination with several conventional methods to examine the role of ubiquitination within the endocytic pathway and in particular focus on the questions of what type of ubiquitin signal was sufficient for sorting into MVB vesicles and whether ubiquitination of ESCRTs was required for their sorting activity. We found that a single Ub was sufficient for membrane protein entry into MVBs in the absence of ESCRT ubiquitination.

The lysosomal-associated protein transmembrane 5 (LAPTM5) is a protein preferentially expressed in the immune cells. LAPTM5 was isolated in our laboratory as an interacting partner of the ubiquitin ligase, Nedd4. The intracellular domains of LAPTM5 contain three PY (L/PPxY) motifs, which bind the WW domains of Nedd4, as well as a ubiquitin-interacting motif (UIM). Here, I show that sorting of LAPTM5 from the Golgi to the lysosomes requires its association with Nedd4 and the clathrin adaptor GGA3. Although the Nedd4-LAPTM5 interaction leads to the ubiquitination of LAPTM5, this event is not necessary for LAPTM5 sorting. Rather, the Nedd4-LAPTM5 complex recruits ubiquitinated GGA3, which binds the UIM of LAPTM5. Hence, I propose a novel mechanism by which the ubiquitin ligase Nedd4, via interactions with GGA3 and cargo (LAPTM5), regulates cargo trafficking to the lysosome without requiring cargo ubiquitination. Because nothing was known about the biological function of LAPTM5, at the beginning of my Ph.D. training, I set out to determine the role of LAPTM5 in the innate immune cells. I demonstrate that LAPTM5 interacts with kinesin, a motor protein previously implicated in the anterograde movement of the late endosomal/lysosomal compartments. In dendritic cells, I show that upon maturation LAPTM5 is present within endolysosomal tubules formed by class II MHC molecules. Although I find that LAPTM5 is dispensable for the translocation of peptide-loaded MHC II molecules to the cell surface, this study extends our knowledge of the repertoire of proteins present within tubules formed by the MHC II compartments in activated dendritic cells. In macrophages, I demonstrate that LAPTM5 acts as a positive regulator of NFκB and MAPK signaling cascades, and promotes efficient proinflammatory cytokine production in response to several inducers of macrophage activation. During TNFα stimulation, LAPTM5 is required for proper initiation of NFκB signaling by acting at the receptor-proximate level. Thus, my findings indicate that LAPTM5 is an important component of inflammatory signaling cascades in macrophages and highlight a role for the endosomal/lysosomal system in regulating these cascades. Collectively, the work presented in this thesis broadens our understanding of lysosomal membrane protein sorting and function.

The lysosomal-associated protein transmembrane 5 (LAPTM5) is a protein preferentially expressed in the immune cells. LAPTM5 was isolated in our laboratory as an interacting partner of the ubiquitin ligase, Nedd4. The intracellular domains of LAPTM5 contain three PY (L/PPxY) motifs, which bind the WW domains of Nedd4, as well as a ubiquitin-interacting motif (UIM). Here, I show that sorting of LAPTM5 from the Golgi to the lysosomes requires its association with Nedd4 and the clathrin adaptor GGA3. Although the Nedd4-LAPTM5 interaction leads to the ubiquitination of LAPTM5, this event is not necessary for LAPTM5 sorting. Rather, the Nedd4-LAPTM5 complex recruits ubiquitinated GGA3, which binds the UIM of LAPTM5. Hence, I propose a novel mechanism by which the ubiquitin ligase Nedd4, via interactions with GGA3 and cargo (LAPTM5), regulates cargo trafficking to the lysosome without requiring cargo ubiquitination. Because nothing was known about the biological function of LAPTM5, at the beginning of my Ph.D. training, I set out to determine the role of LAPTM5 in the innate immune cells. I demonstrate that LAPTM5 interacts with kinesin, a motor protein previously implicated in the anterograde movement of the late endosomal/lysosomal compartments. In dendritic cells, I show that upon maturation LAPTM5 is present within endolysosomal tubules formed by class II MHC molecules. Although I find that LAPTM5 is dispensable for the translocation of peptide-loaded MHC II molecules to the cell surface, this study extends our knowledge of the repertoire of proteins present within tubules formed by the MHC II compartments in activated dendritic cells. In macrophages, I demonstrate that LAPTM5 acts as a positive regulator of NFκB and MAPK signaling cascades, and promotes efficient proinflammatory cytokine production in response to several inducers of macrophage activation. During TNFα stimulation, LAPTM5 is required for proper initiation of NFκB signaling by acting at the receptor-proximate level. Thus, my findings indicate that LAPTM5 is an important component of inflammatory signaling cascades in macrophages and highlight a role for the endosomal/lysosomal system in regulating these cascades. Collectively, the work presented in this thesis broadens our understanding of lysosomal membrane protein sorting and function.

The lysosomal compartment is a major site for intracellular degradation. Lysosomal degradation of the cell’s own constituents, so-called autophagy, not only provides a cell with nutrients, but also removes damaged and potentially dangerous endogenous structures, thus securing intracellular homeostasis. On the other hand, lysosomes have been shown to be involved in the initial stages of apoptosis, and the protective effect of autophagy has been suggested to switch to cell death when excessive. Ageing-related changes of cellular structures result from damage caused by eactive oxygen species (ROS), which are an inevitable by-product of aerobic life. Intracellular turnover of compromised organelles and macromolecules, to which lysosomal degradation is a major contributor, does not function perfectly, even under favourable conditions. This inherent incompleteness of lysosomal degradation is responsible for the accumulation of a variety of nondegraded and functionally inefficient structures, which can be considered biological “garbage”. Biological “garbage” includes damaged non-degraded macromolecules and organelles, as well as intralysosomal non-degradable polymer-like structure called lipofuscin, or age pigment. Although accumulation of biological “garbage” has been suggested harmful, little is known about the mechanisms of its deleterious effects. To gain a better understanding of ageing-related changes of the lysosomal compartment and their influence on cell functions, we focused on studying: (1) the role of macroautophagy in the turnover of organelles and lipofuscin formation; (2) the role of biological “garbage” accumulation in the development of ageing-related changes and eventual death of growth-arrested, postmitotic-like cells; (3) the possible cell-protective effect of mitosis; (4) the influence of lipofuscin on cell survival during complete starvation; and (5) the effects of lipofuscin on lysosomal stability. As a model of induced biological “garbage” accumulation we used confluent human fibroblasts treated with the autophagy inhibitor 3-methyladenine (3MA). Alternatively, lysosomal degradation was suppressed by using the cysteine protease inhibitor leupeptin, or the cathepsin D inhibitor pepstatin A. As a cellular model of aged cells, we used lipofucsin-loaded human fibroblasts. Lipofuscin-loading was achieved by culturing confluent fibroblasts under hyperoxic conditions for 2-4 months. Using these in vitro models, the present study shows that: (1) inhibition of autophagy results in accumulation of lysosome-associated autofluorescent material and mitochondria with low membrane potential; (2) detrimental effect of biological “garbage” accumulation following inhibition of autophagy is prevented by continuous cell division; (3) lipofuscin-loaded cells are more resistant to starvation-induced cell death than control cells; (4) lysosomes of lipofuscinloaded fibroblasts are more resistant to the organelle-targeted stress then lysosomes of control cells. Based on the results of the present study we conclude that properly operating autophagic machinery plays a crucial role in preventing age-related changes associated with accumulation of biological “garbage”. We also suggest that continual proliferation is the natural mechanism by which cells cope with the accumulation of non-degradable material, employing mechanical dilution during the cell division. Finally, we introduce an idea of lipofuscin being a hormetic agent, and possibly possessing some lysosome-stabilising properties. Better understanding of the influence of the age-related accumulation of biological “garbage” on cellular functions may be helpful for future development of anti-ageing therapy and management of age-associated pathologies.

The mammalian target of rapamycin complex 1 (mTORC1) is known to regulate lipogenesis through sterol regulatory element binding proteins (SREBPs), master regulators of cholesterol and fatty acid synthesis. Through an incompletely understood mechanism, mTORC1 triggers translocation of SREBPs, an endoplasmic reticulum (ER) resident protein, to the Golgi, where mature SREBP is proteolytically produced to activate transcription of lipogenic genes. Low ER cholesterol is a well-known trigger for SREBPs activation, which includes translocation, maturation, and transcriptional activation. The study investigated whether mTORC1 activates SREBP by limiting cholesterol delivery to the ER. The findings indicate an increase in mTORC1 activity is accompanied by lower ER cholesterol and by SREBP-2 activation, a transcription factor primarily responsible for cholesterol synthesis. A decrease in mTORC1 activity, on another hand, coincides with higher ER cholesterol and lower SERBP-2 activity. I further report that this ER cholesterol is of lysosomal origin, as blocking the exit of cholesterol from lysosomes by U18666A or NPC1 siRNA prevents ER cholesterol from rising and, consequently, SREBP-2 is activated without mTORC1 activation. I identified two membrane trafficking processes, triggered by low mTORC1 activity, supply the lysosomes with cholesterol: autophagy and re-routing of endosomes to lysosomes. Indeed, a dual blockade by Atg5-/- and rab5 kept the ER cholesterol low even when mTORC1 activity was low, and resulted in SREBP-2 activation. Conversely, over-expressing Atg7, which forces autophagy, raises the ER cholesterol and suppresses SREBP-2 activity even when mTORC1 activity is high. Thus, it can be concluded that mTORC1 actively suppresses the formation of autophagosomes and promotes endosomal recycling, both of which prevents cholesterol to reach the lysosomes, thereby reducing cholesterol levels in the ER and activating SREBP-2.

Macroautophagy (autophagy) is a vesicle trafficking process that targets cytoplasmic cargoes to the lysosome for degradation and underlies multiple human disorders. Pioneering work in Saccharomyces cerevisiae defined the core autophagy machinery, but animals possess autophagy regulators that were not identified in yeast. Autophagic flux occurs when autophagy rate increases or decreases in response to various cellular cues, such as nutrient availability. Indeed, dysregulated autophagy rates contribute to disease, making autophagy- modulation a therapeutic avenue to treat cancer, neurodegenerative disorders, and other diseases. To identify novel regulators of autophagy in animals, I investigated autophagy in the context of animal development using Drosophila. In my dissertation, I screened for phosphoinositide phosphatases that influence autophagy, and identifed CG3530/dMtmr6, a previously uncharacterized phosphatase. CG3530/dMtmr6 is homologous to the human MTMR6 subfamily of myotubularin-related 3-phosphoinositide phosphatases. I showed that dMtmr6 functions as a regulator of autophagic flux in multiple Drosophila cell types, and the MTMR6 family member MTMR8 functions similarly in autophagy of higher animal cells. Decreased dMtmr6 function resulted in autophagic vesicle accumulation, lysosome biogenesis, and impaired both fluid phase endocytosis in the fat body and phagocytosis in embryonic macrophages. Additionally, dMtmr6 is required for development and viability in Drosophila. In human cells, lysosome homeostasis requires both the MTMR8 PH domain and catalytic cysteine residue, but only the PH domain is required to maintain autophagic flux. Collectively, this work identified a role for dMtmr6 and MTMR8 in autophagic flux and lysosome homeostasis.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.