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Bibliography: leaves 1-14 of section Reference / [175] leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, 1985

Glucocorticoids have long been used in the treatment of acute lymphoblastic leukaemia due to their ability to cause cell cycle arrest and apoptosis of lymphoid cells. However, some patients do not respond to glucocorticoid treatment and the majority, who initially respond, may relapse upon prolonged hormone treatment. The inefficiency of the treatment is mainly attributed to the gradual loss of the cellular sensitivity to glucocorticoid-induced apoptosis. Therefore, the need to understand the molecular mechanisms of resistance/ sensitivity of acute lymphoblastic leukaemia cells to glucocorticoid-induced apoptosis is of vital importance, as this will help to develop better prognostic outcomes and improve glucocorticoids therapy. Several mechanisms have been proposed to explain the evasion of glucocorticoid mediated apoptosis in resistant cells. These include post-translational modifications of GR especially phosphorylation which modulates the GR transcriptional activity, and GR mediated signalling thereby affecting gene expression and hence the balance between pro- and anti-apoptotic Bcl-2 family members. In addition the concentration of components of the energy metabolism pathways (i.e. oxidative phosphorylation and glycolysis) and ROS generation are altered in the acute lymphoblastic leukaemia cells. The hypothesis that differentially phosphorylated GR in the resistant versus sensitive ALL cells modulate GR transcriptional activity and target selectively resulting in diverse pro- or anti-apoptotic Bcl-2 family members' gene expression in the two cell lines was tested. Furthermore, in a similar manner, the possibility that differential GR phosphorylation diversely affected gene expression of GR transcriptional target genes that are components of cellular energy production pathways in resistant versus sensitive cells, altering energy and ROS production levels in distinct ways in the two cell lines was explored. GR was found to be predominantly phosphorylated at S211 in the glucocorticoid-sensitive CEM C7-14, and at S226 in the glucocorticoid-resistant CEM C1-15 cells. Differential GR phosphorylation is presumably an indication of dominant p38 MAPK activity in CEM C7-14 and JNK kinase activity in CEM C1-15, which could lead to adverse gene expression of some pro- and anti-apoptotic Bcl-2 family members and particularly Mcl-1, in the two cell lines. Furthermore, differential GR phosphorylation at S211 and S226 in CEM C7-14 and CEM C1-15 affected the gene expression of the Cytochrome C Oxidase assembly factors Surf-1 and SCO2 as well as the nuclear encoded Cytochrome C Oxidase subunit COX-Va and the mitochondrial encoded COX-I, COX-II and COX-III. This effect was more pronounced in the glucocorticoid-sensitive CEM C7-14 cells, probably due to the fact that GR was predominantly phosphorylated at S211 and hence transcriptionally active in these cells. Moreover, in comparison to the resistant CEM C1-15 cells, the CEM C7-14 cells exhibited higher levels of ROS, increased number of active mitochondria and up-regulated glycolysis upon inhibition of oxidative phosphorylation. Glucocorticoids further reduced ROS levels in the CEM C1-15 cells, and increased the NADH/ NAD+ ratio. In conclusion results presented in this thesis provide evidence that differential GR phosphorylation in resistant versus sensitive to glucocorticoid induced apoptosis cells plays essential role in the regulation of programmed cell death and energy metabolism pathways, offering a potential explanation for the molecular events that determine resistance/sensitivity to glucocorticoid-induced apoptosis in ALL cells.

La survie des cellules cancéreuses dépend d’une activité énergétique et biosynthétique accrue et la glutamine participe à de nombreux processus nécessaires à cette adaptation métabolique. Dans les leucémies aiguës myéloïdes (LAM), la croissance et la prolifération sont favorisées par l’activation anormale de plusieurs voies de signalisation, et notamment par la voie mTORC1. Les acides aminés essentiels, et en particulier la leucine, sont indispensables à l’activation de mTORC1. La glutamine est captée par la cellule via le transporteur SLC1A5 et permet ensuite l’entrée de la leucine via le transporteur bidirectionnel SLC7A5. La concentration en glutamine est donc une étape limitante dans l’activation de mTORC1 par la leucine. Nous avons étudié les effets de la privation en glutamine dans les LAM à l’aide de différents outils (milieu sans glutamine, shARN inhibant l’expression du transporteur de la glutamine SLC1A5 et la drogue L-asparaginase, qui a une activité de glutaminase extracellulaire), et observé une inhibition de mTORC1 et de la synthèse protéique. L’inhibition du transporteur SLC1A5 inhibe la pousse tumorale dans un modèle de xénotransplantation. La L-asparaginase inhibe mTORC1 et induit une apoptose de façon proportionnelle à son activité glutaminase et complètement indépendante de la concentration en asparagine. La privation en glutamine induit l’expression de la glutamine synthase et l’autophagie, et ces deux processus peuvent être des mécanismes de résistance intrinsèques ou acquis dans certaines lignées leucémiques. L’apoptose induite par la privation en glutamine n’est cependant pas liée à l’inhibition de mTORC1, puisqu’elle n’est pas diminuée par l’utilisation d’un mutant de mTOR non inhibé par la privation en glutamine. Nous nous sommes donc intéressés à une autre voie dépendante de la glutamine dans de nombreux cancers, la phosphorylation oxydative. L’étape initiale du catabolisme intracellulaire de la glutamine est la conversion de la glutamine en glutamate par des enzymes appelées glutaminases. Différentes isoformes des glutaminases existent qui sont codées chez l’homme par les gènes GLS1 et GLS2. Le glutamate est ensuite transformé en α-cétoglutarate, intermédiaire du cycle TCA. Dans les lignées de LAM, la privation en glutamine inhibe la phosphorylation oxydative mitochondriale. Nous avons observé que la protéine glutaminase C (GAC), une des isoformes de GLS1, est constamment exprimée dans les LAM mais aussi dans les progéniteurs hématopoïétiques CD34+ normaux. L’inhibition d’expression de la GLS1 par des shARN inductibles ou bien par le composé CB-839 réduit la phosphorylation oxydative, conduisant à une inhibition de prolifération et à une induction d’apoptose des cellules leucémiques. L’invalidation génétique de la GLS1 inhibe la formation de tumeur et améliore la survie des souris dans un modèle de xénotransplantation. A l’inverse, le ciblage de la GLS1 n’a pas d’effets cytotoxiques ni cytostatiques sur les progéniteurs hématopoïétiques normaux. Ces effets anti-leucémiques sont inhibés par l’adjonction d’α-cétoglutarate, et ceux induit par le CB-839 sont abrogés lorsqu’est exprimé de façon ectopique un mutant GACK320A hyperactif, attestant du rôle essentiel du maintien d’un cycle TCA actif dans les cellules de LAM. Enfin, nous montrons que l’inhibition de la glutaminolyse active la voie d’apoptose mitochondriale intrinsèque et agit en synergie avec l’inhibition spécifique de BCL-2 par l’ABT-199. Ces résultats démontrent que le ciblage spécifique de la glutaminolyse est une autre façon d’exploiter l’addiction à la glutamine des cellules leucémiques de LAM et que le maintien d’un cycle TCA actif est essentiel à la survie de ces cellules. / Cancer cells survival is dependent on high energetic and biosynthetic activity, and glutamine is involved in many metabolic processes necessary for this adaptation. In acute myeloid leukemia (AML), growth and proliferation are promoted by activation of several signaling pathways, including mTORC1. Essential amino acids, in particular leucine, are required for mTORC1 activation. Glutamine enters into the cell via the SLC1A5 transporter and then allows the input of leucine via the bidirectional SLC7A5 transporter. Therefore, the intracellular glutamine concentration is a limiting step in the activation of mTORC1 by leucine. We studied the effects of glutamine deprivation in AML using different tools (medium without glutamine, shRNA against the SLC1A5 glutamine transporter and the drug L-asparaginase, which has an extracellular glutaminase activity) and observed mTORC1 and protein synthesis inhibition. SLC1A5 transporter knockdown inhibits tumor growth in a xenotransplantation model. L-asparaginase inhibits mTORC1 and induces apoptosis in proportion to its glutaminase activity and independently of asparagine concentration. Glutamine privation induces the expression of glutamine synthase and autophagy, and these two processes are involved in the resistance to glutamine privation in some leukemic cell lines. However, apoptosis induced by glutamine privation is not related to the inhibition of mTORC1, since it is not modified in the presence of a constitutively active mutant of mTOR. We next focused on the oxidative phosphorylation, another glutamine dependent pathway in many cancers. The initial step of the intracellular catabolism of glutamine is the conversion of glutamine to glutamate by enzymes called glutaminases. Different glutaminases isoforms exist that are encoded by the GLS1 and GLS2 genes. Glutamate is then converted to α-ketoglutarate, an essential TCA cycle intermediate. In AML cell lines, we observed that glutamine privation inhibits mitochondrial oxidative phosphorylation. The protein glutaminase C (GAC), an isoform of GLS1, is constantly expressed in AML but also in normal CD34 + hematopoietic progenitors. The knockdown of GLS1 by inducible shRNA or by the CB-839 compound reduced oxidative phosphorylation, leading to proliferation inhibition and apoptosis induction in leukemia cells. Genetic invalidation of GLS1 inhibits tumor formation and improves survival of mice in a xenograft model. Conversely, the targeting of GLS1 has no cytotoxic or cytostatic effects on normal hematopoietic progenitors. These anti-leukemic effects are inhibited by the addition of α-ketoglutarate, and those induced by the CB-839 are suppressed in the presence of an ectopically expressed GACK320A hyperactive mutant, confirming the essential role of maintaining an active TCA cycle in AML cells. Finally, we showed that glutaminolysis inhibition induces the intrinsic mitochondrial pathway of apoptosis and acts synergistically with the specific inhibition of BCL-2 by ABT-199. These results demonstrate that specific targeting of glutaminolysis is another way to exploit glutamine addiction in AML and that an active TCA cycle in essential for AML cell survival.