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Copies of author's previously published articles inserted. / Errata pasted onto verso of back end-paper. / Bibliography: leaves 163-189. / xviii, 203 leaves, [26] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis describes the results of an investigation to determine the molecular basis of an uncharacterised (4;11)(q21;p15) translocation in a patient with T-cell lymphocytic leukemia. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 2000

Thesis (Ph.D.) -- University of Adelaide, Dept. of Medicine, 2000. / Copies of author's previously published articles inserted. Errata pasted onto verso of back end-paper. Bibliography: leaves 163-189.

HSPC1 (Hsp90), a member of the anti-apoptotic Heat Shock Protein (HSP) family appears to play a pivotal role in the development and maintenance of several tumour cell characteristics and as a result has become a target for novel anti-cancer therapies. HSPC1 inhibitors have been tested in clinical trials on a wide variety of cancer types with moderate success. However, despite recent advantages in HSPC1 inhibitor development, the effects of these drugs are not consistent. A number of factors may play a role in determining cell sensitivity to these inhibitors. As Chronic Lymphocytic Leukaemia (CLL) is such a heterogeneous disease with great variation in baseline HSP levels and other proteins amongst the patient cohort, it would not be unreasonable to assume that HSPC1 inhibitors may have varying success as a treatment strategy for this disease. The present study examined the effects of four HSPC1 inhibitors on primary CLL cells, as well as cells from healthy control subjects, and analysed a number of HSPC1 client proteins to assess the efficacy of these inhibitors. Great variation in cellular response to these drugs was observed in both CLL and healthy control subjects. Analysis of HSPC1 client proteins in these cells including ZAP-70, Akt, NF-kB and HSPA1A, revealed that HSPC1 inhibitors do not effect client protein levels in all samples. The results suggest that these inhibitors should not be considered as a universal treatment strategy for CLL and provide a basis for further study into elucidating the mechanisms behind HSPC1 inhibitor resistance. The final aim of this work was to investigate the role of the microenvironment in CLL progression, where a co-culture system was used as an in-vitro tool. Whilst consistent data was obtained using cell lines, and showed that microenvironmental factors promoted resistance to HSPC1 inhibitors, use of primary CLL cells in this model produced inconsistent data, again highlighting the heterogeneity of the disease.

The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling pathway is constitutively active in a variety of cancers, including chronic lymphocytic leukaemia (CLL). The importance of this signalling pathway identifies it as a prime therapeutic target; however, the complexity and potential side effects of inhibiting NF-κB have thus far made the clinical use of NF-κB inhibitors a relatively unexplored resource in this disease. This article discusses the role of NF-κB in CLL as a common crossroad for pathways promoting drug resistance in CLL. We provide the background on how this pathway contributes to both spontaneous and drug-induced apoptosis. Potential new avenues to regulate this pathway in CLL are also discussed.

During stress conditions, glucocorticoids are secreted and exert most of their physiological responses by binding to and modulating the transcriptional activity of the glucocorticoid receptor (GR). Once activated, GR can regulate numerous cellular processes including inflammation, development, growth, metabolism and apoptosis. Although glucocorticoids have been used in the treatment of leukaemia for over 50 years, with the molecular mechanisms by which steroids exert their pro-apoptotic effect, the pathways responsible for the development of resistance to glucocorticoid treatment, as well as their role in the programmed cell death in other tissue types have not been precisely defined. Research has demonstrated that glucocorticoid-induced apoptosis requires a transcriptionally active form of GR and is executed by the induction of the intrinsic pathway of apoptosis. In addition, GR is regulated by diverse types of cytotoxic stress; including UV irradiation and hypoxia, which alter the receptor’s transcriptional activity through multiple mechanisms. These include post-translational modifications, subcellular localisation and interaction of the receptor with co-regulator proteins. The aims of this study are to identify novel members of the Bcl-2 family that are regulated at the transcriptional level by GR in both leukaemia and other tissue types where glucocorticoids promote cell survival. In addition, the molecular crosstalk between signalling pathways activated by cytotoxic stress conditions and the mechanisms by which they differentially modulate the apoptotic response will be investigated. Results obtained in this study have identified putative glucocorticoid response elements in the promoters of the BH3-only pro-apoptotic gene NOXA and the anti-apoptotic gene Mcl-1 and confirmed that both NOXA and Mcl-1 are direct GR transcriptional targets. The glucocorticoid-mediated expression of NOXA and Mcl-1 alters their protein-protein interaction pattern, leading to the subsequent destabilisation of Mcl-1 in cell lines that undergo glucocorticoid-induced apoptosis. Investigation into the effects that other cytotoxic stress pathways have on GR function have revealed that serine 226 phosphorylation of GR by JNK occurs in a rapid and transient manner. Phosphorylation has inhibitory effects on the transcription of GR targets in a gene-specific manner, including the differential regulation of NOXA gene expression. During hypoxia, glucocorticoids differentially regulate the GR and HIF-1 target genes, NOXA and Mcl-1, altering the apoptotic response. This study has provided additional insight into the molecular mechanisms that govern glucocorticoid-induced programmed cell death and revealed mechanisms by which glucocorticoids and cytotoxic stress pathways crosstalk, regulating apoptosis.

Many haematological malignancies are associated with widespread alterations of the transcriptional and epigenetic programmes. Changes in DNA methylation provide the clearest example of epigenetic changes, but the mechanism(s) underlying such changes is unknown. To investigate this I studied DNA methylation across an ~80kb segment of the genome which is not known to be mutated in haematological malignancies. Methylation was perturbed in 35-100% of samples of DNA from individuals with a wide range of haematological malignancies but not in non-malignant haematological disorders. DNA methylation was comprehensively assessed by Southern blot analysis, classical bisulphite sequencing and using a newly developed capture bisulphite sequencing protocol. The results were also compared with analysis by MeDIP, an immunoprecipitation-based technique. These analyses provide methylation status at various levels including individual CpG resolution. This showed both gain and loss of methylation at CpG dinucleotides. Of interest, hypomethylation was most frequently seen in intergenic regions corresponding to transcription factor binding sites and areas of increased chromosome accessibility. These observations suggested that hypomethylation of the genome in haematological malignancies could arise from aberrantly expressed DNA binding proteins which, recruited to sequences in regions of open chromatin, would protect the underlying CpG dinucleotides from the methylation machinery. This, in turn, could lead to passive demethylation accumulating with increasing cell divisions. This hypothesis was tested with electrophoretic mobility shift assays using oligonucleotides representing the DNA underlying one such region. This showed that, compared to nuclear extracts from the lymphocytes of normal individuals, those from patients with CLL were enriched for a protein which binds to oligonucleotides containing the underlying sequence. Using a mass spectrometry approach, I identified a variety of proteins that may bind such regions and account for their passive demethylation in haematological malignancies.

Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia, and despite recent introduction of targeted therapies, remains incurable. An important hallmark of CLL is severe immune deficiency, including the failure to mount effective anti-tumour immune responses. This can partly be explained by insufficient antigen presentation, but also by the existence of complex CLL-induced T-cell defects. Based on the cancer immuno-editing hypothesis that the immune system not only protects a host against tumour formation but can also be compromised to actively provide a pro-tumour microenvironment, modulating cancer-induced T-cell defects could restore the full anti-tumour response and result in more durable clinical responses. The immune checkpoint molecules PD-1 (expressed on activated immune effector cells) and PD-L1 (expressed on antigen-presenting and microenvironmental cells including tumour cells) have emerged as important mediators of T-cell suppression. Several studies suggest that PD-L1/PD-1 inhibitory signalling in CLL might be overcome by the immune modulatory drug lenalidomide. Furthermore, directly targeting PDL-1/PD-1 interactions produces significant responses in solid cancers. However, similar studies are notably absent in CLL, and the effect of PDL-1/PD-1 blockade on restoring cancer-induced immune dysfunction is not understood. Transgenic Eμ-TCL1 mice have been extensively validated as an adequate preclinical model of aggressive human CLL, and our group showed their suitability to mirror T-cell defects observed in human CLL. Using the Eμ-TCL1 model, this dissertation project substantially extends our previous characterization of CLL-induced T-cell dysfunction and evaluates the functional impact of PD-L1/PD-1 inhibitory signalling both in parallel with disease development and in different microenvironments. The findings to be described here demonstrate that developing CLL is associated with specific T-cell subset alterations, phenotypic changes, and functional defects that are very similar in peripheral blood and secondary lymphoid organs. In addition to PD-L1, PD-L2 is identified as a potential mediator of inhibitory signalling in CLL. CD8+ T cells in leukaemic mice are characterised as a functionally heterogeneous population, in which subsets of cells are able to exert effector functions despite PD-1 expression. In vivo lenalidomide treatment repairs selected phenotypic alterations and immune synapse formation, and a PD-L1 IgG blocking antibody effectively controls disease and reverses global T-cell defects even in cells expressing PD-1. In sum, this work provides a strong rationale to explore PD-L1/PD-1 targeting in CLL clinical trials, potentially in combination with novel agents.

Chronic lymphocytic leukaemia (CLL) is a disease characterized by abnormal accumulation of B cells in the blood, bone marrow, lymph nodes and spleen. Several decades ago, it was concluded that CLL lymphocytes might be unable to proliferate in vivo but a recent study performed in vivo in patients with CLL has shown in contrast that these cells proliferate. However, an important and still unanswered question is whether CLL cells proliferate faster or slower compared to their normal counterparts. In this context, the turnover of CLL cell population was compared to the kinetics parameters of normal B lymphocytes after labelling with deuterium glucose. We have also compared the metabolic activity of CLL cells with B lymphocytes from healthy subjects using a new method for measuring RNA turnover in vivo. Based on these observations, we found that leukaemic cells proliferate less frequently than healthy patient and that metabolic activity via measurement of RNA turnover rate is significantly reduced in CLL patients.

Biased immunoglobulin heavy variable (IGHV) gene usage and distinctive B-cell receptor (BCR) features have been reported in chronic lymphocytic leukaemia (CLL), which may reflect clonal selection by antigens during disease development. Furthermore, the IGHV gene mutation status distinguishes two clinical entities of CLL, where patients with unmutated IGHV genes have an inferior prognosis compared to those with mutated IGHV genes. Recently, one subgroup of CLL patients expressing the IGHV3-21 gene was found to display highly similar immunoglobulin (IG) gene features, even within the heavy chain complementarity-determining region 3 (HCDR3). Patients in this subgroup typically had a poor prognosis. In paper I, we aimed to identify further subgroups with restricted BCR features among 346 CLL cases. Six subsets were defined which carried virtually identical BCRs in terms of rearranged heavy and light chain (LC) IG genes and CDR3 length and composition. In paper II, we investigated 90 IGHV3-21 cases from diverse geographical locations. We confirmed the highly restricted HCDR3 characteristics in 56% of patients and a biased usage of the IGLV3-21 gene in 72% of cases. Survival analysis also confirmed the poor outcome of this group, irrespective of IGHV gene mutation status and geographical origin. Papers III and IV involved a large-scale analysis of IGH and IG kappa and lambda (IGK/L) gene rearrangements, to define subsets with ‘stereotyped’ BCRs and also to systematically examine the somatic hypermutation (SHM) features of the IG genes in CLL. We studied a cohort of 1967 IGH and 891 IGK/L gene sequences from 1939 patients from 6 European institutions. Over 5300 IGH and ~4700 IGK/L sequences from non-CLL B cells were used as a control data set. In total, 110 CLL stereotyped subsets were defined according to HCDR3 homology. Striking IGK/L gene biases were also evident within subsets, along with distinctive K/LCDR3 features, such as length and amino acid composition. At cohort level, the patterns of mutation appeared to be consistent with that of a canonical SHM mechanism. However, at a subgroup level, certain stereotyped subsets, e.g. IGHV3-21/IGLV3-21 and IGHV4-34/IGKV2-30 CLL, deviated from this pattern. Furthermore, recurrent ‘stereotyped’ mutations occurred in cases belonging to subsets with restricted HCDR3s, in both IGHV and IGK/LV genes, which were subset- and CLL-biased when compared to non-CLL B cells. In conclusion, our findings implicate antigen selection as a significant factor in the pathogenesis of CLL, particularly in cases carrying stereotyped BCRs. The presence of stereotyped mutations throughout the VH and VL domain also indicates involvement of IG regions other than the CDR3 in antigen recognition. Finally, biased IGK/L gene usage and specific K/LCDR3 features are strong indications that LCs are crucial in shaping the specificity of leukemic BCRs, in association with defined heavy chains.