Traumatic lumbar punctures (TLPs) in children with acute lymphoblastic leukemia are associated with a poorer prognosis. The objective of this study was to determine risk factors for TLPs using a retrospective cohort. We compared and contrasted three different regression methods for the analysis of repeated-measures data. In the multivariable model using generalized estimating equations, variables significantly associated with TLPs were age < l year or ≥ 10 years; body mass index percentile ≥ 95; platelet counts < 100 x 103/µL; fewer days since previous LP, and a preceding TLP. The same variables, with similar estimates and confidence-intervals, were identified by the random-effects model. In a fixed-effects model where each patient was used as their own control, days since prior LP and the effect of using image-guidance were significant. Random-effects and GEE lead to similar conclusions, whereas fixed-effects discards between-subject comparisons and leads to different estimates and interpretation of results.

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.

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.

FDA-approved drugs with previously unrecognized anti-cancer activity could be rapidly repurposed for this new indication. We compiled a library of such off-patent drugs to screen four leukemia cell lines and identified the anti-parasitic agent ivermectin that induced cell death at low micromolar concentrations. In cell death and clonogenic growth assays, low micromolar concentration of ivermectin significantly reduced viability of leukemia cell lines and patient samples compared to normal peripheral blood stem cells. In xenograft mouse models of leukemia, ivermectin decreased tumor volume and weight by up to 72% when compared to control without observable toxicity at pharmacologically achievable dosage. In this study, we further demonstrate that ivermectin activates chloride channels in leukemia cells leading to membrane hyperpolarization and increased reactive oxygen species generation. In addition, it demonstrated synergistic interaction when used in combination with Daunorubicin and Cytarabine. Therefore, this study highlights a potential new therapeutic strategy in repurposing ivermectin for the treatment of AML.

Understanding the nature and timing of leukaemogenic events during the development of childhood acute lymphoblastic leukaemia (ALL) will enable intervention that could prevent ALL in the future. We hypothesised that 9p21 deletion in childhood ALL may unmask predisposing genetic events that would allow us to determine the "nature" of initiating events in childhood ALL; whereas the inclusion, or exclusion, of random �N� nucleotides in normal immunoglobulin gene rearrangements from the developing fetus and the expression of terminal deoxynucleotidyl transferase (TdT) in fetal lymphocytes may allow us to unmask the developmental window during which the first transforming leukaemic event occurs in a pre-leukaemic B cell. The most frequent genetic abnormality in childhood ALL is deletion of chromosome 9p21, with the minimal region of deletion including the CDKN2-locus, making genes at this locus candidates for a predisposing genetic event in ALL. To determine whether genomic imprinting might be involved in ALL at the 9p21 locus we investigated the imprinting status of the candidate genes CDKN2A, CDKN2B and ARF. No evidence for genomic imprinting of ARF was found in this study. Because no expressed polymorphisms could be identified for CDKN2B, and CDKN2A expression was too low in normal tissues, the imprinting status of these genes could not be evaluated. Furthermore investigations in our laboratory have been unable to find genomic imprinting at any of these genes in mice. However, we have shown variation in allelic expression of ARF, which suggests a role for ARF haploinsufficiency in the onset of childhood ALL. A key feature of early human fetal lymphoid development is the absence of random �N� nucleotides between the rearranged V[H], D[H] and J[H] gene segments. The addition of �N� nucleotides at these junctions requires the enzyme terminal deoxynucleotidyl transferase (TdT). TdT is reported to not be expressed during early fetal lymphopoiesis but has been observed by the end of the first trimester, but data are sparse. The reported absence of N nucleotides in the majority of childhood ALLs suggests an early fetal origin. By defining the window-in-time during which TdT-negative B cell development occurs, we will be able to refine the timing of the origin of the B cells that give rise to ALL. Therefore we have sequenced and analysed the V[H]-DJ[H] and D[H]-J[H] junctions from immunoglobulin rearrangements in developing B cells in normal human fetuses aged from 5.1 to 11 weeks gestation. In this study 73 fetal IgH gene rearrangements were amplified from 21 different fetal liver samples. Only eight of the seventy-three rearrangements (11%) analysed in this study had no �N� nucleotides at the N1 (D[H]-J[H]) junction. This finding contrasts with the 24-28% of fetal rearrangements with no �N� nucleotides that have been reported in the literature. Furthermore, �N� nucleotides were shown to be present in the earliest sample, 5.1 weeks gestation. TdT expression was demonstrated by immunohistochemistry at 7.3 weeks and by RT-PCR at 8.3 weeks. B cell development in the fetal liver was detected as early as 6.5 weeks using flow cytometric analysis. Then, IgH gene rearrangements from 99 cases of childhood ALL were analysed. In total, 134 clone-specific IgH gene rearrangements were examined in this study. No association was found between the number of �N� nucleotides from complete and incomplete rearrangements at either the N1 (D[H]-J[H]) or N2 (V[H]-DJ[H]) junctions. Nor was any association observed between ALLs from children [less than or equal to] 3 years of age and those >3 years of age at diagnosis. These findings indicate that ALL IgH rearrangements do not have the paucity of �N� nucleotides that has been previously reported. The findings in this thesis suggest that there is no TdT-negative timepoint during B cell development and that there is no paucity of �N� nucleotides at the N1 junction in either fetal or childhood ALL IgH gene rearrangements.

This thesis is submitted as a PhD by Portfolio of Publications. It represents an integrated examination of the BCR-ABL oncogene as a precise indicator of treatment response, drug resistance and relapse in patients with chronic myeloid leukaemia (CML). / Thesis (PhDBiomedicalScience)--University of South Australia, 2005.

Chronic Myeloid Leukaemia (CML) is a clonal haemopoietic stem cell (HSC) disorder characterised by the presence of a disease-specific gene, BCR-ABL, which leads to the production of a bcr-abl mRNA transcript. CML is an ideal candidate for gene therapy using ribozymes (Rz), catalytic RNA molecules that cleave and inactivate target RNA in a sequence specific manner. Limited data is available on the activity of ribozymes in human CML cells. In this study, hammerhead ribozyme sequences directed against the b3a2 bcr-abl mRNA sequence (Rz6-Rz10) were cloned into several retroviral vectors. Initial experiments using MSCVHSA based retroviral constructs failed to express the sequences in cell lines. Rz cDNA fragments were then cloned into an LNL6 based retroviral vector (LGL1) encoding a GFP reporter gene and stable LGLRz constructs produced. Using cell sorting, high-titre PA317 producer cell line clones were isolated. Transcriptional silencing of the LGLRz6 producer cell line occurred with prolonged culture, with partial reversal on treatment with the demethylating agent 5' azacytidine. To assess the activity of these constructs in human cells, CD34+ HSC were isolated from newly diagnosed b3a2 Ph+ CML patients. Cells were transduced with either control LGL vector or the LGLRz6 construct. Transduced human cells were sorted based on GFP expression and placed into long-term HSC culture (LTC-IC assays). Using a common cDNA, RT-PCR was performed to detect the expression of both the transgene and bcr-abl in individual colonies derived from the LTCIC assay at various time points, allowing assessment of the effect of transgene expression on bcr-abl expression. LGLRz transgene expression was detectable for up to 6 weeks in culture. Colony RT-PCR results from 3 patients showed that expression of the LGLRz6 construct was associated with decreased bcr-abl expression. It also appeared that the reduced bcr-abl expression decreased the proliferation of Ph+ cells leading to their loss from culture. In summary, these results appear to show an effect of a retroviral vector containing a bcr-abl Rz sequence on human CML HSC. Targeting of bcr-abl remains a valid therapeutic goal in the Imatinib era, particularly if problems related to effective ribozyme delivery and targeting can be overcome.

Acute lymphoblastic leukaemia is the most common childhood cancer with cure rates of approximately 80%. This success can be attributed to the introduction of risk stratification for patients and employment of intensified treatment regimes for patients with high risk disease. However, the identification of prognostically important leukaemia subtypes, unfortunately, is an labour-intensive process. In addition, despite the success in treating childhood ALL, specific subgroups of patients nevertheless still have poor survival rates. This is particularly true for leukaemias characterised by chromosomal translocations involving the MLL oncogene on chromosome 11q23. By using a novel bioinformatics approach, GeneRave, a set of 12 classifier genes (PTPRK, FOS, ENG, Lgal-S1, TCFL5, LRMP, CTGF, IGJ, MX1, PENK, CD3D and HBG1) was selected from a publicly available U95 Affymetrix microarray dataset. Real time PCR carried out on a blinded cohort of 58 primary ALL samples yielded an accuracy of 86%. The absence of PENK gene expression in the majority of ALL samples tested appears to have decreased the overall accuracy. Nevertheless, the results indicate that this method of classification can be easily and quickly performed and therefore may be useful as an adjunct to routinely used methodology in the diagnostic classification of childhood ALL. Parellal screening of a 34,000 chemical small molecules library identified 30 ???hits??? that exhibited specificity toward leukaemia, and many of which shared structural similarity. The cytotoxic effect of these compounds was further investigated in a panel of 19 cell lines that included 3 MLL-translocated (MV411, THP-1 and PER-485), 7 non-MLL-translocated leukaemias (REH, Jurkat, K562, HL60, Hal-01, UOB-B, NB4), 2 immortalized normal blood cell lines, 4 non-leukaemic tumour cell lines (Calu6, MCF7, BE(2)-C, and HeLa) and 3 normal cell lines (HSF, MCF10a and MRC5). In particular, two compounds were identified, SM6 and SM7, that were highly effective at killing MLL-translocated cell lines in the low micromolar range while having little or no effect on the other cell lines. Treatment of PER-485 cells with SM6 and SM7 showed mark down-regulation of the MLL chimaeric fusion protein together with its down-stream targets. One other more broadly acting anti-leukaemia compounds were also identified.

Relapsed ALL remams one of the most common causes of death from disease in children. Broad-range drug resistance is often associated with relapse, although its underlying molecular mechanisms remained poorly understood. The aim of this thesis was to establish an in vivo model using the non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse strain, to facilitate the engraftment, expansion and characterisation of childhood ALL cells, obtained from patients at diagnosis or relapse. Mice were inoculated with leukaemia cells from patients' biopsies and engraftment was monitored by the proportion of human CD45+ cells in the blood. Successful leukaemia engraftment was achieved for 20/20 patient biopsies. Continuous passaging of ten xenografts has also been achieved. Immunophenotypic analysis showed only minor changes in cell surface markers after passage in mice. Leukaemia dissemination in murine bone marrow, liver, spleen and blood was consistent with the human disease. The in vivo responses of ten continuous xenografts to dexamethasone and vincristine, but not methotrexate, significantly correlated with patient outcome (p<0.05). Xenograft sub-lines resistant to vincristine, dexamethasone, methotrexate and cytosine arabinoside were also selected by in vivo drug treatments, although these sublines were not found to be cross resistant to structurally unrelated drugs. Resistance to vincristine, either in in vivo selected sub-lines or inherently resistant xenografts, was not associated with increased activity of drug efflux pumps such as P-gp or MRPl. Class I ?? tubulin levels remained unchanged when compared between vincristine resistant sublines and their parental xenografts. Decreased expression of stathmin and increased polymerised tubulin were observed in vincristine resistant sub-lines, suggesting a possible mechanism of counteracting the depolymerising effects of vincristine. In summary, this study has shown that primary ALL cells engraft efficiently into NOD/SCID mice, and indicates that their response to vincristine and dexamethasone mimics the clinical situation. This model appears to be highly relevant for the study of childhood ALL and will provide the foundation to delineate clinically relevant mechanisms of drug resistance.

Errata inserted. / Includes bibliographical references. / xi, 119, [75] leaves, [21] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study demonstrates a conserved structural organisation of the mouse LIF gene that produces three differentially localised proteins. This provides a mechanism for specific control of the sites of LIF action and mechanisms for mediating the variety of putative actions for the LIF gene. Intracellular localisation of the LIF protein provides another example of intracellular cytokine action, mediated by a novel mechanism and provides a system for separate analysis of intracellular and extracellular cytokines. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry,1998?