Currently, the intensive chemotherapy remains the first line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although these regimens have significantly improved patient outcomes, their use is associated with debilitating morbidities and fatal relapses, highlighting the great need in new agents that target essential survival signals in leukemia. Thus, the overall goal of my project was to gain insights into the signaling abnormalities that regulate aberrant proliferation and survival of B-ALL cells in an effort to identify novel targets in this malignancy. This study demonstrated that pre-B cell receptor (pre-BCR)-independent spleen tyrosine kinase (SYK) activity was required for the survival and proliferation of a p53-/-PrkdcSCID/SCID mouse model of B-ALL. I extended this discovery to human disease, demonstrating that SYK was activated in primary B-ALL, independent of the pre-BCR expression. The small molecule SYK inhibitor fostamatinib (fosta) significantly attenuated proliferation of 79 primary diagnostic B-ALL samples at clinically achievable concentrations. Importantly, fosta treatment reduced dissemination of engrafting B-ALL cells into the spleen, liver, kidney and central nervous system (CNS) in a NOD.Prkdcscid/scidIl2rgtm1Wjl/SzJ xenotransplant model of B-ALL. Analysis of signaling abnormalities using a high-throughput phospho-flow cytometry platform demonstrated that pediatric and adult B-ALL samples exhibit variable basal activation of BCR, iii PI3K/AKT/mTOR, MAPK and JAK/STAT pathways. Importantly, we identified that fosta-mediated inhibition of SYK, PLC2, CRKL and EIF4E phosphorylation in B-ALL was predictive of its anti-leukemic activity, and was distinct from the cellular actions of other small molecule inhibitors of key nodal signaling pathways. Examination of molecular mechanism of fosta action by gene expression profiling revealed transcriptional effects of fosta treatment that included, most notably, potent inhibition of pathways involved in lymphocyte activation and inflammation. In conclusion, this study demonstrates that SYK signaling is crucial for B-ALL survival and provides detailed characterization of cellular and molecular mechanisms of fosta action in B-ALL. These data argue in favor of testing small molecule SYK inhibitors in pediatric and adult B-ALL.

New approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Analysis of the effects of pan-PIM protein kinase inhibitors on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 protein kinase levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Gene profiling using a publically available T-ALL dataset demonstrated overexpression of PIM1 in the majority of early T-cell precursor (ETP)-ALLs and a small subset of non-ETP ALL. While the PIM inhibitors blocked growth, they also stimulated ERK and STAT5 phosphorylation, demonstrating that activation of additional signaling pathways occurs with PIM inhibitor treatment. To block these pathways, Ponatinib, a broadly active tyrosine kinase inhibitor (TKI) used to treat chronic myelogenous leukemia, was added to this PIM-inhibitor regimen. The combination of Ponatinib with a PIM inhibitor resulted in synergistic T-ALL growth inhibition and marked apoptotic cell death. Treatment of mice engrafted with human T-ALL with these two agents significantly decreased the tumor burden and improved the survival of treated mice. This dual therapy has the potential to be developed as a novel approach to treat T-ALL with high PIM expression.

Pathological angiogenesis is an essential component of tumor growth, development, and metastasis for which few effective therapeutic options exist. Though many cancer therapies target the function of cell surface receptors, mechanisms regulating membrane receptor crosstalk remain unclear. Two important families of receptors in angiogenesis, the Ties and Integrins, respond to the extracellular environment via outside-in and, in the case of Integrins, also inside- out signaling. Recent reports showed that the endothelial specific tyrosine kinase receptor, Tie2, forms complexes with two of the endothelial Integrin heterodimers, α5β1 and αVβ3, providing a convenient mechanism for the integration of extracellular stimuli. Our data confirm the interaction between Integrins and Tie2 and additionally indicate an interaction with the orphan co-receptor Tie1. To elucidate the biological role of these macromolecular complexes, biochemical and biophysical methods including co-immunoprecipitation, FRET microscopy, and cellular based assays were used to follow receptor/Integrin association in response to the Tie2 ligands Angiopoietin-1 and -2 as well as the Integrin ligand fibronectin. Furthermore, structural analysis by small angle x-ray scattering of Tie2-ligand complexes and specific Integrin and Tie complexes are being used to identify the basis for growth factor receptor and Integrin signal transduction.

OBJECTIVE: Glioblastoma Multiforme (GBM), one of the most malignant forms of primary brain tumors, is characterized by its highly heterogenous genetic composition, aggressive infiltration of surrounding tissue, and resistance to current treatments. Gene expression analysis has characterized GBM into four main types, with a significant portion belonging to the Classical subtype, typified by overexpression and/or mutation of the epidermal growth factor receptor (EGFR). Also common to this subtype of GBM is the loss of crucial tumor suppressor genes Ink4A/ARF and PTEN, which contribute to the invasive nature and unregulated proliferation that underlie the GBM pathology. The high rate of tumor recurrence post treatment with surgical resection, chemotherapy, and radiation has driven the pursuit of more effective molecularly targeted therapies. This study was undertaken to determine the effects of two types of small molecule tyrosine kinase inhibitors on cells overexpressing wild-type EGFR in the context of their respective complements of tumor suppressor genes. METHODS: Several cell lines were established from mouse models of EGFR wild-type (EGFRWT) driven gliomagenesis and treated with 10 μM of type I tyrosine kinase inhibitors Gefitinib (Iressa®, Astra Zeneca), CI-1033 (Canertinib, Pfizer), or Dimethyl Sulfoxide vehicle. Cells were exposed to each drug treatment as part of a time course ranging from 0 to 24 hours and then evaluated by trypan blue exclusion and Western blot analysis for cell viability and molecular and biochemical effects respectively. RESULTS: Evaluation of cell viability indicated that CI-1033 caused a greater increase in cell death than gefitinib when compared to control treated cells regardless of the tumor suppressors lost. Gefitinib was found to cause cell death only in cells expressing the PTEN tumor suppressor whereas CI-1033 showed similar levels of cell death for cells deficient in Ink4A/ARF or both Ink4A/ARF and PTEN tumor suppressors. Western blot analysis revealed that CI-1033 more effectively inhibited EGFR compared to gefitinib. Treatment with both gefitinib and CI-1033 effectively blocked phosphorylation of EGFR, but this effect was less pronounced with gefitinib treatment. Further analysis of downstream signaling molecules showed a greater presence of cleaved caspase 3, a hallmark of apoptosis, in gefitinib treated cells expressing PTEN than in those cells treated with CI-1033. Cells deficient in both Ink4A/ARF and PTEN did not demonstrate any induction of cleaved caspase 3 following either treatment. CONCLUSIONS: Based on the significant differences in cell viability between treatments, CI-1033 is an overall more effective inhibitor of EGFRWT expressing cells lacking PTEN, while gefitinib and CI-1033 were found to be similarly effective in cells expressing PTEN. The results of western blot analysis indicate that total and irreversible EGFR inhibition may be necessary to induce cell death in a manner that effectively terminates downstream cell signaling. It is likely that CI-1033, unlike gefitinib, induces apoptosis in a caspase-independent manner, which may be one of the many differences in downstream effects produced by these two drugs. Further research is necessary to determine the extent to which each inhibitor shuts down proliferative cell signaling pathways such as PI3K-AKT and MEK-ERK signaling pathways downstream of EGFR. Overall, these data indicate that genotype plays an important role in the determination of therapeutic response and may aid in the evaluation of clinical prognoses.

The Receptor Tyrosine Kinase (RTK) Met, overexpressed or mutated in cancer, plays a major role in cancer progression and represents an attractive target for cancer therapy. This study aimed to investigate whether PI3K plays a role in Met oncogenicity. Three cell models were used: (i) NIH3T3 cells expressing WT Met or the constitutively active mutant M1268T Met; (ii) U87MG glioblastoma cells, with endogenous WT Met constitutively activated due to an autocrine loop; (iii) A549 lung cancer cells expressing endogenous WT Met, activated upon binding exogenous HGF. Met dependent Rac1 translocation to the plasma membrane, actin cytoskeleton organisation, cell migration, anchorage independent growth in soft agar and tumour growth were studied in the presence of inhibitors of pan-PI3K / mTOR, various PI3K Class I isoforms, mTOR or Akt, or following siRNA knock-down of PI3K isoforms. We report that PI3K class I (but not class III) regulates Met dependent cell migration. The PI3K class I isoforms required varies among the cell models. Interestingly, the combined inhibition of all p110 Class I isoforms lead to the strongest reduction of Met dependent cell migration. Met dependent phosphorylation of Akt, an effector of PI3K class I, is reduced upon endocytosis inhibition, suggesting that Met signals to PI3K Class I on endosomes. Our results indicate that mTOR is responsible for Met dependent anchorage independent growth and tumour growth in vivo. Surprisingly, PI3K class I (and class III) are not required. Moreover, Rac1 is required for Met dependent mTOR activation, (phosphorylation of mTORC1's effector, p70 S6K) subcellular translocation of mTOR and anchorage independent growth. Finally, our results suggest that this Met-Rac1- mTOR pathway occurs on endosomes. Thus while PI3K class I regulates Met dependent cell migration, mTOR regulates Met driven anchorage independent growth and in vivo tumorigenesis. Thus PI3K Class I / mTOR may be targeted in Met driven cancers.

A biofilm is a complex community of cells enveloped in a self-produced polymeric matrix. Entry into a biofilm is exquisitely controlled at the level of transcription and in Bacillus subtilis it requires the concerted efforts of several major transcription factors including the repressor SinR and activator DegU. I initially identified that these transcriptional regulators control biofilm formation via parallel pathways. Through investigating the regulation of biofilm formation by SinR and DegU, I discovered that biofilm formation is also regulated at the post-translational level. This was achieved by identifying three key proteins which are needed for biofilm formation. These proteins are PtkA, a bacterial tyrosine kinase; TkmA, the cognate modulator of PtkA; and PtpZ, a bacterial tyrosine phosphatase. By introducing amino acid point mutations within the catalytic domains of PtkA and PtpZ it was identified that the kinase phosphatase activities, respectively, are essential function.In addition, PtkA contains a conserved C-tyrosine cluster that is the site autophosphorylation. Investigation of the role of the C-terminal tyrosine cluster tentatively suggests that this domain acts to block access to the active site of PtkA, thus affecting the ability of PtkA to phosphorylate its targets. Deletion of the gene coding for TkmA demonstrated that this modulator was also required for biofilm formation. It was also demonstrated that TkmA may interact with other protein partners, at least in the absence of PtkA, raising the question of how signal specificity is maintained. Finally, a systematic mutagenesis approach was used with the aim of identifying the target(s) of PtkA and PtpZ during biofilm formation but,despite extensive efforts, it remained elusive. The findings presented in this thesis highlight the complexity of biofilm formation by B. subtilis by revealing an additional level of regulation in the form of protein tyrosine phosphorylation.

Amino acid residues that are important for metal binding and catalysis in Grampositive phosphotyrosine phosphatases were identified in Streptococcus thermophilus Wzh/EpsB proteins. The Wzh protein from S. thermophilus MR-1C was purified after heterologous expression and tested for phosphatase activity against synthetic phosphotyrosine and phosphoserine/threonine peptides. The purified Wzh protein was able to remove phosphate from both phosphotyrosine peptides tested and the phosphatase activity of Wzh was dramatically reduced by the presence of the phosphotyrosine phosphatase inhibitor sodium vanadate at concentrations of 1, 5, and 10 mM. Purified Wzh had no activity against the synthetic phosphoserine/threonine peptide. These results established that Wzh functions as a phosphotyrosine phosphatase. By using the yeast two-hybrid system, strong intraspecific protein interactions were detected in S. thermophilus MR-1C, Streptococcus iniae 9066, and Lactococcus lactis subsp. cremoris JRF1 between the putative transmembrane activation protein (Wzd, CpsC, and EpsA, respectively) and the putative protein tyrosine kinase (Wze, CpsD, and EpsB, respectively). Weaker protein interactions take place forming a dimer between two identical protein tyrosine kinases and between the protein tyrosine kinase and phosphotyrosine phosphatase (Wzh, CpsB, and EpsC, respectively) in these species. Protein-protein interactions involving a S. thermophilus MR-1C Wzd/Wze fusion protein and Wzd and Wze indicated that these proteins may form multi-protein complexes. All combinations of the S. thermophilus Wzh, Wzd, Wze, Wzg (regulation), CpsE (glycosyl-1-phosphate transferase), CpsS (polymerization), CpsL (unknown), CpsW (regulation), and CpsU (membrane translocation) proteins were analyzed for protein-protein interactions but no additional interactions were discovered. For each of the intraspecific interactions detected, interspecific interactions were also detected when one protein was from S. iniae and the other was from S. thermophilus. Interactions were also observed between two protein tyrosine kinases when one protein was from either of the Streptococcus species and the other from L. lactis subsp. cremoris. These results and sequence comparisons performed in this study support the conclusion that interactions among the components of the tyrosine kinase/phosphatase regulatory system are conserved in the family Streptococcaceae. Interspecific protein-protein interactions suggest that functional regulatory complexes can be formed in naturally occurring and genetically engineered recombinant strains.

"May 2002." Addendum inside back cover. Bibliography: p. 139-157. Aims to isolate ephrin ligands from Drosophila melanogaster and analyse their involvement in Drosophila deveopment. Also investigates the potential of ephrin B-1 as a causative gene in the human condition Aicardi's syndrome.

"May 2002." / Addendum inside back cover. / Bibliography: p. 139-157. / 174 p. : ill. (some col.), col. plates ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to isolate ephrin ligands from Drosophila melanogaster and analyse their involvement in Drosophila deveopment. Also investigates the potential of ephrin B-1 as a causative gene in the human condition Aicardi's syndrome. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2003

Src Homology 3 (SH3) domains are small protein- protein interaction domains that bind to proline-rich peptides, mediating a range of important biological processes. Because the deregulation of events involving SH3 domains forms the basis of many human diseases, the SH3 domains are appealing targets for the development of potential therapeutics. Previously in the field, no examples of entirely small-molecule ligands for the SH3 domains have been identified. However, in our research group, we have discovered a class of heterocyclic compounds that bind to the Tec SH3 domain at conserved residues in the proline-rich peptide binding site, with weak to moderate affinity. The highest affinity of these was 2- aminoquinoline (Kd = 125 mM). In this thesis, a range of approaches are described, that were intended to contribute towards development of higher affinity small-molecule ligands for the Tec SH3 domain. Preliminary experiments, involving testing a variety of compounds structurally related to 2- aminoquinoline, provided new structure activity information, and led to a better understanding of the 2-aminoquinoline/SH3 domain binding event. The major component of this thesis is a thorough investigation into the synthesis of a range of 2- aminoquinoline derivatives. N-Substituted- 2-aminoquinolines were synthesised, however these compounds bound the SH3 domain with slightly lower affinity than 2-aminoquinoline. 6- Substituted-2-aminoquinolines were subsequently prepared, and ligands were identified with up to six-fold improved affinity relative to 2-aminoquinoline, and enhanced selectivity for the Tec SH3 domain. The techniques used for the ligand binding studies were Nuclear Magnetic Resonance (NMR) chemical shift perturbation and Fluorescence Polarisation (FP) peptide displacement assays. As part of the ligand binding studies, it was intended that the 3D tructure of a 2- aminoquinoline ligand/SH3 complex would be obtained using NMR methods, provided that a ligand was identified that bound the SH3 domain in slow exchange on the NMR timescale. However, this goal was not fulfilled. Despite this, the work presented in this thesis provides a solid foundation for the development of potent 2-aminoquinoline ligands for SH3 domains, with engineered specificity. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2005.