Sir Peter MacCallum Department of Oncology - Theses
Permanent URI for this collection
Search Results
1 - 8 of 8
-
ItemAdvancing the risk prediction and effective management of infection in patients with myeloma in the era of novel therapies( 2016)Infections are a leading cause of morbidity and mortality in patients with myeloma (MM). Increased risk for infections is due to a range of patient-, disease- and treatment-related factors. The major change in the management of MM has been the recent paradigm shift to immunomodulatory drugs (IMiDs) and proteasome inhibitor (PI)-based therapies. In this era, the pattern of infections, risk periods and risk factors for serious infections remains largely undefined. Without this knowledge, effective management of infection and preventative measures that could reduce risk and burden of infections cannot be optimally delivered. Clinical infection risk assessment is becoming unreliable with increasing use of agents with wide-ranging effects on the immune system. This thesis had two primary research aims. The first was to determine the pattern, risk period and clinical determinants for infection in patients managed in the era of novel therapies and to define how these factors vary by treatment period for a range of significant infections. To improve infection management, the second aim of the thesis was to assess the feasibility of immune profiling as a means of predicting future risk for infection. To address the first aim, a longitudinal cohort study was undertaken to establish the epidemiology and risk factors for infection overall and by disease treatment period. Bimodal peaks in incidence of bacterial and viral infections were noted. Treatment with conventional agents and corticosteroids was independently associated with increased risk for infection overall and through key treatment periods, while IMiDs and PIs were not when evaluated against patient and disease factors and conventional agents. This was followed by a series of studies evaluating infections associated with significant burden and mortality, namely varicella zoster, viral respiratory tract infections, bacterial bloodstream infections (BSI) and invasive fungal infections (IFI). Overall, these studies found distinct shifts in patterns of BSI and IFI, identified disease progression to be a high-risk period, multiple lines of therapy (≥ 3) to be an independent risk factor and confirmed the efficacy and duration of antimicrobial prophylaxis. A systematic review and meta-analysis of MM therapy comprehensively established infection rates, contrasted risk for serious infection between the current standard of care (IMiDs and PIs) with the previous standard, and between IMiDs and PIs. In this setting, differential risk for infection by type of IMiD and PI was detected with variation by treatment period. In testing the second research objective, profiling cytokine release in response to mitogen stimulation was feasible and useful for predicting subsequent risk for infection, while numerical evaluation of immune cells was unhelpful. A predominance of T helper-2 cytokine response to mitogen stimulation was associated with future risk for infection. Findings from this body of work have advanced our understanding of infections in this field and enabled the establishment of a future research agenda; studies of preventative measures targeting heavily treated patients with disease progression; evaluation and validation of identified immune profiles in a prospective patient cohort to define its clinical utility; and ultimately, to comprehensively integrate clinical parameters with immunological profiling to advance personalised anti-infective management and prevention in patients with MM.
-
ItemTargeting cyclin-dependent kinase 9 and myeloid cell leukaemia 1 in MYC-driven B-cell lymphoma( 2016)Aggressive B-cell lymphomas include diffuse large B-cell lymphoma, Burkitt lymphoma and intermediate forms. Despite high response rates to conventional immuno-chemotherapeutic approaches, an unmet need for novel therapeutic strategies is required in the setting of relapsed and refractory disease, typified by resistance to chemotherapy and radiotherapy. The proto-oncogene MYC is frequently dysregulated in the aggressive B-cell lymphomas, however, it has proven an elusive direct therapeutic target. A significant body of evidence is accumulating to suggest that MYC-dysregulated disease maintains a ‘transcriptionally-addicted’ state, whereby perturbation of RNA polymerase II activity may indirectly antagonise MYC activity. Furthermore, very recent studies implicate anti-apoptotic myeloid cell leukaemia 1 (MCL-1) as a critical survival determinant of MYC-driven lymphoma. This thesis utilises pharmacologic and genetic techniques in MYC-driven models of aggressive B-cell lymphoma to demonstrate that cyclin-dependent kinase 9 (CDK9) and MCL-1 are oncogenic dependencies of this subset of disease. The cyclin-dependent kinase inhibitor, dinaciclib, and more selective CDK9 inhibitors are used to demonstrate efficient apoptosis induction conferred at least in part by downregulation of MCL1 transcription. Furthermore, a genetic screen identifies other transcriptional cyclin-dependent kinases that are required for viability of MYC-driven lymphoid disease. Finally, having established MCL-1 as a critical oncogenic dependency of MYC-driven lymphoma, this thesis demonstrates the significant activity that is conferred by direct pharmacologic antagonism of MCL-1 using a small molecule BH3-mimetic inhibitor of MCL-1. These findings confirm a druggable pathway of oncogenic cMYC dependency involving CDK9 regulated RNA polymerase II-mediated transcription of MCL-1, and proposes pharmacologic inhibition of CDK9 and MCL-1 as novel anti-lymphoma strategies.
-
ItemInvestigating the role of CRLF2 & JAK2 in B-cell Acute Lymphoblastic Leukemia (B-ALL)( 2016)B-cell acute lymphoblastic leukemia (B-ALL) is a malignant neoplasm of B-lymphoid progenitor cells and accounts for approximately 70% of all childhood ALL (Loh and Mullighan, 2012). Overall clinical outcomes for ALL patients have seen remarkable improvements over the last 50 years predominantly through the optimization of conventional chemotherapeutic regimens (Pui et al., 2008). However, BCR-ABL1-like B-ALL nevertheless remains associated with dismal prognosis, highlighting an urgent clinical need for alternative treatment strategies for this genetic subset of B-ALL (Yeoh et al., 2002). Despite significant advances in the understanding of the genetic basis of BCR-ABL1-like B-ALL, the lack of animal models has significantly debilitated our ability to comprehensively investigate the biology of this devastating disease (Roberts et al., 2012). Accordingly, in this study we pursued the development of novel syngeneic mouse models of BCR-ABL1-like B-ALL to initially establish a clinically relevant setting to unravel and understand the therapeutic vulnerabilities of the disease. Multiple genetic alterations have been shown to frequently co-occur in this subset of B-ALL, including CRLF2 rearrangements, activating JAK2 mutations and/or IKZF1 alterations (Harvey et al., 2010). Using a mosaic approach, we successfully modelled the cooperation of (Eµ-Crlf2 mediated) surface CRLF2 receptor overexpression and B-ALL associated pathogenic JAK2 mutants (JAK2R683G and JAK2P933R mutants) during leukemogenesis. The resulting leukemias were serially transplantable and displayed many of the clinical and molecular attributes exhibited by human CRLF2-rearranged/mutant JAK2-driven B-ALLs. In particular, biochemical characterization of the resulting leukemias demonstrated exquisite similarities with those seen in the human disease, including JAK2-dependent constitutive activation of the STAT5 and PI3K signaling pathways (Tasian et al., 2012). Using an RNAi-mediated approach we subsequently unravelled an unexpected role for oncogenic JAK2 in regulating cell cycle progression independent of apoptosis in both murine and human models of CRLF2/mutant JAK2-driven leukemia. Consequent interrogation for mechanisms underlying JAK2-independent proliferation revealed a potential role of MYC and/or genome wide upregulation of its target genes in driving persistent proliferation upon shRNA-mediated depletion of JAK2 alone.
-
ItemInvestigating the roles of receptor tyrosine kinases in Vemurafenib resistance and phenotype switching in melanoma( 2016)Despite the initial efficacy of Vemurafenib treatment in BRAF-mutant metastatic melanoma, the majority of patients eventually develop progressive disease due to drug resistance. The main mechanisms of resistance reported include reactivation of the RAF-MEK-ERK signalling pathway, commonly through increased activity of upstream receptor tyrosine kinases (RTKs). This study assesses the role of feedback regulation in RTK-mediated Vemurafenib resistance, the influence of RTKs on Vemurafenib-induced phenotype switching of melanoma cells, and the assessment of combination therapies targeting signalling pathways induced by RTK activation. The thesis is mainly focused on in vitro analysis using the A375 cell line model overexpressing EGFR, FGFR1, MET, IGFR and KIT. Prior to this thesis, data generated in our laboratory (Ramsdale and Ferrao et al., unpublished) revealed that ligand activation of EGFR, FGFR1 and cMET in the A375 lines were each able to confer Vemurafenib resistance, sustain cell proliferation during Vemurafenib treatment and enhance activation of ERK and JNK signalling pathways, whereas IGFR activated by IGF was not able to sustain proliferation and enhanced activation of AKT signalling. This thesis first confirmed the reported findings that Vemurafenib treatment resulted in reduced levels of feedback regulator SPROUTY2 (SPRY2). Ligand activation of EGFR, FGFR1 and MET prevented Vemurafenib-induced reduction of SPRY2, whereas IGFR and KIT did not. Using an inducible expression system, enforced expression of SPRY2 to maintain levels during Vemurafenib treatment was demonstrated to reverse the resistance mediated by EGFR, FGFR1 and MET in A375 cells. In multiple BRAF-mutant melanoma cell lines adaptation to longer-term Vemurafenib treatment induced resistance that was associated with reduction of SPRY2 and SPRY4, which was reversible by withdrawal of Vemurafenib. Adaptation to Vemurafenib treatment over 4 weeks for A375 cells also induced a switch to a more mesenchymal-like phenotype associated with elevated expression of EMT-inducing transcription factors (EMT-TFs) and increased cell migration. These Vemurafenib-induced effects were also reversible by drug withdrawal. Through overexpression and knockdown studies, cJUN increased by Vemurafenib treatment was identified as critical mediator of Vemurafenib-induced phenotype switching and cell survival. Activation of Rho-ROCK signalling was demonstrated to contribute to Vemurafenib-induced cJUN. Activation of EGFR in A375 prevented Vemurafenib-induced changes in the expression of cJUN and other EMT-TFs, whereas IGFR did not. Consequently, activation of EGFR but not IGFR prevented the Vemurafenibinduced change to a mesenchymal-like morphology associated with increased cell migration in A375. Immuno-blotting and reverse phase protein array (RPPA) approaches were utilised to assess RTK-mediated signalling. Activated EGFR and IGFR were confirmed to induce different intracellular signalling pathways. EGFR could enhance JNK, Src/STAT3, PI3K/AKT and ERK signalling, whereas IGFR was confirmed to strongly increase PI3K/AKT signalling. The effects of various drug combinations with Vemurafenib including inhibitors to specific RTKs, AKT, JNK, Src and ERK on the A375 cells expressing EGFR, FGFR1, MET, IGFR and KIT were assessed using high throughput cell number assays and high-content imaging analysis. Combination with specific RTK or JNK inhibitors overcame the drug resistance mediated by the specific RTKs, confirming previous dose response assay data (Ramsdale and Ferrao et al, unpublished). Combination with an AKT inhibitor with Vemurafenib was able to reduce the viable cell numbers surviving at the treatment endpoint. Noticeably, immuno-blotting revealed that combination treatment with an AKT inhibitor prevented Vemurafenib-induced reduction of SPRY2 and reduced levels of Vemurafenib-induced cJUN in the RTK-expressing A375 cell lines. Together, findings from this thesis provide direct evidence that disabled feedback regulation by Vemurafenib-induced reduction of SPRY2 contributes to RTKmediated drug resistance. Activation of RTKs can alter Vemurafenib-induced cJUN and EMT-like phenotype switching during Vemurafenib adaptation. Signalling analyses and assessment of combination drug treatments have implications for developing appropriate therapeutic strategies for overcoming RTK-mediated Vemurafenib resistance associated with melanoma progression.
-
ItemAn investigation of type-1 interferon and the immune response against breast cancer metastasis( 2016)Breast cancer is a highly prevalent disease that, like many cancers, lacks effective therapies aimed at treating and preventing metastasis. Harnessing the host immune system to recognise and eliminate malignant cells has recently emerged as an effective therapeutic strategy in many cancers. However, response rates to these approved immunotherapies remain modest in the absence of a more detailed understanding of tumour immunity. The type I interferons are a family of cytokines that have long been understood to enhance the immune response to cancers, though their clinical application has led to underwhelming results in numerous types of cancer. This thesis provides new evidence that proposes the re-visitation of cancer immunotherapies that stimulate the type I interferon pathway. We show that host-derived type interferon is critical for the suppression of breast cancer metastasis through natural- killer cell activation. Induction of a type I IFN response by administering agents that mimic a viral infection (poly(I:C), a double-stranded RNA analog) proved to be powerful anti-metastatic agents in multiple pre-clinical models of triple-negative breast cancer (TNBC). This was linked to widespread immune activation which conferred NK cells with enhanced cytotoxic function to eliminate disseminated tumour cells. The efficacy of this novel immunotherapeutic approach was also found to rely upon the treatment setting in which it was used. Evidence is presented that demonstrates administration prior to primary tumour removal (neo-adjuvant therapy) as the only effective therapeutic regimen. We propose that such immunotherapies are most effective at eliminating circulating and early disseminated cells rather than established metastatic lesions. This provides some explanation to the inefficacy of previous interferon trials that were conducted in patients with late-stage metastatic disease. It also calls into question whether other immunotherapies could be used earlier in cancer treatment to maximise the chances of a clinical response. Finally, we uncover that expression of IRF9, a key transcription factor in the type-I interferon signaling pathway, accurately predicts TNBC patient prognosis. Loss of IRF9 in a patient’s primary tumour predicted significantly poorer overall survival due to metastatic spread. As we show that tumour cells are not directly responsible for the poly(I:C)-induced interferon response, we propose that patients with IRF9-negative TNBC could benefit from neo-adjuvant interferon-based immunotherapy.
-
ItemPerforin biochemistry: function and dysfunction( 2016)Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), collectively referred to as cytotoxic lymphocytes (CLs), are responsible for clearing virus infected and cancerous cells. The predominant manner by which CLs do this is through the delivery of the pore forming protein, perforin, and pro-apoptotic granzymes that synergise to induce apoptosis in a conjugated target cell. Humans who inherit bi-allelic inactivating perforin mutations develop the immunoregulatory disease familial haemophagocytic lymphohistiocytosis (FHL) and/or haematological malignancies, demonstrating the critical importance of expressing functional perforin for the maintenance of immune homeostasis and tumour immune surveillance. While most disease associated perforin mutations are rare, 8-9% of the Caucasian population are carriers of polymorphism A91V (rs35947132, 272C>T). It has been suggested that >50% of individuals homozygous for the A91V allele develop FHL and/or cancer and that individuals heterozygous for A91V have an increased susceptibility to ALL. Despite its frequency and disease association, it remained unknown whether heterozygous inheritance of the A91V allele impairs human CL cytotoxicity and, more broadly, whether perforin is rate limiting in CL cytotoxicity. Here, it has been demonstrated that NK cells from healthy humans heterozygous for the A91V allele show an almost 50% reduction in cytotoxicity compared to individuals homozygous for WT perforin. This reduction in function was due to A91V perforin protein being misfolded within human primary NK cells. Moreover, it was also observed that heterozygous perforin knockout mouse CTLs showed an ~50% reduction in cytotoxicity. Taken together, these data demonstrate that perforin is indeed rate limiting for CLs cytotoxicity and therefore, individuals heterozygous for defective perforin alleles have impaired CL function. Although FHL predominantly presents shortly after birth, a subset of patients present at an age greater than three years, owing to the expression of misfolded perforin variants. Previous studies have shown that when transiently expressed in CTLs, perforin variants associated with late onset disease failed to traffic within CTLs and the cells remained non-functional. Therefore, it was unknown how patient CTLs expressing these variants could avoid FHL in infancy, and maintain a level of immune homeostasis for many years, or even decades. Here, it is shown that perforin variants associated with late onset disease can fold correctly and traffic within CTLs, and thus provide a significant level of cytotoxic function. However, this function was found to be lost if CTLs were cultured at an increased temperature (39 ̊C). Taken together, these data suggest that the CTLs of late onset FHL patients may have sufficient cytotoxicity to delay FHL onset in infancy. However, prolonged fever and, potentially, a more rapid exhaustion of the limited pool of correctly folded perforin mutants may result in the loss of CTL function, leading to FHL and cancer later in life. Prior to its secretion from the CL, the evolutionarily conserved C-terminal residues of perforin are proteolytically cleaved. The functional significance of C- terminal processing has remained controversial. Here it is shown that perforin enriched from human NK cell with an intact and glycosylated C-terminus was not cytotoxic. However, removal of the C-terminal glycan from the protein was found to completely restore function. As full-length deglycosylated perforin has wild type activity, these data suggest that C-terminal cleavage of perforin is permissive for cytotoxic function due to removal of an inhibitory N-linked glycan moiety at the C-terminus of the protein. These findings position the protease(s) responsible for perforin cleavage as critical to CL function. In summary, the studies described in this thesis have added to the understanding to how perforin mutations affect CL cytotoxicity and described a critical final step in perforin maturation. Together, these advancements in perforin biology may contribute to the treatment of disease arising from perforin deficiency and also define new factors critical for CL function and human health.
-
ItemPericytes as microenvironmental regulators of skin tissue regeneration and skin ageing( 2016)This study focuses on how pericytes can contribute to the epidermal cell microenvironment in human skin, using a 3D organotypic co-culture model, I have shown that in the presence of pericytes, epidermal cells regenerate an epithelium that more closely resemble normal healthy skin with respect to basal cell morphology and various biochemical characteristics. Characterization of pericytes in skin ageing and their functionality in organotypic co-culture model suggests a microenvironmental modulatory role of pericytes in skin ageing and epithelial tissue repair.
-
ItemHeterogeneity in gastric cancer and the impact on patient survival( 2016)Gastric cancer (GC) is one of the most common malignancies and is frequently fatal. Epidemiological and biological evidence suggests GC is heterogeneous, with four subtypes of GC identified by the Cancer Genome Atlas (TCGA) in 2014, however currently it is treated as one disease. The aim of this thesis is to investigate how the clinical and biological inter-tumoural heterogeneity of GC affects survival. The clinical predictors of outcome were investigated for a cohort of curatively treated GC patients, revealing that age, stage of disease and Lauren histological subtype predict overall, cancer specific and relapse free survival. The molecular differences between different GC stages, and histological subtype (intestinal gastric cancer (IGC) compared to diffuse gastric cancer (DGC)) were then investigated with the aim of understanding the biological basis underlying survival, relapse and response to treatment. Key molecular pathways were identified as being differentially expressed between DGC and IGC, and having impact on survival. These included the transforming growth factor beta (TGF-β) signalling pathway with the expression of three bone morphogenic protein (BMP) antagonists from this pathway having associations with survival on multivariate analysis, and the mismatch repair (MMR) pathway with four genes from this pathway having impact on survival. BMP and activin membrane bound inhibitor (BAMBI), a gene from the TGF-β pathway was more highly expressed in IGC compared to DGC and was associated with poorer survival in IGC. Molecular analysis of BAMBI in GC cell lines revealed decreased proliferation and invasion, and G0/G1 cell cycle arrest. It was postulated that this change in proliferation and invasion was mediated via alterations in the balance of epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition via a TGF-β mediated process. Further analysis revealed gene expression correlation between BMP antagonists and EMT associated genes, revealing highest correlation with gremlin 1 (GREM1). Higher expression of GREM1 was also associated with poorer survival in IGC. Another key pathway discovered as differentially expressed between DGC and IGC was the MMR pathway. Microsatellite instability was also identified as a molecular subtype of GC by the TCGA. The clinical and histological correlations of the MMR GC subtype were therefore examined. Patients with MMR deficient tumours had improved survival compared to MMR stable tumours, and a higher inflammatory score (IS). The simple IS used in this thesis could be a triaging tool to identify tumours to take forward for formal MMR testing. This thesis adds to the growing body of evidence that demonstrates GC is heterogeneous and elaborates on some of the molecular features that drive this inter-tumour heterogeneity. It identifies a number of genes, pathways and biological associations that impact survival, and potentially response to treatment. This work will help in the personalisation of medical therapy for a disease that can no longer be treated with a one-size-fits-all mentality for therapy. Future work is planned to expand the findings of this work, with the aim of translating these findings to the clinical setting, enabling better prognostication and more rational prescription of treatments for GC patients.