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Medicine & Health
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(2020) Chang, DavidThesisCancer is the leading cause of death globally and a major limitation of conventional treatments is toxicity to normal tissues, restricting the dose deliverable to patients. There has been enormous interest in the application of nanotechnology towards therapeutics for cancer, as many of these therapeutics lack specificity and in some cases, efficacy due to limitations in delivering a therapeutic dose. With the hypothesis that delivering increased therapeutic doses specifically to tumour cells will increase the efficacy of treatment, the aim of this thesis is to develop and evaluate targeted therapies for different types of cancer. Initially, the development of targeted magnetic hyperthermia was examined and demonstrated some of the challenges encountered when working with magnetic iron oxide nanoparticles for this application. This highlighted the need for better control of aggregation under clinically relevant conditions. Next, a modular strategy for targeting a clinically-approved liposomal formulation of doxorubicin towards epidermal growth factor receptor on lung cancer cells, prostate specific membrane antigen on prostate cancer cells and disialoganglioside (GD2) on neuroblastoma cells via bispecific antibodies was developed. Targeting resulted in increased internalisation, apoptosis and cytotoxicity in vitro. While initial in vivo experiments were inconclusive, potential reasons for this are discussed. With insights gained regarding nanoparticle properties for targeting, and to broaden the number of payloads that can be effectively targeted towards cancer cells, novel cross-linked poly amino acid micelles were developed and covalently loaded with several payloads including monomethyl auristatin E, a potent anti-microtubule drug. Micelles had a hydrodynamic diameter of 37 nm and were uniform in size with a polydispersity index of 0.1. When applied with bispecific antibodies, the system demonstrated a remarkable increase in internalisation of payload by cancer cells, microtubule inhibition, G2/M cell cycle arrest, radiosensitisation and significantly enhanced cytotoxicity in both monolayer and 3D spheroid models. These findings broaden our understanding of active targeting with bispecific antibodies. Targeted micellar system may have broad clinical potential as it could be used to deliver a wide array of payloads to various antigens overexpressed by cancer cells.
Development and characterisation of star-shaped nanoparticles to deliver therapeutic siRNA to medulloblastoma(2020) Forgham, HelenThesisBrain cancer kills more children in Australia than any other disease. Medulloblastoma (MB) accounts for approximately 20% of all childhood brain tumours. Chemoresistance, relapse and treatment related toxicity are common for this disease. There is an urgent need to develop new effective and less toxic treatments. Gene therapies which use short-interfering RNA (siRNA) to silence the expression of a target gene have great potential for the treatment of a host of human diseases including cancer. However, a major hurdle for the clinical translation of siRNA drugs is the need for a delivery vehicle to allow siRNA to internalise into cells. Nanoparticles may offer a solution to this problem and can be used as delivery vehicles for siRNA. No studies have examined the potential of star nanoparticles for the treatment of MB. The aims of this study were to: 1) define the biological conditions for di-block copolymer nanoparticles (star nanoparticles) to deliver siRNA to MB cells in vitro to silence the expression of a gene (Polo-Like Kinase 1, PLK1) which is highly expressed in MB cells and plays a major role in promoting tumour growth; 2) determine whether star nanoparticles could deliver siRNA to solid tumours in mice; and 3) examine whether Star nanoparticle-siRNA (star-siRNA) could penetrate a blood-brain barrier (BBB). Results from this thesis demonstrate that star-siRNA form monodisperse nanoparticles with a size of 19 nm. Star-siRNA is internalised into MB cells in vitro and can silence PLK1 expression leading to mitotic arrest, DNA damage and apoptosis. Star-siRNA was non-toxic to mice and siRNA could be effectively delivered to subcutaneous MB tumours to silence PLK1 expression which induced apoptosis. Finally, using in vitro and in vivo models we showed that star-siRNA could penetrate the BBB. Importantly, star nanoparticles delivered siRNA to the brains of mice with growing orthotopic MB tumours. Collectively, results presented in this thesis demonstrate for the first time the potential of star nanoparticles to deliver siRNA to induce apoptosis in MB cells in vitro and in vivo. Star-siRNA nanodrugs may be a novel therapeutic strategy to inhibit MB growth and increase patient survival.
Deciphering mechanisms of neuroblastoma drug resistance using single cell transcriptomics and developing targeted combination therapies for neuroblastoma(2021) Seneviratne, JanithThesisPeripheral neuroblastic tumors (PNTs) represent a spectrum of tumors derived from the neural crest and account for 10% of paediatric malignancies. PNTs comprise four histological subtypes, including neuroblastoma (NB). Half of high-risk NB patients relapse after chemotherapy, and relapsed patients seldom respond to therapy afterward. This study aimed to identify chemoresistance mechanisms and effective combination therapies against chemoresistant NB. In the first results chapter, intratumoural heterogeneity in PNT subtypes were first investigated using single cell RNA sequencing (scRNA-seq) on 7 primary PNTs spanning all histological subtypes. This revealed extensive transcriptome heterogeneity among PNTs and identified malignant neuroblasts differentiating through a novel “transitional” state. This state resembled known chemoresistant cell phenotypes and predicted poor NB patient prognoses. In the second results chapter, drug resistance mechanisms in NB were explored in vitro using longitudinal IMR-32 cisplatin-resistant cell line models. Following scRNA-seq, resistant cells undergoing cytoskeletal remodelling were identified and then characterised in vitro. Additionally, tumour samples from 5 high-risk NB patients at diagnosis and post-chemotherapy were analysed with scRNA-seq, where an enrichment of oncogenic cell states was observed. In the third results chapter, potential combination therapies were identified using a combinatorial drug screen against the chemoresistant SK-N-BE(2)-C cell line. The highest synergy manifested between two histone methyltransferase inhibitors, GSK343 and SGC0946. Mechanistic studies revealed the induction of ER stress programs by the combination treatment. The administration of this combination therapy in vivo reduced NB tumour growth. In the fourth results chapter, an integrative approach was used to identify mitochondrial drug targets in NB, whereby the mitochondrial translocase SLC25A5 was prioritized. Targeting SLC25A5 with the selective inhibitor, PENAO, reduced cell viability in NB cell lines, however TP53 mutations conferred chemoresistance. The histone deacetylase inhibitor SAHA combined with PENAO overcame mutant TP53 mediated chemoresistance to synergistically reduce NB cell and tumour growth. Collectively, this thesis has identified transcriptional cell states that drive chemoresistance and combination therapies that overcome chemoresistant NB, thereby contributing novel therapeutic targets and treatment avenues for NB patients.
(2021) Khan, AaminahThesisDiffuse intrinsic pontine glioma (DIPG) is an aggressive paediatric brainstem tumour with no active systemic therapies and a 5-year survival of less than 1%. Polyamines are small organic polycations that are essential for DNA replication, translation and cell proliferation, and are often upregulated in cancer. Difluoromethylornithine (DFMO) is an FDA-approved inhibitor of the enzyme ornithine decarboxylase (ODC1) which is a key driver of polyamine synthesis. This thesis investigates the efficacy of polyamine pathway inhibitors as a therapeutic strategy against DIPG. High expression levels of key players of the polyamine pathway were observed in DIPG samples by qPCR and western blotting. Alamar blue cytotoxicity and soft-agar clonogenic assays, showed that DFMO inhibited the proliferation of DIPG neurospheres. However, DIPG cells compensated for DFMO inhibition by increasing expression of the polyamine transporter SLC3A2. Addition of polyamine transporter inhibitor AMXT 1501 to DFMO led to synergistic inhibition of DIPG proliferation. Western blotting and flow-cytometric analysis of Annexin V-stained cells showed that combination treatment enhanced apoptosis. Consistent with the in vitro results, the combination of DFMO and AMXT 1501 significantly prolonged the survival of mice bearing DIPG orthografts. DIPG tumours contained higher polyamine concentrations compared to healthy brain tissue, treatment with the combination therapy led to significantly lower levels of intratumoral polyamines. This combination therapy significantly extended the survival of DIPG engrafted mice, with 6/9 mice surviving until the humane endpoint of 160 days. Examination of RNA expression levels in a cohort of high-risk childhood cancers showed that the polyamine transporter, SLC3A2, was significantly overexpressed in DIPG and other paediatric high-grade gliomas compared with all other high-risk childhood cancers. Together these results suggest that this strategy of dual polyamine inhibition may be potent novel therapy for these paediatric brain tumours. AMXT 1501 is currently in clinical development and following completion of an adult Phase 1 trial a clinical trial for DIPG patients is planned. Tumour cells are dependent upon arginine, a semi-essential amino acid, metabolised by arginase enzymes into ornithine, a pivotal precursor to the polyamine pathway. Pegylated arginase (BCT-100) has recently been shown to significantly delay tumour development, prolonging survival of neuroblastoma-prone Th-MYCN mice. Arginine depletion therapy as a single agent and in combination with polyamine pathway inhibitors in DIPG was investigated. We found that ARG2, the gene encoding for arginase II, is significantly over-expressed in DIPG tumours compared to normal brain. Arginine depletion via BCT-100 reduced DIPG cell proliferation and colony formation in patient-derived cell lines. Treatment of orthotopic patient-derived xenograft models of DIPG, with BCT-100 (4x/week) significantly delayed tumour development and increased the survival of the mice (p<0.0001). The combination of BCT-100 with DFMO led to significant enhancement in DIPG survival (p<0.005 compared to single agent treatments). Triple combination therapy with the addition of the polyamine transport inhibitor AMXT 1501 led to a potent and profound improvement in survival. These data show that arginine depletion therapy using BCT-100 combined with dual polyamine inhibitory agents represents a potentially exciting new approach for the treatment of DIPG. Collectively, this study has provided a firm foundation for future investigations into the important roles of polyamines in DIPG / paediatric brain tumour biology and metabolism and has also led to the development of an international clinical trial for DIPG patients.
(2020) Fordham, AshleighThesisEpithelioid inflammatory myofibroblastic sarcoma (eIMS) is an aggressive variant of inflammatory myofibroblastic tumour characterised by ALK staining that is perinuclear or cytoplasmic with perinuclear accentuation, CD30 expression and early relapse despite crizotinib treatment [1, 2]. This study aimed to generate and utilise models of eIMS to assess molecular-targeted therapeutic combinations to prevent and/or treat ALK inhibitor relapse. Two approaches were pursued to develop clinically relevant eIMS models. Malignant ascites from an eIMS patient at diagnosis and at relapse were used to establish cell and xenograft models. Patient-derived models were validated by confirmation of RANBP2-ALK rearrangement and expression of ALK and CD30. RANBP2-ALK transduced cell models did not recapitulate eIMS features despite expression of RANBP2-ALK at the RNA and protein levels. In vitro assays and in vivo dose response studies were undertaken to characterise the eIMS diagnosis and relapse models. Survival extension in the eIMS diagnosis xenografts compared to the relapse xenografts indicated the relapse model was less sensitive to ALK inhibition with crizotinib or ceritinib in vivo. However, this was not reflected by in vitro assays. Therefore, in vivo studies were used for the remainder of the project to validate novel therapeutic strategies identified in vitro. The CD30-targeted agent, brentuximab vedotin (BV) was investigated for therapeutic potential in eIMS. BV treatment resulted in tumour shrinkage in eIMS xenografts. However, reduced CD30 expression and induction of ABCB1 was associated with tumour regrowth during therapy. Therefore, combination therapies identified by rational selection and high-throughput screening were considered. BV resistance was reversed in vitro by combination with tariquidar, an ABCB1 inhibitor. The combination of rigosertib and BV, identified by high-throughput screening, was found to be synergistic in vitro. However, both these combinations only slowed growth of eIMS xenografts. In contrast, combining BV with crizotinib or ceritinib resulted in marked tumour shrinkage and improved survival in both eIMS diagnosis and relapse xenografts. This project established patient-derived in vitro and in vivo models of eIMS and identified therapeutic combinations for the treatment of eIMS. Prolonged tumour-free survival in vivo provides rationale to trial combinations of ALK inhibitors and BV for treatment of eIMS.
(2020) Saha, AyanThesisAlthough long-term survival rates for childhood acute lymphoblastic leukaemia (ALL) are now approaching 90%, children with genetically specified subgroups remain at enhanced risk of treatment failure and reduced rates of survival. Among these subtypes, Philadelphia (Ph)-like ALL is correlated with the clinical features of higher risk, inadequate response to chemotherapy, high levels of minimal residual disease (MRD), early relapse and poor survival. The cytokine receptor like factor 2 (CRLF2) rearrangement and IKAROS family zinc finger 1 (IKZF1) deletion drive oncogenesis in a high proportion of paediatric Ph-like ALL patients; both CRLF2 and IKZF1 alterations drive stem cell renewal, cause abnormal bone marrow (BM) adhesion, and result in decreased chemosensitivity. The BM is the most frequent site of relapse in ALL. Therefore, this study focuses on how the BM microenvironment becomes supportive for the survival of Ph-like ALL cells following chemotherapy. Preclinical in vivo ALL patient-derived xenograft (PDX) models have shown in the present research that, when compared to other organs, induction chemotherapy is less effective in BM regions. Moreover, relapse-initiating MRD cells were higher in the BM regions of the Ph-like ALL PDX-bearing mice than was the case for the low-risk BCP-ALL PDXs, further suggesting substantial chemoresistance mechanisms of Ph-like ALL MRD cells in the BM. Accordingly, this study adopted a single-cell (sc)-RNA sequencing approach to identify how Ph-like ALL cells develop chemoresistance in the BM. Induction chemotherapy resulted in the suppression of TP53 and subsequent cell cycle alterations of Ph-like ALL cells, although this was not seen in low-risk BCP-ALL. In addition, both altered IKZF1 and CRLF2 rearrangement in Ph-like ALL confers glucocorticoid (GC) resistance. GCs form a critical component of chemotherapy regimens for paediatric ALL, with initial resistance to GC therapy being predictive of poor outcome. Interestingly, Ph-like ALL cell lines cocultured with BM stromal cells (BMSCs) exhibited increased resistance against GCs. However, IGF1R inhibitors, in combination with GCs, demonstrated remarkable in vitro efficacy when tested and compared to a Ph-like ALL cell line cocultured with BMSC. Overall, the data presented in this thesis provide novel insights into BM-induced chemoresistance in MRD cells, and could be used to facilitate the development of future therapeutic approaches in the treatment of Ph-like ALL.
Molecular characterisation of lower-grade glioma and investigation of its genomic aberrations influencing patient prognosis and therapeutic response(2022) Noor, HumairaThesisLower-grade gliomas (LGGs) are tumours of the Central Nervous System affecting young adults aged 17-44 years. The overall survival of LGG patients has not improved significantly over the last 30 years, and new therapeutic approaches are warranted to effectively treat this cancer. In this thesis, I have molecularly characterised a retrospective cohort of LGG specimen from 102 patients, including 29 patients with matched primary and recurrent tumours, in order to identify prognostic biomarkers, molecular subgroups with unfavourable prognosis and investigate their potential as therapeutic targets through pre-clinical studies. Specific hotspot tumour protein 53 (TP53) codon 273 mutations occurred in 33% of astrocytoma, which were associated with significantly improved survival in both univariate (p<0.05) and multivariate analysis (p<0.05). TP53 wildtype tumours were associated with the most unfavourable clinical outcome. Analysis of publicly available LGG datasets validated these findings and elucidated the mechanism of this prognostication involving enhanced chemosensitivity of TP53 codon 273 mutations. Integrated analysis uncovered interactions between Yes-associated protein 1 (YAP1) and TP53 mutation potentially plays a role in inducing this chemosensitivity. In silico analysis identified the over-expression of carbonic anhydrase 12 (CA12) mRNA in the TP53 wildtype astrocytoma sub-group with the most unfavourable prognosis. I attempted to establish LGG patient-derived neurosphere cell lines for in vitro drug efficacy studies with the CA12 inhibitor U-104 combined with temozolomide treatment. Drug efficacy studies were conducted on a panel of TP53 wildtype and TP53 mutant glioblastoma cell lines where U-104 monotherapy, but not combination therapy, was efficacious in both subgroups. U-104 monotherapy also showed efficacy in LGG neurospheres. Molecular characterisation revealed that 72.9% of astrocytomas and 63.2% of oligodendrogliomas followed alternative lengthening of telomere (ALT) mechanism to maintain their telomere lengths. Neither ALT nor telomerase mechanisms were prognostic factors in astrocytoma, while, ALT was a significantly associated with a longer progression-free survival in oligodendroglioma. Cyclin a1 (CCNA1) was identified as a potential ALT-associated gene in the analysis of in-house RNA sequencing data. While CCNA1 methylation was not found to be directly associated with ALT upon investigation, CCNA1 methylation showed a strong trend for association with ALT-associated aberrations in ATRX, and progression-free survival in both the in-house astrocytoma cohort and TCGA dataset. Moreover, CCNA1 methylation levels were significantly associated with increasing astrocytoma grade, and it may be involved in tumour progression and aggressiveness. In conclusion, this thesis identified therapeutically actionable biomarkers, determinant of chemosensitivity, and prevalence of TMMs in LGG.
(2021) Kleynhans, AneThesisAdvanced neuroblastoma is associated with MYCN amplification and gene amplification of MYCN has been shown to correlate with poor prognosis in patients. The MYC family of proteins have been widely researched due to their important roles in the development of various cancers including neuroblastoma. Pharmaceutical companies and researchers have attempted to develop novel small molecule inhibitors which are able to target c-MYC and MYCN directly. However there has been limited success in the development of these inhibitors, and to date no MYCN direct inhibitors are in clinical use or clinal trials. It is known that MYCN regulates the transcription of several genes that are involve in cell proliferation and cell death including both tumour suppressor and oncogenes. Therefore, is has been suggest that rather targeting MYCN directly, it may be a better approach toidentify and target proteins which regulate or directly bind to MYCN as potential therapeutic targets. The hypotheses for this thesis is that USP5 acts in an oncogenic manner by directly interacting with MYCN and regulate MYCN stability to promote neuroblastoma tumorigenesis and that novel small molecule compounds are potent and selective in targeting MYCN and USP5 in MYCN-driven neuroblastoma tumorigenesis. Three aims have been established to assess these hypotheses. The first aim is to determine the functional role of USP5 in neuroblastoma tumorigenesis using various phenotypical assays in vitro and in vivo models. The rationale of this aim is to investigate whether USP5 is a potential therapeutic target for the treatment of neuroblastoma. Several studies have reported that USP5 acts in an oncogenic manner in various adult cancers by regulating different cellular pathways. Therefore, aim two of the study is to determine whether USP5 function is in MYCN dependent manner and to study the mechanism of USP5 as an oncogenic protein in neuroblastoma. By addressing this aim this thesis will explore USP5’s role in neuroblastoma tumorigenesis and provide insight into the molecular mechanism which used by USP5 to stabilize MYCN. The third aim of this thesis is to evaluate the efficacy and study the drug action of novel USP5 and MYCN inhibitors for the treatment of neuroblastoma. This aim will demonstratethe importance of developing novel compounds with clinical potential for better targeted treatment options. In summary, the purpose of this thesis is to investigate the role of USP5 in MYCN-drive neuroblastoma and highlight a novel therapeutic option for the treatment of MYCN amplified neuroblastoma by targeting the interaction between USP5 and MYCN.