Identification and characterisation of novel therapeutic targets for the control of medulloblastoma
Creators: name: Sedeeq, Mohammed
Publisher: name: University of Tasmania
Year: 2024

description: Medulloblastoma (MB) is the most common paediatric malignant brain tumour, representing 20% of all childhood brain cancers. MB is a highly heterogeneous tumour and is genetically divided into four main subgroups: WNT, SHH, Group 3, and Group 4. The overall 5-year survival of MB is 60-70%. However, children often suffer severe and lifelong adverse effects from standard medical therapies. Considering the limitations of current MB treatments and their detrimental impact on the patient’s quality of life, finding new therapeutic targets against MB is crucial. Most currently available high-throughput drug screens in MB research rely on assessing cell proliferation using 2D models. These simplistic monolayer cultures lack cell-cell or cell-extracellular matrix interactions and are poorly representative of <i>in vivo</i> nutrient gradients or physiological oxygen concentrations. This results in a failure or a poor selection of drug candidates. We developed a novel 384-well-based colony formation assay to assess the growth of Group 3 MB cell lines in a 3D matrix. In this assay, MB cells were seeded in an agar matrix and allowed to grow as 3D colonies. To facilitate high-throughput screening and rapid result procurement, the cell growth was measured by multiplexing two fluorescent substrates that independently quantified the growth of viable cells without any detected signal interference. This approach also avoided manual counting of colonies, which is a source of investigator bias. We successfully used this assay to screen a panel of plasma membrane calcium (Ca<sup>2+</sup>) channel modulators as potential drug targets. We identified T-type (CaV3) channel inhibitors, mibefradil and NNC-55-0396 (NNC) as selective inhibitors of MB cell growth. In the following study, we characterised the effects of mibefradil and NNC on MB cells and elucidated their mechanism of action. My investigation demonstrated that the inhibition of proliferation caused by mibefradil and NNC was selective to the T-type Ca2+ channel, as L-type inhibitors failed to produce a similar response. Adding CaV3 inhibitors to Vincristine increased the response of MB cells to this chemotherapeutic agent, which suggested an additive effect. Mechanistically, mibefradil and NNC showed dual actions on MB cells: (i) induction of cell apoptosis, as demonstrated through increased expression of two apoptosis markers, caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP), (ii) altered mitochondrial function demonstrated by inhibition of the OXPHOS pathway, and reduction in ATP level. The latter action was also accompanied by marked dissipation of the mitochondrial electrochemical potential gradient and increased production of superoxide. This study offers new insights into the molecular action of mibefradil and NNC. It will pave the way to test these molecules or their analogues in pre-clinical MB models alone and in combination with Vincristine to assess their suitability as a potential MB therapy. The 3D agar colony formation assay also showed that the selective activation of the ORAI1 Ca<sup>2+</sup> Channel by IA65, increased MB cell growth in soft agar. IA65 is a selective ORAI1 channel activator known to increase store-operated calcium entry (SOCE) response and calcium influx. SOCE is the primary pathway of Ca<sup>2+</sup> entry into mammalian cells, and altered SOCE has been linked to many tumour phenotypes, such as cancer cell proliferation, migration, and metastasis. The results from the screening analysis and our previous data showed altered expression of several members of the SOCE pathway in MB tissue. This prompted us to investigate this pathway in MB cell lines. In the third study, I explored and characterised SOCE pathway in MB cells. This study demonstrated significant heterogenicity in the expression of SOCE genes and SOCE activity among different MB cells belonging to different MB subgroups. For example, ORAI1, a key regulator of SOCE, was highly expressed in MB cells from the invasive MB subgroup 3 compared to the less invasive SHH cell line. This difference was also accompanied by a higher SOCE activity in these cells compared to cells from MB subgroups with lower invasion ability. These results highlight that distinct MB subgroups may rely on different genes to maintain their SOCE activity. For the first time, this study provides critical knowledge about this important calcium influx pathway in MB. This information is essential to assess the detailed role of SOCE in MB and the potential of targeting this pathway as a therapeutic strategy for MB. This thesis investigated novel pathways to find drug candidates/targets against MB. It also shows the importance of studying calcium signalling in MB brain tumours, a pathway that remained largely unexplored in this type of cancer.