Targeting the Ubiquitin-Proteasome System in Glioblastoma

Glioblastoma is the most prevalent as well as aggressive type of brain tumour in humans, yet very limited progress
has been made in effective treatment. The ubiquitin-proteasome system poses a promising target with interest fuelled
by efficacy achieved by the first-in-class proteasome inhibitor bortezomib, currently used as treatment for myeloma
and mantle cell lymphoma. Our lab has a strong interest in ubiquitin-dependent cell signalling. Within the ubiquitinproteasome
system, E3 ubiquitin ligases take a primary role. This highly heterogenous protein family poses as
ultimate checkpoint of protein fate and function, in particular in the context of cell cycle progression making it an
attractive target for therapeutic intervention.
The project focusses on the role of E3 ubiquitin ligase HECTD1 in glioblastoma. E3 ubiquitin ligases of the HECT
family have already been implicated in gliomas - in 2015, Pan et al. (Nature) showed that UBE3C directly interacted
and induced degradation of the tumour suppressor Annexin A7 (ANXA7). In accordance, preliminary data generated
in our lab showed the involvement of HECTD1 in cell cycle progression, with depletion resulting in prolonged mitosis.
Subsequently, potential novel substrate and interactors of HECTD1 were identified. In combination with ONCOMINE
data revealing increased HECTD1 mRNA levels in glioblastoma, this builds the foundation of the research project.
In order to further elucidate the role of HECTD1 in the regulation cell growth and proliferation, knockdown models will
be generated in the glioblastoma cell lines U87MG, U251MG and U118MG. This will be expanded to and validated in
patient-derived glioblastoma cell lines. A dual approach is utilised, HECTD1 is targeted via both shRNA and CRISPR/
Cas9. Hence, the first months will focus on design and construction of effective lentiviral particles for shRNA delivery
and CRISPR/Cas9 constructs. Adequate HECTD1 depletion in glioblastoma cell lines will aid to establish t he
enzyme's impact on cell proliferation and cell cycle with rescue assays further contributing to validation of HECTD1
as potential drug target. Simultaneously, In-Fusion cloning of full-length HECTD1 will enable identification of its
temporal interactome/substratome. A quantitative proteomics approach will be used to facilitate the elucidation of
underlying molecular mechanisms.
Through the collaboration with co-supervisor Florian Siebzehnrubl (Cardiff), findings can be extrapolated and further
validated in an orthotopic xenograft in vivo glioblastoma mouse model. Injection with luciferase-labelled glioblastoma
cell lines paired with IVIS and histopathology analysis will allow testing of our hypothesis that HECTD1 depletion
decreases glioblastoma tumour growth, mechanistically due to loss of ubiquitin ligase activity. Data will be paired with
IHC, western blot and qRT-PCR analysis of fully consented human primary and secondary glioblastoma and normal
matched patient samples (co-supervisor Kathreena Kurian, Bristol). In addition, correlative