Profiling and targeting HOX-PBX dimers in adult and paediatric glioblastoma as a novel therapy

Abstract

HOX genes encode a family of transcription factors that play an essential role in embryonic patterning during foetal development. These genes are reported to be aberrantly expressed in numerous cancers, including glioblastoma (GBM). Previous research indicates that HOX genes are overexpressed in GBM tumours compared to normal human astrocytes (NHA). Three Amino Acid Loop Extension Homeobox (TALE) proteins act as important co-factors for HOX proteins, modulating their binding affinities to genomic targets. TALE members Pre-B-cell leukaemia homeobox (PBX) 1-4, bind anterior HOX proteins, facilitating cellular entry, while also limiting their degradation. HXR9, a small hexapeptide drug, has previously been shown to inhibit HOX-PBX dimers, causing rapid cell death in numerous solid and haemopoietic malignancies both in vitro and in vivo. HTL00-1 is a 2nd generation peptide based on HXR9, while ICT9119 is a small molecular inhibitor with the same binding site as the peptide inhibitors. We found clear evidence of overexpression of both HOX and TALE genes, in both adult and paediatric GBM cell lines. A further elevation in expression of these genes was visible in GBM cancer stem cells (CSCs). Normal adult brain tissue expressed low to undetectable levels of HOX genes, while paediatric brains expressed high levels between 0-4 years of age, before dramatically reducing from ages 5 onwards. HOX-PBX inhibitors are shown to be both cytostatic and cytotoxic when used to treat adult and paediatric GBM cells both in vitro and in vivo. GBM CSCs were more sensitive to HOX-PBX inhibition, alluding to their increased reliance on these genes. HTL00-1 and ICT9119 were shown to provide improved pharmacological effects compared to their predecessor HXR9. All HOX-PBX inhibitors cause apoptosis through induction of Fos Proto-Oncogene AP-1 Transcription Factor Subunit (C-Fos) expression, which results in the dimerization of C-Fos and Jun Proto-Oncogene AP-1 Transcription Factor Subunit (C-Jun) to form the Activator protein 1 (AP-1) complex. This directly and indirectly represses B-cell lymphoma 2 (Bcl-2) expression leading to the release, and subsequent cleavage of caspase 3 and 7. This triggers the caspase cascade that concludes with cellular apoptosis. Radiotherapy (RT), combined with ICT9119 treatment proved highly synergistic in terms of cell kill when applied in combination when cancer cells were exposed to RT 24 hours prior to ICT9119. When ICT9119 was delivered 24 hours prior to RT antagonistic effects were observed. Attempts to identify the molecular mechanisms behind differing sequential treatment outcomes proved inconclusive. However, our work did suggest a mixture of cell cycle sequestration, C-Jun induction and the opposing effects of dual specific phosphatase 1 (DUSP1) and Mitogen-activated protein kinases (MAPK) were at least partly involved. This study highlights the important role of HOX genes in paediatric and adult gliomas, the potential utility of HOX/TALE gene expression in the identification of CSC population in patients and potential for HOX-PBX inhibition as a highly targeted effective treatment for adult and paediatric GBM patients. This treatment can be administered in the form of a peptide, as in HXR9 or HTL00-1, or as a small molecular agent in the form of ICT9119, either as single agents, or in combination with RT. HOX-PBX disruption as described in this thesis could potentially be moved rapidly into the clinic, in an area of very high unmet need.