Investigation and validation of the functional role of sMEK1 acetylation in DNA damage repair and HDACi-mediated radiosensitisation

Thesis

Muscle-invasive bladder cancer (MIBC) most commonly affects the elderly with more than half of new diagnoses in the over 75-year age group. This means there is a need for less toxic treatments to improve treatment outcomes. Histone deacetylase inhibitors (HDACi) have emerged as effective, low toxicity radiosensitising agents, although the mechanism of radiosensitisation is not fully defined. This study investigates the radiosensitising effects of the HDAC inhibitor panobinostat (PAN) via a mechanism involving increased acetylation of non-histone proteins.

This project aimed to validate acetylation sites in non-histone proteins which were identified in a mass spectrometry screen following PAN treatment, and to investigate their functional roles in radiosensitisation. I hypothesised that specific acetylation sites in non-histone proteins could have vital roles in the DNA damage response (DDR). Of the identified acetylated non-histone proteins, suppressor of MEK1 (sMEK1), the regulatory subunit of serine/threonine-protein phosphatase 4 complex, was the primary focus, as it can act as a phosphatase for gamma H2A.X (γH2AX) upon completion of DNA repair. Cell lines were transfected with sMEK1 and subjected to PAN treatment and/or ionising radiation (IR) to detect time-course changes of DDR protein markers. A delayed DDR was observed in PAN-treated cells with persistently increased levels of phospho-ATM and γH2AX at 4 hours after recovery from IR, consistent with the radiosensitising activity of PAN. Endogenous sMEK1 protein was downregulated independent of PAN concentration between 8-24 hours, potentially through the regulation of mRNA expression. Reciprocal co-immunoprecipitations using anti-acetyl lysine and anti-HA antibodies confirmed the acetylation status of sMEK1 following PAN treatment. A transient interaction with the most abundant bromodomain protein, BRD4, was also detected in the chromatin compartment. Subcellular fractionation indicated significant nuclear localisation of sMEK1 immediately after IR in control DMSO cells, followed by migration to the chromatin fraction 4 hours later for γH2AX elimination. However, this was disrupted by PAN which resulted in less chromatin localisation at 4 hours after IR and higher levels of𝛾γH2AX than control. Downregulating endogenous sMEK1 in the cells via siRNA transfection not only resulted in an increase in radiosensitivity, but also a delay in the induction of the DDR, as indicated by a slower γH2AX signal intensity achieved at 2 hours. This supports the hypothesis of sMEK1 being a key protein in PAN radiosensitisation through its loss-offunction in the DDR pathway and changes in subcellular localisation.

In conclusion, I have shown that sMEK1 is regulated by PAN treatment and is acetylated. sMEK1 could therefore represent a clinically promising new radiosensitising drug target or biomarker to be further validated.

Authors: Yeo, X

• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford