Tipping the scales in cancer : novel mechanisms of inhibiting protein degradation

Abstract

Protein homeostasis (in short, ‘proteostasis’) requires the timely degradation of proteins to retain control on protein quality, amount and function. Two main proteolytic systems, the ubiquitin-proteasome system (UPS) and autophagy, complement each other to fulfill this regulatory role. Defective proteostasis is linked prominently to age-related disease, including neurodegenerative disorders and cancer. Components of the UPS and autophagy are often mutated or dysregulated during cancer progression, a phenomenon linked to sustained cell proliferation, tumor growth and resistance to therapy. Hence, cancer cells display an increased sensitivity towards drugs that reduce the function of these proteolytic systems, as illustrated by the clinical success of inhibitors of the proteasome, and the clinical trials of lysosome neutralizers to inhibit autophagy in cancer. However, severe side effects, therapy-induced resistance and a lack of efficacy hamper their use, underscoring the need for more effective and tumor-selective compounds blocking the UPS and autophagy.

The work presented in this thesis set out to identify novel inhibitors of the UPS. Despite being rich in potentially druggable proteins, it is challenging to identify, a priori, a suitable target for drug development due to extensive functional redundancy across the pathway. Thus, we took two different approaches to find novel mechanisms for inhibition of protein degradation. In papers I and II, we used a cancer cell line stably expressing a fluorescent UPS reporter and employed a forward chemical genetic screening approach to interrogate the pathway in an unbiased manner, seeking new targets and/or new therapeutics to inhibit proteolysis in cancer. In paper III, we explored whether inhibiting the turnover of specific UPS reporters can be achieved by modulating the delivery of proteins to the proteasome.

In paper I, we characterized CBK77, a first-in-class UPS inhibitor that requires the enzymatic activity of the oxidoreductase NQO1 to be activated in cells. CBK77 impairs the degradation of ubiquitin-dependent substrates, leading to the accumulation of ubiquitylated proteins followed by caspase-mediated cell death. We found that activated CBK77 binds to ubiquitin and hinders deubiquitylating activity in vitro, providing a plausible mechanism for CBK77-induced UPS impairment. We propose that bioactivation can be exploited as a new means to increase cancer selectivity of UPS inhibitors. In paper II, we describe CBK79, a promising novel small molecule inhibitor of proteostasis that simultaneously impairs both the UPS and autophagy and induces profound proteotoxic stress in cancer cells. Whilst this leads to the activation of several stress responses to counteract the disruptive effect of CBK79 on proteolysis, these are not successful in restoring homeostasis or preventing cell death. This work shows the potential of dual targeting of the UPS and autophagy for the development of inhibitors that can overcome cellular compensatory mechanisms and could therefore result in more efficient targeting of cancer cells. In paper III, we studied the mechanisms controlling the delivery of proteins to the proteasome through the ubiquitin-dependent Cdc48/VCP/p97 pathway. We used the turnover of Cdc48-dependent fluorescent reporters in yeast to study these processes and found that the ubiquitin shuttle protein Rad23 is itself ubiquitylated prior to substrate delivery. Modulating this step affected the degradation of Cdc48 model substrates. Overall, our findings reveal an additional layer of regulation in the UPS that could be explored for pharmacological intervention.

In conclusion, by employing reporter substrates of the UPS, we have uncovered new compounds and highlighted novel regulatory processes amenable to targeting with the ultimate goal of expanding the army of proteolysis inhibitors with anti-cancer properties.