Small molecule inhibitor of deubiquitinating enzymes exerts potent anticancer effects through ER stress exacerbation

The combination of rapid growth, relative nutrient deprivation and dysregulation of protein synthesis makes tumor cells especially prone to be exposed to endoplasmic reticulum (ER) stress. The resulting accumulation of unfolded or misfolded proteins leads to the activation of the ubiquitin proteasome system (UPS) and to the unfolded protein response (UPR) to alleviate this stress through a triple transcription factor system. Recently, we identified in a drug discovery program, a family of compounds endowed with the capacity to trigger protein poly-ubiquitination in a large variety of human cancer cells. These compounds inhibited cell proliferation (through the induction of caspase-mediated apoptosis), in particular in multiple myeloma cells that are known to be heavily reliant for survival on the UPR. Further work showed that cell exposure to AP0132, the most active compound of the series with IC50s ranging from10 to 100 nM, led to a significant increase in major UPR markers, namely BiP, phospho-eIF2 and CHOP. To examine the mechanisms beyond the AP0132-driven accumulation of ubiquitinated proteins and increased UPR, we next focused on the ubiquitin proteasome system. Unexpectedly, AP0132 failed to inhibit the protease activity of the proteasome and even slightly increased the caspase-like activity component of the proteasome. By contrast, using different in vitro assays including ubiquitin chain disassembly assay, DUB labeling assay and Ub-AML cleavage, we found that AP0132 directly inhibited the deubiquitinase (DUB) activity of the proteasome, thereby preventing the entrance of ubiquitinated proteins into the 26S subunit. We next extended this finding by documenting that AP0132 could inhibit a large number of DUBs, thereby further supporting the accumulation of polyubiquitinated proteins in treated cancer cells. Analysis of the ubiquitinated proteins upon AP0132 treatment through quantitative diGly proteomics identified that most of them were either ER residents or ER-associated proteins. This finding was supported by the rapid accumulation of AP0132 in the ER as shown by taking advantage of the natural fluorescence of AP0132. We finally examined the therapeutic potential of AP0132 in mouse tumor models and found that its daily administration (40 mg/kg i.p.) led to a significant tumor growth delay without signs of toxicity. Altogether, these data indicate that AP0132 is a pan-inhibitor of DUBs with an ER selectivity that exerts potent cytotoxic activity in a large variety of tumor cells. More generally, this works also points towards the double-hit advantage of such pan-inhibitors of DUBs that lead to the accumulation of polyubiquitinated proteins but also prevent their degradation by inhibiting the necessary deubiquination of proteins before processing by the proteasome.