(635e) Characterization of PMI5011 on the Regulation of Deubiquitinating Enzyme Activity in Multiple Myeloma

Aberrations in the ubiquitin proteasome system (UPS) contribute to the pathological states of several clinical disorders, specifically cancer, thus indicating that targeting proteolytic and regulatory components of the UPS is an efficient strategy for cancer treatment. While inhibition of the proteasome has proven to be effective in the treatment of multiple myeloma (MM), a significant challenge with these proteasome-targeted therapeutics is the heterogeneity associated with cancer cells. This has led to the development of inhibitors for other enzymes associated with the UPS such as the deubiquitinating enzyme (DUBs). While recent studies have suggested that controlled positive regulation of certain DUBs can stabilize autophagy and levels of protein degradation, there are currently no targeted therapeutics that can positively regulate DUB activity. In this work, a botanical extract from Artemisia dracunculus L (herein referred to as PMI5011) was characterized for its effects on DUB activity in two model MM cell line (OPM2 and MM.1S) extracts. Prior studies have been informative on the potential of PMI5011 as a potential therapeutic for diabetes; however, they have not investigated the role of the plant extract on DUB activity in cancer cells. The total extract PMI5011 contains several bioactive components of which five have been identified to exhibit bioactivity: DMC-1, DMC-2, davidigenin, sakuranetin and 6-demethoxycapillarisin. An in-depth enzymology analysis was performed using a commercially available fluorescent DUB reporter to demonstrate a significant effect of PMI5011 and three bioactive components (DMC-1, DMC-2, and davidigenin) on DUB activity. Concentration-dependent studies and mathematical modeling revealed that DUB kinetics in the presence of PMI5011, in both OPM2 and MM.1S lysates, followed a heterotropic co-operativity model where PMI5011 bound with the active domains of DUBs to directly or indirectly enhance their activity without affecting the cellular viability. A similar kinetic profile was observed for DMC-1, DMC-2, and davidigenin; however, their influence on DUB activity was found to be cell line dependent at higher concentrations. Interestingly, davidigenin was found to inhibit DUB activity in MM.1S cells but enhance in OPM2 cells. Whereas, a knock-out extract (KOE) missing both DMC-1 and DMC-2 was observed to enhance DUB activity in both OPM2 and MM.1S lines suggesting that these two bioactive components were not the primary compounds to stimulate DUB activity in the PMI5011 plant extract. An mRNA profile revealed that this cell dependent effect of PMI5011 and its components on OPM2 and MM.1S was due to differential expression of specific DUBs such as USP7, USP15, USP14, Rpn11 in OPM2 and MM1S. These findings demonstrating the complexity of the PMI5011 plant extract and its various bioactive components on DUB regulation reveal their therapeutic properties, but also shine light on the allosteric nature and structural specificity of these enzymes.