Advancements in molecularly targeted therapeutics have shown significant promise in treatment of multiple myeloma including the proteasome inhibitors Bortezomib and Carfilzomib. However, challenges in drug efficacy frequently occur due to tumor heterogeneity, drug resistance, and personalized responses. As such, tremendous attention has shifted towards personalized medicine and high throughput biochemical assays for analyzing single cell behavior. In the case of proteasome inhibition, recent studies have found that tandem therapies targeting both the proteasome and deubiquitinating enzymes (DUBs) have allowed cells to overcome drug resistance. DUBs are part of the ubiquitin-proteasome system (UPS) and are responsible for removing the polyubiquitin chain that targets proteins for proteasomal degradation. In this project, a microfluidic droplet trapping array was coupled with a cell permeable, peptide-based reporter to quantify DUB activity in intact single cells. The peptide reporter consists of an N-terminal β-hairpin ‘protectide’ that both confers stability onto the reporter but also acts as a cell penetrating peptide (CPP). The β-hairpin is conjugated to the N-terminus of a DUB recognizing sequence (LRGG) with a C-terminal fluorophore (AFC, 7-amino-trifluoromethyl coumarin).  Thus, an increase in intracellular fluorescence corresponds to increased DUB activity. To achieve high-throughput screening of DUB activity and cellular resistance to DUB inhibitors, we utilized a microfluific droplet trapping array. First, we demonstrated that capabilities of the microfluidic device to examine cellular heterogeneity by characterizing CPP uptake in cancer cells. The fluorescent images were processed automatically using a Python algorithm followed by hierarchical cluster analysis using SAS enterprise guide to identify the distinct subpopulations present in each sample. As expected, the microfluidic platform was able to identify highly heterogeneous uptake of CPPs that depended upon the location and number of arginine residues. Once the microfluidic platform was optimized, it was then used to examine DUB activity in model multiple myeloma cell lines using the peptide-based reporter to quantify both heterogeneous enzyme activity and resistance to the DUB inhibitor PR-619.  This microfluidic platform has the potential to help with the discovery and diagnosis personalized therapies targeting DUBs in small heterogeneous sample, like patient biopsies.<br/>

High throughput screening of deubiquitinating enzyme activity in single intact cells using a cell permeable peptide-based reporter and a microfluidics droplet trapping array

255th National Meeting (2018)

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