Abstract 1738: Decreases in ubiquitin levels post proteasome inhibition identify FKBP4 immunophilin as a novel target for potentiating the anti-myeloma activity of carfilzomib

Abstract

Abstract Proteasome inhibitors have become the standard of care in relapsed/refractory multiple myeloma (MM) with the FDA approval of bortezomib (BTZ) in 2004 and carfilzomib (CFZ) in 2012. As the cell's center for protein degradation, the proteasome impacts many pathways which made it a challenge to determine the key molecular sequelae post proteasome inhibition (PI). The addition of ubiquitin (Ub) to a protein can trigger targeting to the proteasome or modulation of signaling potential. We hypothesized that monitoring Ub modulation on proteins may reveal pathways that are critical for the biology of PI in MM. We conducted mass spectrometry based Ub profiling that utilizes an antibody to the di-glycine motif that characterizes Ub linkages (Cell Signaling). U266 and NCI-H929 MM cell lines were treated with CFZ or BTZ for 1 hr and samples were collected at 1 and 4 hrs for ubiquitome analysis. A concentration of 125 nM was chosen to reflect physiologically achieved target inhibition levels, which correlates with ∼80% cell death after 48 hrs. An order of magnitude more changes in ubiquitination was observed at 4 hrs compared to 1 hr, demonstrating a kinetic progression in Ub conjugation correlating with an increase in cellular stress post PI. In addition to the expected increase in ubiquitination post PI, we also observed a significant decrease in Ub in ∼20% of the total modulations detected. While studies (Kaiser et al, Nat Methods 2011) have reported that deubiquitination may be a mere redistribution of Ub to proteins prioritized by the cell for inactivation under stress, it may also be a signal to stabilize proteins prioritized for activation. Candidate proteins were identified by those that showed decreased Ub in CFZ as well as BTZ treated U266 and H929 samples after 4 hrs, as well as a decrease in at least one cell line at 1 hr. Of these proteins, ubiquitination of FKBP4 on lysine 28 decreased 5 fold at 1 hr and 33 fold at 4 hrs in H929 and decreased 150 fold in U266 at 4 hrs with no change at 1 hr. FKBP4, an immunophilin also known as HSP56, is involved in protein folding and trafficking which could point to its role in response to PI. Transcript analysis has revealed an increase of FKBP4 transcript in U266 cells treated with CFZ compared to DMSO at 4 and 8 hrs post treatment, which supports the hypothesis that deubiquitination post PI may unveil proteins required for survival. Tacrolimus, an approved immunomodulatory therapy known to bind FKBP4, was used as a chemical modulator for drug combination experiments with CFZ. Favorable bliss synergy scores were observed, with values exceeding 0.4 for both U266 and H929 cell lines when cell viability was measured after 48 hrs. Overexpression studies for FKBP4 are currently on-going to genetically validate it as a resistance protein to proteotoxic stress in MM. These data suggest that a Tacrolimus and CFZ combination could be efficacious in MM. Citation Format: Eric Lowe, Shirin Arastu-Kapur. Decreases in ubiquitin levels post proteasome inhibition identify FKBP4 immunophilin as a novel target for potentiating the anti-myeloma activity of carfilzomib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1738. doi:10.1158/1538-7445.AM2015-1738