Inhibition Of The Deubiquitinating Enzymes UCHL5 and USP14 Is Lethal To Waldenströms Macroglobulinemia Cells
Authors: Chitta Kasyapa S. , Paulus Aneel , Akhtar Sharoon , Kuranz Maja , Roy Vivek , Ansell Stephen M , Novak Anne J. , Martin Peter , Furman Richard R. , Coleman Morton , Linder Stig , Chanan-Khan Asher 
Published: October 2019

Abstract

Background
Waldenströms macroglobulinemia (WM) is a lymphoplasmacytic subtype of Non-Hodgkins lymphoma in which specific molecular changes lead to unrestricted cell proliferation and overproduction of immunoglobulin-M. These processes require exquisite coordination between growth promoting transcription factors (NFkB) and cell homeostatic systems. The bioavailability of various transcription factors and cell cycle proteins is primarily regulated by the ubiquitin proteasome system (UPS). Moreover, the proteolytic machinery ensures proper elimination of unwanted or misfolded proteins, which when disrupted leads to protein accumulation and apoptotic cell death. The 26S proteasome is a barrel shaped structure with a 20S catalytic core that is flanked by 19S caps on either side. 26S proteasomes are critical for WM cell survival, which is clinically evident, as up to 80% of WM patients treated with the proteasome inhibitor (PI) bortezomib have derived significant benefit. Traditional PI such as bortezomib and carfilzomib have been designed to target the B5 catalytic core within the 26S proteasome. However, proteasomes are capable of altering conformation of their proteolytic subunits in response to inflammation and stress, which can lower the effectiveness of these PI. The 19S cap is the regulatory complex and functions in unfolding and deubiquitinating the proteins before their entry into the 20S complex using constitutive deubiquitinating enzymes (DUBS). Recently, it has been demonstrated that inhibition of the DUBS, UCHL5 and USP14 results in cell death in a variety of tumor types but more so in cancer cells that rapidly divide or have a high protein turnover rate.

Aim
To define the anti-WM activity of the novel DUB inhibitor, b-AP15 in preclinical models of WM.

Methods
The WM cell lines BCWM.1, RPCI-WM1 and MWCL-1 were used for experiments in this analysis. Proteasomal activity was measured using synthetic fluorogenic peptide substrates. Apoptosis was determined by annexin-V/PI staining and mitochondrial membrane permeability (MOMP) was assessed using TMRM followed by flow cytometry. Protein profiling was done via immunoblot analysis.

Results
As reported by D’Arcy et al in their characterization of the b-AP15 molecule, inhibition of UCHL5 and USP14 by b-AP15 does not disrupt proteasomal activity conferred by the b-catalytic subunits. We confirmed these findings by treating WM cells with single agent bortezomib (10nM), carfilzomib (10nM), b-AP15 (100nM) or all three agents in combination to assess chymotrypsin-like, caspase-like and trypsin-like proteasomal activity using fluorogenic peptide substrates. Further, when used in combination with the other PI, PI-mediated chymotrypsin-like activity remained inhibited in the presence of b-AP15. Next, we sought to determine if b-AP15 treatment could induce WM cell death. Annexin-v staining showed that b-AP15 (500uM) induced apoptosis in all WM cell lines (28 – 60% cell death) with variable responses noted among the 3 models. Treatment with b-AP15 increased MOMP while also inducing the cleavage of caspases-9 and 3 and PARP-1 suggesting that the anti-WM activity of b-AP15 is mediated through the intrinsic apoptotic pathway. As components of the NFkB signaling cascade lie downstream to the UPS, we examined the effects of b-AP15 on this pathway. When treated with the DUB-inhibitor, nuclear translocation of NFkB was reduced in a time dependent manner. Interestingly, treatment with b-AP15 increased the phosphorylation of the mitogen activated protein kinase-p38 (p38) in all the WM cell lines tested. Treatment of WM cells with b-AP15 in presence of the p38 inhibitor, SB203580, resulted in a synergistic induction of cell death.
Conclusion
b-AP15 is a novel DUB inhibitor that disrupts two regulatory enzymes (UCHL5 and USP14) present in the 19S proteasomal cap. Results presented here demonstrate that b-AP15 induces apoptosis in in vitro models of WM and with in vivo analysis underway, is a potential therapeutic in WM.
We would like to acknowledge the Waterfall Waldenström Macroglobulinemia Research Fund, the Leukemia and Lymphoma Society and the International Waldenström Macroglobulinemia Foundation for their continued support.

Disclosures:
No relevant conflicts of interest to declare.