Selective Inhibition of the Chymotrypsin-Like Activity of the Immunoproteasome and Constitutive Proteasome Represents a Valid Anti-Tumor Strategy in Waldenstrom Macroglobulinemia.
Authors: Roccaro Aldo M. , Sacco Antonio , Aujay Monette , Ngo Hai , Azab Feda , Azab Abdel Kareem , Melhem Molly , Runnels Judith M. , Quang Phong , Demo Susan , Ghobrial Irene 
Published: October 2019

Abstract
Abstract 4911

Introduction
Proteasome inhibition represents a valid therapeutical approach in several tumors and its use has been validated in Waldenstrom macroglobulinemia (WM), where single-agent Bortezomib has been tested in phase 2 clinical trials, achieving 40% to 80% responses. Nevertheless, a significant fraction of patients relapse, or develop significant neuropathy. Therefore preclinical evaluation of new proteasome inhibitor is needed in order to improve patient outcome. We tested PR-047, a new orally bioavailable analog of carfilzomib which selectively inhibits the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome, in WM.

Methods
WM and IgM secreting low-grade lymphoma cell lines (BCWM.1, MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Expression of imunoproteasome and constitutive proteasome subunits (beta1, beta2, beta5; LMP2, MECL1, LMP7) were detected primary WM cells and cell lines by an ELISA-based assay. Cytotoxicity, DNA synthesis, cell cycle and apoptosis were measured by thymidine uptake, MTT, PI staining/flow cytometry analysis and DNA fragmentation, respectively. NF-kB activity has been evaluated on nuclear proteins using a DNA-binding ELISA-based assay. Cell signaling and apoptotic pathways were determined by Western Blot. Determination of the additive or synergistic effect of drugs combination was calculated using the CalcuSyn software based on the Chou-Talalay method.

Results
Primary bone-marrow derived WM cells are characterized by higher expression of the immunopreoteasome as compared to the constitutive proteasome. PR-047 inhibited the chymotrypsin-like activity of both the immunoproteasome (LMP7) and the constitutive proteasome (beta5) and in WM cells, leading to induction of cytotoxicity in primary WM cells; as well as to programmed cell death in a caspase-dependent and -independent manner, as shown by activation of c-jun-N-terminal kinase; inhibition of NF-kB; and initiation of the unfolded protein response. PR-047 induced cytotoxicity and inhibited DNA synthesis in primary WM cells (IC50: 50-80nM), as well as in IgM secreting low grade lymphoma cells (IC50: 30-50nM). Importantly, PR-047 exerted cytotoxicity in WM cells, even in the context of bone marrow milieu, by inhibiting IL-6- and IGF1-BMSC secretion and BMSCs-induced phosphorylation of Akt and ERK in WM cells. Moreover, combination of PR-047 and bortezomib induced synergistic cytotoxicity in WM cells, as shown by enhanced caspases-, PARP-cleavage; NF-kB inhibition; and synergy in inhibiting the chymotrypsin- and caspase-like activities of the immunoproteasome and constitutive proteasome.

Conclusion
These preclinical findings demonstrate that PR-047 targets WM cells, due to its anti-CT-L activity of both immunoproteasome and constitutive proteasome, providing the framework for testing PR-047 in this disease.

Disclosures
Aujay: Proteolix: Employment, Equity Ownership. Demo:Proteolix: Employment, Equity Ownership. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.