Ixazomib, An Investigational Orally Bioavailable Proteasome Inhibitor, Increases p21 Expression Inducing Caspase-Dependent Cell Death, Cell-Cycle Arrest, and In B-Cell Lymphoma Pre-Clinical Models

Pharmacological inhibition of the proteasome with bortezomib (BTZ) has translated into an improved clinical outcome in patients with multiple myeloma and mantle cell lymphoma. Despite the observed clinical activity, BTZ anti-tumor activity in B-cell lymphoma has been partially hindered by treatment-related toxicities (peripheral neuropathy) preventing further dose escalation and emergence of acquired resistance. To further develop therapeutic strategies targeting the proteasome system, we studied the anti-tumor activity and mechanisms-of-action of ixazomib (MLN2238), a reversible proteasome inhibitor, in pre-clinical lymphoma models. Previously we demonstrated that ixazomib is active in various lymphoma pre-clinical in vitro models and that is capable of inducing cancer cell death in a caspase-independent manner. To further explore the effects of ixazomib, we investigated its anti-tumor activity in murine lymphoma models and investigated the mechanisms responsible for cell death observed in our pre-clinical models. For in vivo studies, 6-8 week old severe combined immunodeficiency (SCID) mice were inoculated via tail vein injection (iv) with mantle cell lymphoma Granta cells (day 0) and assigned to observation, ixazomib (iv) (at 6mg/kg/dose on days +1, 4, 8, 11, 15 and 18) or BTZ (ip) (at 0.8mg/kg/dose on days +1, 4, 8, 11, 15, and 18). Differences in survival (measured as the time to limb paralysis development) were evaluated by log-rank test across treatment arms. In addition, we studied the role of p21 in the anti-tumor activity of ixazomib. A panel of rituximab-sensitive (RSCL) and -resistant cell lines (RRCL) was exposed to ixazomib. Changes in cell cycle distribution and expression levels of key cell cycle regulatory proteins were evaluated by Western blotting and flow cytometry respectively. To further define the role of p21 in ixazomib activity, transient p21 knock down was achieved using electroporation with a pooled p21 siRNA. Down regulation of p21 was confirmed by Western blotting. Following transient p21 knock down, RSCL or RRCL were exposed to ixazomib and changes in cell viability were determined using the cell titer glo assay. Finally, RSCL and RRCL were exposed to ixazomib (10nM) +/- the cell cycle inhibitor roscovitine (10nM) and viability was determined by measuring changes in ATP content. As single agent, ixazomib prolonged the survival of Granta-bearing SCID mice when compared to control or BTZ (median=24 vs. 27 vs. 35 days; P = 0.012). In addition, in vitro exposure of lymphoma cell lines to ixazomib resulted in p21 and cell cycle arrest in G1 (RSCL) or G2/M (RRCL). Transient knock down of p21 rescued both Raji RSCL and RRCL from the cytotoxic effects of ixazomib when compared to controls. Moreover, in vitro exposure of RSCL to ixazomib in the presence to roscovitine resulted in synergistic effects on cell viability. Together our data suggests that ixazomib is more effective than BTZ in controlling mantle cell lymphoma growth in vivo. In addition, MLN2238 anti-tumor activity appears to be mediated partially by the stabilization of p21. (Ixazomib was obtained from Millennium Pharmaceuticals, Inc. Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund.)