Preclinical Evaluation of the Oral Proteasome Inhibitor Ixazomib in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL), the most common human lymphoma, comprises a genetically and clinically diverse group of aggressive B cell non-Hodgkin lymphomas (NHL-B) among a small group of important human cancers increasing in incidence in the US over the last four decades. Current frontline DLBCL therapy although fairly successful (~70-80% remission rate with frontline R-CHOP chemo-immunotherapy), frequently relapses (~40% within 2-3yrs.) often with refractory (r/r) DLBCL leaving only very poor salvage therapy options (<20% PR/CR) and short survival. Of note, 2-3% of DLBCL patients with rearrangements of MYC and either BCL2 or BCL6 gene, so called double-hit lymphoma (DHL), are associated with an even more dismal outcome. Investigation of novel therapeutic approaches for refractory DLBCL is still in progress. Ixazomib (MLN9708), an investigational, orally bioavailable, small-molecule, reversible inhibitor of the 20S proteasome, was pre-clinically evaluated in refractory DLBCL models.  We initially screened the activity of ixazomib in 28 DLBCL cell lines using viability assays with increasing doses of ixazomib (0-1000 nM) for 72 hours.  Exposure of ixazomib resulted in a dose-dependent inhibition of cell viability with IC50s ranging from as low as 20 nM up to 200 nM.  When we compared the IC50 values of ixazomib in DLBCL cell lines with other proteasome inhibitors such as bortezomib (BZ) and carfilzomib (CFZ), the IC50s of ixazomib (average 120 nM) is 16-fold higher than BZ (average 7.4 nM) and 8.8-fold higher than CFZ (average 13.5 nM). We have also evaluated the activity of ixazomib in primary cells from patients with refractory DLBCL (7 cases).  The IC50 values of ixazomib in primary refractory DLBCL ranges from 40 nM to 200 nM.  Similar drug concentrations of ixazomib were tested on normal peripheral blood mononuclear cells, but showed no significant cellular toxicity.  The inhibition effect of ixazomib was noticeable in two representative DLBCL cell lines, MZ (GCB subtype) and RC (double hit-DLBCL) with IC50 well below 50 nM.  Thus, we selected these cell line to test whether ixazomib can induce apoptosis.  Annexin V assays showed that ixazomib effectively induces apoptosis in both MZ and RC cells in a dose- and time-dependent manner. To assess the functional significance of ixazomib-induced cell growth inhibition and apoptosis in DLBCL, proteomic approach by reverse phase protein array (RPPA) was used.  RC and MZ cells were treated with ixazomib (IC75) for 12 hrs and 24 hrs, and protein extracts were analyzed by a customized RPPA with 285 validated antibodies.  The profiles for RC and MZ after ixazomib treatment were quite different in that few common proteins get down/up-regulated.  This could be due to the fact that RC is a double hit (BCL-2 x Myc) DLBCL cell line and utilizes different growth/survival pathways than MZ cells, particularly activation of protein synthesis pathway (IGF-PI3K-PDK1-ATK-mTOR-S6K-S6) and the growth/survival pathway (Fibronectin-FAK-MEK1-p38-ELK1). Ixazomib treatment significantly down-regulated these two pathways in RC cells.  Several common proteins that were significantly up-regulated after 24 hrs of ixazomib treatment in both RC and MZ cells are the apoptotic related proteins (Caspase 3, cleaved caspase 7, and cleaved PARP) and the DNA damage related proteins (H2AX, Histone H-3, DM-Histone-H3, and HM-K9-Histone-H3).  These results suggest that DNA damage could be one of ixazomib’s mechanisms of action. In conclusion, our results indicate that ixazomib is an active proteasome inhibitor in refractory DLBCL cells, and warrants further investigation as a potential therapy for treatment of therapy-resistant DLBCL.