Combinatorial Ixazomib and Belinostat Therapy Induce Nuclear Factor Erythroid 2-Related Factor 2 (NRF2)-Dependent Apoptosis in Hodgkin and T-cell Lymphoma

Ixazomib has documented pre-clinical activity in T cell (TCL) and Hodgkin (HL) lymphoma models with transcriptomic analyses predicting synergistic activity in combination with histone deacetylase (HDAC) inhibition. In the present study, we determined the mechanistic basis for synergism with ixazomib in combination with the HDAC inhibitor, belinostat, in HL and TCL cells lines (ixazomib-sensitive and -resistant clones) and primary tumor cells. In ixazomib-treated TCL and HL cells, we observed induction of proteasomal gene expression and NRF2 as a prominent upstream regulator. Ixazomib treatment in TCL and HL cells resulted in transient inhibition followed by full recovery of proteasomal activity within 72 hours. Furthermore, transcriptomic analysis following belinostat treatment resulted in downregulation of both NRF2 and proteasomal gene expression (validated by qPCR) without direct impact on proteasomal activity in Jurkat and L428. In addition, CRISPR/Cas9 mediated knockdown of NRF2 in Jurkat cells resulted in a significant decrease in cell viability with ixazomib compared with untreated control cells. Using transcriptomic and proteosomal activity evaluation of ixazomib, belinostat or ixazomib+belinostat treated cells, we observed that NRF-2, proteasome gene expression and functional recovery were abrogated by the combination. We also confirmed that ixazomib/belinostat resulted in synergistic activity in ixazomib-sensitive and -resistant cell lines and primary cells. Altogether, these results suggest that the synergistic activity of ixazomib and belinostat is mediated via inhibition NRF2-dependent proteasomal recovery and extended proteasomal inhibition culminating in increased cell death. Further investigation is warranted to determine the full biological role for NRF2 as a response modifier in proteasomal targeted therapies. Jurkat, HH, L428, L540 cells were authenticated (by ATCC STR profiling) and maintained under 5% CO2 and at 37OC in culture medium RPMI1640 (Corning, Manassas, VA) with 10% heat inactivated Fetal Bovine Serum (FBS) and antibiotic-antimycotic (200 I.U. Penicillin/Streptomycin, 25µg Amphotericin; Corning, Manassas, VA). Primary lymphoma cells were obtained from cells isolated from ascites fluid of a patient with enteropathy-associated T-cell lymphoma (EATL) refractory to standard chemotherapy, via Institutional Review Board approved consent process and made available for research through Tufts tumor biorepository. The obtained de-identified specimen labeled as EL-8, were cultured using RPMI1640 complete medium through successive passages. Ixazomib resistant cell lines (Jurkat-R and L428-R) were established by culturing in increasing concentrations of ixazomib, 5-50 nM for several months. Ixazomib resistance was determined by comparing IC50 in parental and resistant cells using MTT based cell viability assay. Total RNA was isolated from Jurkat cells treated with ixazomib, belinostat or ixazomib+belinostat for 24 hours, using RNeasy Mini Kit (Qiagen, Valencia, CA). Transcriptomic analysis was performed using Human HT 12 Genechip Illumina (Yale Center for Genomic Analysis). Data normalization (using R), statistical analysis, Gene set enrichment analysis (GSEA) as defined by Subramanian et al. and Ingenuity Pathway Analysis (IPA) were performed as described previously.