Introduction: NF-κB has been shown to be critical for HL cell survival. Ixazomib is a new oral PI with favorable pharmacodynamic and pharmacokinetics; we recently reported activity of ixazomib in HL cells and in vivo SCID xenografts (Ravi et al. Cancer Research. 2016). SBA favored tumor inhibition via downregulation of MYC and CHK1 with CHK1 involved in Myc alteration through chromatin modification & histone H3 acetylation. Continued dissection of cell-specific transcriptome changes with PIT is needed and potential combination therapy with ixazomib should be explored.
Methods: We examined ixazomib-induced transcriptome changes in L540 & L428 cells that were ixazomib sensitive and also with generated resistance. Key genes were derived from overlapping Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA). SBA were used to identify genes related to treatment response; and based upon the aforementioned results, we studied the HDAC inhibitor (HDACi), belinostat, combined with ixazomib.
Results: On GSEA (Figure A), ixazomib induced downregulation of mitotic cell cycle in L540 cells, while L428 cells had noted upregulation. Chromatin architecture was downregulated in L540, while L428 had no change. Catabolic activity and vesicular function linked to protein localization and transport were upregulated in L540 & L428 cells. Furthermore, analysis of “key genes” predicted greater ‘tumor promotion’ in ixazomib-resistant L428 vs ixazomib-sensitive L428 and L540 cells (Figure B & C). Among 66 common key genes in L540 & L428, the most connectivity was seen between CDKN1A (p21), JUN, GADD45A, ATF4, HSPA8, SQSTM1 (p62), CSF2, CEBPB and its inhibitory binding partners DDIT3 and TRIB3 (associated with cell cycle, apoptosis, response to oxidative stress & autophagy). For combination studies, ixazomib + belinostat was highly synergistic with combination index <0.5. On Western blot, ixazomib + belinostat resulted in increased p21, cleaved caspases, decreased autophagy (ie, proteins p62 and cathepsin) and also decreased Myc, which had previously been implicated with ixazomib.
Conclusions: Ixazomib alone has prominent in vitro and in vivo activity in HL, and via SBA, we identified rational synergistic therapy with HDACi. In addition, we identified HL cell-specific transcriptome changes affecting varied and distinct pathways involving cell cycle, autophagy and apoptosis. Continued understanding of cell and drug-specific omic alterations are warranted.