Ibrutinib inhibits BTK-driven NF-κB p65 activity to overcome bortezomib-resistance in multiple myeloma

Abstract

Abbreviations: MM -multiple myeloma; PI -proteasome inhibitor; NF-kB -nuclear factor-kappa B; BMSC -bone marrow stromal cells; BTK -Bruton's tyrosine kinase.Multiple Myeloma (MM) is a haematologic malignancy characterized by the accumulation of clonal plasma cells in the bone marrow. Over the last 10-15 y the introduction of the proteasome-inhibitor bortezomib has improved MM prognosis, however relapse due to bortezomib-resistance is inevitable and the disease, at present, remains incurable. To model bortezomib-resistant MM we generated bortezomib-resistant MM cell lines (n D 4 ) and utilised primary malignant plasma cells from patients relapsing after bortezomib treatment (n D 6 ). We identified enhanced Bruton's tyrosine kinase (BTK) activity in bortezomib-resistant MM cells and found that inhibition of BTK, either pharmacologically with ibrutinib (0.5 mM) or via lenti-viral miRNA-targeted BTK interference, re-sensitized previously bortezomib-resistant MM cells to further bortezomib therapy at a physiologically relevant concentration (5 nM). Further analysis of pro-survival signaling revealed a role for the NF-kB p65 subunit in MM bortezomib-resistance, thus a combination of BTK and NF-kB p65 inhibition, either pharmacologically or via further lenti-viral miRNA NF-kB p65 interference, also restored sensitivity to bortezomib, significantly reducing cell viability (37.5 § 6 .9 %, ANOVA P 0 .001). Accordingly, we propose the clinical evaluation of a bortezomib/ibrutinib combination therapy, including in patients resistant to single-agent bortezomib.

TrxR1 inhibition overcomes both hypoxia-induced and acquired bortezomib resistance in multiple myeloma through NF-кβ inhibition

“…Higher NF-kb expression is seen in bortezomib refractory primary MM patients 18 and bortezomib resistance in MM is associated with increased basal nuclear localization of NF-kb p65 in bortezomib-resistant myeloma cells. 13 Here, we also show increased nuclear NF-kb p65 levels in bortezomib-resistant U266 cells compared to the parent counterpart. We showed that TrxR1 inhibition using auranofin reduced NF-kb p65 mRNA and protein expression, reduced the mRNA levels of downstream NF-kb-regulated genes including anti-apoptotic and proliferation genes in bortezomib-resistant U266 cells (Fig.…”

“…Increased nuclear p65 protein levels have been correlated with the acquisition of drug resistance in cancer cells. 13,29 Our results showed that auranofin-induced TrxR1 inhibition reduced the hypoxia-induced nuclear accumulation of NF-kb p65 protein, reduced the mRNA levels of p65 and downstream NF-kb-regulated genes, Survivin and Cyclin D1 (Fig. 3).…”

“…[27][28][29] Moreover, increased NF-kb expression has been linked to bortezomib resistance in myeloma cells. 13 Therefore, we first aimed to examine the effect of hypoxia on NF-kb p65 expression in myeloma cells. U266 cells were cultured under hypoxia for different time-periods (0-24 hours) and NF-kb p65 protein levels were analyzed by western blot analysis.…”

“…Acquisition of bortezomib resistance has been attributed to different mechanisms including increased growth factor expression, 11 a mutated proteasome subunit PSMb5 and overexpression of PSMb5 protein, 5 an upregulated NF-kb signaling pathway, 12,13 and overexpressed antioxidant molecules.…”

“…[18][19][20] This upregulation of NF-kb expression and its signaling pathway has been associated with acquisition of bortezomib resistance due to prolonged drug exposure in MM. 13,18 However, the role of the NF-kb signaling pathway in hypoxia-induced bortezomib resistance in MM is unknown.…”