Covalent cysteine targeting of Bruton’s tyrosine kinase (BTK) family by withaferin-A reduces survival of glucocorticoid-resistant multiple myeloma MM1 cells

Multiple myeloma (MM) is a hematological malignancy characterized by plasma cells’ uncontrolled growth. The major barrier in treating MM is the occurrence of primary and acquired therapy resistance to anticancer drugs. Often, this therapy resistance is associated with constitutive hyperactivation of tyrosine kinase signaling. Novel covalent kinase inhibitors, such as the clinically approved BTK inhibitor ibrutinib (IBR) and the preclinical phytochemical withaferin A (WA), have, therefore, gained pharmaceutical interest. Remarkably, WA is more effective than IBR in killing BTK-overexpressing glucocorticoid (GC)-resistant MM1R cells. To further characterize the kinase inhibitor profiles of WA and IBR in GC-resistant MM cells, we applied phosphopeptidome- and transcriptome-specific tyrosine kinome profiling. In contrast to IBR, WA was found to reverse BTK overexpression in GC-resistant MM1R cells. Furthermore, WA-induced cell death involves covalent cysteine targeting of Hinge-6 domain type tyrosine kinases of the kinase cysteinome classification, including inhibition of the hyperactivated BTK. Covalent interaction between WA and BTK could further be confirmed by biotin-based affinity purification and confocal microscopy. Similarly, molecular modeling suggests WA preferably targets conserved cysteines in the Hinge-6 region of the kinase cysteinome classification, favoring inhibition of multiple B-cell receptors (BCR) family kinases. Altogether, we show that WA’s promiscuous inhibition of multiple BTK family tyrosine kinases represents a highly effective strategy to overcome GC-therapy resistance in MM.Ministry of Education (MOE)Ministry of Health (MOH)National Medical Research Council (NMRC)National Research Foundation (NRF)Published versionThis research was funded by Foundation Against Cancer (Belgium), grant number 7872, Hercules Foundation, grant number AUHA/13/012 and Research Foundation Flanders, grant number FWO G1179120N, G059713N. N.K.V. acknowledges funding support from the Singapore Ministry of Education (MOE) under its MOE Academic Research Fund (AcRF) Tier 2 Grant (grant number MOE2017-T2-2-004) and the National Research Foundation Singapore under its Open Fund Large Collaborative Grant (grant number OFLCG18May-0028) and administered by the Singapore Ministry of Health’s National Medical Research Council (NMRC)