Abstract 317: Vecabrutinib inhibits C481 mutated Bruton's tyrosine kinase and its downstream signaling in vitro

Inhibition of Bruton’s Tyrosine Kinase (BTK) by ibrutinib, an irreversible inhibitor, has dramatically improved the outcomes in both previously treated and naive chronic lymphocytic leukemia (CLL) patients. Ibrutinib inactivates BTK through covalently binding to the cysteine 481 residue (C481) which then leads to the inhibition of autophosphorylation of BTK and the inactivation of downstream survival nexus. Although ibrutinib demonstrated >90% overall and event-free survival, about 25% of patients discontinue ibrutinib due to leukemia progression and intolerance. Patients that initially respond to treatment may develop resistance and the most prevalent resistance mechanism of ibrutinib is described as the point mutation affecting the C481 residue of BTK that results in disruption of ibrutinib binding and acquired ibrutinib resistance. Most common mutations are C481S and C481R. Vecabrutinib is a potent reversible BTK inhibitor that binds to BTK through noncovalent interactions. As vecabrutinib does not require binding to C481 residue, it retains its efficacy with mutant BTK in vitro . To better understand differential biology of Wild-Type (WT) and serine and arginine substituted BTK, we labelled MEC-1 cell line with green fluorescence protein (GFP) and overexpressed either BTK WT , BTK C481S or BTK C481R that contributes to ibrutinib resistance. We selected MEC-1 cell line as it represents CLL disease and the phenotype of the cells share several characteristics of ex vivo CLL cells. We performed Reverse Phase Protein Array (RPPA) and mRNA-sequencing to determine and compare the proteomic and transcriptomic profiles of the MEC-1 cells harboring WT and mutant BTK. Ingenuity Pathway Analysis (IPA) of RPPA data revealed that overexpression of BTK WT leads to the enrichment of protein alterations involved in cell cycle regulation, B cell receptor signaling, PI3K/AKT signaling, PTEN signaling and ERK/MAPK signaling. IPA of RNAseq data upon BTK WT overexpression unraveled the top canonical pathways that include signaling through axonal guidance, ephrin receptor, c-AMP mediated, CXCR4 signaling and PTEN signaling. Comparative analysis of MEC-1 cells with mutant BTK (C481S vs C481R) using IPA distinguished the activated pathways in BTK C481S harboring cells from cells that express BTK C481R . These results are being validated by western blot and qRT-PCR assays. Immunoblotting results showed that following 24, 48, and 72 hour of vecabrutinib (at 1 µM) treatment reduced p-BTK (Tyr223), p-PLCG2 (Tyr759) and p-ERK (Thr 202/Tyr 204) levels in MEC-1 cells with mutant BTK. These data indicate that vecabrutinib effectively inhibits BTK and its downstream signaling in the presence of mutant BTK, suggesting that vecabrutinib treatment may be a rational approach to overcome ibrutinib resistance. Citation Format: Burcu Aslan, Mikhila Mahendra, Michael D Peoples, Joe R. Marszalek, Christopher P Vellano, Xiaofeng Zheng, Jing Wang, Pietro Taverna, Varsha Gandhi. Vecabrutinib inhibits C481 mutated Bruton9s tyrosine kinase and its downstream signaling in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 317.