Acquisition of BTK C481S Produces Resistance to Ibrutinib in MYD88 Mutated WM and ABC DLBCL Cells That Is Accompanied By ERK1/2 Hyperactivation, and Is Targeted By the Addition of the ERK1/2 Inhibitor Ulixertinib

Activating mutations in MYD88 mutations trigger BTK (Yang et al, Blood 2013; Wilson et al, Nat. Med. 2015) and HCK (Yang et al, Blood 2016) activation in Waldenström's macroglobulinemia (WM) and ABC-DLBCL, which activate multiple downstream pro-survival signaling cascades that include NFkB, AKT, and ERK1/2. Ibrutinib targets BTK and HCK, and shows high levels of activity in MYD88 mutated WM and ABC DLBCL. Resistance to ibrutinib has been observed in CLL, MCL, and in WM (Xu et al, submitted) due to acquisition of mutations that impact the binding of ibrutinib to BTK at Cysteine 481, which include the C481S mutation. To explore the functional impact of the BTK C481S mutation on ibrutinib treatment in MYD88 mutated WM and ABC DLBCL cells, we transduced MYD88 L265P mutated BCWM.1 and MWCL-1 WM, and TMD-8 and HBL-1 ABC DLBCL cell lines with either lentiviral vector alone, or lentiviral vectors expressing BTK wild-type (BTK WT) or BTK with C481S (BTK C481S) mutation. Transduction with BTK C481S led to a 1-3 log fold increase in EC₅₀ for ibrutinib versus vector only or BTK WT transduced cells. BTK C481S expressing cells showed hyperphosphorylation of PLCγ2 (Tyr 1217) which is immediately downstream of BTK, and ERK1/2 (Thr 202/Tyr 204) versus vector only or BTK WT cells in all four MYD88 mutated cell lines following ibrutinib (0.1, 0.5 uM) treatment for 2 hrs. In contrast, no changes in the phospho-IKBa (Ser 32), the gatekeeper of NFkB, nor phospho-AKT (Ser 473) or p38MAPK (Thr 180/Tyr 182) were observed between vector only, BTK WT or BTK C481S transduced cells following ibrutinib treatment. Given the hyperactivation of ERK1/2 in BTK C481S transduced cells treated with ibrutinib, we next evaluated the activity of ulixertinib (BVD-523, VRT752271), a highly selective ERK1/2 inhibitor that is currently under clinical investigation. Ulixertinib blocked ERK1/2 activity as evidenced by reduction of phospho-p90RSK (Ser 380) by western blot analysis in BTK C481S transduced BCWM.1 and TMD-8 cells. The combination of ulixertinib with ibrutinib produced higher levels of tumor cell killing than either agent alone, and showed synergistic interactions with combination index (CI) values below 1.0 at pharmacologically relevant concentrations. Our data indicate that acquisition of BTK C481S produces resistance to ibrutinib in MYD88 mutated WM and ABC DLBCL cells that is accompanied by ERK1/2 hyperactivation. The addition of the ERK1/2 inhibitor ulixertinib to ibrutinib at pharmacologically relevant concentrations produced synergistic tumor cell killing in BTK C481S ibrutinib resistant cells. The findings provide rationale for the investigation of ERK1/2 inhibitors in ibrutinib resistant MYD88 driven WM and ABC DLBCL disease mediated by BTK mutations.