Abstract 1963: ARQ 531, a potent reversible BTK inhibitor, exhibits potent antitumor activity in ibrutinib-resistant diffuse large B-cell lymphoma
Authors: Eathiraj Sudharshan , Yu Yi , Savage Ron , Woyach Jennifer A. , Reiff Sean D. , Johnson Amy J. , Schwartz Brian 
Published: August 2018

Abstract
Background: B-cell receptor (BCR)-mediated signaling plays an important role in the pathogenesis of a subset of diffuse large B-cell lymphoma (DLBCL). Despite major advances in the treatment, ~40% of the relapsed/refractory DLBCL patients still experience early treatment failure after initial response to chemotherapy. ARQ 531, a reversible inhibitor of BTK and BTK-C481S mutant, also potently suppresses BCR signaling. Here we demonstrate that ARQ 531 targets additional kinases and suppresses multiple oncogenic pathways, this inhibitory potency is coupled to broad anti-tumor activity in DLBCL subtypes including tumors resistant to BCR targeted therapy.
Methods: Biochemical inhibition and broad kinase profiling were assessed using recombinant proteins. Binding kinetics and the residence time with BTK and BTK-C481S were measured by Surface Plasmon Resonance (SPR) assay. Binding mode of ARQ 531 with BTK was determined by protein crystallography. Pathway inhibition assessments, in vivo efficacy and in vivo target inhibition were performed in DLBCL tumor models derived from TMD8, SUDHL-4 and DOHH-2 cell lines.
Results: ARQ 531 potently inhibited BTK (IC50 = 0.85 nM), the binding potency was accompanied by long residence time (51 min). Crystal structure of BTK/ARQ 531 complex showed that ARQ 531 occupies the ATP-binding pocket. Kinase selectivity profile suggested that ARQ 531 inhibits sub-families of Tec, Src, Trk kinases. Significant anti-proliferative activity (GI50 = &lt; 1µM) was observed in hematological malignant cell lines characterized by addiction to BTK signaling and primarily resistant to ibrutinib. Pathway inhibition analysis suggested that ARQ 531 targets multiple oncogenic signaling pathways in both ABC- and GCB-DLBCL cell lines. Unlike ibrutinib, ARQ 531 suppressed both the upstream activating signals (via inhibition of a select member of Src kinase family) and the downstream signaling pathways (via pAKT and pERK kinases). In GCB-DLBCL cell lines (SUDHL-4 and DOHH-2), ARQ 531 potently suppressed expression of anti-apoptotic c-Myc and BCL6 oncoproteins in a dose dependent fashion, and concomitantly induced apoptotic cleavage of PARP protein. In the ibrutinib-resistant SUDHL-4 mouse xenograft model, ARQ 531 potently suppressed tumor growth (&gt;80% inhibition) compared to the control group when dosed orally at 75 mg/kg. Additionally, preliminary data suggest that ARQ 531 crosses the blood, brain-barrier.
Conclusion: ARQ 531 is a potent reversible inhibitor of BTK, its distinct kinase selectivity profile offers significant advantage for simultaneous inhibition of multiple therapeutically relevant targets. ARQ 531 attenuated the expression of key oncogenic drivers via inhibition of downstream BCR activating kinases. These results highlight the therapeutic potential of inhibition of BCR signaling inhibition by ARQ 531 in the treatment of DLBCL.
Citation Format: Sudharshan Eathiraj, Yi Yu, Ron Savage, Jennifer A. Woyach, Sean D. Reiff, Amy J. Johnson, Brian Schwartz. ARQ 531, a potent reversible BTK inhibitor, exhibits potent antitumor activity in ibrutinib-resistant diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1963.