Data from Antitumor Activity of the Novel BTK Inhibitor TG-1701 Is Associated with Disruption of Ikaros Signaling in Patients with B-cell Non–Hodgkin Lymphoma

<div>AbstractPurpose:<p>Despite the remarkable activity of BTK inhibitors (BTKi) in relapsed B-cell non–Hodgkin lymphoma (B-NHL), no clinically-relevant biomarker has been associated to these agents so far. The relevance of phosphoproteomic profiling for the early identification of BTKi responders remains underexplored.</p>Experimental Design:<p>A set of six clinical samples from an ongoing phase I trial dosing patients with chronic lymphocytic leukemia (CLL) with TG-1701, a novel irreversible and highly specific BTKi, were characterized by phosphoproteomic and RNA sequencing (RNA-seq) analysis. The activity of TG-1701 was evaluated in a panel of 11 B-NHL cell lines and mouse xenografts, including two NF-κB- and BTK<sup>C481S</sup>-driven BTKi-resistant models. Biomarker validation and signal transduction analysis were conducted through real-time PCR, Western blot analysis, immunostaining, and gene knockout (KO) experiments.</p>Results:<p>A nonsupervised, phosphoproteomic-based clustering did match the early clinical outcomes of patients with CLL and separated a group of “early-responders” from a group of “late-responders.” This clustering was based on a selected list of 96 phosphosites with Ikaros-pSer442/445 as a potential biomarker for TG-1701 efficacy. TG-1701 treatment was further shown to blunt Ikaros gene signature, including <i>YES1</i> and <i>MYC</i>, in early-responder patients as well as in BTKi-sensitive B-NHL cell lines and xenografts. In contrast, Ikaros nuclear activity and signaling remained unaffected by the drug <i>in vitro</i> and <i>in vivo</i> in late-responder patients and in BTK<sup>C481S</sup>, BTK<sup>KO</sup>, and noncanonical NF-κB models.</p>Conclusions:<p>These data validate phosphoproteomic as a valuable tool for the early detection of response to BTK inhibition in the clinic, and for the determination of drug mechanism of action.</p></div>