ANTITUMORAL ACTIVITY OF THE NOVEL BTK INHIBITOR TG‐1701 IS ASSOCIATED WITH DISRUPTION OF IKAROS SIGNALING AND IMPROVEMENT OF ANTI‐CD20 THERAPY IN B‐CELL NON‐HODGKIN LYMPHOMA

Introduction: Covalent Bruton's tyrosine kinase inhibitors (BTKis) have transformed the treatment of B-cell non-Hodgkin lymphoma (B-NHL). However, their activity has been often limited by 1) acquired resistance due to the development of a cysteine to serine mutation at the BTK catalytic site (BTKC481S) or over-activation of the NF-kB pathway, and 2) off-target activity that precluded their use in combination with anti-CD20 antibodies. TG-1701 is a novel irreversible and highly specific BTKi currently under study in patients with relapsed/refractory (R/R) B-NHL alone and in combination with ublituximab, a new glycoengineered anti-CD20 antibody, and umbralisib, a dual PI3Kδ and casein kinase-1ε inhibitor (also referred to as the U2 regimen). Methods: A set of 6 B-NHL clinical samples from TG-1701 phase 1 trial were characterized by phosphoproteomic and RNA-seq analysis, followed by biomarker validations using real-time PCR and western blot. Activity of TG-1701 either alone or in combination with U2 regimen was evaluated by proliferation, antibody-dependent cell cytotoxicity (ADCC) and phagocytosis (ADCP) assays in a panel of n = 10 B-NHL co-cultures and in two mouse xenografts, including non-canonical NFκB and BTKC481S resistant models. Biomarker validation and signal transduction analysis were conducted through immunofluorescence, immunohistochemical studies and gene knock-out (KO) experiments. Results: Phosphoproteomic analysis of tumoral lymphocytes from B-NHL patients receiving TG-1701 led to a non-supervised clustering that matched the early clinical outcomes and separated a group of early “responders” from a group of “non-responders”. This clustering was based on a selected list of 96 phosphosites, with Ikaros-Ser442/445 phosphorylation as a potential biomarker for TG-1701 efficacy. RNA-seq analysis revealed that TG-1701 treatment blunted the Ikaros gene signature, including YES1, MYC and IRF4, in responder patients as well as in BTKi-sensitive B-NHL cell lines and xenografts. On the contrary, Ikaros nuclear activity and Ikaros-dependent gene regulation remained unaffected by the drug in non-responder patients and in BTKC481S , BTKKO and non-canonical NFκB models in vitro and in vivo. Interestingly, and in contrast with the first-in-class BTKI, ibrutinib, TG-1701 did not impair FcγR-driven ADCC and ADCP triggered by the anti-CD20 antibodies rituximab and ublituximab in different B-NHL co-culture system, and cooperated with U2 in reducing the tumor growth in both ibrutinib-sensitive and ibrutinib-insensitive mouse models of B-NHL. Conclusions: Altogether, 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. Our results also support the use of TG-1701-U2 combination in R/R B-NHL patients, irrespective of prior response to ibrutinib. EA – previously submitted to regional or national meetings (up to 1000 attendees). The research was funded by: TG Therapeutics, Fondo de Investigación Sanitaria PI18/01383, European Regional Development Fund (ERDF) “Una manera de hacer Europa” (to GR). Keywords: Molecular Targeted Therapies, Aggressive B-cell non-Hodgkin lymphoma Conflicts of interests pertinent to the abstract G. Roue Employment or leadership position: H. Miskin and E. Normant are employees with stock ownership in TG Therapeutics, Inc Research funding: G.Roué received research funding from TG Therapeutics