Tp-5809, A Novel Tnk1 Inhibitor, Suppresses Tnk1 Dependent Signaling And Tumor Growth In A Preclinical Model Of Hodgkin'S Lymphoma.

Abstract TNK1 (thirty-eight-negative kinase 1) is an ACK non-receptor tyrosine kinase expressed during developmental hematopoiesis. Expression in adult tissues has been implicated in hematologic malignancies and in mediation of cellular stress and apoptosis. A truncating mutation leading to overexpression and hyperactivation of TNK1 drives growth and proliferation of L540 cells, a Hodgkin's lymphoma-derived cell line. TNK1 knockdown is known to reduce phosphorylation of downstream effectors STAT3 and STAT5 in L540 cells, to sensitize cancer cells to stress. However, there are no specific TNK1 inhibitors currently approved for use in hematological malignancies. We propose that pharmacological inhibition of TNK1 phosphorylation can suppress TNK1-driven pathology in preclinical tumor models. TP-5809 is a novel small molecule that potently inhibits TNK1 with an IC50 of 1.5 nM in a biochemical kinase assay. TP-5809 shows cellular target engagement at low nanomolar concentrations (IC50 = 11.3 nM) using a TNK1 Nanobret assay in HEK293 cells. In addition, TP-5809 inhibits TNK1-dependent Ba/F3 cell line growth with an IC50 less than 10 nM. In cell culture viability assays, TP-5809 has highest potency against the L540 Hodgkin's lymphoma cell line that expresses truncated TNK1 (IC50 = 7.2 nM), with nearly 1000-fold higher potency compared to K562 cells that express full length TNK1 and are not dependent on TNK1 for growth (e.g. IC50 = 2800 nM). TP-5809 inhibits STAT5 phosphorylation in L540 cells with an IC50 of 1 nM, whereas no pSTAT5 inhibition is observed in K562 cells. Importantly, TP-5809 efficiently inhibits growth of L540 Hodgkin's lymphoma in a mouse xenograft model but has no effect on K562 tumor growth. Daily dosing with TP-5809 inhibits tumor growth by 65.5% at 25 mg/kg, and by 78.7% at 50 mg/kg in the L540 xenograft mouse model, with a concurrent dose and time-dependent STAT5 and TNK1 phosphorylation inhibition. Pharmacokinetic and toxicology studies with TP-5809 have shown that exposure at biologically relevant doses is well-tolerated in mice, rats, and dogs. Taken together, these data show that TP-5809 potently inhibits TNK1 kinase function in biochemical and cellular assays. The data also show that TP-5809 actively inhibits TNK1 function in a TNK1-dependent xenograft model. We conclude that TP-5809 is a biologically active TNK1 inhibitor, which could allow further exploration of TNK1 as a clinically relevant therapeutic target in lymphoma and/or other TNK1-dependent cancers. Targeting aberrant TNK1 phosphorylation using TP-5809 can potently and specifically inhibit pathology driven by hyperactive and mutant forms of TNK1. Citation Format: Tetyana V. Forostyan, Ethika Tyagi, Jason M. Foulks, Matthew Lalonde, Joshua L. Andersen, Adam Siddiqui, Clifford J. Whatcott, David Bearss, Steve Warner. TP-5809, a novel TNK1 inhibitor, suppresses TNK1 dependent signaling and tumor growth in a preclinical model of Hodgkin's lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1478.