Abstract B174: The non-covalent HCK/BTK inhibitor TTX-114 displays potent in vitro and in vivo anti-tumor activity in leukemia and lymphoma

• Published in: Molecular cancer therapeutics 2023-12, Vol.22 (12_Supplement), p.B174-B174
• Main Authors: Rauh, Ulrike, Serrano-Wu, Michael, Kaushik, Virendar, Pilari, Sabine, Ogahara, Ikuko, Kaneko, Akiko, Arribas, Alberto J, Cannas, Eleonora, Bertoni, Franceso, Javidi-Sharifi, Nathalie R, Davids, Matthew S, Fukami, Takehiro, Saito, Yoriko, Ishikawa, Fumihiko, Letai, Anthony, Hubbard, Brian
• Format: Article
• Language: English
• ISSN: 1538-8514; 1538-8514
• DOI: 10.1158/1535-7163.TARG-23-B174

Background: The SRC family kinase Hematopoietic Cell Kinase (HCK) is upregulated in cycle–quiescent and chemotherapy-resistant leukemic stem cells (LSCs), and inhibition of HCK reduces the survival and self-renewal capacity of LSCs. As such, targeting HCK has emerged as a potential therapeutic strategy in acute myeloid leukemia (AML). In lymphoma, HCK phosphorylates and activates Bruton Tyrosine Kinase (BTK) downstream of Toll-like receptor (TLR) or the IL6 receptor. In conjunction with BTK, HCK engages essential pro-survival signaling through the NF-kB, mTOR, AKT and STAT pathways. Meanwhile, in lymphoma, HCK can take over pro-survival signaling to confer resistance to BTK inhibition. A dual HCK/BTK inhibitor thus provides a promising therapeutic approach to target leukemias and lymphomas. Methods: TTX-114 was identified through a structure-based medicinal chemistry campaign and characterized in cells using Western Blot and Cell Titer Glow for signaling and viability read-outs, respectively. For in vivo efficacy analysis, NSG newborn mice were irradiated prior to bone-marrow reconstitution with patient-derived primary AML LCSs. Mice were treated orally with TTX-114 after AML was established. Results: Here, we describe TTX-114, a non-covalent HCK/BTK inhibitor with anti-tumor activity in leukemia and lymphoma. In biochemical kinase profiling TTX-114 displayed sub-nM activity against BTK with a KD = 0.58 nM and the SRC-kinase cluster with a KD = 0.23nM for HCK. By inhibiting HCK and BTK activity in cells, TTX-114 prevented autophosphorylation of HCK at Y411 and BTK at Y223 in a dose-dependent manner in human peripheral blood mononuclear cells (PBMCs), enabling a starting point for clinical demonstration of target engagement. TTX-114 had anti-tumor activity across a larger panel of leukemia and lymphoma cell lines, with diffuse large B cell lymphoma (DLBCL) cell lines being among the most sensitive (IC50, ≤4 nM). In marginal zone lymphoma (MZL), the activity was slightly higher in the presence of MYD88 L265P mutation. In vivo, TTX-114 treatment significantly reduced the tumor burden in a dose and time-dependent manner in AML-derived PDX mice. A systematic pharmacokinetic/pharmacodynamic (PK/PD) study identified distinct PK driver for efficacy and tolerability, respectively enabling a well-tolerated dosing strategy for TTX-114 which induced complete response in AML-xenograft bearing mice with no impact on body weight. Conclusion: TTX-114 is an orally bioavail