First-in-Class Oral Small Molecule Against the Tyrosine Kinase ROR1 (KAN0439834) Induced Significant Apoptosis of Chronic Lymphocytic Leukemia Cells

Background: ROR1 is a transmembrane protein of the receptor tyrosine kinase (RTK) family normally expressed during embryogenesis, but repressed in most normal adult tissues. ROR1 is of importance for cell proliferation, survival, differentiation, metabolism, and polarity. In chronic lymphocytic leukemia (CLL), ROR1 is upregulated and constitutively phosphorylated. Inhibition of ROR1 by siRNA and monoclonal antibodies (mAbs) abrogated downstream kinase activities as well as induced apoptosis of CLL cells. KAN0439834, a small molecule inhibitor (535 Da) was synthesized with the aim to bind to the TK domain of ROR1 and inhibit phosphorylation.

Aim: In the present study we investigated the effects of KAN0439834, on survival of CLL cells and signaling, and in vivo effects in NOD-SCID mice xenografted with human CLL cells.

Methods: MTT and Annexin V/PI assays were used to analyse cytotoxicity of KAN0439834. Fresh CLL cells were also co-cultured with the human stromal cell line HS-5 (ROR1 negative) and incubated in vitro with KAN0439834 and apoptosis measured (Annexin V/PI). CLL cells were also cultured with KAN0439834 (50-250 nM) for 2 h and recombinant Wnt5a (25-50 µg/mL) was added. The cells were further incubated for 30 min before lysate preparation and cytotoxicity assayed after 24 h. Proximity ligation assay was used to investigate the effects of KAN0439834 on ROR1/LRP6 heterodimerization. Phosphorylated RTKs in CLL cells and the effects of KAN0439834 on phosphorylation of RTKs were analysed by the Human-phospho-RTK array and WB. Finally, in two mice experiments, fresh CLL cells were grafted into NOD-SCID mice and oral KAN0439834 treatment was started 7 days after transplantation and continued for 7 days in the first study and 14 days in the second study.

Results: KAN0439834 effectively induced apoptosis of CLL cells (EC₅₀=250 nM) with a 60-fold selectivity for CLL cells compared to normal PBMC. The molecule induced significant apoptosis of CLL cells from all tumor compartments. Importantly, KAN0439834 was significantly more effective than an anti-ROR1 mAb (against the external part of ROR1) in inducing apoptosis of CLL cells. When CLL and HS-5 cells were co-cultured, HS-5 cells could partially prevent induction of apoptosis of CLL cells at low concentrations of KAN0439834, while at higher concentrations of KAN0439834 the presence of stromal cells had no effect. Wnt5a increased phosphorylation of ROR1 in a dose dependent manner. KAN0439834 dephosphorylated both ROR1 and SRC as well as inhibited Wnt5a-induced ROR1 phosphorylation in this system. KAN0439834 dephosphorylated ROR1, LRP6 and associated downstream signaling molecules as GSK3β, PKCδ, MAPK, PI3K, AKT, mTOR, and CREB. KAN0439834 was highly specific for inhibition of ROR1 phosphorylation as compared to other kinase inhibitors in clinical use for cancer treatment. KAN0439834 could be administered in a dose-schedule to mice giving a plasma conc. of > 600 nM for at least 6 h which according to in vitro pharmacokinetic analyses should be sufficient to induce irreversible apoptosis of CLL cells. Oral administration of KAN0439834 to NOD-SCID mice significantly inhibited growth of xenografted human CLL cells. No major side effects were observed in the KAN0439834 treated mice. Immunohistochemistry staining of spleens from the two studies showed a significant decrease of CLL cells in mice treated with KAN0439834 but not in controls.

Conclusions: KAN0439834 is the first generation of a novel class of ROR1-inhibiting small molecule drugs developed by phenotypic screening using CLL cells. The small molecule was much more effective in inducing CLL apoptosis than the anti-ROR1 antibody. KAN0439834 inactivated various signaling pathways in the leukemic cells suggested to be associated with ROR1 signaling. The development of new anti-cancer drugs with other mechanisms of action than those clinically available when existing drugs fail is warranted to improve the prognosis of CLL. KAN0439834 may not only be potent in CLL but also in other ROR1 expressing tumors; such studies are in progress. Our results support the further development of ROR1 inhibitors as a new therapeutic principle.