First-in-Class ROR1 Small Molecule Inhibitor (KAN0439834) Downregulated Wnt-Canonical and Non-Canonical Signaling Pathways and Induced Apoptosis of CLL Cells

Background: The receptor tyrosine kinase (RTK) ROR1 is detected during embryogenesis but downregulated in adult normal tissues. However, it is expressed in several solid tumors and hematological malignancies. Targeting ROR1 with specific siRNAs in chronic lymphocytic leukemia (CLL) induced apoptosis of the leukemic cells. Moreover, ROR1 specific monoclonal antibodies (mAbs) dephosphorylated ROR1 followed by apoptosis of the CLL cells. Furthermore, ROR1 tyrosine kinase inhibitors (ROR1-TKI) (small molecule inhibitors) have been shown to dephosphorylate ROR1, downregulate the activated PI3K/AKT/mTOR signaling pathway and induce specific apoptosis of CLL cells.

Aim: In the present report we analysed the effects of a ROR1-TKI drug candidate (KAN0439834) on other intracellular signaling pathways involved in cell survival, differentiation and migration in addition to the PI3K/AKT/mTOR pathway in CLL cells.

Methods: KAN0439834 ROR1-TKI was derived from a high-throughput screening (HTS) and a chemical synthesis program, including cellular assays for CLL specific cytotoxicity. The compound was also tested for ADME and in vivo pharmacokinetics characteristics. Peripheral blood mononuclear cells (PBMC) were derived from patients with CLL and normal healthy donors. Intracellular signaling molecules were analysed by Western blot (WB) after 30 min incubation of the cells with the ROR1-TKI (50-1000 nM). Apoptosis/necrosis was determined by the MTT cytotoxicity assay and Annexin V/PI staining in flowcytometry after 24 h of incubation.

Results: CLL cells expressed ROR1 as determined by WB and flowcytometry. ROR1 was shown to be phosphorylated using a polyclonal anti-phospho-ROR1 (pROR1) antibody (WB). After 30 min of incubation with 50-1000 nM of KAN0439834, ROR1 was dephosphorylated in a dose-dependent manner. KAN0439834 also dephosphorylated LRP6, GSK3β, JNK, MAPK/ERK/p42,44, PKC, Src, and c-Jun and decreased the β-catenin concentration as well as deactivated BCL-2 and Bax proteins. KAN0439834 had no effect on Bruton tyrosine kinase (Btk) phosphorylation involved in B-cell receptor (BCR) signaling. Incubation of CLL cells with KAN0439834 (50-1000 nM) showed a dose dependent induction of apoptosis/necrosis of leukemic cells with more than 80% specific killing of CLL cells after 24 h and an IC₅₀ value of 250 nM.

Conclusions: Our data show that KAN0439834 downregulated the activity of various signaling pathways in CLL cells suggested to be connected with ROR1 signaling, including the Wnt-canonical associated molecule as LRP6, GSK3β and β-catenin as well as several Wnt non-canonical associated proteins as Src, MAPK/ERK p42,44, JNK, and PKC and inactivation of c-Jun that was followed by apoptosis of the CLL cells in a dose dependent manner. Further studies are ongoing to study the effects of the ROR1 specific TKIs on ROR1 downstream signaling as well as in preclinical in vivo animal models using human fresh tumors and cell lines to evaluate the anti-tumor effects. Available data suggest a specific ROR1-mediated cytotoxic effect of KAN0439834 on CLL cells, which represents a first-in-class of a novel CLL drug candidate targeting ROR1.