A receptor tyrosine kinase ROR1 inhibitor (KAN0439834) induced significant apoptosis of pancreatic cells which was enhanced by erlotinib and ibrutinib

Abstract

There is a great unmet medical need in pancreatic carcinoma (PC) for novel drugs with other mechanisms of action than existing. PC cells express the onco-fetal RTK ROR1, absent on most normal post-partem cells. ROR1 is involved in proliferation, survival, EMT and metastasis of tumor cells in various malignancies. A small molecule inhibitor (KAN0439834) (530 Da) targeting the TK domain of ROR1 was developed and the activity in ROR1 expressing human PC cell lines (n = 8) evaluated. The effects were compared to a murine mAb against the external part of ROR1, gemcitabine, erlotinib and ibrutinib. KAN0439834 induced significant apoptosis of the tumor cells. EC50 values for KAN0439834 varied between 250-650 nM depending on the cell line. The corresponding values for erlotinib and ibrutinib were 10-40 folds higher. KAN0439834 was much more effective in inducing tumor cell death than the ROR1 mAb although both inhibited ROR1 phosphorylation and downstream non-canonical Wnt pathway molecules. Combination of KAN0439834 with erlotinib or ibrutinib had significant additive effects on tumor cell death. A first-in-class small molecule ROR1 inhibitor (KAN0439834) showed promising in vitro activity against a number of human PC cell lines. Interesting is the additive effects of erlotinib and ibrutinib which warrants further studies as both these agents are in clinical trials for pancreatic carcinoma.

Fig 1. Expression of ROR1 in 8 human pancreatic cancer cell lines.

(A) ROR1 expression by Western blot (The fully glycosylated ROR1 protein is 130 kDa) [11]. β-actin was used as loading control. (B) Frequency (mean±SEM) (%) of surface ROR1⁺ cells (flow cytometry). [Statistically significant difference (p-values≤0.01) were: BxPC-3 vs AsPC-1 <0.01; CFPAC-1 vs Capan-1 <0.01; Capan-1 vs AsPC-1 <0.01; AsPC-1 vs Capan-2 = 0.01]. (C) Expression of phosphorylated ROR1 protein (pROR1) (130 kDa). ROR1 and pROR1 expression in surface ROR1 positive (+) and negative (-) PaCa-2 cells (cell sorting). (D) A 130 kDa ROR1 protein was detected (Western blot) in both the ROR1⁺ and ROR1^- fractions. (E) ROR1⁺ and ROR1^- fractions expressed a pROR1 protein (130 kDa).

Fig 2

(A) Apoptosis/(Annexin V/PI) (mean±SEM) of KAN0439834 (500 nM) (■) and anti-ROR1 mAb (10 μg/ml) (□) in pancreatic cancer cells after 72 h of incubation. P-values refer to the comparison between anti-ROR1 mAb and KAN0439834 incubated cells. *p = 0.05; **p = 0.01; ***p = 0.001 and ns = not significant. Effects of KAN0439834 (B) and anti-ROR1 mAb (C) on the apoptosis associated proteins MCL-1, Bcl-xL, caspase-3 and PARP proteins after 24 h of incubation. MCL-1 and Bcl-xL proteins were downregulated and caspase-3 and PARP cleaved.

Fig 3. Cytotoxicity (MTT) of anti-ROR1 mAb and KAN0439834 in surface ROR1 positive (+) and negative (-) fractions of PaCa-2 cells.

(A) Anti-ROR1 mAb (48 h) (B) KAN0439834 (48 h).

Fig 4. Cytotoxicity (MTT) (mean±SEM) of KAN0439834, gemcitabine and anti-ROR1 mAb in pancreatic cancer cell lines.

(A) KAN0439834 in PaCa-2 cell line was significantly more effective compared to gemcitabine (p = 0.0001). KAN0439834 and gemcitabine in combination induced significantly higher cytotoxicity compared to KAN0439834 or gemcitabine alone (KAN0439834 plus gemcitabine vs KAN0439834, p = 0.0033, KAN0439834 plus gemcitabine vs gemcitabine, p = 0.0001). No statistically significant difference comparing KAN0439834 with KAN0439834 plus anti-ROR1 mAb. (B) Cytotoxicity of Capan-1 cells (KAN0439834 vs gemcitabine, p = 0.0483, KAN0439834 plus gemcitabine vs KAN0439834, p = 0.0029, KAN0439834 plus gemcitabine vs gemcitabine, p = 0.0009, KAN0439834 plus anti-ROR1 CRD mAb vs KAN0439834, p = 0.0039, KAN0439834 plus anti-ROR1 mAb vs anti-ROR1 mAb, p = 0.0001) (C) Cytotoxicity of BxPC-3 cells (KAN0439834 vs gemcitabine, p = 0.0044, KAN0439834 plus gemcitabine vs KAN0439834, p = 0.1481, KAN0439834 plus gemcitabine vs gemcitabine, p = 0.0011, KAN0439834 plus anti-ROR1 mAb vs KAN0439834, p = 0.0034, KAN0439834 plus anti-ROR1 mAb vs anti-ROR1 mAb, p = 0.0001). (D) Cytotoxicity of AsPC-1 cells (KAN0439834 vs gemcitabine, p = 0.0106, KAN0439834 plus gemcitabine vs KAN0439834, p = 0.0010, KAN0439834 plus gemcitabine vs gemcitabine, p = 0.0001, KAN0439834 plus anti-ROR1 mAb vs KAN0439834, p = 0.0007, KAN0439834 plus anti-ROR1 mAb vs anti-ROR1 mAb, p = 0.0001).

Fig 5. Cytotoxicity (%) (mean±SEM) of three pancreatic cell lines cultured (72 h) with KAN0439834 and erlotinib alone and in combination using EC₃₀ value concentrations for the different drugs and cell lines respectively.

Statistics are shown at the top.

Fig 6. Cytotoxicity (%) (mean±SEM) of three pancreatic cell lines cultured (72 h) with KAN0439834 and ibrutinib alone and in combination using EC₃₀ value concentrations for the different drugs and cell lines respectively.

Statistics are shown at the top.

Fig 7. KAN0439834 and anti-ROR1 mAb inhibited ROR1 phosphorylation in pancreatic carcinoma cells (PaCa-2).

Total ROR1 (ROR1) and phosphorylated ROR1 (pROR1) after 2 h incubation with KAN439834 (A) and (B) anti-ROR1 mAb. Intensity values of pROR1 relative to total ROR1 are shown. Effects on phosphorylation of RTKs (Phospho-RTK array) in PaCa-2 cells by KAN0439834. (C) PaCa-2 cells were treated with 500 nM of KAN0439834 for 2 h ((■) or untreated (UT) for 2 h (□)). EGFR, ROR1 and IGF-1R were detected in this specific array. Inhibition of ROR1 phosphorylation could only be noted after exposure to KAN0439834. (D) Relative intensity values of pEGFR, pIGF-1R and pROR1 to total EGFR, IGF-1R and ROR1.

Fig 8. Effects of KAN0439834 and anti-ROR1 mAb on ROR1 associated signaling molecules in the pancreatic cancer cell line PaCa-2.

Western blots showed inhibition of ROR1, LRP6, SRC, PI3Kδ, AKT, mTOR and CREB phosphorylation after incubation (2 h) with (A) KAN0439834 (500 nM) and (B) anti-ROR1 mAb (10 μg/ml). (+) treated cells and (-) untreated cells.