Diffuse Large B Cell Lymphoma (DLBCL) Expresses ROR1 and a ROR1 Small Molecule Inhibitor (KAN0441571C) Induced Significant Apoptosis of Tumor Cells
Authors: Hojjat-Farsangi Mohammad , Ghaderi Amineh , Daneshmanesh AmirHossein , Lehto Jemina , Moshfegh Ali , Kokhaei Parviz , Vågberg Jan , Olsson Elisabeth , Löfberg Charlotta , Norström Charlotta Carina , Schultz Johan , Norin Martin , Olin Thomas , Drakos Elias , Rassidakis George Z. , Österborg Anders , Mellstedt Håkan 
Published: November 2019

Background: Receptor tyrosine kinase (RTK) (ROR1) is normally expressed during embryogenesis but absent in most normal tissues. However, ROR1 is overexpressed in several cancers (onco-fetal RTK) and of importance for various tumor cell functions such as proliferation and survival. In patients with diffuse large B-cell lymphomas (DLBCL) there is a great medical need to develop new treatment alternatives for those not responding to primary treatment as well as for patients with relapse as effective treatments are warranted. Inhibition of ROR1 by a small molecule ROR1 inhibitor (KAN0439834) abrogated downstream kinase activities as well as induced apoptosis of various tumor cells as CLL and pancreatic carcinoma (Leukemia, Oct;32(10):2291-2295, 2018) (PLoS One. 13(6): e0198038, 2018). A 2nd generation of ROR1 inhibitor (KAN0441571C) has been synthesized with the aim to bind to the ROR1-TK domain and inhibit ROR1 signaling.

Aim: To examine the expression of ROR1 in DLBCL cell lines (RC-KB, SUDHL4, MS, OCL-LY3, U2932) and in patients´ samples at different stages of DLBCL as well as effects of KAN0441571C on survival of DLBCL cells and ROR1 signaling.

Methods: Flow cytometry, tissue microarray and immunohistochemistry assays were used to check ROR1 expression. MTT and Annexin V/PI assays were applied to analyse cytotoxicity and apoptosis of KAN0441571C alone or in combination with ibrutinib (BTK inhibitor) and venetoclax (BCL-2 inhibitor) on DLBCL cell lines. Western blot was performed to evaluate ROR1 phosphorylation and associated signaling pathways. DLBCL cells were also cultured with HS-5 stromal cells (ROR1 neg.) to evaluate the apoptosis inhibitory effects of stromal cells.

Results: ROR1 expression was significantly more frequently noted in patients with advanced disease (Richter´s, transformation, transformed follicular lymphoma and refractory DLBCL) compared to less advanced disease (recurrent or de novo DLBCL) (p=0.0001). In primary refractory and relapsing DLBCL 5-years survival was 45% in ROR1- patients (n=17) while in ROR1+ patients (n=16) the corresponding figure was <10% (p= 0.0335).

KAN0441571C induced a dose-dependent cytotoxic effect in all ROR1+ DLBCL cell lines (EC50=50-100 nM) while no effect could be noted in the ROR1- U2932 cell line (EC50>10000 nM). EC50 for venetoclax in the ROR1+ DLBCL cell lines varied between 100 and 500 and 5000 - 10000 nM for ibrutinib. In comparison to venetoclax, KAN0441571C induced a similar or significantly higher cytotoxic effect. KAN0441571C and venetoclax seemed to be the most promising drug combination approaching 100% killing at the EC50 dose for each drug.

Apoptosis was confirmed by Annexin V/PI staining as well as by downregulation of BCL-2 and MCL-1 as well as cleavage of PARP and caspase 3. KAN0441571C dephosphorylated ROR1 as well as the co-receptor LRP6 and the SRC protein which binds to phosphorylated ROR1. The downstream molecules PI3Kδ/AKT/mTOR was also dephosphorylated and the transcription factor CREB. CK1δ and GSK3B were also dephosphorylated and β-catenin downregulated indicating involvement of both the non-canonical and canonical Wnt pathways.

When DLBCL and HS-5 cells (ROR1 neg.) were co-cultured, HS-5 cells could partially prevent induction of apoptosis of DLBCL cells at low concentrations of KAN0441571C, while at higher concentrations the presence of stromal cells was less effective.

Zebrafish embryos transplanted with the OCI-Ly3 cell line were treated for 3 days with KAN0441571C (25-1000 nM). No toxic effects of the drug could be noted. A significant dose and time-dependent decrease in the tumor area were noted.

Conclusion: KAN0441571C is the 2nd generation of a novel class of ROR1-inhibiting small molecule drugs. The molecule was more effective in inducing apoptosis of DCBCL cells than venetoclax or ibrutinib. New anti-cancer drugs with other mechanisms of action than those clinically available for DLBCL are warranted to improve the prognosis. ROR1 inhibitors in combination with other targeted drugs as venetoclax and ibrutinib might improve the therapeutic effects. KAN0441571C may be a novel drug candidate which needs further exploration in DLBCL.

Disclosures

Lehto: Kancera AB: Employment. Vågberg:Kancera AB: Employment. Olsson:Kancera AB: Employment. Löfberg:Kancera AB: Employment. Norström:Kancera AB: Employment. Schultz:Kancera AB: Employment, Equity Ownership. Norin:Kancera AB: Employment. Olin:Kancera AB: Employment, Equity Ownership. Österborg:Kancera AB: Research Funding; Janssen: Research Funding; Abbvie: Research Funding; Gilead: Research Funding; BeiGene: Research Funding. Mellstedt:Kancera AB: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.