ALBERT

Beschreibung: 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. Disclosures Moshfegh: Kancera AB: Employment. Vågberg:Kancera AB: Employment. Styrbjörn:Kancera AB: Employment. Schultz:Kancera AB: Employment. Olsson:Kancera AB: Employment. Löfberg:Kancera AB: Employment. Norström:Kancera AB: Employment. Norin:Kancera AB: Employment. Olin:Kancera AB: Employment, Equity Ownership. Österborg:Janssen Cilag: Research Funding.

Beschreibung: 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 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.