Crosstalk between ROR1 and BCR pathways defines novel treatment strategies in mantle cell lymphoma by Hanna Karvonen, David Chiron, Wilhelmiina Niininen, Sara Ek, Mats Jerkeman, Elaheh Moradi, Matti Nykter, Caroline A. Heckman, Olli Kallioniemi, Astrid Murumägi, and Daniela Ungureanu BloodAdv Volume 1(24):2257-2268 November 14, 2017 © 2017 by The American Society of Hematology

Hanna Karvonen et al. Blood Adv 2017;1:2257-2268 © 2017 by The American Society of Hematology

Expression levels of Wnt5a-, ROR1-, and ROR2-signaling pathways in MCL cell lines and patient samples. Expression levels of Wnt5a-, ROR1-, and ROR2-signaling pathways in MCL cell lines and patient samples. (A) Immunoblot analysis (western blot [WB]) of ROR1- and ROR2-immunoprecipitated (IP) samples or of total cell lysates of MCL cell lines, CLL-like MEC-1 and MEC-2 cells (ROR1−), and Hela cells (ROR2+). (B) Percentage of ROR1+ cells in MCL patient samples used in this study, as described in Table 1. Error bar represents median with interquartile range. Relative ROR1 (C), Wnt5a (D), and ROR2 (E) mRNA expression levels in normal, MCL cell lines and patient samples, and CLL patient samples as derived from the National Center for Biotechnology Information Genomic Spatial Event (GSE) database, data collection number GSE86322 (for MCL cell lines) and the primary samples from GSE50006, GSE19243, GSE35426, GSE16455, GSE36000, GSE21452, and GSE70910. Comparison is done across sample within the same probe set. Error bars represent plus or minus standard deviation (SD). Hanna Karvonen et al. Blood Adv 2017;1:2257-2268 © 2017 by The American Society of Hematology

Analysis of ROR1 silencing in MCL cell lines and primary samples. Analysis of ROR1 silencing in MCL cell lines and primary samples. (A) Cell-viability counts of shRNA-expressing MCL cells plus or minus DOX for the indicated time (days). The percentage of viable cells was normalized to (−) DOX for each time point. Data are representative of 3 independent experiments. (B) Immunoblot analysis of shRNA-expressing cell lysates (cytoplasmic and nuclear) plus or minus DOX for 4 and 7 days. (C) Analysis of ROR1 expression by flow cytometry after siRNA nucleofection of primary MCL cells. (D) Immunoblot analysis of NF-κB p65 and Wnt5a levels in primary cell lysates. Quantifications shown for NF-κB p65 and Wnt5a protein levels were normalized to β-tubulin levels and compared with siCtr for each sample. (E) Luciferase activity (fold induction) of Mino cells cotransfected with ROR1-HA and NF-κB p65 reporter plasmids for 24 hours. Anti-HA immunoblotting shows ROR1 protein levels. Error bars represent mean plus or minus SD of triplicates. Statistical significances compared with sample without ROR1-HA transfection were calculated by a 2-tailed paired Student t test; *P < .05. Ctr, control; HA, hemagglutinin; PE, phycoerythrin. Hanna Karvonen et al. Blood Adv 2017;1:2257-2268 © 2017 by The American Society of Hematology

NF-κB activation modulates ROR1-targeted treatments in MCL cell lines and primary samples. NF-κB activation modulates ROR1-targeted treatments in MCL cell lines and primary samples. (A) Percentage of viable cells (compared with control) after 48-hour incubation with different concentrations of ROR1 2A2 mAb as indicated. Error bars represent mean plus or minus SD of triplicates. (B) Percentage of AnnexinV/PI+ cells (early and late apoptosis) and negative (live cells) after 48-hour incubation with 10 µg/mL ROR1 2A2. Data are representative of 3 independent experiments. (C) Immunoblot analysis of cytoplasmic and nuclear cell lysates of MCL cell lines plus or minus 5 µg/mL ROR1 2A2 mAb for 24 hours. (D) Percentage of viable Mino cells (compared with untreated control) pretreated or not with recombinant TNF-α (100 ng/mL) or BAFF (500 ng/mL) followed by ROR1 2A2 mAb (5 µg/mL) for 48 hours. Error bars represent mean plus or minus SD of triplicates. (E) Immunoblot analysis of NF-κB p65 and p52 levels in nuclear cell lysates of Mino cells treated as in panel D. (F) Percentage of viable cells (compared with untreated control) pretreated or not with recombinant Wnt5a (200 ng/mL) followed by ROR1 2A2 mAb (5 µg/mL) for 48 hours. Error bars represent mean plus or minus SD of triplicates. (G) Immunoblot analysis of NF-κB p65 levels in nuclear cell lysates of MCL cells treated as in panel F. (H) Percentage of viable cells (compared with untreated control) cultured alone or in coculture with L-CD40L fibroblasts plus or minus ROR1 2A2 mAb (5 µg/mL for Jeko-1 and Mino, 7.5 µg/mL for other cell lines) for 48 hours. Error bars represent mean plus or minus SD of triplicates. (I) Percentage of viable cells (compared with untreated control) of primary cells cocultured with L-CD40L fibroblasts and the cytokines cocktail plus or minus 5 µg/mL ROR1 2A2 mAb for 24 hours. Error bars represent mean plus or minus SD of triplicates. (J) Immunoblot analysis of ROR1 and NF-κB p65 levels in primary cell lysates. Cells were treated with 5 µg/mL ROR1 2A2 mAb for 24 hours. Quantifications shown for ROR1 and NF-κB p65 protein levels were normalized to β-tubulin levels and compared with untreated control for each sample. Statistical significances: *P < .05, **P < .01, ***P < .001, calculated by a 2-tailed paired Student t test. Hanna Karvonen et al. Blood Adv 2017;1:2257-2268 © 2017 by The American Society of Hematology

Targeting ROR1 expression augments drug responses for BCR and Bcl-2 inhibitors. Targeting ROR1 expression augments drug responses for BCR and Bcl-2 inhibitors. (A) DSS values for shRNA Ctr-transduced cells were correlated with the corresponding DSS values for shRNA ROR1-transduced cells to depict differential drug sensitivities and resistance patterns. ● indicates Bcl-2–targeted drugs navitoclax and venetoclax as well as BTK inhibitor ibrutinib for each cell line. (B) Heatmap of DSS values for different apoptotic modulators tested in Jeko-1 and Z-138 shRNA-expressing cells by unsupervised clustering based on average DSS values for each row. (C) Heatmap of DSS values for selective BCR inhibitors functionally relevant in shRNA-expressing Jeko-1 cells. (D) Percentage of cell viability (compared with controls) of untreated or treated cells with ROR1 2A2 mAb (5 µg/mL) for 24 hours followed by addition of indicated drugs for 24 hours. Error bars represent mean plus or minus SD of triplicates. (E) ROR1 silencing enhances venetoclax cytotoxicity in MCL#2 primary cells. Cell viability was measured by PI staining after 24 hours and normalized to the untreated siRNA Ctr sample. (F) Percentage of primary cell viability (compared with controls) of untreated or treated cells with ROR1 2A2 mAb (5 µg/mL) for 24 hours followed by addition of venetoclax for 24 hours. Error bars represent mean plus or minus SD of triplicates. (G) Percentage of viable cells (compared with untreated control) of primary MCL#5 cells cultured alone or in coculture with L-CD40L fibroblasts and the cytokines cocktail and treated with ROR1 2A2 mAb or venetoclax, alone or in combination as indicated. Error bars represent mean plus or minus SD of triplicates. Statistical significances: *P < .05, **P < .01, ***P < .001, calculated by a 2-tailed paired Student t test. ATP, adenosine triphosphate; Conc., concentration; EGFR, epidermal growth factor receptor; FAK, focal adhesion kinase; mTOR, mammalian target of rapamycin; Nav, navitoclax; TKI, tyrosine kinase inhibitor; Ven, venetoclax. Hanna Karvonen et al. Blood Adv 2017;1:2257-2268 © 2017 by The American Society of Hematology

BCR inhibition by ibrutinib downregulates ROR1 levels and impairs ROR1-targeted therapies. BCR inhibition by ibrutinib downregulates ROR1 levels and impairs ROR1-targeted therapies. (A) Effect of ibrutinib (1 µM) treatment on MCL cell line proliferation after 3 days. Fold change is compared with starting cell count. Error bars represent mean plus or minus SD of triplicates. (B) Immunoblot analysis of cytoplasmic and nuclear cell lysates of MCL cell lines plus or minus 1 µM ibrutinib for 3 days. *Loss of protein in ibrutinib-treated Mino nuclear cell lysates. Quantifications shown for ROR1 protein levels were normalized to β-tubulin levels and compared with untreated control for each cell line. (C) Flow cytometry analysis of ROR1 levels (in live cells) after 0.5 µM (Jeko-1 and Mino) or 1 µM (Maver-1 and Z-138) ibrutinib for 3 days compared with untreated sample. (D) Percentage of viable cells (compared with sample without mAb) left untreated or pretreated with ibrutinib (1 µM) for 3 days followed by ROR1 2A2 mAb (5 µg/mL) for 48 hours. Error bars represent mean plus or minus SD of triplicates. (E) Table summary of ROR1 levels as measured by flow cytometry in live cells of primary samples untreated or treated ex vivo with ibrutinib (1 µM for 48 hours). (F) Comparison of ROR1 expression in MCL samples from panel E plus or minus ibrutinib. Statistical significances: *P < .05, **P < .01, ***P < .001, calculated by a 2-tailed paired Student t test. Hanna Karvonen et al. Blood Adv 2017;1:2257-2268 © 2017 by The American Society of Hematology