2017-01-18 JeeHeun Kim (AK)

Introduction: Wnt, RNF43 and PORCN inhibitor The canonical Wnt/β-catenin signalling and inhibitors Upstream Wnt pathway inhibitors

Introduction: Wnt, RNF43 and PORCN inhibitor RNF43 and ZNRF3 reduce Wnt signals by selectively ubiquitinating frizzled receptors, thereby targeting these Wnt receptors for degradation. R-spondin enhances Wnt signalling by inhibiting ZNRF3 Paracrine Wnt secretion is an essential driver of RZ−/− tumour growth and imply that a therapeutic window exists for the use of porcupine inhibitors for RZ-mutant cancers.

Introduction: Genome-wide CRISPR screening Design of sgRNA library for genome-scale knockout of coding sequences in human cells

Introduction: Genome-wide CRISPR screening Rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell. High level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9. Establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells.

Result: CRISPR-Cas9 Screen for vulnerabilities of RNF43-mutant PDAC cells Fig. S1 HPAF-II-Cas9 cell generation Validating sensitivity to the PORCN inhibitor (LGK974)

Result: CRISPR-Cas9 Screen for vulnerabilities of RNF43-mutant PDAC cells Fig. 1 a-c Bays factor (BF) : a measure of the confidence that knockout of a specific gene causes a decrease in fitness High BF = ↑confidence that KO of the gene result in a decrease in fitness CTNNB1: gene encoding β-catenin

Result: CRISPR-Cas9 Screen for vulnerabilities of RNF43-mutant PDAC cells Fig. 1 d-e

Result: FZD5 is required for the growth of RNF43-mutant PDAC cells Fig. 2 a-b / Suppl. Table 4 Cell line: HPAF-II Supplementary Table 4 Primer pair Amplicon length (bp) gRNA-1 Expected cleavage product lengths (bp) gRNA-2 Expected cleavage product lengths (bp) FZD4 exon 1 577 196, 381 NA FZD4 exon 2 880 351, 529 FZD5 1297 192, 1105 341, 956 FZD7 614 143, 471 213, 401 FZD8 913 277, 636 338, 575

Result: FZD5 is required for the growth of RNF43-mutant PDAC cells Fig. 2 c-f HPAF-II, PaTu8988S, AsPC-1 - LGK974 sensitive Cell line PANC-1, BxPC-3, YAPC - LGK974 insensitive Wnt target gene - AXIN2 - NKD1 Differentiation marker - MUC5AC

Result: FZD5 dependency is not explained by cell-specific mRNA or protein expression Fig. 3 a-c CRD: Cysteine-rich domains

Result: FZD5 dependency is not explained by cell-specific mRNA or protein expression Fig. 3 d-e

Result: FZD5 dependency is not explained by cell-specific mRNA or protein expression Fig. 3 f-h Wnt3A conditioned media recue Cell line: HPAF-II Wnt target gene - AXIN2 - NKD1

Result: Anti-FZD5 antibodies inhibit the growth of RNF43-mutant PDAC Fig. 4 a-f OMP-18R5: FZD7-derived antibody currently in clinical trials Cross reactivity to FZD1, FZD2, FZD5, FZD7 and FZD8

Result: Anti-FZD5 antibodies inhibit the growth of RNF43-mutant PDAC Fig. S6 HPAF-II AsPC-1 PaTu8988S BxPC-3 PANC-1

Result: Anti-FZD5 antibodies inhibit the growth of RNF43-mutant PDAC Fig. 4 g-h / Fig. S7-8

Result: Anti-FZD5 antibodies inhibit the growth of RNF43-mutant PDAC Fig. 4 I / Fig. S7

Result: Anti-FZD5 antibodies inhibit the growth of RNF43-mutant PDAC Fig. 5 a-c 1mg/kg  46% tumour growth inhibition 2mg/kg 73% tumour growth inhibition No changes in body weight and visual sign of toxcity Alacin Blue – Mucin staining

Result: Anti-FZD5 antibodies inhibit the growth of RNF43-mutant PDAC Fig. 5 d-e HPAF-II orthotopic tumour IgG-2919 (2mg/kg) – 74% growth inhibition OMP-18R5 (2mg/kg) – 42% growth inhibition No weight loss

Result: Anti-FZD5 antibodies inhibit the growth of RNF43-mutant PDAC Fig. 5 f-i AsPC-1 orthotopic tumour IgG-2919 (2mg/kg) – 60% growth inhibition OMP-18R5 (2mg/kg) – 32% growth inhibition No weight loss

Result: Anti-FZD5 antibodies block the growth of RNF43-mutant CRC-patient-derived organoids Fig. 6 a-d RNF43 mutations frequently occur in colorectal cancer P19Tb (colorectal cancer organoid) – sensitive to PORCN inhibitors

Summary Discovered unique requirement for a Wnt signalling circuit: engaging FZD5 FZD5 functional specificity cannot be explained by protein expression patterns FZD5 and FZD8 binding antibodies inhibited the growth and proliferation of RNF43 mutant PDAC cell in vitro and in vivo CRISPR-based genetic screening tool to identify and validate cell surface targets for antibody development and therapy