1.
JeeHeun Kim (AK)

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

3. 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.

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

5. 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.

6. 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)

7. 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

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

9. 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

10. 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

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

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

13. 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

14. 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

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

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

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

18. 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

19. 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

20. 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

21. 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

22. 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