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RAF signalling in cancer: Biology and therapeutic opportunities

RAF signalling in cancer: Biology and therapeutic opportunities. The Cancer Genome Project. In 2002, Mike Stratton, Andy Futreal and their colleagues reported the first high-throughput re-sequencing study aimed at identifying unknown somatic cancers in human cancer (Davies et al, 2002)

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RAF signalling in cancer: Biology and therapeutic opportunities

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  1. RAF signalling in cancer: Biology and therapeutic opportunities

  2. The Cancer Genome Project • In 2002, Mike Stratton, Andy Futreal and their colleagues reported the first high-throughput re-sequencing study aimed at identifying unknown somatic cancers in human cancer (Davies et al, 2002) • The coding regions for all of the components of the RAS/RAF/ MEK/ERK signalling pathway from 545 cell lines, 340 cancer samples were sequenced • Mutations were found in RAS in the expected frequency (15%) • Unexpectedly, mutations were also found in B-RAF in 7% of human cancers • B-RAF was subsequently found to be mutated in • 50-70% of melanoma samples • ~30% of thyroid cancers • ~30% of low-grade ovarian cancers • ~15% of colorectal cancers

  3. The RAS/RAF signalling pathway Growth Factor Over-expressed in cancers Receptor Mutated/amplified in cancers Ras Mutated in 15-20% of cancers B-RAF • Mutated in 7% of cancers MEK ERK proliferation, differentiation, death, senescence

  4. RAF PROTEINS • Serine/ threonine specific protein kinases • Their only widely accepted substrate is MEK • 3 paralogues in humans • A-RAF - single splice variant • B-RAF - multiple (>10) splice variants • C-RAF - single splice variant CR1 CR2 CR3/ Kinase Regulatory Catalytic

  5. B-RAF is mutated in ~7% of human cancers Activation segment Glycine rich-loop CR1 CR2 CR3/ Kinase K A R E E A G V E L ISV V CE VLRR IDE B-RAF: VGQRIGSGSFGTV…………DFGLATVKSRWS

  6. Glycine-rich loop activation segment B-RAF Kinase Domain Wan et al 2004, Cell

  7. Glycine-rich loop activation segment B-RAF Kinase Domain Wan et al 2004, Cell

  8. GLR GLR B-RAF activation by mutation activation segment inactive constitutively active

  9. Glycine rich-loop Activation segment K A R E E A G V E L ISV V CE VLRR IDE B-RAF: VGQRIGSGSFGTV…………DFGLATVKSRWS A-RAF: -------------…………--------T--- C-RAF: -------------…………------------ A-RAF/C-RAF mutations in cancer

  10. Glycine rich-loop Activation segment K A R E E A G V E L ISV V CE VLRR IDE B-RAF: VGQRIGSGSFGTV…………DFGLATVKSRWS A-RAF: -------------…………--------T--- C-RAF: -------------…………------------ A-RAF/C-RAF mutations in cancer • 546 cancer cell lines screened- 45 mutations in B-RAF, none in A-RAF, 4 in C-RAF • No V452EA-RAF or V492EC-RAF mutations (equivalent of V600EB-RAF)

  11. Relative kinase activity Kinase activity 100 C-RAF Kinaseactivity(fold WT) 50 ~600 fold 0 WT V492E WT + RAS

  12. P P P CR2 CR3 CR1 B-RAF has elevated kinase activity due to the N-region RBD CRD CR3 C-RAF: QRDSSYYWEIE B-RAF: RRDSSDDWEIP N-region: Negative-charge regulatory region Marais et al, 1997 JBC

  13. The N-region determines RAF responses to mutation C-RAF Kinase activity B-RAF kinase activity 500 1000 B-RAF kinase activity (fold WT) Kinase activity (fold WT) 250 500 0 0 WT DD WT AAAA/ V600E V600E V492E DD/V492E

  14. Structure of B-RAF

  15. GLR GLR P GLR GLR B-RAF and C-RAF mutations B-RAF C-RAF N-region inactive activation segment GLR const. active

  16. B-RAF in cancer

  17. Proliferation 20,000 [3H]-thymidine incorporation (cpm) 10,000 0 Scr. B-RAF Control siRNA: Human melanoma lines: siRNA WM-266.4 cells: Melanoma cells with V600EB-RAF mutation ERK activity Control B-RAF A-RAF C-RAF Scr. A-RAF B-RAF C-RAF ppERK Total ERK

  18. parental vector WTB-RAF V600EB-RAF myc-tag B-RAF ppMEK ppERK MEK ERK Expression of B-RAF in melan-a cells B-RAF expression in mouse melanocytes ERK signalling Growth in nude mice

  19. V600EB-RAF MEK ERK survival proliferation Oncogenic B-RAF stimulates proliferation and survival in cancer • V600EB-RAF • 500 fold activated • stimulates constitutive signalling • stimulates proliferation • stimulates survival • is an excellent therapeutic target Karasarides et al (2004) Wellbrock et al (2004a) Wan et al (2004) Garnett and Marais (2004) Wellbrock et al (2004b)

  20. CF3 O Cl O N H O N N H N H Sorafenib (Nexavar), a multi-kinase inhibitor • ONYX Pharmaceuticals/Bayer Corporation • Orally available multi-kinase inhibitor (C-RAF, B-RAF, VEGF receptor, etc) • Inhibits V600EB-RAF: IC50 ~40nM • However, sorafenib is ineffective against melanoma • 10 patients treated at the Royal Marsden Hospital • 5 with V600EB-RAF- 4 progressive disease, 1 stable disease • 5 with WTB-RAF- 4 progressive disease, 1 stable disease • December 05, sorafenib was licensed for use in renal cell carcinoma (VEGFR)

  21. B-RAF inhibitors • High throughput screen- 24,000 compounds focused against kinases • Several hit compounds, many of which were pyrazines • Hit was low µmolar inhibitor in vitro (IC50= 3.5µM), and best compound has an IC50 of 800nM H3C

  22. Pyrazines binds to the active conformation Sorafenib binds to the inactive conformation Different modes of binding

  23. A mouse model of melanoma

  24. 15 16 17 18 V600EB-RAF inducible mouse V600E 14 NeoR 15 16 loxP loxP loxP B-RAF minigene Txn terminator Mutant allele cre recombinase V600E loxP 14 15 16 17 18 Mutant allele

  25. V600EB-RAF inducible mouse • Tyrosinase-Cre • melanocyte specific promoter • comes on at ~E9.5 • B-RAF is on chromozome 7, the Tyr::Cre on the X-chromosome • However in over 200 live births, we did not found the double Tyr::Cre, B-RAF targeted mutants • Tyrosinase promoter is leaky and is active in the brain

  26. Cell proliferation Cell numbers (x 104/ml) 100 V600EB-RAF 300 WTB-RAF 200 0 0 5 10 15 20 25 days 100 V600EB-RAF MEK activity Tumour growth WTB-RAF 0 B-RAF Tumour volume (mm3) C-RAF 0 10 20 30 40 50 pMEK MEK1 Days from inoculation

  27. M-MITF C-kit Cells are neuronal, but not melanocytes control PD184352 Time (hrs): 0 6 24 Pax-3 Sox-10 A-MITF tyrosinase Trp-2 GAPDH

  28. Inactivating B-RAF mutations in cancer

  29. BRaf V600E 0.2ng Unexpected inactivating mutations in B-RAF in cancer Inactive in vitro …but active in vivo 480 ACTIVITY 1 0 G466V B-RAF V600E

  30. WM266.4 (V600D- Activated) H1666 (G466V- Impaired) B-RAF SCRx2 SCRx2 C-RAF C-RAF B-RAF B-RAF C-RAF ppERK1/2 ERK2 Impaired activity mutants C-RAF activation 30 20 Fold activity (compared to WTBRAF) 10 0 G466V BRAF vector

  31. B-RAF signalling in cells activated mutants impaired mutants Normal B-RAF B-RAF* B-RAF† B-RAF C-RAF C-RAF MEK MEK MEK ERK ERK ERK

  32. Summary • B-RAF is a mutated in 7% of human cancers (70% melanoma) • The mutations destabilize the inactive conformation • C-RAF and A-RAF are not mutated because their regulation is fundamentally different • Mutant B-RAF stimulates proliferation and survival and is a validated target • B-RAF drug discovery programme- different binding modes • Mouse model of melanoma • B-RAF signalling through C-RAF is a new paradigm in pathway regulation

  33. Gene and Oncogene Targeting Team Lawrence Davies Harmen Djikstra Frank Friedlos Catherine Gaulon Douglas Hedley Jan Martin Dan Niculescu-Duvaz Ion Niculescu-Duvaz Lesley Ogilvie Esteban Roman Ian Scanlon Caroline Springer Structural Biology Team Paul Wan Mark Roe Val Good David Barford Signal Transduction Team Annette Affolter Tanya Ahmad Vicky Emuss Vanessa Gray-Schopfer Robert Hayward Sonja Heidorn Ruth Kirk Sareena Rana Silvy da Rocha-Diaz Slike Schepelmann Simone Walker Steven Whittaker Claudia Wellbrock Royal Marsden Hospital Tim Eisen Martin Gore The Sanger Institute Richard Wooster Andy Futreal Mike Stratton Leicester University Katherine Mercer Susan Giblet Catrin Pritchard Institut Curie, Paris Veronique Delmas Lionel Larue

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