1. Melanoma: treatment update in metastatic disease Maher SALAMOON MD Congresso siro-libanese d’oncologia medica, Beirut, jennaio, 23 2014

2. RAS-RAF Pathway The RAS-RAF (rat sarcoma-rapidly accelerated fibrosarcoma) pathway is a series of protein kinases that transmit signals to control proliferation, differentiation, and survival 1 Under normal physiologic conditions, the RAS-RAF signaling pathway is under the control of mitogenic signals 11 –Growth factors bind to surface receptor tyrosine kinases to activate the membrane-bound protein, RAS 1 –RAS activates the protein kinase BRAF, which subsequently activates a second protein kinase, MEK (mitogen-activated protein kinase [MAPK] or extracellular signal―regulated kinase [ERK] kinase), by phosphorylation. In turn, MEK activates yet another protein kinase, ERK, by phosphorylation 2 Activated ERK can enter the nucleus and phosphorylate transcription factors such as E- twenty-six (ETS), which mediate gene transcription, ultimately stimulating cell survival and proliferation 3 Normal RAS-RAF pathway signaling (adapted from Garnett MJ, et al) 11 ERK BRAF Activated RAS Normal cell proliferation and survival MEK P P Growth factors RTK RAS-GTP Normal activation of RAS by extracellular factors P P 2

3. RAS-RAF Pathway in Melanoma ERK BRAF Activated RAS Normal cell proliferation and survival MEK P P Oncogenic BRAF signaling (adapted from Wan PTC, et al) 16 Normal RAS-RAF pathway signaling (adapted from Garnett MJ, et al) 11 Growth factors RTK Mutated BRAF Mutated BRAF Excessive cell proliferation and survival MEK RAS-GTP Constitutive activation is independent of extracellular factors Not responsive to normal regulatory signals Normal activation of RAS by extracellular factors P P P P P P

4. BRAF Mutations Affect Kinase Activity Ninety percent of BRAF mutations in melanoma result in substitution at position V600 11 –All tested mutations at V600 are classified as high- activity mutants when compared with BRAF wild-type (WT) 16,22 –These mutations constitutively stimulate ERK phosphorylation 16 A unifying feature of the high- and intermediate- activity BRAF mutants is that they disrupt the hydrophobic interaction between the P loop and the activation segment of the kinase domain 22 –This results in destabilization of the inactive conformation of BRAF, thus stimulating its kinase activity and leading to increased ERK phosphorylation 4 BRAF kinase domain structure. Position 600 is indicated in yellow. The BRAF activation segment is shown in purple; dashes indicate the region that was not resolved. The P loop is shown in green (adapted from Garnett MJ, et al). 11

5. Ninety percent of BRAF mutations at V600 in melanoma result in substitution V600E 3 –The substitution of valine for glutamate arises from the point mutation T1796A at codon 600 in exon 15 2 T1796A is not a common UV-induced base change 23 The glutamate residue acts as a phosphomimetic 24 –Activation of BRAF requires phosphorylation of the conserved residues Thr599 and Ser602 25 –Replacing the medium-sized hydrophobic valine side chain at position 600 on the activation loop with the larger and charged glutamate residue is believed to destabilize the inactive conformation, flipping the activation segment into the active position 16 The kinase activity of BRAF V600E is greatly elevated compared with BRAF WT 5,16 –The resulting hyperactivity of the RAS-RAF pathway promotes tumor development 24 BRAF V600E 5 BRAF WT —INACTIVE 3,26 BRAF WT —ACTIVATED by addition of negatively charged phosphates 25 BRAF V600E —CONSTITUTIVELY ACTIVATED by negatively charged glutamate (E) 24

6. Differential effects of BRAF inhibition in BRAF V600 mutant melanoma and BRAF wild type cells CRAF MEK1/2 ERK P P BRAF V600 BRAF V600 mutant melanomaBRAF wild type cells Modeled from Hatzivassiliou et al. Nature 2010, Heidorn et al. Cell 2010, Poulikakos et al. Nature 2010 MAPK signaling CRAF MEK1/2 ERK P P BRAF V600 PLX4032 MAPK signaling CRAF MEK1/2 ERK P P BRAF MAPK signaling RAS CRAF MEK1/2 ERK P P BRAF PLX4032 MAPK signaling RAS

7. B-Raf wild typeB-Raf mutation

8. 100 90 80 70 60 50 40 30 20 10 0 Overall survival (%) 0123456789101112 No. of patients in follow up Dacarbazine Vemurafenib Months 336 283 320 192 266 137 210 98 162 64 111 39 80 20 35 1616 1111 9 14 Hazard ratio 0.37 ( 95% CI; 0.26 - 0.55 ) Log-rank P<0.0001 Overall survival (BRAF mutation) Chapman et al. NEJM 2011 = 63% decrease in the risk of being dead compared to chemotherapy

9. BRIM2: Toxicities with vemurafenib All grades n (%) Grade 3 n (%) Grade 4 n (%) Overall130 (99)79 (60)5 (4) † Arthralgia78 (59)8 (6)– Rash69 (52)9 (7)– Photosensitivity reaction69 (52)4 (3)– Fatigue56 (42)2 (2)– Alopecia48 (36 )–– Pruritus38 (29)3 (2)– Skin papilloma38 (29)–– cuSCC / KA ‡ 34 (26) – Nausea30 (23)2 (2)– Elevated liver enzymes23 (17)8 (6) § 4 (3) ¶ Includes AEs reported in ≥20 patients † One patient with 2 grade 4 AEs ‡ Cases of cuSCC/KA were generally managed with simple excision and did not generally require dose modification § Managed with dose reduction; one removed from study ¶ Led to discontinuation of therapy Ribas et al. ASCO 2011

10. Immunotherapy, a reality for patient benefit in melanoma Immunotherapy is the only treatment that can reproducibly result in cures in (few) patients with metastatic melanoma FDA-approved immunotherapies for melanoma: –Adjuvant treatment: High dose interferon alpha 2b Pegylated interferon alpha 2b –Metastatic melanoma: High dose IL-2 Ipilimumab

11. High dose IL-2 and Ipilimumab: The major benefit is in durable tumor regressions

13. Study 024: Duration of Response (DoR) Data shown for patients with a confirmed complete response (CR) or partial response (PR) Median DoR 19.3 vs 8.1 months p-value 0.03 IPI + DTIC vs Placebo + DTIC Robert C et al. N Engl J Med 2011;[Epub ahead of print]. Copyright © 2011 Massachusetts Medical Society. All rights reserved. IPI + DTIC Placebo + DTIC

14. = 1 st tumor assessment as per protocol Ipilimumab Improves Progression Free Survivial Compared to Control Ipi + gp100 (A) Ipi alone (B) gp100 alone (C) 1234 Years Comparison Hazard Ratio (C.I.) p-value Comparison Hazard Ratio (C.I.) p-value Arms A vs C 0.81 (0.66–1.00) 0.0464 Arms B vs C 0.64 (0.50–0.83) 0.0007 Arms A vs B 1.25 (1.01–1.53) 0.0371

15. Summary Positive impact in overall survival in two randomized clinical trials using different schedules and combinations: –FDA approval as single agent at 3 mg/kg x 4 doses The major benefit is evident in a small population of patients (10- 15%, most probably cured) Clinically-significant inflammatory and immune toxicities in approximately 15-20% of patients Responses usually take time (1-4 months) to declare, and may go through a period of uncertainty about response or progression Vemurafinib produces high response rate and prolongs survival in BRAF mutant metastatic melanoma patients

16. Melanoma cell lines We have esatablished 4 cell lines at the institute of biotechnology: Cell lines wit B-Raf wild type Cell lines with B-raf mutated Cell line with P53 wild type Cell line with mutated P53

17. D0 D1 D3 D7 sb-95---- DMSO The case of non- functional B-Raf

18. disclosure No conflict of interest

19. 1-Sekulic A, et al. Malignant melanoma in the 21st century: the emerging molecular landscape. Mayo Clin Proc 2008;83:825–46. PMID: 18613999PMID: 18613999 2-Flaherty KT, et al. BRAF, a target in melanoma: implications for solid tumor drug development. Cancer 2010;116:4902–13. PMID: 20629085PMID: 20629085 3-Forbes SA, et al. COSMIC (the Catalogue of Somatic Mutations in Cancer): a resource to investigate acquired mutations in human cancer. Nucleic Acids Res 2010;38:D652–7. PMID:19906727PMID:19906727

20. Thank you