1. Benefits of JAK2 Inhibitor Therapy: Why Do They Work in Patients With and Without JAK2 Mutation Alessandro M. Vannucchi Section of Hematology, University of Florence, Italy

2. JAK2 V617F is the Commonest Mutation Causing Abnormal JAK/STAT Signaling in MPN Vannucchi et al., CA Cancer J Clin. 2009; 59:171-91 Survival Differentiation Proliferation Oncogenesis

3. A Myeloproliferative Disorder is Induced by JAK2 V617F in Mice PV MF Zaleska, Plos One 2006;e18

4. Binding to receptors Chaperoning Stabilising at membrane Inhibits basal activity Signaling through P transfer JH7 JH6 JH4 JH2 JH1 V617F JH3JH5 FERM SH2 PseudoKinase Kinase James et al. Nature 2005; 434: 1144-8 ; Baxter et al. Lancet 2005; 365: 1054-61; Levine et al. Cancer Cell 2005; 7:387-97; Kralovics et al. NEJM. 2005: 352:1779-90 Activation loop V617 ATP site Kinase site JAK inhibitors target the ATP binding site of JAK2 at the tyrosine kinase domain and not the pseudokinase domain Therefore, both mutated and wild-type JAK2 are inhibited by JAK2 inhibitors JAK2 inhibitors are not specific for the JAK2 V617F mutation

5. Geron I et al, Cancer Cell, 13; 2008 321 - 330 Inhibition of PV Progenitor Erythroid Differentiation by the JAK2 Inhibitor TG101348

6. Effects of Treatment with Ruxolitinib in a JAK2 V617F-Driven Murine Model Quintás-Cardama et al., Blood 2010;115:3109-3117.

7. JAK1 and JAK2, or JAK2 Only, Inhibitors Drug JAK1Other targets Ruxolitinib/INC424  none known TG101348/SAR302503  FLT3, Ret CYT387JNK1, CDK2 CEP-701FLT3, TrkA AZD1480  Aurora A, TrkA, FGFr1 SB1518FLT3 LY2784544na BMS911543 Verstovsek et al. N Engl J Med. 2010; 363:1117-27. Pardanani et al. JCO. 2011; online Jan 10. Pardanani et al. ASH Abstract 2010; Blood 2010; 116:460. Santos et al. Blood. 2010; 115:1131-6.La Fave LM, Trends Pharm Sciences 2012; 33:564-582.

8. Do they work? If yes, Why?

9. Do they work? If yes, Why?

10. 056112168224280 336 JAK mutation POSITIVE; N = 33 JAK mutation NEGATIVE; N = 6 Time on Therapy (days) Spleen length, cm 0 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 2.5 The Effects of Ruxolitinib on Spleen Size is Independent of JAK2 V617F Mutation In the Phase I/II study with TG101348/SAR302503, 8 of 59 pts were JAK2 wild-type 3 of 4 pts who completed six cycles had >50% reduction of splenomegaly (CI per IWG-MRT) Verstovsek S et al. NEJM 2010; 363:1117-1127; Pardanani A et al, JCO 2011; 29:789-796

11. No significant difference in response rates was observed between patients with the JAK2V617F mutation compared with those without the mutation, although the trend was towards a greater response rate in JAK2 V617F mutated Effect of JAK2 V617F Mutation on the Proportion of Patients Obtaining a >35% Spleen reduction * * By MRI Harrison C et al, ASH 2011; 279

12. The Effects of Ruxolitinib on Symptomatic Control Is Independent of JAK2 V617F Mutation Kiladjian JJ et al, ASCO 2012: 451A

13. Verstovsek S et al. ASH 2011, 378A The Impact of Ruxolitinib on Survival Is Independent of JAK2 V617F Mutation

14. Do they work? If yes, Why?

15. Anand S et al. Blood 2011;118:1610-1621 Similarly Activated Signaling Pathways in JAK2 V617F Mutated and Wild-type MPN Cells

16. Anand S et al. Blood 2011;118:1610-1621 Similar Inhibition of Signaling in JAK2 V617F and Wild-type patients with a Selective JAK2 inhibitor

17. Tefferi A et al, JCO 2011;29:1356-1363 A Cytokine Storm in PMF Patients Fold-increased over controls         JAK2 V617F correlated

18. Vannucchi AM, N Engl J Med. 2010; 363:1180-2. The Significance of JAK1 and JAK2 Inhibition

19. Ruxolitinib-Induced Normalisation of Inflammatory Cytokines in Phase I/II Trial This effect was observed regardless of JAK2 mutational status or MF subtype Verstovsek et al. N Engl J Med. 2010; 363:1117-27. Baseline, Patients with Myelofibrosis vs. Healthy Controls Patients with Myelofibrosis, Day 28 vs. Baseline

20. Predicted Effects of JAK1 and JAK2 inhibition JAK2wt (± JAK1) inhibition affects a variety of cytokine signaling pathways JAK2 inhibitors are not specific for mutated protein THUS they are effective regardless of the JAK2V617F mutated status JAK2 inhibitors are not specific for mutated protein THUS they are effective regardless of the JAK2V617F mutated status Concurrent Inhibition of JAK2wt might result in anemia and thrombocytopenia % of patients Placebo BATRuxolitinib Anemia Thr’penia 19.2 43.2 1.3 12.9 31 42 7 8 A marked reduction of pro-inflammatory cytokines was coincident with improvement in constitutional symptoms Verstovsek S et al. N Engl J Med. 2010; 363:1117-27;Verstovsek S et al. NEJM 2012; 366:799-807; Harrison C et al. NEJM 2012; 366:787-98

21. Conclusions Abnormal JAK/STAT signaling is a common pathogenetic mechanism in MPN cells independent of the JAK2 mutational status Current JAK2 inhibitors are not specific for the mutated protein, and target the wild-type JAK2 as well JAK2 inhibitors are similarly effective in JAK2 mutated and wild- type patients Inhibition of wild-type JAK1 signaling contributes to the clinical efficacy of JAK1/JAK2 inhibitors