Mechanisms of resistance to JAK inhibitors. L. Knoops

Transcription

1 Mechanisms of resistance to JAK inhibitors L. Knoops

2 1 : Resistance to tyrosine kinase inhibition in cancer

3 are related in sequence and structure. The main diagram illustrates the similarity between the protein sequences of these catalytic domains. Each kinase is at the tip of a branch, and the similarity between various kinases is inversely related to the distance between their positions on the tree diagram. Most kinases fall into small families of highly related sequences, and most Tyrosine kinases THE FGFR2 FGFR3 EphB2 EphA3 FGFR4 TRKA EphB1 EphA5 FGFR1 TRKB DDR2 DDR1 EphA4 EphA6 Yes IRR Ros ALK LTK Fyn FLT1 KDR Fms/CSFR Kit Ret Tyro3/ Sky RYK EphA8 CCK4/PTK7 TK Ack Tnk1 HER3 EphA2 BLK ITK FRK CTK CSK Arg Abl Srm Brk HER4 Tyk2 JAK1 JAK2 JAK3 ANKRD3 SgK288 ZAP70 PYK2 Syk FAK EphA1 TEC TXK BTK Etk/BMX EGFR HER2 TIE2 TIE1 Fgr Lck FLT4 PDGFR" FLT3 PDGFR% Met Ron EphB4 EphA7 Tyrosine Kinase inhibitors Mer Axl IGF1R InsR Src Lyn HCK ROR2 ROR1 MuSK EphB3 H Tyrosine Kinase inhibitors TRKC Lmr1 Lmr2 ZA EphB6 RIPK2 Lmr3 IRAK3 IRAK1 EphA10 RIPK3 LIMK1 LIMK2 TESK1 TESK IRAK2 Fes Fer ANP" ANP% JAK3~b JAK2~b Tyk2~b JAK1~b RIPK1 LRRK2 LRRK1 SuRTK106 IRAK4 MOS HSER DYRK2 SgK496 PBK

4 Tyrosine kinase inhibitors cabl kinase domain cristal strucure with imatinib Adapted from Nagar et al, Cancer Res, 2002

5 Tyrosine kinase inhibitors cabl kinase domain cristal strucure with imatinib Adapted from Nagar et al, Cancer Res, 2002

6 Tyrosine kinase inhibitors in cancer Mutant Clone Resistant clone WT TKI Resistance time

7 are related in sequence and structure. The main diagram illustrates the similarity between the protein sequences of these catalytic domains. Each kinase is at the tip of a branch, and the similarity between various kinases is inversely related to the distance between their positions on the tree diagram. Most kinases fall into small families of highly related sequences, and most Tyrosine kinases THE FGFR2 FGFR3 EphB2 EphA3 FGFR4 TRKA EphB1 EphA5 FGFR1 TRKB DDR2 DDR1 EphA4 EphA6 Yes IRR Ros ALK LTK Fyn FLT1 KDR Fms/CSFR Kit Ret Tyro3/ Sky TIE2 TIE1 CCK4/PTK7 Fgr TK Ack Tnk1 HER3 EphA2 BLK ITK FRK CTK CSK Arg Abl Srm Brk Resistance? HER4 Tyk2 JAK1 JAK2 JAK3 ANKRD3 SgK288 ZAP70 PYK2 Syk FAK EphA1 TEC TXK BTK Etk/BMX EGFR HER2 RYK EphA8 Lck FLT4 PDGFR" FLT3 PDGFR% Met Ron EphB4 EphA7 Tyrosine Kinase inhibitors Mer Axl IGF1R InsR Src Lyn HCK ROR2 ROR1 MuSK EphB3 H Tyrosine Kinase inhibitors TRKC Lmr1 Lmr2 ZA EphB6 RIPK2 Lmr3 IRAK3 IRAK1 EphA10 RIPK3 LIMK1 LIMK2 TESK1 TESK IRAK2 Fes Fer ANP" ANP% JAK3~b JAK2~b Tyk2~b JAK1~b RIPK1 LRRK2 LRRK1 SuRTK106 IRAK4 MOS HSER DYRK2 SgK496 PBK

8 JAK2 kinase domain ABL kinase domain JAK2 kinase domain cabl kinase domain cristal strucure with imatinib JAK2 kinase domain cristal structure with CP Adapted from Nagar et al, Cancer Res, 2002;Williams et al, J Mol Biol, 2009

9 JAK2 kinase domain ABL kinase domain JAK2 kinase domain cabl kinase domain cristal strucure with imatinib JAK2 kinase domain cristal structure with CP Adapted from Nagar et al, Cancer Res, 2002;Williams et al, J Mol Biol, 2009

10 JAK2 kinase domain ABL kinase domain JAK2 kinase domain cabl kinase domain cristal strucure with imatinib JAK2 kinase domain cristal structure with CP Adapted from Nagar et al, Cancer Res, 2002;Williams et al, J Mol Biol, 2009

11 JAK Kinases FERM PSEUDO- KINASE DOMAIN KINASE DOMAIN N C Adapted from Giordanette F et al, Protein Eng, 2002 V617F

12 JAK2 V617F model JAK2 JH1-JH2 domains model Adapted from Giordanetto et al, Protein Eng, 2002

13 Cytokine Cytokine receptors signal transduction Cytokine Cytokine Y JAK JAK Y P! Y JAK JAK Y P! P! Y JAK JAK Y P! Y Y Y Y P! Y Y P! Y Y Y Y P! Y Y P! STAT P!Y Y P! STAT JAK-STAT STAT P!Y Y P! STAT

14 Cytokine receptors signal transduction Cytokine RAS JAK JAK P! Y Y P! P! Y Y P! P! Y Y P! MAPKinase PI3Kinase STAT P!Y Y P! STAT JAK-STAT STAT P!Y Y P! STAT

15 Cytokine receptors signal transduction Cytokine 50 cytokine receptors P! Y JAK JAK Y P! P! Y Y P! 4 JAKs P! Y Y P! Homo or heterodimers

16 Cytokine receptors using JAK2 Homodimeric ßc Type II IFN EPO, TPO, GH, PRL IL-3, 5, GM-CSF IFNγ ßc JAK2 JAK2 JAK2 JAK2 JAK1 JAK2 STAT5 STAT5 STAT1

17 Cytokine receptors using JAK1 γc gp130 Type I IFN Type II IFN IL-2, 4, 7, 9, 15, 21 IL-6, G-CSF, OSM IFNa, ß IFNγ γc gp130 JAK1 JAK3 JAK1 TYK2 JAK1 TYK2 JAK1 JAK2 STAT5 STAT3 STAT6 STAT3 STAT1 STAT2 STAT1

18 1 : Resistance to tyrosine kinase inhibition in cancer - JAK inhibitors are ATP-competitive inhibitors - JAK inhibitors are not imatinib!. Not specific for mutated vs WT JAKs. JAK inhibition will interfere with many cytokine receptors 2 : In vitro mechanisms of JAK inhibitor resistance

19 IL-9 R signal transduction IL- 9R signal transduc3on

20 IL-9 R phe116 receptor

21 In vitro model of tumorigenesis JAK1 overexpression JAK1 mutations IL-9 IL-9 / / BaF3 phe116 IL-9 responding Autonomous

22 JAK1 JH1-JH2 domains model JAK1 activating mutations

23 JAK1 activating mutations Mutated residues V658F, L, I JAK1 JH1-JH2 domains model

24 JAK1 activating mutations Mutated residues JAK1 JH1-JH2 domains model

25 JAK1 activating mutations Mutated residues JAK1 JH1-JH2 domains model

26 JAK1 activating mutations Mutated residues Mutated residues in ALL JAK1 JH1-JH2 domains model Flex et al., 2008, Asnafi et al., 2008, Jeong et al., 2008, Mullighan et al., 2009

27 JAK1 activating mutations : kinase domain JAK1 kinase domain cristal structure with CP Adapted from Williams et al., J.Mol.Biol, 2009

28 JAK1 F958V is resistant to JAK inhibitor I Western Blot Hornakova et al, Haematologica, 2011

29 JAK1 F958V is resistant to Ruxolitinib 120 Proliferation (% of control) 100 M-RAS Aut(F958V) 40 Aut(V658F) Ruxolitinib (µm) BaF3 cells - H3 thymidine incorporation Hornakova et al, Haematologica, 2011

30 Prolonged treatment with JAK inhibitor induces JAK1 overexpression Quantitative PCR Hornakova et al, Haematologica, 2011

31 JAK1 and ABL1 hinge regions P960T/S F958V/C/S/L (JAK inhibitors) JAK1 925 KLIMEFLPSGSLKEYLPK-NKN 973.:. :.. :.:.: :. ABL1 312 YIITEFMTYGNLLDYLRECNRQ 333 T315I/A/S (Imatinib) E316D F317L/I M318A G321V Crystal structure of JAK1, JAK2 and ABL Hornakova et al, Haematologica, 2011

32 The JAK2 Y931C mutation induces resistance to ruxolitinib 120 Proliferation (in % of control) V617F Y931C V617F/Y931C Ruxolitinib (um) BaF3 cells - H3 thymidine incorporation Hornakova et al, Haematologica, 2011

33 JAK2 kinase domain mutations resistant to JAK inhibitors Activating and resistant mutations Resistant mutations E864A Y931C G935R Hornakova et al, Haematologica, 2011 Weigert et al, J Exp Med, 2012 JAK2 kinase domain cristal structure with CP Adapted from Williams et al., J.Mol.Biol, 2009

34 1 : Resistance to tyrosine kinase inhibition in cancer - JAK inhibitors are ATP-competitive inhibitors - JAK inhibitors are not imatinib!. Not specific for mutated vs WT JAKs. JAK inhibition will interfere with many cytokine receptors 2 : In vitro mechanisms of JAK inhibitor resistance - JAK inhibitors are able to kill JAK-dependent cells - JAK kinase domain mutation and overexpression can explain resistance 3 : Clinical resistance to JAK inhibitors in MF

35 COMFORT-II : Percent change from baseline in spleen volume 80 At Week 48 Patient characteristics? % Change from baseline at week Ruxoli3nib P <.0001 Primary endpoint - 80 Ruxoli3nib BAT Harrison C, ASH 2011, Abstract 279

36 1 : Resistance to tyrosine kinase inhibition in cancer - JAK inhibitors are ATP-competitive inhibitors - JAK inhibitors are not imatinib!. Not specific for mutated vs WT JAKs. JAK inhibition will interfere with many cytokine receptors 2 : In vitro mechanisms of JAK inhibitor resistance - JAK inhibitors are able to kill JAK-dependent cells - JAK kinase domain mutation and overexpression can explain resistance 3 : Clinical resistance to JAK inhibitors in MF - Clinical resistance exists Mainly primary resistance (short follow-up) 4 : Molecular resistance to JAK inhibitors in MF

37 Tyrosine kinase inhibitors in cancer Mutant Clone Resistant clone WT TKI Resistance time

38 JAK kinases inhibitors in PMF WT JAK2 V617F + JAK2 V617F + WT TKI time

39 JAK kinases inhibitors in PMF WT JAK2 V617F + JAK2 V617F + WT TKI time

40 Potential cause of JAK2 V617F persistance Is the JAK2 V617F MPN clone addicted to JAK-STAT signaling? 120 Proliferation (in % of control) JAK2 V617F M Ras Ruxolitinib (um)

41 JAK kinases inhibitors in PMF JAK2 V617F + JAK2 V617F + WT WT TKI time

42 Potential cause of JAK2 V617F persistance 120 Proliferation (in % of control) JAK2 V617F M Ras Normal hematopoiesis JAK2 V617F persistance Ruxolitinib (um) Toxic dose

43 1 : Resistance to tyrosine kinase inhibition in cancer - JAK inhibitors are ATP-competitive inhibitors - JAK inhibitors are not imatinib!. Not specific for mutated vs WT JAKs. JAK inhibition will interfere with many cytokine receptors 2 : In vitro mechanisms of JAK inhibitor resistance - JAK inhibitors are able to kill JAK-dependent cells - JAK kinase domain mutation and overexpression can explain resistance 3 : Clinical resistance to JAK inhibitors in MF - Clinical resistance exists Mainly primary resistance (short follow-up) 4 : Molecular resistance to JAK inhibitors in MF

44 1 : Resistance to tyrosine kinase inhibition in cancer - JAK inhibitors are ATP-competitive inhibitors - JAK inhibitors are not imatinib!. Not specific for mutated vs WT JAKs. JAK inhibition will interfere with many cytokine receptors 2 : In vitro mechanisms of JAK inhibitor resistance - JAK inhibitors are able to kill JAK-dependent cells - JAK kinase domain mutation and overexpression can explain resistance 3 : Clinical resistance to JAK inhibitors in MF - Clinical resistance exists Mainly primary resistance (short follow-up) 4 : Molecular resistance to JAK inhibitors in MF - No JAK kinase signaling addiction vs effective dose too toxic

45 JAK2 V617F + Better inhibitors? WT WT TKI time

46 Thanks J.C. Renauld T. Hornakova L. Springuel A. Saumet SN Constantinescu C. Hermans E. Van den neste L. Michaux M.C. Vekemans V. Havelange X. Poiré C. Lambert A. Ferrant