1. Mechanisms of Ibrutinib Sensitivity and Resistance in CLL Y. Lynn Wang, MD, PhD, FCAP Dept. of Pathology University of Chicago October 24, 2014

2. Outline Ibrutinib targets in vivo CLL proliferation through inhibition of B-cell receptor signaling activity BTK C481S confers resistance to ibrutinib as a consequence of loss of irreversible drug binding PLC  2 mutations confers resistance to ibrutinib via re-activation of BCR-signaling Overcome ibrutinib resistance: Exploration of other kinase inhibitors

3. Outline Ibrutinib targets in vivo CLL proliferation through inhibition of B-cell receptor signaling activity BTK C481S confers resistance to ibrutinib as a consequence of loss of irreversible drug binding PLC  2 mutations confers resistance to ibrutinib via re-activation of BCR-signaling Overcome ibrutinib resistance: Exploration of other kinase inhibitors

4. BTK is more active in treatment-naive CLL patients than in normal individuals 4 Normal donors 259 CLL patients 89 92 95 96 123 1103 127 2078 208 45 64 67 748276 1103 127 2078 208 t-BTK GAPDH t-BTK GAPDH A Controls ly7+IgM CLL patientsNormal donors ly7 45 64 677476 4771 4778 2078 208 259 ly7 89 92 9596123 4771 4778 2078 208259 82 p-BTK GAPDH p-BTK GAPDH C B CLL (N=11) Normal (N=5) p-BTK relative expression CLL (N=1 Normal (N=5) D Cheng S…Wang YL. Leukemia, 2014, 28, 649-57

5. Study Design In vivo: Prospective collection of serial samples from patients undergoing clinical trial of ibrutinib Enrolled a total of 16 patients –12 previously treated –4 treatment naïve

6. What is the effect of ibrutinib on apoptosis induction?

7. Does ibrutinib induce apoptosis? -An ex vivo study “Apoptosis induced by PCI-32765 in primary CLL cells was significant compared with vehicle treatment alone at 10 uM PCI-32765” Blood, 117, 6287, 2011 The Cmax values in humans range from 80-200 nM Cytopenia was uncommon in patients treated at 420 mg/day. Grade 3&4 neutropenia was more common in the 840 mg group-ASCO 2011

8. In vivo apoptosis was largely absent in the peripheral blood of CLL patients treated with ibrutinib 8 BCL2 XIAP MCL1 PARP Cleaved Caspase-3 GAPDH CLL 085 D28 CLL 195CLL 043CLL 011 pre4h4h D28preD28preD28 Again, apoptosis induction may not be the predominant effect of ibrutinib in CLL patients Cutoff 15% Cheng S…Wang YL. Leukemia, 2014, 28, 649-57

9. What is the effect of ibrutinib on CLL cell proliferation?

10. In vivo CLL proliferation is targeted by BTK inhibition: Ki-67 was reduced over the treatment course 10 Cheng S…Wang YL. Leukemia, 2014, 28, 649-57

11. 11 CLL proliferation is targeted by BTK inhibition in an in vitro model of cell proliferation Cheng S…Wang YL. Leukemia, 2014, 28, 649-57

12. 12 CLL proliferation is targeted by BTK inhibition in an in vitro model of cell proliferation Cheng S…Wang YL. Leukemia, 2014, 28, 649-57

13. What is the effect of ibrutinib on signal transduction?

14. B-cell Receptor Signaling

15. 15 Activity of BTK (pY223) was inhibited in treated patients Cheng S…Wang YL. Leukemia, 2014, 28, 649-57

16. BCR signaling in CLL CLL in vivo proliferation is mediated by the pathway LYN-SYK-BTK- PLC  2 and downstream signaling through AKT and ERK. The data highlight the role of cell proliferation in CLL that has been thought mainly as a disease with apoptotic defects.

17. Outline Ibrutinib targets in vivo CLL proliferation through inhibition of B-cell receptor signaling activity BTK C481S confers resistance to ibrutinib as a consequence of loss of irreversible drug binding PLC  2 mutations confers resistance to ibrutinib via re-activation of BCR-signaling Overcome ibrutinib resistance: Exploration of other kinase inhibitors

18. The Case The patient is a 61 year old woman first diagnosed with CLL in 2000 when she presented with lymphadenopathy. In September of 2010, she was enrolled in the phase Ib trial (NCT01105247) of ibrutinib. She achieved an excellent response, with normalization of her CBC, (ALC=3600), but some residual lymphadenopathy remained on CT scan. In March of 2012, 19 months after ibrutinib initiation, her ALC and lymphadenopathy rapidly increased. In May of 2012, the ibrutinib dose was escalated from 540 to 840 mg. Over the next four weeks, the patient continued to demonstrate disease progression.

19. Four specimens collected 19 Relapsed S2 Pre Responding Relapsed S1 Ibrutinib initiation Furman RR…Wang YL, NEJM, 2014, 370, 2352-4

20. A novel BTK mutation is identified by RNA-Seq which is present only in relapse samples 20 Relapsed Before relapse Pre-Rx Responding Relapse 1 Relapse 2 GC T GCCTC GC A GCCTC Responding Pre-RxRelapse s1 Relapse s2 Nucleotide composition at position 1634 of the BTK gene CLL Numbers of the reads %A Specimens ACGT Pre-Rx00084 0 Responding00054 0 Relapse S1102806 88 Relapse S267501 92 Furman RR…Wang YL, NEJM, 2014, 370, 2352-4

21. What is the impact of the mutation on ibrutinib binding?

22. The mutation disrupts the covalent binding of ibrutinib to BTK 22 Probe-labeled BTK Ibrutinib (nM) - 1 10 100 - 1 10 100 - 1 10 100 No DNA Total BTK  -actin WTC481SC481A WT C481S Covalent bond Furman RR…Wang YL, NEJM, 2014, 370, 2352-4 WT BTK (IC50 =2.2nM) BTK C481S (IC50 =1006nM) Inhibition of BTK phosphorylation (%) 1010100 1000 Ibrutinib (nM)

23. What is functional consequence of BTK C481S at the molecular level?

24. The mutation restores the BCR signaling pathway 24 p-BTK (Y223) p-ERK1/2 (T202/Y204) Before relapse Relapsed Pre-Rx Responding 1 2 p-AKT (S473) GAPDH 3210-2-3 Pre-RxResponding 12 RelapsedBefore relapse EGR1 PAICS DUSP2 KLF10 CD83 CASP3 NFKBIE DDX21 GFI1 IRF4 CTLA4 GNPDA1 Ly9 RGS10 CCL4 LPL PDGFA EGR3 SLAMF7 NAB2 LILRA4 NR4A3 DDIT3 OAS3 TXNRD1 BCR signature BCR Signature Score *** * * Pre-Rx RespondingRelapse1 Relapse2 Pre-Rx Responding Relapse 1 Relapse 2 Cheng S…Wang YL, Leukemia, 2014, epub

25. What is the functional consequence of BTK C481S at the cellular level?

26. The mutation leads to increased cellular proliferation in vivo 26 CD19 Ki67 Pre-Rx Responding Before Relapse 1 Relapsed 2 Cheng S…Wang YL, Leukemia, 2014, epub

27. The mutation leads to increased cellular proliferation in vitro that did not respond well to ibrutinib 27 Iso0 nM250nM500nM 7-AAD BrdU +CLL+Stroma + ibrutinib Before Relapse Relapsed 1 Responding 2 Pre-Rx Cheng S…Wang YL, Leukemia, 2014, epub

28. BTK Mutation Is Recurrent Woyach JA…Byrd JC. NEJM, 2014, 370, 2286-94

29. Outline Ibrutinib targets in vivo CLL proliferation through inhibition of B-cell receptor signaling activity BTK C481S confers resistance to ibrutinib as a consequence of loss of irreversible drug binding PLC  2 mutations confers resistance to ibrutinib via re-activation of BCR-signaling Overcome ibrutinib resistance: Exploration of other kinase inhibitors

30. Reactivation of BCR signaling in patient with PLC  2 R665W mutation Woyach JA…Byrd JC. NEJM, 2014, 370, 2286-94

31. Reactivation of BCR signaling in patient with PLC  2 R665W mutation Woyach JA…Byrd JC. NEJM, 2014, 370, 2286-94

32. Outline Ibrutinib targets in vivo CLL proliferation through inhibition of B-cell receptor signaling activity BTK C481S confers resistance to ibrutinib as a consequence of loss of irreversible drug binding PLC  2 mutations confers resistance to ibrutinib via re-activation of BCR-signaling Overcome ibrutinib resistance: Exploration of other kinase inhibitors

33. Ibrutinib-resistance CLL cells remain sensitive to other BCR inhibitors 33 Cheng S…Wang YL, Leukemia, 2014, epub

34. 34 Ibrutinib-resistance CLL cells remain sensitive to idelalicib Cheng S…Wang YL, Leukemia, 2014, epub

35. Acknowledgement Wang Lab at Cornell Shuhua Cheng, PhD Chunyan Yang, MD Pin Lu, MD, PhD Ailin Guo, MD, PhD Annie Ma, PhD Zibo Song, PhD Joelle Racchumi

36. Acknowledgement Cornell CLL Research Center Dr. Morton Coleman Dr. Richard Furman MSKCC Dr. Christina Leslie Menu Setty Pharmacyclics Dr. Betty Chang Harvard University Dr. Hao Wu

37. Thank you!