1. ALK e meccanismi di resistenza Lucio Crinò S.C. di Oncologia Medica Azienda Ospedaliera di Perugia

2. Outline Current evidence with ALK-TKIs in naïve patients ALK-TKIs in crizotinib-refractory disease Challenging brain mets in ALK+ disease Resistance to crizotinib: focus on ‘unknown’ mechanisms

3. Outline Current evidence with ALK-TKIs in naïve patients ALK-TKIs in crizotinib-refractory disease Challenging brain mets in ALK+ disease Resistance to crizotinib: focus on ‘unknown’ mechanisms

4. Crizotinib for ALK+ advanced NSCLC: RR appr. 60%, PFS 7.7-9.7 mos StudyNo. of patientsRR (%)PFS (mos.) A808100114360.89.7 A808100526159.8 ∞ 8.1 A808100717365 * 7.7 French Temporary Authorization for use of Crizotinib 23056.5Not reported ∞ 259 pts evaluable for response *Independent radiologic review Camidge, et al. Lancet Oncol 2012 Kim, et al. ASCO 2012 Shaw, et al. NEJM 2013 Perol, et al. ECCO 2013

5. [TITLE] Presented By Fabrice Barlesi, MD, PhD at 2013 ASCO Annual Meeting

7. DrugInhibition of secondary L1196M ‘gatekeeper’ mutation CompanyClinical stage AP-26113YesAriad Pharmaceuticals Phase I/II LDK378YesNovartisPhase II/III AlectinibYesChugai Pharmaceuticals Phase I/II TSR-011YesTesaroPhase I NMS-E628YesNerviano MedicalPhase I ASP-3026YesAstellasPhase I X-376 and -396YesXcoveryPhase I CEP-28122YesCephalonPreclinical 2 nd generation ALK-inhibitors in clinical development

8. RR with 2 nd generation ALK-inhibitors in Crizotinib-naïve patients AuthorDrugNo. of ptsRR (%) Camidge (ECCO 2013) AP26113 * 3100 Shaw (ASCO 2013) LDK378 ** 3560 Seto (Lancet Oncol 2013) Alectinib *** 4693.5 *60-300 mg/d **400-750 mg/d ***300 mg x 2/d; Asiatic (Japanese) patients

9. Outline Current evidence with ALK-TKIs in naïve patients ALK-TKIs in crizotinib-refractory disease Challenging brain mets in ALK+ disease Resistance to crizotinib: focus on ‘unknown’ mechanisms

10. CHEMOTHERAPY TKI beyond PD or 2 nd generation TKI ± local therapy TKI beyond PD + brain RT

11. ALK rearrangement Progression on Crizotinib after response or SD > 3 mo No prior pemetrexed N=114 Pemetrexed alone Pemetrexed + ongoing Crizotinib RANDOMIZERANDOMIZE Co-primary endpoint: progression-free survival Respnse rate, pemetrexed alone Chemotherapy +/- ongoing crizotinib for acquired resistance in ALK-positive NSCLC SWOG 1300 (in development) PI: Ross Camidge

12. Crizotinib beyond progression Ou, et al. IASLC 2013

13. AP26113 in ALK+ NSCLCs (N=34) 13 All patients received prior crizotinib unless otherwise indicated; Doses ranged from 60-240 mg/d (23 pts ≥180mg/d); a TKI-naïve; b Received prior crizotinib and LDK378; c PD by RECIST 1.1 due to 2nd primary tumor of melanoma; d Crizotinib-intolerant Response duration 8+ to 40+ weeks 14 confirmed, 4 awaiting confirmation, 4 not confirmed Data as of 6 Sept 2013 65% (22/34) objective response rate (95% CI: 47-80%) 61% (19/31) post-crizotinib (incl. 1 criz intolerant) 100% (3/3) in TKI-naïve (incl. 1 CR) Camidge, et al. IASLC 2013

15. Alectinib in crizotinib-refractory patients Overall RR 54.5% across all cohorts for all patients Waterfall plot % tumor shrinkage Days on study Duration on study (*: off study, data cut-off Sept. 12, 2013) * * * * * * * * * * * * * * Overall RR 59.5% for cohorts of 460 mg dose or higher 24 of the 47 patients received the drug for 120 days or longer Gadgeel, et al. IASLC 2013

16. Outline Current evidence with ALK-TKIs in naïve patients ALK-TKIs in crizotinib-refractory disease Challenging brain mets in ALK+ disease Resistance to crizotinib: focus on ‘unknown’ mechanisms

17. 13/28 (46%) patients at U. of Colorado with first progression in CNS - 2/13 had synchronous systemic progression Weickardt et al. JTO 2013 Decreased CSF:plasma (0.0026) ratio suggestive of pharmacological failure of Crizotinib Costa et al. JCO 2011 The problem of CNS progression to Crizotinib in ALK+ patients

18. Crizotinib antitumour activity in patients with or without brain metastasis (BM) at baseline Previously untreated for BM (n=109) Previously treated for BM (n=166) No BM detected (n=613) nOutcome n n DCR at 12 weeks, % (95%CI) IC10956 (46−66) 16662 (54−70) NA Systemic10963 (54−72) 16665 (57−72) 61371 (68−75) ORR, % (95% CI) IC1097 (3−14) 1667 (4−12) NA Target-lesion BM2218 (5−40) 1833 (13−59) NA Systemic10953 (43−63) 16646 (39−54) 61355 (51−59) Median time to tumor response (range), a weeks IC86.0 (4.9−12.4) 126.4 (5.9−17.7) NA Systemic586.1 (2.0−31.4) 776.1 (3.1−35.3) 3366.1 (3.0−49.1) Median duration of response b (range), a weeks IC826.4 (6.1−59.3) 12 NR (6.0−59.9) NA Systemic5847.9 (5.3−55.0) 7755.6 (4.4−95.3) 33649.0 (4.1−96.1) Median systemic PFS, b (95% CI), c mo 1098.3 (6.7−14.0) 16613.5 (6.2−16.5) 6139.9 (8.8−12.2) NR, not reached a In patients with confirmed objective response; b Kaplan−Meier method; c Brookmeyer−Crowley method Costa, et al. IASLC 2013

19. Example of a complete response of a brain lesion in response to crizotinib treatment Before initiation of crizotinib 6 weeks after crizotinib 250 mg BID This patient presented with a brain lesion previously treated with chemotherapy and palliative IC radiation, which was classified as a target lesion. A CR in this lesion was observed after 6 weeks of treatment and had persisted for 54 weeks by data cutoff (courtesy of J-Y Han, National Cancer Center, Goyang, South Korea)

20. Systemic progression-free survival (PFS) by presence or absence of brain metastases (BM) at baseline (BL) The presence of BM at BL does not significantly affect systemic PFS to crizotinib Costa, et al. IASLC 2013

21. CNS activity of alectinib ORR of the 21 patients as determined by central image review 9/21 patients with baseline CNS metastasis had measurable CNS lesions and received no prior radiation within 4 weeks from first dose of alectinib –No CR. 5/9 achieved CNS PR (≥ 30% reduction in sum of largest dimension). 2/9 had CNS stable disease and 2/9 had CNS progression. Brain RT > 4 wks No brain RT 91+ 203+ 42 224+ 287+ 98+ 196+ 294+ Days on study CRPRSDPD Total N=216582 %29%24%38%10% PR Progressing CNS mets Ou, et al. IASLC 2013

22. Outline Current evidence with ALK-TKIs in naïve patients ALK-TKIs in crizotinib-refractory disease Challenging brain mets in ALK+ disease Resistance to crizotinib: focus on ‘unknown’ mechanisms

23. Unraveling the mechanisms of resistance to EGFR-TKIs and Crizotinib Katayama, et al. Sci Trans Med. 2012 Doebele, et al. Clin Cancer Res. 2012 Unknown Bypass tracks EGFR MT KRAS MT Amplified L1196M G1269A S1206Y G1202R 1151Tins L1152R C1156Y No ALK amp or mut ALK amp ALK mut ALK+

24. IGF-1R and IRS-1 are up-regulated in ALK TKI resistant cells pIGF-1R Y1131 IRS-1 pY941 AKT pAKT S473 S6 pERK ERK IRS-1 IGF-1R pS6 DMSO5005000 ALK TKI [nM] TKI sensitive DMSO5005000 TKI resistant pALK Y1604 ALK References: Lovly, C et al 2011 Cancer Research and Lovly, C et al submitted Generation of isogenic pairs of ALK TKI sensitive and ALK TKI resistant cells

25. pIGF-1R and IRS-1 are up-regulated in patient biopsy samples at the time of acquired resistance to crizotinib Reference: Lovly, C et al submitted pIGF-1R and IRS-1 are up-regulated in patient biopsy samples at the time of acquired resistance to crizotinib

26. Key: - X-376: ALK TKI - MAb391: IGF-1R MAb IGF-1R inhibitors sensitize H3122 ALK TKI resistant cells to the anti-proliferative effects of ALK inhibitors -Soft agar assay in H3122 X-376 resistant cells -Similar results seen in H3122 crizotinib resistant cells -Analogous results seen with IGF-1R TKIs in addition to IGF-1R MAbs Reference: Lovly, C et al submitted

27. Rationale for double ALK/HSP90 inhibition in ALK+ advanced NSCLC From Crystal and Shaw, Clin Cancer Res 2012

28. Hsp90 inhibitor AUY922: activity in ALK+ patients (n=19/22) Felip, et al. ESMO 2012

29. Tumor #6 transplanted H2228R tumor xenografts treated with AT13387 AT13387 treatment inhibits the growth of an ALK-dependent tumor xenograft with acquired resistance to crizotinib Continuous daily crizotinib treatment H2228 tumor xenografts treated with crizotinib Tumor volume (mm 3 ) Wallis, IASLC 2013 The Hsp90i AT13387 overcomes acquired resistance to crizotinib

30. ALK: (e.g. ALK-TKI + Hsp90 inhibitor; ALK-TKI + IGFR-1R inhibitors ) TherapyTherapyTherapy Resistance Selection for genetic instability Selection for resistance Rationale for the upfront use of combination regimens

31. Sang et al. Cancer Discov 2013 Preclinical evidence for UPFRONT dual ALK/Hsp90 inhibition: ALK-TKI + Ganetespib

32. Combination of AT13387 and crizotinib shows improved inhibition of tumor growth over monotherapies Combining crizotinib upfront with AT13387 delays the emergence of resistance in vivo Preclinical evidence for UPFRONT dual ALK/Hsp90 inhibition: ALK-TKI + AT13387 Wallis, IASLC 2013

33. RegimenPhaseClinicalTrials.gov Identifier Crizotinib + AT13387I/IINCT01712217 Crizotinib + GanetespibI/IINCT01579994 LDK378 + AUY922IbNCT01772797 Clinical attempt to better Crizotinib: trials of ALK/HSP90 inhibition

34. ALK-inhibition in ALK+ NSCLC Platform networks required tools for optimization of NSCLC treatment 2 nd generation ALK-TKIs may break the boundary of 60% response rate observed with Crizotinib Appr. 60% of RR with 2 nd generation ALK-TKIs in Crizotinib- refractory patients 2 nd generation ALK-TKIs may prevent CNS pharmacological failure Hsp90 activity and IGF-1R upregulation may be partly responsible for the non-ALK dominant mechanisms of resistance to crizotinib Robust preclinical evidence of anticancer efficacy with ALK- TKIs + HSP90 or IGF-1R inhibitors in ALK+ advanced NSCLC

35. lucio.crino@ospedale.perugia.it Thanks for your attention