1. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy Molecular Mechanisms of Resistance to Imatinib (STI571, CGP57148B)

2. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy Structure of Imatinib (STI571, CGP 57148B) Class: Phenylaminopyrimidines, 589.7 mw CH 3 SO 3 H O

3. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy

4. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy Cellular Selectivity of STI571: IC 50 [µM] Kinases InhibitedKinases Not Inhibited v-ABL0.1–0.3Flt-3 >10 p210 bcr-abl 0.25c-Fms, v-Fms >10 p185 bcr-abl 0.25EGF receptor >100 TEL-Abl0.35c-erbB2 >100 PDGF receptor 0.1Insulin receptor >100 TEL-PDGF IGF-1 receptor >100 receptor 0.15v-Src >10 c-kit0.1JAK-2 >100 Druker BJ et al. Nat Med. 1996;2:561-566; Personal communication 4/00. B Druker, E Buchdunger.

5. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy Effect of STI571 on Growth of Bcr-Abl–Positive and –Negative Cell Lines Gambacorti-Passerini C et al. Blood Cells Mol Dis. 1997;23:380. *Bcr-Abl-negative cell lines Bcr-Abl-positive cell lines U937* KG1* KCL22* K562 KU812 SU DHL1 STI571 Concentration ( M) % Control CPM 00.10.31 310 0 20 40 60 80 100 120

6. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy

7. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy LAMA84 Control LAMA84 24h 1uM LAMA84 44h 1 uM

8. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy

9. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy

10. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy

11. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy

12. National Cancer Institute Milan, Italy

13. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy

14. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy

15. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy

16. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy Resistance to Imatinib

17. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy

18. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy I Discontinuous Inhibition

19. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy

20. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy

21. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy bcr/abl anti-phosphotyrosine anti-abl i.p. 2h p.o. i.p. 5h p.o. i.p. 9h p.o.

22. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy

23. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy

24. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy

25. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy II Insufficient Tissue Levels (Beware of Plasma Concentrations)

26. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy 1 2 3 4 alpha 1 acidic glycoprotein

27. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy

28. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy 205 kd Anti-Abl 205 kd Anti-Ptyr Day 1Day 5 Patient 001 205 kd 47 kd Day 1 Anti-Ptyr Day 2 Anti-Actin Patient 504

29. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy Day 1Relapse No STI571 in vivo > 3µM STI571 in vivo + 3 µM STI571 In vitro + 3µM STI571 in vitro 205 kd Anti-Abl Anti-Ptyr Patient 001 205 kd Patient 505 Anti-Abl Anti-Ptyr Patient 002 Anti-Abl Anti-Ptyr 205 kd

30. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy >3 µM STI571 in vivo + 100 µM Erythromycin in vitro >3µM STI571 in vivo 3 µM STI571 in vitro 205 kd Patient 008 Anti-Abl Anti-Ptyr Patient 009 Anti-Abl Anti-Ptyr >3 µM STI571 in vivo + 100 µM Erythromycin in vitro >3µM STI571 in vivo 3 µM STI571 in vitro 205 kd

31. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy Effect of the administration of Clindamycin (900 mg i.v. over 20 minutes) on STI571 plasma concentrations 0102030 0.0 0.1 0.2 0.3 0.4 Patient 008 Patient 00213 Patient 00241 2 3 4 5 6 7 8 Time (min) STI571 Plasma concentration ( g/ml) Clindamycin

32. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy Effect of Clindamycin (C) administration on steady state STI571 plasma levels

33. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy BCR/ABL STI571

34. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy BCR/ABL AGP STI 571 AGP STI 571 AGP STI 571 AGP STI 571 In the presence of alpha 1 acidic glycoprotein (AGP) STI571

35. National Cancer Institute Milan, Italy University of Milano Bicocca, Monza, Italy Trattamento con STI571 + Eritromicina (E) BCR/ABL STI 571 AGP E E E E