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MOLECULAR TARGETS

MOLECULAR TARGETS. Edward A. Sausville, M.D., Ph.D. Developmental Therapeutics Program National Cancer Institute. % Alive. Treatment B or no R. x. Time. R. x. Untreated. R. Cytostatic. x. Tumor Size. Time. CANCER DRUGS: HOW DO WE KNOW WE HAVE A WINNER?. Treatment A.

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MOLECULAR TARGETS

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  1. MOLECULAR TARGETS Edward A. Sausville, M.D., Ph.D. Developmental Therapeutics Program National Cancer Institute

  2. % Alive Treatment B or no R x Time R x Untreated R Cytostatic x Tumor Size Time CANCER DRUGS:HOW DO WE KNOW WE HAVE A WINNER? Treatment A - PHASE III CLINICAL TRIAL = WINNER - PHASE II = POTENTIAL WINNER ; Time? - PRECLINICAL MODEL (e.g., mouse or rat) Cytotoxic

  3. CANCER DRUGS: HOW TO PICK A WINNER? “VALIDATED” CANCER TARGETS • DNA • Tubulin • Receptors • Oncogene Proteins - Alkylators - Antimetabolites - Topo I / II - Nuclear - Cell Surface(Immune?)

  4. “EMPIRICAL” DRUG DISCOVERY SCREEN PHARMACOLOGY + ANTI-TUMOR ACTIVITY!! (in vitro/in vivo) CHEMISTRY OPTIMIZED SCHEDULE (in vivo) IND-DIRECTED TOX/FORMULATION PHASE I: DOSE/SCHEDULE HUMAN PHARM/TOX PHASE II: ACTIVITY = SHRINKAGE PHASE III: COMPARE WITH STANDARD

  5. KRN5500 Cell Membrane Deacylation SAN-Gly H3C(CH2)8CH=CHCH=CHCO2H Protein Synthesis

  6. vehicle 13.5 qdx5 33.5 q4dx3 22.4 q4dx3 20 qdx5 50 q4dx3 30 qdx5 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 + X 8 12 15 18 22 26 29 33 36 41 EFFECT OF KRN5500 ON COLO-205 ATHYMIC MOUSE XENOGRAFTS Median Tumor Weight (mg) Day Posttumor Implantation

  7. KRN5500 PLASMA CONCENTRATIONS ON EFFECTIVE SCHEDULE(20 MG/KG/D) IN MICE Plasma Concentration (M) Time (days)

  8. SUMMARY OF KRN-5500 PHASE I • 26 patients as IV once per day over 5 days • Dose limiting toxicity = interstitial pneumonitis • MTD = 2.9 mg/M2/d x 5 • Achieve only 0.75 - 1 M at 3.7 mg/M2/d x 5 • 4/6 patients with >25% incr Cmax have grade 4 toxicity Data of J. P. Eder, DFCI

  9. PROBLEMS WITH EMPIRICAL MODELS • Lack of predictive power in vivo • Poor correlation of non-human with human pharmacology • Divorced from biology • Inefficient: many compounds screened; developed, but have “late” = clinical trials outcome at Phase III to define “validation” of compound action

  10. % IN VIVO ACTIVITY vs CLINICAL ACTIVITY(39 AGENTS)

  11. TARGET-DEPENDENT IN VIVO MODEL IND DIRECTED TOX/FORM PHASE I: DOSE/SCHEDULE: HUMAN PHARM/TOX; ? AFFECT TARGET PHASE II: ACTIVITY = ? AFFECT TARGET PHASE III: SURVIVAL/TIME TO PROGRESSION “RATIONAL” DRUG DISCOVERY MOLECULAR TARGET SCREEN PHARMACOLOGY (to affect target) Biochemical Engineered cell Animal (yeast/worm/fish) CHEMISTRY

  12. Self-sufficiency in growth signals Insensitivity to anti-growth signals Evading apoptosis Sustained angiogenesis Tissue invasion & metastasis Limitless replicative potential SIX ESSENTIAL ALTERATIONSIN CELL PHYSIOLOGY IN MALIGNANCY Targets for classical drugs? Targets for novel drugs? Hanahan & Weinberg, Cell 100:57 (2000)

  13. MOLECULAR TARGET DEFINITION - HOW TO? • BIOLOGY: • “ RETROFIT” ACTIVE MOLECULES: • “CLASSICAL:” • CHEMICAL GENETICS: * Cytogenetics Breakpoints Molecules (bcr-abl) * “Positive” selection from tumor DNA Active oncogenes (signal transduction) * Tumor gene expression profiling (CGAP) * Binding partners (geldanamycin, rapamycin, fumagillin) * Computational algorithm (molecule target) * Cell metabolism / Cell cycle effects * Suggest single targets Inefficient * Libraries of molecules and precisely defined organisms - COMPARE - Cluster analysis

  14. bcr-abl AS TARGET: RATIONALE • Apparently pathogenetic in t9:Q22 (Ph+) CML/ALL • Absence in normal tissues • Modulate signal transduction events downstream Maintenance of chronic phase Adjunct to bone marrow transplantation

  15. bcr-abl FUSION PROTEIN DNA Actin bcr SH2 SH2 V SH2/SH3 kinase NT bcr autophosphorylation Phosphorylation of other substances McWhirter JR, EMBO 12:1533, 1993

  16. EXAMPLE OF “RATIONAL” APPROACH:bcr-abl directed agents Natural product empiric lead 1st generation synthetic 2nd generation synthetic; in clinic erbstatin lavendustin piceatannol AG957 AG1112 CGP 57148B = STI571

  17. STI571: An oral in vivo bcr-abl kinase inhibitor (days) (hrs) (days) Antitumor activity in vivo Tyr phosphorylation in vivo le Coutre et al, JNCI 91:163, 1999

  18. EFFICACY AND SAFETY OF A SPECIFIC INHIBITOR OF THE BCR-ABLTYROSINE KINASE IN CHRONIC MYELOID LEUKEMIA Ph Chromosome + Cells White Cell Count 100 100 80 60 10 % in Metaphase (cells x 10-3 / mm3) 40 20 1 0 30 60 90 120 150 0 0 100 200 300 400 Duration of Treatment with STI571 (Days) Duration of Treatment with STI571 (Days) BRIAN J.DRUKER,M.D.,MOSHE TALPAZ,M.D.,DEBRA J.RESTA,R.N.,BIN PENG,PH.D., ELISABETH BUCHDUNGER,PH.D.,JOHN M.FORD,M.D.,NICHOLAS B.LYDON,PH.D.,HAGOP KANTARJIAN,M.D., RENAUD CAPDEVILLE,M.D.,SAYURI OHNO-JONES,B.S.,AND CHARLES L.SAWYERS,M.D. NEJM 344: 1031, 2001

  19. Imatinib 100 Combination therapy 80 Progression-free survival (%) 60 40 20 p<0.001 0 Months after Randomization 0 2 12 543 498 3 7 38 530 442 6 12 73 518 376 9 18 94 505 334 12 29 108 487 302 15 41 119 392 255 18 42 125 162 99 21 42 125 7 7 24 27 PROGRESSION-FREE SURVIVALFOR IMATINIB VS. INTERFERON AND LOW-DOSE CYTARABINE IN CHRONIC-PHASE CML # of Events # at Risk Imatinib Combination Imatinib Combination Druker et al, NEJM 348: 994, 2003

  20. 100 80 60 40 20 0 0 3 6 9 12 15 18 21 24 27 TIME TO A MAJOR CYTOGENETIC RESPONSEFOR IMATINIB VS. INTERFERON AND LOW-DOSE CYTARABINE IN CHRONIC-PHASE CML Imatinib Major Cytogenetic Response (%) Combination therapy p<0.001 Months after Randomization Druker et al, NEJM 348: 994, 2003

  21. 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Myeloid (n=21) Lymphoid (n=14) 0 100 200 300 400 ACTIVITY OF A SPECIFIC INHIBITOR OF THE BCR-ABL TYROSINE KINASEIN THE BLAST CRISIS OF CHRONIC MYELOID LEUKEMIA AND ACUTELYMPHOBLASTIC LEUKEMIA WITH THE PHILADELPHIA CHROMOSOME BRIAN J.DRUKER,M.D.,CHARLES L.SAWYERS,M.D.,HAGOP KANTARJIAN,M.D.,DEBRA J.RESTA,R.N., SOFIA FERNANDES REESE,M.D.,JOHN M.FORD,M.D.,RENAUD CAPDEVILLE,M.D.,AND MOSHE TALPAZ,M.D. Time to Relapse in Patients with Myeloid or Lymphoid Blast Crisis Who Had a Response to STI571 Probability of Relapse Day • Yellow arrows indicate patients still enrolled in the study and in remission at the • time of the last follow-up • White arrows indicate the day on which patients were removed from the study NEJM 344: 1038, 2001

  22. Clinical Resistance to STI-571 Cancer Therapy Caused by BCR-ABL Gene Mutation or Amplification Mercedes E. Gorre,1, 3 Mansoor Mohammed,2 Katharine Ellwood,1 Nicholas Hsu,1 Ron Paquette,1 P. Nagesh Rao,2 Charles L. Sawyers1, 3* Science 293: 876, 2001

  23. Clinical Resistance to STI-571 Cancer Therapy Caused by BCR-ABL Gene Mutation or Amplification STI-571 STI-571 THR315 ILE315 STI-571 (M) 0 0 0.1 0.1 0.5 0.5 1 1 5 5 10 10 BCR-ABL BCR-ABL -P-TYR -ABL Mercedes E. Gorre,1, 3 Mansoor Mohammed,2 Katharine Ellwood,1 Nicholas Hsu,1 Ron Paquette,1 P. Nagesh Rao,2 Charles L. Sawyers1, 3* BCR-ABL Wild Type BCR-ABL T3151 Mutant Science 293: 876, 2001

  24. Cancer Genome Anatomy ProjectPROCESS • Tumor material (archival) • “Laser capture microdissection” of tumor cells • Creation of tumor-derived cDNA libraries • Sequence to establish uniqueness • Deposit in public domain from defined sections

  25. Http://cgap.nci.nih.gov/

  26. Genes and Cancer Treatment Caron, H, et al., New England Journal of Medicine, 1996 PubMed. Often, patients suffering from tumors that by traditional criteria are indistinguishable, nevertheless can experience very different outcomes despite having received the same treament. The research results displayed in this graph demonstrate that for patients suffering from the cancer neuroblastoma, the presence or absence of a specific set of genes found on Chromosome 1 strongly correlates with patient outcome. Therefore, in the future this characteristic of the tumor can be used to identify those patients that would benefit from more aggressive treatment, and those best served by the current treatment protocol. Http://cgap.nci.nih.gov

  27. Gene Expression: The Cell’s Fingerprint Normal Cell Cancer Cell Establishing for a cell the repertoire of genes expressed, together with the amount of gene products produced for each, yields a powerful "fingerprint". Comparing the fingerprints of a normal versus a cancer cell will highlight genes that by their suspicious absence or presence (such as Gene H ) deserve further scientific scrutiny to determine whether such suspects play a role in cancer, or can be exploited in a test for early detection. Http://cgap.nci.nih.gov

  28. Cancer Genome Anatomy ProjectPRODUCT • Genes “associated” with tumor type • How to define “drugs” associated with gene products? - Binding partners/Biochemical assays - “Pathway effect” in engineered organisms - Computational algorithms

  29. MOLECULAR TARGET DEFINITION - HOW TO? • BIOLOGY: • “ RETROFIT” ACTIVE MOLECULES: • “CLASSICAL:” • CHEMICAL GENETICS: * Cytogenetics Breakpoints Molecules (bcr-abl) * “Positive” selection from tumor DNA Active oncogenes (signal transduction) * Tumor gene expression profiling (CGAP) * Binding partners (geldanamycin, rapamycin, fumagillin) * Computational algorithm (molecule target) * Cell metabolism / Cell cycle effects * Suggest single targets Inefficient * Libraries of molecules and precisely defined organisms - COMPARE - Cluster analysis

  30. benzoquinone ansa ring carbamate NSC R Geldanamycin 17-AAG 122750 330507 OMe NHCH2CH=CH2 GELDANAMYCIN STRUCTURE

  31. BENZOQUINOID ANSAMYCINSINITIAL CELL PHARMACOLOGY - I • “Reverse” transformed phenotype of src-transformed rat kidney cell line • decrease tyrosine phosphorylation of pp60src • not inhibit pp60 immune complex kinase directly but these were inhibited from drug-treated cells • thus alter “intracellular environment” of src • Decrease steady state phosphorylation levels to 10% of control • decrease steady state level of pp60src by 30% • accelerate turnover of pp60src (Uehara et al, MCB 6: 2198, 1986) (Uehara et al, Cancer Res 49: 780, 1989)

  32. 150 100 50 0 MDA MB 453 p185 Protein p185 PY % Control 0 2 4 6 8 Hours BENZOQUINOID ANSAMYCINSINITIAL CELL PHARMACOLOGY - II • Reduce levels or inhibit transformation by a large number of PTKs: src, yes, fps, erbB1, lck • e.g., 17AAG decrease erbB2 under conditions where overall transcription/translation little affected Effect of 6 hr, 0.35 M herbimycin A on SKBr3 cells % of control Parameter p185 protein p185 Y-P erbB2 RNA Prot syn RNA syn ATP ATP/ADP 35 5 130 84 90 99 108 (Miller et al, Cancer Res 54: 2724, 1994)

  33. BENZOQUINOID ANSAMYCINSCELL PHARMACOLOGY - III • Insulin receptor degradation induced by herbimycin • inhibited by 20S proteasome inhibitors • - drug induced degradation does not occur in cells • defective in ubiquitination function • Mature erbB2 is ubiquitinated after geldanamycin exposure; • immature erbB2 sequestered • - lactacystin, a 20S proteasome inhibitor, prevents • instability of immature form and induces higher • GMW forms of mature consistent with retarded • proteasome processing of erbB2 “marked” for • destruction by drug Sepp-Lorenzino et al, JBC 270: 16580, 1995 Mimnaugh et al, JBC 271: 22796, 1996

  34. Herbimycin A (nM) Geldanamycin (nM) Cell Line IC50 IC95 IC50 IC95 CHP-100 TC-32 SKNMC D283 Med D341 Med IMR-32 SKNSH NIH3T3 HL-60 CEM 62 31 62 62 10 236 236 710 >472 >472 236 236 236 236 30 >472 >472 3780 >472 >472 5 5 8 ND ND ND ND 80 >40 >40 15 20 25 ND ND ND ND 250 >40 >40 Tumors (Formed/Inoculated) Mean weight mg (SE) Tumors (Formed/Inoculated) Mean weight mg (SE) 10/10 5/10 7/10 357 (49) 302 (97) 221 (49) 10/10 6/10 ND 436 (93) 252 (53) ND Potent inhibition of pediatric neural tumor cell lines GELDANAMYCIN: BASIS FOR NCI INTEREST Evidence of in vivo effect Topical Systemic DMSO Control Geldanamycin Herbimycin A p=0.037 p=0.042 p=0.014 Whitesell et al, Cancer Res 52: 1721, 1992

  35. Dose (mg/kg) Drug Deaths % Opt T/C (D) Growth Delay # Schedule 20 6 6 6 0 3.4 2.3 1.5 qd x 5 (9) qd x 5 (9) qd x 5 (9) qd x 5 (9) 0 3 1 0 --- Toxic 33(22) 90(15) --- --- 50 3 DTP, NCI IN VIVO EVALUATIONOF GELDANAMYCIN IN PC3 PROSTATE CA Early Stage, Athymic Mouse Xenograft Route of administration – i.p. Conclude: Narrow therapeutic index on this schedule Solubility of agent major problem for other schedules

  36. MECHANISM OFBENZOQUINOID ANSAMYCIN ACTION • Geldanamycin does NOT potently inhibit tyrosine kinase in in vitro immune complex kinase assays • In some cell types (e.g., CHP100), inhibition of cell growth and reversion of cellular transformation occur WITHOUT discernible effect on tyrosine phosphorylation • Raises hypothesis that there must be an additional target or means of influencing cellular transformation BESIDES “simply” inhibiting tyrosine kinases Whitesell et al, PNAS 91: 8324, 1994

  37. Bead 18 Atom Spacer

  38. R. Lysate 1 2 3 4 p90 GELDANAMYCIN BEADSIDENTIFY HSP90 AS BINDING PARTNER 1) Bead-Geld 2) Bead-Geld + Geld 3) Bead-Geld + Geldampicin 4) Bead Neckers et al, PNAS 91:8324, 1994

  39. * degradation G0 X-mRNA X erbB2 EGFR lck, met, etc raf ER folding X X hsp 90 nucleus Hsp 90 EIF2 kinase pAKT Immature X Mature X * Cyclin D hsp 90 ER PR etc telomerase nucleus hsp 90 * hsp 90

  40. THREE DIMENSIONAL VIEW OF GELDANAMYCIN BINDING POCKET IN AMINO TERMINUS OF HSP90 Stebbins et al, Cell 89:239, 1997

  41. 120 120 100 100 0.03 0.1 0.3 0.5 2 0.03 0.1 0.3 0.5 2 0.03 0.1 0.3 0.5 2 0.03 0.1 0.3 0.5 2 80 80 Raf-1 p185erbB2 control control 60 60 40 40 20 20 (M) 17-AAG GA (M) 17-AAG GA 0 0 17-AAG GA 17-AAG GA 0 0 1 1 10 10 100 100 1000 1000 10000 10000 17-AAG BINDS TO HSP90 & SHARES IMPORTANT BIOLOGIC ACTIVITIES WITH GELDANAMYCIN erbB2 (% of base line) Raf-1 (% of base line) dose (nM) dose (nM) Schulte & Neckers, Cancer Chemother Pharmacol 42: 273, 1998

  42. MOLECULAR TARGET DEFINITION - HOW TO? • BIOLOGY: • “ RETROFIT” ACTIVE MOLECULES: • “CLASSICAL:” • CHEMICAL GENETICS: * Cytogenetics Breakpoints Molecules (bcr-abl) * “Positive” selection from tumor DNA Active oncogenes (signal transduction) * Tumor gene expression profiling (CGAP) * Binding partners (geldanamycin, rapamycin, fumagillin) * Computational algorithm (molecule target) * Cell metabolism / Cell cycle effects * Suggest single targets Inefficient * Libraries of molecules and precisely defined organisms - COMPARE - Cluster analysis

  43. NCI IN VITRO DRUG SCREEN 1985 Hypothesis: Emerging Realities: • Cell type specific agents • Activity in solid tumors • Unique patterns of activity, cut across cell types • Correlations of compound activity AND Cell type selective patterns found - relate to molecular “target” expression - generate hypothesis re: molecular target

  44. NCI IN VITRO CANCER CELL LINE SCREEN • 60 cell lines • 48 hr exposure; protein stain O.D. (8 breast, 2 prostate, 8 renal, 6 ovary, 7 colon, 6 brain, 9 lung, 8 melanoma, 6 hematopoietic) Control “GI50” = 50% inhibit O.D. “TGI” = 100% inhibit “LC50” = 50% kill Time

  45. National Cancer Institute Developmental Therapeutics Program Dose Response Curves NSC: 643248-Q/2 (a rapamycin) Exp. ID: 9503SC35-46 All Cell Lines 100 50 0 Percentage Growth -50 -100 -7 -9 -8 -6 -5 -4 Log10 of Sample Concentration (Molar)

  46. PATTERN RECOGNITION ALGORITHM:COMPARE • Goal: COMPARE degree of similarity of a new • Calculate mean GI50, TGI or LC50 • Display behavior of particular cell line as deflection • Calculate Pearson correlation coefficient: compound to standard agents 1 = identity ; 0 = no correlation from mean resistant mean sensitive

  47. AGENTS WITH SIMILAR MECHANISMS HAVESIMILAR MEAN GRAPHS Leukemia NSCLC Small Cell Lung Colon CNS Melanoma Ovarian Renal Taxol Halichondrin B Daunorubicin Topoisomerase II Tubulin

  48. THE COMPARE ALGORITHMSeed: Rubidazone 164011 82151 123127 665934Discreet Discreet 267469 305884 665935 668380 639659 644946 254681 Discreet Discreet 180510 Discreet Discreet 1.000 0.921 0.915 0.891 0.880 0.867 0.865 0.865 0.864 0.861 0.854 0.850 0.848 0.847 0.843 0.842 0.837 0.833 Rubidazone Daunomycin Adriamycin Epipodophyllotoxin analogue Gyrase-To-TOPO analogue AMSA analogue Deoxydoxorubicin Acodazole HCL Epipodophyllotoxin analogue Azatoxin analogue Adriamycin analogue Epipodophyllotoxin analogue Daunomycin analogue Epipodophyllotoxin analogue Epipodophyllotoxin analogue Daunomycin analogue Epipodophyllotoxin analogue Gyrase-To-TOPO analogue

  49. COMPARE LINKS DRUG STRUCTURES WITH COMMON TARGET Cucurbitacin E NSC 106399 Jasplakinolide NSC 613009

  50. CUCURBITACIN E:COMPARE ANALYSIS OF GI50s CORRELATION COEFFICIENT COMPOUND Cucurbitacin E Cucurbitacin I Cucurbitacin K Cucurbitacin D Jasplakinolide Cytochalasin E 1.000 0.934 0.891 0.686 0.620 0.598

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