Why are the same drugs active in B-cell not active in T-cell lymphomas?

1. Why are the same drugs active in B-cell not active in T-cell lymphomas? William Plunkett, Ph.D. Department of Experimental Therapeutics The Univ. of Texas M. D. Anderson Cancer Center Houston, Texas

2. A Friendly Game of Bologna Hold ’emThe stakes: most challenging title for the speaker The PlayerThe Bet • Dr. O’Conner Innovative treatments • Dr. Foss New, new drugs • Dr. Cheson Other new, new drugs • Dr. Zinzani Why are the same drugs active in B-cell not active in T-cell? • Dr. Plunkett I’ll see your bet, pair of twos

3. Why are the same drugs active in B-cell not active in T-cell lymphomas? Underlying etiology of tumorigenesis Biological diversity of disease Interactions with the microenvironment Different clinical manifestations Prevalence of the diseases

4. Development of Therapeutics for T-Cell Malignancies – Plan A • Diversity of diseases reflects differences in underlying genetic defects • Search for the genetic basis of disease • Develop cancer-specific therapeutics • Validate that drug inactivates the target • Credential the target in therapeutic trials

5. Plan B – Plan A will Require Complementary Strategies • Expect that tumors will acquire resistance to Plan A • Continue to identify phenotypes of potential sensitivity to cytotoxic therapeutics • Develop mechanism-based combinations of drugs that are active targeting tumor-specific sensitivities

6. What are the Leads for T-cell Therapies? Nucleoside analogues Fludarabine Nelarabine Gemcitabine Clofarabine (?) DNA damaging agents Alkylating agents Platinum compounds Topoisomerase II agents Can we develop a rationales for combining agents from each class?

7. Actions Small alkyl adducts, crosslinks in DNA, DNA strand breaks Platinum compounds cisplatin carboplatin Platinum adducts on DNA, DNA crosslinks DNA damaging drugs doxorubicin mitoxantrone bleomycin Double strand DNA breaks DNA Damaging Agents Alkylating agents cyclophosphamide CCNU, BCNU procarbazine psoralen & UVA

8. How do Malignancies Respond to These Insults? Activation of DNA repair processes

9. DNA adducts Direct repair (O6-methyl-G) Base excision Nucleotide excision DNA crosslinks Crosslink repair DNA breaks Non-homologous recombination What DNA Repair Processes? To be successful, these processes all remove portions of damaged DNA and require re-synthesis of DNA

10. Hypothesis – Nucleoside analogues will be synergistic with DNA damaging agents Activation of excision DNA repair processes will allow incorporation of nucleotide analogs into DNA of quiescent cells. Agents administered in this fashion will act by a different mechanism of action. Tumors resistant to either agent could be sensitive to this new mechanism of action.

11. Why are combinations of DNA damaging agents and nucleoside analogues likely to be active in lymphoid malignancies? • Highly capable of excision DNA repair • Readily anabolize nucleoside analogues to triphosphates • Triphosphates of newer nucleosides are long lived in lymphoid cells • Apoptotic machinery is intact and primed

12. 4-HC washout Cyclophosphamide-induced DNA Repair in CLL Cells Clin Cancer Res 7:3580, 2001

13. 0 4 8 hr Double Strand Single Strand Removal of Oxaliplatin Interstrand Crosslinks by CLL Lymphocytes M. Moufarij et al. , Blood, Dec 2006

14. 1.4 100 1.2 Fludarabine 75 1.0 0.8 % ICL remaining 50 0.6 0.4 25 0.2 0.0 0 0 4 8 0 400 200 Hr after drug removal Oxaliplatin-induced Interstrand Cross Links in DNA of CLL CellsAction of Fludarabine Fludarabine ICL/10 kb Oxaliplatin, mM M. Moufarij et al. , Blood Dec 2006

15. Oxaliplatin-Induced Interstrand Cross Links in CLL DNAActions of Nucleoside Analogues

16. Nucleoside analogues that inhibit DNA repair Fludarabine Clofarabine Nelarabine Gemcitabine

17. Fludarabine DNA-directed actions DNA synthesis inhibitor Ribonucleotide reductase RNA synthesis inhibitor Active in B-cell diseases Orally bioavailable Sem. Oncol., 20 (Suppl. 7): 2-12, 1993

18. Demethoxylation of Nelarabine by Adenosine Deaminase Generates ara-G Lambe, Cancer Res. 55:3352-56, 1995

19. Arabinosylguanine (ara-G) Generated from nelarabine Phosphorylated by dCK and dGK Triphosphate active metabolite -inhibits DNA synthesis -long lived - PK related to response Active in T-cell lymphomas & leukemias Gandhi, Curr. Opin. Oncol., 2006

20. Active metabolite --triphosphate ribonucleotide reductase DNA polymerases Triphosphate is long lived Active in adult & ped leukemias Oral formulation under development Clofarabine Gandhi, Curr. Opin. Oncol., 2006

21. Gemcitabine Phosphorylated by dCK -Concentration-sensitive Active nucleotides are long-lived Diphosphate ---Inhibits RNR Triphosphate -Inhibits DNA synthesis Active alone in CTCL and in combinations in NHL Sem. Oncol., 23 (Suppl. 10):3-15, 1996

22. Synergistic Interaction of Nucleoside Analogs with DNA Damaging Agents Damage to DNA Excision of damaged DNA DNA re-synthesis in repair patch Analog-TP incorporation to inhibit repair synthesis Analog-TP Signals for cell death

23. 20 15 F-ara-A --> 4-HC 10 mean tail moment 5 4-HC 0 F-ara-A alone 0 1 2 3 4 hours wash Inhibition of 4-HC-induced DNA Damage Repair by Fludarabine Kawai, Blood, 2000

24. Fludarabine – Cytoxan for CLL Fludarabine 30 mg/m2 d 1, 2, 3, Q 28 days Cytoxan 250-300 mg/m2 d 1, 2, 3, Q 28 days Day 1 2 3

25. Prior Therapy Overall Response (% CR + PR) Alkylators Fludara Alkylators Fludara Fludara & Cytoxan Fludarabine and Cyclophosphamide in CLL - Response by Prior Treatment - none none 43 79 85 resistant none low 38 60 none resistant 20 low 41 resistant resistant low low 41 O’Brien, JCO, 19:2142, 2001

26. Median TTP, months Fludarabine 29 Flud & Cytox 45 Fludarabine Therapy of Untreated CLLThe Impact of Cyclophosphamide 70 MJ Keating, 2005

27. Structure and Actions of Oxaliplatin Cisplatin Oxaliplatin

28. Untreated m 2.5 M fludarabine m 5 M oxaliplatin Combination Propidium Iodide Annexin V-FITC Synergistic Killing of CLL by Oxaliplatin and Fludarabine F 100 80 60 O Apoptotic Cell Death (%) O + F 40 20 0

29. OFAR in Richter Syndrome & Refractory CLL Fludarabine:↑ ara-CTPaccumulation in leukemic cells Fludarabine & ara-C triphosphates: inhibit the resynthesis step of excision repair Platinum compounds: • Synergistic with ara-C and fludarabine • Activate excision DNA repair mechanisms Oxaliplatin • Synergistic with fludarabine in vitro • Minimal renal/auditory toxicity

30. Oxaliplatin, Fludarabine, Ara-C, Rituximab Therapy of Richter’s Transformation, PLL, & Fludarabine-Refractory CLL (OFAR) • Oxaliplatin 22.5 mg/m2 days 1-4 • Fludarabine 30 mg/m2 days 2, 3 • Ara-C 1 g/m2 days 2, 3 • Rituximab 375 mg/m2 day 3 Wierda et al., ASH abstract, 2006

31. OFAR Therapy for Richter SyndromePhase I Trial % response % response Oxaliplatin N CR PR Or (mg/m2/day) 17.5 2 0 0 0 20 5 2 2 80 25 5 1 2 60 overall 12 3 4 58 Wierda, Tsimberidou, Keating

32. OFAR Therapy for Refractory CLLPhase I Trial % response % response Oxaliplatin N CR PR Or (mg/m2/day) 17.5 0 0 0 0 20 3 0 0 0 25 19 1 5 32 overall 23 1 5 26 Wierda, Tsimberidou, Keating

33. Conclusions • Interactions between DNA-damaging agents and some nucleoside analogues create new mechanisms of action. • Inhibition of DNA repair likely activates new signaling pathways for cell death. • Combination of nelarabine, gemcitabine, and clofarabine with DNA damaging agents may offer advantages in therapy of T-cell diseases.

34. Michael J. Keating, M.D. Varsha Gandhi, Ph.D. Special Thanks!