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 ’em The stakes: most challenging title for the speaker
The Player The 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?
DNA damaging agents
Alkylating agents
Platinum compounds
Topoisomerase II agents
Nucleoside analogues
Fludarabine
Nelarabine
Gemcitabine
Clofarabine (?)
Can we develop a rationales for combining agents from each class?

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

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

9. What DNA Repair Processes?
DNA adducts Direct repair (O6-methyl-G)
Base excision
Nucleotide excision
DNA crosslinks Crosslink repair
DNA breaks Non-homologous recombination
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. Cyclophosphamide-induced DNA Repair in CLL Cells
4-HC
washout
Clin Cancer Res 7:3580, 2001

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

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

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

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

17. Fludarabine DNA-directed actions RNA synthesis inhibitor
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: , 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. Clofarabine Active metabolite --triphosphate ribonucleotide reductase
DNA polymerases
Triphosphate is long lived
Active in adult & ped leukemias
Oral formulation under development
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
Analog-TP incorporation to inhibit repair synthesis
Signals for cell death

23. Inhibition of 4-HC-induced DNA Damage Repair by Fludarabine1 2 3 4 5 10 15 20
F-ara-A --> 4-HC
4-HC
F-ara-A alone
hours
mean tail moment
wash
Kawai, Blood, 2000

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

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

26. Fludarabine Therapy of Untreated CLL The Impact of Cyclophosphamide70
Median TTP, months
Fludarabine 29
Flud & Cytox 45
MJ Keating, 2005

27. Structure and Actions of Oxaliplatin
Cisplatin
Oxaliplatin

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

29. OFAR in Richter Syndrome & Refractory CLL
Fludarabine: ↑ ara-CTP accumulation 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
Fludarabine, ara-C and platinum compounds have been extensively used in the treatment of lymphoproliferative disorders.
After infusion into the blood stream, fludarabine is phosphorylated to the respective nucleoside.
Upon entering the cell it is anabolized to its triphosphate form, which is incorporated into DNA or RNA and blocks further synthesis.
Fludarabine and cytarabine act synergistically to inhibit excision repair of DNA cross-links.
Platinum compounds act by activating mechanisms that excise DNA and they are synergistic with AraC and fludarabine.
Oxaliplatin is a platinum compound with a favorable toxicity profile.

30. Oxaliplatin 22.5 mg/m2 days 1-4 Fludarabine 30 mg/m2 days 2, 3
Oxaliplatin, Fludarabine, Ara-C, Rituximab Therapy of Richter’s Transformation, PLL, & Fludarabine-Refractory CLL (OFAR)
Oxaliplatin 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 Syndrome Phase I Trial
% response
% response
Oxaliplatin N CR PR Or
(mg/m2/day)
overall
Wierda, Tsimberidou, Keating

32. OFAR Therapy for Refractory CLL Phase I Trial
% response
% response
Oxaliplatin N CR PR Or
(mg/m2/day)
overall
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. Special Thanks!
Michael J. Keating, M.D.
Varsha Gandhi, Ph.D.