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

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

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

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

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

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?

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

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

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

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.

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

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

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

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

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

Nucleoside analogues that inhibit DNA repair Fludarabine Clofarabine Nelarabine Gemcitabine

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

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

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

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

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

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

Inhibition of 4-HC-induced DNA Damage Repair by Fludarabine 1 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

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

Fludarabine and Cyclophosphamide in CLL - Response by Prior Treatment - Prior Therapy Overall Response (% CR + PR) Alkylators Fludara Alkylators Fludara Fludara & Cytoxan 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

Fludarabine Therapy of Untreated CLL The Impact of Cyclophosphamide 70 Median TTP, months Fludarabine 29 Flud & Cytox 45 MJ Keating, 2005

Structure and Actions of Oxaliplatin Cisplatin Oxaliplatin

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

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.

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 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

OFAR Therapy for Richter Syndrome Phase 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

OFAR Therapy for Refractory CLL Phase 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

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.

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