1. Targeting Survival and DNA Repair Pathways in Chronic Lymphocytic Leukemia Dr. Lawrence Panasci

2. Potential Conflict of Interest Research Grant – Luitpold Pharmaceuticals / 2007-2009 – Novartis Pharmaceuticals / 2009-

3. Raquel Aloyz PhD Dr Lawrence Panasci MD Department of Oncology & Program in Cancer Genetics McGill university Targeting Survival And DNA Repair Pathways In Chronic Lymphocytic Leukemia

4. Chronic Lymphocytic Leukemia (CLL) CLL is characterized by the accumulation in the blood of affected patients quiescent B-lymphocytes in the G0/G1 phase of the cell cycle. At an early stage of the disease, B-lymphocyte accumulation occurs likely as a consequence of an undefined defect in the apoptotic machinery rather than an increased proliferation of leukemic cells. While the patients often initially respond to conventional treatment with chlorambucil or fludarabine, they eventually become resistant to the drugs.

5. B-Cell Chronic Lymphocytic Leukemia (CLL) 1) Is a disease characterized by the proliferation of abnormal, developmentally regulated immature B lymphocytes that accumulate in the blood of affected patients 2) The nitrogen mustard (NM), chlorambucil (CLB), was commonly used as fist line therapy for CLL with an initial response rate of 60-80%, often to low dose CLB therapy, but eventually (usually after years of therapy), all CLL patients become resistant to CLB 3) Thus CLL is an excellent clinical model of NM drug resistance since:  an homogenous population of malignant B lymphocytes is easily obtainable  these malignant B lymphocytes are representative of the clinical status, i.e. in-vitro/in-vivo NM resistance and  the chronic nature of the drug treatment allows for the development of drug resistance

6. Resistance to therapy Front-line therapy for chronic lymphocytic leukemia (CLL) with alkylating agents is associated with low rates of complete remission and no improvement in overall survival. The ability of CLL cells to efficiently repair alkylator-induced damage to DNA might explain this lack of response. Novel strategies that inhibit DNA repair, such as combinations of alkylating agents, purine nucleoside analogues, and immunotherapy, have pro- duced durable clinical and molecular remission in both untreated and relapsed CLL De novo and acquired resistance to chemotherapy have been associated with alterations in apoptosis and/or DNA repair

8. DNA INTERSTRAND CROSSLINK (ICL) NER Double Strand Break DSB Homologous Recombinational Repair DNA Interstrand Crosslink Repair Chlorambucil Interstrand Crosslink Removal Non Homologous End joining Repair Non Limiting Step Cyclophosphamide

9. Copyright ©1999 American Association for Cancer Research Christodoulopoulos, G. et al. Clin Cancer Res 1999;5:2178-2184 Fig. 1

10. LINEAR REGRESSION Rad51 and Xrcc-3 Protein Level vs. LD 50 Chlorambucil In Chronic Lymphocytic Leukemia

11. summary Resistance to DNA crosslinking agents is associated with: a)Accelerated repair of interstrand crosslinks induced by these agents. b)Increased drug-induced-Rad51 foci density. c)In CLL primary lymphocytes increased protein levels of xrcc3/Rad51 and in epithelial cell lines increased levels of XPD and xrcc3. 3) Overexpression of xrcc3 results in DNA cross linking agent drug resistance.

12. Untreated B-lymphocyte CLB treated B-Lymphocytes RAD51 NUCLEAR FOCI DNA INTERSTRAND CROSSLINK (ICL) NER Double Strand Break DSB Homologous Recombinational Repair c-Abl BRCA1 Sensitization to ICL-inducing agents Decreased Rad51 Foci

13. c-Abl ATM RAD51 DNA DAMAGE Activation Tyr 315 RAD52 RAD51 c-Abl RAD51 BRCA1

14. 1-c-Abl positively regulates Rad51-related Homologous Recombinational Repair 2-Homologous recombinational Repair is implicated in CLB drug sensitivity in CLL We investigate the effect of the c-abl inhibitor Imatinib/STI571 in CLB cytotoxicity in CLL lymphocytes

15. STI571 REGULATED Tyr 412 Tyr 245 ACTIVE

16. IC 50 CLB + [ STI571] IC 50 STI571 = 0.46 I = Determination of Drug Synergy Using the MTT Assay I 1 Antagonism IC 50

17. STI571(Imatinib) Sensitizes CLL cells to CLB (Chlorambucil) Independently of the clinical status

18. Summary of the mechanisms of action of Gleevec alone or in combination with Chlorambucil in malignant CLL- lymphocytes Death Survival

19.  A phase I-II Clinical Trial is in process to assess the effect of Gleevec in combination with CLB

20. A phase I-II trial of Gleevec (imatinib mesylate) in combination with chlorambucil in previously treated chronic lymphocytic leukemia (CLL) patients Study Protocol Sponsor: Novartis Pharmaceuticals Group/Participating Institutions: Jewish General Hospital Hopital Notre-Dame, CHUM Hopital Charles Lemoyne Investigators: Jonathan Hebb, MD, MSc Sarit Assouline, MD Lawrence Panasci, MD Pierre DesJardins, MD Stephen Caplan, MD Raquel Aloyz, PhD

21. Rationale There is a synergistic effect of imatinib on CLB mediated cytotoxicity in CLL cells in vitro. This effect occurred at concentrations of imatinib (<10mm) that are clinically achievable. Drug sensitivity in CLL lymphocytes is determined in part by the repair capacity of nitrogen mustard-induced DNA interstrand cross links (ICLs). The regulation of this repair mechanism has been associated with a c-abl mediated phosphorylation of Rad51 which is involved in repair of CLB – induced ICLs. Imatinib inhibits c-abl activity; imatinib may sensitize CLL cells to CLB through inhibition of c-abl mediated DNA-repair pathways. Imatinib in vitro inhibits c-abl, with a resultant decrease in c-abl mediated Rad51 phosphorylation in CLB-treated CLL lymphocytes. Encouraging results from these in vitro studies provide a basis for initiating a phase I/II clinical study.

22. STUDY OBJECTIVES Primary Objectives To determine the maximum tolerated dose of Gleevec in combination with chlorambucil(CLB). To determine the toxicities of Gleevec in combination with CLB. Secondary Objectives To determine the efficacy of Gleevec at the MTD in combination with CLB. To determine the peak/steady state plasma concentration of Gleevec at each level, but mainly at the MTD. To determine the amount of Gleevec sensitization of CLB in-vitro in pretreatment lymphocytes and correlate these results with in-vivo anti-tumor activity. To determine the duration of response in patients who respond to Gleevec and CLB at the MTD.

23. SAMPLE SIZE Up to 18 patients will be enrolled in the phase I portion of this study, with three patients tested at each dose level of Gleevec. Cohorts will be expanded to 6 patients if there is one dose limiting toxicity in the first three patients enrolled in a given cohort. Once the maximum tolerated dose has been determined, a total of 16 patients will be enrolled in the phase II component of the study.

24. PATIENT POPULATION Patients with CLL in whom treatment is clinically indicated and who have been previously treated with one or more of the following regimens: CLB, with a progression free survival of at least 6 months. Fludarabine or any fludarabine containing regimen. Any other treatment regimen including monoclonal antibodies, corticosteroids, immunotherapies, or radiation.

25. Patient Eligibility Patients with B-cell chronic lymphocytic leukemia (a) Rai Stage 0-II with indication for treatment by NCI Working Group Criteria; or (b) Rai Stage III or IV. The diagnosis of CLL must be pathologically verified according to the WHO classification of hematological malignancies. Received a minimum of one prior chemotherapy regimen. Additionally, prior treatment with corticosteroids, immunotherapies, monoclonal antibodies or radiation therapy is permitted. WBC count of > 25 x 109/L.

26. DOSAGE REGIMEN Gleevec at three dose levels: 300mg, 400mg, or 600mg daily for 10 days (from day 1 to day 10), and a total fixed dose of CLB 8 mg/m2 daily x 5 days (day 3 to day 7) will be administered. The treatment will be administered every 28 days. Patients may receive up to 6 cycles of therapy. Dose LevelGleevecCLBNo of patients -1300mg6mg/m23* 1300mg8mg/m23* 2400mg8mg/m23* 3600mg8mg/m23*

27. Lymphocyte counts for CLL patients on protocol GL-CLB-001 (treatment started at Week 0) 0 20 40 60 80 100 120 123456781216202436 Lymphocyte counts (x10^9/L) 03-01 03-02 03-03 0 50 100 150 200 250 300 123456781216202436 0 50 100 150 200 250 300 350 123456781216202436 01-03 03-05 03-06 01-04 01-05 Dose Level 1 (300 mg Gleevec) 03-04 01-02 Dose Level 2 (400 mg Gleevec) Dose Level 3 (600 mg Gleevec) Weeks

29. DNA INTERSTRAND CROSSLINK (ICL) NER Double Strand Break DSB Homologous Recombinational Repair DNA Interstrand Crosslink Repair Chlorambucil Interstrand Crosslink Removal Non Homologous End joining Repair Non Limiting Step Cyclophosphamide

30. Non Homologous End Joining (NHEJ) Pathway DNA damage recognition and processing: H2X2 Ku70/80 DNA-PKcs Artemis Ligation: Ku70/80 DNA-PKcs Ligase IV/xrcc4

31. LINEAR REGRESSION Ku86 Protein Levels and DNA-PK activity vs LD 50 Chlorambucil In Chronic Lymphocytic Leukemia    DNA - PK Activity (Arbitrary Units) 0 10 20 30 40 5060 70 1 2 3 4 5 6 y=7.51x+4.0 r=0.875 y = 0.024x + 0.625 0 0.5 1 1.5 2 2.5 0102030 Ku86 Levels r=0.5225 Chlorambucil IC 50 (  M) Muller C et al. Blood. 1998 Oct 1;92(7):2213-9

32. NHEJ in CLL NM drug resistance KU80 protein levels and DNA-PK activity correlate directly with CLB drug resistance in-vitro in CLL lymphocytes These results suggest that NHEJ may play a role in CLB drug resistance in CLL In order to investigate this, we utilized relatively specific inhibitors of DNA-PK

33. NU7026 Wortmannin, a nospecific DNA-PK inhibitor’ sensitizes CLL lymphocytes to chlorambucil. Wortmannin is a noncompetitive, irreversible inhibitor of DNA-PK, whereas NU7026 (2-(morpholin-4-yl)- benzo[h]chomen-4-one) is competitive inhibitor of the ATP site of DNA-PK Although Wortmannin is primarily a PI 3-K inhibitor, being 90-fold more active against PI 3-K than DNA-PK or ATM, NU7026 is more selective for DNA-PK with a 60- fold greater potency against this enzyme than PI 3-K and inactive against both ATM and ATR. Thus, in contrast to Wortmannin, NU7026 demonstrates excellent specificity for DNA-PK.

34. Effects of NU7026 and CLB on survival and DNA-PK phosphorylation

35. IC 50 CLB + [ NU7026] IC 50 Nu7026 I = I 1 Antagonism We determine the IC  of CLB or NU7026 alone and CLB in combination with 1, 5 or 10  M NU7026 in vitro in a cohort of 19 B-CLL patients (14 untreated and 5 treated patients) The IC50 (  M) range for the drugs was: CLB7.14 to 61.17 (I value: 0,4-2,0) NU702617.35 to 67.48 (non toxic (>100  M) in 50 % of patients) NU7026 sensitizes the B-CLL lymphocytes to chlorambucil in all the patients but one. The effect of 1, 5 or 10  M NU7026 on chlorambucil sensitivity was synergistic (I value 1). NU7026 (a DNA-PK inhibitor) Sensitizes Primary B-CLL Lymphocytes to CLB in Vitro Amrein L et al J Pharmacol Exp Ther. 2007 Jun;321(3):848-55.

36. Patients IC 50 (  M )CLB alone IC 50 (  M) NU7026 alone IC 50 (  M) CLB+1  M NU7026 Synergy Value I IC 50 (  M) CLB+5  M NU7026 Synergy Value I IC 50 (  M) CLB+10  M NU7026 Synergy Value I U116 ±4.3 17± 3.2 11 ± 3.5 (1.4) a 0.76 ±0.17 6 ±2.9 (2.6) a 0.68 ±0.10 3 ±0.4 (4.6) a 0.79 ±0.14 U227 ±3.7 > 10023 ± 1.3 (1.2) a 0.86 ±0.14 12 ±2.8 (2.2) a 0.46 ±0.21 4 ±0.3 (7.6) a 0.13 ±0.03 U317 ±3.1 > 10017 ± 6.4 (1.0) a 0.99 ±0.19 17 ±1.1 (1.0) a 1.00 ±0.12 13 ±3.6 (1.4) a 0.72 ±0.16 U447 ±5.4 41 ±0.2 42 ±4.9 (1.1) a 0.92 ±0.21 34 ±6.4 (1.4) a 0.84 ±0.20 7 ±1.3 (6.3) a 0.40 ±0.09 U525 ±2.0 65 ±4.7 29 ±2.3 (0.9) a 1.17 ±0.16 26 ±1.1 (1.0) a 1.11 ±0.18 11 ±2.4 (2.2) a 0.60 ±0.06 U624 ±6.3 67 ±2.7 11 ±3.2 (2.2) a 0.47 ±0.05 2.6 ±0.3 (9.2) a 0.18 ±0.04 2.3 ±0.8 (10.1) a 0.25 ±0.05 U752 ±2.8 48 ±5.6 29 ±7.8 (1.8) a 0.58 ±1.13 19 ±3.8 (2.7) a 0.47 ±0.07 17 ±3 (3.0) a 0.55 ±0.03 U89 ±0.8 20 ±2.0 24 ±2.3 (0.4) a 2.73 ±0.22 34 ±1.5 (0.3) a 4.05 ±0.20 16 ±4.1 (0.6) a 2.27 ±0.57 U944 ±3.5 > 10029 ±1.4 (1.5) a 0.65 ±0.05 31 ±2.8 (1.4) a 0.71 ±0.14 28 ±1.3 (1.6) a 0.63 ±0.05 U1061 ±3.7 39 ±4.8 25 ±1.3 (2.5) a 0.43 ±0.04 9.6 ±4.4 (6.4) a 0.28 ±0.02 6.8 ±1.1 (9.0) a 0.37 ±0.01 U117.1 ±2.3 20 ±1.7 5.1 ±1.0 (1.4) a 0.77 ±0.19 4.4 ±0.9 (1.6) a 0.87 ±0.04 2.9 ±0.07 (2.4) a 0.92 ±0.15 U1232 ±3.3 33 ±3.9 22 ±1.7 (1.5) a 0.72 ±0.02 13 ±1.7 (2.5) a 0.55 ±0.01 12 ±1.6 (2.7) a 0.67 ±0.01 U1331 ±3.1 > 10026 ±2.5 (1.2) a 0.83 ±0.14 27 ±0.2 (1.1) a 0.89 ±0.07 21 ±1.1 (1.4) a 0.70 ±0.11 U1412 ±2.8 > 1005.9 ±0.2 (2.1) a 0.48 ±0.12 8.8 ±1.8 (1.4) a 0.72 ±0.18 6.3 ±1.6 (1.9) a 0.52 ±0.02 T124 ±5.5 24 ±6.2 12 ±3.4 (2.0) a 0.54 ±0.15 6.9 ±1.4 (3.5) a 0.49 ±0.09 5.9 ±1.9 (4.0) a 0.66 ±0.18 T259 ±2.8 > 10049 ±5.2 (1.2) a 0.84 ±0.05 40 ±2.7 (1.5) a 0.68 ±0.03 17 ±4.5 (3.4) a 0.30 ±0.05 T332 ±2.2 > 10030 ±3.1 (1.1) a 0.95 ±0.13 22 ±3.7 (1.4) a 0.70 ±0.07 14 ±1.3 (2.2) a 0.45 ±0.01 T443 ±6.0 > 10066 ±5.8 (0.6) a 1.54 ±0.12 48 ±12 (0.9) a 1.11 ±0.37 29 ±2.3 (1.5) a 0.67 ±0.20 T516 ±0.7 > 10010 ±0.8 (1.4) a 0.71 ±0.07 6.9 ±0.4 (2.1) a 0.48 ±0.05 5.3 ±1.0 (2.7) a 0.37 ±0.07 Effect of NU7026 on CLB cytotoxicity in lymphocytes from CLL patients Using the MTT assay, we evaluated the effect of NU7026 on CLB cytotoxicity in malignant B lymphocytes from CLL patients. The I-value, I 1, indicates that the CLB and NU7026 act synergistically or antagonistically, respectively. a Ratio between CLB IC50 alone/CLB IC50 in the presence of NU7026. The results are expressed as the mean value ± s.d. Amrein L et al J Pharmacol Exp Ther. 2007 Jun;321(3):848-55.

37. Copyright ©2001 American Association for Cancer Research Panasci, L. et al. Clin Cancer Res 2001;7:454-461 Fig. 1

38. Dr Aloyz and myself would like to Acknowledge the following scientists Lilian Amrein Annette Hollmann Tiffany A Hernandez David Davidson CIHR- “ Dasatinib Cytotoxicity and sensitization to standard therapy in CLL” Operating Grant to R Aloyz & Leukemia & Lymphoma Society “Inhibition of DNA-PK to improve the efficacy of CLB in CLL”. Translational Research Grant to L Panasci James Johnston Spencer Gibson Manitoba CLL tissue bank