Activity of the clinical-stage CK2-specific inhibitor CX against chronic lymphocytic leukemia

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1 Activity of the clinical-stage CK2-specific inhibitor CX-4945 against chronic lymphocytic leukemia Leila R. Martins, Paulo Lúcio, Alice Melão, Inês Antunes, Bruno A. Cardoso, Ryan Stansfield, Maria Teresa Sabrina Bertilacio, Paolo Ghia, Denis Drygin, Maria G. Silva, João T. Barata SUPPLEMENTAL DATA Supplementary Materials and Methods Supplementary Tables 1-2 Supplementary Figures 1-5 Supplementary References 1

2 Supplementary Materials & methods Patient samples and tumor cell isolation. CLL patients not previously treated (n = 12) were diagnosed according to the National Cancer Institute-sponsored Working Group (NCI-WG) updated guidelines 1. Peripheral blood (PB) samples from CLL patients were collected in accordance with the Declaration of Helsinki after informed consent and ethical approval of Instituto Português de Oncologia (Lisbon, Portugal). Table 1 summarizes the clinical and biological characteristics of the patients analyzed. CLL cells were isolated from PB using RosetteSep human B cell enrichment cocktail (StemCell Technologies) as indicated by the manufacturer. The purity of CLL cells was always higher than 90%, as evaluated by staining with anti-cd5, CD3 and CD19 fluorochrome-conjugated antibodies (ebioscience) followed by flow cytometry analysis using a FACSCanto cytometer (Becton Dickinson). Phenotypic and genetic features of CLL patients. Flow cytometry analysis was performed as described 2. Neoplastic B cells were phenotypically identified by the co-expression of CD19 and CD5, and light chain restriction. CD38 expression was determined in the neoplastic population using anti-cd38 conjugated either with PE or APC-H7 (Becton Dickinson) and patients were considered as CD38-positive when the percentage of CD38 + cells exceeded 30%. Cytogenetic abnormalities were assessed by fluorescent in situ hybridization using probes for del13q14 (LSI D13S319), tris12 (CEP12), del11q22 (LSI ATM) and del17p13 (LSI p53). Cell culture. Primary CLL cells were cultured as 2 x 10 6 cells/ml in RPMI-1640 culture medium supplemented with 2mM L-glutamine, 100 U/mL penicillin, 0.1mg/mL streptomycin (hereafter referred to as glut-pen/strep) and 1% heat-inactivated FBS (Invitrogen). In some experiments, CLL cells were cocultured with the murine stromal cell line OP9, OP9 cells expressing delta-like 1 or the human bone marrow stromal cell line HS5. Stromal cells were seeded in a 48-well plate as 5 x 10 4 cells/well in DMEM supplemented with 25mM glucose, 1mM sodium pyruvate, glut-pen/strep and 10% heat-inactivated FBS. After overnight culture, the confluence of the stromal layer was assessed, the culture medium was removed, and 3 x 10 5 CLL cells were added to each well, under the culture conditions described above. MEC1, MEC2 and MO1043 cell lines were cultured as 5 x 10 5 cells/ml in RPMI supplemented with glut-pen/strep and 10% heat-inactivated FBS. JVM-3 and WaC3CD5 cell lines were cultured 2

3 as 2 x 10 5 cells/ml in RPMI supplemented with glut-pen/strep and 10% heat-inactivated FBS. Where specified, cells were treated with the indicated concentrations of CX-4945 (Cylene Pharmaceuticals). Immunoblot. Cells were lysed and immunoblotting was performed as described 2, using the following antibodies: actin, PKC (Santa Cruz Biotechnology), P-Akt (S129) (ABGent), P- Akt (S473), Akt, P-PTEN (S380), PTEN, P-PKCβ (S660) and P-PKCδ (T505) (Cell Signaling Technology). Analysis of cell viability. Cell viability was determined by double staining with APCconjugated annexin V (ebioscience) and 7-AAD (Becton Dickinson) followed by analysis on a FACSCanto flow cytometer (Becton Dickinson), as previously described 2. Briefly, cells were washed with PBS and resuspended in 100μL of binding buffer with annexin V and 7- AAD. After 15 min of incubation at room temperature in the dark, 100μL of binding buffer were added and the samples were analyzed by flow cytometry. Fully viable cells were identified as the annexin V and 7-AAD double-negative population. Cytotoxicity determination by Alamar blue assay. Cell lines were seeded in flat bottom 96- wells plates under the culture conditions described above and indicated concentrations of CX were added. Following 4 days of incubation with CX-4945, Alamar Blue (Life Technologies) was added to cell cultures in an amount equal to 10% of the culture volume. Cells were further incubated at 37ºC, 5% CO2 for 2-4 hours. Fluorescence was measured at wavelengths of 530nm excitation and 580nm emission in an Infinite M200 plate reader (Tecan). Synergism analysis. CLL cells were seeded in U-bottom 96-wells plates under the culture conditions described above. Serial dilutions of fludarabine (2-Fluoroadenine-9-β-Darabinofuranoside; Sigma) were added immediately after cell seeding and serial dilutions of CX-4945 at a constant combination ratio were added 16h later. CLL cells were then cultured for 8h in the presence of both drugs, washed and cultured in complete medium for additional 24h and 72h, respectively. A fludarabine:cx-4945 ratio of 1:10 and 2:1 was used for primary CLL cells and CLL cell lines, respectively. Single drug analysis was performed in parallel using the same concentrations and incubation times as in the combination study. Alamar Blue assay was done as described above. Combination index (CI) and dose reduction index (DRI) 3

4 values, and consequent classifications, were determined using CalcuSyn software (Biosoft). The combination index (CI) was calculated using the multiple drug-effect equation of Chou- Talalay 3, in which the value 1 indicates an additive effect; <1 indicates a synergistic effect; and >1 reveals antagonism. The dose-reduction index (DRI) 4 is a measure of how much the dose of each drug in a synergistic combination may be reduced at a given effect level compared to the doses for each drug alone. Mice. Mice were housed and bred in a specific pathogen-free animal facility, treated in accordance with the European Union guidelines and approval of the Institutional Ethical Committee of Instituto de Medicina Molecular. Eight week old Swiss nude mice, full-body irradiated with 600cGy on the previous day 5, were subcutaneously injected in the right flank with 5 x 10 6 MO1043 cells resuspended in 100µL of 50% Matrigel (Becton Dickinson) in PBS. At day 3, all mice presented palpable tumors with mm 3 and were randomly assigned into different groups to receive 75mg/Kg CX-4945 (po, bid) and/or 34mg/Kg fludarabine (ip, 5 treatment days and 2 days of rest per week, Teva Pharma). Mice were monitored every day and weighed frequently to determine treatment-induced toxicity. Tumors were measured every other day with a caliper and tumor volume was calculated using the formula: volume = length x width 2 / 2. Mice were sacrificed when the tumor reached 1500mm 3. In vivo pharmacokinetics. A single dose of CX-4945 solution was given by gavage to Swiss Nude (75, 100, 150, 200, 300 mg/kg) and C57B6 (75, 150 mg/kg) mice. Blood was collected by facial vein puncture at 30 min, 1h and 4h following drug administration. Samples were centrifuged and plasma was collected and stored at -80ºC until analysis. Samples were analyzed by liquid chromatography-mass spectrometry (LC-MS) techniques 6. Statistical analysis. Statistical analyses were performed using GraphPad Prism version 5.00 for Windows (GraphPad Software). Statistical differences between mean values were evaluated using 2-tailed Student s t test (paired or unpaired, as appropriate). Relations between variables were assessed by Pearson correlation. Differences in tumor development over the time were determined by a two-way ANOVA test. Differences and correlations were considered significant for P values less than

5 Supplementary Table 1. Clinical and biological characteristics of the patients analyzed. CLL # Age (years) Gender CD38 status Lymphocyte doubling time (months) CD19+/CD5+ cells (%) Clinical stage (Binet) β2m (mg/l) Time without treatment (months) Cytogenetics 1 87 M Negative >12 50 A 3 33 del13q 2 75 F N/A <12 61 B Normal 3 59 M Positive <12 87 A N/A 13 del13q 4 84 M Positive <12 96 C del13q 5 79 M Negative >12 93 A del13q 6 95 M Negative >12 46 A del13q 7 62 M Positive <12 92 A Normal 8 78 M Positive >12 56 A del11q 9 85 M Negative <12 69 C tri12, del13q M Negative >12 65 B del11q, del13q M Negative >12 24 N/A N/A 31 del13q F Negative A del 17p, del11q, del13q 5

6 Supplementary Table 2. Combination index and dose reduction index at EC75 for fludarabine/cx-4945 combination. CI DRI JVM Nearly additive 1.63 MEC Cell lines Moderate synergism 1.57 MO Moderate synergism 2.59 # Synergism 5.37 # Primary CLL Synergism 3.31 # Synergism 2.43 # Strong Synergism 3.83 CI: combination index. DRI: dose-reduction index. The upper and lower rows of the DRI refer to fludarabine and CX-4945, respectively. Similar results were obtained for EC50 and EC90 (not shown). 6

7 Supplementary Figure 1 Figure S1. CLL cell lines are sensitive to the CK2 inhibitor CX (A) Cell viability of MO1043 cell line was assessed at 48h 72h by flow cytometry analysis after annexin V/7- AAD staining. (B) Percentage of MO1043 cells in S-phase was determined by FACS analysis of BrdU incorporation at 48h. (C) CLL cell lines were cultured with increasing concentrations of CX IC50 was determined for each cell line at 96h with an Alamar blue assay. Results indicate mean ± SD and are representative of 3 independent experiments. 7

8 Supplementary Figure 2 Figure S2. Viability upon CX-4945 treatment in CLL primary samples with different cytogenetic profiles. CLL cells were incubated with the indicated concentrations of CX-4945 for 8h, washed and cultured for additional 40h before viability analysis by FACS. Results are shown as mean of duplicates or triplicates for each concentration. 8

9 Supplementary Figure 3 Figure S3. Sensitivity of primary CLL cells to CX-4945 correlates with higher tumor burden, higher proliferation rate and advanced stage disease. (A) Correlation between percent CLL cells in the PB and viability index of CLL cells cultured with 20μM CX-4945 for 48h (n = 11). (B,C) Viability index of CLL cells, after culture with 20μM CX-4945 for 48h, from patients characterized by: (B) clinical stage A (n = 6) or stage B and C (n = 4), and (C) < 3.5mg/L (n = 4) or > 3.5mg/L (n = 5) β2 microglobulin. Viability was assessed by annexin V/7-AAD staining and flow cytometry analysis. 9

10 Supplementary Figure 4 Figure S4. Oral bioavailability of CX-4945 in Swiss nude mice. Plasma concentration of CX-4945 after single dose administration by gavage was measured at 0.5, 1 and 4h. Four to five mice were used to determine the mean ± SEM for each dosage. 10

11 Supplementary Figure 5 Figure S5. Treatment with the CK2 inhibitor CX-4945 does not induce major toxic effects in a subcutaneous CLL mouse model. Previously irradiated Swiss nude mice were injected subcutaneous in the right flank with 5 x 10 6 MO1043 cells. When tumors reached 150mm 3 (day 3) mice were randomly distributed into 4 groups (n=6 per group): control (vehicle alone), CX-4945 (75mg/Kg, bid, po), fludarabine (34mg/Kg, ip, 5 days + 2 days rest, every week) and CX Fludarabine (same schedules combined). Mice were weighed at the indicated time points. No statistically significant differences were found between groups as determined by two-way ANOVA. 11

12 Supplementary References 1. Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Dohner H, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood 2008 Jun 15; 111(12): Martins LR, Lucio P, Silva MC, Anderes KL, Gameiro P, Silva MG, et al. Targeting CK2 overexpression and hyperactivation as a novel therapeutic tool in chronic lymphocytic leukemia. Blood 2010 Oct 14; 116(15): Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984; 22: Tallarida RJ. Drug synergism: its detection and applications. The Journal of pharmacology and experimental therapeutics 2001 Sep; 298(3): Loisel S, Ster KL, Quintin-Roue I, Pers JO, Bordron A, Youinou P, et al. Establishment of a novel human B-CLL-like xenograft model in nude mouse. Leuk Res 2005 Nov; 29(11): Pierre F, Chua PC, O'Brien SE, Siddiqui-Jain A, Bourbon P, Haddach M, et al. Discovery and SAR of 5-(3-chlorophenylamino)benzo[c][2,6]naphthyridine-8- carboxylic acid (CX-4945), the first clinical stage inhibitor of protein kinase CK2 for the treatment of cancer. J Med Chem 2011 Jan 27; 54(2):