Chk1 inhibition significantly potentiates activity of nucleoside analogs in TP53-mutated B-lymphoid cells

Treatment options for TP53-mutated lymphoid tumors are very limited. In experimental models, TP53-mutated lymphomas were sensitive to direct inhibition of checkpoint kinase 1 (Chk1), a pivotal regulator of replication. We initially tested the potential of the highly specific Chk1 inhibitor SCH900776 to synergize with nucleoside analogs (NAs) fludarabine, cytarabine and gemcitabine in cell lines derived from B-cell malignancies. In p53-proficient NALM-6 cells, SCH900776 added to NAs enhanced signaling towards Chk1 (pSer317/pSer345), effectively blocked Chk1 activation (Ser296 autophosphorylation), increased replication stress (p53 and γ-H2AX accumulation) and temporarily potentiated apoptosis. In p53-defective MEC-1 cell line representing adverse chronic lymphocytic leukemia (CLL), Chk1 inhibition together with NAs led to enhanced and sustained replication stress and significantly potentiated apoptosis. Altogether, among 17 tested cell lines SCH900776 sensitized four of them to all three NAs. Focusing further on MEC-1 and co-treatment of SCH900776 with fludarabine, we disclosed chromosome pulverization in cells undergoing aberrant mitoses. SCH900776 also increased the effect of fludarabine in a proportion of primary CLL samples treated with pro-proliferative stimuli, including those with TP53 disruption. Finally, we observed a fludarabine potentiation by SCH900776 in a T-cell leukemia 1 (TCL1)-driven mouse model of CLL. Collectively, we have substantiated the significant potential of Chk1 inhibition in B-lymphoid cells.


Chk1 inhibition significantly potentiates activity of nucleoside analogs in TP53-mutated B-lymphoid cells
SUPPLEMENTARY METHOD S1: SYNTHESIS OF INHIBITOR SCH900776 1

General information
All chemicals were purchased from commercial suppliers and used without further purification. Anhydrous solvents were purchased as extra dry and stored over 4Å molecular sieves. Unless stated otherwise, the reactions were carried out in oven-dried glassware under atmosphere of nitrogen.

Compound 3
Thionyl chloride (1.9 mL, 26.2 mmol, 1.20 equiv) was added dropwise to a stirred solution of Boc-protected acid 1 (5.01 g, 21.9 mmol, 1 equiv) and pyridine (4.41 mL, 54.8 mmol, 2.50 equiv) in dichloromethane (30 mL) at room temperature. The resulting solution was stirred at this temperature for 30 min. Then, 4-dimethylaminopyridine (6.95 g, 56.9 mmol, 2.60 mmol) and Meldrum´s acid (3.55 g, 24.6 mmol, 1.12 equiv) were added at room temperature and the resulting mixture was stirred for another 1 h before it was diluted with diethyl ether (200 mL). The organic phase was washed with 1M aqueous solution of hydrochloric acid (3 × 50 mL) and brine (50 mL). Then, the organic phase was dried over anhydrous magnesium sulfate, the dried solution was filtered and the filtrate was concentrated. Obtained residue was dissolved in methanol (60 mL) and it was heated to reflux and stirred for 16 h. Then, the mixture was allowed to cool down to room temperature, the solvent was evaporated and the obtained residue was passed through a short pad of silica gel (dichloromethane-ethyl acetate 10:1) to provide ketoester 2 as a yellowish oil.
The intermediate 2 (3.51 g, 12.3 mmol, 1 equiv) was dissolved in toluene (11 mL) and 3-aminopyrazole (1.02 g, 12.3 mmol, 1.00 mmol) was added in one portion at room temperature. The resulting mixture was heated to reflux and stirred for 24 h. Then, the mixture was allowed to cool down to room temperature, the solvent was evaporated and the obtained residue was purified by flash-column chromatography (gradient elution with dichloromethanemethanol 20:1 → 12:1) to provide product 3 (3.11 g, 45% from 1) as a yellowish solid. 1

Compound 4
N,N-Dimethylaniline (3.84 mL, 30.3 mmol, 3.1 equiv) was added to a stirred solution of compound 3 (3.11 g, 9.77 mmol, 1 equiv) in phosphorus oxychloride (6.90 mL) at room temperature. The resulting mixture was stirred at this temperature for 4 days. Then, excess of phosphorous oxychloride was evaporated. The obtained residue was carefully poured into saturated aqueous solution of sodium bicarbonate (200 mL) and the aqueous phase was extracted with dichloromethane (3 × 80 mL). The combined organic extracts were dried over anhydrous magnesium sulfate, the dried solution was filtered, and the filtrate was concentrated. The residue was purified by flash-column chromatography (dichloromethaneethyl acetate 8:1) to provide product 4 (2.30 g, 70%) as a yellow wax. 1

Compound 5
Compound 4 (2.30 g, 6.83 mmol, 1 equiv) was mixed with 2M solution of ammonia in propan-2-ol (14 mL) and 29% aqueous solution of ammonia (1.4 mL) at room temperature in a pressure tube. The resulting mixture was stirred at 70°C for 30 h. Then, the mixture was allowed to cool down to room temperature, the solvents were evaporated and the obtained residue was purified by flash-column chromatography (dichloromethane-methanol 10:1) to provide product 5 (1.97 g, 90%) as a white solid.

Compound 6
N,N-diisopropylethylamine (7.58 mL, 43.5 mmol, 7.0 equiv) and chloromethyl(ethyl) ether (2.02 mL, 21.7 mmol, 3.5 equiv) were added to a stirred solution of compound 5 (1.97 g, 6.21 mmol, 1 equiv) in 1,2-dichloroethane (20 mL) at room temperature. The resulting mixture was stirred at 70°C for 4 h. Then, the mixture was allowed to cool down to room temperature and it was diluted with dichloromethane (50 mL) and saturated aqueous solution of sodium bicarbonate (50 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (2 × 50 mL). The combined organic extracts were dried over anhydrous magnesium sulfate, the dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography (dichloromethane-ethyl acetate 2:1) to provide product 6 as a yellow solid (2.29 g, 85%). 1

Compound 7
N-Iodosuccinimide (778 mg, 3.46 mmol, 1.0 equiv) was added to a stirred solution of compound 6 (1.50 g, 3.46 mmol, 1 equiv) in acetonitrile (18 mL) at room temperature. The resulting mixture was stirred at this temperature for 90 min in the dark. Then, the reaction mixture was concentrated and the residue was purified by flash-column chromatography (dichloromethane-ethyl acetate 15:1) to provide product 7 as a yellow wax (1.74 g, 90%). 1

Compound 10
An aqueous solution of hydrochloric acid (3M, 15 mL) was added to a stirred solution of compounds 9 (734 mg, 1.43 mmol, 1 equiv) in ethanol (15 mL) at room temperature. The resulting solution was heated to 60 °C and stirred at this temperature for 2 h. Then, the solution was allowed to cool down to room temperature, the solvents were evaporated and the obtained residue was dissolved in dichloromethane-methanol mixture (5:1, 15 mL). To the mixture was added solid sodium carbonate (2.0 g) and the suspension was stirred at room temperature for 30 min. The mixture was then passed through a short pad of silica gel (dichloromethane-7M solution of ammonia in methanol 6:1) to provide product 10 as a white solid (365 mg, 86%). 1

Compound 11
Di-tert-butyl dicarbonate (264 mg, 1.21 mmol, 1.2 equiv) was added to a stirred solution of compound 10 (300 mg, 1.01 mmol, 1 equiv) in dichloromethane (6 mL) and triethylamine (1.2 mL) at room temperature. After stirring at this temperature for 18 h, the mixture was diluted with dichloromethane (30 mL) and saturated aqueous solution of sodium bicarbonate (40 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (2 × 30 mL). The combined organic extracts were dried over anhydrous magnesium sulfate, the dried solution was filtered, and the filtrate was concentrated. The residue was purified by flash-column chromatography (dichloromethane-methanol 20:1) to provide product 11 as a yellow solid (321 mg, 80%). 1

Compound 12
A solution of bromine (25 μL, 0.478 mmol, 1 equiv) in dichloromethane was added to a stirred solution of compound 11 (190 mg, 0.478 mmol, 1 equiv) in tertbutylamine (4 mL) and dichloromethane (2 mL) at room temperature. The resulting mixture was stirred at this temperature for 20 h. Then, the reaction mixture was concentrated in a vacuum and the residue was purified by flash-column chromatography (dichloromethane-ethyl acetate 1:1) to provide product 12 as a white solid (192 mg, 84%). 1

Racemic SCH900776
Trifluoroacetic acid (2 mL) was added to a stirred solution of compound 12 (181 mg, 0.380 mmol, 1 equiv) in dichloromethane (2 mL) at room temperature. The resulting mixture was stirred at this temperature for 1 h. Then, the solvent was evaporated and the obtained residue was dissolved in methanol (4 mL). To the mixture was added solid sodium carbonate (500 mg) and the suspension was stirred at room temperature for 30 min. The mixture was then passed through a short pad of silica gel (dichloromethane-7M solution of ammonia in methanol 15:1) to provide racemic SCH900776 as a white solid (135 mg, 94 %). 1  The enantiomers were separated using Chiralcel ® OJ™ column; flow: 20 mL/min; injection: 5 mL of racemate solution in EtOH (4 mg/mL); mobile phase: n-hexane/ethanol 80:20 + 0.5 % diethylamine. SCH900776 is the faster eluting enantiomer (retention time: 10. 04 min).

SUPPLEMENTARY METHOD S2: GATING OF LEUKEMIC CELLS IN THE FLOW CYTOMETRY ANALYSIS IN TCL1-MICE
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Oncotarget, Supplementary Materials 2016
To demonstrate the difference between wt C57BL/6 animals and adult Eμ-TCL1 C57BL/6 mice with progressed B-cell leukemia and to explain the process of leukemic B-cell quantification, we describe respective flow cytometry analysis. Peripheral blood samples were analyzed using BD Accuri C6 flow cytometer as follows: Red blood cells (RBC) were lysed by NH 4 Cl lysis buffer and the remaining white blood cells (WBC) were washed and stained according to the manufacturer´s instructions using CD5, B220 (CD45R) and CD3 primary antibodies conjugated with fluorochromes. Stained cells were washed by PBS and resuspended in the final volume of 100 μL PBS. Absolute quantification of the cell counts was achieved by calibration of the measurement by flowcytometry absolute count standard (Bangs Laboratories). Singlets were determined and then lymphocytes were gated according to their morphology (images i, iv; low FSC and SSC). T-cells were excluded using CD3 staining (ii, v) and then leukemic (B220 dim ) and normal B-cells were discriminated using B220 and CD5 staining (iii, vi). Mice with progressed leukemia typically lack the normal B-cell population, while the B220 dim CD5 + cells are dominant.