CHRONIC LYMPHOCYTIC LEUKEMIA CELLS ARE HIGHLY SUSCEPTIBLE TO DIRECT INHIBITION OF CHECKPOINT KINASE 1

Background

Chronic lymphocytic leukemia (CLL) is incurable disease with as yet no optimal treatment options, particularly for patients with mutations in TP53 or ATM genes. Checkpoint kinase 1 (Chk1) is involved in molecular pathways preserving genome integrity in all cell cycle checkpoints (G1/S, intra-S, G2/M) and also in non-dividing cells. SCH900776 (Schering-Plough) is highly specific Chk1 inhibitor currently tested in clinical trials for selected solid tumors and leukemia. We have prepared its potentially more metabolically stable analog OH209EN1, which possesses highly unusual N-trifluoromethyl pyrazole moiety. OH209EN1 phenocopies short interfering RNA-mediated Chk1 ablation and greatly sensitizes tumor cells to nucleoside analogs.

Aims
(1) to assess the impact of direct Chk1 inhibition on CLL cells´ viability using our innovative inhibitor, and (2) to correlate observed effects with the presence of TP53 and ATM defects.

Methods
TP53 and ATM mutations were identified by next-generation sequencing and Sanger sequencing. Deletions 17p and 11q were detected by I-FISH. Peripheral blood mononuclear cells (CLL cells´ proportion over 85%) were treated in 96-well plates using 500 000 cells/well. Cell lines were tested the same way using 50 000 cells/well. The final viability after 72h inhibitor exposure was assessed using metabolic WST-1 assay. Apoptosis (Annexin-V/PI) was measured by flow-cytometry in full blood samples in lepirudin treated with OH209EN1 for 24 h at 37^oC, 5% CO2. 

Results
Firstly we determined appropriate concentration range of OH209EN1 for viability testing using cell lines with known genetic status and healthy cells. The LC50 values after 72h treatment were following: 80 nM for GRANTA-519 (MCL, ATM mutation), 250 nM for MEC-1 (CLL/PL, p53 mutation), and 300 nM for NALM-6 (B-cell precursor leukemia, ATM/p53-wt). Negligible effect on three healthy PBMNC cultures and three non-cancerous fibroblast cell lines was noted up to 400 nM of the inhibitor. Further, Chk1 autophosphorylation on Ser-296 (Chk1 activation marker) was readily eliminated (on western blot) using 200 nM OH209EN1 in the cell line with high baseline autophosphorylation level (NALM-6). According to these results, we used concentrations 100, 200, 300 and 400 nM of OH209EN1 for the testing in CLL cells. Fifty-four CLL cultures were used and a clear concentration-dependent decrease of viability was noted in the very most of them. At the highest concentration 400 nM, the final median viability in individual genetic categories was following (listed according to sensitivity): 33% in samples with ATM inactivation (mutation and 11q-; n=15), 44% in samples with sole 11q- (n=10), 46% in samples with p53 inactivation (mutation with/without 17p-; n=13), and 62% in samples having no ATM or TP53abnormality (n=16). Interestingly, the inhibitor SCH900776 tested in parallel in 44 cultures showed substantially weaker effects with final median viability 80%. The concentration-dependent (OH209EN1, 100-400 nM) induction of apoptosis was clearly noted in two full blood samples from CLL patients, while no apparent effect was observed in one tested healthy control blood.

Summary
Although CLL cells from peripheral blood are non-dividing and exhibit rather low Chk1 expression, they manifest surprisingly high intrinsic sensitivity to specific Chk1 inhibitor. This good response concerning also ATM and TP53 mutated samples seems to be in contrast to non-cancerous cells and thus provides potential clinical utility. Supported by grants NT13519-4 and MUNI/A/1180/2014.

Keyword(s): ATM, Chronic lymphocytic leukemia, DNA damage, P53

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