PF361 ATR-CHK1 SIGNALING IS ACTIVE AND TARGETABLE IN QUIESCENT CLL CELLS
Authors: M. Boudny, P. Khirsariya, M. Borský, Y. Brychtová, K. Paruch, M. Trbušek
Journal: HemaSphere (2019)

Background: Introduction of small-molecule inhibitors of B-cell receptor signaling and BCL2 protein significantly improved therapeutic options in chronic lymphocytic leukemia (CLL). However, some patients suffer from adverse effects mandating treatment discontinuation, and those with TP53 defects more frequently experience early progression of the disease. Development of alternative therapeutic approaches is therefore of critical importance. ATR-CHK1, an essential pathway for cell survival, is a key component of DNA damage response representing a fundamental anti-cancer barrier. Inhibition of CHK1 showed interesting pre-clinical single-agent activity against diverse tumors. CHEK1 was found to be an essential gene for 557 out of 558 cancer cell lines using CRISPR elimination, according to the DepMap database (depmap.org). Concerning CLL, recent work showed that ATR is expressed in non-dividing CLL cells and can be activated after DNA damage. Therefore, we focused on analysis of CHK1, an ATR downstream target, in primary CLL cells. Aims: (a) To analyze ATR-CHK1 signaling in primary CLL cells. (b) To evaluate CHK1 as a therapeutic target in CLL cells using RNA interference and specific CHK1 inhibitors. Methods: Genetic aberrations in CLL cells were assessed by FISH and NGS.Cell viability was measured by WST-1 (Roche), or flow cytometry using DiOC6 (Thermo Fisher Scientific) and propidium iodide (Sigma-Aldrich). Levels of proteins were analyzed by western blot. The cells were transfected by electroporation using Neon Transfection System (Thermo Fisher Scientific). Results: We detected a low baseline CHK1 protein level in all primary CLL samples. Moreover, we detected CHK1 phosphorylation at S345 after fludarabine treatment suggesting that CHK1 plays a role after DNA damage in non-dividing CLL cells. Importantly, we observed viability decrease in CLL cells after transfection with siRNA targeting CHEK1, confirming an important role of CHK1 in CLL cells. Next, we tested three CHK1 specific inhibitors in CLL cells: SCH900776, CHIR-124 and our novel compound MU380. CLL cells responded to all three inhibitors by concentration-dependent viability decrease regardless of TP53 mutational status. MU380 and CHIR-124 showed best response with mean IC50 = 392 nM and 385 nM, respectively. SCH900776 was less active with IC50 = 1944 nM. Finally, we analyzed the effect of MU380 on CLL cells. MU380 led to blockade of CHK1 autophosphorylation at S296, and to total CHK1 level reduction in most samples (17 out of 23 tested samples). Moreover, we noticed reduction of the CDC25C protein level; however, it was not detectable in all tested samples. The mechanism of cell death caused by the MU380 treatment included apoptosis (PARP protein cleavage was detected in 19/22 tested samples), which was probably a consequence of DNA damage accumulation (γH2AX rise in 8/10 samples). Apoptosis induction was already apparent at 100 nM concentration of MU380. Concerning the impact of MU380 on apoptosis-associated proteins, we noted frequent decrease of MCL1 (7/9 samples) and NF-κB (7/12 samples), whilst there was no change in the level of BCL2 (12 samples tested). MU380 also reduced the MYC protein level in 4/6 samples. Summary/Conclusion: Non-dividing primary CLL cells express CHK1 protein and are able to phosphorylate CHK1 upon DNA damage. CHK1 inhibition may represent an interesting therapeutic option for high-risk CLL. Supported byprojects MH CR AZV 15–33999A, FNBr 65269705 and MUNI/A/1105/2018.