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Single-agent CHK1 inhibition in CLL
TP53-wt OSU-CLL cells manifested extensive sub-G1 peak (Figure 3C and Online Supplementary Figure S5). In MEC-1 cells, we also recorded greatly reduced DNA syn- thesis rate and concurrently apparent apoptosis induction (cleavage of PARP protein) after MU380 treatment (Figure 3D and Online Supplementary Figure S6). Apoptosis was also detected in all other tested cell lines using western blot analysis of cleaved PARP and Caspase-3 proteins (Figure 3E). In contrast, no significant apoptosis induction was observable in non-cancerous human cell lines and pri- mary fibroblasts (Online Supplementary Figure S7). Cell death was likely a consequence of enhanced DNA damage as evidenced by gH2AX accumulation in MU380-treated cells (Figure 3F).
All aforementioned results indicate that MU380 activity is not dependent on p53 status, which is further supported by absence of p53 protein accumulation after treating the p53-wt NALM-6 cell line with MU380 (Figure 3G), as well as by negligible induction of p53-downstream target genes CDKN1A (p21), PUMA, BAX, and GADD45 in this cell line; interestingly, the inhibitor further increased the expression elicited by fludarabine (Figure 3H).
MU380-mediated CHK1 inhibition affects transition of MEC-1 cells into mitosis
CHK1 protein inhibition abrogates the intra-S and G2/M cell cycle checkpoints.33,34 In p53-deficient cells lack- ing a functional G1/S checkpoint, CHK1 suppression can result in premature mitosis involving unrepaired DNA damage.34 We hence employed a TP53-mutated MEC-1 cell line, in which MU380 significantly affected the cell cycle profile, and analyzed mitoses, specifically mitotic index (MI) and integrity of mitotic chromosomes. In addi- tion to CHK1 inhibition, we also examined parallel block- ing of ATR to assess contribution of this closely co-oper- ating kinase to the studied phenotypes; in a recent study, ATR/CHK1 co-inhibition exhibited a surprising synergy in cancer cells, which was attributed to accentuated replica- tion collapse.35 For ATR depletion, we used selective inhibitor VE-821,35 which blocked ATR-mediated CHK1 phoshorylations (pS317/pS345) in MEC-1 cells (Online Supplementary Figure S8).
The results of two independent experiments are sum- marized in Table 2. After treatment with MU380 alone,
the MI sharply decreased, and a proportion of mitotic cells manifested chromosome damage in some cells resembling pulverization (Online Supplementary Figure S9). Remarkably, with cells co-treated by CHK1 and ATR inhibitors we measured a higher MI compared to the CHK1 inhibitor alone. In line with this observation, we also recorded a higher proportion of cells with chromo- some damage.
MU380 induces cell death in dividing and non-dividing primary chronic lymphocytic leukemia lymphocytes
We subsequently tested MU380 single-agent activity in primary CLL cells using vitally frozen clinical samples. Since CLL lymphocytes obtained from patient peripheral blood manifest only weak ATR/CHK1 pathway activi- ty,23,36 we initially stimulated proliferation of CLL cells using the anti-CD40/IL-4 system.24,28 This stimulation shifted a significant part of the CLL cells to post-G1 phas- es of the cell cycle, which was apparent from both the DNA content analysis and enhanced expression of prolif- eration markers, MKi67 and BIRC5 (coding survivin) (Online Supplementary Figure S10). The stimulation also results in upregulation of activated CHK1 protein as we previously reported24 and in enhanced anti-apoptotic sig- naling.37
In the stimulated CLL cells, MU380 effectively elicited RS (pS345 CHK1) and abrogated CHK1 activation (pS296) (Figure 4A). The impact on cell viability was tested in 13 stimulated CLL cultures harboring adverse genetic fea- tures including TP53 mutations, ATM mutations, and/or complex karyotype (Online Supplementary Table S2). Notably, MU380 reduced viability of these samples with different genetic background to a similar extent; the IC50 value was approximately 1 μM in all but one sample (Figure 4B). Cell death mechanism included apoptosis as evidenced by the PARP protein cleavage (Figure 4C).
Consequently, we investigated non-stimulated primary CLL cells, which manifest a low but detectable CHK1 pro- tein level (Figure 5A). Historically, the ATR pathway was considered to be inactive in quiescent lymphocytes,38 such as those from CLL patients. However, a recent study36 reported that ATR is active in primary CLL cells, and accordingly we detected a rise in pS345 CHK1 level after treatment with fludarabine (Figure 5B). We also confirmed that MU380 leads to blockade of CHK1 autophosphoryla- tion at S296 (Figure 5C). Moreover, we noticed reduction of the CDC25C protein level; however, it was not detectable in all tested samples (Online Supplementary Figure S11).
To address MU380 impact on cell viability, we tested 96 non-stimulated CLL cultures (Online Supplementary Table S3). A vast majority of these non-dividing CLL cells responded to the inhibitor (100-400 nM) by clear concen- tration-dependent viability decrease, with insignificant differences among studied genetic groups: TP53-mutated samples, mean IC50 = 337 nM; ATM-mutated samples 385 nM; 11q- deleted samples (the other ATM allele intact) 355 nM; and ATM-wt/TP53-wt samples 414 nM; healthy PBMNC cultures were virtually inert to MU380 (Figure 5D). Apoptosis induction was already apparent at 100 nM MU380 (Figure 5E). We also recorded similar response when our CLL cultures were clustered according to the presence of SF3B1 mutations, NOTCH1 mutations, IGHV status, complex karyotype presence, or their therapy sta- tus (Online Supplementary Figure S12).
Table 2. Cytogenetic analysis in MEC-1 cell line.
Experiment 1
Control
CHK1i
ATRi
CHK1i + ATRi
Experiment 2
Control
CHK1i
ATRi
CHK1i + ATRi
Mitotic Analyzed index (%) mitoses
11.5 50
3.0 50 10.8 50 5.7 50
Mitotic Analyzed index (%) mitoses
18.0 50
2.2 54
18.5 55
3.9 52
Cells with breaks
0
7 7 19
Cells with breaks
0
4
19
27
The cells were treated for 24 hours with the following agents: Experiment 1: 200 nM MU380 (CHK1i); 1 μM VE-821 (ATRi); respective combination. Experiment 2: 400 nM MU380, 2 μM VE-821; respective combination.
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