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M. Boudny et al.
the inhibitor on CHK1 protein. MU380 effectively blocked CHK1 activation (autophosphorylation pS296), while at the same time enhancing signaling from (presum- ably) ATR kinase towards the CHK1 (pS317 and pS345) reflecting RS potentiation (Figure 2A). MU380 also impacted the CHK1 downstream activity, which was demonstrated by reduced level of total CDC25A and CDC25C, one pS216 CDC25C isoform, pY15 CDK1, cyclin B1, and cyclin E1 (Figure 2B). The effect was more pronounced when MU380 was combined with gemc- itabine.
Since effective CHK1 inhibitors should sensitize cancer cells to RS inductors, we tested the potential of MU380 in combination with gemcitabine using 10 cell lines harbor- ing TP53 gene disruption and 7 TP53-wt cell lines. MU380 (100 nM) significantly potentiated gemcitabine’s efficacy in all tested cell lines (median half-maximal inhibitory concentration (IC50) = 20.5 nM for gemcitabine vs. 6.5 nM for gemcitabine + MU380) (Table 1, Figure 2C, Online
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Supplementary Table S1 and Online Supplementary Figure S2). As expected, MU380 enhanced the chemotherapy- induced DNA damage level, as evidenced by pS139 H2AX (gH2AX) accumulation (Figure 2D). Altogether, MU380 effectively inhibited CHK1 in lymphoid cancer cells.
MU380 manifests single-agent activity in both p53-wt and p53-mutated cell lines
Certain cancer cell lines including those of hematopoiet- ic origin have been shown to be sensitive to single-agent CHK1 inhibition.20,32 Along these lines, we tested MU380 alone in 10 leukemia and 9 lymphoma cell lines; an addi- tional four non-cancerous cell cultures were also tested. The cancer cell lines responded with concentration-depen- dent viability decrease, which was similar in the TP53-wt (n=8) and TP53-mutated (n=11) samples (median IC50 = 330 and 392 nM, respectively) (Table 1 and Figure 3A). Interestingly, leukemia cell lines were significantly more sensitive than lymphoma lines (median IC50 = 238 and 401
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Figure 2. MU380 is effective in lymphoid tumor cells. (A) Effects on the phosphorylation status of CHK1. The cells were treated for the indicated time with MU380 (200 nM), gemcitabine (MEC-1 cell line: 10 ng/mL; NALM-6 cell line: 5 ng/mL) or combination of the agents. (B) Blocking of CHK1 downstream targets after 24 hours (h) treatment with MU380. Gemcitabine: 10 ng/mL. (C) Synergy with gemcitabine. The combined treatment of MU380 (100 nM) with gemcitabine affected viability (measured by WST-1) of the cell lines significantly more than gemcitabine alone (P<0.001). Note: the graph does not involve the JEKO-1 cell line, in which IC50 for gemcitabine alone was not reached. ***P<0.001. (D) MU380 (200 nM; 24 h) potentiates DNA damage in MEC-1 cells treated with nucleoside analogs. Fludarabine (Flu): 5 μg/mL; gemcitabine (Gem): 5 ng/mL; cytarabine (Cyt): 100 ng/mL; CTR: untreated control.
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haematologica | 2019; 104(12)