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M. Boudny et al.
mice) compared to control animals (n = 8 mice) on average by approximately 44% (Figure 6B and C). In experiment II, we administered ten doses of MU380 between days +14 and +25 post-transplant (inhibitor group n = 15 mice; control group n = 8 mice), which again resulted in pro- nounced tumor growth suppression on average by ~ 61% in this case (Figure 6D).
The tumor cells from mice treated with MU380 exhib- ited a significantly increased RS level as evidenced by accumulation of gH2AX (Figure 6E and F) and pS345 CHK1 (Figure 6F), while also manifesting moderately increased apoptosis (Figure 6E).
No apparent adverse effects were observed in either experiment.
Discussion
Chronic lymphocytic leukemia has long been consid- ered a disease caused by gradual accumulation of malig- nant B lymphocytes with disabled apoptosis induction. However, this static view changed dramatically when Messmer et al.42 reported data on CLL cell kinetics in vivo, revealing that malignant cell turnover is much higher than appreciated, and may reach over 1% of a total clone per day. Lymph nodes are the primary site of CLL cell prolif- eration in vivo;43,44 approximately one-fourth of leukemic cells in this compartment possess proliferative potential.45 Beside a simple cell renewal, proliferation is a prerequisite for clonal selection of new genetic variants, and this phe- nomenon is currently well-documented in CLL.46 Furthermore, CLL genome is characterized by deregulat- ed expression of genes involved in DNA replication, repair and recombination.47 Taking these observations into account, we surmised that targeting dividing leukemic cells may represent a therapeutic strategy in CLL and therefore hypothesized that CHK1 kinase, which is essential for DNA replication and recombina- tion-based repair, can be a suitable target.
We initially focused on obtaining sufficient quantities of the metabolically robust CHK1 inhibitor MU380. Although structurally related clinical candidate SCH900776 can certainly be considered one of the most specific CHK1 inhibitors with excellent selectivity for CHK1 over CHK2 or cyclin-dependent kinases,24,25 its metabolic profile may not be optimal. Specifically, SCH900776 contains the N-methylpyrazole motif, which undergoes oxidative demethylation resulting in the for- mation of significantly less selective metabolite and a rapid decrease of active concentration in plasma.26 In con- trast, MU380 contains highly unusual N-trifluo- romethylpyrazole pharmacophore, which provides sub- stantially better metabolic robustness and pharmacoki- netic profile.26
The newly developed enantioselective synthesis of MU380 described here provides access to gram quantities of enantiomerically pure substance, which enables thor- ough in vivo testing of the compound.
In our in vivo experiments with xenotransplanted MEC- 1 cells, MU380 elicited strong and reproducible tumor growth suppression that was accompanied by an ade- quate molecular phenotype, namely the RS accumulation. Although the induction of apoptosis was rather modest, encouraging in vivo activity of MU380 opens up further opportunities to test more intense administration of the
compound and/or its combination with additional appro- priate agents.
MU380 exhibited interesting single-agent activity in tested leukemia and lymphoma cell lines that responded via viability decrease with IC50 values between 142 and 500 nM. By virtue of this relatively uniform good reac- tion, we were not able to find determinants that would further stratify the response, except that leukemia cell lines were more sensitive than lymphoma ones. Although we hypothesized that a distinct RS level could justify this observation, baseline CHK1 phosphorylations and gH2AX, standard markers of RS, did not correlate with the leukemia/lymphoma status.
Throughout our study, we focused on MU380 effects in TP53-mutated lymphoid cells. Hypothetically, CHK1 inhibition should be effective in a p53-deficient back- ground due to dysfunction of all major cell cycle check- points and consequently complete impairment of cell cycle control potentially resulting in mitotic catastrophe. Nevertheless, our study indicates that this concept of “inducing death by releasing the breaks”34 may not be completely straightforward. Initially, certain p53 mutated cells surprisingly manifest G1-phase accumulation upon CHK1 inhibition, which we consistently observed, for example, in SU-DHL-4 cell line. Moreover, even the cells responding to CHK1 inhibition by more forthcoming S- phase accumulation and G2/M phase decrease are proba- bly equipped with relevant mitotic entry control. This was apparent from the co-inhibition of ATR with CHK1, which resulted in increased MI and chromosome damage compared to the sole CHK1 inhibition. In this respect, recently recorded synergy between CHK1 and ATR inhi- bition may not only be a consequence of more pro- nounced replication collapse,35 but of increased mitotic damage as well.
Beside the proliferative fraction, the CLL cell popula- tion also consists of non-dividing cells arrested either at the G0 (quiescent cells) or G1 phase of the cell cycle.45 Intuitively, such cells might not respond to CHK1 inhibi- tion due to low ATR23,38 and CHK1 levels.11,48 Nevertheless, a recent study49 reported apoptosis induction in non- dividing CLL cells caused by treatment with a dual CHK1/CHK2 inhibitor AZD7762. Moreover, we found that CHEK1 is targetable in CLL cells using siRNA trans- fection. Another recent work by Beyaert et al.36 concluded that ATR, despite its low level, is active in quiescent CLL cells and phosphorylates downstream targets upon DNA damage induction. Here, to our knowledge for the first time, we document that non-stimulated CLL cells also phosphorylate CHK1 upon DNA damage, despite the fact that their CHK1 level is low. Notably, we also observed significant MU380 single-agent activity in non-dividing CLL cells. Although the set of samples was enriched by those with therapeutically unfavorable genetics, only a few were resistant to our inhibitor. In fact, only 5 of 96 samples showed viability ≥ 80% after 72 h treatment. Interestingly, 3 of 5 samples harbored complete ATM inactivation (2 others were ATM-wt/TP53-wt). Thus, although the ATM-mutated samples on average did not manifest resistance, some of them were particularly refractory. It is intriguing that non-cancerous cells with ATM inactivation (fibroblasts from ataxia-telangiectasia patient) also manifested strong resistance to MU380.
Overall, our results support the concept that CHK1 is a critical protein for B-cell lymphomagenesis and that even
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haematologica | 2019; 104(12)