A cell-based screen identifies ATR inhibitors with synthetic lethal properties for cancer-associated mutations

Abstract

SUMMARYOncogene activation has been shown to generate replication-born DNA damage, also known as replicative stress (RS). Notably, the ATR kinase -and not ATM-is the primary responder to RS. One limitation for the study of ATR is the lack of potent inhibitors. We here describe a cell-based screening strategy that has allowed us to identify compounds with ATR inhibitory activity in the nanomolar range. Pharmacological inhibition of ATR generates RS, leading to chromosomal breakage in the presence of conditions that stall replication forks. Moreover, ATR inhibition is particularly toxic for p53 deficient cells, this toxicity being exacerbated by RS-generating conditions such as the overexpression of cyclin E. Importantly, one of the compounds is NVP-BEZ235, a dual PI3K/mTOR inhibitor that is currently being tested for cancer chemotherapy, but which we now show is also very potent against ATM, ATR and DNA-PKcs.

Replication stress and cancer: It takes two to tango

“…It is expected that most mutations that sensitize to ATR inhibitors will be related to the metabolism of replication forks and these studies will help in the design of combined drug treatments that would benefit from the use of these compounds. In fact, our early works revealed that ATR inhibitors enhance the toxicity of Chk1 inhibitors [81]. Given that drugs never achieve a full inhibition of their targets, we think that the addition of ATR inhibitors further decreases Chk1 activity in Chk1 inhibitor-treated cells, resulting in a higher accumulation of replicative stress than with any of the individual treatments.…”

“…The rationale for targeting ATR in cancer stems from the study of mouse models with reduced ATR activity that showed a synthetic lethal effect between ATR deletion or hypomorphism and the loss of p53 [80,81] suggesting that the inhibition of ATR could be particularly useful for the treatment of p53-deficient tumours. The analysis of tissues deficient in ATR and p53 revealed that the absence of p53 further promoted the accumulation of RS in ATR mutant cells, likely due to a more promiscuous S-phase entry driven by p53 deficiency.…”

“…One limitation of this strategy is the lack of appropriate biomarkers of RS that work on clinically relevant samples. So far, pan-nuclear histone H2AX phoshorylation has been used as a surrogate marker, given that it is restricted to S-phase cells and is detected in cells that show RPA foci [10,[81][82][83]. An interesting alternative is the detection of 53BP1 bodies, large nuclear 53BP1 foci which accumulate preferentially at fragile sites in G1 cells after exposure to RS [84,85].…”

“…Selective ATR inhibitors only started to emerge in 2011 and were derived by independent strategies [81,[86][87][88]. In our case, we employed a screening based on a cellular model of "at will" ATR activation upon the addition of an inert derivative of tamoxifen [81].…”

“…In our case, we employed a screening based on a cellular model of "at will" ATR activation upon the addition of an inert derivative of tamoxifen [81]. In agreement with the previous mouse data, ATR inhibitors were particularly toxic for cells expressing Sphase promoting oncogenes such as cyclin E, specially in the context of p53-deficiency [81]. Additionally, ATR inhibition was toxic for ATM deficient cells [87], a synthetic lethal effect that had already been described with mouse models [9].…”