Two papers have recently been published by Petrocchi et al in Bioorg Med Chem Lett and Kettle et. al., in J Med Chem describing compounds which are potent and selective MTH1 inhibitors, the compounds bind to the MTH1 enzyme in cells but do not kill cancer cells. Both papers provide interesting observations and novel insights into MTH1 biology. Importantly, both studies validate that our published compounds are potent MTH1 inhibitors and demonstrate that our compounds effectively kill broad panels of cancer cells. There is nothing in their reports disputing our compounds have interesting anti-cancer properties. In the Kettle paper, they demonstrate that siRNA depletion of MTH1 is ineffective in killing cancer cells. This is in line with our published results where we demonstrated large variability in the efficiency of siRNA in killing cancer cells, and the inability of MTH1 siRNA to kill certain cancer cells. Our interpretation was that even low levels of remaining MTH1 protein were sufficient to maintain cell survival. Overall, there is no experiment performed in either the Petrocchi or Kettle papers that are not in line with our findings published in Gad et al 2014 Nature, except one experiment, Fig 9a in the Kettle paper. We reported that MTH1 siRNA#3 in U2OS cells triggered the DNA damage response, exemplified by phosphorylation of ATM and p53. In the Kettle report, they could not see any induction of phosphorylation of ATM or p53 on Western Blot using the same MTH1 siRNA#3 in U2OS cells. However, Kettle et al show a phosphorylation of ATM and p53 in all conditions tested (even using non-targeting siRNA’s), hence the DNA damage response seems constitutively activated in the U2OS cells used. One additional experiment performed by Kettle and co-workers which is concerning to us is their finding that MTH1-/-/- CRISPR-Cas9 cells survive and that these are more or less equally sensitive to the MTH1 inhibitors tested. This certainly warrants further in-depth study and the AstraZeneca team has generously shared their MTH1-/-/- clone, which we will investigate in detail.
In our paper we demonstrate that our compound inhibits MTH1 activity and elevates 8-oxo-dG incorporation into DNA, triggering DNA responses. We cannot (and do not) exclude other, as yet unknown, interactions which could act synergistically to produce the impressive cell-killing effects we observe with our compounds. Similarly, the selectivity of other reported ligands which do not kill cancer cells cannot be guaranteed. Does this completely disqualify MTH1 as a target for anti-cancer treatment? We certainly do not think so. We would like to point out that other independent laboratories have successfully used siRNA’s (e.g. Prai, Superti-Furga and Ho Jin You and Bayer AG) confirming MTH1’s role in cancer cell survival. We have overwhelming evidence that our MTH1 inhibitor TH588 kills cells through preventing MTH1-mediated sanitation of 8-oxodGTP (see Bräutigam et al., Cancer Research 2016). Our compounds exert their toxicity by increasing incorporation of oxidized nucleotides into DNA. This is consistent with inhibition of MTH1 and MutT rescue further supports a pivotal mechanistic role for MTH1 inhibition. The Kettle and Petrocchi reports serve to highlight how complex uncovering new biology can be. This demonstrates the necessity to incorporate front-line basic science teams in any successful MTH1 drug discovery program and highlights the advantage in using open innovation to effectively progress drug discovery.
The MTH1 biology is highly reminiscent of the complexities in PARP biology uncovered thus far. By analogy, in the PARP-BRCA story, siRNA approaches did not recapitulate the effects we observed with small molecule PARP inhibitors. This underscores the difficulties in using RNAi when validating completely novel targets in DNA repair. Furthermore, some PARP inhibitors do not kill BRCA defective cells at all, even though they are low nM inhibitors which engage and inhibit PARP activity in cells. Other inhibitors are extremely potent in killing BRCA defective cells while some are intermediate. The underlying reason behind this behavior was uncovered much later by Dr’s Junko Murai and Yves Pommier who found that PARP trapping correlated with killing BRCA mutated cells, and not inhibition of PARP1. More than a decade later, a biochemical understanding of the PARP trapping mechanism is still missing. This has not hindered a PARP inhibitor being approved by the FDA and EMA for treatment of BRCA mutated ovarian cancers. More recently the FDA have given it breakthrough status in castration resistant prostate cancer. We believe that open innovation was the key which enabled PARP inhibitors finding their utility as useful drugs, and this will also be the path for MTH1 inhibitors. The underlying MTH1 biology is in my view much more complex than the PARP biology, but I am convinced that by connecting scientists across the world, we can solve this complex puzzle together, in time.