SummaryActivated Ras GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small-molecules, such as SCH51344, but their molecular mechanism of action remains generally enigmatic. Here, using a chemical proteomic approach we identify the target of SCH51344 as the human mutT homologue MTH1, a nucleotide pool sanitising enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells whereas MTH1 overexpression mitigated sensitivity toward SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys, and MTH1 co-crystal structures of both enantiomers provided a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation and small molecule inhibitors of MTH1 in general as a promising novel class of anti-cancer agents.

Genotoxic chemotherapy is the most common cancer treatment strategy. However, its untargeted generic DNA-damaging nature and associated systemic cytotoxicity greatly limit the therapeutic applications. Here, we employed a haploid genetic screen in human cells to discover an absolute dependency of the clinically evaluated anti-cancer compound YM155 on SLC35F2, an uncharacterized member of the solute carrier protein family that is highly expressed in a variety of human cancers. YM155 generated DNA damage through intercalation, which was contingent on the expression of SLC35F2 and its drug importing activity. SLC35F2 expression and YM155 sensitivity correlated across a panel of cancer cell lines and targeted genome editing verified SLC35F2 as the main determinant of YM155-mediated DNA damage toxicity in vitro and in vivo.These findings suggest a novel route to targeted DNA damage by exploiting tumor and patientspecific import of YM155.

SummaryExploitation of embryonic stem cells (ESC) for therapeutic use and biomedical applications is severely hampered by the risk of teratocarcinoma formation. Here, we performed a screen of selected epi-modulating compounds and demonstrate that a transient exposure of mouse ESC to MS-275 (Entinostat), a class I histone deacetylase inhibitor (HDAC), modulates differentiation and prevents teratocarcinoma formation. Morphological and molecular data indicate that MS-275-primed ESCs are committed towards neural differentiation, which is supported by transcriptome analyses. Interestingly, in vitro withdrawal of MS-275 reverses the primed cells to the pluripotent state. In vivo, MS275-primed ES cells injected into recipient mice give only rise to benign teratomas but not teratocarcinomas with prevalence of neural-derived structures. In agreement, MS-275-primed ESC are unable to colonize blastocysts. These findings provide evidence that a transient alteration of acetylation alters the ESC fate.