In our lab, we generated mitotic MTH1 inhibitors TH588 and TH1579 and showed that these have anti-cancer properties, selectively killing transforming cancer cells and being well-tolerated in non-transformed cells [1,2]. Currently, TH1579 is being evaluated in several clinical trials (Eudnr 2016-00262480 and 2019-001221-27), and here we wanted to understand the molecular mechanism for the cancer selectivity of TH588 and TH1579. TH588 and TH1579 are mitotic MTH1 inhibitors and act via a dual mechanism, (i) causing mitotic arrest by disturbing microtubule polymerization [3] (likely both dependent on and independent of MTH1 [4]), which altogether increases ROS, and (ii) by inhibiting MTH1 in promoting the incorporation of 8-oxodGTP into DNA during mitotic replication [4,5]. In humans, the MTH1 enzyme is involved in protection against reactive oxygen species (ROS), where it hydrolyzes oxidized dNTPs, such as 8-oxodGTP into 8-oxodGMP, preventing the incorporation of this oxidized nucleotide into DNA [6]. Moreover, recent data have shown that the MTH1 protein binds tubulin and promotes microtubule polymerization and mitotic progression to avoid formation of oxidative DNA damage [4]. Different MTH1 inhibitors differentially affect microtubule polymerization. TH588 and TH1579 cause incorporation of oxidized dNTPs during mitosis in mitotic replication, an effect linked to MTH1 inhibition [5]. While ROS have been suggested to contribute to replication stress [7], treatment with TH588 or TH1579 does not result in incorporation of oxidized nucleotides during the S phase of the cell cycle [5]. Even more surprising is that when replacing the normal DNA polymerase δ (PolD) with an error-prone variant that also readily incorporates 8-oxodGTP, no replication stress or incorporation of oxidized nucleotides are observed in the S phase of the cell cycle [5]. In contrast, injection of 8-oxodGTP is toxic to cells when treated with TH588, TH1579 [8] or an MTH1 inhibitor that is normally not toxic [4]. Altogether, this suggests that oxidative stress levels are too low in S-phase cells to generate 8-oxodGTP, and also in cancer cells under oncogene pressure. This is further supported by ROS being generated in cells under prolonged mitotic arrest, resulting in mitophagy causing ROS [9]. A model is emerging where TH588 or TH1579 arrest cancer cells in mitosis by disrupting microtubule polymerization (likely also independently of MTH1 [3]) (Figure S1). It is unclear why TH588 and TH1579 do not perturb microtubule polymerization in non-transformed cells, which are also not arrested in mitosis [4]. The mitotic arrest results in ROS accumulation, damaging the nucleotide pool generating 8-oxodGTPs, which in the presence of TH588 or TH1579 are incorporated during mitotic DNA synthesis (MIDAS), also contributing to cell death [5] (Figure S1). Mitotic replication (MIDAS) is essentially a repair synthesis process of unrepaired replication stress S-phase lesions that when carried over to G2/M are repaired by processes such as homologous recombination [10,11]. The objective in this study is to understand the molecular reason(s) by which TH588 and TH1579 can kill cancer but not non-transformed cells. Our hypothesis is that cancer cells are sensitive to these inhibitors, owing to high levels of oncogene-induced replication stress [12,13], causing DNA lesions to persist into mitosis, resulting in chromosomal instability [14], mitotic arrest, ROS production and incorporating 8-oxodGTP in cancer cells. Supporting this theory are previous reports linking replication stress to oxidative stress, activating DNA damage response in gliomas [15].Here, we wanted to generate an isogenic system to study the effect of oncogene-induced replication stress on response to TH588 and TH1579. The single oncogene that is amongst those that most efficiently induce DNA replication fork stress is the transcription factor c-Myc [16,17], which drives cancer and is associated with poor prognosis and unfavorable survival in patients with cancer such as renal cancer, urothelial cancer and ovarian cancer [18,19]. The mechanism underlying c-Myc-induced replication stress is complex and involves both the transcriptional and non-transcriptional roles of c-Myc [16,20]. Elevated c-Myc increases the expression of Cdks (e.g., Cdk4) and cyclins (D1/D2/B) [21,22] which trigger progression through G1, likely causing replication stress. Furthermore, c-Myc represses Cdk inhibitors such as p21 and p15INK4, preventing the p53 response and allowing replication on damaged DNA [23]. dNTP levels are also increased following c-Myc expression, which likely increases replication fork speed and stress [24]. The individual contributions to generate replication stress by these individual factors are not yet established. Further to this, emerging data demonstrate that R-loop and replication collisions are key events underlying replication stress [25]. Indeed, collisions between replication–transcription complexes, alterations of nucleotide pools or metabolic processes resulting in increased levels of ROS that induce DNA damage are likely all contributing to c-Myc-induced replicative stress [17]. The increased load of ROS observed in cancer can result in direct oxidation of DNA or, preferentially, cause damage within the free dNTP pool. One of the major products of nucleotide oxidation is 8-oxo-2′-deoxyguanosine-triphosphate (8-oxodGTP), that upon mispairing with adenine once incorporated into DNA, results in mutations and cell death.