Abstract
The mut-T homolog-1 (MTH1) inhibitor TH588 has shown promise in preclinical cancer studies but its targeting specificity has been questioned. Alternative mechanisms for the anti-cancer effects of TH588 have been suggested but the question remains unresolved. Here, we performed an unbiased CRISPR screen on human lung cancer cells to identify potential mechanisms behind the cytotoxic effect of TH588. The screen identified pathways and complexes involved in mitotic spindle regulation. Using immunofluorescence and live cell imaging, we showed that TH588 rapidly reduced microtubule plus-end mobility, disrupted mitotic spindles, and prolonged mitosis in a concentration-dependent but MTH1-independent manner. These effects activated a USP28-p53 pathway – the mitotic surveillance pathway – that blocked cell cycle reentry after prolonged mitosis; USP28 acted upstream of p53 to arrest TH588-treated cells in the G1-phase of the cell cycle. We conclude that TH588 is a microtubule-modulating agent that activates the mitotic surveillance pathway and thus prevents cancer cells from re-entering the cell cycle.
Highlights
The mut-T homolog 1 (MTH1, known as NUDT1) inhibitor TH588 has shown promise as an anticancer compound in preclinical studies[1,2]
Independent studies have confirmed that TH588 inhibits mut-T homolog-1 (MTH1) at nanomolar concentrations[4,5,6,7,8], accumulating evidence suggests that its anti-cancer effect is mediated by other mechanisms: First, the concentrations of TH588 required for its cytotoxic effect are several fold higher than those required for MTH1 inhibition[1,5]
CRISPR screen of TH588-treated cells identified complexes and pathways associated with mitotic spindle regulation
Summary
The mut-T homolog 1 (MTH1, known as NUDT1) inhibitor TH588 has shown promise as an anticancer compound in preclinical studies[1,2]. A number of alternative MTH1 inhibitors have been developed, none of which are toxic when tested on human cancer cell lines[4,5,6,7]. CRISPR-mediated knockout of MTH1 failed to reproduce the cytotoxic effect of MTH1 knockdown that was previously reported in human cancer cell lines[5,9]. We performed a pooled lentiviral CRISPR screen to identify gene knockouts that rescue H460 human lung cancer cells from the cytotoxic effect of TH588. Data from such screens can identify mechanisms behind drug effects and drug resistance mechanisms
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