Mechanistically we show that CRL CDT is the

Mechanistically, we show that CRL4CDT2 is the primary target of inactivation by pevonedistat in melanoma and that its toxicity is dependent primarily on the stabilization of the CRL4CDT2 substrates SET8 and p21 both in vitro and in vivo. The lack of a therapeutic response on melanoma cells with hypomorphic expression of SET8 or p21 provides solid genetic evidence that the main anti-melanoma activity of pevonedistat is associated with the drug\'s ability to promote rereplication and permanent growth inhibition through preventing SET8 and p21 proteolytic degradation. Pevonedistat however, also transiently inhibits melanoma proliferation through an unknown mechanism. We speculate that this may be dependent on the accumulation of other cullin substrates and may include the CDK2 inhibitor p27, an SCFSKP2 ubiquitylation substrate, which is also stabilized by pevonedistat (Fig. 4a and b). This however, is insufficient to halt melanoma proliferation in the absence of SET8- and p21-mediated cytotoxicities (Fig. 6c). Whereas the CRL4CDT2 substrates CDT1 and p21 are independently required to induce rereplication and senescence in melanoma cells with inactivated CRL4CDT2, SET8 is both necessary and sufficient to promote rereplication and the ensuing senescence. The exact mechanism by which increased SET8 protein stability promotes rereplication is currently unclear, but histone H4K20 methylation may be critical for this activity (Abbas et al., 2010; Tardat et al., 2010). The main role of p21 on the other hand, appears to halt thip progression (thus permitting rereplication) and induce senescence. This is supported by the finding that p21 is critical for the induction of rereplication and senescence in response to CRL4CDT2 inactivation (by si-CDT2 or pevonedistat), and is also upregulated in rereplicating cells following the ectopic expression of CDT1 or SET8ΔPIP, or following EMI1 depletion, but is insufficient to induce rereplication. Although CDT1 promotes rereplication in cancer cells as demonstrated by the robust induction of rereplication through geminin depletion, we did not observe such a role in melanoma cells, likely because CDT1 activity is restrained by cyclin A-dependent SCFSKP2 activity. Non-physiological overexpression of CDT1 however, was sufficient to induce rereplication in melanoma cells, but this is likely to also require SET8 and p21. This conclusion is supported by the fact that although pevonedistat induced significant rereplication in melanoma cells, it failed to do so in cells with hypomorphic expression of SET8 or p21, despite significant increases in CDT1 protein. The anti-melanoma activity of pevonedistat and its dependence on CRL4CDT2 inhibition and the induction of SET8- and p21-dependent rereplication provide a stronger link between presumed drug target (NAE) and biology than is available for many other “targeted therapies”. Our study also demonstrates that CDT2 is significantly overexpressed in melanoma, and its elevated expression correlates significantly with poor overall and disease-free patient survival. Because elevated CDT2 expression correlates with, and renders melanoma cells more susceptible to, pevonedistat-induced rereplication in vitro, and given that rereplication appears to play a major role in mediating its efficacy in vivo, we speculate that pevonedistat would be most efficacious in tumors with elevated CDT2 expression. This includes not only melanoma, but also potentially other malignancies with elevated CDT2 expression (Figure S1). CDT2 is not likely to function as a classical oncogene, but may act as a cancer-associated gene to which cancer cells become “addicted”. This is supported by the finding that while CRL4CDT2 inactivation by pevonedistat induces rereplication in melanoma cells, it failed to do so in non-cancer melanocytic cells. Similarly, CDT2 depletion in non-cancer cells failed to induce rereplication in non-cancer cells, but did so following the ectopic expression of KRAS (Olivero et al., 2014). We propose that CDT2 is overexpressed in melanoma cells to alleviate replication stress that may be induced by melanoma oncogenes.