The
cullin-based CRL4-CDT2
ubiquitin ligase is emerging as a master regulator of cell proliferation. CRL4-CDT2 prevents re-initiation of DNA replication during the same cell cycle "rereplication" through targeted degradation of CDT1, SET8 and p21 during S-phase of the cell cycle. We show that CDT2 is overexpressed in cutaneous
melanoma and predicts poor overall and disease-free survival. CDT2 ablation inhibited a panel of
melanoma cell lines through the induction of SET8- and p21-dependent
DNA rereplication and senescence.
Pevonedistat (
MLN4924), a specific inhibitor of the NEDD8 activating
enzyme (NAE), inhibits the activity of
cullin E3
ligases, thereby stabilizing a vast number of
cullin substrates and resulting in
cancer cell inhibition in vitro and
tumor suppression in nude mice. We demonstrate that
pevonedistat is effective at inhibiting the proliferation of
melanoma cell lines in vitro through the induction of rereplication-dependent permanent growth arrest as well as through a transient, non-rereplication-dependent mechanism. CRISPR/Cas9-mediated heterozygous deletion of CDKN1A (encoding p21) or SET8 in
melanoma cells demonstrated that the rereplication-mediated cytotoxicity of
pevonedistat is mediated through preventing the degradation of p21 and SET8 and is essential for
melanoma suppression in nude mice. By contrast,
pevonedistat-induced transient growth suppression was independent of p21 or SET8, and insufficient to inhibit
tumor growth in vivo.
Pevonedistat additionally synergized with the
BRAF kinase inhibitor
PLX4720 to inhibit BRAF
melanoma, and suppressed PLX4720-resistant
melanoma cells. These findings demonstrate that the CRL4-CDT2-SET8/p21 degradation axis is the primary target of inhibition by
pevonedistat in
melanoma and suggest that a broad patient population may benefit from
pevonedistat therapy.
RESEARCH IN CONTEXT: The identification of new molecular targets and effective inhibitors is of utmost significance for the clinical management of
melanoma. This study identifies CDT2, a substrate receptor for the CRL4
ubiquitin ligase, as a prognostic marker and therapeutic target in
melanoma. CDT2 is required for
melanoma cell proliferation and inhibition of CRL4(CDT2) by
pevonedistat suppresses
melanoma in vitro and in vivo through the induction of
DNA rereplication and senescence through the stabilization of the CRL4(CDT2) substrates p21 and SET8.
Pevonedistat also synergizes with
vemurafenib in vivo and suppresses
vemurafenib-resistant
melanoma cells. These findings show a significant promise for targeting CRL4(CDT2) therapeutically.