Polyubiquitylation leading to proteolytic degradation

Polyubiquitylation leading to proteolytic degradation by the 26S proteasome is involved in all aspects of cell physiology. The highly coordinated process ensures the selective and timely turnover of proteins, thereby controlling cellular activity and maintaining cell and tissue homeostasis (Glickman and Ciechanover, 2002). The ullin ING E3 ubiquitin igase (CRL4) is a master regulator of genome stability and orchestrates a variety of physiological processes, particularly those related to chromatin regulation (Jackson and Xiong, 2009). Along with the substrate receptor CDT2 (also known as DCAF2, DTL/RAMP), the CRL4CDT2 ligase promotes the ubiquitin-dependent degradation of several proteins essential for hippo pathway progression as well as for DNA replication and repair (Abbas and Dutta, 2011; Abbas et al., 2013). One of the main functions of CRL4CDT2 is to prevent re-initiation of DNA replication (rereplication), both during S-phase of the cell cycle and following DNA damage, through the ubiquitylation and degradation of the replication licensing protein CDT1 (unrelated to CDT2), the CDK inhibitor p21, and the histone methyltransferase SET8 (Abbas and Dutta, 2011; Abbas et al., 2013). DNA rereplication is deleterious to cells and promotes cellular senescence and apoptosis due to replication fork stalling and the accumulation of toxic replication intermediates. Cullin-dependent E3 ligases, including CRL4, are activated by NEDD8 modification, which is catalyzed by an enzyme cascade system similar to ubiquitylation (Merlet et al., 2009). Pevonedistat (MLN4924), an inhibitor of the NEDD8-activating enzyme (NAE), induces cytotoxicity in a variety of cancer cell types in vitro and in preclinical mouse models (Jazaeri et al., 2013; Lin et al., 2010; Soucy et al., 2009; Wei et al., 2012). It is currently in clinical trials for hematologic (NCT00722488, NCT00911066) and solid malignancies including melanoma (NCT01011530), but its effects on melanoma cells have not been thoroughly examined. There is also little to no preclinical data on pevonedistat efficacy in the context of the various genetic mutations associated with melanoma or resistance to front line therapies (Garcia et al., 2014; Tan et al., 2013). Consistent with its activity as a general inhibitor of protein neddylation, pevonedistat was shown to inhibit multiple signal transduction pathways in addition to inhibiting cullin-mediated signaling, including the NFκB, AKT and the mTOR signal transduction pathways (Godbersen et al., 2014; Gu et al., 2014; Li et al., 2014a; Li et al., 2014b; Lin et al., 2010; Milhollen et al., 2011; Milhollen et al., 2010; Soucy et al., 2009). Although pevonedistat exerts these wide inhibitory activities, it remains unclear which, if any, mediates its anti-tumor activity. We here show that CDT2 is frequently overexpressed in melanoma, and its elevated expression predicts poor overall and disease-free survival. CDT2 knockdown or deletion inhibits the proliferation of melanoma cells in vitro through the induction of rereplication and senescence, and via a mechanism that is dependent on the stabilization of the CRL4CDT2 substrates SET8 and p21. Pevonedistat exerts significant anti-melanoma activity, irrespective of the BRAF mutational status, and through the induction of SET8- and p21-dependent rereplication and senescence. In vivo studies using melanoma cells with hypomorphic expression of p21 or SET8 show that both of these proteins are required for the anti-melanoma efficacy of pevonedistat, demonstrating that inhibition of the CRL4CDT2-SET8/p21 degradation axis is the primary mechanism by which pevonedistat inhibits melanoma. Finally, we show that pevonedistat synergizes with the BRAF kinase inhibitor PLX4720 to suppress BRAF mutant melanoma in vivo, and suppresses PLX4720-resistant melanoma cells.
Materials & Methods
Results
Discussion We identified the CRL4CDT2 E3 ubiquitin ligase as a molecular therapeutic target in melanoma. CDT2 knockdown or the pharmacological inhibition of CRL4CDT2 activity by the neddylation inhibitor pevonedistat inhibits melanoma cell proliferation in vitro and in vivo through the induction of SET8- and p21-dependent aberrant DNA replication and the induction of p21-dependent cellular senescence. This occurs irrespective of the BRAF or NRAS mutational status. Although p16 may also be involved in rereplication-induced senescence, it is not essential for pevonedistat-induced toxicity, and thus, mutational inactivation of the CDKN2A or oncogenic activation of BRAF or NRAS, all common genetic defects in melanoma, do not present an obstacle for the therapeutic efficacy of pevonedistat. Pevonedistat was also efficacious in suppressing melanoma cells that are resistant to vemurafenib treatment in vitro and synergized with PLX4720 to suppress mutant BRAF melanoma. It remains to be determined however, whether CRL4CDT2 inhibition, or the stabilization of SET8 or p21, contributes to the synergistic activity of pevonedistat with PLX4720. Nevertheless, our findings suggest that pevonedistat may be most efficacious if administered along with other melanoma inhibitors, such as vemurafenib (for BRAF melanomas), and may also be considered as a second line therapeutic for vemurafenib- or other BRAF kinase inhibitor-relapsed melanoma patients.