title: Inactivation of the CRL4-CDT2-SET8/p21 ubiquitylation and degradation axis underlies the therapeutic efficacy of pevonedistat in melanoma.
abstract: 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.
  
  
  BACKGROUND
  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.
authors.affiliation: Department of Radiation Oncology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Mouadh
authors.initials: M
authors.lastname: Benamar
authors.affiliation: Department of Radiation Oncology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Fadila
authors.initials: F
authors.lastname: Guessous
authors.affiliation: Department of Radiation Oncology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Kangping
authors.initials: K
authors.lastname: Du
authors.affiliation: Department of Radiation Oncology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Corbett
authors.affiliation: Department of Surgery, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Obeid
authors.affiliation: Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Gioeli
authors.affiliation: Department of Surgery, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Craig L
authors.initials: CL
authors.lastname: Slingluff
authors.affiliation: Department of Radiation Oncology, University of Virginia, Charlottesville, VA 22908, USA; Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA; Center for Cell Signaling, University of Virginia, Charlottesville, VA 22908, USA. Electronic address: ta8e@virginia.edu.
authors.forename: Tarek
authors.initials: T
authors.lastname: Abbas
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D016922:Cellular Senescence / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology* / Q000627:therapeutic use; D004195:Disease Models, Animal; D005260:Female; D015870:Gene Expression; D020868:Gene Silencing; D011905:Genes, ras; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D053208:Kaplan-Meier Estimate; D008545:Melanoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000401:mortality; D051379:Mice; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011379:Prognosis; D055550:Protein Stability / Q000187:drug effects; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D011743:Pyrimidines / Q000494:pharmacology* / Q000627:therapeutic use; D034622:RNA Interference; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D023041:Xenograft Model Antitumor Assays; D053722:Receptors, Interleukin-17
chemical_list: D000970:Antineoplastic Agents; D050759:Cyclin-Dependent Kinase Inhibitor p21; D003517:Cyclopentanes; D011495:Histone-Lysine N-Methyltransferase; D009687:Nuclear Proteins; D048493:Proto-Oncogene Proteins B-raf; D011743:Pyrimidines; D044767:Ubiquitin-Protein Ligases; C429530:DTL protein, human; C408922:IL17RB protein, human; D053722:Receptors, Interleukin-17; C470730:KMT5A protein, human; C539933:pevonedistat
keywords: BRAF; CDT2; Cullin; DTL; MLN4924; Melanoma; NRAS; Pevonedistat; Ubiquitylation