Structural basis for the recognition of nectin-like protein-5 by the human-activating immune receptor, DNAM-1.

Nectin and nectin-like (Necl) adhesion molecules are broadly overexpressed in a wide range of cancers. By binding to these adhesion molecules, the immunoreceptors DNAX accessory molecule-1 (DNAM-1), CD96 molecule (CD96), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT) play a crucial role in regulating the anticancer activities of immune effector cells. However, within this axis, it remains unclear how DNAM-1 recognizes its cognate ligands. Here, we determined the structure of human DNAM-1 in complex with nectin-like protein-5 (Necl-5) at 2.8 Å resolution. Unexpectedly, we found that the two extracellular domains (D1-D2) of DNAM-1 adopt an unconventional "collapsed" arrangement that is markedly distinct from those in other immunoglobulin-based immunoreceptors. The DNAM-1/Necl-5 interaction was underpinned by conserved lock-and-key motifs located within their respective D1 domains, but also included a distinct interface derived from DNAM-1 D2. Mutation of the signature DNAM-1 "key" motif within the D1 domain attenuated Necl-5 binding and natural killer cell-mediated cytotoxicity. Altogether, our results have implications for understanding the binding mode of an immune receptor family that is emerging as a viable candidate for cancer immunotherapy.

UHRF1/UBE2L6/UBR4-mediated ubiquitination regulates EZH2 abundance and thereby melanocytic differentiation phenotypes in melanoma.

Cellular heterogeneity in cancer is linked to disease progression and therapy response, although mechanisms regulating distinct cellular states within tumors are not well understood. We identified melanin pigment content as a major source of cellular heterogeneity in melanoma and compared RNAseq data from high-pigmented (HPCs) and low-pigmented melanoma cells (LPCs), suggesting EZH2 as a master regulator of these states. EZH2 protein was found to be upregulated in LPCs and inversely correlated with melanin deposition in pigmented patient melanomas. Surprisingly, conventional EZH2 methyltransferase inhibitors, GSK126 and EPZ6438, had no effect on LPC survival, clonogenicity and pigmentation, despite fully inhibiting methyltransferase activity. In contrast, EZH2 silencing by siRNA or degradation by DZNep or MS1943 inhibited growth of LPCs and induced HPCs. As the proteasomal inhibitor MG132 induced EZH2 protein in HPCs, we evaluated ubiquitin pathway proteins in HPC vs LPCs. Biochemical assays and animal studies demonstrated that in LPCs, the E2-conjugating enzyme UBE2L6 depletes EZH2 protein in cooperation with UBR4, an E3 ligase, via ubiquitination at EZH2's K381 residue, and is downregulated in LPCs by UHRF1-mediated CpG methylation. Targeting UHRF1/UBE2L6/UBR4-mediated regulation of EZH2 offers potential for modulating the activity of this oncoprotein in contexts in which conventional EZH2 methyltransferase inhibitors are ineffective.