Posttranslational neddylation of cullins in the Cullin-Ring E3 ligase (CRL) complexes is needed for proteolytic degradation of CRL substrates, whose accumulation induces cell-cycle arrest, apoptosis, and senescence. The Nedd8-activating enzyme (NAE) is critical for neddylation of CRL complexes and their growth-promoting function. Recently, the anticancer small molecule MLN4924 currently in phase I trials was determined to be an inhibitor of NAE that blocks cullin neddylation and inactivates CRL, triggering an accumulation of CRL substrates that trigger cell-cycle arrest, apoptosis, and senescence in cancer cells. Here, we report that MLN4924 also triggers autophagy in response to CRL inactivation and that this effect is important for the ability of MLN4924 to suppress the outgrowth of liver cancer cells in vitro and in vivo. MLN4924-induced autophagy was attributed partially to inhibition of mTOR activity, due to accumulation of the mTOR inhibitory protein Deptor, as well as to induction of reactive oxygen species stress. Inhibiting autophagy enhanced MLN4924-induced apoptosis, suggesting that autophagy is a survival signal triggered in response to CRL inactivation. In a xenograft model of human liver cancer, MLN4924 was well-tolerated and displayed a significant antitumor effect characterized by CRL inactivation and induction of autophagy and apoptosis in liver cancer cells. Together, our findings support the clinical investigation of MLN4924 for liver cancer treatment and provide a preclinical proof-of-concept for combination therapy with an autophagy inhibitor to enhance therapeutic efficacy. Cancer Res; 72(13); 3360-71. Ó2012 AACR.

Our study highlights the overactivated neddylation pathway in lung cancer development and as a promising therapeutic target.

Transforming growth factor β (TGF-β) is a potent antiproliferative factor in multiple types of cells. Deregulation of TGF-β signaling is associated with the development of many cancers, including leukemia, though the molecular mechanisms are largely unclear. Here, we show that Casitas B-lineage lymphoma (c-Cbl), a known proto-oncogene encoding an ubiquitin E3 ligase, promotes TGF-β signaling by neddylating and stabilizing the type II receptor (TβRII). Knockout of c-Cbl decreases the TβRII protein level and desensitizes hematopoietic stem or progenitor cells to TGF-β stimulation, while c-Cbl overexpression stabilizes TβRII and sensitizes leukemia cells to TGF-β. c-Cbl conjugates neural precursor cell-expressed, developmentally downregulated 8 (NEDD8), a ubiquitin-like protein, to TβRII at Lys556 and Lys567. Neddylation of TβRII promotes its endocytosis to EEA1-positive early endosomes while preventing its endocytosis to caveolin-positive compartments, therefore inhibiting TβRII ubiquitination and degradation. We have also identified a neddylation-activity-defective c-Cbl mutation from leukemia patients, implying a link between aberrant TβRII neddylation and leukemia development.

Truncated D-4'-thioadenosine derivatives lacking the 4'-hydroxymethylene moiety were synthesized starting from D-mannose, using cyclization to the 4-thiosugar and one-step conversion of the diol to the acetate as key steps. At the human A3 adenosine receptor (AR), N6-substituted purine analogues bound potently and selectively and acted as antagonists in a cyclic AMP functional assay. An N6-(3-chlorobenzyl)purine analogue 9b displayed a Ki value of 1.66 nM at the human A3 AR. Thus, truncated D-4'-thioadenosine is an excellent template for the design of novel A3 AR antagonists to act at both human and murine species.