The UBE2F-CRL5ASB11-DIRAS2 axis is an oncogene and tumor suppressor cascade in pancreatic cancer cells.

UBE2F, a neddylation E2, neddylates CUL5 to activate cullin-RING ligase-5, upon coupling with neddylation E3 RBX2/SAG. Whether and how UBE2F controls pancreatic tumorigenesis is previously unknown. Here, we showed that UBE2F is essential for the growth of human pancreatic cancer cells with KRAS mutation. In the mouse KrasG12D pancreatic ductal adenocarcinoma (PDAC) model, Ube2f deletion suppresses cerulein-induced pancreatitis, and progression of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia. Mechanistically, Ube2f deletion inactivates the Mapk-c-Myc signals via blocking ubiquitylation of Diras2, a substrate of CRL5Asb11 E3 ligase. Biologically, DIRAS2 suppresses growth and survival of human pancreatic cancer cells harboring mutant KRAS, and Diras2 deletion largely rescues the phenotypes induced by Ube2f deletion. Collectively, Ube2f or Diras2 plays a tumor-promoting or tumor-suppressive role in the mouse KrasG12D PDAC model, respectively. The UBE2F-CRL5ASB11 axis could serve as a valid target for pancreatic cancer, whereas the levels of UBE2F or DIRAS2 may serve as prognostic biomarkers for PDAC patients.

Targeting neddylation E2s: a novel therapeutic strategy in cancer.

Ubiquitin-conjugating enzyme E2 M (UBE2M) and ubiquitin-conjugating enzyme E2 F (UBE2F) are the two NEDD8-conjugating enzymes of the neddylation pathway that take part in posttranslational modification and change the activity of target proteins. The activity of E2 enzymes requires both a 26-residue N-terminal docking peptide and a conserved E2 catalytic core domain, which is the basis for the transfer of neural precursor cell-expressed developmentally downregulated 8 (NEDD8). By recruiting E3 ligases and targeting cullin and non-cullin substrates, UBE2M and UBE2F play diverse biological roles. Currently, there are several inhibitors that target the UBE2M-defective in cullin neddylation protein 1 (DCN1) interaction to treat cancer. As described above, this review provides insights into the mechanism of UBE2M and UBE2F and emphasizes these two E2 enzymes as appealing therapeutic targets for the treatment of cancers.

Introduction.

Protein ubiquitylation is one of the most important posttranslational protein modifications, catalyzed by an enzyme cascade of E1/E2/E3. Neddylation, like ubiquitylation, is also catalyzed by an E1/E2/E3 enzyme cascade to covalently attach the ubiquitin-like molecule NEDD8 to a lysine residue of a substrate, not for degradation, but for modulation of substrate activity. The best known neddylation substrates are cullin family members, the scaffold component of the cullin-RING ligase (CRL), which is the largest family of E3 ligases that catalyze the ubiquitylation of ~20% of cellular proteins doomed for the degradation by proteasome system. The activity of CRLs requires cullin neddylation, which facilitates the adaptation of CRLs to an open conformation for easy substrate access. Since the substrates of CRLs are various key molecules that regulate a variety of cellular functions, CRLs and their neddylation activation, therefore, play the essential roles in many biological processes. Indeed, CRLs are abnormally regulated in many human diseases and serve as the therapeutic targets at least for cancer. This book will summarize most recent progress in this field with three sections, covering (1) structure and regulation of CRL and neddylation, (2) biological function of each CRLs, and (3) drug discovery efforts to target CRL/neddylation for cancer therapy.

Cullin RING Ligase 5 (CRL-5): Neddylation Activation and Biological Functions.

Cullin-5 (Cul-5) was originally identified as an arginine vasopressin (AVP) receptor due to its homology to a vasopressin-activated calcium-mobilizing protein 1 (VACM-1). Cul-5 has subsequently gained much attention after being identified as the key component of CRL-5 (Cullin-RING ligase-5) that mediates ubiquitylation and degradation of several key cellular proteins associated with human cancers and viral infections. Structurally, Cul-5 interacts with the Elongin B/C complex, a RING finger protein (RBX2/SAG), and a SOCS protein to form a CRL-5 E3 ubiquitin ligase protein complex. CRL-5, by controlling turnover of a variety of substrates, is implicated in several biological processes and human diseases. Activation of CRL-5 requires Cul-5 neddylation, catalyzed by a neddylation enzyme cascade, consisting of the E1 NEDD8-activating enzyme (NAE), the E2 neddylation conjugating enzyme (UBE2F), and E3 neddylation ligase (RBX2/SAG). RBX2/SAG, therefore, serves as both Cul-5 neddylation E3 and CRL-5 ubiquitylation E3. Here, we review the current knowledge on CRL-5, its activation by the UBE2F-SAG, its regulation of various signaling pathways via substrate degradation, and its implications in human cancers.

The CUL5 ubiquitin ligase complex mediates resistance to CDK9 and MCL1 inhibitors in lung cancer cells.

Overexpression of anti-apoptotic proteins MCL1 and Bcl-xL are frequently observed in many cancers. Inhibitors targeting MCL1 are in clinical development, however numerous cancer models are intrinsically resistant to this approach. To discover mechanisms underlying resistance to MCL1 inhibition, we performed multiple flow-cytometry based genome-wide CRISPR screens interrogating two drugs that directly (MCL1i) or indirectly (CDK9i) target MCL1. Remarkably, both screens identified three components (CUL5, RNF7 and UBE2F) of a cullin-RING ubiquitin ligase complex (CRL5) that resensitized cells to MCL1 inhibition. We find that levels of the BH3-only pro-apoptotic proteins Bim and Noxa are proteasomally regulated by the CRL5 complex. Accumulation of Noxa caused by depletion of CRL5 components was responsible for re-sensitization to CDK9 inhibitor, but not MCL1 inhibitor. Discovery of a novel role of CRL5 in apoptosis and resistance to multiple types of anticancer agents suggests the potential to improve combination treatments.

UBE2M Is a Stress-Inducible Dual E2 for Neddylation and Ubiquitylation that Promotes Targeted Degradation of UBE2F.

UBE2M and UBE2F are two family members of neddylation E2 conjugating enzyme that, together with E3s, activate CRLs (Cullin-RING Ligases) by catalyzing cullin neddylation. However, whether and how two E2s cross-talk with each other are largely unknown. Here, we report that UBE2M is a stress-inducible gene subjected to cis-transactivation by HIF-1 and AP1, and MLN4924, a small molecule inhibitor of E1 NEDD8-activating enzyme (NAE), upregulates UBE2M via blocking degradation of HIF-1α and c-JUN. UBE2M is a dual E2 for targeted ubiquitylation and degradation of UBE2F, acting as a neddylation E2 to activate CUL3-Keap1 E3 under physiological conditions but as a ubiquitylation E2 for Parkin-DJ-1 E3 under stressed conditions. UBE2M-induced UBE2F degradation leads to CRL5 inactivation and subsequent NOXA accumulation to suppress the growth of lung cancer cells. Collectively, our study establishes a negative regulatory axis between two neddylation E2s with UBE2M ubiquitylating UBE2F, and two CRLs with CRL3 inactivating CRL5.