Inhibition of proliferation and survival of diffuse large B-cell lymphoma cells by a small-molecule inhibitor of the ubiquitin-conjugating enzyme Ubc13-Uev1A

Abstract

IntroductionDiffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous disease comprising at least 3 major subtypes with distinct molecular, biologic, and clinical properties: activated B cell-like DLBCL (ABC-DLBCL), germinal center B cell-like DLBCL (GCB-DLBCL), and primary mediastinal B-cell lymphoma. 1,2 Although the overall cure rate for DLBCL reaches more than 50% with the current therapies such as R-CHOP (rituximab plus cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisone/prednisolone), less than 40% of ABC-DLBCL patients are cured. [2][3][4] Therefore, new therapy approaches efficient for this and other DLBCL subtypes are highly desirable.The transcription factor NF-B controls the expression of a wide range of genes involved in cell proliferation, survival, stress response, angiogenesis, and inflammation. 5,6 NF-B activity is tightly regulated by multiple signaling pathways, and abnormal NF-B activation has been linked to cancer development and progression. [7][8][9] Constitutive NF-B activation has been observed in high frequency in all of the main DLBCL subtypes, especially in ABC-DLBCL, with more than 90% of the tumors showing nuclear NF-B, the hallmark of its activation. 9-14 A recent genomic study revealed that more than 60% of ABC-DLBCLs and approximately 30% of GCB-DLBCLs harbor somatic mutations in multiple components of NF-B signaling pathways, such as the BCR, CD40, and TLR pathways. 15 Significantly, it has been demonstrated that constitutive NF-B signaling is required for the proliferation and survival of ABC-DLBCL cell lines. 11,13,16,17 All of these observations suggest a primary role for constitutive NF-B signaling in the pathogenesis of DLBCL and, therefore, the NF-B signaling pathway may represent a rational therapeutic target in DLBCL. 9,11,18 Ubiquitination, the covalent attachment of the ubiquitin (Ub) molecule to target proteins, regulates diverse cellular processes. 19 Ubiquitination proceeds through a stepwise enzymatic cascade involving 3 classes of enzymes: a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2), and a Ub ligase (E3). The E1 enzyme activates Ub in an ATP-dependent manner and transfers the activated Ub to an E2 enzyme through the formation of a thioester bond between the carboxy terminus of Ub and the active site cysteine of the E2, generating an E2 and Ub thioester conjugate (referred to as E2ϳUb). The E2 then cooperates with an E3 to attach the Ub to a lysine residue of a substrate. Ub itself can serve as a substrate and the process can undergo multiple rounds, resulting in the formation of polyubiquitin chains. 19,20 Because Ub has 7 lysine residues and any one of them can be conjugated to another Ub, polyubiquitin chains of different linkages with distinct functional properties are formed in cells. For example, lysine 48 (K48)-linked polyubiquitin chains typically target substrates for proteasomal degradation, whereas K63-linked polyubiquitin chains function as scaffolds to assemble protein complexes in NF-B signaling and DNA repair. [...

Novel Lys63-linked ubiquitination of IKKβ induces STAT3 signaling

“…Therefore, we used the small molecule inhibitor NSC697923 to inhibit the E2 ubiquitin-conjugating enzyme UBC13-UEV1A complex, selectively inhibiting K63-linked ubiquitination. 44 HEK293T cells expressing WT or K171E were treated with the inhibitor NSC697923 and examined for IKKb S177/S181 phosphorylation. Although some increased phosphorylation was observed for WT IKKb, the activation of IKKb kinase in the K171E mutant protein was largely independent of K63-linked ubiquitination (Fig.…”

Screening for E3-Ubiquitin ligase inhibitors: challenges and opportunities

“…In addition, NSC697923, an inhibitor of the Ubc13–Uev1A E2 enzyme blocks the formation of the E2–Ub thio-ester conjugate. Since Ubc13–Uev1A catalyses the formation of K63-linked poly-Ub chains, this compound has inhibitory activity against NF-κB signalling activation, which ultimately leads to reduced proliferation and viability of cancer cells [132]. …”

MLN4924, an NAE inhibitor, suppresses AKT and mTOR signaling via upregulation of REDD1 in human myeloma cells

“…[6][7][8][9][10][11] Ubiquitination of target proteins is achieved via sequential enzymatic reactions mediated by E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme), and E3 ligases (ubiquitin ligases). Among the known E3 ligases, cullin-ring ligases (CRLs) represent the largest and most frequently used group.…”