HUWE1 is a HECT-domain E3 ubiquitin ligase, plays a pivotal role in regulating critical oncoproteins including c-Myc, p53 and MCL-1 through protein ubiquitination (Kao et al., 2018). Emerging evidence has established a strong association between HUWE1 dysregulation and tumorigenesis, with recent research identifying recurrent HUWE1 mutations in MM patients (Walker et al., 2018). Notably, HUWE1 exhibits significantly elevated expression in MM cells compared to their normal counterparts. Both in vitro and in vivo studies have demonstrated that HUWE1 is essential for sustaining MM proliferation and survival. Mechanistic investigations reveal that HUWE1 depletion induces a striking shift in ubiquitination patterns: reduction in K63-linked polyubiquitination and enhancement in K48-linked polyubiquitination. This ubiquitination switch specifically promotes c-Myc degradation, resulting in potent inhibition of MM cell growth (Crawford et al., 2020). These findings position HUWE1 as both a crucial mediator of MM pathogenesis and a promising therapeutic target. It is worth noting that potential off-target effects of HUWE1 inhibition warrant caution in clinical trial design—as hematopoietic-specific HUWE1 knockout results in bone marrow failure in murine models (King et al., 2016).

The CRL4^CRBN ubiquitin ligase complex represents a critical component of the cellular ubiquitin-proteasome system, composed of three core elements: RING finger domain protein (Roc1/RBX1), cullin4 (CUL4A/B) scaffold protein, and DDB1-CUL4 associated factors (substrate receptor). As the defining substrate recognition subunit of the CRL4 complex, cereblon (CRBN) plays a pivotal role in mediating the therapeutic effects of immunomodulatory drugs (IMiDs) such as lenalidomide and pomalidomide. By binding CRBN, IMiD induces conformational changes in CRBN to alter its substrate specificity. The modified complex selectively mediates ubiquitination and proteasomal degradation of transcription factors IKZF1 and IKZF3, resulting in subsequent depletion of their targets IRF4 and c-Myc. This mechanism ultimately leads to the inhibition of MM cell proliferation (Barankiewicz et al., 2022). Notably, some relapsed MM patients maintain CRBN expression and IKZF1 degradation but progress, implying potential underestimation of CRBN-independent mechanisms such as IL-6/STAT3 pathway activation (Teoh et al., 2025).

The basic zipper leucine transcription factors c-Maf and MafA represent critical oncogenic drivers in MM. Clinical observations reveal that overexpression of c-Maf and MafA are frequently found in MM patients, and are strongly associated with poor prognosis. These oncoproteins play multifunctional role in MM cell proliferation and survival, chemoresistance, and disease progression through transcriptional activation of critical target genes including CCND2, ITGB7, CCR1, ARK5 (Jiang et al., 2022). Contrasting with its oncogenic role in solid tumors (e.g., as a lung cancer biomarker) (Sánchez-Tena et al., 2016), HECT E3 ligase HERC4 demonstrates tumor-suppressive activity in MM through two distinct mechanisms: one way is to mediate the polyubiquitination and proteasomal degradation of c-Maf to specifically reduce c-Maf protein level (Zhang et al., 2016). While the other way is to catalyze K63-linked polyubiquitination of MafA that prevents it from phosphorylation by GSK3β, therefore inhibiting STAT3 activation and transcriptional activity (Zha et al., 2023). These coordinated actions results in the inhibition of MM cell proliferation and retardation of tumor growth. Despite high gene sequence similarity between c-Maf and MafA—both belonging to the MAF transcription factor family—HERC4 exerts differential ubiquitination modifications on these two transcription factors in this research. There is still a lack of structural biological evidence to explain this mechanistic selectivity.

NEDD4-1 is a prototypical HECT-type E3 ubiquitin ligase of the NEDD4 family. In macrophages, lactate-induced lactylation of NEDD4 at K33 disrupts its binding to caspase-11, which diminishes caspase-11 ubiquitination and subsequently enhances non-classical pyroptosis, ultimately exacerbating hepatic injury (Li et al., 2024). It is widely recognized as a key oncogenic regulator in multiple malignancies (Wang et al., 2020). It is involved in the proliferation, apoptosis, cell cycle, autophagy and metastasis in many cancers. However, NEDD4-1 has been reported to recognize and ubiquitinate diverse oncogenic substrates, including N-Myc, c-Myc and RAS proteins, exerts tumor-suppressive functions in cancers such as pancreatic cancer (Zeng et al., 2014). Extensive studies have further established that NEDD4-1 can suppress tumorigenesis by regulating proliferation, apoptosis, metastasis and drug sensitivity (Zhang et al., 2020). In MM, low NEDD4-1 expression is found in patients and it is correlated with poor prognosis. Functionally, NEDD4-1 knockdown results in enhanced cell viability and bortezomib resistance in MM cells, while NEDD4-1 overexpression mediates bortezomib sensitivity through regulation of MM cell apoptosis and cell cycle. Mechanistically, NEDD4-1 mediates tumor-suppressive effects through targeting pAkt-Ser473 for ubiquitination degradation, and suppressing PTEN/PI3K/Akt pathway (Huang et al., 2019).

Another NEDD4 family protein, WWP2, regulates a wide range of biological processes, including DNA repair, gene expression, signal transduction, and cell-fate decisions. As such, WWP2 plays a key role in normal physiology and diseases, such as immune regulation and tumorigenesis (You et al., 2024). In MM, WWP2 is significantly downregulated in the bortezomib-resistant cells. WWP2 overexpression restricts MM tumor growth and enhances cell sensitivity to bortezomib treatment in vitro and in vivo. It has been investigated that WWP2 mediates the ubiquitination and degradation of GLI2, a core transcription factor in the activation of Hedgehog (Hh) pathway. Hence, DKK1-induced WWP2 downregulation leads to the stabilization of GLI2 protein and hyperactivation of Hh signaling, contributing to bortezomib resistance in MM cells (Zhang et al., 2021). However, WWP2-targeted therapeutic strategies present significant clinical challenges. Emerging evidence indicates that WWP2 overexpression suppresses TLR3-mediated immune response, which may facilitate tumor immune evasion (Yang et al., 2013). These immunosuppressive effects pose particularly serious concerns in the context of MM immunotherapy.

SCF ubiquitin ligase complex consists of an adaptor protein SKP1, a scaffold protein cullin1 and a F-box protein (Zhou et al., 2013). The F-box protein serves as the substrate receptor that determines targets specificity for ubiquitination. FBXO9, a member of the F-box protein family, has been identified as a critical regulator in MM. Study shows that FBXO9 exhibits significant overexpression in MM cells and plays dual roles by inhibiting cell proliferation and maintaining cell survival. FBXO9 exerts its biological effects through two distinct pathways. On one hand, FBXO9 is responsible for ubiquitination-mediated degradation of TEL2 and TTI1, which leads to the downregulation of mTORC1 signaling pathway and the inhibition of MM cell growth. On the other hand, PI3K/TORC2/AKT signaling cascade is also activated by FBXO9 to sustain cell survival (Fernández-Sáiz et al., 2013). Studies demonstrate that hyperactivated mTORC1 downstream of insulin/IGF-1 signaling enhances S6K1 activity, leading to phosphorylation of insulin receptor substrate 1 (IRS1) at multiple inhibitory sites. This negative feedback loop suppresses PI3K signaling and attenuates mTORC2 activation (Wang et al., 2024). A similar regulatory mechanism may operate in MM, as suggested by prior findings.

As another prototypical F-box protein, FBXW7 serves as the substrate recognition component of the SCF ubiquitin ligase complex. It plays pivotal roles in maintaining fundamental biological processes including cell cycle, apoptosis and differentiation. This critical tumor suppressor orchestrates the degradation of multiple oncoproteins such as cyclin E, c-Myc, c-JUN, NOTCH, and MCL1. It has been confirmed that FBXW7 gene mutations or downregulations have been found in many types of malignant tumors, which facilitate the uncontrolled proliferation, enhanced invasion and migration, and drug resistance of cancer cells (Qi et al., 2024). While traditionally characterized as a tumor suppressor, FBXW7 exhibits unique functionality in MM. The oncogenic mechanism shows that FBXW7 mediates K48-linked poly-ubiquitination degradation of p100 and triggers proteasomal processing to p52, thus leading the activation of the non-canonical NF-κB signaling pathway to support the growth and survival of MM cells (Busino et al., 2012). The context-dependent nature of FBXW7 function may account for the identification of its non-canonical role in MM.

PIRH2 represents a recently identified ubiquitin ligase induced by p53 activation. It possesses intrinsic RING-dependent ubiquitin ligase activity for polyubiquitination and subsequent proteasomal degradation. PIRH2 functions as oncogenic role through targeting the p53 family proteins, p53, Chk2, p27Kip1, Twist1 and others, participating in such cellular processes as cell proliferation, cell cycle regulation, apoptosis and cellular migration (Daks et al., 2022). Intriguingly, PIRH2 demonstrates tumor-suppressive activity in MM, with emerging evidence establishing it as a promising therapeutic target in this malignancy. Research shows that PIRH2 overexpression enhances bortezomib sensitivity in MM cells. Mechnistic studies demonstrate that PIRH2 reduces phosphorylated IκBα (pIκBα) levels and IκB kinase α (IKKα) expression, subsequent inhibiting the canonical NF-κB signaling pathway in MM cells (Yang et al., 2018). The functional dichotomy of PIRH2 in MM may reflect tissue-specific substrate reprogramming, potentially involving the loss of its p53-binding capacity in this malignancy. While PIRH2 has been shown to downregulate both pIκBα and IKKα, the underlying molecular mechanisms remain to be elucidated, representing an important avenue for future research.

SKP2 is a critical F-box protein of the SCF ubiquitin ligase. It participates in multiple cellular functions such as cell proliferation, cell cycle, metabolism, and tumorigenesis by contributing to the ubiquitination and subsequent degradation of several specific tumor suppressors. Among them, SKP2 is responsible for the degradation of several cell cycle protein-dependent kinase (CDK) inhibitor, such as p27 (Asmamaw et al., 2020). P27 is an important cell cycle regulator that induces a cell cycle arrest and inhibits the G1/S transition through negatively regulating CDK2 and CDK4. SCF^SKP2-mediated K48-linked poly-ubiquitination marks p27 for degradation that permits the CDK-dependent transition from a quiescent to proliferative state. SKP2 interacts with O-GlcNAc transferase and undergoes O-GlcNAcylation at Ser34, which strengthens its binding to SKP1 and prevents its degradation mediated by the anaphase-promoting complex/cyclosome (APC/C)-Cdh1. This post-translational modification enhances SKP2 stability and E3 ligase activity, promoting p21/p27 degradation and accelerating the G1-S phase transition, thereby driving hepatocellular carcinoma (HCC) cell proliferation (Feng et al., 2024). In addition, increased SKP2 expression and reduced p27 levels are frequent in human cancers and are associated with therapeutic resistance (Asmamaw et al., 2020). Particularly in MM, SKP2 is highly expressed in bortezomib-resistant patients and is a negative prognostic indicator for progression-free survival and overall survival. Knockdown of SKP2 stabilizes p27 expression and enhances bortezomib sensitivity of MM cells. Hence, the novel SCF^SKP2 inhibitor DT204 could trigger synergistic anti-myeloma activity with bortezomib and overcame bortezomib resistance, which opens up additional possibilities for the development of novel drug combinations (Malek et al., 2017). Similarly, another SKP2 inhibitor C1 prevents SKP2-mediated ubiquitination and degradation of p27, which results in p27 accumulation and cell cycle arrest, leading to MM cell apoptosis (Yang et al., 2019). It remains unclear whether SKP2 overexpression in MM is regulated through an O-GlcNAcylation-dependent mechanism similar to that observed in HCC, warranting further exploration.

RBX1 serves as an indispensable component of SCF ubiquitin ligase that is vital to ubiquitin ligation. Extensive research has established RBX1’s pivotal oncogenic involvement in cancer cell proliferation, cell senescence, survival, cell cycle and apoptosis (Wei and Sun, 2010). Indeed, experimental evidence reveals that RBX1 knockdown produces potent anti-myeloma effects manifested by growth inhibition in vitro, tumor suppression in vivo models and the decreased chemoresistance. Similarly in mechanism, RBX1 enhances the ubiquitination and degradation of p27 (Shao et al., 2022).

Multiple literature have reported that RFWD2 is engaged in tumorigenesis via meditating several biological processes like transcription, DNA repair, cell cycle and apoptosis. However, RFWD2 plays dualistic oncogenic/tumor-suppressive roles across different cancer types (Song et al., 2020). It demonstrates that MM patients with elevated RFWD2 expression achieve adverse clinical outcomes and drug resistance. Experimental study demonstrates that RFWD2 knockdown hinders cellular growth and triggers apoptosis in MM cells. Mechanism study reveals that RFWD2 controls MM cellular proliferation through regulating the ubiquitination and degradation of p27 via the formation of RFWD2-RCHY1 protein complex (Guo et al., 2021). While p27 degradation typically depends on T187 phosphorylation (mediated by CDK2) (Tsytlonok et al., 2019), whether RFWD2 recognizes this identical phosphodegron remains experimentally unverified.

Lenalidomide is a second-generation immunomodulatory drug (IMiD) that has revolutionized MM treatment. By binding to CRBN, lenalidomide recruits CRL4^CRBN E3 ubiquitin ligase to induce the proteasomal-dependent degradation of transcriptional factors IZKF1 and IKZF3. Degradation of IKZF1/3 downregulates the critical oncoproteins IRF4 and c-MYC, resulting in direct anti-myeloma activity with suppressed proliferation and survival of MM cells (Krönke et al., 2014). Besides, lenalidomide exhibits immunomodulatory effects through enhancing T-cell and NK-cell activity, increasing IL-2 and IFN-γ production, and reducing regulatory T-cell function (Colley et al., 2024). It shows that CRBN expression is required for the anti-myeloma activity of IMiDs. However, during the course of lenalidomide treatment, almost all patients relapse due to the acquisition of drug resistance, which is caused by deregulated CRBN expression, proteasome dysregulation, signal pathway compensation or microenvironment adaptation.

Besides, derivatives of lenalidomide are also being actively explored. Recent studies have demonstrated that modifying the six-position substituent of lenalidomide can enhance substrate selectivity in anti-hematological carcinogenesis while eliminating teratogenic effects associated with certain derivatives. Notably, 6-fluoro-lenalidomide exhibits superior IKZF1 and IKZF3 degradation potency compared to lenalidomide, along with pronounced anti-proliferative activity in MM cell lines. While 6-chlorolenalidomide shows reduced anti-proliferative effects relative to both lenalidomide and its 6-fluoro counterpart, it presents a significant advantage by avoiding degradation of SALL4 and PLZF proteins - the putative mediators of thalidomide’s teratogenicity. These findings establish a promising framework for developing novel thalidomide derivatives with optimized therapeutic efficacy and reduced toxicity profiles for MM treatment (Yamanaka et al., 2023).

Pomalidomide, another second-generation IMiD, is approved for relapsed/refractory multiple myeloma (RRMM). Structurally and mechanistically similar to lenalidomide, pomalidomide exerts direct tumoricidal effects by binding CRL4^CRBN E3 ubiquitin ligase, leading to the degradation of IKZF1/IKZF3, while also enhancing T-cell and NK-cell activation for immunomodulation. However, pomalidomide exhibits distinct anti-proliferative and pro-apoptotic mechanisms compared to lenalidomide. Specifically, it induces caspase-8-dependent apoptosis and upregulates p21^WAF1 (independent of p53), triggering G1/G0 cell cycle arrest and suppressing tumor growth. Within the BM microenvironment, pomalidomide potently inhibits TNF-α, reducing the secretion of growth-promoting cytokines (IL-6, bFGF, VEGF) and promoting MM cell death (Richardson et al., 2013). A key differentiator is pomalidomide’s unique ability to degrade ARID2 (a PBAF complex subunit), which suppresses MYC expression and overcomes lenalidomide resistance (Yamamoto et al., 2020). Given that heavily pretreated MM patients often exhibit immune dysfunction, pomalidomide’s potent anti-proliferative activity makes it an optimal therapeutic choice in this setting. Clinically, the pomalidomide-dexamethasone combination demonstrates strong synergistic tumoricidal effects, offering a promising treatment strategy for RRMM patients (Siegel et al., 2020). The synergistic mechanism likely involves pomalidomide-induced IKZF3 degradation, leading to IRF4 and MYC downregulation, which collectively results in strong, synergistic effects on the induction of apoptosis (Rychak et al., 2016).

The emergence of resistance to traditional IMiDs has driven the development of cereblon E3 ligase modulators (CELMoDs), a novel class of agents characterized by enhanced substrate degradation efficiency and expanded substrate spectrum. Mezigdomide is a novel CELMoD reagent that induces polyubiquitination and degradation of the IKZF1 and IKZF3 more rapidly and effectively than lenalidomide and pomalidomide, resulting in the downregulation of IRF4 and c-MYC expression. Meanwhile, Mezigdomide also significantly promotes the production of IL-2 and IFN-γ, enhancing the activation of T cells and anti-tumor immunity (Patel et al., 2024). Preclinically, mezigdomide exhibits significantly stronger anti-proliferative and tumoricidal effects than lenalidomide or pomalidomide, even in MM cell lines with low or mutated cereblon protein or with acquired resistance to IMiDs (Hansen et al., 2020). These properties make it a promising therapeutic option for RRMM. Clinical trials are currently underway to evaluate its safety and efficacy in both newly diagnosed and RRMM.

Iberdomide represents a clinically significant advancement in the CELMoD class, demonstrating potent anti-proliferative activity against MM cell lines, including those with acquired resistance to lenalidomide and pomalidomide due to CRBN dysregulation (Bjorklund et al., 2020). It exhibits significantly greater potency in inducing cellular degradation of IKZF1 and IKZF3 compared to traditional IMiDs (Matyskiela et al., 2018). In addition to its direct anti-myeloma effects through IKZF1 and IKZF3 degradation, iberdomide exhibits robust immunomodulatory activity including activation of T cells and NK cells in the BM microenvironment of RRMM patients (Van Oekelen et al., 2024). Consistent with this, another study demonstrates that iberdomide’s clinical mechanisms of action are driven by both its cell-autonomous effects overcoming CRBN dysregulation in MM cells, and potent immune stimulation that augments anti-tumor immunity (Amatangelo et al., 2024). This comprehensive mechanism profile positions iberdomide as a promising therapeutic option for RRMM patients, particularly those with IMiDs-refractory disease. Its ability to simultaneously target myeloma cells and enhance anti-tumor immunity offers a unique therapeutic advantage in the evolving MM treatment landscape.

Nutlin-3, the first-in-class MDM2 inhibitor, disrupts the p53-MDM2 interaction by competitively binding to MDM2, thereby stabilizing p53 and activating the p53 signaling pathway. Studies have shown that Nutlin-3 induces apoptotic cell death in wild-type p53 (wt-p53) MM cell lines and primary patient samples. Mechanistically, this is mediated by upregulation of p53, p21, and MDM2 protein levels with a simultaneous increase in pro-apoptotic factors (PUMA, Bax, Bak), downregulation of anti-apoptotic proteins (Bcl2, survivin) and caspase cascade activation. Additionally, Nutlin-3 triggers p53 transcriptional-independent pathways, further contributing to its tumor-suppressive effects (Saha et al., 2010a). These findings highlight the therapeutic potential of nongenotoxic p53 pathway activation in MM. Importantly, Nutlin-3 exhibits synergistic activity with conventional anti-MM agents, including the proteasome inhibitor bortezomib (Ooi et al., 2009), reinforcing its clinical relevance as a promising combination therapy strategy. The addition of MDM2 inhibition to proteasome inhibition not only further stabilizes p53 and increases its protein levels, but also blocks its ubiquitination and transcriptional inactivation, thereby enhancing its functional activity.

RITA is a small-molecule inhibitor that disrupts the p53-MDM2 interaction by directly binding to p53, leading to p53 stabilization and pathway activation. In wt-p53 MM cell lines and primary patient samples, RITA triggers apoptosis through upregulation of proapoptotic NOXA, downregulation of antiapoptotic Mcl-1, and caspases activation. These findings are further corroborated in an MM xenograft mouse model, reinforcing RITA’s therapeutic potential. Interestingly, RITA’s mechanisms extend beyond p53 reactivation. Recent studies reveal that it can also induce G2/M cell cycle arrest, upregulate p53 targets (MDM-2, PUMA, NOXA) and promote PARP cleavage even in MDM2 inhibitor-resistant cells (Jones et al., 2012). Notably, RITA exhibits strong synergistic effects with Nutlin-3, significantly enhancing growth suppression in MM cells (Saha et al., 2010b). The synergistic effect likely results from Nutlin-3’s potent inhibition of p53-MDM2 interaction coupled with RITA’s p53 reactivation and other mechanisms described above. These findings suggest that dual targeting of p53 restoration and MDM2 inhibition represent a rational and promising strategy for MM treatment.