Approximately 60% of primary MM cells in the BM express CXCR4, with its expression inversely correlated with disease activity [15]. Previously, we revealed that high levels of CXCR4 on PC subclones and plasmablasts are associated with prolonged overall survival (OS) and progression-free survival (PFS), while low levels of CXCR4 on PC subclones are linked to inferior PFS and OS outcomes [16]. Contrary to findings suggesting an inverse correlation between CXCR4 expression and disease activity, MM cells exhibit high levels of CXCR4 in peripheral blood. However, in the BM, CXCR4 expression diminishes in response to elevated CXCL12 levels, leading to receptor internalization within MM cells. This internalization into the cytoplasm by CXCL12 signifies poor prognosis, while nuclear localization of CXCR4 indicates a better prognosis [17]. Furthermore, lower methylation frequency of CXCR4 genes in MM patients is linked to improved progression-free survival [18]. Elevated serum levels of CXCL12 are associated with increased osteolytic disease [19]. In our study, we observed a significantly elevated percentage of CXCR4-expressing cells in primary malignant PC of MM patients across premalignant MGUS, SMM, and active MM stages, compared to normal PC from healthy donors. Additionally, we determined CXCR4 overexpression in MM cell lines at both mRNA and protein levels, with the highest expression (> 90%) observed in RPMI-S, OPM-1, and OCI-My5 cells. Surrounding non-PC of the MM microenvironment showed a significantly upregulated percentage of CXCR4-expressing cells compared to non-PC from healthy donors. Furthermore, the disparities in CXCR4 expression between PCs and non-PCs increased in more advanced MM stages, including SMM, NDMM, and RRMM stages. Considering MM’s pathogenesis, the CXCR4/CXCL12 axis emerges as both a prognostic marker and a therapeutic target. This insight fuels the development of novel anti-MM therapeutics, ranging from antagonists to natural or synthetic small molecules, peptides, and monoclonal antibodies.

Understanding the CXCR4/CXCL12 axis has driven the development of anti-CXCR4 therapies. Approved CXCR4 antagonists, such as plerixafor (AMD3100/Mozobil; FDA-approved 2008) and motixafortide (BL-8040; FDA-approved 2023), potently enhance hematopoietic stem/progenitor cell (HSPC) mobilization in MM when combined with G-CSF, achieving 18–20 × 10⁶ CD34 + cells/kg and 76.8-fold HSPC increases over controls, respectively, while plerixafor demonstrates bortezomib chemosensitization (48.5% response rate) despite lacking direct anti-tumor approval [3, 9, 20]. Clinical-stage agents like mavorixafor (X4P-002; orally bioavailable for WHIM syndrome with cancer promise), ulocuplumab (BMS-936565; phase I/II MM trials showing bortezomib synergy), and lilotomab (BKT140) exhibit safety and tumor mobilization but face limitations including hematological toxicities and discontinued development (e.g., F50067) [21,22,23]. Preclinical inhibitors, including AMD3465, panobinostat (LBH589; FDA-approved with anti-MM synergy), PF-06747143, olaptesed pegol, and antibodies like LY2624587 or MDX-1338, further disrupt CXCL12 binding to induce apoptosis, ADCC/CDC, or enable imaging-guided therapies (e.g., 68 Ga/177Lu-pentixafor); however, many have stalled due to toxicity or suboptimal pharmacokinetics [24,25,26,27,28,29]. Thus, the development of novel therapies targeting both direct anti-MM effects and spatial tumor heterogeneity, while inhibiting MM cell dissemination, is crucial.

A small-molecule antagonist of CXCR4, WZ811 binds both the ligand- and agonist-binding sites of the CXCR4 receptor, thereby blocking both the binding and signaling of SDF-1α [30]. Pharmacological evaluations reveal EC50 values of 0.3 nM for binding affinity, 1.2 nM for inhibition of CXCL2-mediated cAMP modulation, and 5.2 nM for suppression of SDF-1-induced Matrigel invasion [13, 31, 32]. In contrast, another study reported very weak anti-CXCR4 inhibitory activity of WZ811 [12]. Despite conflicting reports on its inhibitory activity, WZ811 demonstrates significant anti-tumorigenic potential in diffuse large B-cell lymphoma (DLBCL) and CLL by inhibiting cell proliferation and survival, inducing apoptosis in vitro, and blocking tumor growth in vivo [11, 33]. In our study, we observed the cytotoxic effects of WZ811 on MM cell lines, including those resistant to conventional anti-MM therapeutic agents, with determined EC50 values of 20–40 μM. The pronounced anti-MM activity of WZ811 was corroborated in malignant PC isolated from primary MM patients, exhibiting EC50 values ranging from 21 to 177 μM. The uniform, time- and dose-dependent antiproliferative effects (a twofold viability decrease at 20–40 μM in most lines, such as MM.1S/OPM-1; sustained activity in drug-resistant RPMI variants) confirm that cell line mutations do not bias CXCR4 inhibitor response, validating WZ811’s mechanism via CXCL12 blockade rather than mutation compensation. Primary PC sensitivity (despite patient-specific mutations), versus resistance in non-malignant surrounding/accessory cells, further supports this, as CXCR4 signaling protects malignant PCs in the BM niche without mutation dependency. This consistency across MM cell lines and primary cells strengthens WZ811’s therapeutic potential, bypassing conventional resistance pathways while selectively targeting MM over healthy cells. Furthermore, the reduced tumor burden caused by WZ811 was confirmed in vivo in an MM xenograft mouse model, with notably extended survival.

The extrinsic pathway activates caspase-8, which triggers effector caspases-3 and −7 to cleave substrates like PARP, causing apoptosis and cellular disassembly. In CLL, WZ811 induces apoptosis through the down-regulation of Bcl2, upregulation of Bax, and activation of caspase-3 [11]. In our study, WZ811 treatment induced apoptosis in MM cells, as confirmed by the externalization of transmembrane phosphatidylserine, and was associated with the activation of caspase-3, caspase-7, and caspase-8, as well as the cleavage of PARP protein. In addition, WZ811 triggered upregulation of the pro-apoptotic Bcl-2 family regulator Bax and depolarization of mitochondrial membrane potential, indicating involvement of the intrinsic mitochondrial apoptotic pathway. Mcl-1S, a pro-apoptotic isoform of the Mcl-1 gene, promotes Bax activity, inhibits Mcl-1L survival signals, and is linked to mitotic acceleration, DNA damage, and apoptosis [34]. The simultaneous activation of caspases (particularly −3, −7, and −8), cleavage of PARP, upregulation of Bax, and Mcl-1S expression suggests a well-coordinated apoptotic response driven by both intrinsic and extrinsic pathways.

Increased levels of CXCR4 led to higher cellular concentrations of key signaling proteins associated with autophagy, specifically the protein markers LC3 and beclin [35]. LC3, a key marker of autophagy, transitions from cytosolic LC3-I to lipidated LC3-II to associate with autophagosome membranes and interact with SQSTM1/p62, which regulates autophagy, targets ubiquitinated substrates, and activates the Nrf2 pathway for antioxidant responses and stress management. In LC3 lipidation, ATG7, an E1-like enzyme, facilitates the conjugation of LC3 to phosphatidylethanolamine (PE), while ATG16L1, as part of the ATG5-ATG12 complex, supports this process and aids in targeting ubiquitinated cargo for degradation [36]. Our study demonstrated that WZ8111 significantly increased the levels of LC3A/B, SQSTM1/p62, ATG7, and ATG16L1, highlighting an active autophagic process crucial for autophagosome formation, cellular homeostasis, and the degradation of damaged organelles and protein aggregates. Therefore, the interplay between autophagy and apoptosis appears to drive WZ811’s anti-MM activity.

ATM, a serine/threonine kinase activated by double-strand breaks (DSBs) and recruited by the MRN complex (MRE11-RAD50-NBS1), undergoes autophosphorylation at Ser1981 to initiate DNA damage response signaling. In response to DSBs, ATM phosphorylates H2AX at Ser139, converting it into γH2AX, which marks damage sites, recruits repair proteins, and signals DNA repair and checkpoint activation [37]. Chk2, activated by ATM phosphorylation at Thr68, mediates cell cycle arrest at G1/S and G2/M checkpoints, allowing time for DNA repair before cell division. Elevated p-H2AX levels indicate ongoing DNA repair, marking DSBs, recruiting repair proteins like BRCA1 and MDC1 [38]. Similar to a previous study in CLL [11], we observed a significant increase in the G0/G1 phase in MM cells after exposure to WZ811, along with upregulation of ATM, Chk2, H2AX, and p-H2AX, triggering DNA damage response pathways such as H2AX phosphorylation and Chk2-mediated checkpoint activation to prevent the propagation of damaged DNA.

Pharmacological inhibition of the CXCL12/CXCR4 axis with WZ811 also prevents CD41+ megakaryocyte migration in bleomycin-induced lung fibrosis and reduces T-cell migration [11, 39, 40]. In thyroid carcinoma and NSCLC, WZ811 suppresses tumor invasiveness, inhibits epithelial-mesenchymal transition (EMT), increases E-cadherin, and reduces Twist, vimentin, and Snail expression [41, 42]. In gastric cancer, WZ811 counteracts FER1L4-mediated suppression of CXCR4 and CXCL12, enhancing tumor growth and metastasis [43]. CXCR4 expression correlates positively with mTOR expression, predicting reduced PFS and OS in DLBCL patients co-expressing these markers [44]. The mTOR pathway, often activated in MM, drives tumor growth and survival by promoting protein synthesis and suppressing autophagy, contributing to therapy resistance. SIRT1 (Sirtuin 1), a NAD+-dependent deacetylase, inhibits mTOR signaling via protein deacetylation and enhances autophagy to eliminate damaged components, acting as a stress-response mechanism in MM [45]. In addition, WZ811 reduces osteotropism and activates the mTOR and p70-S6 cell signaling proteins in melanoma cells [35]. In our study, WZ811 treatment similarly resulted in the activation of the mTOR signaling pathway, an increase in SIRT1, and a decrease in c-Myc and Notch1 proteins in MM cells. Reduced c-Myc and Notch1 levels might further promote plasma cell differentiation, shift metabolism toward oxidative phosphorylation, and potentially modulate mTOR activity. Together, these changes suggest a shift toward differentiation and stress adaptation; however, due to the anti-myeloma activity of WZ811, they may also indicate a stress response in MM. Therefore, we hypothesize that targeting CXCR4 with WZ811 may constitute an effective therapeutic approach for MM.

MM plasma cells exhibit phenotypic plasticity, with a subset of MM stem-like cells influencing disease initiation and progression. In the proposed MM stem model, quiescent MM stem cells, serving as tumor initiators, display heightened expression of CXCR4, integrins, and adhesion molecules, facilitating MM cell motility, migration, and adherence to BMSCs. Conversely, proliferative MM stem cells, driving disease progression and the emergence of evolved subclones, exhibit minimal CXCR4 expression, suggesting its downregulation in advanced MM stages associated with relapse [46]. In our previous study, we identified stem-like SP cells expressing CD138 and a high level of ABCG2 transporter activity as an enriched source of tumor-initiating cells in MM. These SP cells demonstrated the ability to regenerate the original population with clonogenic and tumorigenic properties [47]. Besides HSCs, CXCR4 is expressed in SP cells in various cancer stem cells, including those in breast cancer, glioblastoma, and MM, and is linked to increased tumorigenicity, metastatic potential, and resistance to chemotherapy [47]. CXCR4 is also expressed in neural stem cells, mesenchymal stem cells, and cardiac progenitor cells [48]. This highlights the importance of CXCR4 expression in SP cells across various tissues and its role in mediating stem cell functions and therapeutic potential. Moreover, the anti-MM agent lenalidomide decreased the percentage and clonogenicity of SP cells, both in MM cells alone and in the context of BMSCs [47]. Treatment with WZ811 significantly reduced the proportion of stem cell-like SP cells in MM. In the context of BMSCs, although the anti-SP activity of WZ811 was attenuated in both MM cell lines at 24 h, the SP fraction decreased more significantly in RPMI-S cells compared to OPM-1 cells after 72 h of treatment. Our studies show that WZ811 effectively targets the SP fraction in MM cells, establishing a foundation for new therapeutic strategies aimed at eliminating subpopulations of MM cells, including potential stem cells.

CXCR4 is a key marker of bone metastasis in MM, universally upregulated in plasma cells. Its interaction with CXCL12 facilitates MM cell rolling on endothelium, recruitment of CXCL12-expressing stromal cells, and organ-specific homing, including to the liver, lungs, and bone marrow. This axis drives MMP synthesis, enabling ECM degradation, tumor cell detachment, bloodstream egression, and migration to metastatic sites [49]. We showed that WZ811 treatment triggered the upregulated expression of CXCL12 and ECM molecules, including collagen IV and laminin, in MM cells, and these effects were further enhanced in the presence of stromal cells. Treatment with WZ811 can alter signaling pathways within MM cells, potentially triggering feedback mechanisms that upregulate the expression of certain cytokines and chemokines, including CXCL12, either to maintain homeostasis or in response to stress induced by WZ811. Moreover, WZ811 may cause changes in stromal cells that indirectly promote the production of CXCL12 in MM cells. WZ811 may activate key signaling pathways that regulate ECM production, such as TGF-β, Wnt, or integrin signaling, leading to increased synthesis and deposition of ECM components, including collagen IV and laminin. By targeting SP cells or affecting cytokine production (such as CXCL12), WZ811 might modulate interactions between MM cells and stromal cells, leading to ECM remodeling and increased production of ECM proteins.

Combination drug therapy can target multiple pathways involved in cancer cell growth and survival by employing drugs with different mechanisms of action. Moreover, the likelihood of cancer cells simultaneously developing resistance to all agents is significantly reduced. Certain drug combinations exhibit synergistic interactions, where the combined therapeutic effect is greater than the sum of the individual effects, thus targeting different but complementary cellular pathways. The combination of the CXCR4 inhibitor WZ811 with conventional anti-MM agents demonstrates synergistic effects with DOX, an anthracycline that intercalates DNA, inhibits topoisomerase II, and generates free radicals leading to DNA damage and apoptosis, as well as with DEX, a corticosteroid that induces apoptosis. The combination of WZ811 with the alkylating agent MEL demonstrates a range of outcomes, from synergistic to additive to antagonistic, in various MM cell lines. The combination of WZ811 with proteasome inhibitors and IMIDs demonstrates synergistic effects with BTZ, LEN, and POM, while it exhibits antagonistic interactions with CFZ. Thalidomide further downregulates CXCR4 and CXCL12, while carfilzomib inhibits CXCR4 phosphorylation [50]. Moreover, WZ811 significantly enhances the sensitivity of CLL cells to docetaxel [11] and exhibits a synergistic effect in combination with the mTOR inhibitor everolimus in DLBCL cell lines [44]. For example, DOX and DEX may enhance the apoptotic pathways activated by WZ811, while MEL and CFZ may compete with or disrupt the mechanisms triggered by CXCR4 inhibition, depending on the cellular context. Furthermore, the interplay between WZ811’s inhibition of CXCR4 signaling and the proteasome/immune modulatory pathways targeted by BTZ, LEN, and POM may explain their synergistic effects. Using lower doses of each drug in combination therapy can reduce the incidence of dose-limiting toxicities associated with higher doses of single agents. For example, dexamethasone at lower doses reduces the risk of steroid-related side effects such as hyperglycemia and immunosuppression, doxorubicin at lower doses minimizes the risk of cardiotoxicity, and bortezomib at lower doses can reduce the risk of peripheral neuropathy. To maximize therapeutic outcomes, sequential or concurrent administration of drugs can be tailored to exploit specific vulnerabilities of cancer cells at different stages of the cell cycle or in distinct tumor microenvironments. Combination therapy can address tumor heterogeneity by targeting multiple subpopulations within the tumor, thereby reducing the likelihood of treatment failure due to clonal expansion of resistant cells, such as SP cells.

This study demonstrates the preclinical efficacy of WZ811 against MM through CXCR4 blockade, manifesting as apoptosis induction, autophagy promotion, cell cycle arrest, and modulation of MM-associated signaling pathways across diverse MM cell lines, ex vivo patient-derived plasma cells, and in vivo xenograft models. WZ811 further reduced the stem-like side population fraction and synergized with anti-MM agents, establishing a rationale for combination therapies to overcome stromal protection and resistance. However, reliance on transformed/immortalized cell lines and xenograft models introduces limitations, including incomplete recapitulation of human tumor heterogeneity, bone marrow microenvironment dynamics, and acquired resistance mechanisms prevalent in relapsed/refractory disease. Future investigations should advance WZ811 into Phase I clinical trials for relapsed/refractory MM, prioritizing pharmacokinetic/pharmacodynamic profiling, CXCR4/CXCL12 biomarker stratification, and safety assessments to mitigate potential cytopenias from prolonged blockade. This should be complemented by evaluation of rational combination strategies, multi-omics analyses of stress and survival pathways, and validation in humanized models to support durable anti-myeloma activity.