WITHDRAWN: Canonical NF-κB regulator CYLD mutations reinforce RelB mediated disease pathogenesis in Multiple Myeloma

Multiple myeloma (MM) is a n incurable plasma cell malignancy characterized by aberrant activation of NF-κB signaling pathways. While both the canonical (RelA/p50) and non-canonical (RelB/p52) NF -κB cascades contribute to MM pathogenesis, the mechanisms governing their intersection remain poorl y defined. Here, we identify the tumor suppressor CYLD as a critical modulator of NF-κB crosstalk that enforces RelB-dependent transcriptional programs driving MM progression. Primary CD138⁺ plasma cells from MM patients and patient -derived myeloma cell lines were analyzed using NF -κB DNA binding assays and gene expression studies . Large scale transcriptomic analyses were performed using the Multiple Myeloma Research Foundation (MMRF) dataset s. Combining biochemical and in silico studies, we established that e levated nuclear RelB levels correlated with enhanced RelB expression and poor clinical outcomes. Mutational loss of CYLD, a well characterized negative regulator, increased canonical NF -κB activity and further amplified RelB expression and activity, particularly upon non-canonical pathway stimulation. This CYLD deficiency , causally established by CRISPR-Cas9–mediated CYLD gene deletion , conferred a survival and migratory advantage through RelB -dependent upregulation of pro -survival factors such as BCL2, BIRC2, BIRC3, TRAF1, and c -FLIP & chemokine receptors like CXCR4 and CXCR7 that are involved in cell migration. These findings define a novel CYLD–RelB signaling axis that integrates canonical and non -canonical NF -κB pathways to promote MM cell survival and dissemination. Disruption of CYLD augments RelB - driven transcription and sustains tumor -promoting NF-κB crosstalk. Our study demonstrates that modulating this axis may serve as an effective therapeutic strategy against NF -κB–mediated multiple myeloma progression. WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint 1. Introduction Multiple myeloma (MM) is a fatal plasma cell cancer that involves the clonal expansion of malignant plasma cells in the bone marrow. MM is the second most widespread hematologic malignancy after non -Hodgkin lymphoma, with a global estimate of 188,000 new cases and 121,000 deaths mortalities annually ((Jemal et al., 2009; Mafra et al., 2025) . Various studies estimate that the incidence of MM in India is around 1.2 -1.8 per 100,000 individuals(Roy et al., 2018). MM is characterized by the clonal proliferation of cancerous plasma cells (PCs) in the bone marrow microenvironment (Kyle & Rajkumar, 2004; Mahindra et al., 2010; Palumbo & Anderson, 2011). In the absence of a single unifying genetic event corroborating disease manifestation, studies have focused on understanding signaling pathways deregulated in myeloma cells(Demchenko et al., 2010; Ramakrishnan & D’Souza, 2016). Within the tumor microenvironment, immune cells and stromal cells secrete a diverse array of pro- inflammatory cytokines, which activate key survival pathways in malignant cells (Chauhan et al., 1996; Fairfield et al., 2016; Jourdan et al., 2014) . Cancerous mutations are also known to trigger these cell-signaling pathways in a cell-intrinsic manner. Of particular importance is the “Nuclear Factor--light-chain enhancer of activated B cell” (NF- B) signaling system, which forms a major link between cancer and inflammation (Chatterjee et al., 2019; Sen & Baltimore, 1986) . MM, in fact, provides one of the best examples where several mutations have been mapped onto the NF - B system (Demchenko et al., 2010; Keats et al., 2007; Roy et al., 2018) . The NF- B family of transcription factors functions in a wide range of cell types and orchestrates innate and adaptive immune responses(Hayden & Ghosh, 2004). Not surprisingly, deregulated NF- B activities have been implicated in several human ailments, including hematologic cancers (Baud & Karin, 2009). Extracellular stimuli trigger the canonical (also known as classical) or the non -canonical NF-B pathways to induce translocation of cytoplasmic NF -B dimers into the nucleus, where they mediate the expression of hundreds of immune and stress response genes as well as pro - tumorigenic, survival factors(Roy et al., 2017). Importantly, the canonical and non-canonical NF- B pathways were shown to be interlinked(Chatterjee et al., 2019). However, it remains elusive if such interconnectedness of the NF -B system indeed plays a role in multiple myeloma. The deregulated activity of NF-B factors fuels the cancerous growth of myeloma cells within the bone marrow niche and instigates multiple osteolytic bone lesions. WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint Tumor microenvironment -derived cytokines, such as, B -cell activating factor (BAFF) and Lymphotoxin (LT), signal predominantly through the noncanonical arm to activate the NF-B system in myeloma cells (Annunziata et al., 2007; Demchenko et al., 2010; Keats et al., 2007). In addition, cancer-associated mutations, deletions, and gene rearrangements have been shown to occur in genes involved in NF-B regulations, such as TRAF2, TRAF3, BIRC2, BIRC3, CYLD, NIK, LTBR, CD40, TACI, NFKB1, NFKB2, BTRC, CARD11, IKBIP, IKBKB, MAP3K1, MAP3K14, RIPK4, TLR4, and TNFRSF1A (Chapman et al., 2011; Lohr et al., 2014; Ramakrishnan & D’Souza, 2016; Roy et al., 2018) . Therefore, prevailing literature put forward the NF-B system as an important component in myeloma pathogenesis. MM, alongside many other hematological malignancies, has been studied widely to be associated with deleterious mutations in the NF-B regulatory genes (Annunziata et al., 2007; Demchenko et al., 2010). Most of these mutations led to constitutive activation of primarily the noncanonical NF- κB pathway (Keats et al., 2007) . However, CYLD, which negatively regulates canonical NF -κB signaling, was also found to be mutated in 15-20% of MM cases(van Andel et al., 2017). Molecular mechanism that connects mutational inactivation of CYLD to MM pathogenesis remains poorly characterized. Absence of negative regulation such as mutational inactivation of CYLD also provokes constitutive canonical RelA signaling (Brummelkamp et al., 2003; Kovalenko et al., 2003; Trompouki et al., 2003) . However, none of the existing literature shows if CYLD has an impact on the non-canonical RelB/NF-κB pathway. Interestingly, in a B cell homeostasis study, RelB was shown to be elevated in B cells of CYLDex7/8 mice, which expressed a defective version of CYLD(Hövelmeyer et al., 2007). These studies put forward a hypothesis that CYLD deficiency in MM, may not only hyper -activate canonical NF -κB signaling , but may also aggravate non - canonical RelB -dependent NF -κB responses , thus pointing towards the interconnected NF -κB system. This motivated us to further examine the effect of CYLD mutations on RelB NF -κB signaling. WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint 2. Materials and Methods Multiple myeloma patient samples Bone marrow aspirates from 74 MM patients, registered for biopsy at IRCH, AIIMS-New Delhi, were collected after informed consent adhering to institutional human ethics guidelines . 2-5mL of Bone - marrow biopsy samples were collected from newly diagnosed or treated patients in EDTA Vacuum Blood Collection Tube. Bone-marrow mononuclear cells (BM-MNCs) were isolated from biopsy aspirates by Ficoll density gradient centrifugation. Mononuclear cells isolated from MM patient bone marrow aspirates were positively sorted using anti-CD138 magnetic microbeads via magnetic activated cell sorting (MACS) system following manufacturer’s protocol (Mi ltenyi Biotec, Auburn, CA) to >90% purity as determined by anti -CD138-PE staining and Flow cytometry analysis. For gene expression analyses, patient matched BM -MNCs (CD138 negative fraction) were used as non-tumor tissue control. Human cell lines Human Myeloma Cell Lines (HMCLs ; kind gift from Dr. Michael Kuehl, NCI ) and (Human embryonic kidney cells (HEK293T cell line) were cultured in Roswell Park Memorial Institute medium (Invitrogen) and DMEM respectively. Both types of media were supplemented with 10% (v/v) Fetal Bovine Serum (Gibco), 1% (v/v) of 5,000μg/mL penicillin-streptomycin and 1% (v/v) of 200mM L -glutamine. KP -6 cells are IL -6 dependent and were grown in the presence of additional supplement of 2ng/mL Recombinant Human IL -6 (BD Pharmingen). 90% confluent 293T cells were passaged every two-to-three day using trypsin containing 0.05% EDTA. HMCLs were maintained as suspension culture and were split whenever cell density reached approximately 1 million cells per ml. For biochemical experiments, 0.5 million cells per mL were used. Biochemical analyses HMCLs were treated with 50 -100 ng/mL recombinant human BAFF (Gibco ; Cat # 310 -13R- 50UG) to assess nuclear NF -B activity in EMSA, whole cell protein immunoblots and gene expression analyses cells were harvested, and nuclear or whole-cell extracts were prepared and analyzed by EMSA, super-shift analysis, or Western immunoblotting or immunoprecipitation (for NEMO:IKK Kinase assay ) as described previously (Banoth et al., 2015) . Primary antibodies WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint against RelA (sc-372) RelB (sc-226), c-Rel (sc-71), IBα (sc-371), IKK1 (sc-7184) β-Actin (sc- 1615) and HRP-conjugated secondary antibodies Goat anti-rabbit (sc-2004) and Donkey anti-goat (sc-2020) were procured from Santa Cruz Biotechnologies. Antibody against p50 (BB -AB0080) was purchased from BioBharati Life Sciences Ltd. Antibodies against p100/52 (#4882), C YLD (#8462) and GAPDH (#2118) were from Cell Signaling Technologies. For gene expression studies , cells were harvested in RNAiso Plus reagent (Takara; Cat # 9108) and total RNA from the cells were isolated and followed by cDNA synthesis (Takara; Cat # 6110A) for qRT-PCR based gene expression studies (see Appendix Table S1 for primer descriptions). Cell-Death Assay HMCLs were treated with optimized IC50 of Velcade (Bortezomib, LC laboratories) in a 6 -well plate for cell death studies. IC50 for cell -death studies was measured at 24h time post -Velcade treatment using CyQUANT ™ XTT Cell Viability Assay Kit (Cat # X12223) as per the manufacturer’s guidelines. Subsequently, absorbance was measured at 450 nm in a 96 -well plate reader and percent cell viability was measured. Half maximal inhibitor concentration (IC50) at 50% cell death was calculated using non-linear regression analysis. In the cell-death assay, HMCLs were treated with 100 ng/mL of BAFF for 16h prior to Bortezomib treatment. Apoptosis and necrosis were measured by Flow cytometry -based (BD Canto) quantitation of Annexin-V (FITC/PE) and propidium iodide/7 -AAD (BD Pharminogen) stained cells using cell apoptosis detection kit (BD ; Cat # 559763) following manufacturer’s guidelines. Flow cytometry data were analyzed using FlowJo software v10. Transwell Cell-Migration Assay For migration studies, transwell inserts with 8 μm pores in a 24 -well format (Corning Costar, Cambridge, MA) were used . Around 0.1 million HMCLs in 300 µL of serum -free RPMI 1640 medium were seeded onto the upper chamber and 500 µL of RPMI 1640 medium with 100 ng/mL of SDF-1 (PeproTech Cat # 300-28A-50UG) added in the lower chamber (Figure 4A). After 48 h incubation at 37°C, migrated cells were counted under inverted light microscope. WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint MMRF large metadata Analysis using R The RNA -seq metadata available with the Multiple Myeloma Research Foundation database IA13a were used for in silico analyses. The R programming language was used to write the codes, to be available upon request , for generating data. The statistical analyses were carried out using Welch’s t-test. Statistical analysis All data were presented as mean +/- SEM of 3 –5 biological replicates. Statistical significance between groups were calculated using appropriate two-tailed Student's t-test, Welch’s t-test and two-way ANOVA, where “*”, “**” and “***” indicate P<0.05, P<0.01, and P<0.001, respectively. WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint 3. Results 3.1 Nuclear RelB activity is elevated in plasma cells in myeloma patients. Studies have shown engagement of NF-κB signaling in multiple myeloma for over a decade now(Keats et al., 2007; Roy et al., 2018) . Corroborating on this , we acquired bone marrow aspirates from 74 patients diagnosed with MM . Reflecting global statistics of enhanced male vulnerability, 74% of patients were male and the remainder were female in this cohort ( Figure S1A). Based on the clinical history, we classified these patients into two groups, viz. (a) untreated group with newly diagnosed patients (N=29) and (b) treated group with remission (N=27) and relapsed (N=18) patients ( Figure S1A). Post biopsy, bone marrow aspirates were subjected to ficoll density centrifugation to isolate the layer of Bone Marrow Mono -Nuclear Cells (BM - MNCs). CD138+ plasma cells were isolated using MACS based immune-magnetic cell separation (Figure S1B)(Flores‐Montero et al., 2016). As per the availability of sufficient cell numbers post sort, NF-κB DNA binding assay was performed on native protein fraction from the nuclear extracts from CD138+ cells from 10 newly diagnosed patients and 5 cases on treatment . Importantly, we found that CD138+ cells from almost all cases of newly diagnosed myeloma patients also possessed nuclear RelB (nRelB) activity, suggesting ongoing non -canonical NF-κB signaling in multiple myeloma (Figure 1A). Further quantitation of the nuclear NF-κB activity established that nuclear RelA (nRelA) and nRelB activities were co -existent in these patients’ samples (Figure 1B). Performing statistical measure in UpSet plot, we further revealed that the nRelB activity positively correlated with the nRelA activity in the newly diagnosed myeloma patients ( Figure 1C). Our gene expression analyses revealed that newly diagnosed patients with elevated nuclear RelB activities (see Figure 1A) also possessed significantly higher levels of RelB mRNA (Figure 1D). To further dissect the engagement of NF-B pathways in myeloma pathogenesis, we ventured into the Multiple myeloma research foundation (MMRF) database, which catalogs clinical features and molecular attributes of close to a thousand myeloma patients. Analyzing MMRF data in the R programming platform, MM patients with high RelB levels (n=198 cases) were associated with a significant decrease in the days to overall survival (Figure 1E). Collectively these data established the importance of elevated RelB presence in patients with MM. However, these results also motivated towards the molecular signatures that leads to this state. Many studies have shown the wide presence of CYLD mutations in MM(Keats et al., 2007; van Andel et al., 2017) . However, WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint we wanted to establish the mechanistic details for the interconnectedness of the CYLD mutations on RelB NF-κB signaling. 3.2 Absence of CYLD leads to high RelB levels and heightened RelB NF-κB activity. To address if myeloma -associated CYLD mutations also aggravated non -canonical RelB/NF-κB signaling, we set out to compare CYLD -sufficient and CYLD -deficient HMCLs in our in vitro studies. Myeloma patient derived KP-6 cells harbor a deletion in the gene encoding CYLD, whereas no such mutations in the NF-κB pathway were reported for JIM3 cells, serving as controls in our experiments. As such, CYLD inhibits the canonical NF -κB activity by destabilizing the NEMO-IKK complex. Therefore, we first measured the NEMO -IKK activity in these HMCLs in an in vitro kinase assay. As expected, we noticed a basally high NEMO -IKK kinase activity in KP-6 as compared to JIM3 cells (Figure 2A). In our immunoblot analyses, we then examined the cellular level of IκB, which is degraded upon NEMO:IKK activation. Indeed, there was a marked decrease in the IκB level in KP -6 cells in comparison to JIM3 ( Figure 2B ). There was no discernable difference in the level of RelA, whose abundance is known to be insensitive to canonical signals, between these HMCLs. To confirm that proteasomal degradation upon NEMO:IKK-mediated phosphorylation led to the depletion of IκB in KP-6 cells, we treated these cells with the proteasome inhibitor MG132. Our immunoblot analyses demonstrated that proteasomal inhibition restored the IκB level in KP-6 cells (Figure 2C). Our analyses confirmed that CYLD deficiency triggered basal, cytokine -independent but NEMO -IKK-driven canonical signaling in myeloma cells. Next, we performed immunoblot analyses to further probe the cellular levels of the non-canonical NF-κB signal transducers RelB and p100/p52. KP-6 cells, with the absence of CYLD , have increased accumulation of RelB and p100 ( Figure 2D). To demonstrate if these KP-6 cells also elicited hyperactive non -canonical nuclear RelB/NF-κB activity in response to cytokine stimulation such as BAFF, a well-known non-canonical NF-κB pathway activating cue. BAFF treated KP-6 and JIM3 cells were examined for NF-κB DNA binding activity. Our EMSA analyses revealed a strong basal as well as BAFF -induced nuclear RelB /NF-κB activity in KP -6 cells, whereas JIM3 showed a subdued response to BAFF treatment ( Figure 2E). Importantly, our flow cytometric analyses established that BAFF receptors were expressed in both JIM3 and KP-6 cells (Figure S2A). These studies confirmed that aggravated canonical NF-κB signaling, caused due to WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint the deficiency of CYLD, produced basal RelB activity in the nucleus and also fueled heightened non-canonical RelB NF-κB response. Using the transcriptomic data from MMRF, we could find a cohort of patients with mutated CYLD gene. W e could also corroborate that patients with CYLD mutation (n=23) have significantly higher levels of RelB mRNA (Figure 2F). As a member of key component from the family of transcription factors, RelB has also known be transcriptionally regulating several pro-survival and pro-migration genes (Authier et al., 2014; Chatterjee et al., 2019; Cormier et al., 2013; Eluard et al., 2022; Roccaro et al., 2015; Shen et al., 2018) . Of importance, these CYLD mutated cases showed significantly increased expression of BCL2 and CXCR4 (Figure 2G). 3.3 BAFF provides a selective survival advantage to CYLD deficient cells. Within the tumor microenvironment, cytokine such as BAFF promote myeloma cells’ growth and proliferation and instill therapeutic drug resistance (Moreaux et al., 2004) . The protective role of BAFF was tested against the well-established therapeutic agent Bortezomib, a 26S proteasome inhibitory drug that still finds its place in most of the currently approved multiple myeloma treatment regimes(Neri et al., 2011; Richardson et al., 2003, 2005; Song et al., 2017). By inhibiting the 26S proteasome, bortezomib prevents the activation of the canonical NF -κB pathway. Bortezomib treatment induces apoptosis in metabolically active and rapidly dividing cancerous cells engaged in extensive protein synthesis. Because CYLD deficiency aggravated non-canonical RelB activity and elevated RelB in has been mapped onto poor survival, we asked if CYLD dysfunctions offered survival advantages to HMCLs. To this end, we set out to compare bortezomib-induced death in BAFF-treated JIM3 and KP-6 cells as indicated in the experimental workflow in Figure 3A. Given that we were working with non -isogenic cell lines, half -maximal inhibitory concentration (IC50) of bortezomib were determined individually for KP -6 and JIM3 cells in the XTT cell viability assay ( Figure S3 A). KP -6 and JIM3 cells were treated with Bortezomib at the respective IC50 dose for 16hrs. Alternately, cells were also subjected to BAFF stimulation for 16hrs before being subjected to Bortezomib treatment. We found that BAFF pre-treatment did not protect JIM3 cells from bortezomib -induced death (Figure 3B). There was no significant difference in the quantified numbers of viable cells after the Bortezomib treatment when compared to cells receiving BAFF priming ( Figure 3 C). Flow cytometry analyses revealed the presence of both early and late apoptotic cells as well as necrotic WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint cells in the JIM3 population at 16hrs post -bortezomib treatment; prior BAFF stimulation did not impact either of these compartments ( Figure S3B ). On the contrary, pre-treatment with BAFF substantially rescued CYLD deficient KP-6 cells from the Bortezomib-induced death (Figure 3D & Figure 3E) that was associated with a significant decrease in the apoptotic as well as the necrotic cell population (Figure S3C). Furthermore, we compared KP-6 and JIM3 cells for the expression of RelB mRNA levels and other pro-survival BCL-2 (BCL2), c-FLIP (CFLAR), CIAP1(BIRC2), CIAP2(BIRC3), and TRAF1 (TRAF1), in our gene expression analyses. KP-6 cells have higher levels of RelB expression both at basal and BAFF induced state (Figure 3F). Corroborating the nuclear RelB activity observed in these cells, BAFF stimulation for 16hrs further upregulated the expression of these pro-survival factors in KP -6, but not JIM3, line (Figure 3G). Elevated levels of pro-survival genes were also observed at untreated basal state in KP -6 cells except for CIAP1 & TRAF1 (Figure S3D). This allowed us to conclude that the noncanonical signal inducer BAFF offered a selective survival advantage to myeloma cells bearing inactivating CYLD mutations, particularly in the context of chemotherapeutic drug treatment. 3.4 BAFF reinforces migration potential of KP-6 myeloma cells with deletion of CYLD. Myeloma cell migration promotes multiple bone lytic lesions and underlies cancer cell invasion and metastasis(Chen et al., 2016; Qiang, 2005). Myeloma cells are thought to enter blood vessels in the periphery and then migrate to the bone marrow (BM) microenvironment by extravasating from the vascular endothelium (Chen et al., 2016). Notably, a select set of chemokines, including SDF1 (CXCL12), has been implicated in bone marrow metastasis (Roccaro et al., 2014) . We asked if the strengthened RelB pathway in CYLD-deficient HMCLs was associated with increased cell migration potential. To this end, transwell cell migration assay was performed as per schema discussed in Methods section and shown in Figure 4A. JIM3 and KP-6 cells were treated with BAFF for 16hrs alongside an untreated control and then placed in the transwell insert with recombinant SDF1 (CXCL12) as a chemoattractant in the lower chamber. We noticed that a significantly higher number of KP-6 cells migrated to the lower chamber both basally as well as upon BAFF stimulation ( Figure 4C & Figure 4D). However, the migratory of JIM3 cells was rather timid, and BAFF did not improve the transwell migration of these cells even under the influence of a chemogradient ( Figure 4B & Figure 4D). These results found a positive correlation between the strengthened RelB pathway in CYLD deficient KP-6 cells WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint and the higher migration potential of these cells that was further reinforced upon BAFF stimulation. The mutual interaction between myeloma and bone marrow stromal cells (BMSCs) promotes myeloma cell migration to the secondary sites in the bone marrow (Qiang, 2005) . Chemokines play important roles in tumor metastasis and dissemination. Stromal-derived SDF1 binds to its cognate receptors CXCR4 and CXCR7 on the surface of myeloma cells(Gilbert et al., 2019). Indeed, CXCR4 has been implicated in the metastasis of myeloma cells and in the epithelial-mesenchymal-transition (EMT) (Roccaro et al., 2015) . Because we found a strong correlation between the heightened activity of the transcription factor RelB and increased cell migration in CYLD-deficient HMCLs, we sought to examine the abundance of mRNAs encoding cell migration-promoting factors in KP-6 cells. Our gene expression analyses confirmed a basally upregulated expression of CXCR4, and CXCR7 in KP-6 cells as compared to JIM3 (Figure S4A). Moreover, the expression of CXCR4, but not CXCR7, mRNA was further enhanced upon BAFF stimulation of KP -6 cells (Figure 4F). The expression of mRNAs encoding these chemokine receptors was unchanged upon BAFF stimulation of JIM3 cells (Figure 4E).

from Section 3.3 and 3.4 provided evidence that heightened RelB activity was associated with increased survival and migration of myeloma cells. Further experimental validation involving genetic dissection of isogenic lines was planned to establish the causal role of the CYLD -RelB axis in tumorigenesis. 3.5 Deletion of CYLD in JIM3 cells strengthens BAFF mediated cell survival. In our experiments with HMCLs (Section 3.3 & 3.4 ), we used CYLD -deficient KP-6 cells and CYLD-sufficient JIM3 cells. However, we wanted to further validate our results in isogenic myeloma cell lines. To this end, we subjected JIM3 cells to CRISPR-Cas9-mediated gene editing by targeting exon 5 of the hum an CYLD gene. Additionally, non -target sgRNA was used to generate the control JIM3 sgNT (sgNon-Target) cell line. Immunoblot analysis was performed to confirm the abrogation of CYLD expressions in JIM3 sgCYLD cells (Figure 5A). The purity of transduced JIM3 cells was determined by flow cytometry using the GFP expression (Figure 5B). Next, we compared if JIM3 sgCYLD and JIM3sgNT cells were equally protected from Bortezomib - induced death by BAFF. To this end, cells were stimulated with BAFF (100 ng/ml) for 16hrs alongside an untreated control before being subjected to Bortezomib treatment at IC50 for another 16hrs. Apoptosis and necrosis were measured in our flow cytometry analyses by scoring Annexin- WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint V (PE) and 7 -AminoActinomycin D (7 -AAD) staining of the cell populations ( Figure S5A & Figure S5B). Cells negatively stained for PE and 7-AAD were counted as healthy and viable. Interestingly, BAFF did provide survival advantages to CYLD -deficient JIM3sgCYLD cells against Bortezomib-induced cell death (Figure 5D & Figure 5F) but did not protect JIM3 sgNon-target cells (Figure 5C & Figure 5E). In addition, 16 hrs of BAFF treatment of JIM3sgCYLD cells clearly showed a significant decrease in the apoptotic or the necrotic population upon subsequent Bortezomib insult (Figure S5B). JIM3sgNon-target cells were not rescued from apoptosis or necrosis even after BAFF priming (Figure S5A). Additionally, our mRNA analyses revealed that JIM3sgCYLD cells possessed basally elevated levels of RelB mRNA in comparison to control cells (Figure S5C). BAFF stimulation further augmented the RelB RNA expression in JIM3sgCYLD, but not in JIM3sgNon-target cells (Figure 5G). Finally, BAFF potently upregulated the expression of the panel of RelB-target pro -survival genes in CYLD - deficient cells, but not in CYLD -sufficient JIM3 sgNon-target cells ( Figure 5H). These results confirmed a dominant causal role of CYLD-RelB axis in regulating the expression of pro-survival genes and conferring resistance to CYLD -deficient myeloma cells from cell death -inducing chemotherapeutic drugs. 3.6 BAFF rescues the migration potential of JIM3 myeloma cells with deletion of CYLD. As described earlier, CYLD -deficient KP-6 cells displayed higher migration potential compared to CYLD-sufficient JIM3 cells ( Figure 4). We asked if deletion of CYLD in the isogenic JIM3

led to similar outcomes using the same functional transwell cell-migration assay. JIM3sgCYLD cells or JIM3 sgNon-target cells were treated with BAFF for 16hrs and then seeded in transwell inserts over wells of a 24 well plate. Recombinant SDF1  (CXCL12) was added in the lower chamber as a chemoattractant. We observed that as compared to control JIM3sgNon-target cells, a significantly greater number of JIM3sgCYLD cells migrated to the lower chamber even without the pre-treatment of BAFF (Figure 6A & Figure 6B). BAFF priming augmented this effect further in JIM3sgCYLD cells (Figure 6A & Figure 6B). Like CYLD-deficient KP-6 cells, CYLD-deleted JIM3 cells also displayed BAFF -responsive expressions of chemokine receptors such as CXCR4 and CXCR7, presumably owing to strengthened non -canonical RelB activation upon CYLD inactivation (Figure 6D). BAFF was unable to induce the expression of CXCR4 and CXCR7 in CYLD sufficient JIM3sgNon-target cells (Figure 6C). WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint These findings established a causal role of CYLD in modulating the migration of myeloma cells. Previous studies, including those from our group (Roy et al., 2015), supported a role of RelB in the pathogenesis of MM. Our results provide unambiguous genetic evidence that CYLD inactivation is sufficient to upregulate the expression of RelB and RelB-dependent genes, including those encoding pro-survival and pro-migration factors. 3.7 RelB supports survival and migration specific gene upregulation in CD138+ cells in patients with MM. To establish the role of RelB mediated transcriptionally control in upregulation of pro-survival and pro-migration factors, we used the clinical samples collected from patients with MM. We isolated total RNA from these MACS-purified CD138+ cells; and utilized patient matched BM -MNCs as tumor tissue controls (Figure 7A). Our gene expression analyses revealed that patients possessed a significantly higher levels of RelB mRNA in the CD138+ cell fraction as compared to non-tumor tissue from the CD138 negative fraction (Figure 7B). Almost 10-fold elevated RelB expressions in CD138+ malignant plasma cells, warranted if these cells also produced augmented amounts of mRNAs encoding various RelB -target pro -survival and pro -migration factors. Considering the panel of five RelB-target anti-apoptotic genes, such as CIAP1, CIAP2, TRAF1, c-FLIP, and BCL- 2 (Cormier et al., 2013; Roy et al., 2017, 2018) , we found that compared to MNCs, CD138+ myeloma cells indeed displayed substantially higher accumulation of these mRNAs, as also reflected by the transcript heat -map (Figure 7C). These data strongly support that a heightened RelB NF -κB activity sustained the expression of anti -apoptotic genes resulting in aggravated growth of malignant cells and robust myeloma progression. Patients with high RelB expressions also had significantly higher levels of CXCR4 and CXCR7 mRNAs (Figure 7D). In fact, the bone marrow microenvironment is reported to have high levels of SDF -1, and elevated expression of its cognate receptors CXCR4 and CXCR7 allows this signaling axis to be constitutively active in MM(Gilbert et al., 2019). WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint

RelB has emerged as a central node integrating genetic lesions and microenvironmental cues to sustain myeloma cell fitness. Consistent with earlier studies showing constitutive RelB activation and upregulation of anti‑apoptotic NF‑κB target genes in a substantial fraction of newly diagnosed multiple myeloma patients (Cormier et al., 2013 ), our clinical metadata and patient sample analyses indicate that RelB is tightly coupled to both survival and migratory behavior of myeloma cells. The exacerbation of this RelB‑dependent phenotype by CYLD loss aligns with the broader concept that disruption of negative NF‑κB regulators amplifies oncogenic signaling in plasma cell malignancies including multiple myeloma (Roy et al., 2018). The observed correlation between RelB abundance in CD138⁺ myeloma cells and the expression of major pro‑survival genes such as BCL2, as well as pro‑migration genes like CXCR4, reinforces the notion that non‑canonical NF‑κB signaling extends beyond classical function of regulating organogenic chemokines to encompass a wider anti‑apoptotic and chemotactic gene program. Previous genomic surveys have highlighted gain‑of‑function events in alternative NF‑κB components (e.g. NIK activation, TRAF2/3 and cIAP1/2 loss) in myeloma, providing one route to sustained RelB activity (Demchenko et al., 2010) . Our data complement these findings by demonstrating that inactivating mutations in CYLD, a deubiquitinase that restricts canonical NF‑κB, can secondarily reinforce non‑canonical RelB signaling, thereby creating a feed‑forward loop that promotes tumor growth and dissemination (Figure 8). From a therapeutic standpoint, these results support targeting the RelB/NF‑κB axis as a strategy to disrupt both cell‑intrinsic survival circuitry and the CXCL12–CXCR4‑dependent interactions with the bone marrow niche that foster myeloma persistence. Given that NF‑κB activity also modulates responses to proteasome inhibitors and other backbone agents, selective attenuation of RelB‑driven transcription could synergize with existing therapies while sparing broader immune functions governed by canonical NF‑κB signaling. Together, the conv ergence of genetic lesions in CYLD and alternative NF‑κB components on a RelB‑dominated transcriptional landscape underscores RelB as a rational, disease‑defining target in multiple myeloma. Our results involving patients’ samples, in vitro mechanistic details and in silico clinical large data analyses advocated that RelB plays a pivotal role in the survival and migration of myeloma cells and mutational inactivation CYLD exacerbates this disease associated RelB phenotype. In this WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint study, we show how inactivating mutations in the negative regulators of canonical NF-κB pathway also promotes tumor growth by reinforcing non-canonical signaling and elucidate a novel CYLD- RelB signaling axis that could be convincingly crucial for several malignancies including MM. WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint Authors’ contribution UAS carried out the in vitro experiments and in silico analyses with conceptualization, data analysis, and manuscript writing. LK contributed as the Co -PI for study on the clinical samples. SB supervised the research in the lab. WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint Acknowledgments The authors thank Michael Kuehl, NCI, for providing the panel of HMCLs. UAS acknowledges B Chatterjee (NIH) and P Roy (IISc) for helpful discussions. The authors sincerely thank V Kumar (SIL, NII) for technical assistance and A Banerjee (AIIMS) and other personnels at the biopsy center/AIIMS for assistance with patient sample collection. Multiple myeloma-related research in the lab was funded by SERB, Department of Science and Technology, Govt. of India (EMR/2015/000658). Research in PI’s laboratory is funded by an intermediate fellowship (500094/Z/09/Z) to SB from Wellcome Trust DBT India Alliance and NII -Core. UAS thanks DBT-India for research fellowships. WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint

1. Annunziata, C. M., Davis, R. E., Demchenko, Y., Bellamy, W., Gabrea, A., Zhan, F., Lenz, G., Hanamura, I., Wright, G., Xiao, W., Dave, S., Hurt, E. M., Tan, B., Zhao, H., Stephens, O., Santra, M., Williams, D. R., Dang, L., Barlogie, B., … Staudt, L. M. (2 007). Frequent Engagement of the Classical and Alternative NF -κB Pathways by Diverse Genetic Abnormalities in Multiple Myeloma. Cancer Cell , 12(2), 115 –130. https://doi.org/10.1016/j.ccr.2007.07.004 2. Authier, H., Billot, K., Derudder, E., Bordereaux, D., Rivière, P., Rodrigues -Ferreira, S., Nahmias, C., & Baud, V. (2014). IKK phosphorylates RelB to modulate its promoter specificity and promote fibroblast migration downstream of TNF receptors. Proceedings of the National Academy of Sciences , 111(41), 14794 –14799. https://doi.org/10.1073/pnas.1410124111 3. Banoth, B., Chatterjee, B., Vijayaragavan, B., Prasad, M., Roy, P., & Basak, S. (2015). Stimulus-selective crosstalk via the NF -κB signaling system reinforces innate immune response to alleviate gut infection. ELife, 4. https://doi.org/10.7554/eLife.05648 4. Baud, V., & Karin, M. (2009). Is NF-κB a good target for cancer therapy? Hopes and pitfalls. Nature Reviews Drug Discovery, 8(1), 33–40. https://doi.org/10.1038/nrd2781 5. Brummelkamp, T. R., Nijman, S. M. B., Dirac, A. M. G., & Bernards, R. (2003). Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF -κB. Nature, 424(6950), 797–801. https://doi.org/10.1038/nature01811 6. Chapman, M. A., Lawrence, M. S., Keats, J. J., Cibulskis, K., Sougnez, C., Schinzel, A. C., Harview, C. L., Brunet, J. -P., Ahmann, G. J., Adli, M., Anderson, K. C., Ardlie, K. G., Auclair, D., Baker, A., Bergsagel, P. L., Bernstein, B. E., Drier, Y., Fonse ca, R., Gabriel, S. B., … Golub, T. R. (2011). Initial genome sequencing and analysis of multiple myeloma. Nature, 471(7339), 467–472. https://doi.org/10.1038/nature09837 7. Chatterjee, B., Roy, P., Sarkar, U. A., Zhao, M., Ratra, Y., Singh, A., Chawla, M., De, S., Gomes, J., Sen, R., & Basak, S. (2019). Immune Differentiation Regulator p100 Tunes NF - κB Responses to TNF. Frontiers in Immunology , 10. https://doi.org/10.3389/fimmu.2019.00997 8. Chauhan, D., Uchiyama, H., Akbarali, Y., Urashima, M., Yamamoto, K., Libermann, T. A., & Anderson, K. C. (1996). Multiple myeloma cell adhesion-induced interleukin-6 expression in bone marrow stromal cells involves activation of NF-kappa B. Blood, 87(3), 1104–1112. 9. Chen, R., Zhao, H., Wu, D., Zhao, C., Zhao, W., & Zhou, X. (2016). The role of SH3GL3 in myeloma cell migration/invasion, stemness and chemo-resistance. Oncotarget, 7(45), 73101– 73113. https://doi.org/10.18632/oncotarget.12231 10. Cormier, F., Monjanel, H., Fabre, C., Billot, K., Sapharikas, E., Chereau, F., Bordereaux, D., Molina, T. J., Avet-Loiseau, H., & Baud, V. (2013). Frequent Engagement of RelB Activation Is Critical for Cell Survival in Multiple Myeloma. PLoS ONE , 8(3), e59127. https://doi.org/10.1371/journal.pone.0059127 11. Demchenko, Y. N., Glebov, O. K., Zingone, A., Keats, J. J., Bergsagel, P. L., & Kuehl, W. M. (2010). Classical and/or alternative NF -κB pathway activation in multiple myeloma. Blood, 115(17), 3541–3552. https://doi.org/10.1182/blood-2009-09-243535 WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint 12. Eluard, B., Nuan -Aliman, S., Faumont, N., Collares, D., Bordereaux, D., Montagne, A., Martins, I., Cagnard, N., Caly, M., Taoui, O., Lordello, L., Lehmann -Che, J., Tesson, B., Martinez-Climent, J.-A., Copie-Bergman, C., Haioun, C., Tilly, H., Bonsang, B., Vincent- Salomon, A., … Baud, V. (2022). The alternative RelB NF -κB subunit is a novel critical player in diffuse large B -cell lymphoma. Blood, 139(3), 384 –398. https://doi.org/10.1182/blood.2020010039 13. Fairfield, H., Falank, C., Avery, L., & Reagan, M. R. (2016). Multiple myeloma in the marrow: pathogenesis and treatments. Annals of the New York Academy of Sciences, 1364(1), 32–51. https://doi.org/10.1111/nyas.13038 14. Flores‐Montero, J., de Tute , R., Paiva, B., Perez, J. J., Böttcher, S., Wind, H., Sanoja, L., Puig, N., Lecrevisse, Q., Vidriales, M. B., van Dongen, J. J. M., & Orfao, A. (2016). Immunophenotype of normal vs. myeloma plasma cells: Toward antibody panel specifications for MRD detection in multiple myeloma. Cytometry Part B: Clinical Cytometry, 90(1), 61–72. https://doi.org/10.1002/cyto.b.21265 15. Gilbert, W., Bragg, R., Elmansi, A. M., McGee-Lawrence, M. E., Isales, C. M., Hamrick, M. W., Hill, W. D., & Fulzele, S. (2019). Stromal cell-derived factor-1 (CXCL12) and its role in bone and muscle biology. Cytokine, 123, 154783. https://doi.org/10.1016/j.cyto.2019.154783 16. Hayden, M. S., & Ghosh, S. (2004). Signaling to NF -κB. Genes & Development , 18(18), 2195–2224. https://doi.org/10.1101/gad.1228704 17. Hövelmeyer, N., Wunderlich, F. T., Massoumi, R., Jakobsen, C. G., Song, J., Wörns, M. A., Merkwirth, C., Kovalenko, A., Aumailley, M., Strand, D., Br üning, J. C., Galle, P. R., Wallach, D., F ässler, R., & Waisman, A. (2007). Regulation of B cell homeos tasis and activation by the tumor suppressor gene CYLD. The Journal of Experimental Medicine , 204(11), 2615–2627. https://doi.org/10.1084/jem.20070318 18. Jemal, A., Siegel, R., Ward, E., Hao, Y., Xu, J., & Thun, M. J. (2009). Cancer Statistics, 2009. CA: A Cancer Journal for Clinicians, 59(4), 225–249. https://doi.org/10.3322/caac.20006 19. Jourdan, M., Cren, M., Robert, N., Bolloré, K., Fest, T., Duperray, C., Guilloton , F., Hose, D., Tarte, K., & Klein, B. (2014). IL -6 supports the generation of human long -lived plasma cells in combination with either APRIL or stromal cell -soluble factors. Leukemia, 28(8), 1647–1656. https://doi.org/10.1038/leu.2014.61 20. Keats, J. J., Fonseca, R., Chesi, M., Schop, R., Baker, A., Chng, W. -J., Van Wier, S., Tiedemann, R., Shi, C.-X., Sebag, M., Braggio, E., Henry, T., Zhu, Y. -X., Fogle, H., Price- Troska, T., Ahmann, G., Mancini, C., Brents, L. A., Kumar, S., … Bergsagel, P. L. (2007). Promiscuous Mutations Activate the Noncanonical NF -κB Pathway in Multiple Myeloma. Cancer Cell, 12(2), 131–144. https://doi.org/10.1016/j.ccr.2007.07.003 21. Kovalenko, A., Chable -Bessia, C., Cantarella, G., Israël, A., Wallach, D., & Courtois, G. (2003). The tumour suppressor CYLD negatively regulates NF -κB signalling by deubiquitination. Nature, 424(6950), 801–805. https://doi.org/10.1038/nature01802 22. Kyle, R. A., & Rajkumar, S. V. (2004). Multiple Myeloma. New England Journal of Medicine, 351(18), 1860–1873. https://doi.org/10.1056/NEJMra041875 23. Lohr, J. G., Stojanov, P., Carter, S. L., Cruz -Gordillo, P., Lawrence, M. S., Auclair, D., Sougnez, C., Knoechel, B., Gould, J., Saksena, G., Cibulskis, K., McKenna, A., Chapman, M. A., Straussman, R., Levy, J., Perkins, L. M., Keats, J. J., Schumacher, S. E., Rosenberg, WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint M., … Stockerl -Goldstein, K. (2014). Widespread Genetic Heterogeneity in Multiple Myeloma: Implications for Targeted Therapy. Cancer Cell , 25(1), 91 –101. https://doi.org/10.1016/j.ccr.2013.12.015 24. Mafra, A., Laversanne, M., Marcos-Gragera, R., Chaves, H. V. S., Mcshane, C., Bray, F., & Znaor, A. (2025). The global multiple myeloma incidence and mortality burden in 2022 and predictions for 2045. Journal of the National Cancer Institute , 117(5), 907 –914. https://doi.org/10.1093/jnci/djae321 25. Mahindra, A., Hideshima, T., & Anderson, K. C. (2010). Multiple myeloma: biology of the disease. Blood Reviews, 24, S5–S11. https://doi.org/10.1016/S0268-960X(10)70003-5 26. Moreaux, J., Legouffe, E., Jourdan, E., Quittet, P., Rème, T., Lugagne, C., Moine, P., Rossi, J.-F., Klein, B., & Tarte, K. (2004). BAFF and APRIL protect myeloma cells from apoptosis induced by interleukin 6 deprivation and dexamethasone. Blood, 103(8), 3148 –3157. https://doi.org/10.1182/blood-2003-06-1984 27. Neri, P., Ren, L., Gratton, K., Stebner, E., Johnson, J., Klimowicz, A., Duggan, P., Tassone, P., Mansoor, A., Stewart, D. A., Lonial, S., Boise, L. H., & Bahlis, N. J. (2011). Bortezomib- induced “BRCAness” sensitizes multiple myeloma cells to PARP inhibitors. Blood, 118(24), 6368–6379. https://doi.org/10.1182/blood-2011-06-363911 28. Palumbo, A., & Anderson, K. (2011). Multiple Myeloma. New England Journal of Medicine, 364(11), 1046–1060. https://doi.org/10.1056/NEJMra1011442 29. Qiang, Y.-W. (2005). Wnts induce migration and invasion of myeloma plasma cells. Blood, 106(5), 1786–1793. https://doi.org/10.1182/blood-2005-01-0049 30. Ramakrishnan, V., & D’Souza, A. (2016). Signaling Pathways and Emerging Therapies in Multiple Myeloma. Current Hematologic Malignancy Reports , 11(2), 156 –164. https://doi.org/10.1007/s11899-016-0315-4 31. Richardson, P. G., Barlogie, B., Berenson, J., Singhal, S., Jagannath, S., Irwin, D., Rajkumar, S. V., Srkalovic, G., Alsina, M., Alexanian, R., Siegel, D., Orlowski, R. Z., Kuter, D., Limentani, S. A., Lee, S., Hideshima, T., Esseltine, D. -L., Kauffman, M ., Adams, J., … Anderson, K. C. (2003). A Phase 2 Study of Bortezomib in Relapsed, Refractory Myeloma. New England Journal of Medicine , 348(26), 2609 –2617. https://doi.org/10.1056/NEJMoa030288 32. Richardson, P. G., Sonneveld, P., Schuster, M. W., Irwin, D., Stadtmauer, E. A., Facon, T., Harousseau, J.-L., Ben-Yehuda, D., Lonial, S., Goldschmidt, H., Reece, D., San -Miguel, J. F., Bladé, J., Boccadoro, M., Cavenagh, J., Dalton, W. S., Boral, A. L., E sseltine, D. L., Porter, J. B., … Anderson, K. C. (2005). Bortezomib or High -Dose Dexamethasone for Relapsed Multiple Myeloma. New England Journal of Medicine , 352(24), 2487 –2498. https://doi.org/10.1056/NEJMoa043445 33. Roccaro, A. M., Mishima, Y., Sacco, A., Moschetta, M., Tai, Y. -T., Shi, J., Zhang, Y., Reagan, M. R., Huynh, D., Kawano, Y., Sahin, I., Chiarini, M., Manier, S., Cea, M., Aljawai, Y., Glavey, S., Morgan, E., Pan, C., Michor, F., … Ghobrial, I. M. (2015). CXCR4 Regulates Extra-Medullary Myeloma through Epithelial -Mesenchymal-Transition-like Transcriptional Activation. Cell Reports, 12(4), 622–635. https://doi.org/10.1016/j.celrep.2015.06.059 34. Roccaro, A. M., Sacco, A., Purschke, W. G., Moschetta, M., Buchner, K., Maasch, C., Zboralski, D., Zöllner, S., Vonhoff, S., Mishima, Y., Maiso, P., Reagan, M. R., Lonardi, S., WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint Ungari, M., Facchetti, F., Eulberg, D., Kruschinski, A., Vater, A., Rossi, G., … Ghobrial, I. M. (2014). SDF -1 Inhibition Targets the Bone Marrow Niche for Cancer Therapy. Cell Reports, 9(1), 118–128. https://doi.org/10.1016/j.celrep.2014.08.042 35. Roy, P., Mukherjee, T., Chatterjee, B., Vijayaragavan, B., Banoth, B., & Basak, S. (2017). Non-canonical NFκB mutations reinforce pro -survival TNF response in multiple myeloma through an autoregulatory RelB:p50 NFκB pathway. Oncogene, 36(10), 1417 –1429. https://doi.org/10.1038/onc.2016.309 36. Roy, P., Sarkar, U. A., & Basak, S. (2018). The NF -κB Activating Pathways in Multiple Myeloma. Biomedicines, 6(2), 59. https://doi.org/10.3390/biomedicines6020059 37. Sen, R., & Baltimore, D. (1986). Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell, 46(5), 705–716. https://doi.org/10.1016/0092-8674(86)90346-6 38. Shen, F., Guo, Q., Hu, Q., Zeng, A., Wu, W., Yan, W., & You, Y. (2018). RelB, a good prognosis predictor, links cell -cycle and migration to glioma tumorigenesis. Oncology Letters. https://doi.org/10.3892/ol.2018.7894 39. Song, Y., Li, S., Ray, A., Das, D. S., Qi, J., Samur, M. K., Tai, Y. -T., Munshi, N., Carrasco, R. D., Chauhan, D., & Anderson, K. C. (2017). Blockade of deubiquitylating enzyme Rpn11 triggers apoptosis in multiple myeloma cells and overcomes bortezomib res istance. Oncogene, 36(40), 5631–5638. https://doi.org/10.1038/onc.2017.172 40. Trompouki, E., Hatzivassiliou, E., Tsichritzis, T., Farmer, H., Ashworth, A., & Mosialos, G. (2003). CYLD is a deubiquitinating enzyme that negatively regulates NF -κB activation by TNFR family members. Nature, 424(6950), 793–796. https://doi.org/10.1038/nature01803 41. van Andel, H., Kocemba, K. A., de Haan -Kramer, A., Mellink, C. H., Piwowar, M., Broijl, A., van Duin, M., Sonneveld, P., Maurice, M. M., Kersten, M. J., Spaargaren, M., & Pals, S. T. (2017). Loss of CYLD expression unleashes Wnt signaling in multiple myelo ma and is associated with aggressive disease. Oncogene, 36(15), 2105 –2115. https://doi.org/10.1038/onc.2016.368 WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint Figures WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint A nRelA & nRelB Activity [A.U.] 0 3 6 9 nRelA nRelB Figure 1 B C D RelA RelB Oct1 Newly diagnosed cases (n=10) Treated cases (n=5) Nuclear extract nRelB HIGH nRelB LOW nRelA HIGH nRelA LOW Odds Ratio = 16 UpSet Plot 0 0.1 0.2 0.3 0.4 0.5 Fraction Size E 2000 0 500 1000 1500Days to Overall Survival RELB p-value = 8.64x10-5 Gene High (n=198) Gene Low (n=198) nRelB HIGH (n=5) nRelB LOW (n = 5) p-Value = 0.05 0 10 20 30RelB mRNA Levels WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint Figure 1. Charting the RelB pathway in multiple myeloma patients A. EMSA revealing NF-B DNA binding activity in nuclear extracts prepared from CD138+ plasma cells derived from bone marrow aspirate samples of myeloma patients. Oct1 (bottom panel) served as loading control. Nuclear extracts of CD138+ cells from ten Newly diagnosed cases and five Treated cases (diseased control) were examined. B. Quantified signals corresponding to nRelA (pink) and nRelB (green) activities are shown in the bar plot. C. UpSet plot testing the correlation analysis of non-canonical RelB and canonical RelA activities from ten newly diagnosed MM patients. The likelihood of both the pathways being concomitantly activated was determined from the odds ratio. D. qRT-PCR results showing RelB mRNA levels in patients with high and low nuclear RelB NF-B activity. E. Survival analysis charting “Days to overall survival” of patients with high and low levels of gene transcript expression in MMRF dataset. Patients were classified in two strata “Gene High” and “Gene Low” from the first and the fourth quartile of gene transcript levels, respectively. Figure 1 WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint CYLD (110 kDa) WCE RelB (68 kDa) p100 p52 Actin (43 kDa) JIM3 KP-6 MG132 - + - + WCE IB (37 kDa) CYLD (108 kDa) GAPDH (36 kDa) D IP NEMO KA: p-IB IB: IKK1 (85 kDa) Kinase activity assay WCE RelA (65 kDa) IB (37 kDa) GAPDH (36 kDa) CYLD (110 kDa) A B C n.s. ** ** ** E F 0 8 24 JIM3 BAFF Nuclear extract 0 8 24 KP-6 RelA RelB Oct1 hr Figure 2 G CYLD Control n= 23 CYLD Mutated n= 23 1000 500 0 CXCR4 p-value = 3.08 x 10-2 1500 mRNA levels (FPKM) 100 80 60 40 20 0 BCL2 p-value = 1.73 x 10-4 120 CYLD Control n= 23 CYLD Mutated n= 23 100 80 60 40 20 0 mRNA levels (FPKM) RELB p-value = 1.21 x 10-4 CYLD Control n= 23 CYLD Mutated n= 23 WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint Figure 2. Absence of CYLD in HMCLs leads to high RelB levels and heightened RelB NF-B activity A. Kinase assay comparing the constitutive NEMO-IKK activity in KP-6 and JIM3 cells. Briefly, cytoplasmic extracts prepared these cells were subjected to immunoprecipitation using an anti-NEMO antibody. The immunoprecipitates were examined for the NEMO-IKK activity using recombinant GST-IB(1-54) protein as substrate (top panel). Immunoblotting the immunoprecipitates with IKK1 served as loading control. B. Immunoblot analyses for RelA and IB with whole cell lysate prepared from a CYLD deficient KP-6 cells against CYLD sufficient JIM3 cells as control. C. KP-6 and JIM3 cells were treated with the proteasome inhibitor MG132 (10 M) for 4 hrs before being subjected to immunoblot analyses using antibodies against the indicated proteins. D. Immunoblot analyses demonstrating the abundance of p100/p52 and RelB in the whole cell lysates prepared from CYLD sufficient KP-6 and CYLD sufficient JIM3 cells. E. EMSA revealing basal as well as signal-induced NF-B activation upon treatment of CYLD deficient KP-6 and CYLD suffficient JIM3 cells (top panel) with 50ng/ml of BAFF. Oct1 DNA binding served as a loading control (bottom panel). F. In silico analyses using MMRF patient database on R platform showing RelB levels in patients with or without any mutation in CYLD. Using Randomization algorithm, 23 randomly selected patients were catalogued into the equivalently-sized control group lacking mutations in CYLD. G. Transcript level analysis showing levels of BCL-2 and CXCR4 in two strata of patients with mutation in CYLD and randomly selected patients as control group having no mutations in CYLD. Figure 2 WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint AnnexinV – PI Cell Death Assay Bortezomib 16 hrs BAFF 16 hrs BAFF 16 hrs Bortezomib 16 hrs + Untreated A B C Propidium Iodide FITC-Annexin V Bortezomib IC50Untreated JIM3 Figure 3 FITC-Annexin V UntreatedBAFF Bortezomib IC50Untreated KP-6D 100 90 80 70 60 50 40 30 20 10 00 % of total cells (JIM3) Annexin V PI Negative n.s. Untreated BAFF BAFF+ Bortezomib Bortezomib BAFF mediated protection *** 100 90 80 70 60 50 40 30 20 10 00 % of total cells (KP-6)E F G JIM3 BAFFUntreated 0.0 0.5 1.0 1.5 n.s. Relative mRNA levels 0.0 0.5 1.0 1.5 n.s. 0.0 0.5 1.0 1.5 n.s. 0.0 0.5 1.0 1.5 n.s. 0.0 0.5 1.0 1.5 n.s. BCL-2 c-FLIP CIAP1 CIAP2 TRAF1 4 3 2 1 0Relative mRNA levels *** KP-6 JIM3 Untreated RELB ** 0 1 2 3 4 5 0 1 2 3 4 5 ** 0 2 6 4 8 ** 0 2 1 3 * 0.0 0.5 1.0 1.5 2.0 2.5 * Relative mRNA levels KP-6 BAFFUntreated BCL-2 c-FLIP CIAP1 CIAP2 TRAF1 JIM3 0.0 0.5 1.0 1.5 n.s. Relative mRNA levels RELB KP-6 4 3 2 1 0 * RELB WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint Figure 3. BAFF provides a selective survival advantage to CYLD deficient cells A. Experimental design of the AnnexinV – PI Cell Death Assay for addressing the pro-survival role of BAFF in HMCLs. B. & D. Representative flow cytometry analyses of FITC-Annexin V and Propidium Iodide (PI) stained JIM3 (B) & KP-6 (D) cells capturing apoptotic and necrotic death in the indicated treatment conditions. Cells were either left alone or stimulated with BAFF for 16hr and then treated with bortezomib for another 16hrs at IC50. C. & E. Quantification of data from flow cytometry analyses revealing the percentage of viable JIM3 (C) & KP-6 (E) cells under various treatment regime. Data shown are the mean ± SEM of three independent experiments. F. qRT-PCR analyses comparing JIM3 and KP-6 cells for the basal abundance of RelB and BAFF-induced accumulation of RelB (right side panels). G. Gene expression analyses using qRT-PCR showing changes in BAFF-induced levels of mRNAs encoding NF-B-target pro- survival factors, including BCL-2, c-FLIP, CIAP1, CIAP2, and TRAF1 in CYLD sufficient control JIM3 (Green) & KP-6 (Orange) cells. The plot represents Mean ± SEM from three independent biological replicates. Figure 3 WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint Control SDF1 BAFF 16 hr Untreated JIM3 A B C D Figure 4 JIM3 (CYLD sufficient) and KP-6 (CYLD deficient) cells BAFF (50ng/mL) 16 hrs treated cells migrated cells 8 m pore media +SDF1 Migrated cell count KP-6 BAFF 16 hr Untreated E F *** ** Untreated SDF1 BAFF BAFF + SDF1 Untreated SDF1 BAFF BAFF + SDF1 Number of migrated cells JIM3 KP-6 500 400 300 200 100 0 JIM3 JIM3 + BAFF KP-6 KP-6 + BAFF CXCR4 CXCR7 JIM3 0.0 0.5 1.0 1.5 n.s. Relative mRNA levels0.0 0.5 1.0 1.5 n.s. 0.0 0.5 1.0 1.5 2.0 2.5 n.s. Relative mRNA levels 0 2 1 3 ** KP-6 CXCR4 CXCR7 WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint Figure 4. BAFF-stimulation promotes migration of CYLD-deficient myeloma cells A. Schema representing the in vitro transwell migration assay involving HMCLs. B. & C. Representative images from transwell cell migration assays comparing migration potential of JIM3 (B) and KP-6 (C) cells. Cells were either left untreated or priorly treated with BAFF (100 ng/mL) for 16 hrs. SDF1 (100 ng/mL) was used as a chemoattractant. Images were captured in bright field microscope after 48 h; magnification 10x Objective. D. Quantitation of the transwell invasion assay results. Data shown are the mean ± SEM of three independent experiments. E. & F. qRT-PCR analyses demonstrating BAFF-induced expressions of CXCR4 and CXCR7 mRNA in JIM3 (E) and KP-6 (F) cells. The plot represents Mean ± SEM value of three biological replicates. Figure 4 WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint A C B JIM3 WCE CYLD (108 kDa) GAPDH (36 kDa) 101 102 103 104 105 GFP Count (mode normalised) GFP Control sgRNA Non-target sgRNA CYLD JIM3 PE-Annexin V Bortezomib IC50Untreated UntreatedBAFF JIM3sgCYLD 7-AAD PE-Annexin V Bortezomib IC50Untreated JIM3sgNon-Target D Figure 5 E F G 100 90 80 70 60 50 40 30 20 10 00% of total cells (JIM3sgNon-Target ) Annexin V 7-AAD Negative n.s. Untreated BAFF BAFF+ Bortezomib Bortezomib BAFF mediated protection * 100 90 80 70 60 50 40 30 20 10 00% of total cells (JIM3sgCYLD) JIM3sgNon-Target 0.0 0.5 1.0 1.5 n.s. Relative mRNA levels RELB JIM3sgCYLD RELB 0.0 0.5 1.0 1.5 2.0 2.5 * Relative mRNA levels BCL-2 c-FLIP CIAP1 CIAP2 TRAF1 JIM3sgNon-Target BAFFUntreated 0.0 0.5 1.0 1.5 n.s. Relative mRNA levels 0.0 0.5 1.0 1.5 n.s. 0.0 0.5 1.0 1.5 n.s. 0.0 0.5 1.0 1.5 n.s. 0.0 0.5 1.0 2.0 n.s. 1.5 BCL-2 c-FLIP CIAP1 CIAP2 TRAF1 JIM3sgCYLD BAFFUntreated * 0.0 0.5 1.0 2.0 1.5Relative mRNA levels * 0.0 0.5 1.0 2.0 1.5 ** 0.0 0.5 1.0 2.0 1.5 0 1 2 3 4 5 n.s. ** 0 1 2 3 4 5 H WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint Figure 5. Deletion of CYLD in JIM3 myeloma cell lines strengthens BAFF mediated cell survival. A. Immunoblot for CYLD protein in human myeloma cell line JIM3 subjected to CRISPR-Cas9 mediated gene editing. GAPDH was used as loading control. B. Flow cytometry showing GFP expression of transduced JIM3sgNon-Target(light blue) and JIM3sgCYLD(violet) cells. Untransduced JIM3(grey) cells were used as control. C. & D. Representative flow cytometry panels of PE-Annexin V and 7-aminoactinomycin D (7-AAD) stained JIM3sgNon-Target (C) cells and JIM3sgCYLD (D) cells capturing apoptotic and necrotic death in the indicated treatment conditions. Cells were either left untreated or stimulated with BAFF for 16hr and then treated with bortezomib for another 16hr at IC50. D. & F. Quantitation of results from the cell death assay revealing the percentage of viable JIM3sgNon-Target (E) cells and JIM3sgCYLD (F) and under different treatment conditions as indicated. E. qRT-PCR analyses revealing BAFF-induced expressions of RelB mRNA in JIM3sgNon-Target (orange) and JIM3sgCYLD (green) cells. F. Gene expression analyses using qRT-PCR demonstrating BAFF-induced expressions of pro-survival NF- target genes, such as BCL-2, c-FLIP, CIAP1, CIAP2 and TRAF1 in JIM3sgNon-Target (Orange) cells and JIM3sgCYLD (Green) cells . The plot represents Mean ± SEM value of three biological replicates. Figure 5 WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint A BAFF 16 hr Untreated JIM3sgNon-Target JIM3sgCYLD B JIM3sgNon-Target JIM3sgNon-Target + BAFF JIM3sgCYLD JIM3sgCYLD + BAFF Figure 6 C D *** **300 200 100 0 Number of migrated cells CXCR4 CXCR7 JIM3sgNon-Target 0.0 0.5 1.0 1.5 n.s. Relative mRNA levels0.0 0.5 1.0 1.5 n.s. JIM3sgCYLD 4 3 2 1 0 * Relative mRNA levels 0.0 0.5 1.0 2.0 1.5 ** CXCR4 CXCR7 WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint Figure 6 Figure 6. BAFF reinforces migration potential of JIM3 myeloma cells with deletion of CYLD A. Representative images of transwell migration assay comparing migration abilities of CYLD sufficient JIM3sgNon-Target cells and CYLD deficient JIM3sgCYLDcells. Cells were either left untreated or treatment with BAFF(100 ng/mL) for 16 hrs was given. SDF1 (100 ng/mL) was used as chemoattractant. Images were captured using bright field microscope at 48 hr after cell seeding in the transwell inserts; magnification 10x Objective. B. Quantitation of the transwell cell migration assay results. Data shown are the mean ± SEM of three independent experiments. C. and D. Gene expression analyses using qRT-PCR showing BAFF induced mRNA levels of pro-migration RelB target genes such as CXCR4 and CXCR7 in CYLD sufficient control JIM3sgNon-Target cells (C) and CYLD sufficient control JIM3sgCYLD cells (D) . The plot represents Mean ± SEM value of four independent biological replicates. WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint CIAP1 CIAP2 TRAF1 c-FLIP BCL-2 CIAP1 CIAP2 TRAF1 c-FLIP BCL-2 BM MNC CD 138+ 0 1 2 5 10 Color scale RelB status Fold change C Figure 7 A B Bone marrow aspirates Bone Marrow Mono- Nuclear Cells CD138+ Myeloma cells CD138- BM MNCs Ficoll Density Centrifugation CD138+ MACS Sorting Negative fraction p-Value < 0.0001 CD138+ plasma cells (N=42) Patient matched BM MNCs (N=42) Relative mRNA Level RelB 40 20 15 10 5 0 RelB HIGH RelB LOW p-Value = 0.03 RelB HIGH RelB LOW p-Value = 0.01 ED WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint Figure 7. RelB supports survival and migration specific gene upregulation inCD138+ cells in patients with MM A. Workflow for the gene expressions charted out in CD138+ cells in comparison to the CD138- BM MNCs. B. Gene expression analysis using qRT-PCR comparing abundance of RelB mRNA in tumor specific CD138+ plasma cells and patient matched control bone marrow-mononuclear cells (BM-MNCs) from 42 myeloma patients. C. Heat map illustration showing the mRNA expression levels of various RelB-target pro-survival genes CIAP1, CIAP2, TRAF1, c- FLIP and BCL-2 in CD138+ myeloma cells from 42 patients. Patient matched BM-MNCs were used as control. D. & E. Gene expression analysis using qRT-PCR showing mRNA expression levels of CXCR4 and CXCR7 (pro-migration genes) in tumor specific CD138+ plasma cells in comparison to patient matched BM-MNCs in total RNA from 42 MM patients. Figure 7 WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint Figure 8 Figure 8. Model Figure WITHDRAWN see manuscript DOI for details .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 3, 2026. ; https://doi.org/10.64898/2026.02.01.703152doi: bioRxiv preprint