Hypoxia induces chemoresistance to proteasome inhibitors through orchestrating deSUMOylation and ubiquitination of SRC-3 in multiple myeloma

The bone marrow microenvironment in multiple myeloma (MM) is hypoxic and provides multi-advantages for the initiation of chemoresistance, but the underlying mechanisms and key regulators are still indistinct. In the current study, we found that hypoxia stimulus easily induced chemoresistance to proteasome inhibitors (PIs), and the steroid receptor coactivator 3 (SRC-3) expression was remarkably augmented at posttranslational level. Protein interactome analysis identified SENP1 as a key modifier of SRC-3 stability, as SENP1-mediated deSUMOylation attenuated the K11-linked polyubiquitination of SRC-3. SENP1 depletion in the SENP1fl/flCD19Cre/+ B cells showed impaired SRC3 stability, and knockdown of SENP1 in MM cells by CRISPR/cas9 sgRNA accelerated the degradation of SRC-3 and remarkably overcame the resistance to PIs. In the Vk*Myc and 5TGM1 mouse models as well as patient-derived xenograft (PDX) of myeloma, SENP1 inhibitor Momordin Ιc (Mc) increased the sensitivity to PIs in MM cells. Importantly, SENP1 level was positively correlated with SRC-3 level in the tissues from refractory/relapsed MM, as well as in xenograft tissues from mice treated with bortezomib and Mc. Taken together, our findings suggest that hypoxia-induced SENP1 is a crucial regulator of chemoresistance to PIs, and shed light on developing therapeutic strategies to overcome chemoresistance by using small molecules targeting SENP1 or SRC-3.


INTRODUCTION
Multiple myeloma (MM) is a hematologic malignancy of plasma cells accumulating mainly in the bone marrow, and results in secretion of excessive parafunctional monoclonal immunoglobulin protein and end-organ damage [1]. Investigations in molecular mechanisms on bench and the successful application of proteasome inhibitors in the clinic have led to the significant overall survival of MM patients [2,3]. However, MM remains incurable and fatal, which is mainly caused by the occurrence of drug-resistant subclones during therapy [4]. A better understanding of the mechanisms of drug resistance in MM cells is urgently required.
MM cells originate and reside in the bone marrow (BM) [5]. It has been reported that the BM niche confers survival and chemoresistance of MM cells by a complex interplay of cytokines, chemokines, adhesion molecules, proteolytic enzymes, and other components of the extracellular matrix [6][7][8]. Besides, BM has long been regarded as a naturally hypoxic organ [9,10]. A study using the 5T33 MM murine model found that the myelomatous BM was more hypoxic than the normal bone marrow [11]. During MM progression disease, hypoxia BM niche exert strong selective pressure that shapes tumor evolution, make them adapt to reduced oxygen availability, become highly aggressive and resistant to treatment [12]. For instance, our previous study revealed that hypoxia promotes disease progression and bone lesion through upregulating DKK1 expression [13]. Given that hypoxia promotes tumor progression in solid malignancies, for example, live and breast cancers [14,15], it is likely that MM are strongly influenced by hypoxia. However, the role of hypoxia in MM drug resistance is still elusive, and it is pivotal to clarify the mechanisms by which hypoxia promotes the pathogenesis and chemoresistance of MM.
Highly dynamic post-translational modifications (PTMs), including acetylation, phosphorylation, methylation, ubiquitination, SUMOylation, and NEDDylation, control accumulation and functions of proteins and are pivotal for carcinogenesis and disease progression [16,17]. Actually, post-translational modifications such as ubiquitination and SUMOylation play even critical roles in MM than other solid tumors, since the most effective drugs for management of MM in clinic, such as proteasome inhibitors (PIs) and immunomodulatory drugs (iMiDs), disturb the ubiquitin-proteasome system, thus control NF-κB signaling, epigenetic regulations, DNA damage repair, and drug resistance [18]. For example, we have discovered that histone methyltransferase NSD2 stabilizes the steroid receptor coactivator-3 (SRC-3) to prevent degradation of SRC-3 and enhance chemoresistance in the t(4;14) positive MM cells [19]. SRC-3 belongs to the SRC/p160N coactivator family, which consists of three members: SRC-1/NCOA1, SRC-2/TIF2/GRIP1/ NCOA2, and SRC-3/AIB1/NCOA3 [20]. In addition, ubiquitination and SUMOylation modifications regulate both the cellular concentrations and the co-activator activities of SRC-3. Wu et al. reported that estrogen treatment led to decreased SUMOylation and increased phosphorylation, thus regulated the transcriptional activity of SRC-3 [21]. However, key regulators of SRC-3 and its degradation machinery in MM cells, especially under the hypoxic BM environment, are still not well elucidated. Interpretation of these questions will provide a better understanding of MM progression, and shed light on developing new strategies to overcome refractory or replace in the clinic.
In this study, we investigated the effect of hypoxia on chemosensitivity to proteasome inhibitors in MM cells through SENP1-mediated modification of SRC-3 protein, and evaluated the efficacy of newly developed SENP1 and SRC-3 inhibitors in overcoming chemoresistance of immortalized MM cell lines, as well as 5TGM1 and Vk*Myc mouse models of myeloma.

Hypoxia induces bortezomib resistance in MM cells
Hypoxia has been known as a critical factor for drug resistance in ample solid tumors, therefore we investigated whether hypoxia influences the sensitivities of MM cells to effective drugs in clinic, such as Bortezomib (BTZ), carfizomib (CFZ), and melphalan (Mel). The sensitivity to BTZ of two MM cells, MM.1S and LP-1, were all obviously receded under hypoxia stimulus compared to the normoxia control (Fig. 1A, B), as also shown by the significantly augmented half-maximal inhibitory concentration (IC 50 ) values (Fig. 1C), the distinct abrogated cleavage of PARP as a marker of cell apoptosis (Fig. 1D), and the remarkable suppressed apoptotic cell rates (Fig. 1E). Intriguingly, hypoxia yielded similar chemosensitivity alteration when MM were treated with another proteasome inhibitor Carfizomib (Fig. 1F-J), but failed to induce conspicuous consequences of Melphalan in MM cells (Fig. 1K-O). Thus, these results indicate that hypoxia more easily induces chemoresistance to protease inhibitor of MM cells.

Hypoxia upregulates SRC-3 level in MM cells
Our previous study has demonstrated high SRC-3 is associated with poor prognosis in MM, and overexpression of SRC-3 promotes BTZ resistance [19]. To dissert the correlation between SRC-3 and hypoxia, we assessed whether hypoxia affects SRC-3 expression in MM cells at mRNA or protein levels. We confirmed that hypoxia induced the augmentation at SRC-3 protein level in a time-dependent manner, and hypoxia obviously prolonged the half-life of SRC-3 protein in MM cells (Fig. S 1A), but the elevation of SRC-3 protein did not coordinate with the alteration trend of HIF-1α, nor at mRNA level ( Fig. 2A, B). Immunofluorescence assay also validated that hypoxia promoted the accumulation of SRC-3 in the nucleus ( Fig. 2A, B). Similarly, high SRC-3 levels were also found in our previously established bortezomib-resistant (BR) MM.1 S and LP-1 cells compared with their parental cells (Fig. 2C, D) [19,23]. A newly developed small molecule inhibitor SI-2 targeting SRC-3 [24], showed similar inhibitory efficacy with the lentiviral carrying shRNA (SRC3-KD) ( Fig. S 1B), we observed that SI-2 partially rescued the sensitivity to BTZ of MM cells under hypoxia condition ( Fig. 2E-G). Importantly, SI-2 and BTZ only showed synergistic anti-MM effect under hypoxia condition, but not in the normoxia controls, as evidenced by the remarkably augmented apoptotic cell rate and cleavage of PARP (Fig. 2H, I). These data suggest that high SRC-3 protein provoked by hypoxia might be a critical regulator for sensitivity to PIs of MM cells.

Degradation of SRC-3 is ubiquitin-proteasome dependent
Since the augmentation of SRC-3 protein under hypoxia stimulus neither coordinate with HIF-1α, nor occurred at transcriptional level, we speculated that hypoxia may enhance the stability of SRC-3 through another regulators. We assessed pathways governing SRC-3 degradation in MM cells using their specific inhibitors, including MG132 (ubiquitin-proteasome pathway), Baf-A1 (lysosomal proteolysis), and MG101 (calpain system); additionally, we treated MM cells with different doses of MG132, Baf-A1, and MG101 in presence of cycloheximide for 12 h to assess the protein degradation. The results showed that SRC-3 degraded mainly in a proteasome-dependent manner (Fig. S 1B-F), which is similar with the alteration of SRC-3 in BTZ-treated MM cells (Fig. 3A). We further used co-immunoprecipitation to confirm a physical interaction of SRC-3 with ubiquitin, and identified that SRC-3 was degraded mainly through lysine 11 (K11)-linked polyubiquitination, partially through K6-, and K29-linked polyubiquitination (Fig. 3C). However, when the K6−, K11−, and K29− lysine positive mutations of ubiquitin were overexpressed in MM cells together with SRC-3, we found that SRC-3 was modified through K11-linked polyubiquitination, since only dosedependent modification of K11-linked polyubiquitination was elicited (Fig. 3D, E) on SRC-3, we failed to observe the K6-and K29linked polyubiquitination (Fig. 3F, G). Moreover, when the K11 lysine was mutate to arginine, polyubiquitination of SRC-3 could barely be detected (Fig. 3H). Thus, these results indicate that SRC-3 protein is mainly modified through K11-linked polyubiquitination for degradation by the 26S proteasome.

SENP1 stabilizes SRC-3 through deSUMOylation
Next, we sought to deduct the key regulators for SRC-3 protein degradation. We disserted the interactome of SRC-3 in MM.1S cells under hypoxia stimulation using mass spectrometry. Intriguingly, we did not find any E3 ligase in the components of SRC-3 interactome, on the contrary, we discovered a cysteine protease of the sentrin-specific protease (SENP) family, SENP1, which reverses SUMO conjugation on target proteins (Fig. 4A). The interaction between SRC-3 and SENP1 was bilaterally confirmed by coimmunoprecipitation in the HEK293T cells, either using HA-SENP1or Flag-SRC-3 as bait antibodies (Fig. S 2A). Moreover, the endogenous interaction of SRC-3 and SENP1 was also validated in MM cells using anti-SRC-3 or anti-SENP1 antibodies, respectively (Fig. 4B). In addition, protein level of SRC-3 was positively related to the exogenously expressed SENP1 in MM cells (Fig. S 2B), and overexpression of SENP1 obviously prolonged the half-life of SRC-3 protein in MM cells (Fig. 4C). These data suggested that degradation of SRC-3 might be regulated through coordination of SUMOylation and ubiquitination.
Previous studies have shown that transactivation activity of SRC-3 could be regulated by SUMOylation [21]. Actually, convergence of ubiquitination and SUMOylation in modulation of protein functions has emerged as a crucial cellular mechanism in regulating pathogenesis [25]. Our result showed that SRC-3 physically interacted with small ubiquitin-related modifier (SUMO) 1, SUMO2, and SUMO3, but the interaction with SUMO1 was dominant (Fig. S 2C). When SUMO1 was gradually overexpressed in MM cells, SRC-3 degradation was enhanced in a dosedependent manner (Fig. S 2D); on the contrary, SUMO1 depletion resulted in attenuation of the SRC-3 ubiquitination both in HEK293T cells (Fig. S 2E) and in MM cells (Fig. S 2F, G). Thus, we confirmed that SUMOylation was involved in SRC-3 protein degradation. To further investigate the role of SUMOylation in SRC-3 stability in vivo, we generated Sumo1 knockout mouse, and confirmed that SUMO1 has been completely depleted in B cells (Fig. S 2H). In addition, the half-life of SRC-3 protein was markedly increased in the SUMO1 −/− B cells (Fig. S 2I), and ubiquitination of SRC-3 was barely detected in the SUMO1 −/− B cells (Fig. 4H). Taken together, these results suggest that SUMOylation was involved in proteasome-dependent degradation of SRC-3.
To further explore the role of SENP1 in SRC-3 modification, we knockdown SENP1 in the HEK293T cells that have stably expressed SUMO1 and flag-tagged SRC-3, and observed that SUMOylation of SRC-3 was markedly elevated (Fig. S 3A); on the contrary, overexpression of SENP1 (SENP1-OE) remarkably attenuated the ubiquitination modification of SRC-3 compared with the vector controls (Fig. S 3B). Importantly, overexpression of SENP1 dramatically relieved the K11-linked ubiquitination, but not K6-and K29-linked ubiquitination of SRC-3 (Fig. 4D). We further suppressed SENP1 expression using lentivirus carrying shRNA in MM cells, and failed to observe significant attenuation on SRC-3 ubiquitination and SUMOylation, and simultaneously total SRC-3 protein level was unable to elevate under hypoxia stimulation (Fig. 4E, F). To confirm the phenotype of SENP1 in regulating SRC-3 stability in vivo, we generated a SENP1 conditional knockout mouse using SENP1-floxp and CD19-Cre crossed mice, and efficiently deleted SENP1 in the B cells (Fig. S  3C). As a consequence, protein level of SRC-3 in the SENP1 fl/fl; CD19-Cre B cells was obviously downregulated, and administration of MG132 failed to restore the protein level of SRC-3 compared with the B cells from wild type control mice (Fig. S  3D). Moreover, the half-life of SRC-3 protein was markedly shortened in the SENP1 fl/fl; CD19-Cre B cells compared with that in the wild type B cells (Fig. 4H). As expected, endogenous ubiquitination and SUMOylation of SRC-3 was more readily detected in the SENP1 fl/fl; CD19-Cre B cells but not in wild type B cells (Fig. 4I). Collectively, our results indicate that SENP1 protects SRC-3 against degradation by SUMOylation-associated ubiquitination.

SENP1 is a downstream target of HIF-1α in MM cells
We next assessed expressions of the SENP family genes with HIF-1α overexpression (HIF-1α OE) in MM cells, and identified that hypoxia dominantly induced the expression of SENP1 in protein and mRNA level (Fig. 5A, B), the results were similar with that under hypoxia condition in MM cells (Fig. 5C, D). On the contrary, when HIF-1α was knockdown by lentivirus carrying shRNAs in MM cells (Fig. 5E), SENP1 expression was dramatically suppressed both at mRNA and at protein levels, together with the downregulated of SRC-3 only at protein level (Fig. 5F). Interestingly, we verified the similar expression pattern of SENP1 in the BR-MM cells (Fig. 5G, H).
To determine whether HIF-1α is a transcriptional factor for SENP1, we constructed a SENP1-luciferase reporter containing two cisacting elements of HIF-1α at −102~− 111bp and −1684− 1691bp (Fig. 5I), and ectopically expression of HIF-1α resulted in over 10 folds activation of the SENP1-luciferase reporter (Fig. 5J). Importantly, hypoxia triggered significant enrichment of HIF-1α on SENP1 promoter in MM cells when detected by chromatin immunoprecipitation assay (Fig. 5K). These results uncover that SENP1 is a direct target of HIF-1α under hypoxia condition.

SENP1 plays critical role in regulating chemosensitivity to PIs in MM cells
Using an independent cohort of 414 MM patients (GSE4581), we observed that high expression of SENP1 was significantly correlated with worse overall survival (P = 0.0193) (Fig. 6A), and the same conclusion was also confirmed in the CoMMpass (Clinical Outcomes in MM to Personal Assessment of Genetic Profile) dataset (Fig. 6B). To further validate whether SENP1 is an important regulator of chemosensitivity in MM cells, we ectopically overexpressed (OE) or knocked down (KD) the expression of SENP1 in MM.1 S and LP-1 cells using lentivirus carrying expressing or shRNAs vectors, respectively (Fig. S 4A, B). As expected, knockdown of SENP1 expression increased sensitivity to BTZ treatment in a dose-dependent manner, with a significantly reduced IC 50 in both MM.1 S and LP-1 cells (Figs. 6C, S  4C,). Meanwhile, increased cell apoptosis due to BTZ treatment were observed in the SENP1-KD MM cells compared with the pared controls, as evidenced by augmented cleavage of PARP protein (Fig. 6D), and apoptotic cell rates (Fig. 6E). In contrast, overexpression of SENP1 in MM cells led to indolent response to BTZ treatment and inhibited cell apoptosis (Figs. 6F-H, S 4D). Importantly, suppression of SENP1 in the SRC-3 stably expressing MM cells using a specific inhibitor, Momordin Ιc (Mc), remarkably sensitized the anti-MM efficacy of BTZ, but the synergetic effect of Mc was failed to be elicited in the SRC-3-KD MM cells (Figs. 6I, S 4E). In addition, rescue of SRC-3 expression in the SENP1-KD MM cells largely reinstated the resistance to BTZ treatment compared with its paired vector control (Figs. 6J, S 4F). Clinically, we observed that protein levels of HIF-1α, SENP1 and SRC-3 were mutually correlated, and the expressions were all significantly augmented in the refractory/relapsed MM patients (Figs. 6K, S 4G-J), and combination of SENP1 inhibitor Mc with BTZ dramatically augmented cell apoptosis in primary CD138 + plasma cells that have been resistant to BTZ-based regiments (Fig. 6L). In conclusion, these data suggest that SENP1 plays a critical role in regulating chemosensitivity to PIs in MM cells.

Targeting SENP1 suppresses hypoxia-induced SRC-3 and drug resistance in vivo
To assess the effect of targeting SENP1 in overcoming drug resistance in vivo, we established BTZ-resistant MM cell derived xenograft models, RRMM patient CD138 + plasma cell-derived intra-bone growth MM model, as well as the Vk*Myc transgenic and transplant mouse models of MM. The SENP1 inhibitor Mc alone administration had no obvious inhibitory effects on tumor growth, nor did BTZ alone administration, however, the combination of BTZ and Mc considerably suppressed the tumor growth of BR-MM (Fig. 7A), prolonged the survival rate of mice (Fig. 7B). Importantly, more cell apoptosis (Fig. 7C) and decreased expression of SENP1 and SRC-3 were obviously observed in the combined treatment groups (Fig. 7D). Moreover, in unsorted bone marrow mononuclear cells of three relapsed MM patients derived xenograft model, tumor burdens were also noticeably extenuated in the combination treatment groups, as evidenced by significantly suppressed M-protein levels (Fig. 7E) and decreased CD138 + cells percentage in the bone marrow (Fig. 7F). Furthermore, in the successfully constructed Vk*Myc transplant mouse models of MM (Fig. S 5A-D), we further validated the synergistic anti-MM effect of combination of SENP1 inhibitor and BTZ, as shown by the conspicuously reduced M-protein level (Fig. 7G), increased survival rate of mice (Fig. 7H), and nearly disappeared CD138 + plasma cells in the bone marrow (Fig. 7I). Taken together, these in vivo data strongly suggest that pharmacologically targeting SENP1 abrogates chemoresistance to PIs in MM cells.

DISCUSSION
In the current study, we identified an important role of hypoxia in regulating chemosensitivity to PIs of MM cells. Our study showed that hypoxia enhanced SENP1 expression through HIF-1α, and SENP1 deSUMOylates SRC-3 via K11-linked ubiquitination, consequently protects the SRC-3 from 26S proteasome dependent degradation and favors MM cells survival. Translationally, our pre-clinical data suggests that using small molecule targeting SENP1 could re-sensitize resistant MM cells to PIs, which may benefit the strategy development for refractory or relapsed MM patients.
Our previous findings has revealed that the histone methyltransferase NSD2 protects SRC-3 from degradation through forming liquid-liquid phase separation and renders resistance to PIs [19]. SRC-3 promotes numerous aspects of cancer, such as initiation, progression, and chemoresistance [26], and suppression of SRC-3 levels and/or activity are efficient enough to alter its transcriptome [27,28]. Several studies have demonstrated that stimuli could induce multiple posttranslational modifications of SRC-3, including phosphorylation, ubiquitination, SUMOylation, acetylation, and methylation [26,29]. In this study, we report that hypoxia is a new stimulus for SRC-3 expression without affect its transcriptional level. It has been reported that hypoxia triggered activation of NF-кB signaling pathway to enhance bortezomib resistance in MM cells [30,31]. Interestingly, our current study also discover that hypoxia mainly cause chemoresistance towards proteasome inhibitors, but not to other drugs such as Melphalan, a DNA alkylating drug inhibiting DNA and RNA synthesis [32]. Therefore, our study indicates that hypoxia may affect key regulators for chemosensitivity that dependents on the ubiquitin-proteasome system. Since SRC-3 is degraded in a proteasome-dependent manner, thus it is very easily affected by hypoxia. After deciphering the interactome of SRC-3 in MM cells under hypoxia condition, we unexpectedly discovered a deSUMOylation enzyme SENP1, but no ubiquitination-related E3 ligase, interact with SRC-3. SENP1 belongs to SUMO-specific proteases (SENPs), which have a dual function as processing enzymes for pre-SUMO and deconjugates of SUMO conjugates [33]. Overexpression of SENP1 positively correlated with adverse events of tumor such as tumor differentiation, lymph node metastasis, and recurrence [34,35]. Similar to its roles in solid tumors, our study demonstrates that SENP1 is an important regulator for chemoresistance to PIs in MM cells under hypoxia condition. Small ubiquitin-like modifiers (SUMOs) are conservatively expressed in all eukaryotes, engender protein SUMOylation modification, and are essential for the maintenance of genomic integrity and the regulation of gene expression and intracellular signaling [36]. Our present study provides the first evidence that SUMOylation coordinates with ubiquitination in regulating the stability of SRC-3, providing a novel knowledge for understanding the regulation of SRC-3 stability in MM cells under hypoxia condition. In solid tumors, it has been well established that hypoxia has a negative effect on the efficacy of radio-and chemo-therapy, through affecting drug delivery, DNA damage repair, regulation of genes governing drug resistance, as well as cell death pathways [37,38]. The bone marrow which has been invaded by MM cells contains a heterogeneous range of oxygen pressures due to rapidly proliferating cells and angiogenesis, but the average oxygen pressure generally is under the normoxia range. In this study, we provided evidences that under hypoxia, HIF-1α-SENP1 axis modulates SRC-3 stability, but hypoxia does not directly regulate SRC-3 transcription. Thus, we conclude that hypoxia promotes the transcription of SENP1 through HIF-1α, and protects SRC-3 protein from degradation by SUMOylation-mediated ubiquitination. Since bortezomib treatment leads to SRC-3 accumulation, and SRC-3 overexpression confers resistance to PIs, our study suggests a positive feedback leading to MM drug resistance.
In summary, this study provides new knowledge for understanding the chemoresistance of MM cells to PIs under hypoxia condition, and further emphasizes the importance of SRC-3 in regulating sensitivity to PIs via its interaction with the SUMOspecific protease SENP1. Our results also shed light on the development of therapeutic strategies to overcome refractory or relapse in MM patients using the SENP1 inhibitor in clinic.

Patient-derived xenograft (PDX) mouse model for MM
To develop a PDX model, unsorted bone marrow mononuclear cells containing 0.5~1 × 10 6 CD138 + from relapsed MM after bortezomibbased regimen treatment were implanted by intravenous administration (i.v.) into 4-6 weeks old NSG mice (n = 6 mice/per patient). Recipient mice were bled weekly after inoculation and detected by the use of immunoglobulin ELISA Kits (ThermoFisher) in order to monitor engraftment. Twice a week for three weeks from the 2nd week after inoculation, solo bortezomib or in combination with Mc was administered, and the human CD138 + cells from mice bone marrow were analyzed by flow cytometry after the treatments were completed.

Vk*Myc transgenic and transplant mouse models of MM
The Vk*Myc transgenic and transplant mouse model were established according to Dr. Bergsagel PL's report [22]. Briefly, 0.5 × 10 6 cells/mouse were injected via tail vein of 5-to 12-week-old C57BL/6 wild-type recipient mice (n = 6 per group). One Week after transplantation, mice were divided into 4 group randomly and treated with Mc (10 mg/kg, third weekly, n = 6), BTZ (1 mg/Kg, third weekly, n = 6), the combination of both drugs (n = 6) or vehicle control (DMSO, n = 6) for 4 weeks. One Week after medication for Serum Protein Electrophoresis (SPEP) analysis, recipient mice were bled weekly. Their time to engraftment (TTE), or time to first appearance of the M-protein spike, was calculated by the Kaplan-Meier survival curve and log-rank test.

Statistical analysis
Data were shown as mean ± SD for at least n = 3 independent experiments except otherwise explanation. Differences between groups were determined using paired two-sided Student's t-test or two-way ANOVA. Pearson correlation test was used to determine the correlations between gene expressions, and survival analysis and a log-rank test was done by GraphPad Prism 5.0. A P-value less than 0.05 was considered statistically significant compared with the controls, respectively.
Other detailed methods could be found in the supplementary methods.

DATA AVAILABILITY
All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Source data are provided with this paper. Requests for any materials in this study should be directed to Zhiqiang Liu and obtained through an MTA. inoculated NSG mice tail venous blood after treated with BTZ or combined with Mc (n = 6 mice/patient, n = 3 mice/group). F Percentage of human CD138 + cells in the bone marrow of NSG mice after receiving 3 weeks of BTZ or BTZ combined with Mc treatment. n = 6 mice/patient sample, n = 3 mice/group. G Vk*MYC mice were treated with Mc (10 mg/kg, third weekly, n = 6), BTZ (1 mg/Kg, third weekly, n = 6), the combination of both drugs (n = 6) or vehicle control (DMSO, n = 6) for 4 weeks. Serum paraprotein was assessed on day 1 and then weekly for 5 weeks and presented as mean change from levels on day 0 (mean SEM). H Survival of Vk*MYC mice treated with Mc, BTZ, the combination of both drugs and DMSO. I FACS analysis of CD138 + /B220 -PCs in the BM of Vk*MYC mice after 5 days of treatment with Mc, BTZ, the combination of both drugs or DMSO. Values are normalized to the percentage of PCs in vehicle control treated BM (100%). *P < 0.05, **P < 0.01, ***P < 0.001, two-sided P value determined by Student's t-test; mean ± SD of 3 independent experiments.