Expression of SUMO E1 is upregulated in multiple myeloma and associated with poor prognosis

mRNA profiling of myeloma patient cohort GSE5900 showed that SUMO E1 (Ubiquitin Like Modifier Activating Enzyme 2(UBA2)/SUMO activating enzyme 2 (SAE2)) gene expression was significantly upregulated at the premalignant myeloma stage (MGUS) and in the asymptomatic myeloma phase (smoldering MM) compared to healthy plasma cells (Supplemental Fig. S1A). Moreover, higher SUMO E1 (UBA2) gene expression in MM plasma cells was associated with significantly shorter survival in both GSE2658 (n = 559) and CoMMpass (clinical trial NCT01454297, n = 764) database (Supplemental Fig. S1B&C) [15, 16]. These data suggest that SAE2 might be associated with therapy resistance in MM.

SUMOylation inhibition enhances Dex anti-MM activity in primary patient samples and cell lines

Considering Dex is the most widely used therapeutic drug in MM treatment, we started with investigation of SAE2/UBA2 expression and sensitivity to Dex treatment. We correlate expression of UBA2 mRNA levels with the sensitivity of Dex in primary MM cells isolated from relapsing MM patients. We treated CD138+ MM cells from bone marrow aspirates of 15 patients with different Dex concentrations and assayed the cell viability post 48 h treatment. The half maximal inhibitory concentrations (IC50s) were calculated and compared with individual UBA2 mRNA expression across all primary samples by Pearson correlation coefficients (Fig. 1A). We observed that the expression level of UBA2 showed a significant positive correlation with Dex IC50s, indicating UBA2 expression is associated with Dex resistance in MM cells.

Fig. 1
figure1

SUMOylation inhibition synergizes with Dex in decreasing cell viability in primary multiple myeloma cells and MM cell lines. A SAE2 expression is associated with Dex sensitivity in primary MM cells. SAE2 (UBA2) mRNA expression was assessed by q-PCR and presented as relative level normalized to GAPDH. IC50 values were calculated by GraphPad Prism 8. Pearson correlation analysis showed a significant positive correlation between SAE2 mRNA and IC50 for Dex in 15 primary MM. B TAK-981 inhibits global SUMOylation and induces cleaved PARP in MM1S cells. Western blot presents SUMO-1, SUMO-2,3-modified protein in MM1S cells after exposure to TAK-981 at the indicated concentrations for 16 h. GAPDH serves as loading control. Relative protein level was quantified and labeled below each blot. C Cell viability assay showing 1 of 6 primary CD138+ cells from bone marrow aspirates of MM patients treated with TAK-981 or Dex or both (combo) with indicated concentration. Cell viability was assessed by Cell-Titer-Glo after 48 h of treatment. *Combination Index (CI) < 1 determined by CompuSym. D TAK-981 synergizes with Dex cytotoxicity in sensitive line (MM1S) and resistant line (MM1R). MM1S and MM1R cells were treated with indicated concentration of TAK-981 or Dex or both (combo) and cell viability was determined by Cell-Titer-Glo post-48 h treatment. *Combination Index (CI) < 1 determined by CompuSym. E TAK-981 enhances cytotoxicity of Dex in sensitive and resistant MM. MM1S and MM1R cells were treated with Vehicle (Veh), 0.1 μM TAK-981 (TAK), 1 μM Dex (Dex), or 0.1 μM TAK-981 with 1 μM Dex (combo). Apoptosis was measured by flow cytometry using Annexin V/PI staining. F Knockdown of SAE2 and UBC9 enhances MM1R Dex sensitivity. MM1R cells were transfected with siRNA targeting SAE2 (SiSAE2) or UBC9 (SiUBC9), or non-targeting control (SiCtrl) for 48 h then treated with different concentration of Dex for 24 h. Cell viability was measured and normalized to untreated control cells. G IC50 values of Dex in MM1R cells calculated from data in F. Data were analyzed using unpaired Student t tests: Data presented as mean ± SD. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001

TAK-981, a novel, selective small molecule inhibitor of SUMO E1 enzyme, is currently in Phase 1 trials in adult patients with metastatic solid tumors and lymphomas [17]. We treated MM1S cells with TAK-981, detected SUMOylated proteins using anti-SUMO-1 and anti-SUMO-2,3 antibodies (Fig. 1B). TAK-981 inhibited global SUMOylation in a dose-dependent manner along with induction of apoptosis marker cleaved PARP. We then tested the impact of TAK-981 as a single agent and in combination with Dex in MM. In primary MM cells isolated from relapsing myeloma patients, combination treatment of TAK-981 with Dex resulted in significantly enhanced killing compared to the single agents alone (n = 6) (Fig. 1C). Consistent synergistic effects were observed in all 6 tested MM patient samples and the combination index was calculated (Supplemental Table S1).

MM1R cell line is resistant to Dex and its parental sensitive counterpart is MM1S. We conducted cytotoxicity assay in MM1S and MM1R cell lines, TAK-981 synergized with Dex in both cell lines (Fig. 1D). Dex showed limited effects on inducing apoptosis or inhibiting cell proliferation in MM1R cells. In contrast, TAK-981 induced cell apoptosis and decreased ell viability in both cell lines and the effects were further enhanced in combination with Dex (Fig. 1E). The synergistic effects of combination TAK-981 and Dex were observed in other MM cell lines H929 and RPMI8226 (Supplemental Fig. S2A).

To verify the effect of TAK-981 is specific through regulating SUMOylation, we conducted knockdown experiments. Transient (siRNA) knockdown of SUMO E1, SAE2 and SUMO E2, UBC9 significantly increased Dex sensitivity in MM1R cells (Fig. 1F, Supplemental Fig. S2B-C). Furthermore, we established stable cell lines that can be induced by doxycycline (dox) to express shRNA targeting SAE2 in the MM cell line RPMI8226. Two individual shRNAs (shSAE2#1 and shSAE2#5) were used to rule out nonspecific shRNA effects. Cells were treated with Dox for 3 days to achieve knockdown of SAE2 expression in both stable lines (Supplemental Fig. S3D), and then cell viability was measured. SAE2 knockdown significantly reduced MM cell viability (Supplemental Fig. S3E). More importantly, cells with stably knockdown of SAE2 showed a significant response to Dex treatment with decrease of IC50 values in both stable lines (Supplemental Fig. S3F). These data indicated SUMOylation is associated with Dex resistance and SUMOylation inhibition enhances Dex sensitivity.

TAK-981 shows anti-MM activity in vivo

To evaluate the in vivo effects of TAK-981 on MM tumor growth, we generated two murine xenograft models. NSG mice were i.v. injected with MM1S-Luc cells and tumor burden was determined by mice whole body luminescence imaging. TAK-981 treated mice exhibited a significant lower myeloma burden compared to vehicle group (Fig. 3A-B). In the other model, NSG mice subcutaneously (s.c.) injected with MM1R cells and tumor growth was monitored by tumor volume and weight. Consistent with our in vitro experiment, Dex alone did not affect tumor growth, but TAK-981 treatment greatly suppressed tumor growth and further reduced tumor burden when in combination with Dex (Fig. 3C, Supplemental FigureS3). IHC staining of cleaved PAPR was carried out to determine the apoptosis in tumor tissue. Similarly, Dex treatment alone showed almost no induction of apoptosis, but TAK-981 treatment group exhibited a significant induction of cleaved PARP expression and even higher level of cleaved PARP in combination with Dex (Fig. 2D). The results demonstrated that TAK-981 has potent anti-MM activity in vivo.

Fig. 2
figure2

TAK-981 inhibits MM tumor growth and synergizes with Dex effect in vivo. (A) TAK-981 treatment suppresses tumor growth in MM1S-ffGFP xenograft NSG mice. MM1S-ffGFP cells were intravenously (iv) injected to NSG mice via tail vein (1.5 × 106 cells/mouse). After bioluminescence imaging to confirm tumor engraft, mice were randomly assigned to two groups (n = 6) and treated with TAK-981 at 7.5 mg/kg or vehicle twice a week. Tumor growth was monitored by weekly imaging and quantified by Aura software. A Bioluminescence image representative 2 out of 6 mice from each group on Day 10 of treatment. B Tumor growth curve determined by the bioluminescence signal was measured as total photon flux normalized for exposure time and surface area and expressed in units of photons (p) per second per cm2 per steradian (sr). C TAK-981 treatment suppresses tumor growth in MM1R xenograft NSG mice model with synergistic effect in combination with Dex. NSG mice were xenografted by subcutaneously injection of MM1R cells (4 × 106 cells /mouse). Mice were treated with either vehicle, or Dex (3 mg/kg), TAK-981 (10 mg/kg) or combination of both Dex and TAK-981(combo). All agents were treated twice weekly (BIW). Tumor growth was evaluated weekly by caliper measurement and represented as tumor volume (millimeters cubed). Comparison of tumor volume on end point was plotted. D IHC staining of apoptosis marker cleaved PARP and myeloma marker CD138 expression in xenograft tumor tissues. Red bar represents 50 μm. Data were analyzed using unpaired Student t tests: Data presented as mean ± SD. ns, not significant; *, p < 0.05

SUMOylation inhibition enhances Dex sensitivity by upregulating GR through downregulating miR-130b

It has been reported that GR expression is a major mechanism of Dex response and is associated with better patient outcome in MM [3, 16, 18]. Consistent with previous studies, we found that GR expression showed negative correlation with Dex IC50 values in primary MM cells (n = 15) (Fig. 3A). Based on our finding that SUMOylation inhibition enhances Dex sensitivity, we evaluated the effect of SUMOylation inhibition on GR expression. Using primary MM cells obtained from relapsing patients (n = 5), we observed a dose-dependent GR (NR3C1) mRNA upregulation upon TAK-981 48 h treatment (Fig. 3B). miR-130b, which has been reported downregulating GR mRNA level by targeting 3′-UTR [19], showed a dose-dependent reduction upon TAK-981 treatment in same primary MM cells (Fig. 3C). GR protein levels were also induced in TAK-981 treated primary MM cells (Fig. 3D). Similar induction of GR level and decrease of miR-130b were observed in MM1S and H929 cell lines (Fig. 3E&F). Transient (siRNA) knockdown of SAE2, UBC9 and stable (shRNA) knockdown of SAE2 both induced NR3C1 mRNA expression and decreased miR-130b levels (Supplemental Fig. S4A-D), indicating the effect of TAK-981 on GR expression is SUMOylation pathway specific. We then evaluated the effects on GR target genes expression. Ribonucleoside-diphosphate reductase subunit M2 (RRM2) has been identified as GR direct target in MM. The expression of RRM2 is repressed by Dex-induced GR activation and the repression of RRM2 is required for Dex-induced apoptosis [20]. TAK-981 decreased RRM2 level and synergistically further reduced the decrease of RRM2 by Dex in both MM1S and H929 cells. There is corresponding induction of apoptosis marker cleaved PARP (Fig. 3G, Supplemental Fig. 4E), indicating the enhancement of TAK-981 to Dex cytotoxicity is related to GR-regulated RRM2 repression. We also observed similar significant changes in the expression of a Dex-activated gene Ras dexamethasone induced 1(RASD1) [21] (Supplemental Fig. 4E), proving the upregulation of GR function upon TAK-981 treatment. To validate our findings, we analyzed public datasets of primary myeloma patient samples. Analysis of cohort (GSE2658) of 559 MM patients (Supplemental Fig. S1B) showed UBA2 level positively correlates with RRM2 and negatively correlates RASD1 expression (Supplemental Fig. S1F). Patients with low SAE2 level (SUMOylation low) showed lower GR-repressed gene RRM2 expression and higher GR-activated gene RASD1 levels. This analysis supports our finding that SUMOylation inhibition upregulates the GR pathway.

Fig. 3
figure3

TAK-981 upregulates GR expression in primary multiple myeloma and cell lines through downregulation of miR-130b. A GR expression is associated with Dex sensitivity in primary MM cells. GR (NR3C1) mRNA expression was assessed by q-PCR and presented as relative level normalized to GAPDH. IC50 values were calculated by GraphPad Prism 8. Pearson correlation analysis showed a significant negative correlation negative correlation between GR RNA (NR3C1) and IC50 for Dex in 15 primary MM. B NR3C1 mRNA level was decreased and (C) miR-130b level was increased in dose-dependent manner in primary MM cells treated with TAK- 981. Primary MM cells from 5 relapsing patients were treated with TAK-981 at indicated concentrations for 48 h. Total RNA extracted and NR3C1 mRNA and miR-130b level was measured by q-PCR. Data presented as mean ± SD, n = 5 (D) TAK-981 treatment increases GR protein level in primary MM cells. Western blot presents GR protein of 3 primary MM samples treated with or without 0.1 μM TAK-981 for 48 h. Relative protein level was quantified using Image J, normalized to GAPDH, and labeled below each blotting band. E NR3C1 mRNA was increased and miR-130b was decreased in MM1S and H929 cell lines upon TAK-981 treatment. Estimated variation is indicated as SD. P values were derived using a two-tailed Student t test. * p < 0.05, ** p < 0.01, ***p < 0.001 (F) TAK-981 treatment increases GR protein level in MM1S and H929 cell lines. MM1S and H929 cells were treated with or without 20 nM TAK-981 for 48 h. Cell lysates were used for western blot. G GR-repressed gene RRM2 and apoptosis marker cleaved PARP (c-PARP) were measured by western blot in MM1S and H929 cell lines treated with TAK-981, Dex or both for 48 h. GAPDH was used as loading controls. Relative protein level was quantified by Image J and labeled below each blot

Previous studies have shown GR can be SUMO-modified [22,23,24,25]. We then evaluated the effect of SUMOylation inhibition on GR activity upon Dex treatment. GR resides in the cytoplasm. Upon ligand (Dex) binding, GR is phosphorylated, then translocates into the nucleus and functions as a transcription factor [26,27,28,29]. As we observed TAK-981 48 h treatment increased GR expression level, we carried out a short time TAK-981 treatment to determine GR activity without affecting its expression level. We treated MM1S with TAK-981 for 4 h to fully abolish SUMOylation, then added Dex for 2 h. Dex-induced GR phosphorylation (Ser 211 and Ser 226) and nuclear localization were not affected upon TAK-981 treatment, suggesting SUMOylation regulates GR mainly through mRNA level but not post-translational level (Supplemental Fig. S5).

SUMOylation inhibition enhances Dex sensitivity in MM through regulating miR-551b and miR-25

Considering inhibition of SUMOylation by TAK-981 and SAE2 knockdown can enhance Dex sensitivity in MM1R cell line among which GR expression is absent, we presume SUMOylation could regulate Dex resistance through other cellular components except GR. Given recently published articles showing that miRNA deregulation may be involved in Dex resistance [4, 30], we conducted miR-seq and RNA-seq in MM1S and MM1R cell lines treated with Dex, TAK-981 or both to identify potential miRs which are involved in Dex resistance and affected by SUMOylation. We cross compared change of miR expression in Dex and TAK-981 treated MM1S and MM1R and identified two candidate miRs: miR-551b and miR-25 (Supplemental Fig. S6).

To evaluate the role of miR-551b and miR-25 in MM cell growth and Dex resistance, we conducted cell viability assay with overexpression and knockdown of the miRs. Overexpression of miR-551b and miR-25 by transfection of miR mimic promoted MM cell proliferation and reduced Dex sensitivity in MM1S, as IC50 increased ~ 4 fold (Fig. 4A-B). Knockdown of miR-551b and miR-25 by transfection of miR inhibitors suppressed cell growth and enhanced Dex sensitivity in MM1R, as IC50 decreased from 875 μM to 193 and 142 μM (Fig. 4C-D). Western blot of cleaved-PARP further confirmed the regulation of miR-551b and miR-25 on Dex sensitivity in MM (Supplemental Fig. S7). These data indicated that the expression of miR-551b and miR-25 may contribute to the Dex resistance of MM.

Fig. 4
figure4

MiR-551b and miR-25 level affect Dex sensitivity in MM. A Overexpression of miR-551b and miR-25 increases cell proliferation and (B) decreases cell sensitivity to Dex treatment in MM1S cells. (C) Knockdown of miR-551b and miR-25 expression inhibits cell proliferation and (D) increases sensitivity to Dex in MM1R cells. MM1S cells were transfected with microRNAs: miR-551b mimic, miR-25 mimic or non-targeting control (NC), MM1R cells were transfected with anti-miRs: miR-551b inhibitor, miR-25 inhibitor or non-targeting control (NC). Cell proliferation was measured at 24 h, 48 h and 72 h. Transfected cells were treated Dex at indicated concentration for 48 h. Cell viability was measured and IC50 values of Dex were calculated using GraphPad Prism 8 and shown in the right panels. Data were analyzed using unpaired Student t tests: Data presented as mean ± SD. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. E Knockdown of miR-551b expression increased TTP protein level and decreased E2F1 and CyclinD1 in MM1R cells. F miR-551b level negatively correlates with ZFP36 level in in 15 primary MM samples. ZFP36 mRNA expression was assessed by q-PCR and presented as relative level normalized to GAPDH. MiR-551b level was measured by Taqman q-PCR and presented as relative level normalized to RNUB. Pearson correlation analysis was performed. p and r values were labeled. G Knockdown of miR-25 expression increased protein levels of ULK1and p27 in MM1R cells. H miR-25 level negatively correlates with p27/CDKN1B level in in 15 primary MM samples

We applied 3 publicly available algorithms Targetscan, miRRDB and miRDIP to predict the potential miR-551b target genes and the results suggests 3 targets: zinc finger protein 36 homolog (ZFP36), praja-1 (PJA1) and zinc finger protein 280C (ZNF280C) (Supplementary Fig. S8). Because PJA1 and ZNF280C are not abundantly expressed in MM cells, ZFP36 became the only potential target of miR-551b. ZFP36-encoded protein, tristetraprolin (TTP), an RNA binding protein, plays an important role in cancer cell proliferation through destabilizing Cyclin D1 and E2F1 mRNA [31,32,33,34]. Knockdown of miR-551b level by transfection of miR-551b inhibitor caused the increase of TTP level and decrease of Cyclin D1 and E2F1 (Fig. 4E). The expression of ZFP36 is negatively correlated with miR-551b in primary MM samples (Fig. 4F), further indicating miR-551b regulating ZFP36 level.

MiR-25 has been characterized with onco-miR function and has multiple validated targets including Unc-51 Like Autophagy Activating Kinase 1 (ULK1) and Cyclin-dependent kinase inhibitor 1B CDKN1B(p27, 35–39]. Knockdown of miR-25 significantly increased ULK1 levels. Knockdown of miR-25 caused an increased level of p27, which is consistent with the observation that p27 mRNA level is negatively correlated with miR-25 in primary MM samples (Fig. 4G-H).

MM1R cells exhibit higher level of miR-551b and miR-25 than MM1S (Fig. 5A, Supplemental Fig. S6). Dex treatment decreased these two miRs levels in MM1S but showed no effect in MM1R, but TAK-981 treatment decreased them in both MM1S and MM1R cell lines and showed further reduction in combination with Dex (Fig. 5A, Supplemental Fig. S9A). Knockdown of SAE2 or UBC9 by siRNA transient transfection decreased miR-551b and miR-25 levels in MM1R (Fig. 5B). Stable knockdown (shSAE2) of SAE2 exhibited reduced level of miR-551b and miR-25 in RPMI-8226 cell line (Supplemental Fig. S9B). To confirm that miR-551b and miR-25 expression is regulated by SUMOylation in vivo, we examined tumor tissue from MM1R xenograft and primary MM samples. Consistent with cellular experiment results, miR-551b and miR-25 levels were reduced in TAK-981 treatment group and further decreased in combination with Dex treatment in MM1R xenograft tumor tissue (Fig. 5C). We treated 5 primary MM samples with different concentration of TAK-981, miR-551b and miR-25 levels significantly decreased in a dose-dependent manner, indicating that the regulation of miR-551b and miR-25 by SUMOylation is not restricted to cell lines (Fig. 5D).

Fig. 5
figure5

SUMOylation inhibition decreases miR-551b and miR-25 level. A miR-551b and miR-25 levels were decreased by TAK-981 treatment in MM1S and MM1R cells. MM1S and MM1R cells were treated with Vehicle (Veh), 0.1 μM TAK-981 (TAK), 1 μM Dex (Dex), or 0.1 μM TAK-981 with 1 μM Dex (combo) for 48 h. miR level was measured by Taqman q-PCR. B Knockdown of SAE2 or UBC9 decreases the expression of miR-551b and miR-25. MM1R cells were transfected with siRNA targeting SAE2 (SiSAE2) or UBC9 (SiUBC9), or non-targeting control (SiCtrl) for 72 h, miR levels were measured by qPCR. C TAK-981 treatment decreases miR-551b and miR-25 levels in vivo. Tumor tissue from MM1R xenograft mice (in Fig. 2C) was used for RNA extraction and miR measurement. D TAK-981 treatment decreased miR-551b and miR-25 level in primary MM cells in dose-dependent manner in primary MM cells treated with TAK- 981. Primary MM cells from 5 relapsing patients were treated with TAK-981 at indicated concentrations for 48 h. miR-551b and miR-25 level was measured by q-PCR. Data presented as mean ± SD, n = 5. E TAK-981 treatment affects miR-551b and miR-25 downstream genes protein level. Western blot presents protein level in MM1R cells treated with vehicle (Veh) and 0.1 μM TAK-981 (TAK-981) for 48 h. Relative protein level was quantified by Image J and labeled below each blot. F GSEA shows cell cycle regulators are suppressed but apoptosis and autophagy related pathways are upregulated upon TAK-981 treatment. GSEA false discovery rate (FDR)-q values, p-values and normalized enrichment scores (NES) are labeled. G UBA2 level correlates with ZFP36 and ULK1 expression in patient specimens. Analysis of cohort (GSE2658) of 559 MM patients (Supplemental Fig. S1B). Patients with high SAE2 (UBA2; UBA2high group) showed lower ZFP36 levels (left) and ULK1 level (right) than patients with low SAE2 (UBA2; UBA2low group). Data were analyzed using unpaired Student t tests: Data presented as mean ± SD. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001

We then evaluated the impact of SUMOylation inhibition on target gene levels of these miRs. TAK-981 treatment caused induction of miR-551b target gene TTP expression and resulted in Cyclin D1 and E2F1 decrease in MM1R cells (Fig. 5E). Because E2F1 is a transcription factor, GSEA analysis was performed and results indicated that E2F1 target gene sets were suppressed upon TAK-981 treatment (Supplemental Fig. S9C). TAK-981 treatment induced expression of miR-25 target genes ULK1 and p27(Fig. 5E). mRNA levels of ZFP36(TTP), ULK1 and p27 showed reversed changes compared to miR-551b and miR-25 in Dex, TAK-981 and combo treated MM1S and MM1R cells (Supplemental Fig. S9D). The expression levels were increased upon TAK-981 treatment and further enhanced in combination with Dex. Furthermore, TAK-981 caused induction of ZFP36 mRNA level was suppressed by exogenous expression of miR-551b. The increased ULK1 and p27(CDKN1B) mRNA levels upon TAK-981 treatment were also suppressed by overexpression of miR-25, indicating that SUMOylation inhibition increased ZFP36 through miR-551b and regulated ULK1 and p27 through miR-25 (Supplemental Fig. S9E). ULK1 is a major enzyme in autophagy pathway, Cyclin D1 and p27 play important roles in cell cycle regulation and apoptosis. GSEA analysis of RNA-seq indicated TAK-981 treatment affected cell cycle, apoptosis and autophagy gene sets (Fig. 5F, Supplemental Table S2), suggesting the effects may be achieved through these genes targeting microRNAs- miR-551b and miR-25 level. We analyzed a MM patients data set GSE2658 (n = 559), among which SAE2(UBA2) level is associated with poor outcome (Supplemental Fig. S1B). Patients with low SAE2 (UBA2; UBA2low group) showed higher ZFP36 (miR-551b target gene) and ULK1 (miR-25 target gene) levels than patients with high SAE2 (UBA2; UBA2high group) (Fig. 5G). The analysis further supports our finding that SUMOylation regulates miR-551b, miR-25 and their target genes expression.

SUMOylation inhibition decreases the expression miR-551b, miR-25 and miR-130b via c-Myc

Next, we investigated whether SUMOylation inhibition caused decrease of miR-551b, miR-25 and miR-130b levels through mediating the transcription of pri-miRNAs. Upon TAK-981 treatment, pri-miR-551b, pri-miR-25 and pri-miR-130b expression levels significantly decreased in both MM1S and MM1R cells (Supplemental Fig. S10A). Knockdown of SAE2 in RPMI8226 cells caused similar reduction of these pri-miRs (Supplemental Fig. S10B). Therefore, these results indicate SUMOylation regulates miR-551b, miR-25 and miR-130b gene expression at transcriptional level.

To identify the transcription factors involved in regulating miR-551b, miR-25 and miR-130b expression in a SUMOylation-dependent manner, we focused on the transcription factors E2F1, c-Fos, and c-Myc, which were shown to bind to the promoter regions in genome-wide ChIP-seq studies using UCSC Genome Browser (genome.ucsc.edu) [35, 36]. We transfected plasmids expressing E2F1, c-Fos, or c-Myc into 293 T cells, then measured pri-miRs level by qPCR. All three pri-miR levels were significantly increased by overexpression of c-Myc, but were not affected by E2F1 or c-Fos overexpression (Supplemental Fig. S10C). The promoter of miR-551b was subcloned to luciferase reporter plasmid, and miR-551b promoter activity was significantly increased by c-Myc overexpression in a dose-dependent manner (Fig. S10D). Because miR-25 promoter is co-localized with miR-93 and miR-106b and miR-130b promoter is in the same region as miR-301b, the reporter plasmid and assay of miR-25 and miR-130b promoter were not conducted. In MM1S cells, c-Myc induced increase of pri-miR levels of miR-551b, miR-25 and miR-130b can be inhibited by addition of TAK-981 treatment (Fig. 6A). In order to further determine the role of c-Myc on miR-551b, miR-25 and miR-130b expression, knockdown of c-Myc expression was carried out. siRNA transfection of c-Myc (siMyc) in MM1S cells caused significant reduction of pri-miRs and mature miR levels (Fig. 6B). We generated stable cell line that can be induced to express shRNA targeting c-Myc in RPMI8226 cells. The pri-miRs and mature miRs decreased after Dox-induced c-Myc knockdown (Fig. 6C and Supplemental Fig. S10E). All these results demonstrated c-Myc as a major transcriptional activator of miR-551b, miR-25 and miR-130b gene expression. Next we evaluated the binding of endogenous c-Myc to miR-551b, miR-25 and miR-130b promoters using ChIP in MM1S and MM1R cells treated with TAK-981 or Dex. TAK-981 treatment significantly decreased c-Myc binding occupancy at the promoter regions of these miRs in both MM1S and MM1R cells (Fig. 6D), proving SUMOylation regulates these miRs transcription via c-Myc binding to the promoter region. Dex showed minor decrease of c-Myc binding in MM1S cells and no effects in MM1R cells, indicating these miRs are involved in Dex sensitivity in MM.

Fig. 6
figure6

c-Myc is a major transcription factor of miR-551b, miR-25 and miR-130b. A Overexpression of c-Myc induced pri-miR-551b, pri-miR-25 and pri-miR-130b and the induction can be inhibited by TAK-981 treatment. MM1S cells were transfected with empty vector (EV) or c-Myc expression plasmid (c-Myc) then treated with 0.1 μM TAK-981 (TAK) or vehicle (Veh) for 24 h. Pri-miR level was measured by q-PCR. B Knockdown of c-Myc decreased miR-551b, miR-25 and miR-130b miR and pri-miR level. MM1S cells were transfected with siRNA targeting c-Myc (siMyc) or non-targeting control (SiCtrl) for 72 h, miR and pri-miR level were determined by qPCR. C RPMI8226 stable cell line was generated with inducible shRNA targeting c-Myc. Dox (5 μg/mL) was added to induce c-Myc knockdown (shMyc+Dox) for 48 h. miRs and pri-miRs levels were measured by qPCR. D ChIP assay identified c-myc binding at the promoter regions of miR-551b, miR-25, and miR-130b. TAK-981 treatment decreases the c-Myc binding occupancy in both MM1S and MM1R cells but Dex only decreases in MM1S cells. ChIP was performed using an anti-c-Myc antibody in MM1S and MM1R cells treated with Vehicle (Veh), 0.1 μM TAK-981 (TAK-981) or 1 μM Dex (Dex). The occupancy was normalized to DNA input and calculated relative to IgG control. Data were analyzed using ANOVA. Data presented as mean ± SD. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. E c-Myc and (F) SAE2 (UBA2) level correlates with miR-551b, miR-25 and miR-130b level in 15 primary MM samples. Pearson correlation analysis was performed. p and r values were labeled

The regulation of c-Myc and SAE2 of miR-551b, miR-25 and miR-130b in MM cell was further confirmed using qPCR by observing a significant positive correlation between c-Myc and SAE2 level and the expression of these miRs in primary MM cells from 15 relapsed patients (Fig. 6E-F). All these demonstrate that SUMOylation regulates miR-551b, miR-25 and miR-130b expression via mediating c-Myc.

SUMOylation inhibition decreases c-Myc level

We then evaluated the impact of SUMOylation inhibition on c-Myc level. Dex decreased c-Myc protein in Dex-sensitive MM1S cells, but showed no effect in MM1R cells. However, TAK-981 treatment showed reduction of c-Myc protein in both MM1S and MM1R cell lines and caused further decrease when combined with Dex (Fig. 7A). Consistent with this, c-Myc level decreased when SAE2 was knockdown in RPMI-8226 cells (Supplemental Fig. S11A). The reduction of TAK-981 on c-Myc level was also observed in another MM cell line H929 (Supplemental Fig. S11B). IHC staining in tumor tissues from MM1R xenograft mice indicated TAK-981 decreased c-Myc level and further reduced when combined with Dex although Dex alone showed no effect on c-Myc in vivo (Supplemental Fig. S11C). GSEA indicated TAK-981 treatment suppressed Myc target gene sets in both MM1S and MM1R cells, TAK-981 and Dex combination treatment showed further suppression of Myc targets compared to Dex treatment alone (Fig. 7B, Supplemental Fig. 11D). We observed a time-dependent decrease of c-Myc protein level upon TAK-981 treatment in MM1R cells (Fig. 7C). Because c-Myc has been identified as SUMO-modified protein [37, 38], we measured protein stability using a cycloheximide (CHX) assa. c-Myc decreased faster in myeloma cells treated with TAK-981 compared to vehicle (Fig. 7D, Supplemental Fig. S11E), indicating SUMOylation inhibition downregulated c-Myc by enhancing their degradation. We performed IP assays to confirm the SUMO modification of c-Myc was reduced by SUMOylation inhibition or SAE2 knockdown. 293 T cells were transfected with His-tagged SUMO-1, His-tagged SUMO-2 and untagged c-Myc expression plasmids. Cells were treated with 0.1 μM TAK-981(TAK) or Vehicle (Veh) for 8 h then Cell lysates were harvested and incubated with Ni-NTA beads to pull down all His-tagged SUMOs then blot c-Myc level (Fig. 7E). The western blot indicated there is much less SUMO-modified c-Myc upon TAK-981 treatment. We conducted IP assay to detect SUMO-modification of endogenous c-Myc in RPMI8226 stable cell line with inducible SAE2 shRNA. The results indicated there was less SUMO modification of c-Myc upon SAE2 knockdown (Supplemental Fig. S11F). These results, along with literature, proved the regulation of c-Myc by direct SUMO-modification.

Fig. 7
figure7

SUMOylation inhibition decreased c-Myc protein level through regulating protein stability. A TAK-981 treatment decreases c-Myc levels in both MM1S and MM1R cell lines and Dex has no effect on c-Myc level in MM1R cell line. MM1S and MM1R cells were treated with TAK-981 or Dex or both for 48 h, c-Myc and cleaved PAPR (c-PARP) levels were determined by western blot. SUMO-2,3 was blotted to determine global SUMOylation and GAPDH was used as loading control. B GSEA analysis of RNA-seq data shows TAK-981 treatment inhibits c-Myc target gene sets in MM1S and MM1R cells. C TAK-981 treatment decreases c-Myc protein level in time-dependent manner. MM1R cells were treated with TAK-981 at 0.1 μM and cells were harvested at indicated time points. C-Myc protein level was determined by western blot. D TAK-981 treatment accelerates c-Myc protein degradation. MM1R cell pre-treated with vehicle or TAK-981 for 2 h were exposed to cycloheximide (CHX) to block protein synthesis, cells were harvested at indicated time points and analyzed for c-Myc level by western blot. c-Myc level was quantified by ImageJ, normalized to GAPDH and plotted. E SUMO modification of c-Myc was reduced upon TAK-981 treatment. 293 T cells were transfected with His-tagged SUMO-1, His-tagged SUMO-2 and untagged c-Myc expression plasmids. Cells were treated with 0.1 μM TAK-981(TAK) or Vehicle (Veh) for 8 h. Cell lysates were incubated with Ni-NTA beads to pull down all His-tagged SUMO-1 and SUMO-2, followed by Western blot analysis with c-Myc antibody (left) or an anti-His-tag antibody (right). Cells transfected with His-tagged SUMO-1 and SUMO-2 but without c-Myc expressing plasmid were used as control (Ctrl). SUMO-modified and unmodified c-Myc were pointed. F Schematic showing the mechanism of SUMOylation inhibition enhance MM sensitivity to Dex. SUMOylation inhibition decreases c-Myc level, causes reduction of miR-130b, miR-551b and miR-25. The decrease of miR-130b results in induction of GR expression. miR-551b and miR-25 reduction causes increase of TTP, ULK1, p27, resulting in dysregulation of cell cycle, apoptosis and autophagy. All these contribute to enhanced sensitivity to Dex

We also observed c-Myc mRNA level decreased upon TAK-981 treatment, which is consistent with our previous finding that SUMOylation regulates c-Myc mRNA level through regulation its targeting microRNA miR-34b/c [39].

Except MM, Glucocorticoids (GCs) are a mainstay of treatment of lymphoid malignancies including Acute Lymphoblastic Leukemia (ALL). We treated Jurkat, a T-ALL cell line, with TAK-981, Dex or both, then measured cell viability. Consistent with previous studies [40, 41], Jurkat cells are resistant to Dex treatment, TAK-981 treatment alone showed potent cytotoxicity and further enhanced cell death when combined with Dex (Supplemental Fig. S12A). Apoptosis assay and cleaved-PARP level further confirmed that TAK-981 enhanced Dex sensitivity in Jurkat cells (Supplemental Fig. S12B-C). Similar as we observed in MM, TAK-981 treatment increased GR and decreased c-Myc level, along with downregulation of miR-130b, miR-551b and miR-25 in Jurkat cells (Supplemental Fig. S12C-E). These findings suggest the mechanism of SUMOylation inhibition enhances Dex sensitivity might not be restricted in MM but also applicable to other types of hematologic cancer.

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