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Myeloma

Bone morphogenetic proteins induce apoptosis in multiple myeloma cells by Smad-dependent repression of MYC

A Corrigendum to this article was published on 09 May 2012

Abstract

Bone morphogenetic proteins (BMPs) have been shown to induce apoptosis and growth arrest in myeloma cells. However, the molecular mechanisms behind these events are not known. The MYC oncogene is a master regulator of cell growth and protein synthesis and MYC overexpression has been proposed to be associated with the progression of multiple myeloma. Here, we show that BMP-induced apoptosis in myeloma cells is dependent on downregulation of MYC. Moreover, the results suggest that targeting the MYC addiction in multiple myeloma is an efficient way of killing a majority of primary myeloma clones. We also found that myeloma cells harboring immunoglobulin (IG)-MYC translocations evaded BMP-induced apoptosis, suggesting a novel way for myeloma cells to overcome potential tumor suppression by BMPs.

Introduction

Multiple myeloma is a cancer of the bone marrow due to malignant transformation and clonal expansion of plasma cells. Despite recent advances in treatment it is still an incurable disease. Multiple myeloma is always preceded by a premalignant condition termed monoclonal gammopathy of undetermined significance.1 In approximately half of the patients with monoclonal gammopathy of undetermined significance, the plasma cells contain a translocation juxtaposing an oncogene to an immunoglobulin enhancer.2 Such translocations may lay a foundation for secondary oncogenic events leading to manifest multiple myeloma. Gene expression profiling of the malignant cells has identified distinct subgroups of the disease, probably reflecting activation of distinct oncogenic pathways in different patients.3 Recent evidence suggests that deregulated MYC may be associated with progression toward multiple myeloma from monoclonal gammopathy of undetermined significance.3, 4, 5, 6 MYC is also central in two other B cell-derived tumors, namely plasmacytomas and Burkitt's lymphoma where translocations of IGH-MYC are major oncogenic events.7 Overexpression of MYC occurs by different mechanisms including gene amplification, translocation and mutation. MYC chromosomal rearrangements are present in many myeloma cell lines,8, 9 and to a varying degree (15–46%) in primary myeloma cells.10, 11 Moreover, 40% of MYC translocations in human myeloma cell lines do not involve an immunoglobulin gene.9 However, overexpression of MYC has been reported in a larger fraction of myeloma patients than the patients carrying MYC translocations.6, 12 Importantly, knockdown of MYC was shown to be toxic to myeloma cell lines,13 indicating that myeloma cell lines may be addicted to MYC expression.

We and others have earlier shown that treatment of myeloma cells with bone morphogenetic proteins (BMPs) may induce growth arrest and apoptosis in vitro.14, 15, 16 BMPs are present in the bone marrow and are produced by stromal cells17 as well as by normal and malignant plasma cells.18, 19 Furthermore, high expression of BMP-6 mRNA in myeloma cells is beneficial for the patient.18 Taken together, these findings suggest that BMPs suppress tumorigenesis in multiple myeloma.

BMPs are members of the transforming growth factor (TGF)-β superfamily. They signal to cells by binding and formation of heterotetrameric receptor complexes consisting of combinations of type 1 (activin receptor-like kinase (ALK)-1, -2, -3 and -6) and type 2 (BMPR2, ACVR2A and ACVR2B) BMP receptors.20 Dependent on the expression of type 1 receptors, myeloma cells tend to be sensitive to either BMP-4 or BMP-6 or both.16 The main intracellular result of BMP receptor activation is phosphorylation of at least one of the closely related Smad1, Smad5 or Smad8 proteins, which together with Smad4 translocate to the cell nucleus to directly or indirectly regulate gene transcription. BMP receptors have also been reported to activate signaling cascades inside cells independently of Smads. Several mechanisms have been proposed for BMP-mediated growth inhibition and apoptosis in various cell types.21, 22, 23 TGF-β has earlier been shown to exert some of its tumor suppressor activity by repressing MYC transcription through interaction of Smad2/3 with a sequence in the proximal MYC promoter.24, 25, 26 Here, we address the mechanisms behind BMP-induced apoptosis in multiple myeloma, and find that it is dependent on downregulation of MYC. Furthermore, the results indicate that the majority of myeloma clones are dependent on MYC expression.

Materials and methods

Cell culture and reagents

The multiple myeloma cell lines used in this study were INA-6 (kind gift from Dr Martin Gramatzki, Erlangen, Germany)27 and IH-1.14 INA-6 cells were grown in 10% heat inactivated fetal calf serum in RPMI-1640 (hereafter described as RPMI) supplemented with recombinant human interleukin (IL)-6 (1 ng/ml) (Biosource, Camarillo, CA, USA). IH-1 cells were maintained in 10% heat inactivated human serum (Blood Bank, St Olav's University Hospital, Trondheim, Norway) in RPMI and IL-6 (2 ng/ml). All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. Other recombinant human cytokines used were IL-15, BMP-4 and BMP-6 (R&D Systems Europe Ltd, Abingdon, UK). Dorsomorphin (Sigma-Aldrich, St Louis, MO, USA) was used to inhibit BMP type 1 receptor-mediated Smad activation.28 Stock solutions (40 mM) of the MYC inhibitor 10058-F4 (Sigma-Aldrich) were made using dimethyl sulphoxide.

Gene expression profiling

Gene expression profiling was performed by the NMC (Norwegian Microarray Consortium) at NTNU, Trondheim, Norway, applying Illumina Human-6 v2 Expression BeadChips (Illumina, Inc., San Diego, CA, USA). In short, IH-1 cells were washed in Hanks’ balanced salt solution, seeded in RPMI with 0.1% bovine serum albumin and treated with BMP-4 and IL-6 or IL-15 for 0.5, 1, 2, 4 and 8 h. Untreated control samples were included at time 0 and 8 h. To isolate total RNA from pelleted cells, we used the High Pure RNA Isolation Kit (Roche Applied Science, Mannheim, Germany) and RNA was kept at −80 °C until further processing. The Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to check both the quality of RNA before and after RNA amplification and cRNA synthesis. cRNA concentrations were measured using NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) to ensure that equal amounts were used for all hybridizations. Raw and normalized microarray data are available through ArrayExpress, accession number: E-MTAB-619.

Primary myeloma cells

CD138+ cells were isolated from multiple myeloma patient samples consecutively included in the Norwegian Myeloma Biobank at St Olav's University Hospital, Trondheim, Norway. The samples were leftover material from bone marrow or peripheral blood taken for diagnostic purposes. Of 21 samples included, 15 were subject to all experiments, whereas for the remaining 6 patients, the immunoblotting was omitted due to scarcity of cells available for study. Patient characteristics are described in Table 1. The project (#4.2007.933) was approved by the Regional Ethics Committee and all patients had given informed consent. CD138+ cells were isolated from bone marrow aspirates using RoboSep automated cell separator and Human CD138 Positive Selection Kit (StemCell Technologies SARL, Grenoble, France). For analysis of BMP response, CD138+ cells were seeded in media containing 2% human serum in RPMI and treated with BMP-4 or BMP-6 as indicated.

Table 1 Patient characteristics and BMP responses

Transfections

For transient gene expression studies INA-6 cells were transfected using the Nucleofector device (Lonza, Basel, Switzerland) as described.29 SMAD4 and negative control Silencer Select siRNAs were from Ambion (Applied Biosystems, Carlsbad, CA, USA). For stable exogenous MYC expression in INA-6 cells, we first transfected the cells with pcDNA3 (Invitrogen Ltd, Paisley, UK) or pcDNA3-cmyc plasmid 16011 from Addgene plasmid repository (deposited by Wafik El-Deiry).30 The pcDNA3-cmyc plasmid does not contain the promoter region of MYC. Following transfection, the cells were subject to selection by G418 (1 mg/ml) (Invitrogen) and cloned by seeding single cells in 96-well plates.

Apoptosis studies

The extent of apoptosis was measured by flow cytometry using APOTEST-FITC Kit (Nexins Research, Kattendijke, The Netherlands). In short, cells were incubated with annexin-V FITC in binding buffer for 1 h. Propidium iodide (PI) (1.4 μg/ml) was added 5 min before the cells were analyzed on a Coulter Epics Cytometer (Beckman Coulter, Inc., Brea, CA, USA). Cells negative for both annexin-V FITC and PI were considered viable.

Immunoblotting

The procedure is described in Supplementary materials and methods.

Fluorescent in situ hybridization

Fluorescent in situ hybridization was used to detect MYC translocations. See Supplementary materials and methods for description of the method, including the probes used.

Real-time RT-PCR

Total RNA was isolated as described above. Complementary DNA was synthesized from total RNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems). PCR was performed using StepOne Real-Time PCR System and Taqman Gene Expression Assays (Applied Biosystems). The comparative Ct method was used to estimate relative changes in gene expression using GAPDH as housekeeping gene. Taqman assays are listed in Supplementary materials and methods.

Statistical analysis

Statistical significance of differences in cell viability was evaluated using one-tailed Student's t-test, where P-values <0.05 were regarded as significant.

Results

BMP-induced apoptosis correlated with downregulation of MYC and MYC target genes in the IH-1 myeloma cell line

To characterize the mechanism behind BMP-induced apoptosis in multiple myeloma cells, we analyzed changes in gene expression levels by microarray technology during the early phase of apoptosis induction in the IH-1 myeloma cell line. Apoptosis, as detected by annexin-V staining, cannot be detected in the cultures before 20 h after BMP addition in this cell line.14 However, when mRNA levels were analyzed 8 h after cytokine addition, 823 probe sets were significantly different in cells treated with BMP-4 compared with untreated cells, that is, in cells later going to die by apoptosis versus in surviving cells (data not shown).

To further narrow down the list to genes more likely to be important for BMP-induced apoptosis, we took advantage of earlier findings that the IH-1 is relatively unique among myeloma cell lines because it responds to a number of cytokines (data not shown). Thus, in a screen for cytokines able to counteract BMP-induced apoptosis in this cell line, we found that IL-15, in contrast to IL-6, counteracted apoptosis induced either by BMP-4 or by BMP-6 (Figure 1a; Supplementary Figure S1). We therefore looked specifically at the genes that were significantly different in cells treated for 8 h with BMP-4 in the presence of IL-6 compared with cells treated with BMP-4 in the presence of IL-15. We then ended up with 222 probe sets, 3 non-coding small nucleolar RNAs, the microRNA host gene MIR17HG, 6 not annotated gene loci, whereas 4 of the genes appeared with 2 different probe sets. (All probe sets are listed in Supplementary Table S1.) We were thus left with 208 protein-coding genes, of which 156 were downregulated and 52 were upregulated in IH-1 cells going into apoptosis versus in non-apoptotic cells (Figure 1b). We expected to find genes previously described in the pro-apoptotic BMP response in other cells. However, it immediately became clear that the most striking common property of many of these genes was that they were known to be regulated by the transcription factor MYC. Thus, of the 208 protein-coding genes regulated by BMP-4 in IH-1 cells going into apoptosis, 129 (62%) had been proposed to be MYC transcriptional targets (108/156 of the downregulated and 21/52 of the upregulated genes) (Figure 1b; Supplementary Table S1).

Figure 1
figure1

BMP-induced apoptosis correlated with downregulation of MYC and MYC target genes in IH-1 cells. (a) IL-15 (20 ng/ml), in contrast to IL-6 (0.1 ng/ml), abrogated apoptosis induced by BMP-4 or BMP-6 in the IH-1 myeloma cell line. Cells were treated with cytokines for 72 h and fractions of viable cells were determined as annexin-V- and propidium iodide-negative cells by flow cytometry. Error bars represent the s.d. of duplicate measurements. (b) Comparison between the total number of genes and MYC target genes with significantly changed expression in IL-6/BMP-4 versus control and IL15/BMP-4 at 8 h. See also Supplementary Table S1.

MYC was downregulated by BMP-4 or -6; involvement of Smads

One well-known transcriptional target of MYC is the MYC gene itself.31 MYC mRNA (Figures 2a and b; Supplementary Figure S2) and protein levels (Figures 2c and d) were reduced in response to BMP treatment in both IH-1 and INA-6 myeloma cell lines under apoptotic conditions. Additionally, under apoptotic conditions, caspase-3 was cleaved and levels of Bcl-xL were downregulated (Figure 2c). In contrast, levels of mRNA encoding ID1, -2 and -3, which are well-established transcriptional targets for activated Smad1/5/8, were elevated by BMP treatment, irrespective if the cells were determined to die or to survive (Supplementary Figure S2).

Figure 2
figure2

BMP induced downregulation of MYC; involvement of Smads. (a) Levels of MYC mRNA in the IH-1 cell line after treatment with BMP-4 for different time periods measured by QRT-PCR. (b) MYC mRNA levels in the IH-1 cell line after treatment with increasing amounts of BMP-4 for 8 h measured by QRT-PCR. (c) MYC immunoblot of IH-1 cells treated for 24 h (two upper panels) or 48 h (three lower panels) with BMP-4 together with IL-6 or IL-15. (d) Time-dependent downregulation of MYC protein levels in IH-1 by BMP-4 (20 ng/ml) (upper panel) and in INA-6 by BMP-6 (300 ng/ml) (lower panel). (e) INA-6 cells were treated with BMP-6 (100 ng/ml) with or without dorsomorphin (2 μM) for measurement of viability by annexin-V and PI staining (72 h) or Smad1/5/8 activation by immunoblotting (20 h). (f) INA-6 cells were transfected with Smad4 or control siRNA. At 40 h after transfection, the cells were incubated with BMP-6 (150 ng/ml) for 4 h. Cells were lysed and subject to immunoblotting using MYC and Smad4 antibodies. A GAPDH antibody was used as loading control for all immunoblots.

Earlier, an oncogenic cycle has been proposed to exist in myeloma cell lines whereby MYC and the transcription factor IRF4 maintain the expression of each other.13 Also in the IH-1 cell line, BMP-4 treatment reduced IRF4 mRNA levels (Supplementary Figure S2), but whereas IL-15 protected the cells from BMP-induced repression of MYC, it did not prevent the reduction of IRF4 mRNA, suggesting that there is no simple relationship between MYC and IRF4 expression and apoptosis in the IH-1 cell line.

The BMP type 1 receptor inhibitor dorsomorphin was used to examine the involvement of Smad signaling in BMP-induced apoptosis in IH-1 cells. Dorsomorphin protected cells from apoptosis concomitant with reduced levels of phosphorylated Smad1/5/8 and sustained MYC expression (Figure 2e). The same effect was observed using INA-6 cells (data not shown). Furthermore, transient knockdown of Smad4 in INA-6 cells using siRNA protected the cells from BMP-induced downregulation of MYC (Figure 2f).

Taken together, the results indicated that in the IH-1 and INA-6 myeloma cell lines there was a correlation between BMP-induced apoptosis and reduction of MYC levels, and that induction of apoptosis and MYC downregulation at least partly depended on Smad activity.

MYC was downregulated by BMP in primary myeloma cells

Multiple myeloma cell lines have been reported to be addicted to MYC expression.13 In order to see if MYC downregulation by BMP was associated with apoptosis also in primary myeloma cells, CD138+ cells isolated from patient bone marrow aspirates or peripheral blood were treated with BMP-4 (20 ng/ml), BMP-6 (300 ng/ml) or without BMP for 3 days before measurement of cell viability (Figure 3a; Supplementary Figure S3). Cells from 15 of the patients, where sufficient number of CD138+ cells were available, were analyzed for protein levels of phospho-Smad1/5/8, MYC and GAPDH (Figure 3a). In parallel, the presence of MYC translocations was investigated by fluorescent in situ hybridization in all patient samples as well as in IH-1 and INA-6 cell lines (Table 1; Supplementary Table S2; Supplementary Figure S3). Neither IH-1 nor INA-6 cells contained immunoglobulin (IG)-MYC translocations.

Figure 3
figure3

BMP-induced apoptosis correlated with activation of Smads and downregulation of MYC protein in primary myeloma cells. (a) Samples from multiple myeloma patients, MM1–MM4, MM6-MM10, MM13-MM16, MM18 and MM21, treated with BMP-4 (20 ng/ml) or BMP-6 (300 ng/ml) for 72 h before measurement of cell viability and for 20 h before immunoblot analysis. Cells were labeled with annexin-V FITC and PI and analyzed by flow cytometry. Cells negative for both annexin-V and PI were considered viable. Error bars represent s.d. of duplicate measurements. Asterisks (*) indicate significantly lower cell viability as compared with control (P<0.05, one-tailed t-test). Western blots were probed with antibodies specific for phospho-Smad1/5/8 (upper panel), MYC (middle panel) or GAPDH (lower panel). (b) Interphase fluorescent in situ hybridization of MM7 and MM9 primary myeloma samples using probes for IGL (green) and MYC (red). See also Table 1 for patient characteristics and BMP responses, Supplementary Figure S3 and Supplementary Table S2 for details regarding fluorescent in situ hybridization analyses.

In 11 out of the 15 patients (MM1, MM2, MM6, MM8, MM10, MM13–16, MM18 and MM21), either BMP-4 or BMP-6 induced phosphorylation of Smad1/5/8, as well as repression of MYC, concomitant with induction of apoptosis. Thus, in a majority of the myeloma clones analyzed, Smad activation correlated with MYC downregulation and induction of apoptosis. Generally, the effects of BMP-6 were more pronounced than the effects of BMP-4. The reason might be that we have used a relatively low concentration of BMP-4 compared with BMP-6, when the ED50 of these cytokines in various cell assays is taken into consideration (data not shown). Alternatively, there could be differences in BMP receptor expression between patients that could explain the differential effects.16

In contrast, two patient samples (MM7 and MM9) showed BMP-induced activation of Smads, but not MYC downregulation, and these cells were protected (MM9) from apoptosis or showed a statistically significant but very minor degree of apoptosis (MM7). Interestingly, these two patients had translocations placing MYC in the proximity of the IGL gene (Figure 3b; Table 1; Supplementary Table S2), suggesting that the presence of a strong immunoglobulin enhancer may override the ability of BMPs to downregulate MYC expression.

In patients MM3 and MM4, we neither observed MYC downregulation, nor apoptosis after incubation with BMP. However, cells from these two patients presented with activated Smad1/5/8 even before treatment with BMPs, suggesting that cells from these patients already were adapted to survival even in the presence of active BMP signaling.

For the six patients where immunoblot data could not be obtained, patients MM5 and MM17 were resistant to both BMP-4- and -6-induced apoptosis (Table 1; Supplementary Figure S3). Interestingly, one of these resistant clones of primary myeloma cells also had an IG-MYC translocation (Table 1), suggesting that such translocations may protect the cells from BMP-induced apoptosis.

Taken together, the results indicate that in the majority of primary myeloma cell samples analyzed there was a correlation between induction of apoptosis and reduction of MYC protein by treatment with either BMP-4 or -6.

Forced expression of MYC protected myeloma cells from BMP-induced apoptosis

The relationship between cell viability and MYC levels has been reported to be complicated in many cell types. Thus, shRNA-mediated downregulation of endogenous MYC mRNA was shown to induce apoptosis in a number of myeloma cell lines.13 However, overexpression of MYC may also induce apoptosis in various cells.32 Furthermore, there are indications that cancer cells may become addicted to a certain level of MYC that may promote cell survival and proliferation.33 We, therefore, stably transfected INA-6 cells with a vector expressing MYC under control of a viral promoter before selection and cloning of surviving cells. In three such clones shown here, MYC expression protected cells from BMP-induced apoptosis (Figure 4a; Supplementary Figure S4). Furthermore, high MYC protein and mRNA levels were maintained despite BMP treatment of the cells (Figures 4b and c). Importantly, the lack of BMP sensitivity was not due to impairment of BMP signaling as the cells showed normal BMP-induced Smad1/5/8 activation (Figure 4b).

Figure 4
figure4

Forced expression of MYC protected myeloma cells from BMP-induced apoptosis. INA-6 cells were stably transfected with pcDNA3-cmyc (MYC) or control vector (CTR). (a) The INA-6 clones CTR and MYC-1-3 were treated with increasing amounts of BMP-6 for 72 h. Fractions of viable cells were determined as annexin-V- and PI-negative cells by flow cytometry. (b) The INA-6 CTR and MYC-1 clones were treated with BMP-6 for 5 h and subject to immunoblotting. The blot was probed with p-Smad1/5/8, MYC and GAPDH antibodies. (c) Measurement of MYC mRNA by RT-PCR in INA-6 MYC-1 or CTR, after treatment of increasing amounts of BMP-6 for 5 h. (d) Cell viability of INA-6 CTR and MYC-1 clones after treatment with the MYC inhibitor 10058-F4 for 24 h. (e, f) Measurement of CDKN1B (e) or IRF4 (f) mRNA by RT-PCR after treatment with increasing amounts of BMP-6 for 5 h. Error bars represent s.d. of duplicate (a, d) or triplicate (c, e, f) measurements.

The low-molecular weight compound 10058-F4 has been identified by its ability to inhibit MYC–MAX interaction and function.34 This inhibitor efficiently induced apoptosis in both control INA-6 cells as well as in cells stably expressing MYC (Figure 4d), indicating that apoptotic effectors are intact in this clone and that the cells still depend on MYC function. CDKN1B, encoding p27, is known to be induced after BMP or TGF-β activation, but is at the same time transcriptionally repressed by MYC. In the INA-6 cells, BMP-6 induced CDKN1B expression; however, forced expression of MYC counteracted this effect (Figure 4e). In contrast, expression of IRF4 mRNA in these cells was downregulated by BMP-6 despite maintenance of MYC levels (Figure 4f), suggesting that also in the INA-6 myeloma cell line, as in IH-1 cells (Supplementary Figure S2), there is no clear-cut relationship between MYC and IRF4 expression. Altogether, the data indicate that at least under some conditions, forced maintenance of MYC levels protected myeloma cells from BMP-induced apoptosis.

Discussion

We and others have earlier reported that BMPs are potent inducers of apoptosis in multiple myeloma cells; however, the molecular mechanisms underlying their tumor suppressive effects in myeloma cells have not been clarified. The most important finding reported here is that BMP-induced apoptosis in myeloma cells is dependent on downregulation of MYC. Overexpression of MYC has earlier been reported in primary myeloma cells.6, 12 Furthermore, the concept of oncogenic addiction to MYC was recently suggested to characterize multiple myeloma cell lines.13 We found that in the majority of primary myeloma samples (11 out of 15), apoptosis induced by BMPs coincided with MYC downregulation. An unexpected by-product of the study is that the results obtained suggest that targeting MYC expression in myeloma cells efficiently kills a majority of the primary malignant plasma cell clones.

MYC has been shown to coordinate a number of cellular functions through transcriptional control of a large fraction of cellular genes.35 It is not entirely clear how downregulation of MYC in MYC addicted cells induce apoptosis at the molecular level. Thus, MYC-mediated perturbation of the activity of pro- and anti-apoptotic Bcl2 proteins, MYC-mediated apoptosis by p53, or by p38- and JNK-MAPK-dependent mechanisms have been proposed to operate in various cell types.35 Furthermore, TGF-β has been shown to induce apoptosis through a TAK1/TRAF6/p38-dependent mechanism in prostate cancer cells.36 However, in our hands, neither siRNA-mediated downregulation of p53, nor incubation of cells with pharmacological inhibitors of p38 or JNK, affected the ability of BMPs to induce apoptosis in myeloma cell lines37 (data not shown). Interestingly, it takes significant time before the first apoptotic markers can be detected after BMP is added to myeloma cells in vitro.14 Thus, in myeloma cells, the time course of BMP-induced apoptosis is more similar to apoptosis by growth factor deprivation and it is very different from the rapid Fas-induced apoptosis that can be observed in myeloma cells.38 An explanation for this delayed apoptosis can be that it takes some time before the diverse effects of downregulation of MYC expression affects the effector machinery involved in apoptosis.

In the study reported here, we found that 16 out of 21 consecutive analyzed patients were sensitive to BMP-induced apoptosis. The generality of our findings for myeloma patients as a group was based on the assumption that the patients who were available for analysis were not selected patients in any way. This may, however, not be the case, as only patients where sufficient amounts of CD138+ cells could be isolated were included in the study.

On the other hand, 5 out of 20 patients had cells that were resistant to BMP-induced apoptosis. Interestingly, in two of these patients (MM5 and MM9) the resistance toward BMP-induced apoptosis and lack of BMP-induced downregulation of MYC correlated with MYC translocations to immunoglobulin light chain genes (IGK and IGL, respectively). However, at the same time, one of the patients with very moderate but significant BMP-induced apoptosis also carried an IGL-MYC translocation (MM7). Light chain translocations to MYC have earlier been reported in primary myeloma cells in 15% of the patients,11 a frequency that fits well with our observation of 3 out of 21 patients having such translocations. Similar translocations are common in Burkitt's lymphomas and plasmacytomas. Interestingly, the lymphoma cell line Daudi, harboring a t(8;14) translocation,39 was resistant to BMP-induced apoptosis despite having proper BMP receptors and functional Smad signaling (TH, unpublished data).

Two other patients who were resistant to BMP-induced apoptosis and MYC downregulation presented with constitutive Smad activation even before addition of BMPs (MM3 and MM4). Constitutively, active Smad signaling in these patients could either be due to production of endogenous BMPs in the cells or to activating mutations in BMP receptors in these patients. Indeed, in both of these patients constitutive Smad activation correlated with endogenous production of BMP-6 mRNA in the myeloma cells (data not shown). As BMPs have been reported to have the potential of either inhibiting or promoting cancer cells we speculated that the cells from these patients in fact could have become addicted to BMP signaling. However, in one of the patients from whom sufficient cells were available for analysis (MM3), incubation of the cells with the BMP receptor kinase antagonist dorsomorphin did not affect growth or survival of the cells, whereas it did inhibit Smad phosphorylation (data not shown), suggesting that additional genetic events had taken place in the cells making them resistant toward BMP-induced downregulation of MYC.

To show that BMP-induced apoptosis was dependent on MYC expression by counteracting apoptosis by overexpression of exogenous MYC in myeloma cell lines turned out to be a difficult task. The reason for this is that not only may downregulation of MYC induce apoptosis as shown here, but overexpression of MYC has also been reported to induce apoptosis in itself. Thus, experiments with transient overexpression of MYC in the INA-6 myeloma cell line were inconclusive due to substantial apoptosis in the transfected cells even before treatment with BMPs (data not shown). In order to overcome these problems, we selected clones of the INA-6 cell line surviving the forced overexpression of MYC from a viral promoter. The clones had similar growth characteristics as a clone transfected with empty vector only and also as compared with untransfected INA-6 cells. Furthermore, the clones had similar BMP receptor expression (data not shown) as the parent INA-6 cell line and Smad signaling were operating normally. However, BMP treatment did not reduce the level of MYC, nor did it induce apoptosis in the cells. The results obtained with the cells from the two patients with IG-MYC translocations also strengthen the hypothesis that BMP-induced apoptosis is dependent on MYC downregulation. Also here, BMPs did not downregulate MYC despite functional Smad signaling, and the cells were protected from BMP-induced apoptosis. In contrast, clones of INA-6 cells expressing MYC from a viral promoter were as sensitive as control INA-6 cells to apoptosis mediated by the MYC–MAX heterodimerization inhibitor, 10058-F4. Thus, even though BMPs were unable to downregulate MYC, inhibition of MYC activity appeared to be sufficient to kill also these cells.

BMPs and other members of the TGF-β superfamily have previously been shown to induce apoptosis by different mechanisms in different cell systems. Interestingly, BMP-induced growth suppression of the colorectal cancer cell line HT-29 was recently shown to be associated with MYC downregulation by a RUNX3-dependent mechanism.40 At least in IH-1 myeloma cells, BMPs only marginally increased RUNX3 expression, and there was no difference in the induction of RUNX3 under apoptotic versus non-apoptotic conditions (data not shown).

Multiple myeloma is an incurable disease; however, the prognosis for patients has improved during the latest years due to the introduction of new treatments such as bortezomib, thalidomide and lenalidomide. Given the MYC dependence in a majority of primary myeloma cells reported here, it will be of interest to see if new treatment modalities directly or indirectly target MYC expression. There is already some evidence along this line, as it has been reported that proteasome inhibitors may induce BMP expression in osteoblasts.41 Taken together, the results presented here indicate that MYC expression is an attractive target for therapy of multiple myeloma.

Accession codes

Accessions

GenBank/EMBL/DDBJ

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Acknowledgements

We are grateful to Berit Fladvad Størdal for excellent technical assistance. We wish to thank the staff at the Department of Hematology, St Olav's University Hospital, Trondheim, Norway and the Norwegian Myeloma Biobank for help obtaining primary myeloma samples. We thank Karin Fahl Wader for valuable help organizing clinical data from patients. We also acknowledge the support provided by the Norwegian Microarray Consortium (NMC), Trondheim, Norway. This work was funded by the Norwegian Cancer Society and the Norwegian Research Council.

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Correspondence to T Holien.

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Holien, T., Våtsveen, T., Hella, H. et al. Bone morphogenetic proteins induce apoptosis in multiple myeloma cells by Smad-dependent repression of MYC. Leukemia 26, 1073–1080 (2012). https://doi.org/10.1038/leu.2011.263

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Keywords

  • multiple myeloma
  • bone morphogenetic protein
  • MYC
  • apoptosis
  • oncogene addiction

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