Expression of BMP6, BMP-receptors and downstream SMADs
Expression of BMPs, BMP-receptors and the members of the downstream signal-transduction chain were evaluated using U133 A+B (Heidelberg/Montpellier-group 1; HM1) and U133 2.0 plus (HM2) Affymetrix microarrays (see , Table S1
Presence of expression of BMP6, BMP receptors and downstream SMADs
is the only BMP expressed by normal and malignant plasma cells both in HM1 and HM2 (, Supplementary Table S2
). The mean BMP6-expression is significantly and by several orders of magnitude higher in BMPCs or MMCs compared to B-cell precursor cells (MBCs and polyclonal plasmablastic cells, PPCs; p<.001). Human myeloma cell lines (HMCLs) show a lower expression of BMP6
compared to BMPCs (p<.001 in HM1, Supplementary Table S1
Of the BMP-receptors, four are expressed in BMPCs and MMCs. BMPR2
is present in most BMPCs and precursors without significant change throughout plasma cell differentiation, as well as in MMCs. ACVR1
is an early plasma cell marker, lacking expression in MBCs (, ), and ACVR2B
is aberrantly expressed in MMCs of 12.5 % of patients (). No significantly different gene expression could be found for BMP6 or BMP-receptors between MMCs from early-stage (monoclonal gammopathy of unknown significance (MGUS) and MMI) and advanced stage (MMII and MMIII) patients (Supplementary Table S1D
Expression of BMP6, BMP-receptors and SMADs and validation of gene expression by flow cytometry and western blotting
Of the downstream signaling cascade, SMAD5 is expressed in all plasma cell precursor-, plasma cell- and 166/168 MMC samples (, ), whereas SMAD1 is aberrantly expressed in 100/233 MMC samples and 39/40 HMCLs. SMAD4 is expressed in 1/13 MBC, 4/12 PPC, 4/14 BMPC, and 147/233 MMC samples as well as 36/40 HMCLs. Thus, transducing SMAD1/SMAD4 or SMAD5/SMAD4 complexes are increasingly present from B-cell precursors over MMCs to cell lines. SMAD8 is not expressed.
Of the populations investigated within the whole bone marrow (WBM), only BMPCs, MMCs, and a sub-fraction of mesenchymal stromal cells (MSCs), express BMP6 (). The low number of BMPCs and MSCs in the BM of normal donors could explain the lack of detectable BMP6-expression. Expression of BMP6 within the WBM correlates significantly (rs=.45, P=.001) with the percentage of plasma cell infiltration.
Validation of gene expression
By quantitative real-time PCR (qRT-PCR), BMP6 is expressed in 9/10 HMCLs (absent in XG-10) and 10/10 primary MMC samples, consistent with results by PANP/GEP (see methods). BMP6-receptors (BMPR2, ACVR1) are expressed in all HMCLs and primary MMC samples investigated. BMP6-expression measured by qRT-PCR and GEP correlates well for HMCLs (rs=0.78, P=.009) and MMCs (rs=0.56, P=.05).
By flow cytometry, intracellular BMP6-expression can be detected in 9/10 HMCLs and 3/3 primary MMC samples. In agreement with PANP/GEP and qRT-PCR, BMP6-expression is absent in XG-10. Exemplary data are shown in .
Performing enzyme-linked immunosorbent assays (ELISA), amounts of BMP6 around the level of the detection limit could be found in supernatants of HMCLs. For the highly BMP6-expressing HMCLs XG-11 and U266 (see ) BMP6 levels of 47.59 and 57.51 pg/ml could be measured, respectively. In agreement with GEP, qRT-PCR and flow cytometry no BMP6 was detectable in supernatants of XG-10.
To determine whether BMP6 induces phosphorylation of downstream SMADs, phosphorylated SMAD1, -5, -8 were investigated by western blotting. Following incubation with BMP6, SMAD activation can be detected within 15 minutes. After pre-incubation with heparin, no SMAD-phosphorylation is detectable (, see below).
Biological and clinical correlations of BMP6-expression
BMP6-expression in MMCs inversely correlates (albeit weakly) with a gene expression based proliferation index (GPI; HM1 r=−0.45, P<.001, HM2 r=−0.35, P<.001). No correlation of the expression of BMP6 or BMP-receptors in MMCs with either the presence of the chromosomal aberration t(11;14), t(4;14), gain of 11q13, hyperdiploidy (as measured by our copy number score, see below), gain of 1q21, deletion of 17p, or deletion of 13q14.3 could be found. Likewise, no correlation could be found with expression of D-type cyclins (CCND1, CCND2, CCND3), or clinical parameters (including B2M, ISS, Durie-Salmon-stage, serum-albumin, data not shown). We likewise found no difference of the genetic or clinical markers in BMP6high and BMP6low patients.
MMCs bind BMP6 via membrane heparan-sulfate proteoglycans
For BMP-family members, a binding to heparin/heparan sulfates has been described (Irie et al. 2003
). At the same time, we have shown heparan-sulfate binding members of the EGF-family to bind syndecan-1 (CD138), the only heparan-sulfate proteoglycan constantly present on the surface of BMPCs and MMCs (Mahtouk et al. 2006
). Therefore, HMCLs were incubated with saturating concentrations of BMP6 and analyzed using flow cytometry. We could demonstrate BMP6 binding to 10/10 HMCLs. Binding in all cases is reduced by incubation of BMP6 with heparin, functioning as a competitor that likely captures away BMP6 (, exemplary data). Together with the fact that BMP6-induced apoptosis is abrogated by heparin (see above), these data suggest BMP6 being a heparan-sulfate binding molecule.
Binding of BMP6 to human myeloma cell lines
BMP6 inhibits proliferation and induces apoptosis in HMCLs and primary MMCs
BMP6 significantly inhibits proliferation of all HMCLs investigated in a dose-dependent manner (). The maximum inhibition at 4 μg/ml ranged from 27.9 % (RPMI-8226) to 91.1 % (OPM-2). For 6/10 cell lines, a 50 % inhibition (IC50) could be obtained ranging from 0.08 (XG-11) - 2.15 (LP-1) μg/ml.
Inhibition of proliferation and induction of apoptosis of myeloma cell lines by BMP6 as well as survival of primary myeloma cells
Next, OPM-2 and XG-11 cells were cultured for 3 days with or without BMP6. Cell viability and apoptosis were determined by flow cytometric analysis of annexin V binding and propidium iodine (PI) uptake. BMP6 induced apoptosis after 8 h (12.1 % vs. 6.2 % control) to 72 h (38.5 % vs. 6.7 % control). Heparin-pretreatment inhibits BMP6-induced apoptosis (, exemplary data), likely explained by the heparin-induced competitive reduction of BMP6-binding to heparan-sulfate chains on plasma cells (see below). Heparin alone did not influence the apoptosis rate.
To test whether the survival of primary MMCs is inhibited as well, these were cultured within their BM microenvironment (negative fraction of plasma cell purification) and exposed to BMP6. After 6 days, cell viability was measured by CD138/PI flow cytometry. As shown in , BMP6 significantly inhibited the survival of 3/3 primary MMC samples. The maximal inhibition was 90 %, 93.6 %, and 94.6 %, respectively.
In terms of apoptosis-induction we could demonstrate increasing levels of cleaved caspase-3 (effector caspase), -8 and -9 (initiator caspases) after BMP6 treatment for 48 and 72 h, respectively. This effect is abrogated by pre-treatment with heparin (, exemplary data).
Next, we tested whether the production of BMP6 by MMCs themselves inhibits their proliferation. We therefore exposed HCMLs with the BMP6-inhibitors noggin (Kersten et al. 2006
) and sclerostin (Kusu et al. 2003
), respectively. Both, sclerostin and noggin exposure yielded a concentration dependant increase of proliferation. Exemplary data for U266 (high endogenous BMP6-production, very low sensitivity to exogenous BMP6; see , ) are shown in . As a control, we co-exposed the HMCLs with exogenous BMP6 (1 μg/ml) and graded concentrations of noggin or sclerostin, showing that indeed noggin and sclerostin significantly and concentration-dependently abrogated BMP6 mediated inhibition of myeloma cell proliferation (). Therefore, production of BMP6 by myeloma cells contributes to the inhibition of their growth in vitro.
Inhibition of in vitro tubule formation by BMP6
The angiogenic potential of BMP6 was investigated in the AngioKit assay with graded concentrations of BMP6. BMP6 significantly inhibited in vitro tubule formation with a strong inhibition already observed at 0.032 μg/ml (, P=.04 and P=.001 in two independent experiments) compared to the medium control. The inhibition was as efficient as that provided by suramin, a usual tubule formation inhibitor.
Inhibition of in vitro induction of angiogenesis by BMP6
Prognostic value of BMP6, BMP-receptors and SMAD-expression
In a Cox-model as single continuous variable, BMP6-expression is significantly predictive for overall survival (OS) in the HM1 (P=.02), HM2 (P=.01) and the LR-data (P=.005). For event-free survival (EFS), it is only predictive in the LR-data (P=.004). In a Cox-model tested with B2M (as continuous variable), BMP6-expression appears as independent prognostic factor for OS in the HM1 (BMP6-expression (P=.02), B2M (P=.7)), HM2 (BMP6-expression (P=.03), B2M (P=.02)), and the LR-data (BMP6-expression (P=.01), B2M (P<.001)). The same holds true if BMP6-expression is tested with ISS in the HM1 (BMP6-expression (P=.04), ISS (P=.9)), HM2 (BMP6-expression (P=.03), ISS (P=.03)) and the LR-data (BMP6-expression (P=.02), ISS (P<.001)). In the LR-data, BMP6 remains an independent prognostic factor for EFS if tested with B2M (BMP6-expression (P=.02), B2M (P<.001)) or ISS (BMP6-expression (P=.01), ISS (P<.001)). Likewise, patients with BMP6high expressing MMCs show a significantly better OS compared to patients with BMP6low-expression (n=168, P=.02, HR 0.4, CI [0.18;0.87], ). No prognostic value could be determined for EFS (P=.9, P=.9, and P=.9, respectively). Similar observations could be made with the patient cohort from the LR-group (n=345, OS, P=.03, HR 0.67, CI [0.46;0.97] and EFS, P=.15, ). Genes coding for BMP-receptors or downstream SMADs had no prognostic value. In the WBM (n=57), BMP6-expression above the median (WBM-BMP6high) delineated a group with better EFS (n=49, P=.03, HR 0.45, CI [0.21;0.95]) and a tendency to better OS (P=.3, ).
Effect of BMP6-expression on event-free and overall survival