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. 2017 May 10;12(5):e0174884.
doi: 10.1371/journal.pone.0174884. eCollection 2017.

Utilizing BMP-2 muteins for treatment of multiple myeloma

Axel Seher  1 , Charlotte Lagler  1 , Thorsten Stühmer  2 , Urs Dietmar Achim Müller-Richter  1 , Alexander Christian Kübler  1 , Walter Sebald  3 , Thomas Dieter Müller  4 , Joachim Nickel  5   6
Affiliations

Utilizing BMP-2 muteins for treatment of multiple myeloma

Axel Seher et al. PLoS One. .

Abstract

Multiple myeloma (MM) represents a haematological cancer characterized by the pathological hyper proliferation of antibody-producing B-lymphocytes. Patients typically suffer from kidney malfunction and skeletal disorders. In the context of MM, the transforming growth factor β (TGFβ) member Activin A was recently identified as a promoter of both accompanying symptoms. Because studies have shown that bone morphogenetic protein (BMP)-2-mediated activities are counteracted by Activin A, we analysed whether BMP2, which also binds to the Activin A receptors ActRII and ActRIIB but activates the alternative SMAD-1/5/8 pathway, can be used to antagonize Activin A activities, such as in the context of MM. Therefore three BMP2 derivatives were generated with modified binding activities for the type II (ActRIIB) and/or type I receptor (BMPRIA) showing either increased or decreased BMP2 activity. In the context of MM these BMP2 muteins show two functionalities since they act as a) an anti-proliferative/apoptotic agent against neoplastic B-cells, b) as a bone-formation promoting growth factor. The molecular basis of both activities was shown in two different cellular models to clearly rely on the properties of the investigated BMP2 muteins to compete for the binding of Activin A to the Activin type II receptors. The experimental outcome suggests new therapeutic strategies using BMP2 variants in the treatment of MM-related pathologies.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. BMP2, but not Activin A, downregulates relative cell number in the L363 and KMS12-BM myeloma cell lines.
(A) Cells were grown in the presence or absence of 125 nM BMP2 or Activin A for 72 h. Relative cell numbers were assessed by WST-1 measurements. (B) For a detailed analysis the dose response curve of BMP2 was determined for L363 (black circle line) and KMS12-BM (black circle) after incubation with BMP2 for 72h via WST1 assay. To allow comparison of the IC50 values, the dose-response curves were normalized with the value measured in the absence of BMP2 set to zero. The figures show the mean values and standard deviation of triplicates. The assays were carried out as three independent experiments.
Fig 2
Fig 2. Activin A inhibits BMP2-mediated cellular responses.
(A) KMS12-BM cells were stimulated with either 125 nM Activin A or 4 nM BMP2 alone or co-stimulated with both growth factors for 72 h. Cell growth was assessed by WST-1 measurements. (B) C2C12 cells were incubated without BMP2, 10 nM BMP2 and 10 nM BMP2 plus increasing concentrations of Activin A. After 72 h, ALP activity was measured by determining p-nitrophenylphosphate conversion using an ELISA reader. In the absence of BMP2 cells showed no ALP activity and the background signal was set as 0%. ALP activity derived from stimulation with 10 nM BMP2 was defined as 100% (dashed line). Addition of Activin A without BMP2 yielded no ALP activity (data not shown). The figures show the mean values and the standard deviation of triplicates. The assays were performed as three independent experiments.
Fig 3
Fig 3. Schematic representation of the BMP2 variants in terms of their receptor binding characteristics.
The arrows demonstrate the relative binding affinities of the individual BMP2 variants to the indicated receptors. High-affinity binding is indicated by thick black arrows, while low-affinity binding is indicated by thin grey arrows. For both “P” variants, there was no binding (indicated by the grey cross) to the type I receptors BRIA and BRIB that could be detected by surface plasmon resonance (SPR) analysis [15,28]. Bold receptor names indicate binding preferences among the two receptor types.
Fig 4
Fig 4. Biological activities of the different BMP2 variants.
(A) ATDC5 cells were stimulated with the indicated concentrations of either wildtype BMP2, BMP2-KD, BMP2-PKD or BMP2-P. ALP activity was determined after 72 h. (B) KMS12-BM and (C) L363 cells were stimulated with the indicated concentrations of the four BMP2 variants. Inhibition of proliferation was assessed after 96 h by WST-1 measurements. The figures show mean values and standard deviation of triplicates. The measurements were performed in three independent experiments.
Fig 5
Fig 5
(A) A collection of different human multiple myeloma cell lines are Activin A-resistant. Ten different cell lines were incubated with or without 125 nM Activin A. After 72 h, cell proliferation was assessed by WST-1 measurements. (B) Expression profile of Activin A receptors in MM cell lines. RNA was isolated from the indicated MM cell lines, and the expression levels of the indicated receptors were determined by qRT-PCR. Expression levels were normalized to the housekeeping gene HPRT (= 100%). (C) Activin A sensitivity of primary CD138+ cells isolated from different MM patients. CD138+ cells were isolated from 8 different donors and stimulated with 125 nM Activin A for 72 h. The control cells were untreated. Cell numbers were assessed by WST-1 assay and related to the untreated cells (control). Assays were performed in duplicate (except for the values marked with a #). The figures show the mean values and standard deviation of duplicates.
Fig 6
Fig 6. BMP2 counteracts Activin A-induced inhibition of cell proliferation in INA6 cells.
INA6 cells were incubated with Activin A alone (10 nM) or co-stimulated by the addition of Activin A and increasing concentrations of either wildtype BMP2 or BMP2-PKD. Cell numbers were assessed by WST-1 measurements. The black line represents the value obtained from the untreated cells (control).The dashed line indicates the IC50 (half maximal biological inhibitor activity. In the experiment the IC50 was determined as 250 nM for BMP2 and as 62.5 nM for BMP2-PKD. The figures show the mean values of triplicates with standard deviation. The measurements were performed as three independent experiments.
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This work was supported by the Deutsche Forschungsgemeinschaft (KFO 216) and the University of Wuerzburg (funding program Open Access Publishing).