FKBP12 is a major regulator of ALK2 activity in multiple myeloma cells.

BACKGROUND: The immunophilin FKBP12 binds to TGF-ß family type I receptors, including the BMP type I receptor ALK2. FKBP12 keeps the type I receptor in an inactive state and controls signaling activity. Removal of FKBP12 with drugs such as the FKBP-ligand FK506 enhances BMP activity in various cell types. In multiple myeloma cells, activation of SMAD1/5/8 leads to apoptosis. We hypothesized that removing FKBP12 from ALK2 in myeloma cells would potentiate BMP-induced ALK2-SMAD1/5/8 activity and in consequence cell death. METHODS: Multiple myeloma cell lines were treated with FK506, or other FKBP-binding compounds, combined with different BMPs before analyzing SMAD1/5/8 activity and cell viability. SMAD1/5/8 activity was also investigated using a reporter cell line, INA-6 BRE-luc. To characterize the functional signaling receptor complex, we genetically manipulated receptor expression by siRNA, shRNA and CRISPR/Cas9 technology. RESULTS: FK506 potentiated BMP-induced SMAD1/5/8 activation and apoptosis in multiple myeloma cell lines. By using FKBP-binding compounds with different affinity profiles, and siRNA targeting FKBP12, we show that the FK506 effect is mediated by binding to FKBP12. Ligands that typically signal via ALK3 in myeloma cells, BMP2, BMP4, and BMP10, did not induce apoptosis in cells lacking ALK3. Notably, BMP10 competed with BMP6 and BMP9 and antagonized their activity via ALK2. However, upon addition of FK506, we saw a surprising shift in specificity, as the ALK3 ligands gained the ability to signal via ALK2 and induce apoptosis. This indicates that the receptor complex can switch from an inactive non-signaling complex (NSC) to an active one by adding FK506. This gain of activity was also seen in other cell types, indicating that the observed effects have broader relevance. BMP2, BMP4 and BMP10 depended on BMPR2 as type II receptor to signal, which contrasts with BMP6 and BMP9, that activate ALK2 more potently when BMPR2 is knocked down. CONCLUSIONS: In summary, our data suggest that FKBP12 is a major regulator of ALK2 activity in multiple myeloma cells, partly by switching an NSC into an active signaling complex. FKBP12 targeting compounds devoid of immunosuppressing activity could have potential in novel treatment strategies aiming at reducing multiple myeloma tumor load. Video Abstract.

BMPR2 inhibits activin and BMP signaling via wild-type ALK2.

TGF-ß/BMP superfamily ligands require heteromeric complexes of type 1 and 2 receptors for ligand-dependent downstream signaling. Activin A, a TGF-ß superfamily member, inhibits growth of multiple myeloma cells, but the mechanism for this is unknown. We therefore aimed to clarify how activins affect myeloma cell survival. Activin A activates the transcription factors SMAD2/3 through the ALK4 type 1 receptor, but may also activate SMAD1/5/8 through mutated variants of the type 1 receptor ALK2 (also known as ACVR1). We demonstrate that activin A and B activate SMAD1/5/8 in myeloma cells through endogenous wild-type ALK2. Knockdown of the type 2 receptor BMPR2 strongly potentiated activin A- and activin B-induced activation of SMAD1/5/8 and subsequent cell death. Furthermore, activity of BMP6, BMP7 or BMP9, which may also signal via ALK2, was potentiated by knockdown of BMPR2. Similar results were seen in HepG2 liver carcinoma cells. We propose that BMPR2 inhibits ALK2-mediated signaling by preventing ALK2 from oligomerizing with the type 2 receptors ACVR2A and ACVR2B, which are necessary for activation of ALK2 by activins and several BMPs. In conclusion, BMPR2 could be explored as a possible target for therapy in patients with multiple myeloma.This article has an associated First Person interview with the first author of the paper.

Activin A inhibits BMP-signaling by binding ACVR2A and ACVR2B.

BACKGROUND: Activins are members of the TGF-ß family of ligands that have multiple biological functions in embryonic stem cells as well as in differentiated tissue. Serum levels of activin A were found to be elevated in pathological conditions such as cachexia, osteoporosis and cancer. Signaling by activin A through canonical ALK4-ACVR2 receptor complexes activates the transcription factors SMAD2 and SMAD3. Activin A has a strong affinity to type 2 receptors, a feature that they share with some of the bone morphogenetic proteins (BMPs). Activin A is also elevated in myeloma patients with advanced disease and is involved in myeloma bone disease. RESULTS: In this study we investigated effects of activin A binding to receptors that are shared with BMPs using myeloma cell lines with well-characterized BMP-receptor expression and responses. Activin A antagonized BMP-6 and BMP-9, but not BMP-2 and BMP-4. Activin A was able to counteract BMPs that signal through the type 2 receptors ACVR2A and ACVR2B in combination with ALK2, but not BMPs that signal through BMPR2 in combination with ALK3 and ALK6. CONCLUSIONS: We propose that one important way that activin A regulates cell behavior is by antagonizing BMP-ACVR2A/ACVR2B/ALK2 signaling.

High concentrations of soluble endoglin can inhibit BMP9 signaling in non-endothelial cells.

Endoglin (ENG) is a single-pass transmembrane protein highly expressed on vascular endothelial cells, although low expression levels can be detected in many other cell types. Its extracellular domain can be found in circulation known as soluble endoglin (sENG). Levels of sENG are elevated in many pathological conditions, in particular preeclampsia. We have shown that while loss of cell surface ENG decreases BMP9 signaling in endothelial cells, knocking down ENG in blood cancer cells enhances BMP9 signaling. Despite sENG binding to BMP9 with high affinity and blocking the type II receptor binding site on BMP9, sENG did not inhibit BMP9 signaling in vascular endothelial cells, but the dimeric form of sENG inhibited BMP9 signaling in blood cancer cells. Here we report that in non-endothelial cells such as human multiple myeloma cell lines and the mouse myoblast cell line C2C12, both monomeric and dimeric forms of sENG inhibit BMP9 signaling when present at high concentrations. Such inhibition can be alleviated by the overexpression of ENG and ACVRL1 (encoding ALK1) in the non-endothelial cells. Our findings suggest that the effects of sENG on BMP9 signaling is cell-type specific. This is an important consideration when developing therapies targeting the ENG and ALK1 pathway.

RNA-Integrity and 8-Isoprostane Levels Are Stable in Prostate Tissue Samples Upon Long-Term Storage at -80°C.

Sampling of prostate tissue (n = 97) was performed in conjunction with planned radical prostatectomies, in collaboration with Biobank1®. The tissue used in this study was collected during the period 2003-2016, quickly frozen, and kept at -80°C until assayed in 2018. RNA extraction was performed with two different protocols (miRNeasy and mirVana™), and RNA quality was determined by measuring the RNA Integrity Number (RIN). The level of isoprostanes is widely recognized as a specific indicator of lipid peroxidation both in vitro and in vivo. The level of 8-isoprostane was measured because it is the main oxidation product of arachidonic acid, the most abundant phospholipid fatty acid. The level of 8-isoprostane was measured using enzyme immunoassay. There was no statistically significant difference in yield between the samples isolated with the mirVana protocol compared to the miRNeasy protocol. Average RIN was 2.8 units higher with the mirVana extraction protocol compared to the miRNeasy protocol (p < 0.001). For miRNeasy extractions, RINs were 7.1 for prostatectomies in 2005-2007 and 6.2 for those in 2018 (p < 0.001). For mirVana extractions, the difference in RIN score between the two groups regarding years of collection was not statistically significant. There was no significant increase in the levels of 8-isoprostane between the 2005-2007 samples and the 2018. The conclusion is that there is no oxidation of phospholipids with increasing storage time up to 15 years.

Activins as Dual Specificity TGF-ß Family Molecules: SMAD-Activation via Activin- and BMP-Type 1 Receptors.

Activins belong to the transforming growth factor (TGF)-ß family of multifunctional cytokines and signal via the activin receptors ALK4 or ALK7 to activate the SMAD2/3 pathway. In some cases, activins also signal via the bone morphogenetic protein (BMP) receptor ALK2, causing activation of the SMAD1/5/8 pathway. In this study, we aimed to dissect how activin A and activin B homodimers, and activin AB and AC heterodimers activate the two main SMAD branches. We compared the activin-induced signaling dynamics of ALK4/7-SMAD2/3 and ALK2-SMAD1/5 in a multiple myeloma cell line. Signaling via the ALK2-SMAD1/5 pathway exhibited greater differences between ligands than signaling via ALK4/ALK7-SMAD2/3. Interestingly, activin B and activin AB very potently activated SMAD1/5, resembling the activation commonly seen with BMPs. As SMAD1/5 was also activated by activins in other cell types, we propose that dual specificity is a general mechanism for activin ligands. In addition, we found that the antagonist follistatin inhibited signaling by all the tested activins, whereas the antagonist cerberus specifically inhibited activin B. Taken together, we propose that activins may be considered dual specificity TGF-ß family members, critically affecting how activins may be considered and targeted clinically.

Correction: MYC amplifications in myeloma cell lines: correlation with MYC-inhibitor efficacy.

[This corrects the article DOI: 10.18632/oncotarget.4245.].

TGF-ß contamination of purified recombinant GDF15.

Purified recombinant proteins for use in biomedical research are invaluable to investigate protein function. However, purity varies in protein batches made in mammalian expression systems, such as CHO-cells or HEK293-cells. This study points to caution while investigating effects of proteins related to the transforming growth factor (TGF)-ß superfamily. TGF-ß itself is a very potent cytokine and has effects on cells in the femtomolar range. Thus, even very small amounts of contaminating TGF-ß in purified protein batches may influence the experimental results given that receptors for TGF-ß are present. When we attempted to characterize possible receptors for the TGF-ß superfamily ligand GDF15, striking similarities between GDF15-induced activities and known TGF-ß activities were found. However, differences between batches of GDF15 were a concern and finally led us to the conclusion that the measured effects were caused by TGF-ß and not by GDF15. Our results emphasize that purified recombinant proteins must be used with caution and warrant proper controls. Notably, some conclusions made about GDF15 in already published papers may not be supported by the results shown. Awareness about this issue in the scientific community may prevent spreading of false positive results.

MYC amplifications in myeloma cell lines: correlation with MYC-inhibitor efficacy.

In multiple myeloma, elevated MYC expression is related to disease initiation and progression. We found that in myeloma cell lines, MYC gene amplifications were common and correlated with MYC mRNA and protein. In primary cell samples MYC mRNA levels were also relatively high; however gene copy number alterations were uncommon. Elevated levels of MYC in primary myeloma cells have been reported to arise from complex genetic aberrations and are more common than previously thought. Thus, elevated MYC expression is achieved differently in myeloma cell lines and primary cells. Sensitivity of myeloma cell lines to the MYC inhibitor 10058-F4 correlated with MYC expression, supporting that the activity of 10058-F4 was through specific inhibition of MYC.