Hanne Hella

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

BackgroundActivins are members of the TGF-β 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.ResultsIn 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.ConclusionsWe propose that one important way that activin A regulates cell behavior is by antagonizing BMP-ACVR2A/ACVR2B/ALK2 signaling.

High expression of BCL3 in human myeloma cells is associated with increased proliferation and inferior prognosis

Background:u2002 BCL3 is a putative oncogene encoding for a protein belonging to the inhibitory κB‐family. We experienced that this putative oncogene was a common target gene for growth‐promoting cytokines in myeloma cell lines.

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

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.

Nonlabeled secondary antibodies augment/maintain the binding of primary, specific antibodies to cell membrane antigens

BACKGROUNDnIn studies on surface membrane antigen expression using immunofluorescence techniques, it is commonly observed that direct staining gives weaker signals than the signals following indirect staining with fluorochrome-conjugated secondary antibodies. This is most marked when cells have also been permeabilized in order to stain intracellular protein. The commonly accepted explanation for this observation is that fluorochrome-conjugated secondary antibodies bind to a higher number of binding sites on the primary antibody, as compared to the binding of conjugated primary antibodies to the membrane antigens. Another hypothesis might be that the antibody/antibody complexes formed on the membranes when using the indirect technique may have an augmented ability to bind the membrane epitopes. The present study was performed in order to check this hypothesis.nnnMATERIALS AND METHODSnPeripheral blood mononuclear cells were stained with fluorochrome-conjugated anti-CD antibodies directly without or with a second-step application of nonconjugated goat anti-mouse IgG antibodies, followed by different fixation and permeabilization methods. The cells were analyzed by flow cytometry.nnnRESULTSnA second-step application of nonconjugated goat anti-mouse IgG antibodies following direct staining with fluorochrome-conjugated anti-CD antibodies gave a significant increase in membrane antigen expression on permeabilized cells as compared to direct staining alone. The secondary antibody must be bivalent, since whole IgG or F(ab)(2) fragments of the goat anti-mouse antibodies showed effects, while Fab fragments did not.nnnCONCLUSIONSnNonlabeled secondary antibodies are able to influence the binding of primary, specific antibodies to cell membrane antigens on cells treated with permeabilizing agents necessary for staining intracellular proteins. The improved membrane antigen expression seems to be due to the formation of a network of primary and secondary antibodies on the cell surface, with increased ability for maintaining binding to CD antigens.

Lymphoma and myeloma cells are highly sensitive to growth arrest and apoptosis induced by artesunate

The use of new drugs has improved the treatment of multiple myeloma and diffuse large B‐cell lymphoma (DLBCL). Nevertheless, over time many patients relapse and develop resistance to treatment, and efforts are needed to overcome drug resistance. The widely used malaria drug artesunate has been reported to have antitumor activity, and we aimed to test the effects of artesunate on a panel of myeloma and lymphoma cells.

CpG-Oligodeoxynucleotide Inhibits Smad-Dependent Bone Morphogenetic Protein Signaling: Effects on Myeloma Cell Apoptosis and In Vitro Osteoblastogenesis

The TLR9 agonist CpG-oligodeoxynucleotide (CpG-ODN) with a phosphorothioate backbone (PTO-CpG-ODN) is evaluated in clinical trials as a vaccine adjuvant or as treatment of cancers. Bone morphogenetic proteins (BMPs) regulate growth and differentiation of several cell types, and also induce apoptosis of cancer cells. Cross-talk between BMP- and TLR-signaling has been reported, and we aimed to investigate whether CpG-ODN influenced BMP-induced osteoblast differentiation or BMP-induced apoptosis of malignant plasma cells. We found that PTO-CpG-ODN inhibited BMP-2–induced osteoblast differentiation from human mesenchymal stem cells. Further, PTO-CpG-ODN counteracted BMP-2– and BMP-6–induced apoptosis of the human myeloma cell lines IH-1 and INA-6, respectively. In contrast, PTO-CpG-ODN did not antagonize the antiproliferative effect of BMP-2 on hMSCs or IH-1 cells. Inhibition of Smad-signaling and p38 MAPK-signaling indicated that apoptosis of IH-1 cells is dependent on Smad-signaling downstream of BMP, whereas the antiproliferative effect of BMP-2 on IH-1 cells also involves p38 MAPK-signaling. Together, the data suggested a specific inhibition by PTO-CpG-ODN on BMP–Smad-signaling. Supporting this we found that PTO-CpG-ODN inhibited BMP-induced phosphorylation of receptor-Smads in human mesenchymal stem cells and myeloma cell lines. This effect appeared to be independent of TLR9 because GpC-ODN and other ODNs with the ability to form multimeric structures inhibited Smad-signaling as efficiently as PTO-CpG-ODNs, and because knockdown of TLR9 by small interfering RNA in INA-6 cells did not blunt the effect of PTO-CpG-ODN. In conclusion, our results demonstrate that PTO-CpG-ODN inhibits BMP-signaling, and thus might provoke unwanted TLR9-independent side effects in patients.

Intracellular glutathione determines bortezomib cytotoxicity in multiple myeloma cells

Multiple myeloma (myeloma in short) is an incurable cancer of antibody-producing plasma cells that comprise 13% of all hematological malignancies. The proteasome inhibitor bortezomib has improved treatment significantly, but inherent and acquired resistance to the drug remains a problem. We here show that bortezomib-induced cytotoxicity was completely dampened when cells were supplemented with cysteine or its derivative, glutathione (GSH) in ANBL-6 and INA-6 myeloma cell lines. GSH is a major component of the antioxidative defense in eukaryotic cells. Increasing intracellular GSH levels fully abolished bortezomib-induced cytotoxicity and transcriptional changes. Elevated intracellular GSH levels blocked bortezomib-induced nuclear factor erythroid 2-related factor 2 (NFE2L2, NRF2)-associated stress responses, including upregulation of the xCT subunit of the Xc- cystine-glutamate antiporter. INA-6 cells conditioned to increasing bortezomib doses displayed reduced bortezomib sensitivity and elevated xCT levels. Inhibiting Xc- activity potentiated bortezomib-induced cytotoxicity in myeloma cell lines and primary cells, and re-established sensitivity to bortezomib in bortezomib-conditioned cells. We propose that intracellular GSH level is the main determinant of bortezomib-induced cytotoxicity in a subset of myeloma cells, and that combined targeting of the proteasome and the Xc- cystine-glutamate antiporter can circumvent bortezomib resistance.

Caspase-8 regulates the expression of pro- and anti-inflammatory cytokines in human bone marrow-derived mesenchymal stromal cells.

Mesenchymal stem cells, also called mesenchymal stromal cells, MSCs, have great potential in stem cell therapy partly due to their immunosuppressive properties. How these cells respond to chronic inflammatory stimuli is therefore of importance. Toll‐like receptors (TLR)s are innate immune receptors that mediate inflammatory signals in response to infection, stress, and damage. Caspase‐8 is involved in activation of NF‐kB downstream of TLRs in immune cells. Here we investigated the role of caspase‐8 in regulating TLR‐induced cytokine production from human bone marrow‐derived mesenchymal stromal cells (hBMSCs).

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

ABSTRACT TGF-β/BMP superfamily ligands require heteromeric complexes of type 1 and 2 receptors for ligand-dependent downstream signaling. Activin A, a TGF-β 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. Summary: Activation of SMAD1/5/8 via endogenous wild-type ALK2 by activin A, activin B and certain BMPs is enhanced when BMPR2 levels are knocked down.

Abstract 3130: Dependency on c-MYC in multiple myeloma.

The oncogene c-MYC is deregulated in multiple myeloma. Importantly, the activity of c-MYC is elevated in plasma cells in patients with myeloma compared to the premalignant state monoclonal gammopathy of undetermined significance, indicating a prominent role for c-MYC in myeloma pathogenesis. We earlier found the bone morphogenetic proteins induce apoptosis in myeloma cell lines and primary cells by SMAD-dependent downregulation of c-MYC. Repression of c-MYC by short hairpin RNA in myeloma cell lines has by others also been shown to induce apoptosis. We wanted to go one step further and study the role of c-MYC in primary myeloma cells. Transcriptional regulation by c-MYC requires heterodimerization of c-MYC and its obligate partner MAX. The small molecular inhibitor 10058-F4 disrupts the formation of such heterodimers and was used in vitro to test the dependency on c-MYC activity in primary myeloma cells and myeloma cell lines. The inhibitor induced apoptosis in a dose-dependent manner in the 22 primary samples tested. Apoptosis was induced in five of six cell lines tested. The sixth cell line, U266, does not express c-MYC, and was not sensitive to the inhibitor. Using the INA-6 myeloma cell line, we found that apoptosis induced by 10058-F4 correlated with downregulation of the anti-apoptotic protein Bcl-xL. We also found that apoptosis was induced by the inhibitor even in the presence of bone marrow stromal cells derived from myeloma patients. Our results further support that c-MYC is a potential target for therapy in multiple myeloma. Citation Format: Toril Holien, Thea K. Vatsveen, Hanne Hella, Anders Waage, Anders Sundan. Dependency on c-MYC in multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3130. doi:10.1158/1538-7445.AM2013-3130