In multiple myeloma, c-MYC is activated and contributes to the malignant phenotype. Targeting MYC by short hairpin RNA induced cell death in myeloma cell lines; however, cell lines are generated from samples taken in advanced stages of the disease and may not reflect patient cells adequately. In this study, we used the selective small molecule inhibitor of MYC-MAX heterodimerization, 10058-F4, on myeloma cell lines as well as primary myeloma cells, and we show that inhibition of c-MYC activity efficiently induces myeloma cell death. Moreover, in cocultures of cell lines with bone marrow stromal cells from myeloma patients, the inhibitor still induces apoptosis. Our results provide further evidence that myeloma cells are addicted to c-MYC activity and that c-MYC is a promising therapeutic target in multiple myeloma.

Activins 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. 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. We propose that one important way that activin A regulates cell behavior is by antagonizing BMP-ACVR2A/ACVR2B/ALK2 signaling.

Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA) is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM). Thus inhibiting PCNA’s protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells’ sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment.

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)-β superfamily. TGF-β itself is a very potent cytokine and has effects on cells in the femtomolar range. Thus, even very small amounts of contaminating TGF-β in purified protein batches may influence the experimental results given that receptors for TGF-β are present. When we attempted to characterize possible receptors for the TGF-β superfamily ligand GDF15, striking similarities between GDF15-induced activities and known TGF-β 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-β 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.

The transforming growth factor (TGF)-β family is a group of structurally related, multifunctional growth factors, or ligands that are crucially involved in the development, regulation, and maintenance of animal tissues. In humans, the family counts over 33 members. These secreted ligands typically form multimeric complexes with two type I and two type II receptors to activate one of two distinct signal transduction branches. A striking feature of the family is its promiscuity, i.e., many ligands bind the same receptors and compete with each other for binding to these receptors. Although several explanations for this feature have been considered, its functional significance has remained puzzling. However, several recent reports have promoted the idea that ligand-receptor binding promiscuity and competition are critical features of the TGF-β family that provide an essential regulating function. Namely, they allow a cell to read and process multi-ligand inputs. This capability may be necessary for producing subtle, distinctive, or adaptive responses and, possibly, for facilitating developmental plasticity. Here, we review the molecular basis for ligand competition, with emphasis on molecular structures and binding affinities. We give an overview of methods that were used to establish experimentally ligand competition. Finally, we discuss how the concept of ligand competition may be fundamentally tied to human physiology, disease, and therapy.