Enhancing Bone Morphogenetic Protein Activity to Target Multiple Myeloma

Abstract

Multiple myeloma is an incurable cancer of the bone marrow. In multiple myeloma cells bone morphogenetic proteins (BMP) can cause apoptosis and growth restriction. BMPs exist in the bone marrow microenvironment, but patients still develop multiple myeloma. We want to explore taking advantage of the resident BMPs and enhance their activity, and we hypothesize that increased BMP activity might be beneficial for patients. BMP ligand-receptor signaling complexes consist of combinations of type I receptors and type II receptors, and their BMP ligands. The complexes also interact with other proteins and co-factors, like the immunophilin FKBP12. When the BMP ligand-receptor complex forms it can activate SMADs, which then move into the nucleus and act as transcription factors, regulating genes that determine the cells’ fate. FKBP12 plays an important role in regulating BMP signaling by interacting with the type I receptors in the region responsible for SMAD activation. Thus, when FKBP12 is bound to the type I receptor it prevents leaky signaling. In theory targeting FKBP12 should enhance BMP signaling and cause increased activation of SMADs and myeloma cell death. To target FKBP12 we have chosen two different approaches. FKBP12 can be targeted with a drug known as Tacrolimus, or FK506, which leads to an increase in SMAD1/5/8 activation. The disadvantage of the drug FK506 is that it is a known immunosuppressant, as it inhibits calcineurin which is important for activation of T-cells. Therefore we treated cells with new drugs, which also interact with FKBP12 but without FK506s immunosuppressive effect. These drugs worked similarly as FK506, and potentiated BMP-induced SMAD1/5 activation. As these are newly developed drugs they have not yet been tested outside the lab. The second approach we used was PROTACs that are based on a technology where you combine an E3 ubiquitin ligase with a linker and a protein binder that binds specifically to your target protein. The result is ubiquitination and degradation of the target protein. We used two novel FKBP12 targeting PROTACs, and show that they work in a similar way as FK506, potently increasing BMP induced SMAD1/5 activity and myeloma cell death. As a third approach to enhance BMP activity we studied selinexor. Selinexor is a newly approved drug for treatment of multiple myeloma, but not much is known about its mechanism of action. Selinexor is a known selective inhibitor of exportin-1, a major nuclear exporter. When nuclear export of transcription factors like SMADs are inhibited they accumulate in the nucleus, and we hypothesized that selinexor treatment combined with BMPs could prolong the BMP effect as the SMADs are withheld in the nucleus and continue to act as transcription factors. We found that combining selinexor and BMPs caused enhanced apoptosis of myeloma cells, but the exact mechanism is still not clear. To summarize we have investigated different approaches to enhance BMP activity in multiple myeloma cell lines. The knowledge gained from this work might be beneficial for patients in the future.