Abstract Background Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells. Although new treatments approved in the last decades have improved patient outcomes considerably, most patients relapse and there is a large unmet medical need for novel MM treatments. Several publications have shown that stimulation of MM cells with bone morphogenetic proteins (BMPs) induce apoptosis or growth arrest in vitro. In addition, it has been shown that BMP signaling is regulated by FK506 binding protein 12 kDa (FKBP12). Binding FKBP12 with the immunosuppressive ligand FK506 potentiates SMAD1/5/8 signaling and can be used to increase the effects observed earlier in MM cells. In this thesis, potentiation of TGF-β family signaling have been investigated with the ligands BMP-6, Activin A and TGFβ in combination with FKBP12-binding compounds, such as FK506. In addition, two nonimmunosuppressive FKBP12-binding compounds, JK096 and JK098, have been characterized to investigate if they can be used to potentiate TGF-β family signaling. Some secondary work looking at migration of MM cells was also performed in this thesis.
Material and methods BMP-6 activity levels were assayed with cell viability assays in the MM cell line INA-6. Next, FKBP12 siRNA was utilized to investigate FKBP12s regulative role. Potentiation of SMAD1/5/8 and SMAD2/3 signaling was investigated directly by using BRE-Luciferase and CAGA-Luciferase transduced reporter cell lines. JK096 and JK098 were tested in the above assays and were also investigated for any immunosuppression by looking at NFAT and mTOR activity with western blotting.
Results The results show that FKBP-binding compounds can significantly potentiate BMP-6 and Activin A signaling through their respective receptors ALK2 and ALK4, but not TGF-β through its receptor ALK5. Furthermore, the results indicate that JK096 and JK098 can potentiate SMAD signaling as strongly as FK506 at varying concentrations. Our results confirm that these compounds do not inhibit NFAT or mTOR, and this suggests that inhibition of these pathways are not important for the potentiation observed with FK506 or rapamycin. FKBP12 siRNA also increases the effect of BMP-6 signaling by lowering cell viability in INA-6, affirming the role of FKBP12 in receptor regulation.
Conclusion With the potentiation observed in this thesis, it is proposed that ALK2 and ALK4 activation can be modulated with FKBP12-binding compounds by binding and removing FKBP12 from the receptor. Even though ALK5 is known to bind FKBP12, it seems FKBP12 either can’t be removed by FKBP12-binding compounds or its removal has no effect on signaling in our assay. Lastly, our results demonstrate that JK096 and JK098 can be useful research tools to investigate binding of the FKBP family in vitro, without the confounding effects of mTOR and NFAT inhibition.