Enhancement by displacement. A single methyl group displaces a water molecule from the binding site of FKBPs, resulting in the most potent binders known, outperforming the natural products FK506 and rapamycin in biochemical and cellular assays.

Background The immunophilin FKBP12 binds to TGF-β 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.

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.