5 research outputs found
FKBP12 is a major regulator of ALK2 activity in multiple myeloma cells
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
Classification of Breast Cancer Based on DCE MRI
This master thesis is a part of a project at the MR group at the department of physics at NTNU. The aim of MR group is to make a model which is able to classify the subtype of breast tumours. In this master thesis the focus has been on dynamic contrast enhanced images
The first part of this master thesis consist of trying out different methods for motion correcting images. The reason for this was some unsatisfactory results of the motion correction done in the specialisation project during the fall of 2016. Some of the patients had images with remaining motion after the motion correction algorithm had been applied. The motion correction was now done using normalised cross correlation with 3, 6, and 9 degrees of freedom, and then using another similarity function, namely normalised mutual information. The result was that for some patients increasing the number of degrees of freedom from 3 to 6 would slightly improve the results of the motion correction.
The next part was to coregister the images to a reference image. This was done to make the future combination of images from different MRI modalities easier. The reference image chosen was a T2 MRI image, because this is taken for all patients for anatomical reasons. The method used for coregistration was a software called CMTK, the same we used for motion correction. The similarity function used was normalised cross correlation with 3 degrees of freedom.
Then regions of interest was found for different parameters, we chose to try to classify air, breast tissue in general, benign tumours and malignant tumours. ROIs was found for the included patients, and the pixel values implemented in a matrix. Then a SVM classifier was used on the matrix, to see if it was possible to classify different kinds of pixels in a test set. When only including benign tumours, not malignant, the SVM classifier classified 99.7 % of the pixels correctly. When including malignant tumours the correctly classified rate dropped slightly to 97.4%
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High concentrations of soluble endoglin can inhibit BMP9 signaling in non-endothelial cells.
Acknowledgements: This work was supported by the British Heart Foundation grants PG/12/54/29734 to W.L., P.D.U., N.W.M, PG/17/1/32532 to W.L. and N.W.M., RG/19/3/34265 to N.W.M, P.D.U and W.L., FS/SBSRF/20/31005 to W.L., and by the Liaison Committee for education, research, and innovation in Central Norway to T.H., and the Joint Research Committee between St. Olav’s Hospital and Faculty of Medicine and Health Sciences, NTNU to T.H. This research has received infrastructure support by the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care.Funder: the Liaison Committee for education, research, and innovation in Central NorwayFunder: the Joint Research Committee between St. Olav’s Hospital and Faculty of Medicine and Health Sciences, NTNUEndoglin (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
Picomolar FKBP inhibitors enabled by a single water-displacing methyl group in bicyclic [4.3.1] aza-amides
Methyl groups can have profound effects in drug discovery but the underlying mechanisms are diverse and incompletely understood. Here we report the stereospecific effect of a single, solvent-exposed methyl group in bicyclic [4.3.1] aza-amides, robustly leading to a 2 to 10-fold increase in binding affinity for FK506-binding proteins (FKBPs). This resulted in the most potent and efficient FKBP ligands known to date. By a combination of co-crystal structures, isothermal titration calorimetry (ITC), density-functional theory (DFT), and 3D reference interaction site model (3D-RISM) calculations we elucidated the origin of the observed affinity boost, which was purely entropically driven and relied on the displacement of a water molecule at the protein–ligand–bulk solvent interface. The best compounds potently occupied FKBPs in cells and enhanced bone morphogenic protein (BMP) signaling. Our results show how subtle manipulation of the solvent network can be used to design atom-efficient ligands for difficult, solvent-exposed binding pockets
Picomolar FKBP inhibitors enabled by a single water-displacing methyl group in bicyclic [4.3.1] aza-amides
Methyl groups can have profound effects in drug discovery but the underlying mechanisms are diverse and incompletely understood. Here we report the stereospecific effect of a single, solvent-exposed methyl group in bicyclic [4.3.1] aza-amides, robustly leading to a 2 to 10-fold increase in binding affinity for FK506-binding proteins (FKBPs). This resulted in the most potent and efficient FKBP ligands known to date. By a combination of co-crystal structures, isothermal titration calorimetry (ITC), density-functional theory (DFT), and 3D reference interaction site model (3D-RISM) calculations we elucidated the origin of the observed affinity boost, which was purely entropically driven and relied on the displacement of a water molecule at the protein–ligand–bulk solvent interface. The best compounds potently occupied FKBPs in cells and enhanced bone morphogenic protein (BMP) signaling. Our results show how subtle manipulation of the solvent network can be used to design atom-efficient ligands for difficult, solvent-exposed binding pockets