Using the Connectivity Map to discover compounds influencing human osteoblast differentiation

Abstract

Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. Identification of factors influencing osteoblast differentiation and bone formation is very important. Previously, we identified parbendazole to be a novel compound that stimulates osteogenic differentiation of human mesenchymal stromal cells (hMSCs), using gene expression profiling and bioinformatic analyzes, including the Connectivity Map (CMap), as an in-silico approach. The aim for this paper is to identify additional compounds affecting osteoblast differentiation using the CMap. Gene expression profiling was performed on hMSCs differentiated to osteoblasts using Illumina microarrays. Our osteoblast gene signature, the top regulated genes 6 hr after induction by dexamethasone, was uploaded into CMap (www.broadinstitute.org/cmap/). Through this approach we identified compounds with gene signatures positively correlating (withaferin-A, calcium folinate, amylocaine) or negatively correlating (salbutamol, metaraminol, diprophylline) to our osteoblast gene signature. All positively correlating compounds stimulated osteogenic differentiation, as indicated by increased mineralization compared to control treated cells. One of three negatively correlating compounds, salbutamol, inhibited dexamethasone-induced osteoblastic differentiation, while the other two had no effect. Based on gene expression data of withaferin-A and salbutamol, we identified HMOX1 and STC1 as being strongly differentially expressed . shRNA knockdown of HMOX1 or STC1 in hMSCs inhibited osteoblast differentiation. These results confirm that the CMap is a powerful approach to identify positively compounds that stimulate osteogenesis of hMSCs, and through this approach we can identify genes that play an important role in osteoblast differentiation and could be targets for novel bone anabolic therapies.

Evaluation of Connectivity Map shows limited reproducibility in drug repositioning

“…In Brum et al, CMap 1 was queried with a signature derived from human mesenchymal stromal cellderived osteoblasts (Brum et al, 2015(Brum et al, , 2018. Among the top compounds recommended were parbendazole, withaferin-A and amylocaine.…”

“…In contrast, known osteogenic glucocorticoids were ranked in the top-20 in both CMaps. Brum et al (2018) were also interested in compounds that could reverse the query signature and validated the dexamethasone-inhibiting activity of salbutamol, which was recommended by CMap 1; CMap 2 failed to recommend this compound, giving an inverse-rank of 61 (τ = -68.41).…”

“…while being rejected by CMap 1 (Zador et al, 2018). On the other hand, there are also anecdotal reports of positive results, in which researchers have experimentally verified the biological effects of candidate drugs recommended by CMap 1 (Braconi et al, 2009;Brum et al, 2015Brum et al, , 2018Byun et al, 2019;Johnstone et al, 2012;Luo et al, 2019;Schanstra et al, 2019;Wang et al, 2019;Wu et al, 2019) or CMap 2 (Ferguson et al, 2018;Leung et al, 2019;Manzotti et al, 2019;Ryals et al, 2018). An attempt to address the reproducibility question was presented by De Abrew et al, in which they generated an independent CMap-style dataset for 34 compounds in four cell lines (De Abrew et al, 2016).…”

Malignant Evaluation and Clinical Prognostic Values of m6A RNA Methylation Regulators in Glioblastoma

“…CMap can identify biomarkers for predicting specific drug reactions, mechanisms of treatment, and ways to overcome them (65)(66)(67). CMap analysis, which is based on a limited number of treated cell lines, accurately identified a number of compounds that have been shown to have an effect on m6A of other tumor types with specificity (33,(68)(69)(70). METTL3 has been reported to promote gastric cancer angiogenesis by secreting HDGF (71).…”

The Effect of the Human Plasma Molecule GHK-Cu on Stem Cell Actions and Expression of Relevant Genes

“…Today, researchers have an unprecedented opportunity to access gene profiling databases and determine which genes are affected during various physiological and pathological processes. These databases, such as The Broad Institute Connectivity Map, allow researchers to analyze gene expression changes caused by various biologically active molecules [9]. Also, it is now discovered that inflammation and the accompanying generation of free radicals of oxygen (ROS) can trigger tissue plasticity by reverting cells to pluripotency [10].…”