The endometrial cancer A230V-ALK5 (TGFBR1) mutant attenuates TGF-β signaling and exhibits reduced in vitro sensitivity to ALK5 inhibitors

The ALK5 (TGFBR1) receptor serine/threonine kinase transduces TGF-β (Transforming Growth Factor beta) signaling to activate SMAD2/3-dependent and -independent pathways. Here, we aimed to determine the functional consequences of ALK5 mutations in human endometrial cancer (EC). Somatic mutation data were retrieved from publicly available databases. Using seven in silico algorithms, 78.5% (11 of 14) of ALK5 kinase domain mutations in EC, including A230V-ALK5, were predicted to impact protein function. For in vitro studies, we focused on A230V-ALK5 because it was the only mutated residue located within the ATP-binding pocket, which is an important region for both ATP-binding and binding of ATP-competitive inhibitors. Constructs expressing wildtype-, constitutively-active-, kinase-dead-, or mutant A230V-ALK5, were transfected into NIH/3T3 cells. Following TGF-β1 stimulation, transient exogenous expression of A230V-ALK5 resulted in attenuated SMAD2/3 signal transduction and reduced AKT activation. We further showed that the A230V-ALK5 mutant had reduced stability resulting from increased ubiquitin-dependent protein degradation. Our structural modeling predicted that SB-431542, a small molecule ATP-competitive inhibitor of ALK5, binds to the A230V-ALK5 mutant with reduced affinity compared to wildtype-ALK5. We therefore examined the inhibitory effect of SB-431542 and galunisertib on wildtype- and mutant-ALK5 activity using a Smad-binding element (SBE) luciferase reporter assay combined with TGF-β1 stimulation, in NIH/3T3 cells and HEC-265 EC cells. SBE luciferase activity in A230V-ALK5 transfected cells was inhibited less by SB-431542 and galunisertib than in wildtype-ALK5 transfected cells indicating that A230V-ALK5 is less sensitive to inhibition by these agents than wildtype-ALK5, potentially due to changes in SB-431542/A230V-ALK5 binding affinity. Our findings are novel and show that A230V-ALK5 is a partial loss-of-function mutant that attenuates TGF-β1 signal transduction and has reduced sensitivity to ALK5 small molecule inhibitors.