Cancer-linked ANKRD26-RET uses an unusual combination of pathomechanisms leading to strongly increased cell proliferation.

Truncated ANKRD26 fused with the kinase domain of the protooncogene product RET had been linked to papillary thyroid carcinoma. Yet, its pathomechanisms remained elusive. Commonly, loss of membrane association is considered as characteristic for pathology of RET fusions. We found that for ANKRD261-1405-RET713-1114 the opposite was true. Derailed RET signaling was strongly promoted by ANKRD26-mediated plasma membrane anchoring. Additionally, ANKRD26's coiled coil domain fragment included in ANKRD261-1405-RET713-1114 was still able to self-associate and interlinked ANKRD261-1405-RET713-1114. Only together, these ANKRD26-mediated molecular functions led to increased RET Y905, Y981, Y1015 and Y1062 phosphorylations. ANKRD261-1405-RET713-1114 severely and constitutively propagated intracellular RET signaling and thereby strongly increased cell proliferation and colony formation. Our results revealed an unfortunate combination of three aspects, membrane-association and self-association provided by ANKRD26 as well as RET kinase domain functionality, as important molecular mechanisms underlying ANKRD261-1405-RET713-1114 signaling and pathophysiology. These findings provide insights into how derailed and constitutively overactivated RET signaling originating from the plasma membrane instead from the cytoplasm, as for other longer-known RET fusions, leads to cell proliferation. Different RET pathogenic variants were reported to show specific efficacy outcomes for RET inhibitors in patients. Our results demonstrate that the enhanced colony formation caused by the distinct ANKRD261-1405-RET713-1114 pathomechanism was fully suppressible by RXDX-105/agerafenib and BLU-667/pralsetinib treatments but the increased cell proliferation responded merely moderately. This may suggest that the ANKRD261-1405-RET713-1114-intrinsic pathomechanisms may not be solely brought about by aberrant RET kinase activity but that putative therapeutic interventions may additionally need to focus on ANKRD26 dysfunctions.