GSK3i combinatorial treatments affect CDK4/6 and compensatory pathways in 3D preclinical models of pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (pNETs) are rare tumors, often detected at advanced stages for which current therapies are mostly unsuccessful. Despite significant progress in the understanding of pathogenic molecular pathways, most single targeted treatments only slow disease progression while patients develop resistance due to compensatory mechanisms. To identify novel combination therapies, we assessed the effects of TNFα, insulin and GSK3 inhibition (GSK3i) in spheroids of the metastatic pNET model BON-1 on subcellular localization and activation of cell cycle components and apoptosis. While cyclin D1 and CDK4 and 6 (cyclin-dependent kinase) stainings, related protein levels and apoptosis were not affected or only weakly affected by single treatments, the combinatorial treatments acted synergistically to induce cell death, as assessed by cleaved caspase-3 immunopositivity. Protein levels of CDK4 and CDK6 were furthermore validated in primary pNET cultures. Compensatory mechanisms under the single treatments might include activation of CDK1/2 and the DNA damage response, as assessed by the activation of phospho-Chk2, since this was perturbed under the combinatorial treatments. Taken together, our work identifies combinatorial treatments that substantially reduce the viability of spheroidal BON-1 cultures and patient-derived primary cultures as potential novel therapies for the treatment of pNETs.