| dc.description.abstract | Abstract
Background and aims: Multiple myeloma (MM) is a form of cancer caused by the proliferation of aberrant plasma cells. Myeloma cells are addicted to the MYC oncogene, and this research group has previously shown that MYC downregulation is potentiated by TGF-β family ligands via intracellular SMAD1/5 signaling. It has been demonstrated that SMAD1/5 signaling is potentiated upon release of the inhibitory FK506 binding protein 12 (FKBP12) molecule from TGF-β type I receptors. Current drugs used for removing FKBP12 from TGF-β type I receptors have immunosuppressive effects, making them unsuitable in cancer therapy. The main aim of this thesis was to investigate the abilities of two FKBP12-degrading PROTACs (Proteolysis Targeting Chimera), dFKBP-1 and RC32, to potentiate BMP-induced SMAD1/5 signaling, with the desired end result of inducing myeloma cell death. Additionally, myeloma clones subjected to CRISPR/Cas9 technology were sequenced to find clones with receptor knockouts, which will be used in future research.
Methods: Western blots, reporter gene assays and cell viability assays were used to characterize the effects of dFKBP-1 and RC32 on FKBP12 protein expression, phosphorylated SMAD1/5 expression and activity, and myeloma cell viability. Western blot analysis was also used to detect PROTAC effects on FKBP51 and FKBP52 expression, and RC32 effects on phosphorylated mTOR and Ikaros expression. Modified myeloma clones were sequenced with both Sanger sequencing and next-generation sequencing with Illumina.
Results and conclusions: RC32 and dFKBP-1 both effected dose-dependent FKBP12 degradation and strongly potentiated ligand-induced phosphorylated SMAD1/5 expression and myeloma cell death. dFKBP-1 potentiated ID1 induction, indicative of SMAD1/5 activity, in a dose-dependent manner, but RC32 did not, possibly because of an unknown effect of RC32. Neither of the PROTACs affected FKBP51 or FKBP52 protein expression with the drug doses used here, showing that these drugs have a higher specificity for FKBP12. RC32 had no clear impact on mTOR or Ikaros activity, suggesting that this drug does not have residual immunomodulatory effects from its component parts, although positive controls in this experiment failed to show significant results. Sanger sequencing showed potential receptor knockout clones that might be useful in future investigations on receptor binding and signaling specificities, and next-generation sequencing results are underway. dFKBP-1 and RC32 might have potential use in MM therapy, but many of their features, including in vivo functioning, still need to be tested. | |