Characterization of POXAPy-macrocycle molecule activity in acute promyelocytic leukemia NB4 cells

In the last years, macrocyclic polyamines gained attention as anticancer agents in consequence of their unique properties such as improved bioavailability, binding affinity, and favorable pharmacokinetics profiles. Similarly, compounds containing 1,3,4-oxadiazole rings showed main biological properties, including potential antitumor activities. Based on this evidence, new polyamine macrocycle molecules containing 1,3,4-oxadiazole rings were synthesized, leading to the selection of a molecule named POXAPy-macrocycle, showing the ability to reduce the survival of cancer cells at micromolar concentrations. The molecule was tested against a panel of 60 cell lines encompassing tumors of different origins, indicating a selectivity against hematopoietic tumors, with the promyelocytic leukemia NB4 cell line being the most sensitive. Thus, the effects of the molecule were investigated at the molecular level by integrating transcriptome and proteome profiling of POXAPy-macrocycle-treated NB4 cells. This approach led to the identification of mTOR signaling as the most significantly affected pathway. mTOR signaling inhibition plays a main role in autophagy and has been recently proposed as a new potential target in different kinds of tumors. mTOR inhibition was confirmed by analyzing its phosphorylation by Western blotting, and the activation of autophagy was also evaluated, confirming the increased expression of main autophagic markers such as LC3B and p62. Due to its role in autophagy and its interplay with mTOR signaling, the MAPK/ERK pathway was also investigated, showing the reduction of ERK phosphorylation. Finally, transmission electron microscopy (TEM) showed mitochondria alteration and their engulfment in autophagic vesicles, suggesting the activation of mitophagy and indicating the mitochondrion as a target of POXAPy-macrocycle activity. In addition, cell-free assays indicated a selective ability of POXAPy-macrocycle to interact with specific G-quadruplex (G4)-forming DNA sequences, such as those within the MYC promoter region, although no conclusive evidence was obtained in cell-based models, suggesting that the observed MYC downregulation may rather result from the inhibition of upstream signaling pathways. Although further studies will be necessary to clarify the molecular mechanism through which POXAPy-macrocycle exerts the biological activities herein described, these results indicate POXAPy-macrocycle as a promising antitumor candidate.