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Published on: January 11, 2019
Cell-penetrating TAT-FOXO3 fusion proteins induce apoptotic cell death in leukemic cells
Makram Essafi1, Alice D Baudot, Xavier Mouska
1INSERM UMR 576, Hôpital de l'Archet, 151 Route de Saint-Antoine de Ginestiére, B.P. 3079, 06202, Nice Cedex 3, France.
Abstract:
FOXO proteins are Akt-regulated transcription factors involved in the control of cell cycle, DNA repair, stress defense, apoptosis, and tumor suppression. We reported that plasmid-based overexpression of constitutively active FOXO3 in cells from chronic lymphocytic leukemia (CLL) reduced their survival, suggesting that increasing FOXO3 activity in hematologic malignancies may represent a promising therapeutic strategy. The transactivating transcription factor (TAT) protein transduction domain (PTD) derived from the HIV TAT protein was shown to efficiently deliver macromolecular cargo in various cell types. In this study, wild-type FOXO3 and FOXO3 mutated on Akt sites [FOXO3 T32A/S253A/S315A or TM (triple mutant)] were fused to the TAT-PTD. Using biochemical techniques, flow cytometry, and microscopy analysis, we found a rapid and dose-dependent cell penetration into leukemic cells of unlabeled and fluorescein isothiocyanate-labeled TAT-FOXO3 fusion proteins followed by their accumulation within nuclear and cytoplasmic compartments. Treatment with TAT-FOXO3 TM-but not wild-type TAT-FOXO3-proteins induced Jurkat and K562 leukemic cell death and affected cell viability of other hematologic malignancies including primary cells from CLL. Cell transduction with TAT-FOXO3 TM induced apoptotic cell death as shown by morphologic changes, Annexin V/7-AAD (7-amino-actinomycin D) staining, activation of effector caspases, and PARP cleavage, caspase blockade through the use of the inhibitor Z-VAD, and expression of Bim and p27(KIP1). By contrast, TAT-FOXO3 TM blocked cell proliferation of primary T cells, without affecting their viability. Together, our data show that cell penetrating TAT-FOXO3 TM fusion proteins constitute novel potential therapeutic agents in the treatment of lymphoproliferative disorders and hematologic malignancies.
Insights
TAT-FOXO3 fusion proteins effectively penetrate leukemic cells, with the TM mutant inducing apoptosis and inhibiting proliferation in hematologic malignancies. This offers a promising therapeutic strategy for these disorders.
Area of Science:
- Molecular Biology
- Cancer Biology
- Cellular Biology
Background:
- FOXO proteins are key regulators of cell cycle, DNA repair, stress defense, apoptosis, and tumor suppression.
- Akt-mediated regulation of FOXO proteins influences their activity.
- Previous studies suggest increasing FOXO3 activity could be a therapeutic strategy for hematologic malignancies.
Purpose of the Study:
- To investigate the therapeutic potential of cell-penetrating TAT-FOXO3 fusion proteins in hematologic malignancies.
- To evaluate the efficacy of wild-type and constitutively active (TM mutant) FOXO3 fused to the TAT protein transduction domain (PTD).
Main Methods:
- Fusion proteins (wild-type FOXO3 and TM mutant FOXO3) were constructed with the TAT-PTD.
- Biochemical techniques, flow cytometry, and microscopy were used to assess protein delivery and localization.
- Cell viability, apoptosis assays (Annexin V/7-AAD staining, caspase activation, PARP cleavage), and proliferation assays were performed on leukemic and primary cells.
Main Results:
- TAT-FOXO3 fusion proteins rapidly and dose-dependently penetrated leukemic cells, localizing to nuclear and cytoplasmic compartments.
- The TAT-FOXO3 TM mutant, but not wild-type TAT-FOXO3, induced cell death in Jurkat and K562 leukemic cells and affected primary chronic lymphocytic leukemia (CLL) cells.
- TAT-FOXO3 TM induced apoptosis via morphological changes, caspase activation, PARP cleavage, and altered expression of Bim and p27(KIP1).
- TAT-FOXO3 TM inhibited primary T cell proliferation without affecting viability.
Conclusions:
- Cell-penetrating TAT-FOXO3 TM fusion proteins demonstrate potent anti-leukemic activity by inducing apoptosis and inhibiting proliferation.
- These fusion proteins represent novel therapeutic agents for lymphoproliferative disorders and hematologic malignancies.
- Targeting FOXO3 activity via TAT-mediated delivery offers a promising strategy for treating these cancers.
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