Cell-Penetrating Peptide Targets Senescent Cells to Restore Tissue Function

Researchers designed a cell-penetrating peptide (CPP) called FOXO4-DRI that selectively targeted senescent cells for apoptosis and restored function in tissues affected by chemotherapy and aging. Results of the study, recently published in Cell, make it possible “to envision a point on the horizon where the disease indications are identified that could benefit most from FOXO4-DRI therapy,” the authors wrote.

Accumulated DNA damage negatively affects an organism’s healthspan—the span during which an organism has optimal health. When a cell’s DNA repair mechanisms are insufficient, cells undergo apoptosis or become senescent. Senescent cells develop a pro-inflammatory senescence-associated secretory phenotype (SASP), which contributes to disease development and accelerated aging. To date, it has been unknown how cells evade apoptosis to become senescent.

CPPs have been developed to target senescent cells. Unlike broad-range inhibitors, CPPs can, in theory, inhibit specific protein-protein interactions and therefore have very selective inhibitory activity. Currently, little research has been done on CPPs, particularly in the field of aging.

FOXO4 is a member of the FOXO transcription factor family. Previous studies have reported FOXOs' roles in aging and tissue homeostasis.

For this current study, authors performed a series of in vitro and in vivo experiments to evaluate FOXO4-DRI.

In vitro experiments

Authors first evaluated FOXO4 in senescent and normal human fibroblasts; senescence was induced by ionizing radiation. Compared with other FOXOs, FOXO4 expression was markedly increased in the senescent fibroblasts. Inhibiting FOXO4 expression promoted apoptotic events and reduced the senescent fibroblasts’ viability and colony density.

Authors observed an interaction between FOXO4 and p53—a protein that regulates apoptosis and senescence. They designed FOXO4-DRI to target this interaction; the ‘DRI’ signifies a peptide modification called ‘D-retro inverso’ that reversed the sequence of D-amino acids to increase potency.

FOXO4-DRI dose-dependently competed with FOXO4 for p53 binding and had a higher affinity for p53. This competition disrupted senescence and excluded active nuclear p53 to the cytosol.

Authors then evaluated FOXO4-DRI’s apoptotic activity. Importantly, incubation with FOXO4-DRI reduced cellular viability only in senescent fibroblasts, not normal fibroblasts. Knockdown of p53, as well as co-incubation with FOXO4-DRI and pan-caspase inhibitors, reduced FOXO4-DRI’s ability to target senescent fibroblasts; the co-incubation suggested FOXO4-DRI’s apoptotic activity is caspase-dependent.

In vivo experiments

Next, authors developed 3 in vivo senescence models: chemotoxicity, accelerated aging, and natural aging. Mice were used for the aging models.

Because chemotherapy can induce senescence, authors induced senescence with doxorubicin, which increased FOXO4 expression in senescent fibroblasts and hepatocytes. Doxorubicin also increased hepatic IL-6 expression and plasma AST levels, and reduced total body weight; IL-6 expression is indicative of the pro-inflammatory SASP in senescent cells. FOXO4-DRI treatment reversed each effect.

The fast-aging mice demonstrated several characteristics, including accelerated hair loss and less exploratory behavior, that improved with FOXO4-DRI treatment. They also had reduced kidney function, indicated by increased plasma urea and increased renal tubular IL-6 expression. FOXO4-DRI treatment increased apoptosis of senescent renal tubular cells, restoring kidney function.

FOXO4-DRI treatment effects were similar in naturally aging mice as in fast-aging mice.

Conclusions

Taken together, study results highlight key aspects of FOXO4-DRI activity:

• FOXO4-DRI selectively targets senescent cells for apoptosis, sparing healthy cells.
• FOXO4-DRI’s apoptotic activity restores tissues injured by chemotherapy and aging.

Authors cautioned against permanent FOXO4 inhibition, given FOXOs’ role in DNA-damage repair. However, they noted, FOXO4-DRI holds promise for the treatment of malignant cancer and other senescent-driven diseases.

Dr. JoAnna Pendergrass received her Doctor of Veterinary Medicine degree from the Virginia-Maryland College of Veterinary Medicine. Following veterinary school, she completed a postdoctoral fellowship at Emory University’s Yerkes National Primate Research Center. Dr. Pendergrass is the founder and owner of JPen Communications, a medical communications company.