• DAF16 homologues FOXO3 and 4 regulated by PI3K/PKB/AKT (Kops Nature. 1999 Apr 15;398(6728):630-4. Brunet Cell. 1999 Mar 19;96(6):857-68.)
• FOXOs involved in cell cycle regulation (Medema Nature. 2000 Apr 13;404(6779):782-7.)
• FOXOs involved in combatting cellular stress (Kops Nature. 2002 Sep 19;419(6904):316-21.)
• FOXOs regulated by cellular stress (Essers EMBO J. 2004 Dec 8;23(24):4802-12.
• FOXOs regulated by cellular stress (Essers Science. 2005 May 20;308(5725):1181-4.)

FOXO4 can physically interact with p53 under conditions of stress. In response to excessive stress signaling, pathways are turned on to counter the negative consequences. FOXO4-p53 signaling is important for this reponse.

FOXO proteins are downstream targets of the senescence response. Oncogene-mutation, as well as other forms of persistent stress can lead to a chronic damage response. As a consequence, anti-stress signaling is activated. This is for instance evident from FOXO4 phosphorylation and activation. This lead us to the following model (e.g. P.L.J. de Keizer, Ph.D. dissertation and de Keizer et al., 2010, Cancer Research):

Identification by our team that FOXO4 is a pivot in maintaining the viability of senescent cells. Using primary senescent cells, we observed that removing FOXO4 could trigger a “clean” cell death response, known as apoptosis. This created a new window of opportunity to eliminate these deleterious cells. As we found FOXO4 to also exert a protective response to chemotherapy, it additionally opened the door towards development of methods to overcome therapy resistance of cancer cells.

Generation of the first and second generation of compounds to eliminate senescent and therapy-resistant cancer cells (e.g. patent US20130288981 A1; 2012). Here, we showed for the first time that not only inhibition of FOXO4 itself, but more specifically, blocking the FOXO4-p53 binding by cell-penetrating peptides could effectively eliminate senescent cells and therapy-resistant cancer cells.

Development of the third generation of FOXO4-based anti-senescence drugs: the FOXO4-DRI peptide, aka Proxofim. This proved to be effective in counteracting signs of chemotoxicity and, excitingly, was able to restore healthspan in models for fast and natural aging, e.g. fur density, behavior and renal function. (Baar et al., Cell, 2017). This research received worldwide media attention, including coverage in numerous TV and radio shows, newspapers and blogs.

Founding of Cleara to continue our evolution of FOXO4-based anti-senescence drugs and generate the fourth generation capable of human translation. Since the selectivity of the third generation compound, FOXO4-DRI, for senescent cells is ~10-fold, it is unfortunately still too dangerous to allow for clinical trials. At Cleara Biotech, it is our mission to improve the safety profile and potency of Proxofim and to do so, we launched the iPROX program to generate “Improved Proxofim”. We are passionate in translating iPROX to humans and combat senescence-driven age-related diseases, including therapy resistant cancer.