# Emerging Non-Canonical Functions and Regulation by p53: p53 and Stemess
## 4 Therapeutic Strategies to Restore _wt_p53 Function in CSCs  
### Small and Large Molecules  
Molecule development to explore novel targets and signaling pathways to restore _wt_p53 function is necessary for probing the mechanisms of p53 inactivation in CSC genesis and tumor development. Loss of _wt_p53 function is prevalent in more than half of all solid and hematologic malignancies and persists as mutations or deletions altering p53 activity levels. _W_p53 found in cancers is nonfunctional in that p53 is either degraded by Mdm2, or secluded from the nucleus inhibiting its function in transcription [34; 233; 234]. Changes in epigenetic regulation of p14/ARF, methylation of the p53 promoter and persistent expression of Mdm2 and Mdmx contribute to _wt_p53 loss of function [136].  
Combinatorial therapies have been employed to target _mut_p53 in CSCs and improve therapeutic strategies, including chemotoxic agents (Figure 4). Many of these small molecules have been shown to be effective in preliminary studies against solid tumors and hematological cancers. These molecules can be classified as _wt_p53 activators (RITA, Nutlin-3, MI-219, BDA, HL198C, Tenovin-1, JJ78:12), _mut_p53 reactivators (CP-31398, PRIMA-1, MIRA-1, ellipticine, P53R3, WR-1065) or _wt_p53 inhibitors (PFT-\(\alpha\), PFT-\(\mu\)) [235; 236; 237; 238; 239; 240; 241; 242; 243; 244; 245; 246; 247; 248; 249].  
[PERSON] et al. (2009) used Ellipticine with 5-FU as a strategy to deplete deficient p53 DLD1 colon CSC. The use of small molecules, such as CP-31398, MIRA1 or PRIMA-1, to induce transactivation activities in _mut_p53 has also been employed [243; 250; 251; 252]. PRIMA-1 and MIRA-1 have unique chemical structures, but both share the ability to restore the p53 conformation from mutant to wildtype [242; 243]. PRIMA-1 converts to a methylated form, PRIMA-1 MET, and modifies thiol groups in the central domain of _mut_p53 to restore tumor suppressor p53 [253]. [PERSON] et al. (2016) [250] used PRIMA-1 to target _mut_p53 in CSC from breast, endometrial and pancreatic cancer cell lines, allowing for the reduction in tumor cell growth and inhibition of tumorsphere formation. In solid tumors, small molecule MIRA-1 can induce apoptosis through p53-dependent processes [243]. The p53 activity of chemoresistant colorectal CSCs can be restored by the small molecule Prodigiosin through c-Jun-mediated \(\Delta\)Np73 downregulation and p73 activation [254].  
Synthetic peptides derived from known p53 binding proteins can restore the conformation of _mut_p53 to _wt_p53, including the transcriptional activity of hotspot p53 mutants p53R273H and p53R175H, such as peptide CDB3 with L-amino acid sequence REDEDEIEW [255]. Peptide 46, a p53 C-terminal peptide that increases tumor efficacy through core domain stabilization leading to p53 reactivation and apoptosis [256; 257; 258]. Stapled peptides or stitched peptides have been retrofitted with a synthetic brace to promote peptides stabilizing an alpha-helix structure that disrupts the p53-Mdm2 interaction and reactivating the p53 tumor suppressor pathway [259]. Positively charged cell-permeable stapled peptides have also been shown to inhibit the p53-Mdm2/Mdmx complex [260]. These peptides can be combined with other targeting approaches, such as polymeric micelles for enhanced tumor penetration and access to the blood brain barrier (BBB) [261]. Bispecific antibodies have also shown promise in targeting the p53-Mdm2 interaction by binding to p53. [PERSON] et al. (2004) [262] demonstrated that a bispecific single-chain antibody can penetrate colon cancer cells with _mut_p53 and restore _wt_p53 function.