Overview of p53 and p53-targeted Therapeutic Strategies

The p53 gene is located on human chromosome 17 and consists of 11 exons and 10 introns, with a length of about 20kb. The p53 gene is transcribed to produce 2.5kb mRNA, which encodes a protein of 393 amino acids with a molecular weight of 43.7kDa. The p53 protein is composed of multiple functional domains, including two transcriptional activation domains (TAD1 and TAD2) at the N-terminus, a proline-rich domain (PRD), a DNA-binding domain (DBD), tetramer oligomerization domain (OD) and a regulatory domain (RD) at the C-terminus. The p53 protein is mainly distributed in the nucleus, and also exists in the mitochondria, nucleolus and other structures, and has an interaction relationship with the cytoskeleton. The expression level and activity of p53 protein are regulated by multiple levels, including transcriptional regulation, post-transcriptional regulation and post-translational modification.

P53 is an important tumor suppressor, known as the "guardian of the genome". P53 can respond to various stressors in cells, such as DNA damage, hypoxia, oxidative stress. By regulating the expression of target genes or independent transcriptional mechanisms, p53 promotes anti-cancer processes such as cell cycle arrest, apoptosis, autophagy, and DNA repair. However, in a variety of human cancers, p53 gene is mutated or deleted, leading to the loss of p53 function or the acquisition of new cancer-promoting functions, thereby promoting the occurrence and development of tumors. Therefore, restoring or activating the function of p53 is a promising cancer treatment strategy.

Gene therapy is a method of using genes or nucleic acid molecules to treat diseases. Gene therapy can be divided into two ways: in vivo and in vitro. In vivo gene therapy is the direct delivery of genes or nucleic acid molecules into target cells or tissues in vivo. In vitro gene therapy is to take target cells or tissues out of the body, perform gene transfection or transduction in vitro, and then transplant them back into the body. Gene therapy has the advantages of strong pertinence, few side effects, and good durability, and has made some progress in the fields of genetic diseases, infectious diseases, and tumors.

Mutation Types and Functions of P53

P53 is the most frequently mutated gene in human cancers, and it is estimated that mutations in the p53 gene exist in more than 50% of cancers. The mutation frequency and distribution of p53 are related to different cancer types, tissue origins, environmental factors and genetic backgrounds. For example, p53 mutations were more frequent in liver, esophageal, and lung cancers, while p53 mutations were less frequent in neuroblastoma, testicular cancer, and childhood leukemia. The mutations of p53 are mainly concentrated in its DNA-binding domain, accounting for more than 90% of all mutations, and exons 5, 6, 7, and 8 are hot spots, accounting for about 75% of all mutations. Mutations in p53 also exist in other domains, such as transcriptional activation, oligomerization, and regulatory domains, but are relatively rare.

Mutations in p53 can be divided into two categories: loss-of-function mutations and gain-of-function mutations. Loss-of-function mutations cause p53 protein to lose its normal transcriptional activation ability, unable to effectively induce the expression of target genes, and thus lose its tumor-suppressing effect. Gain-of-function mutations lead to new or enhanced functions of the p53 protein, thereby promoting tumorigenesis and progression.

There are several types of loss-of-function mutations:

• Truncating mutations: lead to premature termination of p53 protein or deletion of part of the amino acid sequence, affecting its stability or structural integrity.
• Missense mutation: results in a single amino acid substitution that affects its DNA-binding ability or transcriptional activation ability.
• Splicing mutation: Causes abnormal splicing of mRNA, affecting its coding ability or producing defective protein.
• In-frame mutation: results in amino acid insertion or deletion, affecting its reading frame or domain.

Gain-of-function mutations include two types:

• Missense mutations: lead to single amino acid substitutions that show functionally enhanced activity during tumorigenesis, such as p53 R175H and R273H mutations that promote tumor cell invasion and migration.
• Out-of-frame mutations: lead to amino acid insertion or deletion, change its reading frame or generate new fusion proteins, such as the p53 R248Q mutation that fuses with EWSR1 to generate EWSR1-p53 protein.

Different types of p53 mutations have different effects on the structure and activity of p53. Generally speaking, loss-of-function mutations prevent p53 protein from binding to DNA or transcriptionally activating target genes, while gain-of-function mutations make p53 proteins have new or enhanced function, such as interaction with other proteins or transcriptional activation of new target genes. Specifically, loss-of-function mutations can lead to some changes in the structures and activities. First, the stability or degradation rate of p53 protein is affected, such as truncating mutations or splicing mutations. Second, the DNA-binding ability or specificity of the p53 protein is altered, such as missense mutations or in-frame mutations. Third, the transcriptional activation ability or selectivity of p53 protein is affected. Fourth, the oligomerization ability or form of the p53 protein is changed. Gain-of-function mutations can also result in some structural and activity changes. DNA binding sites, transcriptional activation target genes, interaction partners, and post-translational modification sites of p53 protein may be altered, such as missense mutations or out-of-frame mutations.

Cancer Therapeutic Strategies Targeting p53

Small molecule drugs, immunotherapy, and gene therapy approaches targeting p53 are the three main strategies currently being developed or tested.

Small molecule drugs mainly target p53 in two ways, one is to restore the wild-type conformation and activity of mutant p53, and the other is to release the tumor suppressor activity of wild-type p53. The former mainly targets tumors containing TP53 missense mutations, and the latter mainly targets tumors that maintain wild-type p53. At present, a variety of small molecule drugs have entered clinical trials, the most representative of which are APR-246 and AMG-232. APR-246, a compound that reactivates mutant p53, has shown effectiveness in multiple clinical trials against cancers such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). AMG-232 is a compound that can inhibit the interaction between p53 and mouse double minute 2 homolog (MDM2), and has shown effectiveness against osteosarcoma, gastric cancer and other cancers in multiple clinical trials. The advantage of small molecule drugs is that they can be administered orally, have good bioavailability and selectivity, and can induce apoptosis of cancer cells or enhance immune responses by regulating the p53 signaling pathway. However, small molecule drugs may also have problems such as drug resistance, toxic side effects, and non-specific binding.

Immunotherapy also uses p53 mainly in two ways, one is to use p53 as a tumor-associated antigen to induce immune response, and the other is to use p53 to regulate immune cell function to enhance immune response. The former mainly targets tumors containing TP53 mutations, and the latter mainly targets tumors that maintain wild-type p53. So far, many immunotherapies have entered clinical trials, the most representative of which are TG4050 and Pfizer-BioNTech COVID-19 Vaccine. TG4050 is an mRNA-based personalized cancer vaccine that can be designed based on each patient's tumor mutation profile, which includes p53 mutations. The Pfizer-BioNTech COVID-19 Vaccine, an mRNA-based SARS-CoV-2 vaccine already approved for use globally, contains a sequence encoding a fragment of the human p53 protein. Fortunately, immunotherapy can use the body's own immune system to kill cancer cells, has high specificity and persistence, and can avoid problems such as drug resistance and toxic side effects. However, immune escape, autoimmune responses, and individual differences are issues that immunotherapy may address.

P53 gene therapy for cancer is a treatment method that uses genetic engineering technology to introduce normal p53 gene into cancer cells, thereby restoring the tumor suppressor function of p53, inducing apoptosis of cancer cells or enhancing immune response. There are three main programs: gene transfection, gene editing, and gene immunization. Gene transfection refers to the transfer of exogenous p53 gene into cancer cells through vectors (such as viruses or plasmids), so that it can express normal p53 protein, thereby inhibiting the growth and proliferation of cancer cells. For example, Ad-p53 is a recombinant adenovirus vector that can transfect the normal P53 gene into tumor cells, thereby restoring the function of P53 and inducing apoptosis or senescence of tumor cells. Ad-p53 is the first gene therapy product approved in China in 2003, mainly for the treatment of head and neck tumors. Ad-p53 is administered by minimally invasive intratumoral injection, intracavitary perfusion, or intravascular infusion. Ad-p53 combined with chemotherapy and radiotherapy can significantly improve the remission rate and survival period of patients. In 12 years of commercial use, Ad-p53 has treated more than 30,000 patients, and its safety and efficacy have been confirmed by multiple clinical studies. Gene editing refers to the use of technologies such as CRISPR/Cas9 to precisely repair or knock out the mutated P53 gene in tumor cells, restore or eliminate the function of P53, thereby affecting tumor growth. The CRISPR/Cas9 system has been used for the first time in humans in a phase 1 clinical trial in patients with advanced liver cancer. In this experiment, the CRISPR/Cas9 system was used to knock out two mutation sites on the TERT promoter, which can bind to mutated P53 and promote the proliferation of tumor cells. A total of 12 patients were enrolled in the trial, 10 of whom received an infusion of gene-edited cells. The trial showed that the approach is feasible and safe, and can delay the progression of liver cancer. One of the patients remained free of tumor recurrence or metastasis 18 months after the infusion. The principle of genetic immunization is to use the P53 gene or its encoded polypeptide fragment as an antigen to stimulate the body to generate an immune response against P53, thereby killing tumor cells expressing mutant P53. Gene Immunization has been evaluated in a phase 2 clinical trial in patients with advanced non-small cell lung cancer. In this trial, a vaccine (TG4010) containing a humanized p53 polypeptide fragment and an adjuvant was used in combination with chemotherapy. A total of 221 patients were enrolled in the trial, 148 of whom received TG4010 in combination with chemotherapy and 73 who received placebo in combination with chemotherapy. The trial showed that there was no significant difference in progression-free survival between the TG4010 and chemotherapy group and the placebo and chemotherapy group (5.9 months vs 5.1 months, P=0.74), but overall survival There was a significant advantage (11.9 months vs 8.7 months, P=0.012).

Table 1. P53 Gene Targeted Drugs in Clinical Trials or Approved