p53 drugs in cancer: one protein, multiple targets

However, several promising approaches to p53-based therapies have emerged in recent years that both protect p53 from its negative regulators and restore mutant p53 protein function
.
However, many issues remain to be resolved
.

Physiological role of p53 and cancer mechanism

p53 tumor suppressor protein acts as the main barrier
to prevent the initiation and progression of cancer.
Biochemically, p53 functions primarily as a sequence-specific transcription factor, able to bind to and activate transcription of adjacent genes with defined DNA sequences within the genome, called p53 reactive elements or p53 binding sites, as well as transcription of more distant genes regulated by enhancers with p53 binding sites
.

In normal, non-stressed cells, p53 protein levels are kept low by constitutive proteasome degradation, guided by the E3 ubiquitin ligase MDM2, the main inhibitor of
p53.
In addition, the biochemical activity of p53 as a transcription factor is also inhibited by the MDM4 protein (also known as MDMX), which thus acts as an additional physiological p53 inhibitor
.

TP53 gene mutations eliminate the tumor suppressive activity of its encoded protein p53, are the most common single gene alterations in human cancer, and are thought to be the driving event
for many types of tumors.
However, most of these efforts have had limited success: very few p53 drugs have reached late-stage clinical trials, and none have so far made them approved
by the FDA or EMA.

p53, an attractive target in cancer

Since the p53 protein was first described in 1979, its function and its relationship to cancer have been widely understood
.
As a transcription factor, p53 coordinates the expression
of target genes that can promote cell cycle arrest, apoptosis, DNA repair, etc.
In addition, p53 can also exert antiproliferative effects
through transcription-independent mechanisms.
Unlike other tumor suppressor genes observed, cancer-associated TP53 mutations are primarily missense mutations that cause single amino acid substitution
.
Although hundreds of different p53 mutations have been recorded, some of them are particularly common and are therefore known as hotspot mutations
.
There is growing evidence that in addition to the loss of wtp53's cancer-suppressing activity, some p53 mutants can acquire new gain-of-function (GOF) activity, further promoting cancer
.

Figure 1: Tumor suppressive effect of wild-type p53 and carcinogenic effect of mutant p53

Small molecules

Tumors with different TP53 states require different small molecule strategies
.
Therefore, for tumors carrying TP53 missense mutations, small molecule drug development has focused on compounds
that restore wild-type conformation and mutp53 protein activity.
Conversely, for cancers that maintain wtp53, the main approach is to identify small molecules that inhibit p53 from its negative regulators, most notably MDM2, thereby releasing the full p53 activity
.

Figure 2: Number of p53-targeted clinical trials by year and treatment class

Target TP53 missense mutant tumors

TP53 missense mutations are unusually common in human cancers, accounting for about 70% of all TP53 alterations
.
The resulting structural or conformational mutants have been seen as promising targets for small molecules
.
The drug, CP31398, identified by Pfizer through a synthetic compound library, restored the transcriptional activity of p53 and reduced tumor growth
in vivo.
However, later work revealed more complex effects, including nonspecific toxicity caused by drug embedding into DNA and upregulation
of the p53-independent pro-apoptotic protein BAX35.
Therefore, CP31398 was not included in clinical development
.

In 2002, chemical drug screening found that PRIMA-1 (p53 reactivation induces mass apoptosis1), a compound that restores wtp53 function after binding to mutp53, triggers apoptosis in vitro in transducing Saos-2 cells expressing the R273H mutant protein (p53 (R273H)) and inhibits tumor formation
in vivo through these cells 。 Like PRIMA-1, MIRA-1 and STIMA-1 also have Michael receptor activity and can potentially modify cysteine in the p53 protein to stabilize wild-type conformations and prevent mutp53 from unfolding
.
Although both compounds exhibited p53-dependent effects in vitro and in vivo, neither entered clinical trials
due to solubility issues (STIMA-1) and toxicity to normal cells (MIRA-1).

The first to enter clinical trials was PRIMA-1 MET, a methylated derivative of PRIMA-1, also known as APR-246
.
In vitro and preclinical studies, APR-246 has shown better activity than PRIMA-1, with increased
apoptosis in acute myeloid leukemia (AML) cell lines and primary cells in patients.
In addition to its effects when administered as a single agent, APR-246 also increased cancer cell death
in vitro and when combined with chemotherapy in mouse xenograft models of lung and ovarian cancer.

Figure 3: P53-based small molecules for cancer treatment

Clinical trials of APR-246 are currently ongoing
.
Two Phase I/II trials demonstrated significant efficacy
when APR-246 was combined with azacitidine, the first FDA-approved drug to treat myelodysplastic syndrome (MDS), in patients with MDS or AML with p53 mutations.

Target WTP53 tumors

MDM2-mediated ubiquitination and degradation relies on its direct binding to p53, prompting the search for small molecules that inhibit MDM2-p53 binding as a means of
stabilizing p53.
APG-115, an orally bioavailable, potent MDM2 inhibitor, exhibits potent antitumor effects
in AML83 and radiotherapy-sensitive preclinical models of gastric cancer xenograft.
APG-115 is currently being evaluated in multiple clinical trials (e.
g.
, NCT02935907, NCT03611868, NCT04785196, NCT03781986), including monotherapy or in combination with chemotherapy or immune checkpoint suppression
.
AMG 232 is another orally bioavailable MDM2 inhibitor that has been shown to promote wtp53 function and tumor regression
in osteosarcoma cells.
Head-to-head activity of AMG 232 has been reported to be superior to other MDM2 inhibitors, including idasanutlin86.

AMG 232 combined with cytotoxic chemotherapy has a superior antitumor efficacy than AMG 232 or chemotherapy
alone.

In contrast, proteolytic and molecular glue compounds that target chimeras (PROTACs) act as a catalyst and may make them effective
even at low doses.
By correctly localizing the target protein physically close to the E3 ligase, PROTACs enhance the ubiquitination and subsequent proteasome degradation
of the former.
In this way, any particular protein may be selectively eliminated
if a suitable PROTAC is available.

The human papillomavirus (HPV) E6 protein is another therapeutic target associated with wtp53
.
E6 interacts with p53 to promote its degradation by recruiting ubiquitin-protein ligase E3A (E6AP); This is often the case
for HPV-induced cervical cancer.
RITA (reactivation of p53 and induction of apoptosis in tumors) is a p53-binding drug that was first thought to be an MDM2 inhibitor, but has since also been shown to interfere with p53-E6 interactions
.

• Target truncated p53

Although most cancer-associated TP53 mutations are missense mutations, approximately 10% of TP53-mutant tumors carry nonsense mutations that produce truncated proteins that are usually degraded
by senseless-mediated mRNA decay (NMD) mechanisms shortly after translation.
Therefore, two alternative approaches have been proposed to activate the p53 signaling pathway
in cancer cells carrying p53-truncated mutations.
The first method is based on molecules that facilitate translational read-through, enabling translation machines to bypass the RNA terminating codon and produce a full-length p53 protein
.
Another approach requires suppression of the NMD process
.

• Target mutp53 GOF

Although most p53-based drug development efforts are aimed at restoring wild-type p53 activity in cancer cells, there have also been attempts to eliminate mutp53 GOF activity
by targeting mutp53 for rapid degradation.

p53-based cancer immunotherapy

• P53-based vaccines

A synthetic long peptide (SLP) vaccine consisting of 10 overlapping peptides of the wtp53 sequence, injected twice at intervals of 3 weeks, has been shown to cause a CD4+ T cell-dominated T cell response
in metastatic colorectal cancer.
Adverse events were relatively mild: toxicity was limited to grade 1/2 and most occurred at the vaccination site
.
In ovarian cancer patients, low-dose cyclophosphamide treatment before SLP vaccination enhances the immunogenicity
of p53.
However, one clinical trial failed to show a benefit of SLP vaccination over a
control.

The success of mRNA vaccination during the coronavirus disease 2019 (COVID-19) pandemic has brought new hope
for p53 mRNA vaccines.
After autologous TP53 mRNA-transfected DCs were introduced into breast cancer patients, 13 of the 18 patients with high tumor expression of p53 showed p53-specific interferon γ (IFNγ) T cell responses in vitro.
This contrasts with p53-specific IFNγT cell responses in healthy donors and breast cancer patients with low p53 expression (1/10 and 2/18, respectively
).

• p53-specific antibody

T cell receptor mimicry (TCRm) antibodies, also known as TCR-like antibodies, are potential strategies
for targeting intracellular proteins.
These antibodies are typically generated by phage display library screening or hybridoma screening, recognizing epitopes displayed by MHC class I molecules on the cell surface, similar to how T cells recognize such epitopes through their TCRs, capable of recognizing peptides
derived from intracellular proteins.

Bispecific antibodies are a very promising immunotherapy for
cancer.
A single-stranded mutp53-based bispecific antibody has recently emerged that recognizes neoantigens
derived from p53(R175H) hotspot mutants and TCR–CD3 complexes.
This bispecific antibody overcomes the lack of neoantigen presentation and selectively redirects T cells to recognize cancer cells
presenting mutant peptides.

• p53 and tumor microenvironment

In addition to targeted attempts to develop p53-specific immunotherapy modalities, recent research has highlighted the broader link
between p53 and cancer immunotherapy.
Thus, p53 status in cancer cells can affect the immune landscape
in TME.
Therefore, drugs that enhance p53 activity may also enhance the involuntary cancer suppression function
of p53 in TME.

• p53-based genetic therapy

Gendicine, a recombinant human p53 adenovirus developed by Shenzhen GeneTech, was approved for use in HNSCC by the State Food and Drug Administration (CFDA
) in 2003.
Other adenovirus-based p53 gene therapies, including advexin and SCH-58500, have shown promising results
in clinical trials.

SGT-53 is a cationic liposome developed by SynerGene Therapeutics that carries wtp53-encoded DNA and selectively homs to tumor cells through anti-transferrin single-chain antibody fragments, sensitizing glioblastoma cells to temozolomide both in vitro and in vivo, and improving survival in
mouse models of glioblastoma.

summary

The fact that efforts to develop p53-based therapies have been ongoing for nearly three decades raises concerns about
their cost-effectiveness.
As our knowledge grows, it becomes increasingly clear that one p53 drug fits all is bound to fail, just as
KRAS (G12C) inhibitors do not benefit patients with cancer with other KRAS mutations.
Therefore, accurate patient-drug matching will be crucial
.

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