Targeting the P53 Protein for Cancer Therapies: The Translational Impact of P53 Research Open Access

The p53 protein is a transcription factor. Its function is to respond, by increasing its concentration and activity, to intrinsic and extrinsic stresses that may lead to increased rates of mutation, due to replicating damaged DNA or other processes during cellular replication. The increased levels of the p53 protein are regulated by the E3 ubiquitin ligase MDM2 along with several associated proteins. After exposure to a stress, MDM2 is commonly inhibited and modified along with the activation of the p53 protein (phosphorylation, acetylation, methylation) by the epigenetic enzymatic machinery of the cell. Depending upon the cell type, the nature of the stress, and the intensity of that stress, the p53 protein rolls in a transcriptional program resulting in cell-cycle arrest, repair, and return to homeostasis or cell death. The genes transcribed in these programs are redundant. For example, the p53 program controls five different kinds of cell death; (i) apoptosis, (ii) ferroptosis, necroptosis mediated by either (iii) TNF or (iv) FAS ligand and (v) cellular senescence followed by secretion of cytokines that attract the immune system. This redundancy not only assures cell death, it may differentially regulate the presentation of antigens to the immune system and as such enhance the type of immune responses to infections, cancers, inflammation or autoimmune disorders. It is the epigenetic modifiers that inform the choices of p53 transcriptional programs and one of the stresses that the p53 program responds to is a large change in the epigenetic states of cells. The entire pathway is filled with feedback loops that regulate it and ensure homeostasis. The p53 pathway ensures fidelity of cellular duplication by death (1).

Cancer is an extraordinarily heterogeneous disease. There may be no common feature, mutated gene, causative agent, or treatment for these diseases. But the most common spontaneous mutational inactivation event or inhibition by a viral or bacterial etiology of cancer is the inactivation of the p53 protein and the p53 pathway. Fully 50% of spontaneous cancers in humans have Tp53 mutations and most, if not all, cancer cells with a wild-type (WT) Tp53 gene have compromised some p53-related activities. For these reasons, the p53 protein has been an attractive target for cancer therapy for decades, but this has been a difficult task for several reasons; (i) restoring a loss of function is more difficult than inhibiting a gain of function, (ii) every Tp53-mutant allele that has been examined in detail differs from the other p53 mutants, (iii) there are more than 300 Tp53 mutants that contribute to cancers and they occur at different frequencies, (iv) viral initiated cancers inactivate p53 by protein–protein interactions, not by mutations. Yet there are a number of different approaches to these problems and drugs targeting p53 or its associated proteins that are making their way into the clinic.

There have been at least six different approaches to developing translational therapies to the p53 protein and pathway. (i) The use of viruses to either deliver a WT p53 c-DNA to tumor cells or the use of mutant viruses that can not inactivate p53 functions and so replicate only in cells with mutant Tp53 (ONYX-015). (ii) The inhibition of the p53-MDM2 complexes (nutlin like compounds) or the inhibition of other associated proteins (MDM4, PPMID-1, a p53 deubiquitinase, etc.) that raise the levels of WT p53 in cells. (iii) The inhibition of activities that become synthetic lethal functions only in p53-mutant cells. (iv) The degradation or inhibition of possible p53 “gain-of-function activities” caused by p53-mutant proteins. (v) Structural reactivators, which are small molecules or peptides that restore both WT structure and function to the p53 protein with missense mutations. (vi) The activation of the immune system so as to selectively kill Tp53-mutant cells. Some Tp53 mutations are recognized by the B-cell and T-cell repertoire (2) and other p53-mutant peptides are recognized by bispecific antibodies that bring T cells to the tumor (3)

There are a large number of compounds and agents that have been or are being tested in the clinic and have yielded a number of interesting results. This is not an inclusive list and is chosen for its diversity and lessons learned (Table 1).

1. A recombinant WT p53 c-DNA delivered by an adenovirus vector into a head and neck cancer that is then irradiated to activate the p53 protein and kill some of the tumor cells (Shenzhen SiBionoGene Tech). This approach was approved in 2003 by the Chinese FDA. The failure to infect all the tumor cells means the tumor grows back in a short time. But the experiment proves the concept that WTp53 (plus irradiation) can kill cancer cells in vivo (4).

2. Acute promyelocytic leukemia (PML) is driven by a gene fusion that produces a PML protein fused with a retinoic acid-alpha receptor. PML bodies in the nucleus of normal cells are where many proteins, like p53, are modified (acetylation, methylation) so as to function. Treatment with arsenic trioxide plus all-trans-retinoic acid degrades the PML-RAR-alpha nuclear bodies, permitting the WT PML allele to produce normal nuclear bodies, modifying the WT p53 protein from an inactive form (repressor) to initiating a senescence program resulting in cell differentiation of the PML stem cells to neutrophils, or in some cases apoptosis (5). In many cases of this rare cancer, the activated WTp53 protein results in a cure of this leukemia. In the absence of the WT p53 protein, there is no cure.

3. There is a good deal of preclinical evidence that azacitidine or decitabine, which changes epigenetic patterns of cytosine methylation in DNA, inhibits cells with Tp53 mutations better than cells with WT Tp53 (1). A phase I trial with 5 patients with either acute myeloid leukemia (AML) or myelodysplastic syndrome and selected Tp53 mutations (NCT03855371) treated with decitabine and arsenic trioxide resulted in tumor remissions that however rapidly relapsed with resistance to these drugs. Another trial with patients with AML with Tp53 mutations treated with decitabine alone, showed similar results (6). These trials support the idea that WT Tp53 is protective against large epigenetic changes while mutant Tp53 cells preferentially are stressed, fail to replicate, die or differentiate.

4. Small molecules or a stapled peptides (from many companies) that block the p53-MDM2 protein complexes and increases WT p53 protein and activity, result in some patients initiating tumor cell death and tumor regression. These drugs work only with some fraction of cancers with WT p53 and never with cancers containing mutant p53 proteins. Biomarkers, such as amplified MDM2 genes, are not consistent indicators of success. These drugs act on normal cells (hematopoietic cells and intestinal cells are most sensitive) so on target side effects can limit their use. These drugs are presently being tested in clinical studies in patients with AML and a few other tumor types.

5. Treadwell Therapeutics has developed a potential synthetic lethal like drug for cells with mutant p53 and extensive aneuploidy in AML cells (the drug is called 400945). The drug is an inhibitor of PLK-4 (polo-like kinase 4) involved in the fidelity of chromosome segregation (G₂–M checkpoint) by detecting damaged chromosomes (7). The WTp53 protein is known to regulate the number of centrosomes. Cells with a mutant p53 have multiple centrosomes and develop aneuploidy with accompanied high levels of cell death. (NCT04730258).

6. It has been suggested that the wee-1 protein kinase, a G₂–M checkpoint for damaged DNA, could be a synthetic lethal for p53-mutant cells. Many drugs that inhibit wee-1 activity in p53-mutant cells have been tested in several trials. The examples 5 and 6 here are consistent with the idea that synthetic lethal drugs in mutant p53 cells may cluster in the G₂–M checkpoints of the cell.

7. Aprea Therapeutics developed Aprea-Met, (APR-246, eprenetapopt), which is a small molecule that covalently binds to both the WT and mutant p53 proteins and many other proteins. In clinical trials, it has not demonstrated single-agent activity directly against tumors. In a phase III trial of 193 patients with myelodysplastic syndromes containing Tp53 mutations, APR-246 plus azacitidine was compared with azacitidine alone, this drug failed to meet its primary endpoint (NCT03745716). Clinical trials in combinations with other approved products are ongoing with different tumor types.

8. PMV Pharmaceuticals developed an allele specific reactivator drug (mutant converted to WT activity) for the Y220C Tp53 mutation. It is presently in phase I/II clinical trials (NCT04585750). In preclinical studies, the single agent could produce complete regression of tumors in mice and at lower concentrations of the drug it synergized with checkpoint therapy, PD-1 antibody treatment to yield complete regression of tumors. These experiments demonstrated that changing Y220C mutant p53 protein to its WT functions not only resulted in p53-mediated cell death, but also enhanced the killing of the tumors by the immune system of mice.

9. At City of Hope, a vaccinia virus vector with p53 encoding sequences was employed to immunize patients with ovarian cancers against p53 epitopes (p53 MVA) and pembrolizumab is administered to overcome tolerance to p53 epitopes if they are present with the correct HLA types (NCT03113487). With Tp53 mutations that arise spontaneously in some cancers the immune system produces antibodies against p53 epitopes. These antibodies detect WT p53 amino acid sequences from the amino terminal and carboxy-terminal ends of the p53 protein. This occurs only when cancerous Tp53-mutant cells are present. A p53-mutant protein fails to transcribe the MDM2 gene and in the absence of an MDM2 protein the mutant p53 protein levels increase up to ten fold more p53 protein compared with cells with WT p53. Presumably this triggers a B-cell adaptive immune response (8). There is no indication that this B-cell immune response is sufficient to inhibit tumor growth (8).

There is an interesting relationship between drugs that alter epigenetic marks in the DNA and RNA and p53. Azacitidine and decitabine incorporate into DNA and RNA and prevent methylation of cytidine in CpG dinucleotides. Both drugs kill cancer cells with Tp53 mutations better than cancer cells with WT p53 both in cell culture and in tumors in vivo. The Yamanaka factors, which radically reduce methylated CpG residues do so much more efficiently in the absence of p53 than in the presence of WT p53. Indeed, the loss of methylated CpG residues in a normal cell activates WT p53 and kills the cell by apoptosis. Mutant p53 permits these epigenetic changes. The fundamental mechanisms that mediate these observations remain unclear (this topic has been discussed in detail and references given in reference 1).

The lessons learned and the ideas suggested from these early clinical trials may be summarized as follows: (i) Adding p53 to some tumor cells (4) or activating WT p53 in tumor cells (5) can result in cell death, tumor remission or even cures. (ii) PML bodies in the cell nucleus are where epigenetic modifications to WT p53 are important for initiating cell death, senescence, or differentiation in some cell types (5). (iii) Inhibitors of DNA methylation, such as azacytidine or decitabine, kill tumor cells with mutant Tp53 genes better that tumor or normal cells with WT Tp53 (1, 6). (iv) Synthetic lethal inhibitors of Tp53-mutant cells can act upon G₂–M checkpoint proteins, recognizing aneuploidy or copy number intolerance resulting in cell death (7). (v). There is a complex communication between cells with activated WT p53 protein and/or cells with mutant p53 proteins and the adaptive and innate immune system (8).

Drugs have been developed and approved that inhibit the RAS oncogene in an allele specific fashion employing covalent modifiers (9). The development of allele specific RAS inhibitors and allele specific p53 reactivators (NCT04585750), often in the same tumor cells, could lead to very powerful combinations. Furthermore, covalent RAS inhibitors add a haptene like drug to mutant specific RAS protein containing cells, making them potential targets for the drug acting as a haptene for the immune system. At the same time, CD-8 T-cells directed against allele specific Tp53 mutations have been demonstrated in patients with selected HLA types (10). These kinds of cancer specific or allele specific drugs, that do not have activity in normal cells, are the kind of drug combinations that can be employed to focus and enhance immunologic checkpoint therapies because they only function in cells with allele specific RAS or p53 mutations. By being cancer selective (allele specific), they could reduce the side effects of drugs and immunologic treatments and possibly could lead to cures.

A.J. Levine reports grants from NIH, NCI Po1-CA087497-18 during the conduct of the study; personal fees and other support from PMV Pharmaceuticals board, SAB, Genecentric Inc. board, MeiraGTX board, ROME SAB; personal fees from Pharmabody LTD board, Janssen Pharmaceutica SAB, WCG Copernicus SAB, Hattereas SAB; other support from Genotwin SAB, Isoplexsis SAB, Intervenn SAB, AtlasXomics SAB, SU2C SAB, Mark Foundation reviewer, LFSA SAB outside the submitted work.

The author would like to thank S. Christen for her aid with the preparation of this article. Some of the ideas expressed in this short communication were benefited by discussions with K. Shokat.

This work is supported by NIH NCI P01CA087497-20.