Identifying p53 Target Genes Using Hypomorphic Variants

As one of the most studied genes in history, the TP53 tumor suppressor is a critical molecular hub playing a role in preventing cancer development and progression (1). Mutations in TP53 (encoding for the p53 protein) can lead to the loss of its tumor-suppressive functions as well as to gain of oncogenic functions, manifested by an increased risk for cancer (2, 3). Hypomorphic variants of the TP53 gene refer to mutations that do not completely abolish the function of the protein but rather result in a partial reduction in its activity.

As such, hypomorphic variants of the TP53 gene may become instrumental as research models with the attempt to discover novel targets for the wild-type (WT) form of the gene. p53 is suppressing tumorigenesis by acting as a sequence-specific transcription factor with the capability to execute hundreds of transcriptional programs. Despite this well-established notion and the extensive experimental efforts to define p53 target genes with roles in cell-cycle arrest, senescence, and apoptosis, our comprehension of the targeting repertoire of p53 remains incomplete, with gaps still present.

In a recent study published in this issue of Cancer Discovery, Indeglia and colleagues have been investigating the TP53 variant mutated in the Y107H position (4). This variant was reported to be significantly more prevalent in African-­American populations compared with the general population. Notably, this is not the first attempt of this group to identify p53 target genes using a hypomorph variant. Another recent research article revealed that phospholipid transfer protein transactivation is tightly correlated with levels of p53 activity demonstrated using several hypomorphs (including the Y107H variant; ref. 5).

Although this current study exhibits that the Y107H variant maintains many wild-type (WT) p53 activities, the authors established a transgenic mouse model carrying the Y107H allele (as well as its murine analogue Y104H) in which they observe a significant increase in cancer risk. The authors propose that structural misfolding of the hypomorph might be reducing considerable parts of the typical WT function.

The higher cancer incidence in mice, added with The Cancer Genome Atlas (TCGA) data indicating that the Y107H variant is a less effective tumor suppressor compared with the WT, led the authors to ask which genes are not activated by the hypomorph, thus suspected as potential novel downstream molecular associates of WT p53.

To do this, the authors used lines of immortalized human lymphoblastoid cells obtained from human donors in which p53 is either WT or heterozygous (one WT allele and one hypomorph allele) and challenged them with traditional experimental activation of p53—either nutlin or cisplatin. To investigate the differences in transcription programs, RNA sequencing was conducted and compared between the WT and the hypomorph heterozygous. Although no change was observed in the vast majority of gene expression levels in response to both p53-activating agents, some 75 targets were found to be activated less efficiently by the Y107H variant. Among this list of genes, peptidyl arginine deiminase 4 (PADI4) was validated in additional hypomorph systems and chosen by the authors as a promising potential target of the WT form.

PADI4 encodes for an enzyme involved in the posttranslational modification of proteins, specifically the conversion of arginine to citrulline. PADI4 has been linked to several autoimmune diseases, including rheumatoid arthritis and lupus (6). The role played by PADI4 in cancer is controversial and likely to differ between cancer types and cellular context, but interestingly, a study from 2012 showed that citrullination of histone H4 and Lamin C occurs in response to DNA damage through the p53–PADI4 pathway (7). Citrullinated histone H4 and Lamin C localize around fragmented nuclei in apoptotic cells. That suggested a key role for p53 in sensing an “apoptotic histone code” that detects damaged cells and induces nuclear fragmentation, where PADI4 is a major associate.

In a series of cellular assays, the authors validated that PADI4 is indeed regulated by p53 and that transactivation levels are dampened when Y107H is tested. When closely focused on the DNA-binding efficiency, nuclear magnetic resonance spectroscopy combined with crystal structure revealed a modest change in amino acid sequence around Y107H, albeit the structure resembled that of WT p53. Nonetheless, a decrease in the melting temperature of the core domain was noted, thus delineating the thermal instability of Y107H, suggesting self-aggregation and misfolding to a mutant conformation that allowed inefficient binding to PADI4. Although the authors indicated that the hypomorph binding is weaker compared with the WT, this was not specific to the PADI4 response element and was observed in several other known p53 target genes such as CDKN1A (p21).

After the discovery of p53 in the late 1970s and the dawning of its role as the most mutated tumor suppressor in the late 1980s, constant attempts have been constructed to make p53 and its family members druggable. Personalized medicine for cancer is gaining acceptance, but there are only a few examples of truly personalized approaches that take the mutational spectrum of the gene into account. Entering this void, Indeglia and colleagues have capitalized on a library of over 2,000 compounds and screened for compounds that exhibit a higher reduction in cell viability in two separate Y107H clones as opposed to the WT. Out of a set of 24 positive hits, the authors picked CB-839 and BPTES as two candidate compounds specific for the hypomorph.

Experimentally, both molecules were found to inhibit tumor growth in transformed homozygous Y107H HCT116 cells when compared with WT HCT116, concomitant with increased apoptosis and decreased proliferation. By testing CB-839 also in a xenograft mouse model, the authors were able to display a Y107H-specific antitumor effect that was not observed in the WT tumors. This precision medicine approach echoes a previous study published by the group, suggesting use of cisplatin as a primary therapy for African-American patients with tumors carrying the TP53 P47S hypomorph (8). Because CB-839 is an activator of the ATF4 pathway, the group also decided to investigate the expression of ATF4 and observed an increased induction of ATF4 as well as the prodeath protein CHOP, which, again, was corroborated in vivo.

The last part of this work was dedicated to p53–PADI4 interactions in the context of the immune response. The authors demonstrated their findings in melanoma models, being a top immunogenic cancer (potentially due to high levels of tumor mutation burden). Two melanoma cell lines (chosen based on basal PADI4 activity) were overexpressed with PADI4 and treated with nutlin to activate p53. This led the authors to reveal a specific gene set that included signaling genes related to JAK/STAT, IL8, and CXCR4. In addition, these PADI4 melanoma clones were used in vivo to delineate the ability of PADI4-expressing cells to suppress tumors in the presence of CD8⁺ T cells. These findings were further corroborated when PADI4 failed to suppress tumors in immune-deficient NSG mice when compared with immune-intact C57Bl/6 mice. Lastly, the authors scanned TCGA data for genes that are coexpressed with PADI4 but affected by the Y107H presence. Expression of PADI4 along with IL16, IL21R, S1PR4, and CEACAM21 was found to be associated with increased CD8⁺ T-cell infiltration and improved survival in melanoma after immune checkpoint inhibitors.

This research highlights the essential unmet need for health disparities in cancer, now considered a major public health issue requiring urgent attention (9, 10). It is well established that certain populations, including racial and ethnic minorities, low-income individuals, and those living in rural areas, experience higher rates of cancer incidence, morbidity, and mortality compared with their more privileged counterparts. The study by Indeglia and colleagues may offer promising treatment options for people with genetic hypomorphic variants of p53, thus contributing to cancer disparities in individuals of African descent. As depicted by the model displayed in Fig. 1, this work represents a significant contribution toward understanding the downstream effects of the cardinal tumor suppressor TP53 by utilizing hypomorphic variants of the gene, paving the way for future research efforts.