Elucidating the chain of command: our current understanding of critical target genes for p53-mediated tumor suppression.

TP53 encodes a transcription factor that is centrally-involved in several pathways, including the control of metabolism, the stress response, DNA repair, cell cycle arrest, senescence, programmed cell death, and others. Since the discovery of TP53 as the most frequently-mutated tumor suppressor gene in cancer over four decades ago, the field has focused on uncovering target genes of this transcription factor that are essential for tumor suppression. This search has been fraught with red herrings, however. Dozens of p53 target genes were discovered that had logical roles in tumor suppression, but subsequent data showed that most were not tumor suppressive, and were dispensable for p53-mediated tumor suppression. In this review, we focus on p53 transcriptional targets in two categories: (1) canonical targets like CDKN1A (p21) and BBC3 (PUMA), which clearly play critical roles in p53-mediated cell cycle arrest/senescence and cell death, but which are not mutated in cancer, and for which knockout mice fail to develop spontaneous tumors; and (2) a smaller category of recently-described p53 target genes that are mutated in human cancer, and which appear to be critical for tumor suppression by p53. Interestingly, many of these genes encode proteins that control broad cellular pathways, like splicing and protein degradation, and several of them encode proteins that feed back to regulate p53. These include ZMAT3, GLS2, PADI4, ZBXW7, RFX7, and BTG2. The findings from these studies provide a more complex, but exciting, potential framework for understanding the role of p53 in tumor suppression.

NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in Solitary Fibrous Tumors.

The pathogenesis of many rare tumor types is poorly understood, preventing the design of effective treatments. Solitary fibrous tumors (SFTs) are neoplasms of mesenchymal origin that affect 1/1,000,000 individuals every year and are clinically assimilated to soft tissue sarcomas. SFTs can arise throughout the body and are usually managed surgically. However, 30-40% of SFTs will relapse local-regionally or metastasize. There are no systemic therapies with durable activity for malignant SFTs to date. The molecular hallmark of SFTs is a gene fusion between the NAB2 and STAT6 loci on chromosome 12, resulting in a chimeric protein of poorly characterized function called NAB2-STAT6. We use primary samples and an inducible cell model to discover that NAB2-STAT6 operates as a transcriptional coactivator for a specific set of enhancers and promoters that are normally targeted by the EGR1 transcription factor. In physiological conditions, NAB2 is primarily localized to the cytoplasm and only a small nuclear fraction is available to operate as a co-activator of EGR1 targets. NAB2-STAT6 redirects NAB1, NAB2, and additional EGR1 to the nucleus and bolster the expression of neuronal EGR1 targets. The STAT6 moiety of the fusion protein is a major driver of its nuclear localization and further contributes to NAB2's co-activating abilities. In primary tumors, NAB2-STAT6 activates a neuroendocrine gene signature that sets it apart from most sarcomas. These discoveries provide new insight into the pathogenesis of SFTs and reveal new targets with therapeutic potential.

The African-centric P47S Variant of TP53 Confers Immune Dysregulation and Impaired Response to Immune Checkpoint Inhibition.

The tumor suppressor TP53 is the most frequently mutated gene in cancer and is mutationally inactivated in 50% of sporadic tumors. Inactivating mutations in TP53 also occur in Li Fraumeni syndrome (LFS). In addition to germline mutations in TP53 in LFS that completely inactivate this protein, there are many more germline mutant forms of TP53 in human populations that partially inactivate this protein: we call these partially inactivating mutations "hypomorphs." One of these hypomorphs is a SNP that exists in 6%-10% of Africans and 1%-2% of African Americans, which changes proline at amino acid 47 to serine (Pro47Ser; P47S). We previously showed that the P47S variant of p53 is intrinsically impaired for tumor suppressor function, and that this SNP is associated with increased cancer risk in mice and humans. Here we show that this SNP also influences the tumor microenvironment, and the immune microenvironment profile in P47S mice is more protumorigenic. At basal levels, P47S mice show impaired memory T-cell formation and function, along with increased anti-inflammatory (so-called "M2") macrophages. We show that in tumor-bearing P47S mice, there is an increase in immunosuppressive myeloid-derived suppressor cells and decreased numbers of activated dendritic cells, macrophages, and B cells, along with evidence for increased T-cell exhaustion in the tumor microenvironment. Finally, we show that P47S mice demonstrate an incomplete response to anti-PD-L1 therapy. Our combined data suggest that the African-centric P47S variant leads to both intrinsic and extrinsic defects in tumor suppression. Significance: Findings presented here show that the P47S variant of TP53 influences the immune microenvironment, and the immune response to cancer. This is the first time that a naturally occurring genetic variant of TP53 has been shown to negatively impact the immune microenvironment and the response to immunotherapy.

p53 Oligomerization Domain Mutants: A New Class of Mutants That Retain "License to Kill".

SUMMARY: In this issue of Cancer Discovery, companion articles from the Prives and Lozano groups describe functional analyses of a common dimeric mutant of p53 found in Li-Fraumeni disease and sporadic cancer: A347D (AD). The authors show that the AD mutant is completely defective for canonical p53 transcriptional function, but interestingly retains some tumor suppressor function, which they show is manifested as "neomorphic" activities in transcription and the control of mitochondrial metabolism. See related article by Gencel-Augusto et al., p. 1230 (7). See related article by Choe et al., p. 1250 (6).

An African-Specific Variant of TP53 Reveals PADI4 as a Regulator of p53-Mediated Tumor Suppression.

TP53 is the most frequently mutated gene in cancer, yet key target genes for p53-mediated tumor suppression remain unidentified. Here, we characterize a rare, African-specific germline variant of TP53 in the DNA-binding domain Tyr107His (Y107H). Nuclear magnetic resonance and crystal structures reveal that Y107H is structurally similar to wild-type p53. Consistent with this, we find that Y107H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p53 target genes; this includes the epigenetic modifier PADI4, which deiminates arginine to the nonnatural amino acid citrulline. Surprisingly, we show that Y107H mice develop spontaneous cancers and metastases and that Y107H shows impaired tumor suppression in two other models. We show that PADI4 is itself tumor suppressive and that it requires an intact immune system for tumor suppression. We identify a p53-PADI4 gene signature that is predictive of survival and the efficacy of immune-checkpoint inhibitors. SIGNIFICANCE: We analyze the African-centric Y107H hypomorphic variant and show that it confers increased cancer risk; we use Y107H in order to identify PADI4 as a key tumor-suppressive p53 target gene that contributes to an immune modulation signature and that is predictive of cancer survival and the success of immunotherapy. See related commentary by Bhatta and Cooks, p. 1518. This article is highlighted in the In This Issue feature, p. 1501.

Common activities and predictive gene signature identified for genetic hypomorphs of TP53.

Missense mutations that inactivate p53 occur commonly in cancer, and germline mutations in TP53 cause Li Fraumeni syndrome, which is associated with early-onset cancer. In addition, there are over two hundred germline missense variants of p53 that remain uncharacterized. In some cases, these germline variants have been shown to encode lesser-functioning, or hypomorphic, p53 protein, and these alleles are associated with increased cancer risk in humans and mouse models. However, most hypomorphic p53 variants remain un- or mis-classified in clinical genetics databases. There thus exists a significant need to better understand the behavior of p53 hypomorphs and to develop a functional assay that can distinguish hypomorphs from wild-type p53 or benign variants. We report the surprising finding that two different African-centric genetic hypomorphs of p53 that occur in distinct functional domains of the protein share common activities. Specifically, the Pro47Ser variant, located in the transactivation domain, and the Tyr107His variant, located in the DNA binding domain, both share increased propensity to misfold into a conformation specific for mutant, misfolded p53. Additionally, cells and tissues containing these hypomorphic variants show increased NF-κB activity. We identify a common gene expression signature from unstressed lymphocyte cell lines that is shared between multiple germline hypomorphic variants of TP53, and which successfully distinguishes wild-type p53 and a benign variant from lesser-functioning hypomorphic p53 variants. Our findings will allow us to better understand the contribution of p53 hypomorphs to disease risk and should help better inform cancer risk in the carriers of p53 variants.

PLTP is a p53 target gene with roles in cancer growth suppression and ferroptosis.

The tumor suppressor protein p53 suppresses cancer by regulating processes such as apoptosis, cell cycle arrest, senescence, and ferroptosis, which is an iron-mediated and lipid peroxide-induced cell death pathway. Whereas numerous p53 target genes have been identified, only a few appear to be critical for the suppression of tumor growth. Additionally, while ferroptosis is clearly implicated in tumor suppression by p53, few p53 target genes with roles in ferroptosis have been identified. We have previously studied germline missense p53 variants that are hypomorphic or display reduced activity. These hypomorphic variants are associated with increased risk for cancer, but they retain the majority of p53 transcriptional function; as such, study of the transcriptional targets of these hypomorphs has the potential to reveal the identity of other genes important for p53-mediated tumor suppression. Here, using RNA-seq in lymphoblastoid cell lines, we identify PLTP (phospholipid transfer protein) as a p53 target gene that shows impaired transactivation by three different cancer-associated p53 hypomorphs: P47S (Pro47Ser, rs1800371), Y107H (Tyr107His, rs368771578), and G334R (Gly334Arg, rs78378222). We show that enforced expression of PLTP potently suppresses colony formation in human tumor cell lines. We also demonstrate that PLTP regulates the sensitivity of cells to ferroptosis. Taken together, our findings reveal PLTP to be a p53 target gene that is extremely sensitive to p53 transcriptional function and which has roles in growth suppression and ferroptosis.

Targeting ErbB3 and Cellular NADPH/NADP+ Abundance Sensitizes Cutaneous Melanomas to Ferroptosis Inducers.

Melanoma is a serious health challenge. Ferroptosis is a regulated form of oxidative cell death that shows varied efficacy in melanoma. We aimed to better understand the molecular basis for this differential ferroptosis sensitivity. We find that elevated expression of ErbB3 (V-Erb-B2 Avian Erythroblastic Leukemia Viral Oncogene Homologue 3) associates with ferroptosis resistance and that ErbB3 knockdown sensitizes to ferroptosis inducers. ErbB3 depletion also promotes a marked reduction in the cellular ratio of GSH/GSSG (reduced/oxidized glutathione) and that of NADPH/NADP+ (reduced/oxidized nicotinamide adenine dinucleotide phosphate), together with an increase in the abundance of the lipid peroxidation product malondialdehyde (MDA). We identify several small molecule inhibitors targeting ErbB3 signaling pathways that also reduce the NADPH/NADP+ and GSH/GSSG ratios, concomitantly sensitizing the melanomas to ferroptosis activators. These findings point to a previously unrecognized role of ErbB3 in ferroptosis sensitivity and provide new insight into pathways that regulate this cell death process.

Shifting the paradigms for tumor suppression: lessons from the p53 field.

The TP53 gene continues to hold distinction as the most frequently mutated gene in cancer. Since its discovery in 1979, hundreds of research groups have devoted their efforts toward understanding why this gene is so frequently selected against by tumors, with the hopes of harnessing this information toward the improved therapy of cancer. The result is that this protein has been meticulously analyzed in tumor and normal cells, resulting in over 100,000 publications, with an average of 5000 papers published on p53 every year for the past decade. The journey toward understanding p53 function has been anything but straightforward; in fact, the field is notable for the numerous times that established paradigms not only have been shifted, but in fact have been shattered or reversed. In this review, we will discuss the manuscripts, or series of manuscripts, that have most radically changed our thinking about how this tumor suppressor functions, and we will delve into the emerging challenges for the future in this important area of research. It is hoped that this review will serve as a useful historical reference for those interested in p53, and a useful lesson on the need to be flexible in the face of established paradigms.

P53 regulates cellular redox state, ferroptosis and metabolism.

The tumor protein P53 (TP53, or p53) has complex and at times seemingly contradictory roles in the regulation of metabolism and ferroptosis sensitivity. We find that the actions of p53 influence the redox state, which can trigger changes in redox-sensitive proteins, thereby modifying metabolic processes and response to ferroptosis.

Mutant p53 regulates Survivin to foster lung metastasis.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers worldwide and evolves often to lung metastasis. P53R175H (homologous to Trp53R172H in mice) is a common hot spot mutation. How metastasis is regulated by p53R175H in ESCC remains to be investigated. To investigate p53R175H-mediated molecular mechanisms, we used a carcinogen-induced approach in Trp53R172H/- mice to model ESCC. In the primary Trp53R172H/- tumor cell lines, we depleted Trp53R172H (shTrp53) and observed a marked reduction in cell invasion in vitro and lung metastasis burden in a tail-vein injection model in comparing isogenic cells (shCtrl). Furthermore, we performed bulk RNA-seq to compare gene expression profiles of metastatic and primary shCtrl and shTrp53 cells. We identified the YAP-BIRC5 axis as a potential mediator of Trp53R172H -mediated metastasis. We demonstrate that expression of Survivin, an antiapoptotic protein encoded by BIRC5, increases in the presence of Trp53R172H Furthermore, depletion of Survivin specifically decreases Trp53R172H-driven lung metastasis. Mechanistically, Trp53R172H but not wild-type Trp53, binds with YAP in ESCC cells, suggesting their cooperation to induce Survivin expression. Furthermore, Survivin high expression level is associated with increased metastasis in several GI cancers. Taken together, this study unravels new insights into how mutant p53 mediates metastasis.

HSP70 inhibition blocks adaptive resistance and synergizes with MEK inhibition for the treatment of NRAS-mutant melanoma.

NRAS-mutant melanoma is currently a challenge to treat. This is due to an absence of inhibitors directed against mutant NRAS, along with adaptive and acquired resistance of this tumor type to inhibitors in the MAPK pathway. Inhibitors to MEK (mitogen-activated protein kinase kinase) have shown some promise for NRAS-mutant melanoma. In this work we explored the use of MEK inhibitors for NRAS-mutant melanoma. At the same time we investigated the impact of the brain microenvironment, specifically astrocytes, on the response of a melanoma brain metastatic cell line to MEK inhibition. These parallel avenues led to the surprising finding that astrocytes enhance the sensitivity of melanoma tumors to MEK inhibitors (MEKi). We show that MEKi cause an upregulation of the transcription factor ID3, which confers resistance. This upregulation of ID3 is blocked by conditioned media from astrocytes. We show that silencing ID3 enhances the sensitivity of melanoma to MEK inhibitors, thus mimicking the effect of the brain microenvironment. Moreover, we report that ID3 is a client protein of the chaperone HSP70, and that HSP70 inhibition causes ID3 to misfold and accumulate in a detergent-insoluble fraction in cells. We show that HSP70 inhibitors synergize with MEK inhibitors against NRAS-mutant melanoma, and that this combination significantly enhances the survival of mice in two different models of NRAS-mutant melanoma. These studies highlight ID3 as a mediator of adaptive resistance, and support the combined use of MEK and HSP70 inhibitors for the therapy of NRAS-mutant melanoma. SIGNIFICANCE: MEK inhibitors are currently used for NRAS-mutant melanoma, but have shown modest efficacy as single agents. This research shows a synergistic effect of combining HSP70 inhibitors with MEK inhibitors for the treatment of NRAS mutant melanoma.

IspH inhibitors kill Gram-negative bacteria and mobilize immune clearance.

Isoprenoids are vital for all organisms, in which they maintain membrane stability and support core functions such as respiration1. IspH, an enzyme in the methyl erythritol phosphate pathway of isoprenoid synthesis, is essential for Gram-negative bacteria, mycobacteria and apicomplexans2,3. Its substrate, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), is not produced in metazoans, and in humans and other primates it activates cytotoxic Vγ9Vδ2 T cells at extremely low concentrations4-6. Here we describe a class of IspH inhibitors and refine their potency to nanomolar levels through structure-guided analogue design. After modification of these compounds into prodrugs for delivery into bacteria, we show that they kill clinical isolates of several multidrug-resistant bacteria-including those from the genera Acinetobacter, Pseudomonas, Klebsiella, Enterobacter, Vibrio, Shigella, Salmonella, Yersinia, Mycobacterium and Bacillus-yet are relatively non-toxic to mammalian cells. Proteomic analysis reveals that bacteria treated with these prodrugs resemble those after conditional IspH knockdown. Notably, these prodrugs also induce the expansion and activation of human Vγ9Vδ2 T cells in a humanized mouse model of bacterial infection. The prodrugs we describe here synergize the direct killing of bacteria with a simultaneous rapid immune response by cytotoxic γδ T cells, which may limit the increase of antibiotic-resistant bacterial populations.

Identification of Mubritinib (TAK 165) as an inhibitor of KSHV driven primary effusion lymphoma via disruption of mitochondrial OXPHOS metabolism.

KSHV-associated cancers have poor prognoses and lack therapeutics that selectively target viral gene functions. We developed a screening campaign to identify known drugs that could be repurposed for the treatment of KSHV-associated cancers. We focused on primary effusion lymphoma (PEL), which has particularly poor treatment outcomes. We developed a luciferase reporter assay to test the ability of drugs to inhibit DNA binding of the KSHV LANA DNA binding domain (DBD). In parallel, we screened drugs for selective inhibition of a KSHV+ PEL cells. While potent hits were identified in each assay, only one hit, Mubritinib, was found to score in both assays. Mubritinib caused PEL cells to undergo cell cycle arrest with accumulation of sub-G1 population and Annexin V. Mubritinib inhibited LANA binding to KSHV terminal repeat (TR) DNA in KSHV+ PEL cells, but did not lead to KSHV lytic cycle reactivation. Mubritinib was originally identified as a receptor tyrosine kinase (RTK) inhibitor selective for HER2/ErbB2. But recent studies have revealed that Mubritinib can also inhibit the electron transport chain (ETC) complex at nanomolar concentrations. We found that other related ETC complex inhibitors (Rotenone and Deguelin) exhibited PEL cell growth inhibition while RTK inhibitors failed. Seahorse analysis demonstrated that Mubritinib selectively inhibits the maximal oxygen consumption (OCR) in PEL cells and metabolomics revealed changes in ATP/ADP and ATP/AMP ratios. These findings indicate that PEL cells are selectively sensitive to ETC complex inhibitors and provide a rationale for repurposing Mubritinib for selective treatment of PEL.

Increased mTOR activity and metabolic efficiency in mouse and human cells containing the African-centric tumor-predisposing p53 variant Pro47Ser.

The Pro47Ser variant of p53 (S47) exists in African-descent populations and is associated with increased cancer risk in humans and mice. Due to impaired repression of the cystine importer Slc7a11, S47 cells show increased glutathione (GSH) accumulation compared to cells with wild -type p53. We show that mice containing the S47 variant display increased mTOR activity and oxidative metabolism, as well as larger size, improved metabolic efficiency, and signs of superior fitness. Mechanistically, we show that mTOR and its positive regulator Rheb display increased association in S47 cells; this is due to an altered redox state of GAPDH in S47 cells that inhibits its ability to bind and sequester Rheb. Compounds that decrease glutathione normalize GAPDH-Rheb complexes and mTOR activity in S47 cells. This study reveals a novel layer of regulation of mTOR by p53, and raises the possibility that this variant may have been selected for in early Africa.

Functional interplay among thiol-based redox signaling, metabolism, and ferroptosis unveiled by a genetic variant of TP53.

The p53 tumor suppressor protein is a transcription factor and master stress response mediator, and it is subject to reduction-oxidation (redox)-dependent regulation. The P47S variant of TP53, which exists primarily in African-descent populations, associates with an elevated abundance of low molecular weight (LMW) thiols, including glutathione (GSH) and coenzyme A (CoA). Here we show that S47 and P47 cells exhibit distinct metabolic profiles, controlled by their different redox states and expression of Activating Transcription Factor-4 (ATF4). We find that S47 cells exhibit decreased catabolic glycolysis but increased use of the pentose phosphate pathway (PPP), and an enhanced abundance of the antioxidant, NADPH. We identify ATF4 as differentially expressed in P47 and S47 cells and show that ATF4 can reverse the redox status and rescue metabolism of S47 cells, as well as increase sensitivity to ferroptosis. This adaptive metabolic switch is rapid, reversible, and accompanied by thiol-mediated changes in the structures and activities of key glycolytic signaling pathway proteins, including GAPDH and G6PD. The results presented here unveil the important functional interplay among pathways regulating thiol-redox status, metabolic adaptation, and cellular responses to oxidative stress.

A Novel Inhibitor of HSP70 Induces Mitochondrial Toxicity and Immune Cell Recruitment in Tumors.

The protein chaperone HSP70 is overexpressed in many cancers including colorectal cancer, where overexpression is associated with poor survival. We report here the creation of a uniquely acting HSP70 inhibitor (HSP70i) that targets multiple compartments in the cancer cell, including mitochondria. This inhibitor was mitochondria toxic and cytotoxic to colorectal cancer cells, but not to normal colon epithelial cells. Inhibition of HSP70 was efficacious as a single agent in primary and metastatic models of colorectal cancer and enabled identification of novel mitochondrial client proteins for HSP70. In a syngeneic colorectal cancer model, the inhibitor increased immune cell recruitment into tumors. Cells treated with the inhibitor secreted danger-associated molecular patterns (DAMP), including ATP and HMGB1, and functioned effectively as a tumor vaccine. Interestingly, the unique properties of this HSP70i in the disruption of mitochondrial function and the inhibition of proteostasis both contributed to DAMP release. This HSP70i constitutes a promising therapeutic opportunity in colorectal cancer and may exhibit antitumor activity against other tumor types. SIGNIFICANCE: These findings describe a novel HSP70i that disrupts mitochondrial proteostasis, demonstrating single-agent efficacy that induces immunogenic cell death in treated tumors.

A Rare TP53 Mutation Predominant in Ashkenazi Jews Confers Risk of Multiple Cancers.

Germline mutations in TP53 cause a rare high penetrance cancer syndrome, Li-Fraumeni syndrome (LFS). Here, we identified a rare TP53 tetramerization domain missense mutation, c.1000G>C;p.G334R, in a family with multiple late-onset LFS-spectrum cancers. Twenty additional c.1000G>C probands and one c.1000G>A proband were identified, and available tumors showed biallelic somatic inactivation of TP53. The majority of families were of Ashkenazi Jewish descent, and the TP53 c.1000G>C allele was found on a commonly inherited chromosome 17p13.1 haplotype. Transient transfection of the p.G334R allele conferred a mild defect in colony suppression assays. Lymphoblastoid cell lines from the index family in comparison with TP53 normal lines showed that although classical p53 target gene activation was maintained, a subset of p53 target genes (including PCLO, PLTP, PLXNB3, and LCN15) showed defective transactivation when treated with Nutlin-3a. Structural analysis demonstrated thermal instability of the G334R-mutant tetramer, and the G334R-mutant protein showed increased preponderance of mutant conformation. Clinical case review in comparison with classic LFS cohorts demonstrated similar rates of pediatric adrenocortical tumors and other LFS component cancers, but the latter at significantly later ages of onset. Our data show that TP53 c.1000G>C;p.G334R is found predominantly in Ashkenazi Jewish individuals, causes a mild defect in p53 function, and leads to low penetrance LFS. SIGNIFICANCE: TP53 c.1000C>G;p.G334R is a pathogenic, Ashkenazi Jewish-predominant mutation associated with a familial multiple cancer syndrome in which carriers should undergo screening and preventive measures to reduce cancer risk.