Integrins and mutant p53 on the road to metastasis.

Understanding how tumor cells invade tissues is key to developing drugs to block metastasis. In this issue, Muller et al. (2009) report that a mutant form of the tumor suppressor p53 in cancer cells boosts the endocytic recycling of the adhesion molecule integrin alpha5beta1 and of epidermal growth factor receptor, promoting invasion and metastasis.

Mutant p53 gain of function mediates cancer immune escape that is counteracted by APR-246.

BACKGROUND: p53 mutants contribute to the chronic inflammatory tumour microenvironment (TME). In this study, we address the mechanism of how p53 mutants lead to chronic inflammation in tumours and how to transform it to restore cancer immune surveillance. METHODS: Our analysis of RNA-seq data from The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) project revealed that mutant p53 (mtp53) cancers correlated with chronic inflammation. We used cell-based assays and a mouse model to discover a novel gain of function of mtp53 and the effect of the mtp53 reactivating compound APR-246 on the anti-tumour immune response. RESULTS: We found that tumour samples from patients with breast carcinoma carrying mtp53 showed elevated Interferon (IFN) signalling, Tumour Inflammation Signature (TIS) score and infiltration of CD8+ T cells compared to wild type p53 (wtp53) tumours. We showed that the expression of IFN and immune checkpoints were elevated in tumour cells in a mtp53-dependent manner, suggesting a novel gain of function. Restoration of wt function to mtp53 by APR-246 induced the expression of endogenous retroviruses, IFN signalling and repressed immune checkpoints. Moreover, APR-246 promoted CD4+ T cells infiltration and IFN signalling and prevented CD8+ T cells exhaustion within the TME in vivo. CONCLUSIONS: Breast carcinomas with mtp53 displayed enhanced inflammation. APR-246 boosted the interferon response or represses immune checkpoints in p53 mutant tumour cells, and restores cancer immune surveillance in vivo.

Identification and targeting of selective vulnerability rendered by tamoxifen resistance.

BACKGROUND: The estrogen receptor (ER)-positive breast cancer represents over 80% of all breast cancer cases. Even though adjuvant hormone therapy with tamoxifen (TMX) is saving lives of patients with ER-positive breast cancer, the acquired resistance to TMX anti-estrogen therapy is the main hurdle for successful TMX therapy. Here we address the mechanism for TMX resistance and explore the ways to eradicate TMX-resistant breast cancer in both in vitro and ex vivo experiments. EXPERIMENTAL DESIGN: To identify compounds able to overcome TMX resistance, we used short-term and long-term viability assays in cancer cells in vitro and in patient samples in 3D ex vivo, analysis of gene expression profiles and cell line pharmacology database, shRNA screen, CRISPR-Cas9 genome editing, real-time PCR, immunofluorescent analysis, western blot, measurement of oxidative stress using flow cytometry, and thioredoxin reductase 1 enzymatic activity. RESULTS: Here, for the first time, we provide an ample evidence that a high level of the detoxifying enzyme SULT1A1 confers resistance to TMX therapy in both in vitro and ex vivo models and correlates with TMX resistance in metastatic samples in relapsed patients. Based on the data from different approaches, we identified three anticancer compounds, RITA (Reactivation of p53 and Induction of Tumor cell Apoptosis), aminoflavone (AF), and oncrasin-1 (ONC-1), whose tumor cell inhibition activity is dependent on SULT1A1. We discovered thioredoxin reductase 1 (TrxR1, encoded by TXNRD1) as a target of bio-activated RITA, AF, and ONC-1. SULT1A1 depletion prevented the inhibition of TrxR1, induction of oxidative stress, DNA damage signaling, and apoptosis triggered by the compounds. Notably, RITA efficiently suppressed TMX-unresponsive patient-derived breast cancer cells ex vivo. CONCLUSION: We have identified a mechanism of resistance to TMX via hyperactivated SULT1A1, which renders selective vulnerability to anticancer compounds RITA, AF, and ONC-1, and provide a rationale for a new combination therapy to overcome TMX resistance in breast cancer patients. Our novel findings may provide a strategy to circumvent TMX resistance and suggest that this approach could be developed further for the benefit of relapsed breast cancer patients.

Therapeutic targeting of mutant p53 in pediatric acute lymphoblastic leukemia.

Alterations of the tumor suppressor gene TP53 are found in different cancers, in particular in carcinomas of adults. In pediatric acute lymphoblastic leukemia (ALL), TP53 mutations are infrequent but enriched at relapse. As in most cancers, mainly DNA-binding domain missense mutations are found, resulting in accumulation of mutant p53, poor therapy response, and inferior outcome. Different strategies to target mutant p53 have been developed including reactivation of p53's wildtype function by the small molecule APR-246. We investigated TP53 mutations in cell lines and 62 B-cell precursor ALL samples and evaluated the activity of APR-246 in TP53-mutated or wildtype ALL. We identified cases with TP53 missense mutations, high (mutant) p53 expression and insensitivity to the DNA-damaging agent doxorubicin. In TP53-mutated ALL, APR-246 induced apoptosis showing strong anti-leukemia activity. APR-246 restored mutant p53 to its wildtype conformation, leading to pathway activation with induction of transcriptional targets and re-sensitization to genotoxic therapy in vitro and in vivo In addition, induction of oxidative stress contributed to APR-246-mediated cell death. In a preclinical model of patient-derived TP53-mutant ALL, APR-246 reduced leukemia burden and synergized strongly with the genotoxic agent doxorubicin, leading to superior leukemia-free survival in vivo Thus, targeting mutant p53 by APR-246, restoring its tumor suppressive function, seems to be an effective therapeutic strategy for this high-risk group of TP53-mutant ALL.

Modulation of the poly (ADP-ribose) polymerase inhibitor response and DNA recombination in breast cancer cells by drugs affecting endogenous wild-type p53.

Synthetic lethal interactions between poly (ADP-ribose) polymerase (PARP) and homologous recombination (HR) repair pathways have been exploited for the development of novel mono- and combination cancer therapies. The tumor suppressor p53 was demonstrated to exhibit indirect and direct regulatory activities in DNA repair, particularly in DNA double-strand break (DSB)-induced and replication-associated HR. In this study, we tested a potential influence of the p53 status on the response to PARP inhibition, which is known to cause replication stress. Silencing endogenous or inducibly expressing p53 we found a protective effect of p53 on PARP inhibitor (PARPi)-mediated cytotoxicities. This effect was specific for wild-type versus mutant p53 and observed in cancer but not in non-transformed cell lines. Enhanced cytotoxicities after treatment with the p53-inhibitory drug Pifithrinα further supported p53-mediated resistance to PARP inhibition. Surprisingly, we equally observed increased PARPi sensitivity in the presence of the p53-activating compound Nutlin-3. As a common denominator, both drug responses correlated with decreased HR activities: Pifithrinα downregulated spontaneous HR resulting in damage accumulation. Nutlin-3 induced a decrease of DSB-induced HR, which was accompanied by a severe drop in RAD51 protein levels. Thus, we revealed a novel link between PARPi responsiveness and p53-controlled HR activities. These data expand the concept of cell and stress type-dependent healer and killer functions of wild-type p53 in response to cancer therapeutic treatment. Our findings have implications for the individualized design of cancer therapies using PARPi and the potentially combined use of p53-modulatory drugs.

Translating p53-based therapies for cancer into the clinic.

Inactivation of the most important tumour suppressor gene TP53 occurs in most, if not all, human cancers. Loss of functional wild-type p53 is achieved via two main mechanisms: mutation of the gene leading to an absence of tumour suppressor activity and, in some cases, gain-of-oncogenic function; or inhibition of the wild-type p53 protein mediated by overexpression of its negative regulators MDM2 and MDMX. Because of its high potency as a tumour suppressor and the dependence of at least some established tumours on its inactivation, p53 appears to be a highly attractive target for the development of new anticancer drugs. However, p53 is a transcription factor and therefore has long been considered undruggable. Nevertheless, several innovative strategies have been pursued for targeting dysfunctional p53 for cancer treatment. In mutant p53-expressing tumours, the predominant strategy is to restore tumour suppressor function with compounds acting either in a generic manner or otherwise selective for one or a few specific p53 mutations. In addition, approaches to deplete mutant p53 or to target vulnerabilities created by mutant p53 expression are currently under development. In wild-type p53 tumours, the major approach is to protect p53 from the actions of MDM2 and MDMX by targeting these negative regulators with inhibitors. Although the results of at least some clinical trials of MDM2 inhibitors and mutant p53-restoring compounds are promising, none of the agents has yet been approved by the FDA. Alternative strategies, based on a better understanding of p53 biology, the mechanisms of action of compounds and treatment regimens as well as the development of new technologies are gaining interest, such as proteolysis-targeting chimeras for MDM2 degradation. Other approaches are taking advantage of the progress made in immune-based therapies for cancer. In this Review, we present these ongoing clinical trials and emerging approaches to re-evaluate the current state of knowledge of p53-based therapies for cancer.

Protein kinase Cα (PKCα) regulates p53 localization and melanoma cell survival downstream of integrin αv in three-dimensional collagen and in vivo.

Protein kinase C α (PKCα) is overexpressed in numerous types of cancer. Importantly, PKCα has been linked to metastasis of malignant melanoma in patients. However, it has been unclear how PKCα may be regulated and how it exerts its role in melanoma. Here, we identified a role for PKCα in melanoma cell survival in a three-dimensional collagen model mimicking the in vivo pathophysiology of the dermis. A pathway was identified that involved integrin αv-mediated up-regulation of PKCα and PKCα-dependent regulation of p53 localization, which was connected to melanoma cell survival. Melanoma survival and growth in three-dimensional microenvironments requires the expression of integrin αv, which acts to suppress p53 activity. Interestingly, microarray analysis revealed that PKCα was up-regulated by integrin αv in a three-dimensional microenvironment-dependent manner. Integrin αv was observed to promote a relocalization of endogenous p53 from the nucleus to the cytoplasm upon growth in three-dimensional collagen as well as in vivo, whereas stable knockdown of PKCα inhibited the integrin αv-mediated relocalization of p53. Importantly, knockdown of PKCα also promoted apoptosis in three-dimensional collagen and in vivo, resulting in reduced tumor growth. This indicates that PKCα constitutes a crucial component of the integrin αv-mediated pathway(s) that promote p53 relocalization and melanoma survival.

MDM2-PROTAC versus MDM2 Inhibitors: Beyond p53 Reactivation.

SUMMARY: In this issue of Cancer Discovery, Adams and colleagues present the discovery of a potent PROTAC, MDM2 degrader, which activates wild-type p53 leading to cancer cell death. Importantly, in a number of in vitro and in vivo experiments, the authors show that the depletion of MDM2 by PROTAC kills p53-mutant or p53-null cancer cells. See related article by Adams et al., p. 1210 (5).

Inhibition of glycolytic enzymes mediated by pharmacologically activated p53: targeting Warburg effect to fight cancer.

Unique sensitivity of tumor cells to the inhibition of glycolysis is a good target for anticancer therapy. Here, we demonstrate that the pharmacologically activated tumor suppressor p53 mediates the inhibition of glycolytic enzymes in cancer cells in vitro and in vivo. We showed that p53 binds to the promoters of metabolic genes and represses their expression, including glucose transporters SLC2A12 (GLUT12) and SLC2A1 (GLUT1). Furthermore, p53-mediated repression of transcription factors c-Myc and HIF1α, key drivers of ATP-generating pathways in tumors, contributed to ATP production block. Inhibition of c-Myc by p53 mediated the ablation of several glycolytic genes in normoxia, whereas in hypoxia down-regulation of HIF1α contributed to this effect. We identified Sp1 as a transcription cofactor cooperating with p53 in the ablation of metabolic genes. Using different approaches, we demonstrated that glycolysis block contributes to the robust induction of apoptosis by p53 in cancer cells. Taken together, our data suggest that tumor-specific reinstatement of p53 function targets the "Achilles heel" of cancer cells (i.e. their dependence on glycolysis), which could contribute to the tumor-selective killing of cancer cells by pharmacologically activated p53.

Therapeutic targeting of p53 by small molecules.

Aberrant p53 function is one of the major requirements for tumor development. Reactivation of p53 function by small molecules is a promising strategy to combat cancer due to potent tumor suppressor activities of p53. Recent developments in p53 biology reveal that manipulation of p53 function might pave way to a long cancer-free life. A number of small molecules which rescue p53 function by different mechanisms, acting upstream of p53 or targeting the p53 protein itself have been identified. Notably, these molecules trigger different biological outcomes, suggesting that it might be feasible to direct p53-mediated response in a desired way. In this review I discuss the latest developments in the search for small molecules which rescue p53 function by targeting the p53 protein.

Decreased DNA Damage and Improved p53 Specificity of RITA Analogs.

Reactivation of p53 tumor-suppressor function by small molecules is an attractive strategy to defeat cancer. A potent p53-reactivating molecule RITA, which triggers p53-dependent apoptosis in human tumor cells in vitro and in vivo, exhibits p53-independent cytotoxicity due to modifications by detoxification enzyme Sulfotransferase 1A1 (SULT1A1), producing a reactive carbocation. Several synthetic modifications to RITA's heterocyclic scaffold lead to higher energy barriers for carbocation formation. In this study, we addressed the question whether RITA analogs NSC777196 and NSC782846 can induce p53-dependent apoptosis without SULT1A1-dependent DNA damage. We found that RITA analog NSC782846, but not NSC777196, induced p53-regulated genes, targeted oncogene addiction, and killed cancer cells upon p53 reactivation, but without induction of DNA damage and inhibition RNA pol II. Our results might demonstrate a method for designing more specific and potent RITA analogs to accelerate translation of p53-targeting compounds from laboratory bench to clinic.

Enterobacteria impair host p53 tumor suppressor activity through mRNA destabilization.

Increasing evidence highlights the role of bacteria in the physiopathology of cancer. However, the underlying molecular mechanisms remains poorly understood. Several cancer-associated bacteria have been shown to produce toxins which interfere with the host defense against tumorigenesis. Here, we show that lipopolysaccharides from Klebsiella pneumoniae and other Enterobacteria strongly inhibit the host tumor suppressor p53 pathway through a novel mechanism of p53 regulation. We found that lipopolysaccharides destabilize TP53 mRNA through a TLR4-NF-κB-mediated inhibition of the RNA-binding factor Wig-1. Importantly, we show that K. pneumoniae disables two major tumor barriers, oncogene-induced DNA damage signaling and senescence, by impairing p53 transcriptional activity upon DNA damage and oncogenic stress. Furthermore, we found an inverse correlation between the levels of TLR4 and p53 mutation in colorectal tumors. Hence, our data suggest that the repression of p53 by Enterobacteria via TLR4 alleviates the selection pressure for p53 oncogenic mutations and shapes the genomic evolution of cancer.

Novel Allosteric Mechanism of Dual p53/MDM2 and p53/MDM4 Inhibition by a Small Molecule.

Restoration of the p53 tumor suppressor for personalised cancer therapy is a promising treatment strategy. However, several high-affinity MDM2 inhibitors have shown substantial side effects in clinical trials. Thus, elucidation of the molecular mechanisms of action of p53 reactivating molecules with alternative functional principle is of the utmost importance. Here, we report a discovery of a novel allosteric mechanism of p53 reactivation through targeting the p53 N-terminus which promotes inhibition of both p53/MDM2 (murine double minute 2) and p53/MDM4 interactions. Using biochemical assays and molecular docking, we identified the binding site of two p53 reactivating molecules, RITA (reactivation of p53 and induction of tumor cell apoptosis) and protoporphyrin IX (PpIX). Ion mobility-mass spectrometry revealed that the binding of RITA to serine 33 and serine 37 is responsible for inducing the allosteric shift in p53, which shields the MDM2 binding residues of p53 and prevents its interactions with MDM2 and MDM4. Our results point to an alternative mechanism of blocking p53 interaction with MDM2 and MDM4 and may pave the way for the development of novel allosteric inhibitors of p53/MDM2 and p53/MDM4 interactions.

Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound.

The tumor suppressor p53 inhibits tumor growth primarily through its ability to induce apoptosis. Mutations in p53 occur in at least 50% of human tumors. We hypothesized that reactivation of mutant p53 in such tumors should trigger massive apoptosis and eliminate the tumor cells. To test this, we screened a library of low-molecular-weight compounds in order to identify compounds that can restore wild-type function to mutant p53. We found one compound capable of inducing apoptosis in human tumor cells through restoration of the transcriptional transactivation function to mutant p53. This molecule, named PRIMA-1, restored sequence-specific DNA binding and the active conformation to mutant p53 proteins in vitro and in living cells. PRIMA-1 rescued both DNA contact and structural p53 mutants. In vivo studies in mice revealed an antitumor effect with no apparent toxicity. This molecule may serve as a lead compound for the development of anticancer drugs targeting mutant p53.

Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors.

In tumors that retain wild-type p53, its tumor-suppressor function is often impaired as a result of the deregulation of HDM-2, which binds to p53 and targets it for proteasomal degradation. We have screened a chemical library and identified a small molecule named RITA (reactivation of p53 and induction of tumor cell apoptosis), which bound to p53 and induced its accumulation in tumor cells. RITA prevented p53-HDM-2 interaction in vitro and in vivo and affected p53 interaction with several negative regulators. RITA induced expression of p53 target genes and massive apoptosis in various tumor cells lines expressing wild-type p53. RITA suppressed the growth of human fibroblasts and lymphoblasts only upon oncogene expression and showed substantial p53-dependent antitumor effect in vivo. RITA may serve as a lead compound for the development of an anticancer drug that targets tumors with wild-type p53.

p53-Dependent Repression: DREAM or Reality?

p53 is a major tumor suppressor that integrates diverse types of signaling in mammalian cells. In response to a broad range of intra- or extra-cellular stimuli, p53 controls the expression of multiple target genes and elicits a vast repertoire of biological responses. The exact code by which p53 integrates the various stresses and translates them into an appropriate transcriptional response is still obscure. p53 is tightly regulated at multiple levels, leading to a wide diversity in p53 complexes on its target promoters and providing adaptability to its transcriptional program. As p53-targeted therapies are making their way into clinics, we need to understand how to direct p53 towards the desired outcome (i.e., cell death, senescence or other) selectively in cancer cells without affecting normal tissues or the immune system. While the core p53 transcriptional program has been proposed, the mechanisms conferring a cell type- and stimuli-dependent transcriptional outcome by p53 require further investigations. The mechanism by which p53 localizes to repressed promoters and manages its co-repressor interactions is controversial and remains an important gap in our understanding of the p53 cistrome. We hope that our review of the recent literature will help to stimulate the appreciation and investigation of largely unexplored p53-mediated repression.

Pharmacologic Activation of p53 Triggers Viral Mimicry Response Thereby Abolishing Tumor Immune Evasion and Promoting Antitumor Immunity.

The repression of repetitive elements is an important facet of p53's function as a guardian of the genome. Paradoxically, we found that p53 activated by MDM2 inhibitors induced the expression of endogenous retroviruses (ERV) via increased occupancy on ERV promoters and inhibition of two major ERV repressors, histone demethylase LSD1 and DNA methyltransferase DNMT1. Double-stranded RNA stress caused by ERVs triggered type I/III interferon expression and antigen processing and presentation. Pharmacologic activation of p53 in vivo unleashed the IFN program, promoted T-cell infiltration, and significantly enhanced the efficacy of checkpoint therapy in an allograft tumor model. Furthermore, the MDM2 inhibitor ALRN-6924 induced a viral mimicry pathway and tumor inflammation signature genes in patients with melanoma. Our results identify ERV expression as the central mechanism whereby p53 induction overcomes tumor immune evasion and transforms tumor microenvironment to a favorable phenotype, providing a rationale for the synergy of MDM2 inhibitors and immunotherapy. SIGNIFICANCE: We found that p53 activated by MDM2 inhibitors induced the expression of ERVs, in part via epigenetic factors LSD1 and DNMT1. Induction of IFN response caused by ERV derepression upon p53-targeting therapies provides a possibility to overcome resistance to immune checkpoint blockade and potentially transform "cold" tumors into "hot." This article is highlighted in the In This Issue feature, p. 2945.

Tumor-specific induction of apoptosis by a p53-reactivating compound.

The tumor suppressor function of p53 is disabled in the majority of tumors, either by a point mutation of the p53 gene, or via MDM2-dependent proteasomal degradation. We have screened a chemical library using a cell-based assay and identified a low molecular weight compound named MITA which induced wild-type p53-dependent cell death in a variety of different types of human tumor cells, such as lung, colon and breast carcinoma cells, as well as in osteosarcoma and fibrosarcoma-derived cells. MITA inhibited p53-MDM2 interaction in vitro and in cells, which in turn prevented MDM2-mediated ubiquitination of p53 and resulted in a prolonged half-life and accumulation of p53 in tumor cells. Notably, p53 induction by MITA resulted in upregulated expression of p53 target genes MDM2, Bax, Gadd45 and PUMA, on protein and mRNA level. Importantly, neither p53 nor these target genes were induced in normal human fibroblasts (HDFs), which correlated with the absence of growth suppression in fibroblasts after treatment with MITA. However, upon activation of oncogenes in fibroblasts an induction and activation of p53 was observed, suggesting that activation of p53 by MITA occurs predominantly in tumor cells.

Pifithrin-α alters p53 post-translational modifications pattern and differentially inhibits p53 target genes.

Pifithrin-α (PFT-α) is a small molecule which has been widely used as a specific inhibitor of p53 transcription activity. However, its molecular mechanism of action remains unclear. PFT-α has also been described to display potent p53-independent activity in cells. In this study, we addressed the mechanism of action of PFT-α. We found that PFT-α failed to prevent the effects of Mdm2 inhibitor Nutlin-3 on cell cycle and apoptosis in several cancer cell lines. However, PFT-α rescued normal primary fibroblasts from growth inhibition by Nutlin-3. PFT-α displayed a very limited effect on p53-dependent transcription upon its activation by Nutlin-3. Moreover, PFT-α inhibitory effect on transcription was highly dependent on the nature of the p53 target gene. PFT-α attenuated post-translational modifications of p53 without affecting total p53 protein level. Finally, we found that PFT-α can decrease the level of intracellular reactive oxygen species through activation of an aryl hydrocarbon receptor (AHR)-Nrf2 axis in a p53-independent manner. In conclusion, PFT-α inhibits only some aspects of p53 function, therefore it should be used with extreme caution to study p53-dependent processes.