Mutant P53 Gain Of Function Research Articles

A small-molecule inhibitor of TopBP1 exerts anti-MYC activity and synergy with PARP inhibitors.

We have previously identified TopBP1 (topoisomerase IIβ-binding protein 1) as a promising target for cancer therapy, given its role in the convergence of Rb, PI(3)K/Akt, and p53 pathways. Based on this, we conducted a large-scale molecular docking screening to identify a small-molecule inhibitor that specifically targets the BRCT7/8 domains of TopBP1, which we have named 5D4. Our studies show that 5D4 inhibits TopBP1 interactions with E2F1, mutant p53, and Cancerous Inhibitor of Protein Phosphatase 2A. This leads to the activation of E2F1-mediated apoptosis and the inhibition of mutant p53 gain of function. In addition, 5D4 disrupts the interaction of TopBP1 with MIZ1, which in turn allows MIZ1 to bind to its target gene promoters and repress MYC activity. Moreover, 5D4 inhibits the association of the TopBP1-PLK1 complex and prevents the formation of Rad51 foci. When combined with inhibitors of PARP1/2 or PARP14, 5D4 synergizes to effectively block cancer cell proliferation. Our animal studies have demonstrated the antitumor activity of 5D4 in breast and ovarian cancer xenograft models. Moreover, the effectiveness of 5D4 is further enhanced when combined with a PARP1/2 inhibitor talazoparib. Taken together, our findings strongly support the potential use of TopBP1-BRCT7/8 inhibitors as a targeted cancer therapy.

Gain-of-function mutant p53 together with ERG proto-oncogene drive prostate cancer by beta-catenin activation and pyrimidine synthesis

Whether TMPRSS2-ERG fusion and TP53 gene alteration coordinately promote prostate cancer (PCa) remains unclear. Here we demonstrate that TMPRSS2-ERG fusion and TP53 mutation / deletion co-occur in PCa patient specimens and this co-occurrence accelerates prostatic oncogenesis. p53 gain-of-function (GOF) mutants are now shown to bind to a unique DNA sequence in the CTNNB1 gene promoter and transactivate its expression. ERG and β-Catenin co-occupy sites at pyrimidine synthesis gene (PSG) loci and promote PSG expression, pyrimidine synthesis and PCa growth. β-Catenin inhibition by small molecule inhibitors or oligonucleotide-based PROTAC suppresses TMPRSS2-ERG- and p53 mutant-positive PCa cell growth in vitro and in mice. Our study identifies a gene transactivation function of GOF mutant p53 and reveals β-Catenin as a transcriptional target gene of p53 GOF mutants and a driver and therapeutic target of TMPRSS2-ERG- and p53 GOF mutant-positive PCa.

Abstract 2585: Mutant p53 gains oncogenic functions through a cytosolic DNA response

Abstract TP53 mutations are the most common cancer driver mutations among all cancers. Although some TP53 mutations lead to a loss of function of wild-type p53, many other TP53 mutations confer gain-of-function (GOF) activities, which promote cancer cell metastasis and pro-tumorigenic inflammation. Despite that many functional models of GOF mutant p53 (mutp53) have been proposed previously, the mechanisms involved in mutp53 GOF still remain largely elusive. Here we show that by directly targeting minichromosome maintenance complex component 5 (MCM5), a component of the hexametric DNA helicase MCM2-7 complex, GOF mutp53 predisposes cancer cells to replication stress and chromosomal instability, which leads to a tumor cell-autonomous and stimulator of interferon genes (STING)-dependent cytosolic DNA response that activates downstream non-canonical nuclear factor kappa light chain enhancer of activated B cell (NC-NF-κB) signaling. Furthermore, our results demonstrate that GOF mutp53-activated tumor cell-intrinsic STING-NC-NF-κB signaling not only stimulates tumor cell metastasis, but also promotes tumor immune resistance through fostering an immunosuppressive tumor microenvironment. Therefore, our findings that mutp53 exerts its GOF role through pro-tumorigenic MCM5-CIN-STING-NC-NF-κB signaling highlight the importance of TP53 and its inactivation in cancer genome evolution of genomic instability that drives tumor development and progression. Citation Format: Mei Zhao, Tianxiao Wang, Frederico O. Gleber-Netto, Zhen Chen, Daniel J. McGrail, Javier A. Gomez, Wutong Ju, Mayur A. Gadhikar, Wencai Ma, Li Shen, Ximing Tang, Sen Pathak, Maria G. Raso, Jared Burks, Shiaw-Yih Lin, Jing Wang, Asha S. Multani, Curtis R. Pickering, Junjie Chen, Jeffrey N. Myers, Ge Zhou. Mutant p53 gains oncogenic functions through a cytosolic DNA response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2585.

The ubiquitin ligase TRIM21 regulates mutant p53 accumulation and gain of function in cancer.

The tumor suppressor TP53 is the most frequently mutated gene in human cancers. Mutant p53 (mutp53) proteins often accumulate to very high levels in human cancers to promote cancer progression through the gain-of-function (GOF) mechanism. Currently, the mechanism underlying mutp53 accumulation and GOF is incompletely understood. Here, we identified TRIM21 as a critical E3 ubiquitin ligase of mutp53 by screening for specific mutp53-interacting proteins. TRIM21 directly interacted with mutp53 but not WT p53, resulting in ubiquitination and degradation of mutp53 to suppress mutp53 GOF in tumorigenesis. TRIM21 deficiency in cancer cells promoted mutp53 accumulation and GOF in tumorigenesis. Compared with p53R172H knockin mice, which displayed mutp53 accumulation specifically in tumors but not normal tissues, TRIM21 deletion in p53R172H knockin mice resulted in mutp53 accumulation in normal tissues, an earlier tumor onset, and a shortened life span of mice. Furthermore, TRIM21 was frequently downregulated in some human cancers, including colorectal and breast cancers, and low TRIM21 expression was associated with poor prognosis in patients with cancers carrying mutp53. Our results revealed a critical mechanism underlying mutp53 accumulation in cancers and also uncovered an important tumor-suppressive function of TRIM21 and its mechanism in cancers carrying mutp53.

PP01.32 Proteasome Hyperactivity as a Targetable Vulnerability in Oncogenic Missense TP53 Mutated NSCLC

Despite advances in targeted approaches and immunotherapy, lung cancer remains the leading cause of cancer mortality in the United States and worldwide. This is partly because these novel treatments are not applicable to all patients and are often associated with primary or secondary resistance. This highlights the need for continued search for new therapeutic strategies for patients with non-small cell lung cancer (NSCLC). Gain-of-function (GOF) oncogenic TP53 mutations, which are present in 20-30% of NSCLC, are harbingers of poorer prognosis and subdued responses to therapy. To date, strategies aimed at directly targeting mutant p53 have not been successful. Here, we provide evidence of proteasome hyperactivity in GOF mutant p53 NSCLC and show that this could be a targetable vulnerability specific to this subset of NSCLC.

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

Backgroundp53 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.MethodsOur 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.ResultsWe 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.ConclusionsBreast 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.

Abstract 1826: Rapid and specific degradation of mutant p53 proteins by cupric diethyldithiocarbamate, the active cytotoxic component of disulfiram-copper repurposing in human cancers

Abstract Background: Disulfiram (DSF) in combination with copper pills has emerged as a safe and exciting anticancer strategy and is undergoing clinical trials in multiple human cancers. However, DSF breaks down in the gut or blood immediately and chances of cupric diethyldithiocarbamate (Cu-DDC), the active cytotoxic principle being formed at tumor sites is very low. Therefore, we have found that synthetic Cu-DDC is directly and potently cytotoxic against p53 mutant cancers. We report here for the first time the differential effects of Cu-DDC in robustly eliminating the mutant p53 proteins from tumor cells in contrast to its augmentation of the wild-type p53 protein and activation. Destabilization of Gain Of Function (GOF) p53 mutant proteins for overcoming oncogenic addiction and improving the therapeutic efficacy has been a long-sought, but unmet anticancer avenue. Methods: A panel of 7 P53 mutant cell lines (R248W, R273H,R173H), and p53 wt tumor and normal cells (HPBLs, dental pulp stem cells, IMR90 and HUVEC) were used. Immunoprecipitation, Western blotting, Annexin-V apoptotic assays, flow cytometry, immunocytochemistry were used to quantitate p53 protein. Mia-Paca2 xenografts developed in nude mice were administered Cu-DDC/BSA. Results: Cu-DDC generated peroxides but not superoxides in tumor cells. The GOF mutant p53 proteins, R248W and R273H in three different tumor cell lines were eliminated after Cu-DDC treatment in a time- and concentration-dependent manner. The loss of mutant p53 proteins was confirmed by three different procedures. The degradation occurred rapidly in 8-10 h at 100 nM Cu-DDC and was inhibited by proteasome blockers. The expression of the GRO-α oncogene, positively regulated by GOF R248W was decreased in Cu-DDC treated Mia-Paca2 cells. Cellular proteasome activity was not inhibited by Cu-DDC. Ubiquitination of mutant p53 proteins was not altered by Cu-DDC. In contrast, the wild-type p53 present in HCT116 and MCF-7 cells, and normal cell counterparts (HPBL, IMR-90 lung cells) was significantly upregulated and activated with enhanced p21cip1 expression. Cu-DDC triggered strong apoptosis irrespective of the p53 status, although the mutant p53 containing cells were 2 to 3-fold more sensitive in cytotoxicity assays (IC50 range 0.25-0.66 µM). A significant tumor growth delay accompanied by loss of mutant p53 protein was observed in MiaPaca2 xenografts. Conclusions: In moving the DSF anticancer repurposing forward, we describe the unique ability of Cu-DDC to rapidly eliminate oncogenic p53 mutant proteins and confer potent antitumor efficacy. The degradation was unrelated to the proteostasis involving the inactivation of NPL4 by Cu-DDC described previously (supported by a Cancer Prevention and Research Institute of Texas [CPRIT] grant RP170207 to KSS). Citation Format: Viswanath Arutla, Kalkunte S. Srivenugopal. Rapid and specific degradation of mutant p53 proteins by cupric diethyldithiocarbamate, the active cytotoxic component of disulfiram-copper repurposing in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1826.

Altered Lipid Raft Composition Correlates with Oncogenic Potential

IntroductionMutant gain‐of‐function (GOF) p53 acts as an epigenetic regulator to promote oncogenesis. Many cancers with a GOF mutation accumulate cholesterol and inhibition of cholesterol synthesis with statin treatment reverses some of the pro‐oncogenic properties of GOF p53. Our central hypothesis is that cholesterol accumulation alters lipid raft signaling, contributing to oncogenesis. To address the relationship between cholesterol and oncogenic properties, we measured cholesterol levels in a panel of cell lines and correlate these levels with both GOF p53 expression and oncogenic properties. To directly tie lipid raft composition to oncogenic properties, we utilized the SW13 cell line with two epigenetically distinct cell subtypes with differing oncogenic properties. The SW13‐ subtype has an epithelial morphology and is highly proliferative, while the SW13+ subtype has a mesenchymal‐like phenotype and a higher metastatic potential. When treated with histone deacetylase inhibitors (HDACi), SW13‐ cells appear to adopt a SW13+ phenotype. Therefore, we treated with both HDACi to control the subtype conversion and shRNA to knock‐down GOF p53 expression. This experimental approach allows the composition of raft fractions to be correlated to the oncogenic properties of each SW13 subtype, with and without GOF p53 expression.MethodsProliferation rate and MMP expression were quantitated by EdU assays and in situ zymography respectively. Lipid raft fractions were isolated using the detergent‐resistant enrichment technique from SW13 cells of each subtype, with and without GOF p53 expression, and analyzed by mass spectrometry. Cholesterol assays were used to quantify cholesterol levels after treatment, and western blots were performed to assess levels of p53 and raft markers.ResultsCell lines expressing GOF p53 have 2 ‐ 4 fold increase in normalized levels of cholesterol when compared to a p53‐null cell line (p=0.02). Within the SW13 cell line subtypes, each subtype appears to have different oncogenic properties. First, p53 knock‐down alters cell behavior by decreasing MMP activity in SW13+ (p = 0.05) but not SW13‐ cells. Second, an increased rate of proliferation was measured for SW13‐ (p = 0.01) but not for SW13+ cells. We observed differential expression of genes coding for lipid raft components including GPI anchors, cholesterol biosynthesis, and sphingolipids; additionally, multiple raft‐related proteins are also differentially expressed. Suggestive of a functional difference in rafts, our data suggest a shift from planar rafts in the SW13‐ to caveolar rafts in the SW13+ line. Although the contribution of GOF p53 to this shift is under study, evidence suggests that raft composition is altered when GOF p53 is knocked down.ConclusionsOur data support the hypothesis that cholesterol levels and lipid raft signaling are significantly altered in cell lines expressing GOF p53 mutations. These changes correlate with distinct oncogenic properties and may correlate to oncogenic potential. Ongoing studies are aimed at delineating the link between GOF mutant p53 and raft function.

Triptolide promotes degradation of the unfolded gain-of-function Tp53R175H/Y220C mutant protein by initiating heat shock protein 70 transcription in non-small cell lung cancer.

BackgroundThe mutation rate of the tumor protein P53 (TP53) has been reported to be greater than 50% in non-small cell lung cancer (NSCLC), and gain-of-function (GOF) mutations in unfolded P53 (TP53R175H and TP53Y220C) have been associated with poor prognosis. However, the best treatment for patients with NSCLC harboring unfolded mutant P53 (mutp53) remains unclear. Triptolide is a natural compound derived from Tripterygium wilfordii that has shown a strong antitumor effect in a variety of cancers. Our study aimed to explore the GOF mutations in unfolded mutp53 (TP53R175H and TP53Y220C) and to clarify the molecular mechanisms by which triptolide regulates the degradation of unfolded mutp53 proteins in NSCLC.MethodsTwo unfolded proteins harboring TP53R175H and TP53Y220C mutations were selected to explore their functions in NSCLC progression. NCI-H1299 cells (TP53-null) were transfected with wild-type TP53 (TP53WT), TP53R175H, or TP53Y220C genes and treated with triptolide or a vehicle. Wound healing and transwell assays were performed to measure cell migration and invasion in vitro. Lung metastasis models were constructed through tail vein injection of mutant cells into BALB/c nude mice to evaluate the effect of triptolide on metastasis in vivo. Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), immunoprecipitation, and dual-luciferase reporter assays were performed to explore the relevant molecular mechanisms.ResultsOur study revealed that triptolide treatment reduced TP53R175H levels and that the TP53Y220C mutation enhanced the invasion and migration of NCI-H1299 cells. Mechanistically, triptolide promoted TP53R175H and TP53Y220C protein proteasomal degradation mediated through the E3 ligase murine double minute 2 (MDM2) by directly interacting with heat shock protein 70 (HSP70). Moreover, by upregulating HSP70 transcription, triptolide contributed to the protein degradation of the GOF mutp53.ConclusionsOur study reports, for the first time, the mechanism underlying triptolide-regulated protein degradation of TP53R175H or TP53Y220C, which offers new insight into developing a better therapeutic strategy for patients with NSCLC who express the unfolded mutp53 GOF protein.

Cross-talk between mutant p53 and p62/SQSTM1 augments cancer cell migration by promoting the degradation of cell adhesion proteins

Missense mutations in the p53 tumor suppressor abound in human cancer. Common (“hotspot”) mutations endow mutant p53 (mutp53) proteins with oncogenic gain of function (GOF), including enhanced cell migration and invasiveness, favoring cancer progression. GOF is usually attributed to transcriptional effects of mutp53. To elucidate transcription-independent effects of mutp53, we characterized the protein interactome of the p53R273H mutant in cells derived from pancreatic ductal adenocarcinoma (PDAC), where p53R273H is the most frequent p53 mutant. We now report that p53R273H, but not the p53R175H hotspot mutant, interacts with SQSTM1/p62 and promotes cancer cell migration and invasion in a p62-dependent manner. Mechanistically, the p53R273H-p62 axis drives the proteasomal degradation of several cell junction–associated proteins, including the gap junction protein Connexin 43, facilitating scattered cell migration. Concordantly, down-regulation of Connexin 43 augments PDAC cell migration, while its forced overexpression blunts the promigratory effect of the p53R273H-p62 axis. These findings define a mechanism of mutp53 GOF.

NAMPT Inhibitor and P73 Activator Represses P53 R175H Mutated HNSCC Cell Proliferation in a Synergistic Manner.

The p53 family has the following three members: p53, p63 and p73. p53 is a tumor suppressor gene that frequently exhibits mutation in head and neck cancer. Most p53 mutants are loss-of-function (LoF) mutants, but some acquire some oncogenic function, such as gain of function (GoF). It is known that the aggregation of mutant p53 can induce p53 GoF. The p73 activators RETRA and NSC59984 have an anti-cancer effect in p53 mutation cells, but we found that p73 activators were not effective in all head and neck squamous cell carcinoma (HNSCC) cell lines, with different p53 mutants. A comparison of the gene expression profiles of several regulator(s) in mutant HNSCC cells with or without aggregation of p53 revealed that nicotinamide phosphoribosyltransferase (NAMPT) is a key regulator of mutant p53 aggregation. An NAMPT inhibitor, to reduce abnormal aggregation of mutant p53, used in combination with a p73 activator, was able to effectively repress growth in HNSCC cells with p53 GoF mutants. This study, therefore, suggests a potential combination therapy approach for HNSCC with a p53 GoF mutation.

Abstract P5-09-01: Using isogenic model systems to determine mechanisms regulating mutant p53 protein stability in breast cancer cells

Abstract Two of the most common events in triple-negative breast cancers (TNBC) are mutation of the tumor suppressor gene TP53 and development of aneuploidy. This is often the case in aggressive and molecularly heterogeneous cancer subtypes such as TNBC, where ~80% of cases harbor a mutation in TP53. In addition to losing wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. Mutant p53 proteins can accumulate to high levels in tumors, and this stabilization is thought to be a pre-requisite for mutant p53 to exert its GOF effects. Mechanisms underlying the accumulation of mutant p53 are not well understood. To study mutant p53 GOF mechanisms and phenotypes, we used CRISPR/Cas9-mediated genome editing and developed two isogenic TNBC cell line models (one non-transformed and one tumor-derived) that express the most frequently occurring p53 missense mutations (R175H and R273H), are deficient for functional p53 protein (null), or retain the wild-type (WT) protein. In these engineered models, endogenous p53 expression is regulated by the native p53 promoter, thus providing a controlled system for rigorous functional experimentation across different p53 states. Through functional genomics analyses comparing isogenic TNBC cell lines, which initially differed only by the TP53 genotype, we have evaluated the relationship between mutant p53 and development of aneuploidy and determined that development of aneuploidy and not TP53 genotype contributed to several previously reported mutant p53 GOF phenotypes in vitro and in vivo1. Immunoblotting, immunofluorescence, and immunohistochemistry experiments revealed that mutant p53-expressing cell lines display a range of p53 protein levels, with several maintaining low levels of p53, similar to the cell lines which contain WT p53. In addition, mutant p53 levels correlate with aneuploidy, but not mean nuclear area or whole-genome doubling status, and protein levels increase over time in cell lines that become aneuploid. Using our various clonal cell lines that exhibit differing p53 protein stability, we are currently determining if p53 stability is dysregulated in aneuploid cell lines after copy number gain or loss of genes encoding critical p53 regulators, leading to the accumulation of the mutant protein. Analysis of cancer cell line genetic dependencies and human tumor data from The Cancer Genome Atlas (TCGA) has identified genetic and clinical correlates with increased mutant p53 protein levels. Utilizing biochemical and bioinformatic approaches, we aim to uncover mechanisms for increased mutant p53 stability and identify novel regulators of both WT and mutant p53 proteins. The dissection of mechanisms that contribute to stabilization of p53 protein will not only give insight into the regulation and tumor suppressive function of WT p53, but it has the potential for clinical translation in human cancers that have high-frequency p53 mutation and high levels of mutant p53 protein, as targeting mutant p53 through pharmacologic inhibition or antibody-based therapies is an active area of investigation. 1Article in press in Nature Communications Citation Format: Lindsay Redman-Rivera, Hailing Jin, Brian D Lehmann, Jennifer A Pietenpol. Using isogenic model systems to determine mechanisms regulating mutant p53 protein stability in breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-09-01.

Regulation of p53 and Cancer Signaling by Heat Shock Protein 40/J-Domain Protein Family Members.

Heat shock proteins (HSPs) are molecular chaperones that assist diverse cellular activities including protein folding, intracellular transportation, assembly or disassembly of protein complexes, and stabilization or degradation of misfolded or aggregated proteins. HSP40, also known as J-domain proteins (JDPs), is the largest family with over fifty members and contains highly conserved J domains responsible for binding to HSP70 and stimulation of the ATPase activity as a co-chaperone. Tumor suppressor p53 (p53), the most frequently mutated gene in human cancers, is one of the proteins that functionally interact with HSP40/JDPs. The majority of p53 mutations are missense mutations, resulting in acquirement of unexpected oncogenic activities, referred to as gain of function (GOF), in addition to loss of the tumor suppressive function. Moreover, stability and levels of wild-type p53 (wtp53) and mutant p53 (mutp53) are crucial for their tumor suppressive and oncogenic activities, respectively. However, the regulatory mechanisms of wtp53 and mutp53 are not fully understood. Accumulating reports demonstrate regulation of wtp53 and mutp53 levels and/or activities by HSP40/JDPs. Here, we summarize updated knowledge related to the link of HSP40/JDPs with p53 and cancer signaling to improve our understanding of the regulation of tumor suppressive wtp53 and oncogenic mutp53 GOF activities.

Regulation of miRNAs Expression by Mutant p53 Gain of Function in Cancer.

The p53 roles have been largely described; among them, cell proliferation and apoptosis control are some of the best studied and understood. Interestingly, the mutations on the six hotspot sites within the region that encodes the DNA-binding domain of p53 give rise to other very different variants. The particular behavior of these variants led to consider p53 mutants as separate oncogene entities; that is, they do not retain wild type functions but acquire new ones, namely Gain-of-function p53 mutants. Furthermore, recent studies have revealed how p53 mutants regulate gene expression and exert oncogenic effects by unbalancing specific microRNAs (miRNAs) levels that provoke epithelial-mesenchymal transition, chemoresistance, and cell survival, among others. In this review, we discuss recent evidence of the crosstalk between miRNAs and mutants of p53, as well as the consequent cellular processes dysregulated.

Selected Articles from This Issue

Radium-223 dichloride is a radiopharmaceutical used to treat cancer that has metastasized to the bones, and whose effects are derived from both direct action of radiation on tumor cells as well as bystander effects caused by irradiation of both tumor and non-tumor cells in the bone microenvironment. Here, Canter and colleagues quantify the biological effects derived from both of these mechanisms of action. Direct effects from alpha particle irradiation (yellow arrows in the image at left) contribute to biological changes detected histologically in disseminated tumor cells and osteocytes within the range of the alpha particles. Bystander effects also contribute to biological changes in tumor cells beyond the range of the alpha particles emitted from the endosteal surface of the bone. These radiation-induced bystander effects can arise from tumor cells (white outlined arrow) and marrow cells (pink outlined arrow) within the range of alpha particles, and from bone cells (brown outlined arrow). The data suggest that bystander effects in the bone environment likely contribute to growth delay of disseminated tumor cells in bone marrow.Regulation of cancer stemness has emerged as a key tumor-intrinsic property underlying disease progression and therap. resistance. Recently, gain-of-function (GOF) mutations in p53 have been shown to promote increased stemness characteristics, but the mechanism for this is not well understood. Here, Ghatak and colleagues identify the microRNA miR-324-5p as the key effector of GOF mutant p53 that controls this phenomenon in non–small cell lung cancer (NSCLC) cells. GOF p53 isoforms expressed in NSCLC cells caused upregulation of c-Myc, which bound to and activated transcription of miR-324. The authors demonstrate that expression of CUEDC2—the direct target of miR-324-5p in this context—was ablated in cells expressing GOF p53 isoforms, and the loss of CUEDC2 expression was associated with activation of NF-κB and increased expression of stemness markers. CUEDC2 downregulation was further shown to be characteristic of human tumors bearing GOF p53 mutations. The authors conclude that TP53 mutations coupled with high miR-324-5p expression is associated with worse prognosis in lung cancer patients. Taken together, the data identify miR-324-5p as a novel epigenetic regulator of stemness properties in NSCLC.Balanced MDM2:p53 expression maintains proper cell cycle progression, whereas aberrant p53 activity is associated with developmental defects and pathologic states. Here, Mohibi and colleagues demonstrate that the RNA binding protein Zfp871 is a direct transcriptional target of p53 in vivo. Zfp871 functions to provoke a negative feedback loop regulating p53 activation by binding to the 3′UTR of MDM2 mRNA and stabilizing it. This interaction increases MDM2 expression and, by consequence, promotes degradation of p53. Mice deficient in Zfp871 were prone to early death, and heterozygous mice experienced a shortened lifespan and prominent steatohepatitis. The early death seen in Zfp871-KO mice, however, was ameliorated by ablation of Trp53. Gene expression analyses unveiled that p53 hyperactivation caused by Zfp871 loss was associated with alterations in lipid metabolism and inflammatory signaling as well as key developmental pathways. While the precise mechanism underlying the control of these pathways is yet to be determined, the data clearly demonstrate that precise control of p53 expression and activity—in part through the novel Zfp871-MDM2-p53 axis—is required to prevent the onset of pathologic conditions.Micro-metastatic lesions in the bone are often latent and can persist undetected for years in a quiescent state. However, the mechanisms by which rapidly proliferating tumor cells suddenly become quiescent upon metastasis are poorly defined. Here, Shupp and colleagues demonstrate that soluble signals from the bone metastatic microenvironment may play a key role in regulating this process. In particular, small extracellular vesicles (sEV) released by osteoblasts in proximity to breast cancer cells were shown to harbor factors such as miR-148a-3p, which had implications for breast cancer cell biology. Among other effects, the uptake of these sEV by metastatic breast cancer cells resulted in disruptions of both ERK1/2 signaling and cell cycle progression, thereby slowing cancer cell proliferation and contributing toward their latency. Taken together, the data elucidate a novel mechanism by which stromal cells influence cancer cell progression in the metastatic niche and underscore the importance of extracellular vesicles in mediating tumor–stromal cell interactions.

Acquisition of aneuploidy drives mutant p53-associated gain-of-function phenotypes

p53 is mutated in over half of human cancers. In addition to losing wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. To study mutant p53 GOF mechanisms and phenotypes, we genetically engineered non-transformed and tumor-derived WT p53 cell line models to express endogenous missense mutant p53 (R175H and R273H) or to be deficient for p53 protein (null). Characterization of the models, which initially differed only by TP53 genotype, revealed that aneuploidy frequently occurred in mutant p53-expressing cells. GOF phenotypes occurred clonally in vitro and in vivo, were independent of p53 alteration and correlated with increased aneuploidy. Further, analysis of outcome data revealed that individuals with aneuploid-high tumors displayed unfavorable prognoses, regardless of the TP53 genotype. Our results indicate that genetic variation resulting from aneuploidy accounts for the diversity of previously reported mutant p53 GOF phenotypes.

MicroRNA-324-5p-CUEDC2 Axis Mediates Gain-of-Function Mutant p53-Driven Cancer Stemness.

Regulation of cancer stemness has recently emerged as a new gain-of-function (GOF) property of mutant p53. In this study, we identify miR-324-5p as a critical epigenetic regulator of cancer stemness and demonstrate its role in mediating GOF-mutant p53-driven stemness phenotypes. We report that miR-324-5p is upregulated in human cancer cell lines and non-small cell lung carcinoma (NSCLC) tumors carrying TP53 GOF mutations. Mechanistically, we show that GOF mutant p53 upregulates miR-324-5p expression via c-Myc, an oncogenic transcription factor in cancer cells. Our experimental results suggest that miR-324-5p-induced CSC phenotypes stem from the downregulation of CUEDC2, a downstream target gene of miR-324-5p. Accordingly, CUEDC2 complementation diminishes elevated CSC marker expression in miR-324-5p-overexpressing cancer cells. We further demonstrate that mutant p53 cancer cells maintain a low level of CUEDC2 that is rescued upon miR-324-5p inhibition. Importantly, we identify CUEDC2 downregulation as a novel characteristic feature of TP53-mutated human cancers. We show that activation of NF-κB due to downregulation of CUEDC2 by miR-324-5p imparts stemness in GOF mutant p53 cancer cells. Finally, we provide evidence that TP53 mutations coupled with high miR-324-5p expression predict poor prognosis in patients with lung adenocarcinoma. Thus, our study delineates an altered miR-324-5p-CUEDC2-NF-κB pathway as a novel regulator of GOF mutant p53-driven cancer stemness. IMPLICATIONS: Our findings implicate miRNA-324-5p as a novel epigenetic modifier of human cancer stemness.

Abstract 2489: A functional genomics approach to determine mutant p53 gain-of-function mechanisms and phenotypes in tumorigenesis

Abstract Two of the most common events in human tumors are mutation of the tumor suppressor gene TP53 and development of aneuploidy. In addition to losing their wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. Mechanistic understanding of mutant p53 GOF activities is complicated by the diversity and context-specific nature of reported GOF phenotypes. The study of mutant p53 GOF activities is especially challenging because mutations in p53 are positively correlated with the development of aneuploidy, which can increase heterogeneity through diverse chromosomal alterations and itself contributes to tumorigenesis. To study mutant p53 GOF mechanisms and phenotypes, we used CRISPR/Cas9-mediated genome editing and developed two isogenic epithelial cell line models (one non-transformed and one tumor-derived) that express the most frequently occurring p53 missense mutations (R175H and R273H), are deficient for functional p53 protein (null), or retain the wild-type (WT) protein. In these engineered models, endogenous p53 expression is regulated by the native p53 promoter, thus providing a controlled system for rigorous functional experimentation across different p53 states. Additionally, the use of clonally-derived cell lines originating from the same near diploid parental genetic background allows for assessment of the genomic alterations and resulting molecular heterogeneity following mutation of TP53. Through genomic, transcriptomic, and cellular based assays we have validated our cell line models and found that missense mutant and p53 null cells display loss of p53 function. Through functional genomics analyses comparing isogenic epithelial cells, which initially differed only by the TP53 genotype, we have evaluated the relationship between mutant p53 and aneuploidy and assessed whether our clonal cell lines display several previously reported mutant p53 GOF phenotypes such as altered gene expression, proliferation, metabolism, drug sensitivity, and migration. Further, using lentiviral mediated knockdown of p53 protein we evaluated the dependency of these phenotypes on expression of mutant p53 protein. Finally, data from The Cancer Genome Atlas (TCGA) was used for the analysis of manifestations of clinical mutant p53 GOF phenotypes. The dissection of mutant p53 GOF phenotypes will improve the current understanding of the role of mutant p53 in tumorigenesis. The results generated from these studies have the potential for clinical translation in major types of human cancer that have high-frequency p53 mutation. Citation Format: Lindsay N. Redman-Rivera, Timothy M. Shaver, Hailing Jin, Johanna M. Schafer, Quanhu Sheng, Rachel A. Hongo, Kathryn E. Beckermann, Brian D. Lehmann, Ferrin C. Wheeler, Jennifer A. Pietenpol. A functional genomics approach to determine mutant p53 gain-of-function mechanisms and phenotypes in tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2489.

Abstract 1782: Acidovorax temperans modulates the host immune microenvironment to promote lung cancer development

Abstract Lung cancer is the most common cause of cancer-related death worldwide in both men and women. The overall survival of lung cancer remains poor due to limitations in diagnostic and therapeutic strategies. Therefore, the development of novel biomarkers is urgently needed to improve patient prognosis. Growing evidence has demonstrated alterations of the normal microbiome in lung cancer. Dysbiosis may alter normal microbiota-host interactions and induce immune dysregulation, and therefore contribute to the promotion of tumorigenesis. Although multiple studies show an association between dysbiosis, host immune response and tumorigenesis, the underlying mechanisms are still unclear. We previously characterized a distinct group of taxa that are enriched in lung adenocarcinoma and squamous cell carcinoma. Among these taxa, Acidovorax spp. were associated with cigarette smoking and TP53 mutations, with Acidovorax temperans identified by full-length 16S sequencing. We hypothesized that enrichment of A. temperans in the lung microbial community may modulate the immune microenvironment and therefore contribute to tumor development. We used an autochthonous mouse model of lung adenocarcinoma, in which tumorigenesis is driven by oncogenic K-Ras and a gain-of function mutant p53 under the control of the Cre-lox system. We administered biweekly intranasal instillations of A. temperans to mimic the effects of chronic tobacco smoking. Following bacterial instillation, the lung tumor development was determined by magnetic resonance imaging (MRI) at 13 weeks post Ad-Cre instillation. Mice challenged with A. temperans exhibited significantly larger tumor burden and a deleterious effect on survival compared to the sham group. Given the importance of the immune system in modulating tumor development, we then investigated whether A. temperans exposure induced changes in the immune microenvironment through high parametric flow cytometry post 5th instillation and post MRI. Lungs from mice instilled with A. temperans showed an increased number of total leukocytes. The vast majority of leukocytes were pro-inflammatory cells and predominantly neutrophils. Moreover, we also found an increase in CD4+ T cells and double negative T cells, and an increase in RORγt+/IL17+ subpopulations in both of these T cell groups. Consistent with these results, the IL-17 expression level was upregulated in response to A. temperans instillation. Taken together, these results indicate that A. temperans exposure induces a robust pro-inflammatory response, thereby increasing immune infiltration in the tumor microenvironment and promoting development of lung adenocarcinoma. Citation Format: Chenran Zhang, Natalia von Muhlinen, Araya Romina, Joshua Stone, Romina Goldszmid, Curtis Harris. Acidovorax temperans modulates the host immune microenvironment to promote lung cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1782.

Gain of Function Mutant p53 Drives Clonal Hematopoiesis and the Pathogenesis of Myelodysplastic Syndromes

Clonal hematopoiesis increases with age, where a single mutant hematopoietic stem or progenitor cell (HSPC) contributes to a significant, measurable clonal proportion of mature blood lineages. Evolution of mutant clonal hematopoiesis with age predisposes the elderly to myelodysplastic syndromes (MDS) and other aging-associated diseases, suggesting that mutations identified in HSPCs contribute to disease development. Previous studies using whole exome sequencing of blood DNAs identified a high frequency of TP53 mutations in aged healthy individuals . TP53 mutations are also present in 10% of MDS cases and correlate with worse clinical outcomes. However, the role of TP53 mutations in clonal hematopoiesis and the pathogenesis of MDS is largely unknown. Ample data indicate that mutant p53 proteins not only lose their tumor suppressive functions, but also gain new abilities that promote tumorigenesis. Here, we report that specific gain-of-function (GOF) mutant p53 proteins drive the development of pre-leukemic hematopoietic stem cells (HSCs), predisposing aged mutant p53 mice to MDS development.Pre-leukemic HSCs have been shown to contribute to normal blood development and harbor a selective growth advantage compared to normal HSCs. We found that GOF mutant p53 (p53R248W) enhances the competitive advantage of HSCs without affecting terminal differentiation in steady state. Further, we observed that p53R248W mutant promotes clonal expansion of HSCs following genotoxic stresses. RNA-seq analysis revealed that HSC and leukemia stem cell signatures were significantly enriched in mutantHSCs compared to wild-type HSCs. Thus, HSCs expressing GOF mutant p53 recapitulate several key features of pre-leukemic HSCs. GOF p53 proteins are capable of dramatically altering tumor cell behavior, primarily through their interactions with other cellular proteins and regulation of cancer cell transcriptional programs. We discovered that GOF mutant p53 proteins, including p53R248W, p53R273H, and p53R175H, but not wild-type p53, interact with histone methyltransferase EZH2 and enhance H3K27me3 in HSCs. EZH2, a catalytic component of the Polycomb repressive complex 2 (PRC2), trimethylates histone H3 at lysine 27 (H3K27me3) to repress the transcription of target genes. Indeed, we found that the expression of HSC regulators, including Elf4 and Gadd45g, was significantly downregulated in mutant HSCs compared to wild-type HSCs. ChIP-seq analysis show enrichment of H3K27me3 in the promoter regions of Elf4 and Gadd45g in mutant HSCs. Importantly, we discovered that EZH2 inhibitor treatment decreased the replating potential of mutantbone marrow cells to wild-type cell level. Thus, we demonstrated that GOF mutant p53 drives the development of pre-leukemic HSCs through enhancing EZH2 activity.Given that most homozygous p53-/- and p53R248W/R248Wmice develop spontaneous tumors, including lymphoma, thymoma, and sarcoma, and die within 3 to 6 months after birth, it was not clear whether heterozygous mutant p53 mice (p53R248W/+) would develop myeloid malignancies during aging. To evaluate the role of GOF mutant p53 in the pathogenesis of myeloid malignancies, we maintained p53+/+ and p53R248W/+ mice for more than a year and monitored their survival and tumor development. While p53R248W/+ mice show extended life span compared to p53-/- and p53R248W/R248W mice, most of these mice died within 16-18 months after birth. During the observation period, approximately 30 % of heterozygous mutant p53mice developed MDS based upon pathological analysis of BM, spleen, and peripheral blood smear. Histological assessment revealed hypercellular BM in some aged p53R248W/+ mice. BM of some aged p53R248W/+ mice show neutrophil, erythroid precursor and megakaryocyte dysplasia. We further demonstrated that mutant p53 driven-MDS is transplantable. Thus, we provided evidence that GOF mutant p53 drives MDS development during aging.In summary, we have identified a critical role for GOF mutant p53 in clonal hematopoiesis and the pathogenesis of MDS. DisclosuresNo relevant conflicts of interest to declare.