Loss Of Tumor Suppressor Function Research Articles

Post-Translational Modification of PTEN Protein: Quantity and Activity.

Post-translational modifications play crucial roles in regulating protein functions and stabilities. PTEN is a critical tumor suppressor involved in regulating cellular proliferation, survival, and migration processes. However, dysregulation of PTEN is common in various human cancers. PTEN stability and activation/suppression have been extensively studied in the context of tumorigenesis through inhibition of the PI3K/AKT signaling pathway. PTEN undergoes various post-translational modifications, primarily including phosphorylation, acetylation, ubiquitination, SUMOylation, neddylation, and oxidation, which finely tune its activity and stability. Generally, phosphorylation modulates PTEN activity through its lipid phosphatase function, leading to altered power of the signaling pathways. Acetylation influences PTEN protein stability and degradation rate. SUMOylation has been implicated in PTEN localization and interactions with other proteins, affecting its overall function. Neddylation, as a novel modification of PTEN, is a key regulatory mechanism in the loss of tumor suppressor function of PTEN. Although current therapeutic approaches focus primarily on inhibiting PI3 kinase, understanding the post-translational modifications of PTEN could help provide new therapeutic strategies that can restore PTEN's role in PIP3-dependent tumors. The present review summarizes the major recent developments in the regulation of PTEN protein level and activity. We expect that these insights will contribute to better understanding of this critical tumor suppressor and its potential implications for cancer therapy in the future.

Impacts of cytoplasmic p53 aggregates on the prognosis and the transcriptome in lung squamous cell carcinoma.

The tumor suppressor TP53 gene, the most frequently mutated gene in human cancers, produces the product tumor protein p53, which plays an essential role in DNA damage. p53 protein mutations may contribute to tumorigenesis by loss of tumor suppressive functions and malignancy of cancer cells via gain-of-oncogenic functions. We previously reported that mutant p53 proteins form aggregates and that cytoplasmic p53 aggregates were associated with poor prognosis in human ovarian cancer. However, the prognostic impact of p53 aggregation in other tumors including lung squamous cell carcinoma (SCC) is poorly understood. Here, we demonstrated that lung SCC cases with cytoplasmic p53 aggregates had a significantly poor clinical prognosis. Analysis via patient-derived tumor organoids (PDOs) established from lung SCC patients and possessing cytoplasmic p53 aggregates showed that eliminating cytoplasmic p53 aggregates suppressed cell proliferation. RNA sequencing and transcriptome analysis of p53 aggregate-harboring PDOs indicated multiple candidate pathways involved in p53 aggregate oncogenic functions. With lung SCC-derived cell lines, we found that cytoplasmic p53 aggregates contributed to cisplatin resistance. This study thus shows that p53 aggregates are a predictor of poor prognosis in lung SCC and suggests that detecting p53 aggregates via p53 conventional immunohistochemical analysis may aid patient selection for platinum-based therapy.

Identification of a Novel Germline PPP4R3A Missense Mutation Asp409Asn on Familial Non-Medullary Thyroid Carcinoma.

Familial non-medullary thyroid carcinoma (FNMTC) accounts for 3% to 9% of all thyroid cancer cases, yet its genetic mechanisms remain unknown. Our study aimed to screen and identify novel susceptibility genes for FNMTC. Whole-exome sequencing (WES) was conducted on a confirmed FNMTC pedigree, comprising four affected individuals across two generations. Variants were filtered and analyzed using ExAC and 1000 Genomes Project, with candidate gene pathogenicity predicted using SIFT, PolyPhen, and MutationTaster. Validation was performed through Sanger sequencing in affected pedigree members and sporadic patients (TCGA database) as well as general population data (gnomAD database). Ultimately, we identified the mutant PPP4R3A (NC_000014.8:g.91942196C>T, or NM_001366432.2(NP_001353361.1):p.(Asp409Asn), based on GRCH37) as an FNMTC susceptibility gene. Subsequently, a series of functional experiments were conducted to investigate the impact of PPP4R3A and its Asp409Asn missense variant in thyroid cancer. Our findings demonstrated that wild-type PPP4R3A exerted tumor-suppressive effects via the Akt-mTOR-P70 S6K/4E-BP1 axis. However, overexpression of the PPP4R3A Asp409Asn mutant resulted in loss of tumor-suppressive function, ineffective inhibition of cell invasion, and even promotion of cell proliferation and migration by activating the Akt/mTOR signaling pathway. These results indicated that the missense variant PPP4R3A Asp409Asn is a candidate susceptibility gene for FNMTC, providing new insights into the diagnosis and intervention of FNMTC.

Integrated multi-omic analyses provide insight into colon adenoma susceptibility modulation by the gut microbiota.

Colon cancer onset is strongly associated with the differences in microbial taxa in the gastrointestinal tract. Although recent studies highlight the role of individual taxa, the effect of a complex gut microbiome (GM) on the metabolome and host transcriptome is still unknown. We used a multi-omics approach to determine how differences in the GM affect the susceptibility to adenoma development in a rat model of human colon cancer. Ultra-high performance liquid chromatography mass spectrometry of feces collected prior to observable disease onset identified putative metabolite profiles that likely predict future disease severity. Transcriptome analyses performed after disease onset from normal colonic epithelium and tumor tissues show a correlation between GM and host gene expression. Integrated pathway analyses of the metabolome and transcriptome based on putatively identified metabolic features indicate that bile acid biosynthesis is enriched in rats with high tumors along with increased fatty acid metabolism and mucin biosynthesis. Targeted pyrosequencing of the Pirc allele indicates that the GM alters the mechanism of adenoma development and may drive an epigenetic pathway of tumor suppressor silencing. This study reveals how untargeted metabolomics identifies signatures of susceptibility and integrated analyses uncover pathways of differential mechanisms of loss of tumor suppressor gene function and for potential prevention and therapeutic intervention. IMPORTANCE The association between the gut microbiome and colon cancer is significant but difficult to test in model systems. This study highlights the association of differences in the pathogen-free gut microbiome to changes in the host transcriptome and metabolome that correlate with colon adenoma initiation and development in a rat genetic model of early colon cancer. The utilization of a multi-omics approach integrating metabolomics and transcriptomics reveals differences in pathways including bile acid biosynthesis and fatty acid metabolism. The study also shows that differences in gut microbiomes significantly alter the mechanism of adenoma formation, shifting from genetic changes to epigenetic changes that initiate the early loss of tumor suppressor function. These findings enhance our understanding of the gut microbiome's role in colon cancer susceptibility, offer insights into potential biomarkers and therapeutic targets, and may pave the way for future prevention and intervention strategies.

Abstract 2226: Modeling the role of Tet-deficiency and restoration in Bcl2-driven B cell lymphoma progression

Abstract Loss of function in DNA methylation regulators such as the Ten-eleven translocation (TET1-3) proteins, have been implicated in the progression of lymphoma. TET enzymes constitute a family of iron and αKG-dependent dioxygenases that oxidize 5-methylcytosine in DNA to promote demethylation and the reversal of gene silencing. The TET enzymes require vitamin C for their optimal catalytic activity, however, many patients with hematological malignancies are vitamin C deficient. Enhancing and restoring TET function with high-dose vitamin C has been shown to block the progression of solid tumors and blood malignancies. A combination of genetic and epigenetic diversity in lymphoma can result in the aberrant overexpression of oncogenes, loss of tumor suppressor function, and genomic instability. Our research models Bcl2-driven B cell lymphoma with Tet-deficiency using transgenic RNAi for the knockdown of Tet1 or Tet2. In vitro, our results have shown that this model shows increased self-renewal capacity of early B cell progenitors and precursors that can be suppressed by genetic restoration of Tet expression or upon treatment with vitamin C. In vivo, the B cell compartment of mice with Bcl2 overexpression and Tet1 or Tet2 deficiency exhibit a block in B cell maturation during a long period of pre-malignancy, and a decreased frequency of follicular B cells in the spleen from 3 months of age. Our aim is to determine mechanistically the effects of Tet-deficiency on B cell lymphoma progression and whether vitamin C can restore Tet activity to suppress lymphoma progression. Citation Format: Minh Quang Lam, Luisa Cimmino. Modeling the role of Tet-deficiency and restoration in Bcl2-driven B cell lymphoma progression [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 2226.

The mutational spectrum in whole exon of p53 in oral squamous cell carcinoma and its clinical implications

Mutations in p53 are common in human oral squamous cell carcinoma (OSCC). However, in previous analyses, only detection of mutant p53 protein using immunohistochemistry or mutations in some exons have been examined. Full length mutant p53 protein in many cases shows a loss of tumor suppressor function, but in some cases possibly shows a gain of oncogenic function. In this study, we investigate relationships of outcomes with the mutational spectrum of p53 (missense and truncation mutations) in whole exon in OSCC. Specimens from biopsy or surgery (67 cases) were evaluated using next-generation sequencing for p53, and other oncogenic driver genes. The data were compared with overall survival (OS) and disease-free survival (DFS) using univariate and multivariate analyses. p53 mutations were detected in 54 patients (80.6%), 33 missense mutations and 24 truncation mutations. p53 mutations were common in the DNA-binding domain (43/52) and many were missense mutations (31/43). Mutations in other regions were mostly p53 truncation mutations. We detected some mutations in 6 oncogenic driver genes on 67 OSCC, 25 in NOTCH1, 14 in CDKN2A, 5 in PIK3CA, 3 in FBXW7, 3 in HRAS, and 1 in BRAF. However, there was no associations of the p53 mutational spectrum with mutations of oncogenic driver genes in OSCC. A comparison of cases with p53 mutations (missense or truncation) with wild-type p53 cases showed a significant difference in lymph node metastasis. DFS was significantly poorer in cases with p53 truncation mutations. Cases with p53 truncation mutations increased malignancy. In contrast, significant differences were not found between cases with p53 missense mutations and other mutations. The p53 missense mutation cases might include cases with mostly similar function to that of the wild-type, cases with loss of function, and cases with various degrees of gain of oncogenic function.

Abstract P2-08-18: Evaluation of the activity of key actionable oncogenic driving pathways in triple negative breast cancer using OncoSignal™; a novel molecular assay based on transcriptional profile analysis

Abstract Introduction: Triple negative breast cancer (TNBC), defined by the absence of expression of estrogen receptor (ER), progesterone receptor, and HER2, is a heterogenous subgroup of breast cancer which currently accounts for a significant proportion of the mortality from the disease. Consequently, there is an urgent need to identify more effective therapy for women with this type of breast cancer. We have recently developed a novel assay, OncoSignal™ which is capable of precisely measuring the activity of seven key signaling pathways through utilizing measurements of mRNA. The assay quantitatively determines the specific activity of ER, androgen receptor, PI3K, MAPK, HedgeHog, Notch, and TGFβ signal pathways via measurement and analysis of mRNA expression from transcriptional targets of these pathways. This approach overcomes some of the limitations of NGS and other methods which analyze only partial components of a complex signaling pathway, producing significant risk of false positive and negative results, and resulting in potentially inaccurate diagnosis and treatment selection. In this study we evaluated oncogenic pathway activation in 88 cases of TNBC using the OncoSignal™ assay. Materials and methods: Samples and pathway scores for TNBC tumors were calculated using a publicly available Affymetrix dataset GSE76275. This study included 88 cases of TNBC obtained at Baylor College of Medicine. OncoSignal™ pathway activity scores (PAS) were calculated from the transcriptional profile for each case. In addition, 10 samples of benign breast tissue were available for analysis of PAS and used as controls for this evaluation. The mean pathway activity scores and ranges were calculated from the benign tissues. The PAS results for each of the 7 oncogenic pathways from the TNBC cases were compared with PAS results from benign tissues. Results: The OncoSignal™ PAS were significantly higher in TNBC compared to benign tissues for MAPK, PI3K, and HH pathways. The PAS of ER and TGFβ pathways were significantly lower in TNBC compared to the benign tissue. MAPK, AR, ER, PI3K, and HH were elevated in 86%, 17%, 8%, 95%, and 94% of TNBC cases respectively. TGFβ pathway, for which oncogenic versus tumor suppressive functionality is contextually determined, showed reduced PAS in 85% of TNBC cases compared to benign tissue controls. Conclusion: OncoSignal™ analysis identifies enhanced targetable oncogenic pathway activity in a majority of TNBC breast cancers. Of interest, 7 of 88 cases (8%) classified as TNBC using IHC methods showed evidence of estrogen receptor signal pathway activation, and 15 (17%) showed elevated AR PAS. PI3K and MAPK had high PAS in over 85% of TNBC cases. Results suggest loss of tumor suppressive function of the TGFβ pathway. We conclude that OncoSignal™ analysis may help identify TNBC tumors with targetable signal transformation pathways. Citation Format: Genevra Magliocco, Eveline den Biezen, Diederick Keizer, Martijn Akse, Martijn van Zelst, Dianne van Strijp, Saskia Vermeer, Anja van de Stolpe, Anthony Magliocco. Evaluation of the activity of key actionable oncogenic driving pathways in triple negative breast cancer using OncoSignal™; a novel molecular assay based on transcriptional profile analysis [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 P2-08-18.

Mutated p53 in HGSC-From a Common Mutation to a Target for Therapy.

Simple SummaryOvarian high-grade serous cancer (HGSC), the most common and the deadliest subtype of epithelial ovarian cancer, is characterized by frequent mutations in the TP53 tumor suppressor gene, encoding for the p53 protein in nearly 100% of cases. This makes p53 the focus of many studies trying to understand its role in HGSC. The aim of our review paper is to provide updates on the latest findings related to the role of mutant p53 in HGSC. This includes the clinical outcomes of TP53 mutations in HGSC, upstream regulators and downstream effectors of p53, its function in the earliest stages of HGSC development and in the interplay between the tumor cells and their microenvironment. We summarize with the likelihood of p53 mutants to serve as biomarkers for early diagnosis and as targets for therapy in HGSC.Mutations in tumor suppressor gene TP53, encoding for the p53 protein, are the most ubiquitous genetic variation in human ovarian HGSC, the most prevalent and lethal histologic subtype of epithelial ovarian cancer (EOC). The majority of TP53 mutations are missense mutations, leading to loss of tumor suppressive function of p53 and gain of new oncogenic functions. This review presents the clinical relevance of TP53 mutations in HGSC, elaborating on several recently identified upstream regulators of mutant p53 that control its expression and downstream target genes that mediate its roles in the disease. TP53 mutations are the earliest genetic alterations during HGSC pathogenesis, and we summarize current information related to p53 function in the pathogenesis of HGSC. The role of p53 is cell autonomous, and in the interaction between cancer cells and its microenvironment. We discuss the reduction in p53 expression levels in tumor associated fibroblasts that promotes cancer progression, and the role of mutated p53 in the interaction between the tumor and its microenvironment. Lastly, we discuss the potential of TP53 mutations to serve as diagnostic biomarkers and detail some more advanced efforts to use mutated p53 as a therapeutic target in HGSC.

Abstract 2487: disrupting binding of p53 mutants to PEPD unleashes their tumor suppressor activities

Abstract Background & Goals: The p53 tumor suppressor is one of the most important determinants of cell fate and is critical for tumor suppression. It functions by regulating the expression of a plethora of genes via sequence-specific DNA binding and also by transcription-independent, mitochondria-mediated mechanisms. Not surprisingly, the TP53 gene, which encodes p53, is frequently mutated in human cancer. For example, TP53 is mutated in about 75% of HER2-positive breast cancer and about 85% of triple negative breast cancer. The majority of TP53 mutations are missense mutations, changing one amino acid per mutant, most of which occur in its DNA-binding domain. p53 mutants show loss of tumor suppressor functions or gain of oncogenic functions. We recently found that peptidase D (PEPD), also known as prolidase, binds via its C-terminal sequence to the proline-rich domain (PRD) of wild type p53 and that disrupting the binding activates p53. The enzymatic activity of PEPD is not required for this function. Our objective in the present study is to determine whether PEPD also regulates p53 mutants. We focused on some of the most common oncogenic p53 mutants, including R175H, R248Q, R273H and R280K. Results: Our study shows that 1) each p53 mutant binds directly to PEPD and binds to PEPD in cells in a significant quantity, 2) disrupting their binding in cells by PEPD knockdown reactivates each p53 mutant, unleashing both transcription-dependent and -independent tumor suppressing activities, 3) after leaving PEPD, p53 mutants undergo acetylation, mono-ubiquitination and phosphorylation, which drive their refolding and reactivation, 4) Lysine 373 acetylation is essential for their reactivation, and 5) p53 mutants and p53WT show almost no difference in PEPD-binding and response to PEPD KD. Conclusions & Therapeutic Relevance: In conclusion, we have identified a cellular mechanism by which the tumor suppressor functions of p53 mutants are strongly reactivated, which is neither cell-specific nor mutant-specific. Mainly, p53 mutants bind to PEPD in cancer cells, and disrupting the complex causes posttranslational modifications of mutants freed from PEPD, which drive their refolding and unleash their tumor suppressor activities. This discovery advances our understanding of the function and regulation of p53 mutants and breaks new ground for developing novel cancer treatment strategies. Citation Format: Lu Yang. disrupting binding of p53 mutants to PEPD unleashes their tumor suppressor activities [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 2487.

Chromothripsis, DNA repair and checkpoints defects

Chromothripsis is a unique form of genome instability characterized by tens to hundreds of DNA double-strand breaks on one or very few chromosomes, followed by error-prone repair. The derivative chromosome(s) display massive rearrangements, which lead to the loss of tumor suppressor function and to the activation of oncogenes. Chromothripsis plays a major role in cancer as well as in other conditions, such as congenital diseases. In this review, we discuss the repair processes involved in the rejoining of the chromosome fragments, the role of DNA repair and checkpoint defects as a cause for chromothripsis as well as DNA repair defects resulting from chromothripsis. Finally, we consider clinical implications and potential therapeutic vulnerabilities that could be utilized to eliminate tumor cells with chromothripsis.

NOTCH1 Signaling in Head and Neck Squamous Cell Carcinoma

Biomarker-driven targeted therapies are lacking for head and neck squamous cell carcinoma (HNSCC), which is common and lethal. Efforts to develop such therapies are hindered by a genomic landscape dominated by the loss of tumor suppressor function, including NOTCH1 that is frequently mutated in HNSCC. Clearer understanding of NOTCH1 signaling in HNSCCs is crucial to clinically targeting this pathway. Structural characterization of NOTCH1 mutations in HNSCC demonstrates that most are predicted to cause loss of function, in agreement with NOTCH1’s role as a tumor suppressor in this cancer. Experimental manipulation of NOTCH1 signaling in HNSCC cell lines harboring either mutant or wild-type NOTCH1 further supports a tumor suppressor function. Additionally, the loss of NOTCH1 signaling can drive HNSCC tumorigenesis and clinical aggressiveness. Our recent data suggest that NOTCH1 controls genes involved in early differentiation that could have different phenotypic consequences depending on the cancer’s genetic background, including acquisition of pseudo-stem cell-like properties. The presence of NOTCH1 mutations may predict response to treatment with an immune checkpoint or phosphatidylinositol 3-kinase inhibitors. The latter is being tested in a clinical trial, and if validated, it may lead to the development of the first biomarker-driven targeted therapy for HNSCC.

Aging-Associated Alterations in Mammary Epithelia and Stroma Revealed by Single-Cell RNA Sequencing

SUMMARYAging is closely associated with increased susceptibility to breast cancer, yet there have been limited systematic studies of aging-induced alterations in the mammary gland. Here, we leverage high-throughput single-cell RNA sequencing to generate a detailed transcriptomic atlas of young and aged murine mammary tissues. By analyzing epithelial, stromal, and immune cells, we identify age-dependent alterations in cell proportions and gene expression, providing evidence that suggests alveolar maturation and physiological decline. The analysis also uncovers potential pro-tumorigenic mechanisms coupled to the age-associated loss of tumor suppressor function and change in microenvironment. In addition, we identify a rare, age-dependent luminal population co-expressing hormone-sensing and secretory-alveolar lineage markers, as well as two macrophage populations expressing distinct gene signatures, underscoring the complex heterogeneity of the mammary epithelia and stroma. Collectively, this rich single-cell atlas reveals the effects of aging on mammary physiology and can serve as a useful resource for understanding aging-associated cancer risk.

Distinct TP53 Mutation Types Exhibit Increased Sensitivity to Ferroptosis Independently of Changes in Iron Regulatory Protein Activity.

The tumor suppressor gene TP53 is the most commonly mutated gene in human cancer. In addition to loss of tumor suppressor functions, mutations in TP53 promote cancer progression by altering cellular iron acquisition and metabolism. A newly identified role for TP53 in the coordination of iron homeostasis and cancer cell survival lies in the ability for TP53 to protect against ferroptosis, a form of iron-mediated cell death. The purpose of this study was to determine the extent to which TP53 mutation status affects the cellular response to ferroptosis induction. Using H1299 cells, which are null for TP53, we generated cell lines expressing either a tetracycline inducible wild-type (WT) TP53 gene, or a representative mutated TP53 gene from six exemplary “hotspot” mutations in the DNA binding domain (R273H, R248Q, R282W, R175H, G245S, and R249S). TP53 mutants (R273H, R248Q, R175H, G245S, and R249S) exhibited increased sensitivity ferroptosis compared to cells expressing WT TP53. As iron-mediated lipid peroxidation is critical for ferroptosis induction, we hypothesized that iron acquisition pathways would be upregulated in mutant TP53-expressing cells. However, only cells expressing the R248Q, R175H, and G245S TP53 mutation types exhibited statistically significant increases in spontaneous iron regulatory protein (IRP) RNA binding activity following ferroptosis activation. Moreover, changes in the expression of downstream IRP targets were inconsistent with the observed differences in sensitivity to ferroptosis. These findings reveal that canonical iron regulatory pathways are bypassed during ferroptotic cell death. These results also indicate that induction of ferroptosis may be an effective therapeutic approach for tumor cells expressing distinct TP53 mutation types.

Nuclear Heparanase Regulates Chromatin Remodeling, Gene Expression and PTEN Tumor Suppressor Function.

Heparanase (HPSE) is an endoglycosidase that cleaves heparan sulfate and has been shown in various cancers to promote metastasis, angiogenesis, osteolysis, and chemoresistance. Although heparanase is thought to act predominantly extracellularly or within the cytoplasm, it is also present in the nucleus, where it may function in regulating gene transcription. Using myeloma cell lines, we report here that heparanase enhances chromatin accessibility and confirm a previous report that it also upregulates the acetylation of histones. Employing the Multiple Myeloma Research Foundation CoMMpass database, we demonstrate that patients expressing high levels of heparanase display elevated expression of proteins involved in chromatin remodeling and several oncogenic factors compared to patients expressing low levels of heparanase. These signatures were consistent with the known function of heparanase in driving tumor progression. Chromatin opening and downstream target genes were abrogated by inhibition of heparanase. Enhanced levels of heparanase in myeloma cells led to a dramatic increase in phosphorylation of PTEN, an event known to stabilize PTEN, leading to its inactivity and loss of tumor suppressor function. Collectively, this study demonstrates that heparanase promotes chromatin opening and transcriptional activity, some of which likely is through its impact on diminishing PTEN tumor suppressor activity.

Abstract A66: Repeatome profiling in high-grade serous ovarian cancer reveals abundant repeat noncoding RNA expression

Abstract The lack of consensus on clinically relevant molecular subtypes by gene expression in high-grade serous ovarian carcinoma (HGSOC) creates a barrier to subtype-based clinical investigation for the development of targeted therapies. We previously discovered aberrant expression of noncoding repeat RNAs across epithelial cancers including ovarian (Ting, Science 2011), which can invoke innate immune responses in tumors (Chiappinelli, Cell 2015). Epigenetic alterations and loss of tumor suppressor function, especially p53, can derepress endogenous repetitive elements in cancer. Further, BRCA1 deficiency induces satellite repeat RNAs, which promote genomic instability via interacting with the BRCA1 complex (Zhu, Mol Cell 2018), highlighting their potential significance in HGSOC. Here we aim to comprehensively define the as yet uncharacterized “repeatome” in HGSOC to refine molecular subtypes and identify novel biomarkers and therapeutic targets. We developed a Total RNASeq platform and novel computational pipelines to quantify the repeatome (Solovyov, Cell Rep 2018). Results from RNASeq are validated in cell lines and human tumors with RNA in situ hybridization (RNA-ISH) using probes to specific repeat RNAs, and combined IHC is performed to quantify intratumoral immune subpopulations. Whole-exome sequencing is performed to link the repeatome with somatic mutations. To obtain a global landscape of the ovarian cancer repeatome, Total RNASeq was performed on 32 patient-derived ovarian cancer cell lines, 11 HGSOC PDX, and 11 additional ovarian cancer cell lines. We detect abundant repeat RNA expression from all major subclasses including retrotransposons, endogenous retroviruses, and satellites. HGSOC are enriched for satellite repeats compared with other cancers, most notably in BRCA-mutant HGSOC. Human satellite II (HSATII), a cancer-specific satellite, is strongly upregulated in HGSOC compared with fallopian tube epithelial cells and displays highly variable expression across different models. RNA-ISH for HSATII on human ovarian cancer tissue microarrays confirms the abundance and variation of HSATII. Additionally, the repeatome is altered in HGSOC following exposure to in vitro chemotherapy and epigenetic agents with distinct patterns of expression linked to specific agents. Additional RNAseq analysis using hierarchical clustering and principal component analysis are being used to understand the relationship of repeatome expression patterns with coding gene expression, somatic mutations, in vitro drug sensitivity, and clinical outcomes. Digital image analysis of repeat RNA-ISH expression and immune cell infiltrate quantitation will determine the link between tumor cell repeat RNA levels and the responding immune tumor microenvironment. Overall, these studies will define the undiscovered repeatome in HGSOC and provide a foundation for discovery of novel biomarkers and potential therapeutic strategies, particularly those to increase efficacy of immunotherapies. Citation Format: Rebecca L. Porter, Anna Szabolcs, Niyati Desai, Vishal Thapar, Raghav Mohan, Alexander Solovyov, David Pepin, Joyce Liu, Benjamin Greenbaum, David T. Ting. Repeatome profiling in high-grade serous ovarian cancer reveals abundant repeat noncoding RNA expression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A66.

Fusions involving BCOR and CREBBP are rare events in infiltrating glioma

BCOR has been recognized as a recurrently altered gene in a subset of pediatric tumors of the central nervous system (CNS). Here, we describe a novel BCOR-CREBBP fusion event in a case of pediatric infiltrating astrocytoma and further probe the frequency of related fusion events in CNS tumors. We analyzed biopsy samples taken from a 15-year-old male with an aggressive, unresectable and multifocal infiltrating astrocytoma. We performed RNA sequencing (RNA-seq) and targeted DNA sequencing. In the index case, the fused BCOR-CREBBP transcript comprises exons 1–4 of BCOR and exon 31 of CREBBP. The fused gene thus retains the Bcl6 interaction domain of BCOR while eliminating the domain that has been shown to interact with the polycomb group protein PCGF1. The fusion event was validated by FISH and reverse transcriptase PCR. An additional set of 177 pediatric and adult primary CNS tumors were assessed via FISH for BCOR break apart events, all of which were negative. An additional 509 adult lower grade infiltrating gliomas from the publicly available TCGA dataset were screened for BCOR or CREBBP fusions. In this set, one case was found to harbor a CREBBP-GOLGA6L2 fusion and one case a CREBBP-SRRM2 fusion. In a third patient, both BCOR-L3MBTL2 and EP300-BCOR fusions were seen. Of particular interest to this study, EP300 is a paralog of CREBBP and the breakpoint seen involves a similar region of the gene to that of the index case; however, the resultant transcript is predicted to be completely distinct. While this gene fusion may play an oncogenic role through the loss of tumor suppressor functions of BCOR and CREBBP, further screening over larger cohorts and functional validation is needed to determine the degree to which this or similar fusions are recurrent and to elucidate their oncogenic potential.

TP53 Mutation Status Influences Iron Regulatory Protein RNA Binding Activity and Sensitivity to Ferroptotic Cell Death

Abstract Objectives The tumor suppressor gene TP53 is the most commonly mutated gene in human cancer, but mutations in TP53 do not just result in loss of tumor suppressor function, they can also promote cancer progression by altering cellular iron acquisition and metabolism. A newly identified role for TP53 in the mediation of iron homeostasis and cancer cell survival lies in the ability for TP53 to protect against ferroptosis, a form of iron mediated cell death. The purpose of this study was to determine the extent to which TP53 mutation status effects iron-mediated cell death in response to ferroptosis induction. We also measured TP53 dependent differences in iron regulatory protein (IRP) RNA binding activity to begin to clarify the mechanisms by which TP53 mutation status may influence sensitivity to ferroptosis. Methods Using H1299 cells, which are null for TP53, we generated cell lines expressing either a tetracycline inducible wild-type TP53 gene, or a representative mutated TP53 gene from exemplary “hotspot” mutations in the DNA binding domain (R248, R273, R282, G245, R249 and R175). These six mutation types were selected because they represent 25% of all TP53 mutations in human cancer. To determine the influence of TP53 mutation status on sensitivity to ferroptotic cell death, we treated cells with erastin, a potent inducer of ferroptosis and measured differences in cell viability between these cell lines using PrestoBlue cell viability reagent. To assess mutant TP53-depenent differences in IRP RNA binding activity during ferroptosis we measured differences in IRP RNA binding activity via Electrophoretic Mobility-Shift Assay. Results We found that TP53 mutants (R273, R248, R175, G245, and R249) were significantly less viable (P < 0.05) after initiation of ferroptosis compared to cells expressing WT TP53. Following ferroptosis induction, we observed a significant (P < 0.05) increase in IRP RNA binding in G245, R248, and R175 mutants. Conclusions Our preliminary analyses indicate that TP53 mutants may be more sensitive to ferroptosis, but IRPs do not seem to be solely responsible for the increase in iron during ferroptotic cell death. Furthermore, ferroptosis may be a potential therapeutic target for cancers with these TP53 mutations but further investigation is warranted. Funding Sources Internal funding at Oklahoma State University.

Growth inhibitory role of the p53 activator SCH529074 in non‑small cell lung cancer cells expressing mutantp53.

Mutations of p53 occur in approximately 50% of advanced non‑small cell lung cancer (NSCLC) cases, leading to loss of tumor suppressive function and/or gain of p53oncogenic activity. Reactivation of mutantp53 and consequently induction of apoptosis in cancer cells is the goal of p53‑targeted therapy. Recently, several p53mutant reactivating compounds were discovered including SCH529074. However, the role of SCH529074 in NSCLC has not been fully explored. In the present study, the effects of this compound on cell survival, cell cycle progression, induction of apoptosis and modulation of cell signaling in p53mutant NSCLCcells (H1975, H322 and H157) and p53 wild‑type NSCLCcells (A549), was investigated. Cell‑based functional assays, real‑time RT‑qPCR and western blot assays were used. HCT116 [p53 wild‑type (WT)] and HCT116 p53‑/‑ (p53null) were used as control cells. The results demonstrated that SCH529074 treatment caused significant reduction in cell viability and colony formation activity in p53mutant, p53WT and p53‑deficientcells. The treatment of NSCLC cells with SCH529074 resulted in a dose‑dependent induction of apoptosis and G0/G1 cell cycle arrest, which was associated with the activation of caspases(3and7), p53‑independent upregulation of p21 and PUMA as well as increased LC3II, a biomarker of autophagy. The combination treatment with the autophagy inhibitor chloroquine (CQ) and SCH529074 led to decreased cell viability, colony formation and increased induction of apoptosis. The data indicated that SCH529074 may exert its growth inhibitory function in a p53‑independent manner in NSCLCcells.

Mevalonate Pathway Provides Ubiquinone to Maintain Pyrimidine Synthesis and Survival in p53-Deficient Cancer Cells Exposed to Metabolic Stress.

Oncogene activation and loss of tumor suppressor function changes the metabolic activity of cancer cells to drive unrestricted proliferation. Moreover, cancer cells adapt their metabolism to sustain growth and survival when access to oxygen and nutrients is restricted, such as in poorly vascularized tumor areas. We show here that p53-deficient colon cancer cells exposed to tumor-like metabolic stress in spheroid culture activated the mevalonate pathway to promote the synthesis of ubiquinone. This was essential to maintain mitochondrial electron transport for respiration and pyrimidine synthesis in metabolically compromised environments. Induction of mevalonate pathway enzyme expression in the absence of p53 was mediated by accumulation and stabilization of mature SREBP2. Mevalonate pathway inhibition by statins blocked pyrimidine nucleotide biosynthesis and induced oxidative stress and apoptosis in p53-deficient cancer cells in spheroid culture. Moreover, ubiquinone produced by the mevalonate pathway was essential for the growth of p53-deficient tumor organoids. In contrast, inhibition of intestinal hyperproliferation by statins in an Apc/KrasG12D-mutant mouse model was independent of de novo pyrimidine synthesis. Our results highlight the importance of the mevalonate pathway for maintaining mitochondrial electron transfer and biosynthetic activity in cancer cells exposed to metabolic stress. They also demonstrate that the metabolic output of this pathway depends on both genetic and environmental context. SIGNIFICANCE: These findings suggest that p53-deficient cancer cells activate the mevalonate pathway via SREBP2 and promote the synthesis of ubiquinone that plays an essential role in reducing oxidative stress and supports the synthesis of pyrimidine nucleotide.

GENE-59. NOT ALL p53 MUTATIONS ARE CREATED EQUAL: A MURINE ASTROCYTE MODEL FOR HIGH-THROUGHPUT FUNCTIONAL ASSESSMENT OF p53 MISSENSE MUTATIONS

Abstract The tumor suppressor TP53 (p53) is the most commonly mutated gene in cancer and among the most frequently mutated genes in glioblastoma (GBM). The majority of p53 mutations in GBM are missense mutations in the DNA binding domain that lead to the production of full length mutant p53 protein. In addition to the complete loss of tumor suppressor function, these mutations have gain-of-function (GOF) properties either through attenuation of wild-type function or neomorphic functions. The variability in GOF mutations results in heterogeneity in cancer phenotypes between mutants that remain poorly understood. Here, we developed a murine astrocyte model to functionally assess a library of p53 mutants in parallel. Primary astrocytes were isolated from postnatal day one pups possessing a single copy of wild-type p53 flanked by loxP sites (TRP53f/-). We then built a library of 17 individual alleles of recurring mutations in GBM with flanking loxP sites. When co-transfected into the mouse astrocytes with a plasmid expressing Cre recombinase, the endogenous WT p53 was excised and replaced with a single copy of the mutant allele. In this way, all astrocytes expressed a single copy of mutant p53 from the endogenous p53 locus. As the mutant p53 cells expanded, aliquots of cells were extracted for targeted genomic sequencing of the p53 allele. Comparing the allelic frequencies of each mutant overtime revealed a wide distribution of growth rates between mutants. To validate the screen results, wildtype astrocytes were transduced with mutant p53-IRES-eGFP retrovirus to overexpress one of three mutations with divergent growth phenotypes. As observed in the initial screen, equivalent overexpression of the different p53 mutants was sufficient to induce significant differences in growth phenotype, with astrocytes expressing the Y217C growing the fastest, R172H second, and Y202C growing the slowest. Ongoing studies are evaluating mutation-specific p53 binding partners and transcriptional outputs.