Inactivation Of Tumor Suppressor Research Articles

Abstract A017: Identifying proliferative and immune-modulatory drivers of small cell lung cancer (SCLC)

Abstract Small cell lung cancer (SCLC) is a highly aggressive form of lung cancer that causes an estimated 200,000 patient deaths globally every year. Most (>70%) patients present with extensive stage (ES)-SCLC, and the disease primarily presents with a neuroendocrine (NE), immune-desert phenotype, with low expression of antigen-presenting machinery genes. Despite limited immune infiltration in the tumor, SCLC has reproducibly responded to immune checkpoint blockade. IMpower133 was a pivotal Phase III trial that added atezolizumab (anti-PD-L1) to platinum/etoposide chemotherapy for treatment of first-line ES-SCLC, which led to the first approval of an immune checkpoint blockade strategy in SCLC. While the inclusion of atezolizumab to the treatment regimen has extended the survival of some patients, SCLC remains an unmet medical need with a mOS of 12.3 months. Furthermore, we lack a deep molecular understanding of factors mediating response and resistance to standard of care therapies in SCLC. To understand the genetic drivers of SCLC that could be linked to immunotherapy resistance, we analyzed the matched whole-exome sequencing and RNA-seq data from baseline tumor samples collected from the IMpower133 trial. Previously, we applied the non-negative matrix factorization (NMF) approach to the RNA-seq data, which clustered patients into four subsets of SCLC, with varying degrees of immune infiltration, neuroendocrine program activation, and clinical benefit from immunotherapy. Notably, we identified an inflamed-neuroendocrine (I-NE) subset of patients that exhibit longer overall survival with chemoimmunotherapy compared to chemotherapy alone, and an inflamed-non-neuroendocrine (I-nonNE) subset of patients that did not show a similar benefit from the addition of immunotherapy to chemotherapy. Here, we interrogated the matched WES data for recurrent alterations that may identify subsets of tumors with distinct immunological features. We identified several genes whose alterations significantly affected the enrichment of immune-related gene signatures. In particular, we identified MANF and CSK, genes previously shown to be potential tumor suppressors, as genes whose alteration associates with an “immune desert” transcriptional program. Specifically, genetic deletions or low RNA expression was associated with significantly decreased gene expression of gene signatures associated with lymphocytes, macrophages, and antigen presentation machinery. These data suggest a potential mechanism by which commonly inactivated tumor suppressors may shape the “immune desert” phenotype of SCLC. Further work is needed to determine if and how these genes may directly or indirectly modulate the tumor-immune interaction. Citation Format: Myung Chang Lee, Velimir Gayevskiy, Stefanie Morris, Minu K Srivastava, Namrata S Patil, Stephen V Liu, Martin Reck, Sanjeev Mariathasan, David S Shames, David P MacPherson, Robert J Johnston, Barzin Y Nabet. Identifying proliferative and immune-modulatory drivers of small cell lung cancer (SCLC) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A017.

Expression, DNA methylation pattern and transcription factor EPB41L3 in gastric cancer: a study of 262 cases

PurposeDNA methylation prominently inactivates tumor suppressor genes and facilitates oncogenesis. Previously, we delineated a chromosome 18 deletion encompassing the erythrocyte membrane protein band 4.1-like 3 (EPB41L3) gene, a progenitor for the tumor suppressor that is differentially expressed in adenocarcinoma of the lung-1 (DAL-1) in gastric cancer (GC).MethodsOur current investigation aimed to elucidate EPB41L3 expression and methylation in GC, identify regulatory transcription factors, and identify affected downstream pathways. Immunohistochemistry demonstrated that DAL-1 expression is markedly reduced in GC tissues, with its downregulation serving as an independent prognostic marker.ResultsHigh-throughput bisulfite sequencing of 70 GC patient tissue pairs revealed that higher methylation of non-CpGs in the EPB41L3 promoter was correlated with more malignant tumor progression and higher-grade tissue classification. Such hypermethylation was shown to diminish DAL-1 expression, thus contributing to the malignancy of GC phenotypes. The DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-CdR) was found to partially restore DAL-1 expression. Moreover, direct binding of the transcription factor CDC5L to the upstream region of the EPB41L3 promoter was identified via chromosome immunoprecipitation (ChIP)-qPCR and luciferase reporter assays. Immunohistochemistry confirmed the positive correlation between CDC5L and DAL-1 protein levels. Subsequent RNA-seq analysis revealed that DAL-1 significantly influences the extracellular matrix and space-related pathways. GC cell RNA-seq post-5-Aza-CdR treatment and single-cell RNA-seq data of GC tissues confirmed the upregulation of AREG and COL17A1, pivotal tumor suppressors, in response to EPB41L3 demethylation or overexpression in GC epithelial cells.ConclusionIn conclusion, this study elucidates the association between non-CpG methylation of EPB41L3 and GC progression and identifies the key transcription factors and downstream molecules involved. These findings enhance our understanding of the role of EPB41L3 in gastric cancer and provide a solid theoretical foundation for future research and potential clinical applications.

Identifying actionable synthetically lethal cancer gene pairs using mutual exclusivity.

Mutually exclusive loss-of-function alterations in gene pairs are those that occur together less frequently than may be expected and may denote a synthetically lethal relationship (SSL) between the genes. SSLs can be exploited therapeutically to selectively kill cancer cells. Here, we analysed mutation, copy number variation, and methylation levels in samples from The Cancer Genome Atlas, using the hypergeometric and the Poisson binomial tests to identify mutually exclusive inactivated genes. We focused on gene pairs where one is an inactivated tumour suppressor and the other a gene whose protein product can be inhibited by known drugs. This provided an abundance of potential targeted therapeutics and repositioning opportunities for several cancers. These data are available on the MexDrugs website, https://bioinformaticslab.sussex.ac.uk/mexdrugs.

Inflammaging and prognostic markers of endometriosis

Inflammaging, an age-associated inflammation, is a cellular stress response caused by DNA damage, activation of oncogenes or inactivation of tumor suppressors, oxidative stress, chemotherapy, mitochondrial dysfunction, or epigenetic changes. Damage to macromolecules leads to the cessation of proliferation due to the activation of pathways such as p53/p21CIP1 and p16INK4a/RB. These form the senescence-associated secretory phenotype (SASP), the molecular/cellular manifestations of which in endometrial cells have features similar to those observed in endometriosis. Presently, there are no uniform diagnostic criteria or established molecular markers that can predict the development and course of endometriosis. In this regard, it is relevant to develop new minimally invasive examination methods, statistically based criteria and molecular markers for early diagnosis and prognosis of endometriosis. This review article is devoted to identifying molecular markers that characterize the pathogenesis of endometriosis during inflaming. The aim of the study was to consider modern ideas about the mechanisms of inflaming and its role in the development of endometriosis to determine possible molecular markers for predicting the course of the pathology. We used the PubMed, Scopus and Google Scholar databases to analyze and systematize the literature over the past ten years. Our review reflects the main molecular mechanisms and prognostic criteria that characterize the development of endometriosis during inflaming.

Methylation-regulated tumor suppressor gene PDE7B promotes HCC invasion and metastasis through the PI3K/AKT signaling pathway.

Hepatocellular carcinoma (HCC) has a high mortality rate, and the mechanisms underlying tumor development and progression remain unclear. However, inactivated tumor suppressor genes might play key roles. DNA methylation is a critical regulatory mechanism for inactivating tumor suppressor genes in HCC. Therefore, this study investigated methylation-related tumor suppressors in HCC to identify potential biomarkers and therapeutic targets. We assessed genome-wide DNA methylation in HCC using whole genome bisulfite sequencing (WGBS) and RNA sequencing, respectively, and identified the differential expression of methylation-related genes, and finally screened phosphodiesterase 7B (PDE7B) for the study. The correlation between PDE7B expression and clinical features was then assessed. We then analyzed the changes of PDE7B expression in HCC cells before and after DNA methyltransferase inhibitor treatment by MassArray nucleic acid mass spectrometry. Furthermore, HCC cell lines overexpressing PDE7B were constructed to investigate its effect on HCC cell function. Finally, GO and KEGG were applied for the enrichment analysis of PDE7B-related pathways, and their effects on the expression of pathway proteins and EMT-related factors in HCC cells were preliminarily explored. HCC exhibited a genome-wide hypomethylation pattern. We screened 713 hypomethylated and 362 hypermethylated mCG regions in HCC and adjacent normal tissues. GO analysis showed that the main molecular functions of hypermethylation and hypomethylation were "DNA-binding transcriptional activator activity" and "structural component of ribosomes", respectively, whereas KEGG analysis showed that they were enriched in "bile secretion" and "Ras-associated protein-1 (Rap1) signaling pathway", respectively. PDE7B expression was significantly down-regulated in HCC tissues, and this low expression was negatively correlated with recurrence and prognosis of HCC. In addition, DNA methylation regulates PDE7B expression in HCC. On the contrary, overexpression of PDE7B inhibited tumor proliferation and metastasis in vitro. In addition, PDE7B-related genes were mainly enriched in the PI3K/ATK signaling pathway, and PDE7B overexpression inhibited the progression of PI3K/ATK signaling pathway-related proteins and EMT. PDE7B expression in HCC may be regulated by promoter methylation. PDE7B can regulate the EMT process in HCC cells through the PI3K/AKT pathway, which in turn affects HCC metastasis and invasion.

Targeted Therapy in Mesotheliomas: Uphill All the Way.

Mesothelioma (MM) is an aggressive and lethal disease with few therapeutic opportunities. Platinum-pemetrexed chemotherapy is the backbone of first-line treatment for MM. The introduction of immunotherapy (IO) has been the only novelty of the last decades, allowing an increase in survival compared to standard chemotherapy (CT). However, IO is not approved for epithelioid histology in many countries. Therefore, therapy for relapsed MM remains an unmet clinical need, and the prognosis of MM remains poor, with an average survival of only 18 months. Increasing evidence reveals MM complexity and heterogeneity, of which histological classification fails to explain. Thus, scientific focus on possibly new molecular markers or cellular targets is increasing, together with the search for target therapies directed towards them. The molecular landscape of MM is characterized by inactivating tumor suppressor alterations, the most common of which is found in CDKN2A, BAP1, MTAP, and NF2. In addition, cellular targets such as mesothelin or metabolic enzymes such as ASS1 could be potentially amenable to specific therapies. This review examines the major targets and relative attempts of therapeutic approaches to provide an overview of the potential prospects for treating this rare neoplasm.

Semimethylation is a feature of diffuse large B-cell lymphoma, and subgroups with poor prognosis are characterized by global hypomethylation and short telomere length

BackgroundLarge B-cell lymphoma (LBCL) is the most common lymphoma and is known to be a biologically heterogeneous disease regarding genetic, phenotypic, and clinical features. Although the prognosis is good, one-third has a primary refractory or relapsing disease which underscores the importance of developing predictive biological markers capable of identifying high- and low-risk patients. DNA methylation (DNAm) and telomere maintenance alterations are hallmarks of cancer and aging. Both these alterations may contribute to the heterogeneity of the disease, and potentially influence the prognosis of LBCL.ResultsWe studied the DNAm profiles (Infinium MethylationEPIC BeadChip) and relative telomere lengths (RTL) with qPCR of 93 LBCL cases: Diffuse large B-cell lymphoma not otherwise specified (DLBCL, n = 66), High-grade B-cell lymphoma (n = 7), Primary CNS lymphoma (n = 8), and transformation of indolent B-cell lymphoma (n = 12). There was a substantial methylation heterogeneity in DLBCL and other LBCL entities compared to normal cells and other B-cell neoplasms. LBCL cases had a particularly aberrant semimethylated pattern (0.15 ≤ β ≤ 0.8) with large intertumor variation and overall low hypermethylation (β > 0.8). DNAm patterns could not be used to distinguish between germinal center B-cell-like (GC) and non-GC DLBCL cases. In cases treated with R-CHOP-like regimens, a high percentage of global hypomethylation (β < 0.15) was in multivariable analysis associated with worse disease-specific survival (DSS) (HR 6.920, 95% CI 1.499–31.943) and progression-free survival (PFS) (HR 4.923, 95% CI 1.286–18.849) in DLBCL and with worse DSS (HR 5.147, 95% CI 1.239–21.388) in LBCL. These cases with a high percentage of global hypomethylation also had a higher degree of CpG island methylation, including islands in promoter-associated regions, than the cases with less hypomethylation. Additionally, telomere length was heterogenous in LBCL, with a subset of the DLBCL-GC cases accounting for the longest RTL. Short RTL was independently associated with worse DSS (HR 6.011, 95% CI 1.319–27.397) and PFS (HR 4.689, 95% CI 1.102–19.963) in LBCL treated with R-CHOP-like regimens.ConclusionWe hypothesize that subclones with high global hypomethylation and hypermethylated CpG islands could have advantages in tumor progression, e.g. by inactivating tumor suppressor genes or promoting treatment resistance. Our findings suggest that cases with high global hypomethylation and thus poor prognosis could be candidates for alternative treatment regimens including hypomethylating drugs.

Tumor Suppressor MicroRNAs in Clinical and Preclinical Trials for Neurological Disorders.

Cancers and neurological disorders are two major types of diseases in humans. We developed the concept called the "Aberrant Cell Cycle Disease (ACCD)" due to the accumulating evidence that shows that two different diseases share the common mechanism of aberrant cell cycle re-entry. The aberrant cell cycle re-entry is manifested as kinase/oncoprotein activation and tumor suppressor (TS) inactivation, which are associated with both tumor growth in cancers and neuronal death in neurological disorders. Therefore, some cancer therapies (e.g., kinase/oncogene inhibition and TS elevation) can be leveraged for neurological treatments. MicroRNA (miR/miRNA) provides a new style of drug-target binding. For example, a single tumor suppressor miRNA (TS-miR/miRNA) can bind to and decrease tens of target kinases/oncogenes, producing much more robust efficacy to block cell cycle re-entry than inhibiting a single kinase/oncogene. In this review, we summarize the miRNAs that are altered in both cancers and neurological disorders, with an emphasis on miRNA drugs that have entered into clinical trials for neurological treatment.

Future of epigenetic immunotherapy in kidney cancer

In clinical practice, immune checkpoint inhibition based on the use of antibodies against PD-1 (programmed death 1), PD-L1 (programmed death-ligand 1) and CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) is actively used for treatment of kidney cancer. However, objective response to monotherapy with these drugs is observed only in 9–24 % of patients, and combinations with other anticancer drugs in most cases cause severe adverse reactions. At the same time, there is an increased risk of toxic liver damage, immune-dependent pneumonitis, and rash. Therefore, it is necessary to search for new methods of immunotherapy, the most promising of which is the method of viral mimicry based on epigenetic stimulation of retroelement expression. Double-stranded retroelement transcripts activate antiviral interferon response that induces apoptosis of tumor cells. To achieve this, inhibitors of DNA methyltransferase, deacetylase and histone methyltransferase are used which have been successfully applied to treat various malignant neoplasms. In the experiment, DNA methyltransferase inhibitor 5-aza-2-deoxytidine (decitabine) effectively inhibited clear cell renal cell carcinoma cells proliferation which indicates their potential in treatment of kidney cancer. However, similarly to other neoplasms, activation of retroelements in renal cell carcinoma serves as initiator of the tumor process as it leads to increased expression of oncogenes, inactivation of tumor suppressors, and genomic instability. Therefore, the method of viral mimicry requires a differentiated approach with inhibition of retroelements involved in carcinogenesis and simultaneous stimulation of expression of retrotransposons that are not involved in the mechanisms of tumor development and have immunogenic properties. For this, microRNAs derived from transposons can be used as guides for DNA methyltransferases. An analysis of scientific literature revealed 41 such microRNAs of which decreased expression in kidney cancer was established for miR-95, -887, -652, -585, -511, -502, -495, -493, -487b, -335; increased for miR-1249, -1266, -151a, -211, -2114, -2355, -28, -3144, -340, -342, -374a, -374b, -3934, -421, -545, -576, -582, -584, -616, -769; and specific expression in different tumor subtypes for miR-708, -577, -450b, -326, -3200, -31, -224, -192, -1271. Since activation of retroelements can lead to insertions into new genome loci with formation of new mutations involved in carcinogenesis, a promising direction in integrated immunotherapy of kidney cancer is the use of reverse transcriptase inhibitors.

MiR-939-3p induces sarcoma proliferation and poor prognosis via suppressing BATF2.

Sarcoma is a rare and aggressive malignancy with poor prognosis, in which oncogene activation and tumor suppressor inactivation are involved. Accumulated studies suggested basic leucine zipper transcription factor ATF-like 2 (BATF2) as a candidate tumor suppressor, but its specific role and mechanism in sarcoma remain unclear. The expression levels of BATF2 and miR-939-3p were evaluated by using human sarcoma samples, cell lines and xenograft mouse models. Bioinformatics analysis, qPCR, Western blot, cell proliferation assay, overexpression plasmid construction, point mutation and dual luciferase reporter assay were utilized to investigate the role and mechanism of miR-939-3p in sarcoma. In this study, we demonstrated that the expression of BATF2 was downregulated in human sarcoma tissues and cell lines. The downregulation of BATF2 was negatively associated with the prognosis of sarcoma patients. Subsequent bioinformatic prediction and experimental validations showed that BATF2 expression was reduced by microRNA (miR)-939-3p mimic and increased by miR-939-3p inhibitor. Additionally, miR-939-3p was upregulated in sarcoma tissues and cells, correlating with a poor prognosis of sarcoma patients. Moreover, miR-939-3p overexpression suppressed sarcoma cell proliferation, which was significantly attenuated by the restoration of BATF2, while siRNA-mediated knockdown of BATF2 aggravated the miR-939-3p-induced promotion of sarcoma cell proliferation. Further computational algorithms and dual-luciferase reporter assays demonstrated that miR-939-3p repressed BATF2 expression via directly binding to its 3' untranslated region (3' UTR). Collectively, these findings identified miR-939-3p as a novel regulator of BATF2, as well as a prognostic biomarker in sarcoma, and revealed that suppressing miR-939-3p or inducing BATF2 expression may serve as a promising therapeutic strategy against sarcoma.

Targeted inhibition of SCFSKP2 confers anti-tumor activities resulting in a survival benefit in osteosarcoma

Osteosarcoma(OS) is a highly aggressive bone cancer for which treatment has remained essentially unchanged for decades. Although OS is characterized by extensive genomic heterogeneity and instability, RB1 and TP53 have been shown to be the most commonly inactivated tumor suppressors in OS. We previously generated a mouse model with a double knockout (DKO) of Rb1 and Trp53 within cells of the osteoblastic lineage, which largely recapitulates human OS with nearly complete penetrance. SKP2 is a repression target of pRb and serves as a substrate recruiting subunit of the SCFSKP2 complex. In addition, SKP2 plays a central role in regulating the cell cycle by ubiquitinating and promoting the degradation of p27. We previously reported the DKOAA transgenic model, which harbored a knock-in mutation in p27 that impaired its binding to SKP2. Here, we generated a novel p53-Rb1-SKP2 triple-knockout model (TKO) to examine SKP2 function and its potential as a therapeutic target in OS. First, we observed that OS tumorigenesis was significantly delayed in TKO mice and their overall survival was markedly improved. In addition, the loss of SKP2 also promoted an apoptotic microenvironment and reduced the stemness of DKO tumors. Furthermore, we found that small-molecule inhibitors of SKP2 exhibited anti-tumor activities in vivo and in OS organoids as well as synergistic effects when combined with a standard chemotherapeutic agent. Taken together, our results suggest that SKP2 inhibitors may reduce the stemness plasticity of OS and should be leveraged as next-generation adjuvants in this cancer.

Methylation Status of CDH1 Gene in Gastric Biopsy Specimens and Its Association with H. Pylori Infection.

Helicobacter pylori (H. pylori) have been accepted as having an etiologic role in gastro-duodenal diseases as chronic gastritis, peptic ulcer, and gastric carcinoma. Methylation of CGI has been correlated with the tumorigenic process since it can inactivate tumor suppressor genes. CDH1 is a tumor suppressor gene that encodes the E-cadherin protein, which is preserving cell-cell connections. Early stages of gastric carcinogenesis may be affected by the promoter methylation-mediated inactivation of this gene. This study aimed to investigate the methylation status of CDH1 using Methylation-Specific PCR (MSP) technique in clinical suspected patients with H. pylori infection who undergoing upper gastrointestinal endoscopy and correlated it with H. pylori detection by glmM PCR test. Fifty gastric mucosal biopsies were selected from one hundred and five samples included in this study. The detection of H. pylori was performed with the PCR primers specific to glmM gene. Bisulfite modification was done and the methylation status of the CDH1 gene was detected using MSP reaction. H. pylori was detected in 36% (18/50) of study population using glmM gene PCR test, 89% (16/18) of H. pylori positive cases were CDH1 methylated positive (chi-square, p-value=0.002). CDH1 methylation can be present in cancerous and noncancerous gastric mucosa, where 60% (18/30) of CDH1 methylation positive gastric mucosa showed gastritis as an endoscopy finding and gastric cancer in 6% (2/30). There was a significant correlation between and CDH1 methylation positive results and age group (P-value = 0.02). There was no significant correlation between CDH1 methylation positive results and participants gender (p-value=0.431) and clinical symptoms (all P-value > 0.05). This work suggested strong significance association between H. pylori infection and CDH1 methylation.

Oncogenic context shapes the fitness landscape of tumor suppression

Tumors acquire alterations in oncogenes and tumor suppressor genes in an adaptive walk through the fitness landscape of tumorigenesis. However, the interactions between oncogenes and tumor suppressor genes that shape this landscape remain poorly resolved and cannot be revealed by human cancer genomics alone. Here, we use a multiplexed, autochthonous mouse platform to model and quantify the initiation and growth of more than one hundred genotypes of lung tumors across four oncogenic contexts: KRAS G12D, KRAS G12C, BRAF V600E, and EGFR L858R. We show that the fitness landscape is rugged—the effect of tumor suppressor inactivation often switches between beneficial and deleterious depending on the oncogenic context—and shows no evidence of diminishing-returns epistasis within variants of the same oncogene. These findings argue against a simple linear signaling relationship amongst these three oncogenes and imply a critical role for off-axis signaling in determining the fitness effects of inactivating tumor suppressors.

The role of mycoplasmas as an infectious agent in carcinogenesis

The review presents data on studies of the role of mycoplasmas as infectious agents in carcinogenesis, as well as their participation in cancer drug therapy and the impact on the outcome of treatment. Mycoplasmas are of particular interest because they have unique abilities to readily attach to and enter eukaryotic cells, modulate their functional state, and induce chronic inflammation while evading the host’s immune system. The review will highlight the data confirming the increased colonization of tumor tissue by mycoplasmas compared to healthy ones, describe the molecular mechanisms by which mycoplasmas activate the expression of oncogenes and growth factors, inactivate tumor suppressors, promote NF-κB-dependent migration of cancer cells and modulate apoptosis, which results in abnormal growth and transformation of host cells. The review also presents data on the effectiveness of anticancer drugs in mycoplasmal infections.

Association between Human Papilloma Virus (HPV) genotype and mutant protein 53 (p53) expression in cervical cancer

Link of Video Abstract:https://youtu.be/fff-Unnud2w Background: Human Papilloma Virus (HPV) infection is the main cause of cervical cancer, a primary malignant tumor developing from squamous epithelial cells. HPV expresses oncoproteins E6 and E7, known to inactivate tumor suppressor proteins, one of which is protein 53 (p53). A promising biomarker for diagnosing and prognosis malignancies brought on by the HPV genotype is the identification of p53. This study investigates the association between p53 expression and HPV genotype in cervical cancer patients. Method: This observational cross-sectional study involved patients diagnosed with cervical cancer on histopathological examination who met the requirements. The polymerase chain reaction (PCR) method was used to determine the HPV genotype, and an immunohistochemistry analysis was used to assess the level of p53 expression. A Chi-Square test is utilized to evaluate the association between the two variables. Results: Of the 49 patients, there were 7 (14.3%) patients with HPV type 16, 13 (26.5%) with HPV type 18, 14 (26.5%) other types of HPV and 15 (30.6%) negatives for HPV. Examination of p53 expression showed that 17 (34.6%) samples had &lt;10% expression, 17 (34.6%) had 10-50% expression, and 15 (30.8%) samples had &gt;50% expression. There was no correlation between p53 expression and HPV genotype (p = 0.071). However, an association between p53 expression and cervical cancer's clinical stage was identified (p = 0.028). Conclusion: Increased cervical cancer stage can be associated with increased p53 expression. Thus, p53 can be used as a predictor of cervical cancer stage. However, in cervical cancer, p53 expression cannot be associated with the HPV genotype.

Modeling human brain rhabdoid tumor by inactivating tumor suppressor genes in induced pluripotent stem cells

Atypical teratoid/rhabdoid tumor (ATRT) is a rare childhood malignancy that originates in the central nervous system. Over ninety-five percent of ATRT patients have biallelic inactivation of the tumor suppressor gene SMARCB1. ATRT has no standard treatment, and a major limiting factor in therapeutic development is the lack of reliable ATRT models. We employed CRISPR/Cas9 gene-editing technology to knock out SMARCB1 and TP53 genes in human episomal induced pluripotent stem cells (Epi-iPSCs), followed by brief neural induction, to generate an ATRT-like model. The dual knockout Epi-iPSCs retained their stemness with the capacity to differentiate into three germ layers. High expression of OCT4 and NANOG in neurally induced knockout spheroids was comparable to that in two ATRT cell lines. Beta-catenin protein expression was higher in SMARCB1-deficient cells and spheroids than in normal Epi-iPSC-derived spheroids. Nucleophosmin, Osteopontin, and Ki-67 proteins were also expressed by the SMARCB1-deficient spheroids. In summary, the tumor model resembled embryonal features of ATRT and expressed ATRT biomarkers at mRNA and protein levels. Ribociclib, PTC-209, and the combination of clofilium tosylate and pazopanib decreased the viability of the ATRT-like cells. This disease modeling scheme may enable the establishment of individualized tumor models with patient-specific mutations and facilitate high-throughput drug testing.

The spectrum and significance of secondary (co-occurring) genetic alterations in sarcomas: the hallmarks of sarcomagenesis.

Bone and soft tissue tumors are generally classified into complex karyotype sarcomas versus those with recurrent genetic alterations, often in the form of gene fusions. In this review, we provide an overview of important co-occurring genomic alterations, organized by biological mechanisms and covering a spectrum of genomic alteration types: mutations (single-nucleotide variations or indels) in oncogenes or tumor suppressor genes, copy number alterations, transcriptomic signatures, genomic complexity indices (e.g. CINSARC), and complex genomic structural variants. We discuss the biological and prognostic roles of these so-called secondary or co-occurring alterations, arguing that recognition and detection of these alterations may be significant for our understanding and management of mesenchymal tumors. On a related note, we also discuss major recurrent alterations in so-called complex karyotype sarcomas. These secondary alterations are essential to sarcomagenesis via a variety of mechanisms, such as inactivation of tumor suppressors, activation of proliferative signal transduction, telomere maintenance, and aberrant regulation of epigenomic/chromatin remodeling players. The use of comprehensive genomic profiling, including targeted next-generation sequencing panels or whole-exome sequencing, may be incorporated into clinical workflows to offer more comprehensive, potentially clinically actionable information. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Recurrent Tumor Suppressor Alterations in Primary Pericardial Mesothelioma

Primary pericardial mesotheliomas are extremely rare, accounting for <1% of all mesotheliomas, and their molecular genetic features and predisposing factors remain to be determined. Here, we report the clinicopathologic, immunohistochemical, and molecular genetic findings of 3 pericardial mesotheliomas without pleural involvement. Three cases diagnosed between 2004 and 2022 were included in the study and analyzed by immunohistochemistry and targeted next-generation sequencing (NGS); corresponding nonneoplastic tissue was sequenced in all cases. Two patients were female and 1 was male, aged between 66 and 75 years. Two patients each had prior asbestos exposure and were smokers. Histologic subtypes were epithelioid in 2 cases and biphasic in 1 case. Immunohistochemical staining identified expression of cytokeratin AE1/AE3 and calretinin in all cases, D2-40 in 2 cases, and WT1 in 1 case. Staining for tumor suppressors revealed loss of p16, MTAP, and Merlin (NF2) expression in 2 cases and loss of BAP1 and p53 in 1 case. Abnormal cytoplasmic BAP1 expression was observed in an additional case. Protein expression abnormalities correlated with NGS results, which showed concurrent complete genomic inactivation of CDKN2A/p16, CDKN2B, MTAP, and NF2 in 2 mesotheliomas and of BAP1 and TP53 in 1 mesothelioma each, respectively. In addition, 1 patient harbored a pathogenic BRCA1 germline mutation, which resulted in biallelic inactivation in the mesothelioma. All mesotheliomas were mismatch repair proficient and showed several chromosomal gains and losses. All patients died from disease. Our study demonstrates that pericardial mesotheliomas share common morphologic, immunohistochemical, and molecular genetic features with pleural mesothelioma, including recurrent genomic inactivation of canonical tumor suppressors. Our study adds new insights into the genetic landscape of primary pericardial mesothelioma and highlights BRCA1 loss as a potential contributing factor in a subset of cases, thereby contributing to refined precision diagnostics for this rare cancer.

Abstract A078: Identifying and targeting the genetic determinants of immune suppression and immunotherapy failure in prostate cancer

Abstract The advent of immunotherapies such as immune checkpoint blockade (ICB) has greatly expanded the therapeutic options available to patients with treatment-refractory solid tumors. However, patients with castration-resistant prostate cancer (CRPC) generally remain unresponsive to ICB therapy due to an immunologically “cold” prostate tumor microenvironment (TME) with limited accessibility to cytotoxic lymphocytes such as Natural Killer (NK) and T cells that can mediate potent anti-tumor immunity. During the course of tumor evolution, CRPCs acquire many genomic alterations and copy number changes that can influence therapy outcomes. Yet the impact of such genetic alterations on the immune TME and response to ICB remains largely unknown, in part due to the lack of models to functionally study them in an immune competent setting. To this end, we previously developed a novel electroporation-based genetically engineered mouse model (EPO-GEMM) to rapidly generate genetically defined prostate tumors de novo within an intact prostate TME (Leibold*, Ruscetti* et al., Cancer Discovery 2020). Here, we applied the EPO-GEMM approach to characterize the immune landscapes of distinct genetic subtypes of CRPC and subsequently identify and target tumor intrinsic mediators of immune suppression for prostate cancer immunotherapy. Using transposon systems to overexpress the MYC oncogene and CRISPR/Cas9 vectors to inactivate tumor suppressor genes (TSGs) commonly altered in CRPC (Pten, P53, Rb1, Apc), we generated a suite of genetically distinct prostate cancer-bearing EPO-GEMMs. Histological analysis revealed genotype-specific immune infiltrates, with MYC-driven tumors generally lacking cytotoxic CD8+ T cells and harboring increased numbers of regulatory T cells (Tregs). A similar correlation was also observed between MYC levels and cytotoxic lymphocyte suppression in CRPC patient samples. In particular, tumors with compound MYC overexpression and p53 loss (MP) displayed drastic NK cell suppression. Cytokine and RNA-seq profiling of primary EPO-GEMM CRPCs, tumor-derived cell lines, and isogenic Myc-CaP cells engineered with TSG losses revealed that MP alterations led to not only inhibition of inflammatory cytokine and interferon signaling and antigen presentation/processing gene expression, but also induction of VEGF signaling and increased angiogenesis in the prostate TME. Treatment of MP tumors with a VEGFR2 antibody to block VEGF signaling significantly reduced tumor growth and increased NK and CD8+T cell infiltration and activation, indicating a phenotypic switch from a traditionally “cold” TME. Our results demonstrate that the genetic configuration of prostate cancer shapes its surrounding TME. Additionally, targeting VEGF signaling may be a mechanism to overcome ICB resistance in prostate cancer. More broadly, we believe that by understanding the tumor intrinsic mechanisms driving immune suppression, we can identify rational immunotherapy combinations for CRPC based on the genetic fingerprint of a patient’s tumor for precision medicine. Citation Format: Katherine C. Murphy, Kelly DeMarco, Yvette Lopez-Diaz, Lin Zhou, Marcus Ruscetti. Identifying and targeting the genetic determinants of immune suppression and immunotherapy failure in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A078.

The effect of virus-induced cellular transformation on oncogenesis

Aim to summarize the scientific data presented in the recent publications on tumor-associated processes induced by viruses. We analyzed 23 international publications devoted to the development and course of tumor-related processes associated with oncogenic viruses.&#x0D; The tumor-associated mechanisms are based on the processes of cell transformation, which largely depend on the state of telomeres. No less important are viral and cellular oncogenes, molecular circuits that control cell proliferation. Viral oncogenes encode proteins that increase the concentration of telomerase in the infected cells, and thereby increase the number of cell division cycles. The immune homeostasis, maintaining the integrity of body tissues, is regulated by activating and inhibiting metabolic pathways. The errors in the functioning of these signaling pathways caused by oncogenic viruses can lead to cell transformation and oncogenesis. Guaninenucleotide-binding protein RAS and protein kinase AKT are important components of signaling pathways that contribute to the production of D-type cyclins that control the cell cycle and regulate the activity of metabolic enzymes. Cyclin-dependent kinase is an important factor controlling cell cycles, damage and problems with nucleic acid replication, as well as proper assembly of the mitotic spindle. These processes can be disrupted by the transformation caused by oncogenic viruses. In most cases, viral oncogenes undergo additional changes that contribute to their transformation potential. The transformative activity of viral gene products correlates with binding to specific cellular proteins. In the immunopathogenesis of oncogenesis, an important role belongs to the inactivation of tumor suppressors, and the processes of phosphorylation.