Classical Tumor Suppressor Gene Research Articles

Current data and future perspectives on DNA methylation in ovarian cancer (Review).

Ovarian cancer (OC) represents the most prevalent malignancy of the female reproductive system. Its distinguishing features include a high aggressiveness, substantial morbidity and mortality, and a lack of apparent symptoms, which collectively pose significant challenges for early detection. Given that aberrant DNA methylation events leading to altered gene expression are characteristic of numerous tumor types, there has been extensive research into epigenetic mechanisms, particularly DNA methylation, in human cancers. In the context of OC, DNA methylation is often associated with the regulation of critical genes, such as BRCA1/2 and Ras‑association domain family 1A. Methylation modifications within the promoter regions of these genes not only contribute to the pathogenesis of OC, but also induce medication resistance and influence the prognosis of patients with OC. As such, a more in‑depth understanding of DNA methylation underpinning carcinogenesis could potentially facilitate the development of more effective therapeutic approaches for this intricate disease. The present review focuses on classical tumor suppressor genes, oncogenes, signaling pathways and associated microRNAs in an aim to elucidate the influence of DNA methylation on the development and progression of OC. The advantages and limitations of employing DNA methylation in the diagnosis, treatment and prevention of OC are also discussed. On the whole, the present literature review indicates that the DNA methylation of specific genes could potentially serve as a prognostic biomarker for OC and a therapeutic target for personalized treatment strategies. Further investigations in this field may yield more efficacious diagnostic and therapeutic alternatives for patients with OC.

Abstract 624: Ptpn1 deficiency collaborates with a NUP98::HOXD13 fusion gene to generate B cell precursor acute lymphoblastic leukemia

Abstract Background: Acute lymphoid leukemia is characterized by inherited or acquired mutations that effect critical differentiation and proliferation pathways. We have shown that Mcm2 deficient mice developed T cell acute lymphoblastic leukemia, due to copy number variations, most commonly interstitial deletions throughout the genome. When crossed with mice that expressed a NUP98::HOXD13 (NHD13) fusion gene, Mcm2−/−NHD13 mice developed B cell precursor ALL (BCP-ALL). A majority of these BCP-ALL had acquired homozygous deletions of Ptpn1, a protein tyrosine phosphatase, leading to the hypothesis that Ptpn1 deficiency combined with NHD13 expression leads to BCP ALL. Objective: To investigate the role of Ptpn1 deletion in BCP-ALL development using mouse models. Methods: Mice expressing an NHD13 fusion gene were crossed with Ptpn1 knockout mice, generating 6 possible genotypes. Mice were followed for 18 months. Mice with signs of leukemia were characterized by clinical evaluation, CBC, flow cytometry, and IHC. Primary BCP-ALL and derived BCP-ALL cell lines were also evaluated with RNA-seq and molecular pathway analysis. Results: NHD13+Ptpn1-/- mice developed BCP-ALL with 65% penetrance, characterized by hyperleukocytosis, anemia, thrombocytopenia, and invasion of non-hematopoietic tissues. Similar to human BCP-ALL, NHD13+Ptpn1-/- BCP-ALL had clonal IGH as well as clonal Tcrd gene rearrangements. Flow cytometry revealed CD19 and/or B220 expression. NHD13+Ptpn1+/- mice with BCP-ALL frequently lost the wild-type (WT) Ptpn1 allele in leukemic cells, reinforcing the hypothesis that Ptpn1 can function as a classic tumor suppressor gene in this context. Whole exome sequencing revealed acquired mutations in B-cell differentiation genes (Pax5 or Bcor) and activating mutations in tyrosine kinase genes (Jak1/3 and Flt3). Transcription signature analysis showed significant upregulation of Hoxa/b gene clusters, RNase12 and LncRNAs subsets. Conclusion: This study demonstrates that Ptpn1 loss combined with expression of NHD13 fusion gene leads to highly penetrant BCP-ALL in mice, suggesting a role for Ptpn1 in preventing malignant transformation. These findings present a collaborative model for BCP-ALL in which the NHD13 transgene leads to increased stem cell self-renewal, somatic Bcor or Pax5 mutations block normal B cell differentiation, and somatic signaling mutations (Jak1/3,Flt3) lead to hyperproliferation, which is potentiated by Ptpn1 deficiency. Citation Format: Nupur Nigam, Toshihiro Matsukawa, Ryan Bertoli, Michel L. Tremblay, Mianmian Yin, Peter D. Aplan. Ptpn1 deficiency collaborates with a NUP98::HOXD13 fusion gene to generate B cell precursor acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 624.

Abstract 3241: Elevated H3K27 trimethylation mediates adaptation to DNA demethylation in BRAFV600E-mutated colorectal cancer

Abstract Background: BRAFV600E in colorectal cancer (CRC) defines a unique subgroup of patients with poor prognosis and scarcely benefit from BRAF inhibitor-based treatment. Interestingly, BRAFV600E tumors demonstrated profound CpG island methylator phenotype (CIMP), suggesting extensive epigenetic dysregulation. Thus, we investigated epigenomic regulations in this subset of tumors with treatment-induced DNA demethylation. Methods: We treated six cell lines and two patient-derived xenograft (PDX) models of BRAFV600E CRC with vehicle, 5-azacitidine (DNMTi), vemurafenib (BRAFi), and combination treatment. PDX tumors treated with either vehicle or 5-azacitidine were collected on day 21 and sequenced for DNA methylation (Infinium HumanMethylation 450k BeadChip array), histone marks (ChIP-seq), chromatin accessibility (ATAC-seq), and gene expression (RNA-seq). Comprehensive post-translational modifications (PTMs) in histones with DNMTi or BRAFi treatment were assessed by mass spectrometry (MS). The combinational effect of 5-azacitidine with EZH2 or RNF2 inhibitors (Polycomb repressive complex activity (PRC) inhibition) was assessed through cell viability assays and colony formation assays in BRAFV600E CRC cell lines. Results: Analysis of TCGA COAD/READ tumors demonstrated a higher DNA methylation profile in BRAFV600E tumors than wild-type tumors even among the CIMP population (FDR < 1e-4). The combination of DNMTi with BRAFi showed selective additivity in vitro; however, did not improve efficacies in PDXs. The 5-azacitidine treatment induced profound demethylation in PDXs compared to control samples, predominantly attributed to promoter regions (48%). However, DNA demethylation failed to reactivate classic CIMP markers or tumor suppressor genes in CRC (e.g., CDKN2A, MGMT). Interestingly, demethylated genomic regions gained H3K27 trimethylation, repressive histone mark, in azacitidine-treated samples compared to control (9,431 vs 5,147 peaks respectively). We validated the elevation of H3K27 trimethylation as well as additional histone PTMs following DNMTi in MS-based histone analysis. Finally, combining a demethylating agent with EZH2i or RNF2i, which either deplete H3K27 trimethylation or block the PRC activity respectively, has demonstrated additive growth inhibitory effects in BRAFV600E CRC cell lines (p <0.05). Conclusions: This study unveiled intriguing aspects of demethylation agent treatment, which led to the compensatory engagement of other epigenomic markers, particularly repressive histone marks, resulting in limited efficacies to azacitidine treatment in preclinical BRAFV600E CRC models. This finding suggests the existence of adaptive interactions between epigenetic modifiers and potential clinical strategies in combinational epigenetic therapeutics. Citation Format: Hey Min Lee, Ajay Kumar Saw, Van Karlyle Morris, Anand Kamal Singh, Stefania Napolitano, Alexey Sorokin, Preeti Kanikarla Marie, Oluwadara Coker, Oscar Eduardo Villarreal, Nazanin Esmaeili Anvar, Kunal Rai, Scott Kopetz. Elevated H3K27 trimethylation mediates adaptation to DNA demethylation in BRAFV600E-mutated colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3241.

Abstract 1743: Pan-cancer review of TP53 somatic alterations as a function of tumor type: Sometimes lost, sometimes gained, frequently altered

Abstract Background: TP53 is the gene most frequently disrupted by somatic alterations in cancer. Mutations include single nucleotide variants resulting in functional alterations, as well as premature stop codons, splice variants, and cryptic alterations. TP53 gene dosage is disrupted by heterozygous and homozygous deletions, copy-neutral loss of heterozygosity, and other structural changes such as intragenic deletions and fusions. TP53 is altered by epigenetic events and viral oncogenes, such as E6 expressed by the human papillomavirus. The readout of altered TP53 includes decreased gene expression of either variant or wild-type alleles. Epistatic alteration of TP53 pathway components have also been described, with the E3 ubiquitin ligase MDM2 being the canonical example. Depending on the type of TP53 alteration, there may be differential signaling consequences, ranging from loss of function and dominant negative to oncogenic signaling. Hypothesis: Patterns of TP53 mutations will vary based on tumor type. Differential alterations of TP53 will suggest differential pathway dependence and downstream pathophysiology that may be relevant to signaling and therapeutic potential. Methods: We analyzed extensive cancer genomics repositories, such as the Pan-Cancer Atlas (10,000+ tumors, >30 cancer types) from The Cancer Genome Atlas project and the International Cancer Genome Consortium. Assessing genes in the TP53 pathway, we examined somatic mutations, copy number alterations, structural changes, and gene expression. Epigenetic data was considered when available. Results: A significant pattern amongst sarcomas, prostate cancers, and urothelial carcinomas emerged, revealing that certain tumors exhibited a predominant classic tumor suppressor gene genotype. This genotype featured frequent homozygous deletions, inactivating mutations, and relatively low TP53 gene expression, often accompanied by epistatic MDM2 amplification. By contrast, other tumors demonstrate an atypical pattern in which TP53 mutations were rarely if ever associated with loss of TP53 gene expression with low levels of homozygous TP53 gene deletion. Likewise, atypical TP53 mutant tumor types rarely demonstrated MDM2 amplifications, and in opposition to typical TP53 cases, MDM2 amplification was positively associated with TP53 mutation rather than anti-correlated. Head and neck squamous cell carcinoma, uterine carcinoma, and pancreatic adenocarcinoma followed this pattern. Investigation within selected tumor types such as lung squamous cell carcinoma suggested that both typical and atypical TP53 mutation patterns could be observed. Conclusions: TP53 mutation patterns were strongly associated with tumor type and may suggest differential pathway activation according to the somatic alteration status. Citation Format: Ubaid A. Tanveer, David N. Hayes, Julie George, Jeremiah R. Holt, Martin Peifer, Roman Thomas. Pan-cancer review of TP53 somatic alterations as a function of tumor type: Sometimes lost, sometimes gained, frequently altered [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1743.

CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression

Metastatic prostate cancer (PCa) poses a significant therapeutic challenge with high mortality rates. Utilizing CRISPR-Cas9 in vivo, we target five potential tumor suppressor genes (Pten, Trp53, Rb1, Stk11, and RnaseL) in the mouse prostate, reaching humane endpoint after eight weeks without metastasis. By further depleting three epigenetic factors (Kmt2c, Kmt2d, and Zbtb16), lung metastases are present in all mice. While whole genome sequencing reveals few mutations in coding sequence, RNA sequencing shows significant dysregulation, especially in a conserved genomic region at chr5qE1 regulated by KMT2C. Depleting Odam and Cabs1 in this region prevents metastasis. Notably, the gene expression signatures, resulting from our study, predict progression-free and overall survival and distinguish primary and metastatic human prostate cancer. This study emphasizes positive genetic interactions between classical tumor suppressor genes and epigenetic modulators in metastatic PCa progression, offering insights into potential treatments.

SAT177 Molecular Analysis Of AMBRA1 As A Candidate Tumor Suppressor In Sporadic Parathyroid Adenomas

Abstract Disclosure: S. Chinwo: None. J. Costa: None. A. Arnold: None. J. Bellizzi: None. Primary hyperparathyroidism is a common endocrine disorder that is most often caused by a sporadic single-gland parathyroid adenoma. Currently, the only known and experimentally validated oncoprotein for parathyroid adenomas is cyclin D1. The overexpression of cyclin D1 protein has been reported in 20-40% of cases and about 8 percent of cases harbor a DNA rearrangement involving the cyclin D1 (CCND1) locus. Otherwise, the molecular mechanisms underlying this cyclin D1 overexpression have yet to be identified. Here, we explored a potential parathyroid tumorigenic mechanism that could increase cyclin D1 stability through a defect in the AMBRA1 E3 ligase adaptor, a molecule responsible for its degradation. Evidence that AMBRA1 may be a tumor suppressor gene includes the accumulation of cyclin D with hyperproliferation in AMBRA1-deficient cells, the context-dependent growth of tumors in Ambra1-deficient mice, and the finding that low levels of AMBRA1 correlated with poor clinical outcomes in The Cancer Genome Atlas. Therefore, we proceeded to examine AMBRA1 for evidence of tumor suppressor-type inactivation within a cohort of 98 typical parathyroid adenomas. Genomic DNA samples from these tumors were subjected to PCR-amplification of all 18 coding exons of AMBRA1, followed by Sanger sequencing. Sequences were analyzed by comparison to the normal reference sequence: ENST00000683756.1, seeking to assess any observed intragenic or splice-site variants as potential or likely loss-of-function mutations. Thus far, with 97 percent of the coding region for this 1298-amino acid protein fully sequenced, we have identified an inactivating mutation in 1 of the 98 tumors (1%): c.126G>A in exon 2, resulting in an early stop codon p.Trp42*. Sequencing the patient’s matched germline DNA confirmed the mutation was somatic and showed loss of heterozygosity. We also identified likely-nonpathogenic variants in 5 parathyroid adenomas: missense variant c.3385G>T (p.Ala1129Ser), 3’ UTR variants c.*576A>T and c.*491T>C and synonymous variants c.579C>T and c.2776C>T, each in a single tumor. 4 of 5 were found as germline variants in the normal population and the one missense scored benign by in silico criteria. To conclude, our observations suggest that AMBRA1 may function as a classical tumor suppressor gene in sporadic parathyroid adenomas at very low frequency. Further study may include protein expression analysis of AMBRA1 to investigate influences on gene expression. To further elucidate the mechanisms behind cyclin D1 overexpression in parathyroid adenomas, other molecules that participate in its degradation should be explored as potential tumor suppressors. Presentation: Saturday, June 17, 2023

Abstract 5279: Molecular analysis of AMBRA1 as a candidate tumor suppressor in sporadic parathyroid adenomas

Abstract Primary hyperparathyroidism is a common endocrine disorder that is most often caused by a sporadic single-gland parathyroid adenoma. Currently, the only known and experimentally validated oncoprotein for parathyroid adenomas is cyclin D1. Overexpression of cyclin D1 has been reported in 20-40% of cases; about 8 percent of cases harbor a DNA rearrangement involving the cyclin D1 (CCND1) locus. Other molecular mechanisms underlying this cyclin D1 overexpression have yet to be identified. Here, we explored a potential parathyroid tumorigenic mechanism that could increase cyclin D1 stability through a defect in the AMBRA1 E3 ligase adaptor, a molecule responsible for its degradation. Evidence that AMBRA1 may be a tumor suppressor gene includes: accumulation of cyclin D with hyperproliferation in AMBRA1-deficient cells, context-dependent growth of tumors in Ambra1-deficient mice, and the correlation between low levels of AMBRA1 and poor clinical outcomes in The Cancer Genome Atlas. Therefore, we examined AMBRA1 for evidence of tumor suppressor-type inactivation in a cohort of 98 typical parathyroid adenomas. Tumor genomic DNA was subjected to PCR-amplification of all 18 coding exons of AMBRA1, followed by Sanger sequencing. Sequences were analyzed by comparison to the normal reference sequence (ENST00000683756.1), seeking to assess any observed intragenic or splice-site variants as potential or likely loss-of-function mutations. Thus far, with 97 percent of the coding region for this 1298-amino acid protein fully sequenced, we have identified an inactivating mutation in 1 of 98 tumors (1%): c.126G>A, resulting in an early stop codon p.Trp42. This mutation showed loss of heterozygosity and was confirmed to be somatic by sequencing of the patient’s matched germline DNA. We also identified likely non-pathogenic variants in 5 tumors: missense variant c.3385G>T (p.Ala1129Ser), 3’ UTR variants c.*576A>T and c.*491T>C and synonymous variants c.579C>T and c.2776C>T, each in a single tumor. 4 of 5 were found as germline variants in the normal population and the one missense scored as benign by in silico criteria. To conclude, our observations suggest that AMBRA1 may function as a classical tumor suppressor gene in sporadic parathyroid adenomas at very low frequency. Further study may include protein expression analysis of AMBRA1 to investigate influences on gene expression. To further elucidate the mechanisms behind cyclin D1 overexpression in parathyroid adenomas, other molecules that participate in its degradation should be explored as potential tumor suppressors. Citation Format: Stephanie Chinwo, Justin Bellizzi, Jessica Costa-Guda, Andrew Arnold. Molecular analysis of AMBRA1 as a candidate tumor suppressor in sporadic parathyroid adenomas. [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 5279.

PSAT176 Molecular Analysis of AMBRA1 as a Candidate Tumor Suppressor in Sporadic Parathyroid Adenomas

Abstract Primary hyperparathyroidism is a common endocrine disorder that is most often caused by a sporadic single-gland parathyroid adenoma. Currently, the only known and experimentally validated oncoprotein for parathyroid adenomas is cyclin D1. The overexpression of cyclin D1 protein has been reported in twenty to forty percent of cases. Eight percent of this overexpression can be explained by DNA rearrangement involving the cyclin D1 locus. The remaining molecular mechanisms behind this overexpression have yet to be identified. In this study, we explored a potential parathyroid tumorigenic mechanism that could increase cyclin D1 stability through a defect in the AMBRA1 E3 ligase adaptor, a molecule responsible for its degradation. Evidence that AMBRA1 may be a tumor suppressor gene includes the accumulation of cyclin D with hyperproliferation in AMBRA1-deficient cells, the context-dependent growth of tumors in Ambra1-deficient mice, and the finding that low levels of AMBRA1 correlated with poor clinical outcomes in The Cancer Genome Atlas (TCGA). We have therefore proceeded examine AMBRA1 for evidence of tumor suppressor-type inactivation within a cohort of parathyroid adenomas. Genomic DNA from twenty-six typical sporadic parathyroid adenomas was subjected to PCR-amplification of all eighteen coding exons of AMBRA1, followed by Sanger sequencing. Sequences were analyzed by comparison to the normal reference sequence, seeking to assess any observed intragenic or splice-site variants as potential or likely loss-of-function mutations. Thus far, with ninety-seven percent of the coding region fully sequenced, no intragenic AMBRA1 inactivating mutations were observed. Three known single nucleotide polymorphisms, considered non-pathogenic, were identified: missense variant c.3028G>T, and 3' UTR variants c.*491T>C and c.*576A>T, each in a single tumor. Future study of increased numbers of parathyroid adenomas is needed to exclude the possibility that AMBRA1 inactivation could play a driver role in a small percentage of cases, and the possibility that AMBRA1 might contribute less directly e.g. via secondarily altered expression also warrants investigation. Nonetheless, our current observations strongly suggest that AMBRA1 does not have a frequent role as a classic 2-hit tumor suppressor gene in sporadic parathyroid adenomas. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Inactivation of RB1, CDKN2A, and TP53 have distinct effects on genomic stability at side-by-side comparison in karyotypically normal cells.

Chromosomal instability is a common feature in malignant tumors. Previous studies have indicated that inactivation of the classical tumor suppressor genes RB1, CDKN2A, and TP53 may contribute to chromosomal aberrations in cancer by disrupting different aspects of the cell cycle and DNA damage checkpoint machinery. We performed a side-by-side comparison of how inactivation of each of these genes affected chromosomal stability in vitro. Using CRISPR-Cas9 technology, RB1, CDKN2A, and TP53 were independently knocked out in karyotypically normal immortalized cells, after which these cells were followed over time. Bulk RNA sequencing revealed a distinct phenotype with upregulation of pathways related to cell cycle control and proliferation in all three knockouts. Surprisingly, the RB1 and CDKN2A knocked out cell lines did not harbor more copy number aberrations than wild-type cells, despite culturing for months. The TP53-knocked out cells, in contrast, showed a massive amount of copy number alterations and saltatory evolution through whole genome duplication. This side-by-side comparison indicated that the effects on chromosomal stability from inactivation of RB1 and CDKN2A are negligible compared to inactivation of TP53, under the same conditions in a nonstressful environment, even though partly overlapping regulatory pathways are affected. Our data suggest that loss of RB1 and CDKN2A alone is not enough to trigger surviving detectable aneuploid clones while inactivation of TP53 on its own caused massive CIN leading to saltatory clonal evolution in vitro and clonal selection.

Research Progress of DNA Methylation in Endometrial Cancer.

Endometrial cancer (EC)) is one of the most common malignant tumors of the female genital system, with an increasing incidence and mortality, worldwide. Although the therapeutic strategy of EC is still complicated and challenging, further understanding of carcinogenesis from a gene perspective would allow an effort to improve therapeutic precision in this complex malignancy. DNA methylation is the most widely studied epigenetic alteration in human tumors. Aberrant DNA methylation events, resulting in altered gene expression, are features of many tumor types. In this review, we provide an update on evidence about the roles of aberrant DNA methylation within some classical tumor suppressor genes and oncogenes in endometrial carcinogenesis, and report on recent advances in the understanding of the contribution of aberrant DNA methylation to EC, as well as opportunities and challenges of DNA methylation in EC management and prevention.

Circular RNA NF1-419 Inhibits Proliferation and Induces Apoptosis by Regulating Lipid Metabolism in Astroglioma Cells.

Astroglioma is the most common primary tumor of the central nervous system. Currently, there is no effective treatment for astroglioma. In the present study, the extract (L3) from Ganoderma Lucidum (G. lucidum) was found to inhibit the growth of astroglioma U87 cells and change the expression of circular RNAs (circRNAs). One of these, including the circular NF1-419 (circNF1-419), was of interest because NF1 gene is a classic tumor suppressor gene. The functional role of circ-NF1-419 in the inhibition of astroglioma cells remains unknown. This study focuses on the role of circNF1-419 in functional abnormalities of U87 astroglioma cells and aims to elaborate on its regulatory mechanism. The circNF1-419 overexpressing U87 (U87-NF1-419) cells were constructed. We generated U87-NF1-419 to evaluate the role of circNF1-419 on cell cycle, apoptosis, proliferation, tumor growth and metabolic regulation. Finally, we used docking screening to identify compounds in G. lucidum extracts that target circ-419. U87-NF1-419 can promote cell apoptosis and regulate lipid metabolism through glycerophospholipid metabolism and retrograde endocannabinoid signaling. Further examinations revealed that the expression of metabolic regulators, such as L-type voltage-operated calcium channels (L-VOCC), phospholipase C-β3 (PLCβ3), Mucin1, cationic amino acid transporter 4 (CAT4), cationic amino acid transporter 1 (CAT1) and a kinase (PRKA) anchor protein 4 (AKAP4) was inhibited, while phosphatidylserine synthase 1 (PTDSS1) was enhanced in U87-NF1-419 cells. In vivo experiments showed that circNF1-419 inhibits tumor growth in BALB/C nude mice, and enhanced AKAP4 and PTDSS1 in tumor tissues. The virtual docking screening results supported that ganosporeric acid A, ganodermatriol, ganoderic acid B and α-D-Arabinofuranosyladenine in L3 could activate circNF1-419 in astroglioma treatment. This study indicated that circNF1-419 could be a therapeutic target for the clinical treatment of astroglioma. L3 from Ganoderma Lucidum (G. lucidum) could inhibit astroglioma growth by activating circNF1-419.

BRCA1-Associated Protein 1 (BAP-1) as a Prognostic and Predictive Biomarker in Clear Cell Renal Cell Carcinoma: A Systematic Review

BACKGROUND: The gene that encodes BRCA1-associated protein 1 (BAP1) has been reported to be dysregulated in several human cancers such as uveal melanoma, malignant pleural mesothelioma, hepatocellular carcinoma, thymic epithelial tumors, and clear-cell renal cell carcinoma (ccRCC). The gene is located on the human chromosome 3p21.3, encoding a deubiquitinase and acts as a classic two-hit tumor suppressor gene. BAP1 predominantly resides in the nucleus, where it interacts with several chromatin-associated factors, as well as regulates calcium signaling in the cytoplasm. As newer therapies continue to evolve for the management of RCC, it is important to understand the role of BAP1 mutation as a prognostic and predictive biomarker. OBJECTIVE: We aimed to systematically evaluate the role of BAP1 mutations in patients with RCC in terms of its impact on prognosis and its role as a predictive biomarker. METHODS: Following PRISMA guidelines, we performed a systematic literature search using PubMed and Embase through March 2021. Titles and abstracts were screened to identify articles for full-text and then a descriptive review was performed. RESULTS: A total of 490 articles were initially identified. Ultimately 71 articles that met our inclusion criteria published between 2012–2021 were included in the analysis. Data were extracted and organized to reflect the role of BAP1 alterations as a marker of prognosis as well as a marker of response to treatments, such as mTOR inhibitors, VEGF tyrosine kinase inhibitors, and immune checkpoint inhibitors. CONCLUSIONS: Alterations in BAP1 appear to be uniformly associated with poor prognosis in patients with RCC. Knowledge gaps remain with regard to the predictive relevance of BAP1 alterations, especially in the context of immunotherapy. Prospective studies are required to more precisely ascertain the predictive value of BAP1 alterations in RCC.

Understanding BRCA2 Function as a Tumor Suppressor Based on Domain-Specific Activities in DNA Damage Responses.

BRCA2 is an essential genome stability gene that has various functions in cells, including roles in homologous recombination, G2 checkpoint control, protection of stalled replication forks, and promotion of cellular resistance to numerous types of DNA damage. Heterozygous mutation of BRCA2 is associated with an increased risk of developing cancers of the breast, ovaries, pancreas, and other sites, thus BRCA2 acts as a classic tumor suppressor gene. However, understanding BRCA2 function as a tumor suppressor is severely limited by the fact that ~70% of the encoded protein has not been tested or assigned a function in the cellular DNA damage response. Remarkably, even the specific role(s) of many known domains in BRCA2 are not well characterized, predominantly because stable expression of the very large BRCA2 protein in cells, for experimental purposes, is challenging. Here, we review what is known about these domains and the assay systems that are available to study the cellular roles of BRCA2 domains in DNA damage responses. We also list criteria for better testing systems because, ultimately, functional assays for assessing the impact of germline and acquired mutations identified in genetic screens are important for guiding cancer prevention measures and for tailored cancer treatments.

TRIM28 variants and Wilms' tumour predisposition.

TRIM28 was recently identified as a Wilms' tumour (WT) predisposition gene, with germline pathogenic variants identified in around 1% of isolated and 8% of familial WT cases. TRIM28 variants are associated with epithelial WT, but the presence of other tumour components or anaplasia does not exclude the presence of a germline or somatic TRIM28 variant. In children with WT, TRIM28 acts as a classical tumour suppressor gene, with both alleles generally disrupted in the tumour. Therefore, loss of TRIM28 (KAP1/TIF1beta) protein expression in tumour tissue by immunohistochemistry is an effective strategy to identify patients carrying pathogenic TRIM28 variants. TRIM28 is a ubiquitously expressed corepressor that binds transcription factors in a context‐, species‐, and cell‐type‐specific manner to control the expression of genes and transposable elements during embryogenesis and cellular differentiation. In this review, we describe the inheritance patterns, histopathological and clinical features of TRIM28‐associated WT, as well as potential underlying mechanisms of tumourigenesis during embryonic kidney development. Recognizing germline TRIM28 variants in patients with WT can enable counselling, genetic testing, and potential early detection of WT in other children in the family. A further exploration of TRIM28‐associated WT will help to unravel the diverse and complex mechanisms underlying WT development. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

In This Issue

See article, p. 32.Inflammatory signaling perturbs hematopoietic stem cell (HSC) differentiation through a process termed emergency hematopoiesis. Whether an equivalent process has a role in regulating leukemia stem cells (LSC) is unclear. Here Xie and colleagues demonstrate that sphingosine-1-phosphate receptor 3 (S1RP3), a receptor for proinflammatory and bioactive lipid sphingosine-1-phosphate (S1P), drives myeloid differentiation in both HSC and LSC. S1RP3 expression is high in CD33+ myeloid cells and low in primitive CD34+ cells. When S1RP3 is upregulated, it promotes myeloid differentiation in synergy with proinflammatory signaling pathways in HSC and LSC. In addition, S1RP3 expression level could be utilized to stratify acute myeloid leukemia (AML) patients into more differentiated and more primitive cases. Moreover, pharmacologic disruption of S1P signaling by the S1P prodrug FTY720 reduces LSC frequency and decreases leukemia burden in vivo. The results suggest that S1RP3 could serve as both a biomarker and therapeutic target in human AML.See article, p. 54.Specific alterations in DNA methylation patterns are common in cancer cells across tissue origins and genetic drivers. In this study, Kretzmer and colleagues asked how early cancer-associated methylation patterns emerge during chronic lymphocytic leukemia (CLL) oncogenesis. They have profiled DNA methylomes of single cells and bulk samples across cell types representing key stages of oncogenesis, from normal germinal center B-cell subsets to preneoplastic monoclonal B-cell lymphocytosis (MBL) to serial CLL samples at diagnosis and throughout disease progression and therapy. The authors identify cancer-specific methylation patterns distinguishing normal B-cell subtypes from MBL and CLL. These methylation patterns are shared among all CLL clones in each patient case and are already present in nonmalignant MBL cell state. Persistence of the methylation pattern is particularly remarkable when considered on the backdrop of genetic and transcriptional heterogeneity. These findings shed light on possible CLL cell of origin and provide support to the concept that precancerous methylome may be a prerequisite for oncogenic transformation and maintenance of neoplastic state.See article, p. 70.Activated B-cell–like diffuse large B-cell lymphoma (ABC-DLBCL) is associated with poor prognosis when compared with germinal center B-cell–like (GCB)-DLBCL, and there is an urgent need to develop a preclinical animal model that closely models human ABC-DLBCL for the development of effective targeting therapies. MYD88 mutation and gain of BCL2 copy numbers are two of the most frequent genomic aberrations in ABC-DLBCL. Here Flümann and colleagues characterize the phenotypes of a Myd88/BCL2-driven (MBC) mouse lymphoma model and find that this model closely resembles the morphologic and gene expression signatures of human ABC-DLBCL. Moreover, MBC-derived lymphomas undergo apoptosis in response to venetoclax, revealing their druggable vulnerabilities against BCL2. Furthermore, combined treatment of venetoclax plus anti–PD-1 antibody RMP1-14 extends survival of MBC mice after they develop tumors far longer than either drug alone. These findings validate MBC mice as a tool to study ABC-DLBCL and demonstrate preclinical evidence that targeting both BCL2 and PD-1 could serve as a novel therapy for the treatment of ABC-DLBCL.See article, p. 92.PTEN is the second most frequently mutated tumor suppressor gene in cancer. In this study, Tottone and colleagues identified a distal regulatory element enhancing PTEN expression (PE) as a recurrent deletion in human T-ALL. PE displays enhancer-associated chromatin marks in hematopoietic lineage, but not solid tissues. In several human T-ALL cell lines, the PE region is occupied by multiple transcription factors including Notch, is bidirectionally transcribed, and engages in long-range interactions with the PTEN promoter. PE deletion in these cells results in decreased PTEN mRNA and decreased proliferation. PE is conserved across species, and its germline as well as conditional deletion in mice results in dose-dependent diminished PTEN expression in T cells, but not other lineages. Notch1-driven leukemia development is accelerated on PE-null mouse background, providing functional evidence of PE tumor-suppressive role consistent with its deletion profile in human T-ALL. Cancer-associated genetic inactivation of tumor suppressor genes is mostly documented within their promoter or protein-coding regions. This study reveals that distal enhancer deletion is another mechanism by which a classic and ubiquitous tumor suppressor gene can be inactivated in cancer. This work identifies previously uncharacterized PTEN enhancer activity in normal T cells and in T-ALL. It invites exploration of lineage-specific enhancer regulation of PTEN and other classic tumor suppressors in physiology and oncogenesis.In This Issue is written by Blood Cancer Discovery editorial staff. Readers are encouraged to consult the original articles for full details.

Tumor Suppressors Having Oncogenic Functions: The Double Agents.

Cancer progression involves multiple genetic and epigenetic events, which involve gain-of-functions of oncogenes and loss-of-functions of tumor suppressor genes. Classical tumor suppressor genes are recessive in nature, anti-proliferative, and frequently found inactivated or mutated in cancers. However, extensive research over the last few years have elucidated that certain tumor suppressor genes do not conform to these standard definitions and might act as “double agents”, playing contrasting roles in vivo in cells, where either due to haploinsufficiency, epigenetic hypermethylation, or due to involvement with multiple genetic and oncogenic events, they play an enhanced proliferative role and facilitate the pathogenesis of cancer. This review discusses and highlights some of these exceptions; the genetic events, cellular contexts, and mechanisms by which four important tumor suppressors—pRb, PTEN, FOXO, and PML display their oncogenic potentials and pro-survival traits in cancer.

Effects of eight InDel variants in FHIT on milk traits in Xinjiang brown cattle

In our previous genome-wide association study (GWAS), we identified the fragile histidine triad diadenosine triphosphatase (FHIT) gene in Xinjiang brown cattle (XJBC) as a candidate gene associated with cattle productive traits, with potential application in mark-assisted selection (MAS) in cattle breeding. FHIT is a prototype of a class of tumor suppressor genes that contain genomic loci mapped to common fragile loci. Here, 388 healthy and unrelated XJBC were selected to identify insertion/deletion (InDel) variants in the bovine FHIT and assess their effects on milk traits. Eight of the thirteen InDel loci were found to be polymorphic in FHIT. The polymorphism information content of the eight loci ranged from 0.061 to 0.375. The correlation analysis showed that all the new InDel variants were significantly related to six different milk traits (p < 0.05). The following variants presented a significant relationship with productive traits: P2-23bp with the 305 milk yield (p = 0.005) in the sixth parity; P3-24bp with the milk fat yield (p = 0.009) in the third parity; P5-21bp with the somatic cell score (p = 0.001) in the first parity and with the milk protein percentage (p = 0.002) in the sixth parity; and P7-26bp with the somatic cell score (p = 0.003) in the sixth parity. These findings will help evaluate InDel genotypes, within and between cattle breeds and identify potential target loci to accelerate progress in MAS in cattle breeding.

SUN-909 A Unique Presentation of Multiple Endocrine Neoplasia (MEN) Type 1 Mosaicism Caused by a Novel c.124G&amp;gt;A Variant in the MEN1 Gene

The MEN1 gene is positioned on the long arm of chromosome 11 (11q13) and results in the production of menin. A classical tumour suppressor gene; the spectrum of reported germline MEN1 mutations occur throughout the gene with no strong genotype/phenotype relationship. Mosaicism is extremely rare, a recent report citing only 2 mosaic cases by next generation sequencing1. We describe a 43-year-old female with MEN1 mosaicism associated with parathyroid adenoma and probable gastrinoma.Our patient initially presented with nephrolithiasis and nephrocalcinosis alongside biochemistry of primary hyperparathyroidism. Imaging confirmed a right lower pole parathyroid adenoma and the lesion was removed, pathology confirming the diagnosis (July 2018). Post operatively she remained hypercalcaemic. Repeat imaging suggested a further right sided culprit lesion.One year previously (August 2017) she was admitted with a perforated duodenal ulcer within the first part of the duodenum (D1), ascribed to ibuprofen use, requiring surgical repair. Whilst awaiting repeat parathyroid surgery she represented with a further duodenal ulcer and perforation (February 2019) this time in the second part of the duodenum(D2). A second parathyroidectomy was performed and genetic investigations were instigated, given the presence of classical MEN1 phenotype. Again, pathology was consistent with a parathyroid adenoma (March 2019).DNA was extracted for next generation sequencing which revealed mosaicism with a c.124 G>A variant detected in the MEN1 gene at a level of approximately 15%.Further investigations have shown normal serum prolactin and pituitary and pancreatic MRI imaging. With no other family members testing positive (parents and 2/3 children) this mutation appears to be a de novo index case. Adjusted Calcium is normal 2.53 mmol/L (2.2-2.6 mmol/L) but serum Chromogranin A is elevated (2620 µg/L (ref range <95 µg/L)).The c.124 G>A variant predicts an amino acid substitution p.(Gly42Ser) in menin structure, an amino acid change previously associated with MEN12. Given the paucity of MEN1 mosaicism reported within the literature with the presence of the two cell lines and only 15% mosaicism close monitoring will be crucial to determine the genotype/phenotype relationship in our patient. 1 Choppin et al 2019 Eur J Endocrinol 1; 180(2):L1-L3 2 Itoh et al 2017 Clin Pediatr Endocrinol 26;25-28

Mutational Profiling of Driver Tumor Suppressor and Oncogenic Genes in Brazilian Malignant Pleural Mesotheliomas

Background: Malignant pleural mesothelioma (MPM) is a highly lethal disease comprising a heterogeneous group of tumors with challenging to predict biological behavior. The diagnosis is complex, and the histologic classification includes 2 major subtypes of MPM: epithelioid (∼60% of cases) and sarcomatous (∼20%). Its identification depends upon pathological investigation supported by clinical and radiological evidence and more recently ancillary molecular testing. Treatment options are currently limited, with no known targeted therapies available. Objectives: To elucidate the mutation profile of driver tumor suppressor and oncogenic genes in a cohort of Brazilian patients. Methods: We sequenced 16 driver genes in a series of 43 Brazilian malignant mesothelioma (MM) patients from 3 distinct Brazilian centers. Genomic DNA was extracted from formalin-fixed paraffin-embedded tumor tissue blocks, and the TERT promoter region was amplified by PCR followed by direct capillary sequencing. The Illumina TruSight Tumor 15 was used to evaluate 250 amplicons from 15 genes associated with solid tumors (AKT1, GNA11, NRAS, BRAF, GNAQ, PDGFRA, EGFR, KIT, PIK3CA, ERBB2, KRAS, RET, FOXL2, MET,and TP53). Library preparation with the TruSight Tumor 15 was performed before sequencing at the MiSeq platform. Data analysis was performed using Sophia DDM software. Results: Out of 43 MPM patients, 38 (88.4%) were epithelioid subtype and 5 (11.6%) were sarcomatoid histotype. Asbestos exposure was present in 15 (39.5%) patients with epithelioid MPM and 3 (60%) patients with sarcomatoid MPM. We found a TERT promoter mutation in 11.6% of MM, and the c.-146C>T mutation was the most common event. The next-generation sequencing was successful in 33 cases. A total of 18 samples showed at least 1 pathogenic, with a median of 1.8 variants, ranging from 1 to 6. The most mutated genes were TP53 and ERBB2 with 7 variants each, followed by NRAS BRAF, PI3KCA, EGFR and PDGFRA with 2 variants each. KIT, AKT1, and FOXL2 genes exhibited 1 variant each. Interestingly, 2 variants observed in the PDGFRA gene are classic imatinib-sensitive therapy. Conclusions: We concluded that Brazilian MPM harbor mutation in classic tumor suppressor and oncogenic genes, which might help in the guidance of personalized treatment of MPM.

6PD - The mutational signature of spontaneously developing tumours in MLH1-/- mice: Potential consequences for immunotherapeutic approaches

BackgroundMLH1 knock out mice develop mismatch repair deficient (MMR-D) neoplasias spontaneously and reflect the diverse clinical presentation of MMR-D-driven carcinogenesis in men. The tumor spectrum includes a high prevalence of early Non-Hodgkin T cell lymphomas (NHL), lymphoid skin lesions as well as later developing epithelial tumors of the gastrointestinal tract (GIT). MethodsUsing whole-exome sequencing on MLH1-/- tumors (2x GIT, 1x splenic NHL, 1x skin lymphoma) as well as GIT-derived cell lines (n=2), we focused on identification of (I) shared and (II) mutually exclusive mutations and described the processes of ongoing mutational events in tumor-derived cultures. ResultsMLH1-/- tumors show high tumor mutational burden with 3/4 primary tumor samples even being ultra-hypermutated (> 100 mut/MB). Missense mutations were more frequent than nonsense mutations, base changes were mainly due to transitions (C>T; A>G). The resulting mutational landscape was heterogeneous and in accordance with the human counterpart, MLH1-/- tumors frequently harbor mutations in PIK3CA, EGFR, KRAS, and/or ERBB3. Of note, only a few shared mutations were detectable among different tumor entities, among them were ARID1A and IDH2. Mutations in classical tumor suppressor genes SMAD4 and POLE were mutually exclusive in lymphomas, most likely contributing to a more aggressive in vivo phenotype. Comparing the mutational profile of selected tumors and their corresponding cell line revealed continuous increased numbers of somatic gene mutations. The same was true for coding microsatellite mutations in MMR-D target genes. Partial overlap was detectable, yet recognizing shared antigens. Two promising candidates are AKT3, a RAC-gamma serine/threonine-protein kinase and the endonuclease ERCC5 (Excision Repair 5), involved in DNA excision repair. ConclusionsThe present study is the first reporting results of a comparison between different spontaneously developing tumors as models for MMR-D driven tumorigenesis. Additionally to identifying ARID1A as causative mutation hotspot, this comprehensive characterization of the mutational landscape may be a good starting point to predict antigens for vaccination approaches. Legal entity responsible for the studyThe authors. Fundinggerman research foundation (DFG); grant number: MA5799/2-1. DisclosureAll authors have declared no conflicts of interest.