Showing papers on "BAP1 published in 2023"

BAP1 Loss Is Associated with Higher ASS1 Expression in Epithelioid Mesothelioma: Implications for Therapeutic Stratification

Abstract: The nuclear deubiquitylase BRCA1-associated protein 1 (BAP1) is frequently inactivated in malignant pleural mesothelioma (MPM) and germline BAP1 mutation predisposes to cancers including MPM. To explore the influence on cell physiology and drug sensitivity, we sequentially edited a predisposition mutation (w-) and a promoter trap (KO) into human mesothelial cells. BAP1w-/KO MeT5A cells express less BAP1 protein and phenocopy key aspects of BAP1 loss in MPM. Stable isotope labeling with amino acids in cell culture-mass spectrometry revealed evidence of metabolic adaptation, with concomitant alteration of cellular metabolites. In MeT5A, BAP1 deficiency reduces glycolytic enzyme levels but increases enzymes involved in the tricarboxylic acid cycle and anaplerotic pathways. Notably both argininosuccinate synthase 1 (ASS1), essential for cellular synthesis of arginine, and its substrate aspartate, are elevated in BAP1w-/KO MeT5A cells. Likewise, ASS1 expression is higher in BAP1-altered MPM cell lines, and inversely correlates with BAP1 in The Cancer Genome Atlas MESO dataset. Elevated ASS1 is also evident by IHC staining in epithelioid MPM lacking nuclear BAP1 expression, with improved survival among patients with BAP1-negative/ASS1-expressing tumors. Alterations in arginine metabolism may sensitize cells to metabolic drugs and we find that BAP1-negative/ASS1-expressing MPM cell lines are more sensitive to ASS1 inhibition, although not to inhibition of purine synthesis by mizoribine. Importantly, BAP1w-/KO MeT5A become desensitized to arginine deprivation by pegylated arginine deiminase (ADI-PEG20), phenocopying BAP1-negative/ASS1-expressing MPM cell lines.Our data reveal an interrelationship between BAP1 and arginine metabolism, providing a potential means of identifying patients with epithelioid MPM likely to benefit from ADI-PEG20.

Genetics and RNA Regulation of Uveal Melanoma

Abstract: Simple Summary Uveal melanoma (UM) is a rare eye cancer with a high mortality rate due to metastases, leading to death in up to 50% of patients within 10 years from UM diagnosis. Moreover, patients show a median survival of 6 to 12 months after metastasis diagnosis. UM and cutaneous melanoma (CM) have the same melanocytic origin; however, they are very different in terms of molecular alterations and biological behavior. In this review, we will discuss the complex genetic and non-coding RNA-based epigenetic landscapes underlying the transformation, progression, and dissemination of UM. This knowledge will pave the way for the future identification of new biomarkers of the pathology and therapeutic targets. Abstract Uveal melanoma (UM) is the most common intraocular malignant tumor and the most frequent melanoma not affecting the skin. While the rate of UM occurrence is relatively low, about 50% of patients develop metastasis, primarily to the liver, with lethal outcome despite medical treatment. Notwithstanding that UM etiopathogenesis is still under investigation, a set of known mutations and chromosomal aberrations are associated with its pathogenesis and have a relevant prognostic value. The most frequently mutated genes are BAP1, EIF1AX, GNA11, GNAQ, and SF3B1, with mutually exclusive mutations occurring in GNAQ and GNA11, and almost mutually exclusive ones in BAP1 and SF3B1, and BAP1 and EIF1AX. Among chromosomal aberrations, monosomy of chromosome 3 is the most frequent, followed by gain of chromosome 8q, and full or partial loss of chromosomes 1 and 6. In addition, epigenetic mechanisms regulated by non-coding RNAs (ncRNA), namely microRNAs and long non-coding RNAs, have also been investigated. Several papers investigating the role of ncRNAs in UM have reported that their dysregulated expression affects cancer-related processes in both in vitro and in vivo models. This review will summarize current findings about genetic mutations, chromosomal aberrations, and ncRNA dysregulation establishing UM biology.

Novel prognostication biomarker adipophilin reveals a metabolic shift in uveal melanoma and new therapeutic opportunities

Abstract: Metastatic uveal melanoma remains incurable at present. We previously demonstrated that loss of BAP1 gene expression in tumour cells triggers molecular mechanisms of immunosuppression in the tumour microenvironment (TME) of metastatic uveal melanoma. Adipophilin is a structural protein of lipid droplets involved in fat storage within mammalian cells, and its expression has been identified in uveal melanoma. We comprehensively evaluated adipophilin expression at the RNA (PLIN2) and protein levels of 80 patients of the GDC‐TCGA‐UM study and in a local cohort of 43 primary uveal melanoma samples respectively. PLIN2 expression is a survival prognosticator biomarker in uveal melanoma. Loss of adipophilin expression is significantly associated with monosomy 3 status and nuclear BAP1 losses in uveal melanoma tumours. Integrative transcriptomic and secretome studies show a relationship between transient loss of adipophilin expression and increased levels of tumour‐associated macrophages and hypoxia genes, suggesting PLIN2‐dependent changes in oxygen and lipid metabolism in the TME of low and high‐metastatic risk uveal melanoma. We designed four adipophilin‐based multigene signatures for uveal melanoma prognostication using a transcriptomic and secretome survival‐functional network approach. Adipophilin‐based multigene signatures were validated in BAP1‐positive and BAP1‐negative uveal melanoma cell lines using a next‐generation RNA sequencing approach. We identified existing small molecules, mostly adrenergic, retinoid, and glucocorticoid receptor agonists, MEK, and RAF inhibitors, with the potential to reverse this multigene signature expression in uveal melanoma. Some of these molecules were able to impact tumour cell viability, and carvedilol, an adrenergic receptor antagonist, restored PLIN2 levels, mimicking the expression of normoxia/lipid storage signatures and reversing the expression of hypoxia/lipolysis signatures in co‐cultures of uveal melanoma cells with human macrophages. These findings open up a new research line for understanding the lipid metabolic regulation of immune responses, with implications for therapeutic innovation in uveal melanoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

BAP1 is a novel regulator of HIF-1α

Abstract: Significance BAP1 modulates crucial cellular pathways that regulate genomic stability and cell death. BAP1 mutations on the one hand favor malignant transformation and mesothelioma development; on the other hand, they reduce mesothelioma aggressiveness. Investigating this apparent paradox, we discovered that BAP1 deubiquitylates and stabilizes HIF-1α in hypoxia; thus, BAP1 inactivating mutations significantly reduce HIF-1α. Given the critical role of HIF-1α in promoting tumor invasion, we propose that: 1) Reduced BAP1 in the tumor cells and tumor microenvironment of individuals carrying germline BAP1 mutations may contribute to the reduced invasion and the significantly improved prognosis of mesothelioma; 2) targeting wild-type BAP1 after tumor development could be a novel effective strategy to reduce HIF-1α protein levels in hypoxic tissues and impair tumor growth.

Increased Histological Tumor Pigmentation in Uveal Melanoma Is Related to Eye Color and Loss of Chromosome 3/BAP1

Abstract: Heavy pigmentation is known to be a prognostic risk factor in uveal melanoma (UM). We analyzed whether genetic tumor parameters were associated with tumor pigmentation and whether pigmentation should be included in prognostic tests.Retrospective comparison of clinical, histopathological, and genetic features and survival in UM with different pigmentation.A total of 1058 patients with UM from a White European population with diverse eye colors enucleated between 1972 and 2021.Cox regression and log-rank tests were used for survival analysis; the chi-square test and Mann-Whitney U test were used for correlation analysis.Uveal melanoma-related survival based on tumor pigmentation and chromosome status, correlation of tumor pigmentation with prognostic factors.The 5-year UM-related mortality was 8% in patients with nonpigmented tumors (n = 54), 25% with lightly pigmented tumors (n = 489), 41% with moderately pigmented tumors (n = 333), and 33% with dark tumors (n = 178) (P < 0.001). The percentage of tumors with monosomy 3 (M3) or 8q gain increased with increasing pigmentation (31%, 46%, 62%, and 70% having M3 [P < 0.001], and 19%, 43%, 61%, and 63% having 8q gain [P < 0.001] in the 4 increasing pigment groups, respectively). BRCA-associated protein 1 (BAP1) loss (known for 204 cases) was associated with increased tumor pigmentation (P = 0.001). Cox regression analysis on survival showed that when chromosome status and pigmentation were both included, pigmentation was not an independent prognostic indicator. Preferentially expressed antigen in melanoma (PRAME) expression was a significant prognostic marker in light tumors (P = 0.02) but not in dark tumors (P = 0.85).Patients with moderately and heavily pigmented tumors showed a significantly higher UM-related mortality than patients with unpigmented and light tumors (P < 0.001), supporting prior reports on the relation between increased tumor pigmentation and a worse prognosis. Although we previously showed that a dark eye color was associated with tumor pigmentation, we now show that the tumor's genetic status (chromosome 3 and 8q/BAP1 status) is also related to tumor pigmentation. When pigmentation and chromosome 3 status are both included in a Cox regression analysis, pigmentation is not an independent prognostic factor. However, evidence from this and previous studies shows that chromosome changes and PRAME expression have a stronger association with survival when they occur in light tumors than in dark ones. Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references.

Highlighting the immunohistochemical differences of malignant mesothelioma subtypes via case presentations

Abstract: Malignant mesothelioma (MM) is a rare tumor of mesothelial cells, with an increasing incidence both in developed and developing countries. MM has three major histological subtypes, in order of frequency, according to the World Health Organization (WHO) Classification of 2021: epithelioid, biphasic, and sarcomatoid MM. Distinction may be a challenging task for the pathologist, due to the unspecific morphology.

Comprehensive Genomic Profiling of NF2-Mutated Kidney Tumors Reveals Potential Targets for Therapy

Abstract: Abstract Genomic alterations (GA) in NF2 tumor-suppressor gene have been associated with aggressive behavior in kidney tumors. We used comprehensive genomic profiling (CGP) to evaluate the frequencies of NF2 GA in histologic subtypes of kidney tumors and co-occurring GA in other genes and biomarkers. Advanced kidney tumors included 1875 clear cell (ccRCC), 405 papillary (pRCC), 108 chromophobe (chRCC), 171 sarcomatoid (sRCC), 61 collecting duct (cdRCC), 49 medullary (mRCC), 134 unclassified (uRCC), 906 urothelial carcinoma of renal pelvis (UC), and 147 Wilms tumors underwent hybrid-capture based CGP to evaluate all classes of GA. 192 (4.9%) of kidney tumors featured NF2 GA which were predominantly structural variant mutations (89%), followed by copy number alterations (9%). Gender and age were similar between NF2-mutant (NF2mut) and NF2-wild type (NF2wt) cohorts with male preponderance. NF2 GA frequency was highest in cdRCC (30%), sRCC (21%), uRCC (15%), and pRCC (12%) while lowest in ccRCC (3%), UC (3%) Wilms tumor (1%), and chRCC (0%). NF2 mutational status was associated with loss of Ch 22 (P < .001). NF2mut RCC harbored co-occurring GA including CDKN2A, CDKN2B, SETD2, and BAP1. VHL, PBRM1, PTEN, and FGFR3 GA were significantly more frequent in NF2wt than in NF2mut tumors. MTOR pathway GAs were uncommon in NF2mut tumors. No NF2 mutated RCC featured MSI-high or high TMB. sRCC was associated with high PD-L1 expression. PD-L1 SP142 tumoral (P = .04) and immune cells (P = .013) were more frequent in NF2mut as compared to NF2wt group. Among histologic subtypes of RCC, cdRCC, sRCC, pRCC, and uRCC are enriched in NF2 GA. Co-occurrent GA in CDKN2A/B, SETD2, and BAP1 may represent potential therapeutic targets. Higher level of PD-L1 expression in NF2mut cohort suggests that these tumors might be sensitive to immune checkpoint inhibitor therapies.

Mutational signature of extracranial meningioma metastases and their respective primary tumors

Abstract: Abstract Extracranial metastases of intracranial meningiomas are rare. Little is known about the mutational pattern of these tumors and their metastatic seeding. Here, we retrospectively explored the molecular alterations of these metastatic lesions and their respective intracranial tumor manifestations. Histology and genome sequencing were performed in intracranial meningiomas and their extracranial metastatic lesions operated upon between 2002 and 2021. Next-generation DNA/RNA sequencing (NGS) and methylome analysis were performed to determine molecular alterations. We analyzed the tumors of five patients with clinically suspected metastases of a meningioma using methylome analysis and next generation panel sequencing of the primary tumors as well as the metastatic lesions. Metastases were found in the spinal cord and one in the lung. In four of these patients, molecular analyses confirmed metastatic disease, while the fifth patient was found to harbor two molecularly distinct meningiomas. On pathological assessment, the primary lesions ranged from CNS WHO grades 1 to 3 (integrated molecular-morphologic meningioma classification scores 2 to 6). Of the four true metastatic cases, three out of the four metastasizing tumors harbored alterations in the BAP1 gene, comprising a stop-mutation combined with copy-number loss (WHO grade 1), copy number loss (WHO grade 3) and a frameshift mutation (WHO grade 2). Furthermore, the latter was confirmed to harbor a BAP1 tumor predisposition syndrome. The fourth metastasizing tumor had copy-number losses in NF2 and PTEN. Only one of four showed CDKN2A homozygous deletion; none showed TERT promotor mutation. Our results molecularly confirm true metastatic disease in four meningioma patients. BAP1 gene alterations were the most frequent. Larger cohorts, most likely from multicenter studies are necessary to evaluate the role of BAP-1 alterations to further understand the metastatic spread in meningiomas. for metastatic spread and might indicate patients at risk for metastatic spread. Further explorations within larger cohorts are necessary to validate these findings which might influence the clinical management in the future.

Epigenetic and transcriptomic characterization reveals progression markers and essential pathways in clear cell renal cell carcinoma

Abstract: Identifying tumor-cell-specific markers and elucidating their epigenetic regulation and spatial heterogeneity provides mechanistic insights into cancer etiology. Here, we perform snRNA-seq and snATAC-seq in 34 and 28 human clear cell renal cell carcinoma (ccRCC) specimens, respectively, with matched bulk proteogenomics data. By identifying 20 tumor-specific markers through a multi-omics tiered approach, we reveal an association between higher ceruloplasmin (CP) expression and reduced survival. CP knockdown, combined with spatial transcriptomics, suggests a role for CP in regulating hyalinized stroma and tumor-stroma interactions in ccRCC. Intratumoral heterogeneity analysis portrays tumor cell-intrinsic inflammation and epithelial-mesenchymal transition (EMT) as two distinguishing features of tumor subpopulations. Finally, BAP1 mutations are associated with widespread reduction of chromatin accessibility, while PBRM1 mutations generally increase accessibility, with the former affecting five times more accessible peaks than the latter. These integrated analyses reveal the cellular architecture of ccRCC, providing insights into key markers and pathways in ccRCC tumorigenesis.

Genomic profiling of rare undifferentiated sarcomatoid subtypes of pancreatic carcinomas for potential response to immunotherapy.

Abstract: 741 Background: While pancreatic adenocarcinoma (PDAC) remains a leading cause of cancer-related deaths, the highly aggressive PDAC subtype of undifferentiated sarcomatoid carcinoma (USC) remains poorly characterized as it comprises only 2-3% of all PDAC histology. Previous case reports suggest that immune checkpoint inhibitors could be a promising treatment strategy for USC, but the prevalence of established predictive biomarkers of response are largely unknown in this unique subpopulation. We identified PDAC USC patient samples from a large dataset and performed comprehensive genomic profiling to determine the prevalence of biomarkers associated with response to immunotherapy. Methods: PDAC USC patient samples (N=43) underwent central pathology review to confirm this diagnosis and were compared to non-USC PDAC patient samples (N=5562). Next-generation sequencing of DNA and RNA was performed at Caris Life Sciences (Phoenix, AZ). PD-L1 expression was tested by IHC (SP142; Positive (+): ≥ 2+, ≥%5). A combination of IHC, NGS, and fragment analysis was used to assess deficient mismatch repair/microsatellite instability high (dMMR/MSI). High tumor mutational burden (TMB-High) was defined as ≥10 mutations/MB. Immune cell fractions of the tumor microenvironment were estimated by RNA deconvolution analysis using quanTIseq. Chi-square tests with Bonferoni correction for multiple comparisons were used to determine significance. Results: Among PDAC USC samples, KRAS (86% USC vs 90% non-USC, p = 0.31, q = 1), TP53 (86% vs 77%, p = 0.16, q = 1), and CDKN2A (18% vs 23%, p = 0.45, q = 1) were the most commonly mutated genes with a similar prevalence compared to non-USC histologies; however, KRAS was amplified more frequently (7% USC vs <1% non-USC, q = 0.006). Furthermore, more USC tumors were PD-L1+ (63% vs 16%, q < 0.001), while few USC samples were dMMR/MSI (2% USC vs 1% non-USC, p = 0.45, q = 1) or TMB-High (2% vs 2%, p = 0.89, q = 1). All USC tumors were deplete of lymphocytes, but many were differentially enriched (>5%) with neutrophils (85% vs 57%, q = 0.03) or M2 macrophages (52% vs 28%, p = 0.006, q = 0.06). Conclusions: This work represents the largest molecular analysis of PDAC USC samples to date. Our analysis uncovered a different prevalence of amplified KRAS and PD-L1 expression in USC as compared to other PDAC histologies amidst an immune desert lacking lymphocytes in the USC tumor microenvironment. These findings provide evidence for further investigation into combination therapy of KRAS inhibitors with immune checkpoint inhibitors to target these immune-imbalanced microenvironment landscapes.

BAP1 Malignant Pleural Mesothelioma Mutations in Caenorhabditis elegans Reveal Synthetic Lethality between ubh-4/BAP1 and the Proteasome Subunit rpn-9/PSMD13

Abstract: The deubiquitinase BAP1 (BRCA1-associated protein 1) is associated with BAP1 tumor predisposition syndrome (TPDS). BAP1 is a tumor suppressor gene whose alterations in cancer are commonly caused by gene mutations leading to protein loss of function. By CRISPR-Cas, we have generated mutations in ubh-4, the BAP1 ortholog in Caenorhabditis elegans, to model the functional impact of BAP1 mutations. We have found that a mimicked BAP1 cancer missense mutation (UBH-4 A87D; BAP1 A95D) resembles the phenotypes of ubh-4 deletion mutants. Despite ubh-4 being ubiquitously expressed, the gene is not essential for viability and its deletion causes only mild phenotypes without affecting 20S proteasome levels. Such viability facilitated an RNAi screen for ubh-4 genetic interactors that identified rpn-9, the ortholog of human PSMD13, a gene encoding subunit of the regulatory particle of the 26S proteasome. ubh-4[A87D], similarly to ubh-4 deletion, cause a synthetic genetic interaction with rpn-9 inactivation affecting body size, lifespan, and the development of germ cells. Finally, we show how ubh-4 inactivation sensitizes animals to the chemotherapeutic agent Bortezomib, which is a proteasome inhibitor. Thus, we have established a model to study BAP1 cancer-related mutations in C. elegans, and our data points toward vulnerabilities that should be studied to explore therapeutic opportunities within the complexity of BAP1 tumors.

New Markers for Management of Mesothelioma

Abstract: Abstract In this review, we provide an update on the status of cancer biomarkers for the clinical management of pleural mesothelioma, an aggressive cancer associated with asbestos exposure. Mesothelioma can be difficult to diagnose, and response to treatment is transient, even with recently adopted immune checkpoint inhibitor (ICI) combinations. Identification of mesothelioma-specific biomarkers could facilitate early diagnosis and tailor treatment strategies. Mesothelioma is characterized by frequent loss or alteration of the tumor suppressor genes cyclin-dependent kinase inhibitor 2A ( CDKN2A ) and BRCA1-associated protein-1 ( BAP1 ) . Accumulating data show these genes and/or their related protein products will be valuable tissue-based biomarkers for mesothelioma. Loss of BAP1, CDKN2A, p16, or methylthioadenosine phosphorylase provide pathologists with a reliable means of differentiating between mesothelioma and reactive mesothelial cell proliferations. This can aid diagnosis in difficult cases and is requisite for the identification of the new pathological entity malignant mesothelioma in situ. However, limited progress in identifying clinically useful soluble biomarkers in this cancer type has been made, with mesothelin remaining the benchmark. To date, results from studies to identify predictive biomarkers for ICI response have been disappointing. A recent retrospective study demonstrated BAP1 loss was predictive of improved survival following combination pemetrexed- and platinum-based chemotherapy. Validation of this result could have important clinical implications. Clinical trials aimed at targeting therapy based on biomarker expression are generally in the early phase setting, with overall results being moderate. The identification of biomarkers for mesothelioma remains a key research question due to their potential to improve patient outcomes in this deadly cancer.

Abstract 926: Genomics-based personalized oncology of advanced thymic epithelial tumors

Abstract: Introduction: Thymic epithelial tumors (TETs) are very rare. Thymoma A and AB have a better prognosis than more aggressive thymoma B, thymic carcinoma (TC) and neuroendocrine tumors of the thymus (NET). While previous efforts such as TCGA have mainly characterized thymomas (Radovich et al., Cancer Cell 2018), the molecular landscape of TCs and NETs is still elusive. Patients and Methods: Between 03/2014 and 07/2020, we enrolled 44 TET patients (27 TCs, 11 thymomas, 6 NETs) in a prospective observational study (MASTER) conducted by the National Center for Tumor Diseases (NCT) Heidelberg, NCT Dresden and the German Cancer Consortium (DKTK). MASTER applied whole genome/exome sequencing (WGS, n=22; WES, n=22), transcriptome (n=40) and germline analysis to inform therapy recommendations by a dedicated molecular tumor board (MTB). We systematically gathered follow-up data to evaluate outcome and compared progression-free survival (PFS) of the first treatment according to an MTB recommendation (PFS2) to the last prior systemic treatment (PFS1) in each patient (PFS ratio). Results: Tumor mutational burden (TMB) was low (median=0.99 mutations/Mb, range 0.08-3.48) but higher than in TCGA (p<0.05). TMB was higher in TCs than in thymoma (p<0.05). Most frequently mutated genes were TP53 (30%), CYLD (16%), SETD2 (14%) and KIT (14%). Germline analysis revealed (likely) pathogenic germline alterations in 25% of patients (MUTYH, n=3; BRCA1, n=2; BRCA2, BAP1, CHEK2, FANCA, TP53, MEN1, n=1). A comprehensive analysis of candidate biomarkers of homologous recombination repair (HRR) defects revealed a subgroup of TET patients with a rationale for PARP inhibitor therapy. Unsupervised clustering of RNA sequencing data mainly revealed clustering that correlated with WHO classification. Additionally, TCs clustered in two subgroups that we identified as immunologically hot and cold tumors using immunohistochemistry validation. Overall survival of patients with hot tumors was significantly longer (p<0.05). The MTB recommended therapies for 42 patients (95%), which were implemented in 24 cases (57%). Five patients had a PFS2 > 6 months and a PFS ratio > 1.3. The best outcome was achieved using imatinib in a patient with a KIT mutation (p.W557R). After progression, the MTB recommended ponatinib based on a secondary KIT mutation (p.V654A). The patient was still on ponatinib when the observation period ended. Conclusion: We demonstrate that comprehensive molecular analysis provides clinically relevant information in a subgroup of TET patients. Thymoma, TCs, and NETs present with different molecular characteristics. Distinction between immunologically hot and cold TCs may have value for risk stratification and therapeutic strategies. PARP inhibition could be a potential new treatment option in a small subgroup of TETs. Molecular testing of KIT, germline analysis and genetic counseling should be recommended for all patients with advanced TETs. Citation Format: Lino Möhrmann, Lysann Rostock, Małgorzata Oleś, Arne Jahn, Marie Arlt, Nagarajan Paramasivam, Korinna Jöhrens, Luise Rupp, Marc Schmitz, Daniela Richter, Sebastian Uhrig, Martina Fröhlich, Barbara Hutter, Jennifer Hüllein, Elena E. Wolf, Dorothea Hanf, Laura Gieldon, Simon Kreutzfeldt, Christoph E. Heilig, Veronica Teleanu, Daniel B. Lipka, Andreas Mock, Ivan Jelas, Damian T. Rieke, Marcel Wiesweg, Melanie Boerries, Anna L. Illert, Alexander Desuki, Thomas Kindler, Angela M. Krackhardt, C. Benedikt Westphalen, Heidrun Grosch, Leonidas Apostolidis, Albrecht Stenzinger, Irina A. Kerle, Christoph Heining, Daniel Hübschmann, Evelin Schröck, Stefan Fröhling, Hanno Glimm. Genomics-based personalized oncology of advanced thymic epithelial tumors [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 926.

BAP1 as a guardian of genome stability: implications in human cancer

Abstract: BAP1 is a ubiquitin C-terminal hydrolase domain-containing deubiquitinase with a wide array of biological activities. Studies in which advanced sequencing technologies were used have uncovered a link between BAP1 and human cancer. Somatic and germline mutations of the BAP1 gene have been identified in multiple human cancers, with a particularly high frequency in mesothelioma, uveal melanoma and clear cell renal cell carcinoma. BAP1 cancer syndrome highlights that all carriers of inherited BAP1-inactivating mutations develop at least one and often multiple cancers with high penetrance during their lifetime. These findings, together with substantial evidence indicating the involvement of BAP1 in many cancer-related biological activities, strongly suggest that BAP1 functions as a tumor suppressor. Nonetheless, the mechanisms that account for the tumor suppressor function of BAP1 have only begun to be elucidated. Recently, the roles of BAP1 in genome stability and apoptosis have drawn considerable attention, and they are compelling candidates for key mechanistic factors. In this review, we focus on genome stability and summarize the details of the cellular and molecular functions of BAP1 in DNA repair and replication, which are crucial for genome integrity, and discuss the implications for BAP1-associated cancer and relevant therapeutic strategies. We also highlight some unresolved issues and potential future research directions.

Molecular Targets in Salivary Gland Cancers: A Comprehensive Genomic Analysis of 118 Mucoepidermoid Carcinoma Tumors

Abstract: Introduction: Salivary gland carcinomas (SGC) are histologically diverse cancers and next-generation sequencing (NGS) to identify key molecular targets is an important aspect in the management of advanced cases. Methods: DNA was extracted from paraffin embedded tissues of advanced SGC and comprehensive genomic profiling (CGP) was carried out to evaluate for base substitutions, short insertions, deletions, copy number changes, gene fusions and rearrangements. Tumor mutation burden (TMB) was calculated on approximately 1.25 Mb. Some 324 genes in the FoundationOne CDX panel were analyzed. Results: Mucoepidermoid carcinoma (MECa) mutations were assessed. CDKN2A and CDKN2B GA were common in mucoepidermoid carcinoma (MECa) (52.5 and 30.5%). PIK3CA was also common in MECa (16.9%). ERBB2 amplification/short variants (amp/SV) were found in MECa (5.9/0%). HRAS GA was common in MECa (14.4%) as well. Other targets, including BAP1, PTEN, and KRAS, were noted but had a low incidence. In terms of immunotherapy (IO)-predictive markers, TMB > 10 was more common in MECa (16.9%). PDL1 high was also seen in MECa (4.20%). Conclusion: SGC are rare tumors with no FDA-approved treatment options. This large dataset reveals many opportunities for IO and targeted therapy contributing to the continuously increased precision in the selection of treatment for these patients.

Exploration of Morphological Features of Clear Cell Renal Cell Carcinoma With PBRM1, SETD2, BAP1, or KDM5C Mutations.

Abstract: The last decade has seen great advances in genomic profiling and prognosis-associated factors of clear cell renal cell carcinoma (RCC), the most common entity in kidney cancer. Following VHL, PBRM1, SETD2, BAP1, and KDM5C have been validated as the most common co-occurring gene mutations in clear cell RCC by multicenter studies. However, the morphological features of clear cell RCC with co-occurring gene mutations remain unclear. In this study, we presented 20 clear cell RCCs that underwent next-generation sequencing, of which 1 tumor was reclassified as ELOC-mutated RCC. PBRM1, SETD2, BAP1, and KDM5C were the most common mutations, following VHL. Morphologically, clear cell RCC with PBRM1 or KDM5C mutation usually displayed a low-grade pattern. Cystic changes and hyalinized stroma were often observed. The Ki67 index was <10%. These observations indicated good prognosis. However, mutated SETD2 may increase the malignancy of clear cell RCC with PBRM1 mutation. Two clear cell RCCs with mutated PBRM1 and SETD2 developed local or distant metastases. Clear cell RCC with BAP1 mutations always had high-grade patterns, and rhabdoid differentiation was also observed, indicating that BAP1 mutation was associated with poor outcomes. Papillary architecture was often a feature of BAP1 mutation, which is uncommon in clear cell RCC. PDL1 was positive in only one tumor with BAP1 mutation, and the positivity rate was limited to 5%. B7H3 was negative in all tumors. Morphologic findings in this small cohort may suggest why PBRM1 mutation does not correlate with decreased survival, whereas BAP1 mutation usually predicts poor outcomes.

Supplementary Table 10-12 from DNA Methylation Signature Reveals Cell Ontogeny of Renal Cell Carcinomas

Abstract: <p>Supplementary Table 10: List of samples misclassified among the clear-cell renal cell carcinomas dataset with the corresponding new classification. Among those, many of them have been demonstrated to belong to the C4 cluster of long non-coding RNA classification. The first table reports the 5 misclassified TCGA clear-cell RCC (KIRC) using gene expression and which turned out to be chromophobe RCC (chRCC). The second table reports on misclassified TCGA KIRC cases with DNA methylation available. Supplementary Table 11: List of importance scores for genes used in the statistical model for predicting outcome of patients with clear-cell RCC. Supplementary Table 12: Associations (both training and validation datasets) between mutational load, TCGA clear-cell renal cell carcinomas transcriptomic subtypes classification and recurrent somatic mutations (VHL, PBRM1, BAP1 and KDM6A) identified in clear-cell renal cell carcinoma TCGA dataset with our 56 genes epi-signature. P-values less than 0.05 is considered as statistically significant.</p>

Supplementary Table S2 from BAP1 Loss Is Associated with DNA Methylomic Repatterning in Highly Aggressive Class 2 Uveal Melanomas

Abstract: <p>TCGA_Methyl_DMP: All significantly methylated probes (FDR < 0.05) in Class 2 uveal melanomas compared to Class 1 uveal melanomas from the 80 TCGA cases. JWH_Methyl_DMP: All significantly methylated probes (p < 0.05) in Class 2 uveal melanomas compared to Class 1 uveal melanomas from 12 cases of the senior author (JWH). TCGA_D12_PC1_PC2_Methyl: The 67,261 probes shared between the top 20% most variable methylated probes from the TCGA and JWH datasets used in the principal component analyses. The percentage contribution to PC1 and PC2 are displayed for all probes. TCGA_D12_Probes_Top5000PC1: The 204 probes shared between the TCGA and JWH datasets out of the Top 5000 shared most variable methylated probes contributing to PC1 in the principal component analyses.</p>

Supplementary Video 3 from Deubiquitination of γ-Tubulin by BAP1 Prevents Chromosome Instability in Breast Cancer Cells

Abstract: <p>Live cell microscopy of MDA-MB-468 inducibly expressing empty expression plasmid and transiently transfected with H2B-GFP. The movie shows anaphase bridge.</p>

Supplemental Table 2 from Inactivation of &lt;i&gt;Bap1&lt;/i&gt; Cooperates with Losses of &lt;i&gt;Nf2&lt;/i&gt; and &lt;i&gt;Cdkn2a&lt;/i&gt; to Drive the Development of Pleural Malignant Mesothelioma in Conditional Mouse Models

Abstract: <p>Differences in survival of mice succumbing to malignant mesothelioma after IT injection with Adeno-Cre in different conditional knockout mouse groups.</p>

Supplementary Figure S1 from Modeling Renal Cell Carcinoma in Mice: &lt;i&gt;Bap1&lt;/i&gt; and &lt;i&gt;Pbrm1&lt;/i&gt; Inactivation Drive Tumor Grade

Abstract: <p>Validation of Pax8-Cre;Vhl;Bap1 mice.</p>

Blood-based molecular profiling of Chinese patients with advanced renal cell carcinoma.

Abstract: e16550 Background: The therapeutic landscape of renal cell carcinoma (RCC) has rapidly expanded, and there is an urgent need to develop noninvasive biomarkers that can select an optimal therapy in real time. Next-generation sequencing (NGS)-based profiling of circulating tumor DNA (ctDNA) shows promise for noninvasive detection. To evaluate the clinical utility of ctDNA analysis in RCC, we aimed to analyze the molecular characteristics of RCC using ctDNA by NGS. Methods: A total of 189 plasma samples and 26 matched tumor tissues from Chinese patients with advanced renal cell carcinoma (n = 189) were analyzed by Acornmed panel with 808 cancer-related genes, and matched white blood cell as germline comparators. Results: Overall, 29 germline mutations were detected in 14.3% (27/189) patients, such as TSC2 (5/189, 2.65%), FH (3/189, 1.6%) and VHL (2/189, 1.1%), of which 10 germline aberrations in DNA damage repair (DDR) genes were detected in 5.3% patients (10/18). In addition, a total of 1590 somatic variations were identified in 96.8% (183/189) ctDNA, including 1278 nonsynonymous single nucleotide variants, 126 small insertions or deletions, 75 copy number variations. And the median blood tumor mutation burden (bTMB) was 4.36 mut/Mb. Mutations in VHL (36/189, 19.4%) were the most prominent variation, followed by those in TP53 (19/189, 10.0%), BAP1 (12/189, 6.3%), PBRM1 (10/189, 5.3%) and SETD2 (7/189, 3.7%). Moreover, totals of 154 clinically actionable mutations from 92 patients (48.7%) were identified. In 26 patients with both tumor tissues and plasma samples, sequencing detected alterations in 100% (26/26) tumor tissue DNA (tDNA) and 96.2% (25/26) ctDNA, respectively. Furthermore, 29.6% of mutations of tissues were founded in ctDNA, and 21.6% of mutations of ctDNA were detected in tissues. Among them, 73.1% (19/26) patients had at least one overlapping variant called by tDNA and ctDNA. The most common shared mutation was VHL (5/26, 19.2%). Meanwhile, four shared copy number variants were detected in two patients (15.4%). Conclusions: Our study contributes to understand the molecular characteristics of RCC using ctDNA, which is a viable option for tissue when tissue is difficult to obtain. Furthermore, ctDNA testing by NGS is helpful for drug selection and provides more effective treatment options for RCC, particularly for hereditary RCC.

Data from BAP1 Loss Is Associated with Higher ASS1 Expression in Epithelioid Mesothelioma: Implications for Therapeutic Stratification

Abstract: <div>Abstract<p>The nuclear deubiquitylase BRCA1-associated protein 1 (BAP1) is frequently inactivated in malignant pleural mesothelioma (MPM) and germline <i>BAP1</i> mutation predisposes to cancers including MPM. To explore the influence on cell physiology and drug sensitivity, we sequentially edited a predisposition mutation (<i>w-</i>) and a promoter trap (<i>KO</i>) into human mesothelial cells. <i>BAP1^w-/KO</i> MeT5A cells express less BAP1 protein and phenocopy key aspects of BAP1 loss in MPM. Stable isotope labeling with amino acids in cell culture–mass spectrometry revealed evidence of metabolic adaptation, with concomitant alteration of cellular metabolites. In MeT5A, BAP1 deficiency reduces glycolytic enzyme levels but increases enzymes involved in the tricarboxylic acid cycle and anaplerotic pathways. Notably both argininosuccinate synthase 1 (ASS1), essential for cellular synthesis of arginine, and its substrate aspartate, are elevated in <i>BAP1^w-/KO</i> MeT5A cells. Likewise, ASS1 expression is higher in BAP1-altered MPM cell lines, and inversely correlates with BAP1 in The Cancer Genome Atlas MESO dataset. Elevated ASS1 is also evident by IHC staining in epithelioid MPM lacking nuclear BAP1 expression, with improved survival among patients with BAP1-negative/ASS1-expressing tumors. Alterations in arginine metabolism may sensitize cells to metabolic drugs and we find that BAP1-negative/ASS1-expressing MPM cell lines are more sensitive to ASS1 inhibition, although not to inhibition of purine synthesis by mizoribine. Importantly, <i>BAP1^w-/KO</i> MeT5A become desensitized to arginine deprivation by pegylated arginine deiminase (ADI-PEG20), phenocopying BAP1-negative/ASS1-expressing MPM cell lines.</p>Implications:<p>Our data reveal an interrelationship between BAP1 and arginine metabolism, providing a potential means of identifying patients with epithelioid MPM likely to benefit from ADI-PEG20.</p></div>

Supplementary Table S4 from BAP1 Loss Is Associated with Higher ASS1 Expression in Epithelioid Mesothelioma: Implications for Therapeutic Stratification

Abstract: <p>Metabolomics MeT5A isogenics</p>

Supplementary Table S1 from BAP1 Loss Is Associated with Higher ASS1 Expression in Epithelioid Mesothelioma: Implications for Therapeutic Stratification

Abstract: <p>STR profiling MPM cell lines</p>

Supplementary Data from Alterations in &lt;i&gt;BAP1&lt;/i&gt; Are Associated with Cisplatin Resistance through Inhibition of Apoptosis in Malignant Pleural Mesothelioma

Abstract: <p>Suppl Figures, Tables, Methods</p>

Germline pathogenic variants in patients with high-grade gastroenteropancreatic neuroendocrine neoplasms

Abstract: High-grade gastroenteropancreatic (HG-GEP) NEN are highly aggressive cancers. The molecular etiology of these tumors remains unclear and the prevalence of pathogenic germline variants in patients with HG-GEP-NEN is unknown. We assessed sequencing data of 360 cancer genes in normal tissue, from 240 patients with HG GEP-NEN; 198 patients with NEC and 42 with NET G3. Applying strict criteria, we identified pathogenic germline variants and compared the frequency with previously reported data from 33 different cancer types. We found a recurrent MYOC variant in 3 patients and a recurrent MUTYH variant in 2 patients, indicating that these genes may be important underlying risk factors for HG-GEP-NEN, when mutated. Further, germline variants were found in canonical tumor suppressor genes, such as TP53, RB1, BRIP1 and BAP1. Overall, we found that 4.5% of patients with NEC and 9.5% of patients with NET G3 carry germline pathogenic or highly likely pathogenic variants. Applying identical criteria for variant classification in-silico to mined data from 33 other cancer types, the median percentage of patients carrying pathogenic or highly likely pathogenic variants was 3.4% (range: 0-17%). The patients with NEC and pathogenic germline variants had a median overall survival of 9 months, similar to what is generally expected for metastatic GEP-NEC. A patient with NET G3 and pathogenic MUTYH variant had much shorter overall survival than expected. The fraction of HG GEP-NEN with germline pathogenic variants is relatively high, but still <10%, meaning that that germline mutations cannot be the major underlying cause of HG GEP-NEN.