Putative Tumor Suppressor Gene Research Articles

ARHGAP26 deficiency drives the oocyte aneuploidy and early embryonic development failure.

Aneuploidy, the presence of a chromosomal anomaly, is a major cause of spontaneous abortions and recurrent pregnancy loss in humans. However, the underlying molecular mechanisms still remain poorly understood. Here, we report that ARHGAP26, a putative tumor suppressor gene, is a newly identified regulator of oocyte quality to maintain mitochondrial integrity and chromosome euploidy, thus ensuring normal embryonic development and fertility. Taking advantage of knockout mouse model, we revealed that genetic ablation of Arhgap26 caused the oocyte death at GV stage due to the mitochondrial dysfunction-induced ROS accumulation. Lack of Arhgap26 also impaired both in vitro and in vivo maturation of survived oocytes which results in maturation arrest and aneuploidy, and consequently leading to early embryonic development defects and subfertility. These observations were further verified by transcriptome analysis. Mechanistically, we discovered that Arhgap26 interacted with Cofilin1 to maintain the mitochondrial integrity by regulating Drp1 dynamics, and restoration of Arhgap26 protein level recovered the quality of Arhgap26-null oocytes. Importantly, we found an ARHGAP26 mutation in a patient with history of recurrent miscarriage by chromosomal microarray analysis. Altogether, our findings uncover a novel function of ARHGAP26 in the oocyte quality control and prevention of aneuploidy and provide a potential treatment strategy for infertile women caused by ARHGAP26 mutation.

Abstract PO-035: Genome-wide CRISPR knockout screen identifies sialylation as a tumor suppressive mechanism in B-cell malignancies

Abstract Ba/F3 is a murine pro-B cell line that is dependent on interleukin-3 (IL-3) for its survival and proliferation. It has been widely used for identifying oncogenes and mechanisms of drug-resistance. Using this cell line, we conducted a genome-wide CRISPR knockout screen to identify genes whose loss can confer IL-3 independence, hence may be implied as tumor suppressor genes (TSGs). We identified several genes that map to human chromosome 6q, a frequent deletion hotspot in B-cell malignancies, that may act as TSGs. One of these putative novel TSGs is Slc35a1, which encodes the membrane solute carrier that facilitates the transport of nucleotide sugar (CMP-sialic acid) into the Golgi apparatus for subsequent sialylation. Interestingly, in addition to Slc35a1, pathway analysis of hits from the CRISPR screen identified several genes in the sialic acid metabolism pathway, Nans, Cmas, and St3gal5. We confirmed that their silencing led to decreased protein sialyation and could confer IL-3-independent cell survival and growth in Ba/F3 cells. We continued to characterize Slc35a1 since its homozygous and heterozygous deletions are seen in 7-40% of different types of B-cell malignancies, including approximately 40% of patients with Diffuse Large B-cell Lymphoma. Slc35a1 deletion in murine and human cell-lines conferred cytokine-independent growth. Tail-vein injection of Slc35a1-silenced Ba/F3 cells resulted in allograft tumor growth presenting with splenomegaly and lymphadenopathy. Using Maackia amurensis lectin II (MAL II) to pull down sialylated proteins for mass spectrometry, we identified proteins that were differentially sialylated in Slc35a1 wild-type and knockout cells, among which are the α and β subunits of the integrin receptor, lymphocyte functional antigen 1 (LFA-1). As integrin desialylation can promote its constitutive activation, we are currently studying the impact of LFA-1 sialylation on B-cell function and transformation. We have also developed mouse models with B-cell specific ablation of Nans and Slc35a1 to further evaluate the role of sialylation on B-cell development and tumorigenesis in vivo. Overall, our study demonstrates that sialylation may function as a tumor suppressive mechanism and serve as a therapeutic target in B-cell cancers. Citation Format: Namratha Sheshadri, Rongrong Li, Muhammad Usama Tariq, Jianliang Shen, Jaeyong Jung, Junrong Yan, Kevin Lu, Zhiyuan Shen, Ping Xie, Wei-Xing Zong. Genome-wide CRISPR knockout screen identifies sialylation as a tumor suppressive mechanism in B-cell malignancies [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-035.

Involvement of N4BP2L1, PLEKHA4, and BEGAIN genes in breast cancer and muscle cell development.

Patients with breast cancer show altered expression of genes within the pectoralis major skeletal muscle cells of the breast. Through analyses of The Cancer Genome Atlas (TCGA)-breast cancer (BRCA), we identified three previously uncharacterized putative novel tumor suppressor genes expressed in normal muscle cells, whose expression was downregulated in breast tumors. We found that NEDD4 binding protein 2-like 1 (N4BP2L1), pleckstrin homology domain-containing family A member 4 (PLEKHA4), and brain-enriched guanylate kinase-associated protein (BEGAIN) that are normally highly expressed in breast myoepithelial cells and smooth muscle cells were significantly downregulated in breast tumor tissues of a cohort of 50 patients with this cancer. Our data revealed that the low expression of PLEKHA4 in patients with menopause below 50years correlated with a higher risk of breast cancer. Moreover, we identified N4BP2L1 and BEGAIN as potential biomarkers of HER2-positive breast cancer. Furthermore, low BEGAIN expression in breast cancer patients with blood fat, heart problems, and diabetes correlated with a higher risk of this cancer. In addition, protein and RNA expression analysis of TCGA-BRCA revealed N4BP2L1 as a promising diagnostic protein biomarker in breast cancer. In addition, the in silico data of scRNA-seq showed high expression of these genes in several cell types of normal breast tissue, including breast myoepithelial cells and smooth muscle cells. Thus, our results suggest their possible tumor-suppressive function in breast cancer and muscle development.

Computational Analysis of Single Nucleotide Polymorphisms in Human HIC1 Gene.

Background A putative tumor suppressor gene called HIC1 (hypermethylated in cancer) is situated at 17p13.3, a locus where the allelic loss occurs often in human malignancies, including breast cancer. Hypermethylated in cancer 1 protein is a protein that in humans is encoded by the HIC1 gene and it's aHomo sapiens(Human). This gene functions as a growth regulatory and tumor repressor gene. The molecular function of HIC1 gene includes DNA-binding transcription factor activity, sequence-specific DNA binding, DNA binding, histone deacetylase binding, protein binding, metal ion binding, nucleic acid binding, DNA-binding transcription repressor activity, RNA polymerase II-specific, DNA-binding transcription factor activity, RNA polymerase II-specific. The biological process of HIC1 gene includes multicellular organism development, negative regulation of Wnt signaling pathway, positive regulation of DNA damage response, signal transduction by p53 class mediator regulation of transcription, DNA-templated, negative regulation of transcription by RNA polymerase II, Wnt signaling pathway, transcription, DNA-templated, intrinsic apoptotic signaling pathway in response to DNA damage, cellular response to DNA damage stimulus. The study aimed to predict the stability and structure of the protein that will arise from single nucleotide polymorphisms (SNPs) in the human HIC1 gene. Methodology To investigate the possible negative effects associated with these SNPs, bioinformatic analysis is typically essential. The following tools were employed for forecasting harmful SNPs: scale-invariant feature transform (SIFT), Protein Analysis Through Evolutionary Relationships (PANTHER), nonsynonymous SNP by Protein Variation Effect Analyzer (PROVEAN), and nonsynonymous SNP by Single Nucleotide Polymorphism Annotation Platform (SNAP). Results The present study identified a total of 36 SNPs using the SIFT approach, which were shown to have functional significance. Twenty-six were determined to be tolerable, whereas 10 were shown to be detrimental. Out of 20 SNPs, seven (P370A, P646S, R654P, A476T, S400S, D666N, D7V) SNPs were predicted as "Possibly damaging" and seven (L9F, G468R, G490R, L482R, S12W, G489D, S12P) were identified as "probably benign", and six (R725G, G620S, A56V, E463D, D394N, L338V) were identified as "probably damaging" according to the predictions made by PANTHER tools. The majority of the pixels on the strip were red, indicating that the gene changes may have dangerous consequences. These results highlight the need for more research to fully comprehend how these mutations affect the hic1 protein's function, which is essential for the emergence of different types of cancer. Conclusion The current research has provided us with essential information about how SNPs might be used as a diagnostic marker for cancer, given that SNPs may be candidates for cellular changes caused by mutations linked to cancer.

Familial bilateral macronodular adrenal hyperplasia due to a novel ARMC 5 germline mutation: Clinical status and possible association with other neoplasms

Introduction/ObjectivesMutations in the ARMC5 (armadillo repeat containing 5, OMIM 615549) gene, a putative tumor suppressor gene, have recently been identified as a common cause of sporadic and familial bilateral macronodular adrenal hyperplasia (BMAH). Familial BMAH is thought to be caused by two mutations, one germline and the other somatic, as suggested by the 2-hit theory. The objective is to describe a new mutation and develop its clinical characteristics and implications. Methods, Results and ConclusionsWe present an affected family with 11 members carrying a novel mutation of the ARMC5 gene (NM_001288767.1): c.2162T>C p. (Leu721Pro). Two of the carriers developed clinical Cushing's syndrome (CS), two mild autonomous cortisol secretion (MACS) and one presented with autonomous cortisol secretion (ACS). Four patients developed other tumors, three of whom died from this cause. It is not known whether these tumors could be related to the described mutation.

Familial bilateral macronodular adrenal hyperplasia due to a novel ARMC 5 germline mutation: Clinical status and possible association with other neoplasms

Introduction/ObjectivesMutations in the ARMC5 (armadillo repeat containing 5, OMIM 615549) gene, a putative tumor suppressor gene, have recently been identified as a common cause of sporadic and familial bilateral macronodular adrenal hyperplasia (BMAH). Familial BMAH is thought to be caused by two mutations, one germline and the other somatic, as suggested by the 2-hit theory. The objective is to describe a new mutation and develop its clinical characteristics and implications. Methods, Results and ConclusionsWe present an affected family with 11 members carrying a novel mutation of the ARMC5 gene (NM_001288767.1): c.2162T>C p. (Leu721Pro). Two of the carriers developed clinical Cushing's syndrome (CS), two mild autonomous cortisol secretion (MACS) and one presented with autonomous cortisol secretion (ACS). Four patients developed other tumors, three of whom died from this cause. It is not known whether these tumors could be related to the described mutation.

CLEC19A overexpression inhibits tumor cell proliferation/migration and promotes apoptosis concomitant suppression of PI3K/AKT/NF-κB signaling pathway in glioblastoma multiforme.

GBM is the most frequent malignant primary brain tumor in humans. The CLEC19A is a member of the C-type lectin family, which has a high expression in brain tissue. Herein, we sought to carry out an in-depth analysis to pinpoint the role of CLEC19A expression in GBM. To determine the localization of CLEC19A, this protein was detected using Western blot, Immunocytochemistry/Immunofluorescence, and confocal microscopy imaging. CLEC19A expression in glioma cells and tissues was evaluated by qRT-PCR. Cell viability, proliferation, migration, and apoptosis were examined through MTT assay, CFSE assay, colony formation, wound healing assay, transwell test, and flow cytometry respectively after CLEC19A overexpression. The effect of CLEC19A overexpression on the PI3K/AKT/NF-κB signaling pathway was investigated using Western blot. An in vivo experiment substantiated the in vitro results using the glioblastoma rat models. Our in-silico analysis using TCGA data and measuring CLEC19A expression level by qRT-PCR determined significantly lower expression of CLEC19A in human glioma tissues compared to healthy brain tissues. By employment of ICC/IF, confocal microscopy imaging, and Western blot we could show that CLEC19A is plausibly a secreted protein. Results obtained from several in vitro readouts showed that CLEC19A overexpression in U87 and C6 glioma cell lines is associated with the inhibition of cell proliferation, viability, and migration. Further, qRT-PCR and Western blot analysis showed CLEC19A overexpression could reduce the expression levels of PI3K, VEGFα, MMP2, and NF-κB and increase PTEN, TIMP3, RECK, and PDCD4 expression levels in glioma cell lines. Furthermore, flow cytometry results revealed that CLEC19A overexpression was associated with significant cell cycle arrest and promotion of apoptosis in glioma cell lines. Interestingly, using a glioma rat model we could substantiate that CLEC19A overexpression suppresses glioma tumor growth. To our knowledge, this is the first report providing in-silico, molecular, cellular, and in vivo evidences on the role of CLEC19A as a putative tumor suppressor gene in GBM. These results enhance our understanding of the role of CLEC19A in glioma and warrant further exploration of CLEC19A as a potential therapeutic target for GBM.

DOK1 facilitates the advancement of ccRCC.

Background: Renal cell carcinoma (RCC) is one of the most common human cancers. Clear cell renal cell carcinoma (ccRCC) is a major subtype of RCC. However, the molecular mechanisms underlying ccRCC oncogenesis require further investigation. Docking protein 1 (DOK1) is a putative tumor suppressor gene; however, its role in ccRCC remains unclear. Methods: Bioinformatic analysis was used to illustrate the poor prognosis associated with DOK1 expression and its role in tumor development in ccRCC in patients. qPCR (quantitative polymerase chain reaction) and western blotting assays were used to validate DOK1 expression in ccRCC cells. In vitro experiments were performed to further elucidate the biological role of DOK1 in ccRCC. Results: DOK1 was overexpressed in ccRCC tissues and cells at both mRNA and protein levels. High DOK1 expression closely correlated with poor survival in patients with ccRCC. DOK1 expression significantly accelerated ccRCC proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Through PI3K (phosphatidylin-ositol-3-kinase)/AKT (protein kinase B)/GSK3β (glycogen synthase kinase 3 beta) signaling, DOK1 may control the progression of ccRCC. Conclusion: DOK1 has the potential to serve as a valuable biomarker and target for treatment in ccRCC through its regulation of PI3K/AKT/GSK3β signaling to promote ccRCC progression.

Functional Characterization of Genes Residing in Chromosomal Regions with “High-Risk” Lesions in Multiple Myeloma

The molecular landscape of DNA copy number variants (CNV) in multiple myeloma (MM) has been extensively studied, especially for lesions associated with adverse clinical outcome, e.g., 17p13(del) and 1p32(del) and 1q21(gain/amp). However, for many genes residing in those loci, their functional roles have not been systematically dissected. We thus assessed the functional impact of specific genomic perturbations for these genes in a collection of CRISPR gene editing studies, to define if these genes are recurrently essential or, conversely, serve as tumor suppressor genes (TSGs) for MM cells. For genome-scale CRISPR studies in 19 MM lines, CHRONOS scores (22Q2 DepMap dataset) ≤-0.4 and ≥0.4 were considered, respectively, as consistent with essentiality or putative TSG role. We focused on 254, 33 and 66 genes with available CHRONOS scores residing in chr1q21, chr17p13 (minimal common deleted region based on MMRF CoMMpass data), and chr1p32, respectively. Genes essential in >90% of tumor lines across all lineages were considered as “core” (pan-cancer) dependencies. Among non-core essential genes, MM-preferential dependencies (more potently and frequently essential in MM vs. all non-MM lines) were defined (based on de Matos Simoes et al. Nat Cancer 2023). Non-core/non-preferential genes essential in ≥3 MM lines were considered recurrent dependencies. While most genes in these loci are typically non-essential for any MM line, core, MM-preferential or MM-recurrent dependencies represent a total of 41% (104/254), 18% (6/33) and 18% (12/66) of genes residing in 1q21, 17p13 and 1p32, respectively. For instance, 1q21 contains 14 core essential genes (e.g. RPS27, H3C13, PSMB4), 89 recurrent MM dependencies (e.g. MCL1, DPM3, UBE2Q1) and one MM preferential dependency ( RPRD2). The large number of recurrent MM dependencies residing throughout 1q21, most of which are essential in both lines with and without 1q21(gain/amp), provides functional evidence supporting the notion that the biological and clinical implications of 1q21 involve concomitant activity of multiple genes, rather than a singular driver. Notably, CKS1B, a gene often used as surrogate marker for 1q21(amp) in MM, was essential in a subset of MM lines (8/19), including 4 lines without 1q21(gain), indicating that it may not be the main/specific driver for the biology of 1q21(amp). Based on CHRONOS scores and transcript levels, genes on 1q21 did not exhibit properties consistent with candidate role as recurrent TSGs. On the other hand, in MM lines which retain copy(-ies) of 17p13 or 1p32, CRISPR-based gene editing of TP53 or CDKN2C led to recurrent enrichment (CHRONOS>0.4) of these gene KOs (in 3 and 5 lines, respectively), consistent with the TSG role of these genes in MM. Notably, these 2 loci also contain essential genes, e.g., 17p13 contains core essential genes (e.g. EIF4A1 and TRAPPC1) as well as MM-preferential ( MPDU1) and recurrent ( HES7) dependencies; while chr1p32 contains 4 core ( PRPF38A, BTF3L4, ORC1, SSBP3) and 8 recurrently essential genes. CNV losses of these 2 loci is typically monoallelic vs. biallelic, respectively, e.g., CoMMpass IA22 data reveal TP53 loss in 11.5% of pts, all monoallelic vs. CDKN2C loss in 12% of pts, with 15% (23/152) biallelic events. This may be explained at least partly by the proximity of TP53 to multiple recurrent MM dependencies, while CDKN2C has fewer essential genes at its vicinity and longer genomic distances from them. For example, MPDU1 is within 97kbp of TP53, while the closest essential gene to CDKN2C ( BTF3L4) is >1Mbp away. In summary, the large number of functionally-defined MM driver genes in 1q21, including core and MM preferential/recurrently essential genes, can explain the propensity for CNV gains of this site in clinically aggressive MM; highlights several known or previously understudied therapeutic targets residing on 1q21, but also suggests that targeting any of these dependencies individually may not be sufficient to abrogate the biological significance of 1q21(gain/amp) in MM. This study also confirms the role of TP53 and CDKN2C as key drivers of the biology of 17p13(del) and 1p32(del), and points to the proximity of TP53 to multiple essential genes as possible reason why complete loss-of-function of TP53 typically involves monoallelic 17p13(del) and mutation of the other TP53 allele, while CKDN2C, which is more distant from essential genes, exhibits more frequent biallelic deletion.

Comprehensive molecular characterization of adenoid cystic carcinoma reveals tumor suppressors as novel drivers and prognostic biomarkers.

Adenoid cystic carcinoma (ACC) is a MYB-driven head and neck malignancy with high rates of local recurrence and distant metastasis and poor long-term survival. New effective targeted therapies and clinically useful biomarkers for patient stratification are needed to improve ACC patient survival. Here, we present an integrated copy number and transcriptomic analysis of ACC to identify novel driver genes and prognostic biomarkers. A total of 598 ACCs were studied. Clinical follow-up was available from 366 patients, the largest cohort analyzed to date. Copy number losses of 1p36 (70/492; 14%) and of the tumor suppressor gene PARK2 (6q26) (85/343; 25%) were prognostic biomarkers; patients with concurrent losses (n = 20) had significantly shorter overall survival (OS) than those with one or no deletions (p < 0.0001). Deletion of 1p36 independently predicted short OS in multivariate analysis (p = 0.02). Two pro-apoptotic genes, TP73 and KIF1B, were identified as putative 1p36 tumor suppressor genes whose reduced expression was associated with poor survival and increased resistance to apoptosis. PARK2 expression was markedly reduced in tumors with 6q deletions, and PARK2 knockdown increased spherogenesis and decreased apoptosis, indicating that PARK2 is a tumor suppressor in ACC. Moreover, analysis of the global gene expression pattern in 30 ACCs revealed a transcriptomic signature associated with short OS, multiple copy number alterations including 1p36 deletions, and reduced expression of TP73. Taken together, the results indicate that TP73 and PARK2 are novel putative tumor suppressor genes and potential prognostic biomarkers in ACC. Our studies provide new important insights into the pathogenesis of ACC. The results have important implications for biomarker-driven stratification of patients in clinical trials. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Epigenetic reactivation of tumor suppressor genes with CRISPRa technologies as precision therapy for hepatocellular carcinoma

BackgroundEpigenetic silencing of tumor suppressor genes (TSGs) is a key feature of oncogenesis in hepatocellular carcinoma (HCC). Liver-targeted delivery of CRISPR-activation (CRISPRa) systems makes it possible to exploit chromatin plasticity, by reprogramming transcriptional dysregulation.ResultsUsing The Cancer Genome Atlas HCC data, we identify 12 putative TSGs with negative associations between promoter DNA methylation and transcript abundance, with limited genetic alterations. All HCC samples harbor at least one silenced TSG, suggesting that combining a specific panel of genomic targets could maximize efficacy, and potentially improve outcomes as a personalized treatment strategy for HCC patients. Unlike epigenetic modifying drugs lacking locus selectivity, CRISPRa systems enable potent and precise reactivation of at least 4 TSGs tailored to representative HCC lines. Concerted reactivation of HHIP, MT1M, PZP, and TTC36 in Hep3B cells inhibits multiple facets of HCC pathogenesis, such as cell viability, proliferation, and migration.ConclusionsBy combining multiple effector domains, we demonstrate the utility of a CRISPRa toolbox of epigenetic effectors and gRNAs for patient-specific treatment of aggressive HCC.

Abstract C056: Prognostic relevance of SUV420H2 (KMT5C) gene expression in clear cell renal cell carcinoma: Implications for patients of African ancestry

Abstract African Americans are second only to Native Americans in having the highest incidence of renal cancer. Despite this statistic, very few studies have examined whether there are racial/ethnic differences in gene and protein expression. Since higher stage/grade ccRCC is uniformly lethal, it is imperative that candidate genes and their corresponding protein products be found that can identify patients across various demographics who are at greater risk of progressing to advanced stage/grade, drug-resistant clear cell renal carcinoma; and that can point to the drivers of ccRCC progression in different demographic groups. Using KIRC (clear cell renal carcinoma) TCGA dataset, we observed that SUV420H2 (KMT5C), a Lysine N-Methyltransferase, is significantly overexpressed in ccRCC (p&amp;lt;1E-12). Moreover, SUV420H2 transcripts were significantly elevated in higher stages and grades (p&amp;lt;.001). Higher SUV420H2 transcripts levels were also correlated with metastasis (p&amp;lt;0.01) and were associated with the more aggressive ccB Renal Csrcinoma phenotype (p&amp;lt;0.001). Overexpression of SUV420H2 was also associated with worst overall patient survival (p&amp;lt;0.0001; HR = 2.05 (1.5151 to 2.79) with a mean survival of 2208 days for the high expression group and 3256 days for the low expression group. Elevated levels were also associated with lower Disease Specific Survival [p&amp;lt;0.0001 HR= 2.7658 (1.8406 to 4.1560)] with a mean survival for the high expression group of 2208 versus 3871 days for the low expression group. Significantly shorter progression free disease times [p&amp;lt;0.0001, HR= 1.9830 (1.4389 to 2.7328) were also observed in the higher expression group (2189 days) relative to the low expression group (2971 days). In addition, in silico data analysis revealed that African Americans exhibited higher expression of SUV420H2 transcripts relative to Whites (p=5.94E-09) and that expression was associated with lower survival outcome (p&amp;lt;0.01, HR=4.33 (1.29 to 14.51). Taken collectively, our data suggests that higher levels of SUV420H2(KMT5C) is associated with cancer progression and the more aggressive ccRCC subtypes. Moreover, SUV420H2 behaves in a manner that suggest it may be a possible oncogene and that it is an unfavorable ccRCC biomarker. Analysis of CPTAC database and Human Protein Atlas failed to yield any information of protein expression in relationship to ccRCC. Given that SUV420H2 functions as a histone methyltransferase that specifically trimethylates histone H4, it is possible that dysregulation could result in altered epigenetic processes and inappropriate repression of genes in ccRCC. Lastly, our data underscores the need for proteomics studies that analyze protein expression differences of putative oncogenes or tumor suppressor genes and proteins across racial and ethnic demographic groups. Citation Format: Romonia Renee Reams, Simone Heyliger, Marilyn Saulsbury. Prognostic relevance of SUV420H2 (KMT5C) gene expression in clear cell renal cell carcinoma: Implications for patients of African ancestry [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C056.

Genome-Wide Association Study Identifies Variants Associated with Clinical Outcomes in Pediatric Patients with Newly Diagnosed Acute Myeloid Leukemia

Genetic aberrations in leukemic blasts are associated with clinical outcomes in patients with AML. However, the role of inherited genetic variants with regards to variation in AML treatment outcome remains obscure. To that end, we conducted a genome wide association study (GWAS) on 400 newly diagnosed pediatric AML patients to identify single nucleotide polymorphism (SNPs) that may be associated with minimal residual disease (MRD1) after induction 1 chemotherapy, event-free survival (EFS) and overall survival (OS). In this study, we utilized whole-genome genotyping with Illumina's Omni2.5Exome-8 BeadChip array to investigate SNPs for association with clinical outcome in 2 pediatric AML cohorts: the multi-site AML02 [NCT00136084, n=167] and AML08 [NCT00703820, n=233] trials. Post GWAS quality control, 1,365,439 unphased SNP genotypes were selected for imputation using TOPMed Imputation Server. Following imputation and additional quality control filtering, 6,741,200 SNPs evaluated for association with clinical outcomes (MRD1, EFS and OS). To improve statistical rigor, two GWAS approaches were utilized: i) A one-stage GWAS with both clinical trial cohorts combined (n=400) and ii) A two-stage GWAS where we utilized a two-step iterative resampling procedure (PMID: 26377241) by splitting the cohort at a 70:30 ratio into discovery and replication cohorts, balanced by clinical trial, treatment arm and initial risk group at diagnosis (100 randomizations performed). For each of the 100 iterations of the two-stage GWAS, we used the discovery cohort to perform a genome-wide analysis, followed by a replication screen. SNPs replicated in at least 20 of 100 rounds of discovery-replication were prioritized. A comparison of the two approaches showed all significant SNPs in the two-stage GWAS were also captured in the one-stage GWAS. For EFS, a Bonferroni-adjusted genome-wide significant association was observed for a SNP in CSMD1 and this SNP was replicated in 83 out of 100 discovery-replication rounds for two-stage GWAS. For CSMD1 SNP, presence of variant A allele has significantly reduced EFS (HR=3.29, 95% CI (2.177-4.975), P=1.61x10-8, Figure 1A). CSMD1 is a membrane-bound complement inhibitor that has been shown to act as a putative tumor suppressor gene in other malignancies (PMIDs:27764775, 35623420). Another significant marker worth mentioning was an intronic SNP in CDKN2B-AS1 that associated with poor outcome [EFS: HR=2.37, 95% CI (1.700-3.317), P=4.40x10-7] and [OS: HR=2.87, 95% CI (1.954-4.225), P=8.06x10-8, Figure 1B]. This marker is in high linkage disequilibrium with 26 other SNPs within the CDKN2B and CDKN2B-AS1 cluster. The product of this gene is an RNA molecule that interacts with polycomb repressive complex-1 (PRC1) and -2 (PRC2) involved in silencing other genes within this cluster through epigenetic mechanisms (PMID: 20716771). CDKN2B-AS1 has been reported to play a role in tumorigenesis and chemoresistance in pediatric T-cell acute lymphoblastic leukemia and with the risk of chronic lymphocytic leukemia (PMIDs: 32976214/23770605). Though limited by numbers, this is one of the largest GWAS conducted in pediatric AML to identify SNPs associated with clinical outcomes. Our top results show that genetic variation in several genes of biological relevance may impact MRD1, EFS and OS. Future studies are aimed at looking at SNP/gene and association with gene expression collected from leukemic cells at time of diagnosis to establish AML-specific eQTLs and functional validation of the most promising candidates. Additionally, future analyses will include other subgroups such as cytogenetic risk group and validation of these findings in larger cohort of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Trisomy-6: The Ying and Yang of Chromosome 6 Aberrations

Karyotypic aberrations of chromosome 6 (chr.6), include duplications (trisomy 6; +6), deletion of its short arm (del6p), uniparental disomy 6 (UPD6p), as well as various microduplications and microdeletions. Among many important genes, the HLA locus is located on 6p, and loss of heterozygosity and allelic loss have been described as means of immune escape in immune-mediated aplastic anemia (AA) and in relapse of myeloid neoplasia following allogeneic bone marrow transplant. The pathogenesis of duplication of chr.6 appears more complex and might involve deletion of a putative tumor suppressor gene (TSG) causing duplication of the HLA locus with increased HLA expression. We have encountered several patients with +6 who inspired us to investigate the clinical and molecular phenotypes of +6. A total of 29 +6 MDS patients were identified, of whom 16 had isolated +6 (Iso +6). When compared to normal karyotype (NK) MDS (n=420), median age was younger in Iso +6 (70 vs 42 years, P<.0001). While no differences were noted for white blood counts, Iso +6 patients had lower platelets (median 27 vs. 112 x 103/uL, P=.19), were more frequently anemic (median hemoglobin 8.4 vs. 9.5 g/dL, P=.04) and showed higher frequency of hypocellularity at marrow evaluation (62.5% vs. 9.5%, P=0.0016) as opposed to NK MDS. Indeed, 67% of Iso +6 patients had a clinical picture reminiscent of AA or MDS phenotype which might have evolved from an antecedent AA phase. However, 67% of Iso +6 MDS had high risk MDS (IPSS-R ≥3.5) and 37% transformed to AML. Thus, our cohort indicated a phenotypic dichotomy which include hypocellular MDS/AA and high-risk disease with a poor prognosis. Of all +6 MDS, mutations most commonly affected TP53 (69%) followed by EZH2 (19%). Recurrent copy number variations (CNV) such as micro-duplications and microdeletions may be used to identify culprit genes. Indeed, microdeletions involving HLA have previously hinted towards the pathogenic role of HLA deletions. We obtained CNV data and compared them to conventional germline chromosome 6 CNV gains publicly available in the Database of Genomic Variants (DGV). Genes pertaining to significantly duplicated segments were then identified using the UCSC Genome Browser (https://genome.ucsc.edu/cgi-bin/hgGateway). In our cohort, in addition to 833 cases with del6p, we have identified microduplications involving 6p locus in 115 (11.5%) NK MDS cases. Overall, a total of 1198 6p copy gains were found in these patients, ranging in size 0.09-7.8Mb and with a median of 0.12Mb. The genomic architecture of each segment was investigated using the UCSC Genome Browser, from which only complete gene duplications that mirror an extra chromosomal copy were considered. Complete duplications were most commonly seen in loci containing genes of DUSP22 (12.2%) and most importantly HLA-DRB1 (10.4%), HLA-DRB5 (10.4%), HLA-DQA1 (9.6%) and MICA (9.6%). All of these five genes were significantly enriched in NK MDS as compared to healthy controls (DUSP22 3% P<0.0001, HLA-DRB1 0.4% P<0.0001, HLA-DRB5 0.5% P<0.0001, HLA-DQA1 0.5% P<0.0001 and MICA 0.2% P<0.0001) (Fig 1). Interestingly, while checking the Cosmic Cancer Browser database (https://cancer.sanger.ac.uk), we noticed that copy gains involving HLA-DRB1 and HLA-DRB5 were also more common in primary AML than in healthy controls from DGV (3.6 vs 0.4%, P<0.0001 and 3.6 vs 0.5%, P<0.0001, respectively). While the involvement of the HLA genes hints towards an immune-mediated mechanism underlying the pathogenesis of +6 MDS, further involvement in primary AML may suggest a common mechanism through which CN gains of the HLA locus promote myelo-neoplasia. We stipulate that while clonal deletion or UPD of HLA locus can be a result of immune pressure as seen in AA and during GvL, clonal duplication may lead to increased autoantigens presentation beyond the TCR activation threshold, triggering immune reactions ultimately resulting in hypocellular marrow and AA-like presentation. Alternatively, +6 and related lesions may lead to risk allele duplication and consequential more efficient presentation of a specific antigenic peptide. In more advanced diseases, deletion of TSG may also play a role, with progression reflecting a somatic pressure to overcome immune inhibition. HLA constellation and mutational studies will be presented in the context of the findings of 6p duplications and microdeletion in MDS and AA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Expression of the methylcytosine dioxygenase ten-eleven translocation-2 and connexin 43 in inflammatory bowel disease and colorectal cancer.

Inflammatory bowel disease (IBD) constitutes a substantial risk factor for colorectal cancer. Connexin 43 (Cx43) is a protein that forms gap junction (GJ) complexes involved in intercellular communication, and its expression is altered under pathological conditions, such as IBD and cancer. Recent studies have implicated epigenetic processes modulating DNA methylation in the pathogenesis of diverse inflammatory and malignant diseases. The ten-eleven translocation-2 (TET-2) enzyme catalyzes the demethylation, hence, regulating the activity of various cancer-promoting and tumor-suppressor genes. To investigate Cx43 and TET-2 expression levels and presence of 5-hydroxymethylcytosine (5-hmC) marks under inflammatory conditions both in vitro and in vivo. TET-2 expression was evaluated in parental HT-29 cells and in HT-29 cells expressing low or high levels of Cx43, a putative tumor-suppressor gene whose expression varies in IBD and colorectal cancer, and which has been implicated in the inflammatory process and in tumor onset. The dextran sulfate sodium-induced colitis model was reproduced in BALB/c mice to evaluate the expression of TET-2 and Cx43 under inflammatory conditions in vivo. In addition, archived colon tissue sections from normal, IBD (ulcerative colitis), and sporadic colon adenocarcinoma patients were obtained and evaluated for the expression of TET-2 and Cx43. Expression levels were reported at the transcriptional level by quantitative real-time polymerase chain reaction, and at the translational level by Western blotting and immunofluorescence. Under inflammatory conditions, Cx43 and TET-2 expression levels increased compared to non-inflammatory conditions. TET-2 upregulation was more pronounced in Cx43-deficient cells. Moreover, colon tissue sections from normal, ulcerative colitis, and sporadic colon adenocarcinoma patients corroborated that Cx43 expression increased in IBD and decreased in adenocarcinoma, compared to tissues from non-IBD subjects. However, TET-2 expression and 5-hmC mark levels decreased in samples from patients with ulcerative colitis or cancer. Cx43 and TET-2 expression levels were also investigated in an experimental colitis mouse model. Interestingly, mice exposed to carbenoxolone (CBX), a GJ inhibitor, had upregulated TET-2 levels. Collectively, these results show that TET-2 levels and activity increased under inflammatory conditions, in cells downregulating gap junctional protein Cx43, and in colon tissues from mice exposed to CBX. These results suggest that TET-2 expression levels, as well as Cx43 expression levels, are modulated in models of intestinal inflammation. We hypothesize that TET-2 may demethylate genes involved in inflammation and tumorigenesis, such as Cx43, potentially contributing to intestinal inflammation and associated carcinogenesis.

Abstract 1274: Prognostic relevance of ZnF 649 in clear cell carcinoma

Abstract Clear cell renal carcinoma (ccRCC) is the most common form of neoplastic disease affecting the kidneys that accounts for at least 70% of all kidney cancers. Given its resistance to medications and high mortality at later stages, there is a need to identify biomarkers that can detect ccRCC. KRAB Zinc finger proteins (KRAB-ZFPs) are the largest family of mammalian transcription regulators. They are differentially expressed in various tissues during cellular development and phenotypic differentiation. Though poorly understood, these transcription factors, are also differentially expressed in several cancers though very few have been reported to be implicated in ccRCC. Using bioinformatics techniques, we have demonstrated that ZNF649 and its paralog ZNF613 are suppressed in clear cell renal carcinomas (p&amp;lt;0.001) relative to normal tissues. Moreover, The ZNF649 and ZNF613 transcripts were downregulated across all histological grades and pathological stages (p&amp;lt;0.001). In addition, both ZNF649 and ZNF613 downregulation was associated with metastasis and unsatisfactory patient survival (p&amp;lt;0.001). Univariate and multivariante Cox regression analysis of age, gene expression, gender, pathological stage, histological grade, and pathological N in relationship to overall survival in ccRCC patients revealed that ZNF649 expression was predictive of overall survival. Gene Set Enrichment Analysis of the Gene Ontology Biological Processes database indicate that the top pathways enriched with genes correlated to ZNF649 expression were associated with angiogenesis, cellular motility and mitochondrial regulation (p&amp;lt;0.001 and FDR &amp;lt;0.05). Taken collectively, our observations indicate that ZNF649 is a prognostic marker and is a putative tumor suppressor gene in clear cell renal carcinoma. Citation Format: Renee Reams, Simone O. Heyliger, marilyn Saulsbury, Karam F. Soliman. Prognostic relevance of ZnF 649 in clear cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1274.

Abstract 2196: Oncogenic context drives the landscape of tumor suppression in lung cancer

Abstract Cooperative interactions between genomic alterations in oncogenes and tumor suppressors are a hallmark of cancer. While molecular characterization of tens of thousands of primary tumors has solidified estimates of how frequently specific genomic alterations occur, identifying genetic interactions from co-mutation rates alone remains limited by the vast number of possible combinations as well as inherent biases and confounding effects. Conversely, genetically engineered mouse models can reliably uncover causal effects of specific genetic alterations but are not sufficiently high throughput to generate broad insights into the interplay between genetic alterations in tumors. Therefore, how the effects of one alteration on tumor growth change in the context of other alterations remains largely unknown. We previously addressed these technical limitations by combining CRISPR/Cas9-mediated gene editing, tumor barcoding, and ultra-deep barcode sequencing methods with genetically engineered mouse models of human lung cancer, which together enable multiplexing of tumor genotypes in individual mice and precise, high-throughput quantification of in vivo tumor fitness. Here, we leverage this platform to systematically quantify the growth effects of over 45 known and putative tumor suppressor gene alterations across different common oncogenic point mutations in Kras and Braf. While the set of genes we identified as tumor suppressive in KrasG12D- and KrasG12C-driven lung cancers was largely consistent, inactivation of those genes typically resulted in larger tumor growth advantages in the KrasG12D model. Inactivation of most genes in BrafV600E-driven lung tumors had a strikingly different growth effect than in tumors with either Kras variant, and effects were generally far less pronounced in the BrafV600E context. Interestingly, loss of genes upstream of Braf within the Ras pathway often had strong effects in the KrasG12D or KrasG12C models—loss of Nf1, a negative regulator of Kras, or loss of the wildtype Kras allele enhanced oncogenic Kras-driven tumor growth, while loss of Shp2, a positive regulator of Kras, was mildly detrimental—but had little or no effects in the BrafV600E model. We assessed translatability of our experimental cause-and-effect data through comparisons to correlative data from publicly available human lung cancer genomics databases, and found that genetic interactions predicted by our models and gene co-mutation rates in patient tumors largely align. Together, these findings underscore the need for a contextual understanding of the ways in which tumors are influenced by their genetics and highlight the utility of high-throughput, quantitative autochthonous mouse models in pursuing this endeavor. Citation Format: Ian Winters, Lily Blair, Lafia Sebastian, Gabriel Grenot, Edwin Apilado, Wensheng Nie, Vy Tran, Ian Lai, Gregory Wall, Dmitri Petrov, Monte Winslow, Joseph Juan, Michael Rosen. Oncogenic context drives the landscape of tumor suppression in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2196.

Hypermethylation-Mediated Silencing of CIDEA, MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies.

Gene expression is controlled by epigenetic deregulation, a hallmark of cancer. The DNA methylome of canine diffuse large B-cell lymphoma (cDLBCL), the most frequent malignancy of B-lymphocytes in dog, has recently been investigated, suggesting that aberrant hypermethylation of CpG loci is associated with gene silencing. Here, we used a multi-omics approach (DNA methylome, transcriptome and copy number variations) combined with functional in vitro assays, to identify putative tumour suppressor genes subjected to DNA methylation in cDLBCL. Using four cDLBCL primary cell cultures and CLBL-1 cells, we found that CiDEA, MAL and PCDH17, which were significantly suppressed in DLBCL samples, were hypermethylated and also responsive (at the DNA, mRNA and protein level) to pharmacological unmasking with hypomethylating drugs and histone deacetylase inhibitors. The regulatory mechanism underneath the methylation-dependent inhibition of those target genes expression was then investigated through luciferase and in vitro methylation assays. In the most responsive CpG-rich regions, an in silico analysis allowed the prediction of putative transcription factor binding sites influenced by DNA methylation. Interestingly, regulatory elements for AP2, MZF1, NF-kB, PAX5 and SP1 were commonly identified in all three genes. This study provides a foundation for characterisation and experimental validation of novel epigenetically-dysregulated pathways in cDLBCL.

LZTS2: A novel and independent prognostic biomarker for clear cell renal cell carcinoma

PurposeLeucine zipper tumor suppressor 2 (LZTS2), a putative tumor suppressor gene, has been demonstrated to be a negative regulator of microtubule severing during cytokinesis and a negative regulator of the Wnt signaling pathway. In a genetically modified mouse model, deletion of Lzts2 altered normal ureteric bud branching morphogenesis and caused cystogenesis in mice. Cyst-lining cells demonstrated atypical features, closely resembling those observed in mouse models of human clear cell renal cell carcinoma (ccRCC), which could represent a preneoplastic lesion. These findings suggest that LZTS2 may play a role in ccRCC tumorigenesis. The aim of this study was to establish an association between LZTS2 differential expression and clinicopathological features of ccRCC and to investigate its prognostic value as well as the underlying mechanisms in ccRCC progression. Materials and MethodsGene expression data by RNA-sequencing for cohorts of 510 ccRCC cases with clinical outcome data were extracted from The Cancer Genome Atlas (TCGA) using cBioPortal. Chi-square test of independence, Kaplan-Meier curves, and Cox regression models were used to investigate the possible relationship between LZTS2 mRNA expression levels and clinicopathological parameters as well as patient survival to establish its prognostic values. To examine its cellular localization, we performed LZTS2 antibody staining and scored the expression levels in a pilot study on a tissue microarray (TMA) containing 31 clear cell RCCs, 32 chromophobe RCCs, 12 papillary RCCs, and 20 adjacent benign renal tissue, as well as placental tissue diagnosed between 2001 and 2007 at the University of California San Francisco Medical Center. Staining was subsequently repeated in 15 ccRCCs on whole section slides to confirm the results. ResultsOur analysis of TCGA data demonstrated that LZTS2 expression levels were associated with tumor grade (p = 0.005), T stage (p < 0.001), metastasis status (p < 0.001), and overall clinical stage (p < 0.001). High level of expression was correlated with worse overall survival (p < 0.001), disease-specific survival (p < 0.001), progression-free survival (p < 0.001), and disease-free survival (p < 0.001) compared to low level of expression. Multivariate Cox regression analysis revealed that high LZTS2 expression was an independent poor prognostic factor for overall survival (HR = 2.083, p < 0.001), disease-specific survival (HR = 2.298, p < 0.001), and progression-free survival (HR = 1.896, p < 0.001) in patients with ccRCC. A few known driver mutations in ccRCC pathogenesis, including BAP1, NF2, and RELN, were enriched in LZTS2 high expression tumors. In particular, LZTS2 expression level could be a biomarker for risk stratification of the prognosis of BAP1-mutated ccRCCs. Immunohistochemical staining with anti-LZTS2 antibody was performed to examine its cellular localization in ccRCC and demonstrated centrosomal and acentrosomal distribution in tumors of various Fuhrman nuclear grades. Furthermore, high LZTS2 cytoplasmic expression was associated with centrosomal amplification (p = 0.030) in this small-scale study. ConclusionsThe current study established an independent prognostic value of LZTS2 expression in ccRCC and explored the molecular mechanisms of LZTS2 in predicting the prognosis of ccRCC. Further studies are needed to validate our analysis and to provide a precise understanding of the function of LZTS2 in ccRCC.

Prognostic Relevance of ZNF844 and Chr 19p13.2 KRAB-Zinc Finger Proteins in Clear Cell Renal Carcinoma.

Clear-cell renal cell carcinoma (ccRCC) is the most common and aggressive form of all urological cancers, with poor prognosis and high mortality. Despite growing evidence of involvement in carcinogenesis, the role of KRAB-ZFP in ccRCC has not been fully explored. KRAB Zinc finger proteins (KRAB-ZFPs) are the largest family of mammalian transcription regulators. They are differentially expressed in various tissues during cellular development and phenotypic differentiation. In this study, the levels of transcripts of ccRCC from The Cancer Genome Atlas (TCGA) dataset were used to identify prognostic biomarkers in this disease. Using bioinformatics techniques, we demonstrate that approximately 60% of KRAB zinc finger proteins located on chromosome 19p13.2 are differentially expressed, with all but two being down-regulated in ccRCC. Moreover, ZNF844, a paralog of ZNF433, was the most down-regulated across all histological grades and pathological stages (p<0.001). In addition, the decrease in ZNF844 expression was associated with poor patient survival (HR=0.41; 95% CI=0.3-0.56; p<0.0001). Gene Set Enrichment Analysis of genes inversely co-expressed with ZNF844 revealed that enriched pathways were consistently related to immune and translation processes (p<0.05, FDR <0.05). Lastly, ZNF844 expression showed moderate, inverse correlation to Helper T-cell (CD4 or Th1) subtype 1 (R=-0.558, p=5.15×10-39) infiltration and with the exhausted T-cell phenotype (R=-0.37; p=4.1×10-21). Down-regulation of KRAB-ZFPs at 19p13.2 may represent a signature for ccRCC. Moreover, ZNF844 is a prognostic marker for ccRCC and may serve as a putative immune-related tumor suppressor gene.