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Abstract Background The normal growth and function of mammary epithelial cells depend on interactions with the supportive stroma. Alterations in this communication can lead to the progression or expansion of malignant growth. The human mammary gland contains two distinctive types of fibroblasts within the stroma. The epithelial cells are surrounded by loosely connected intralobular fibroblasts, which are subsequently surrounded by the more compacted interlobular fibroblasts. The different proximity of these fibroblasts to the epithelial cells suggests distinctive functions for these two subtypes. In this report, we compared the gene expression profiles between the two stromal subtypes. Methods Fresh normal breast tissue was collected from reduction mammoplasty patients and immediately placed into embedding medium and frozen on dry ice. Tissue sections were subjected to laser capture microscopy to isolate the interlobular from the intralobular fibroblasts. RNA was prepared and subjected to microarray analysis using the Affymetrix Human Genome U133 GeneChip®. Data was analyzed using the Affy and Limma packages available from Bioconductor. Findings from the microarray analysis were validated by RT-PCR and immunohistochemistry. Results No statistically significant difference was detected between the gene expression profiles of the interlobular and intralobular fibroblasts by microarray analysis and RT-PCR. However, for some of the genes tested, the protein expression patterns between the two subtypes of fibroblasts were significantly different. Conclusion This study is the first to report the gene expression profiles of the two distinct fibroblast populations within the human mammary gland. While there was no significant difference in the gene expression profiles between the groups, there was an obvious difference in the expression pattern of several proteins tested. This report also highlights the importance of studying gene regulation at both the transcriptional and post-translational level.

The Cancer Genome Atlas (TCGA) network study of 12 cancer types (PanCancer 12) revealed frequent mutation of TP53, and amplification and expression of related TP63 isoform ΔNp63 in squamous cancers. Further, aberrant expression of inflammatory genes and TP53/p63/p73 targets were detected in the PanCancer 12 project, reminiscent of gene programs comodulated by cREL/ΔNp63/TAp73 transcription factors we uncovered in head and neck squamous cell carcinomas (HNSCCs). However, how inflammatory gene signatures and cREL/p63/p73 targets are comodulated genome wide is unclear. Here, we examined how the inflammatory factor tumor necrosis factor-α (TNF-α) broadly modulates redistribution of cREL with ΔNp63α/TAp73 complexes and signatures genome wide in the HNSCC model UM-SCC46 using chromatin immunoprecipitation sequencing (ChIP-seq). TNF-α enhanced genome-wide co-occupancy of cREL with ΔNp63α on TP53/p63 sites, while unexpectedly promoting redistribution of TAp73 from TP53 to activator protein-1 (AP-1) sites. cREL, ΔNp63α and TAp73 binding and oligomerization on NF-κB-, TP53- or AP-1-specific sequences were independently validated by ChIP-qPCR (quantitative PCR), oligonucleotide-binding assays and analytical ultracentrifugation. Function of the binding activity was confirmed using TP53-, AP-1- and NF-κB-specific REs or p21, SERPINE1 and IL-6 promoter luciferase reporter activities. Concurrently, TNF-α regulated a broad gene network with cobinding activities for cREL, ΔNp63α and TAp73 observed upon array profiling and reverse transcription-PCR. Overlapping target gene signatures were observed in squamous cancer subsets and in inflamed skin of transgenic mice overexpressing ΔNp63α. Furthermore, multiple target genes identified in this study were linked to TP63 and TP73 activity and increased gene expression in large squamous cancer samples from PanCancer 12 TCGA by CircleMap. PARADIGM inferred pathway analysis revealed the network connection of TP63 and NF-κB complexes through an AP-1 hub, further supporting our findings. Thus, inflammatory cytokine TNF-α mediates genome-wide redistribution of the cREL/p63/p73, and AP-1 interactome, to diminish TAp73 tumor suppressor function and reciprocally activate NF-κB and AP-1 gene programs implicated in malignancy.

DNA methyltransferase inhibitors (DNMTi), most commonly cytidine analogs, are compounds that decrease 5'-cytosine methylation. DNMTi are used clinically based on the hypothesis that cytosine demethylation will lead to re-expression of tumor suppressor genes. 5-Aza-4'-thio-2'-deoxycytidine (Aza-TdCyd or ATC) is a recently described thiol-substituted DNMTi that has been shown to have anti-tumor activity in solid tumor models. In this study, we investigated the therapeutic potential of ATC in a murine transplantation model of myelodysplastic syndrome. ATC treatment led to the transformation of transplanted wild-type bone marrow nucleated cells into lymphoid leukemia, and healthy mice treated with ATC also developed lymphoid leukemia. Whole-exome sequencing revealed 1,000 acquired mutations, almost all of which were C>G transversions in a specific 5'-NCG-3' context. These mutations involved dozens of genes involved in human lymphoid leukemia, such as Notch1, Pten, Pax5, Trp53, and Nf1. Human cells treated in vitro with ATC showed 1,000 acquired C>G transversions in a similar context. Deletion of Dck, the rate-limiting enzyme for the cytidine salvage pathway, eliminated C>G transversions. Taken together, these findings demonstrate a highly penetrant mutagenic and leukemogenic phenotype associated with ATC. Significance: Treatment with a DNA methyltransferase inhibitor generates a distinct mutation signature and triggers leukemic transformation, which has important implications for the research and clinical applications of these inhibitors., (©2024 American Association for Cancer Research.)

Sarcomas are a diverse group of rare malignancies composed of multiple different clinical and molecular subtypes. Due to their rarity and heterogeneity, basic, translational, and clinical research in sarcoma has trailed behind that of other cancers. Outcomes for patients remain generally poor due to an incomplete understanding of disease biology and a lack of novel therapies. To address some of the limitations impeding preclinical sarcoma research, we have developed Sarcoma_CellMinerCDB, a publicly available interactive tool that merges publicly available sarcoma cell line data and newly generated omics data to create a comprehensive database of genomic, transcriptomic, methylomic, proteomic, metabolic, and pharmacologic data on 133 annotated sarcoma cell lines. The reproducibility, functionality, biological relevance, and therapeutic applications of Sarcoma_CellMinerCDB described herein are powerful tools to address and generate biological questions and test hypotheses for translational research. Sarcoma_CellMinerCDB (https://discover.nci.nih.gov/SarcomaCellMinerCDB) aims to contribute to advancing the preclinical study of sarcoma., Competing Interests: The authors declare no competing interests.

Colorectal cancers (CRCs) are prevalent worldwide, yet current treatments remain inadequate. Using chemical genetic screens, we identify that co-inhibition of topoisomerase I (TOP1) and NEDD8 is synergistically cytotoxic in human CRC cells. Combination of the TOP1 inhibitor irinotecan or its bioactive metabolite SN38 with the NEDD8-activating enzyme inhibitor pevonedistat exhibits synergy in CRC patient-derived organoids and xenografts. Mechanistically, we show that pevonedistat blocks the ubiquitin/proteasome-dependent repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) induced by TOP1 inhibitors and that the CUL4-RBX1 complex (CRL4) is a prominent ubiquitin ligase acting on TOP1-DPCs for proteasomal degradation upon auto-NEDD8 modification during replication. We identify DCAF13, a DDB1 and Cullin Associated Factor, as the receptor of TOP1-DPCs for CRL4. Our study not only uncovers a replication-coupled ubiquitin-proteasome pathway for the repair of TOP1-DPCs but also provides molecular and translational rationale for combining TOP1 inhibitors and pevonedistat for CRC and other types of cancers., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Proliferating cells must enact a telomere maintenance mechanism to ensure genomic stability. In a subset of tumors, telomeres are maintained not by telomerase, but through a homologous recombination-based mechanism termed Alternative Lengthening of Telomeres or ALT. The ALT process is linked to mutations in the ATRX/DAXX/H3.3 histone chaperone complex. This complex is responsible for depositing non-replicative histone variant H3.3 at pericentric and telomeric heterochromatin but has also been found to have roles in ameliorating replication in repeat sequences and in promoting DNA repair. In this review, we will discuss ways in which ATRX/DAXX helps to protect the genome, and how loss of this complex allows ALT to take hold.

A small percentage of bladder cancers in the general population have been found to harbor DNA viruses. In contrast, up to 25% of tumors of solid organ transplant recipients, who are at an increased risk of developing bladder cancer and have an overall poorer outcomes, harbor BK polyomavirus (BKPyV). To better understand the biology of the tumors and the mechanisms of carcinogenesis from potential oncoviruses, we performed whole genome and transcriptome sequencing on bladder cancer specimens from 43 transplant patients. Nearly half of the tumors from this patient population contained viral sequences. The most common were from BKPyV (N=9, 21%), JC polyomavirus (N=7, 16%), carcinogenic human papillomaviruses (N=3, 7%), and torque teno viruses (N=5, 12%). Immunohistochemistry revealed variable Large T antigen expression in BKPyV-positive tumors ranging from 100% positive staining of tumor tissue to less than 1%. In most cases of BKPyV-positive tumors, the viral genome appeared to be clonally integrated into the host chromosome consistent with microhomology-mediated end joining and coincided with focal amplifications of the tumor genome similar to other virus-mediated cancers. Significant changes in host gene expression consistent with the functions of BKPyV Large T antigen were also observed in these tumors. Lastly, we identified four mutation signatures in our cases, with those attributable to APOBEC3 and SBS5 being the most abundant. Mutation signatures associated with an antiviral drug, ganciclovir, and aristolochic acid, a nephrotoxic compound found in some herbal medicines, were also observed. The results suggest multiple pathways to carcinogenesis in solid organ transplant recipients with a large fraction being virus-associated., Competing Interests: GS, KY, MP, DP, MD, AI, BH, TT, IC, LG, CM, SH, CL, LP, PM, CB, EE No competing interests declared, YG, PL is affiliated with Leidos Biomedical Research Inc. The author has no financial interests to declare, RH is a co-founder, shareholder, and consultant of ApoGen Biotechnologies Inc

Background: DNA methylation aberrations are widespread among the malignant B lymphocytes of patients with chronic lymphocytic leukaemia (CLL), suggesting that DNA methylation might contribute to the pathogenesis of CLL. Aim: We aimed to explore the differentially methylated positions (DMPs) associated with CLL and screen the differentially methylated and expressed genes (DMEGs) by combining public databases. We aimed to observe the direction of each DMEG in CLL based on the DMPs in the promoter and the body region respectively to narrow down DMEGs. We also aimed to explore the methylation heterogeneity of CLL subgroups and the effect of B cells maturation on CLL. Methods: In this population-based case control study, we reported a genome-wide DNA methylation association study using the Infinium HumanMethylation450 BeadChip, profiling the DNA methylation of CD19 + B Cells from 48 CLL cases and 28 healthy controls. By integrating methylation data and expression data from public databases, gene sets were jointly screened, and then the relationship between methylation sites in promoter and body region and expression of each gene was explored. In addition, support vector machine (SVM) classification algorithm was used to identify subgroups of CLL cases based on methylation pattern, and the effect of B-cell differentiation related methylation sites on CLL-related sites was observed. Results: We identified 34,797 DMPs related to CLL across the genome, most of which were hypomethylated; the majority were located in gene body regions. By combining these DMPs with published DNA methylation and RNA sequencing data, we detected 26,244 replicated DMPs associated with 1,130 genes whose expression were significantly different in CLL cases. Among these DMEGs, nine low expressed DMEGs were selected with hypermethylated in promoter and hypomethylated in body region, and 83 high expressed DMEGs were selected with both hypomethylated in promoter and body region. The 48 CLL cases were divided into 3 subgroups based on methylation site by SVM algorithm. Over 92% of CpGs associated with B cell subtypes were found in CLL-related DMPs. Conclusion: The DNA methylation pattern was altered across the genome in CLL patients. The methylation of ZAP70 , FMOD , and ADAMTS17 was significantly different between CLL cases and controls. Further studies are warranted to confirm our findings and identify the underlying mechanisms through which these methylation markers are associated with CLL., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Gao, Lin, Goldin, Wang, Stevenson, Edelman, Killian, Marti, Meltzer, Xiang and Caporaso.)

Germline mutations within the Krebs cycle enzyme genes fumarate hydratase (FH) or succinate dehydrogenase (SDHB, SDHC, SDHD) are associated with an increased risk of aggressive and early metastasizing variants of renal cell carcinoma (RCC). These RCCs express significantly increased levels of intracellular fumarate or succinate that inhibit 2-oxoglutarate-dependent dioxygenases, such as the TET enzymes that regulate DNA methylation. This study evaluated the genome-wide methylation profiles of 34 RCCs from patients with RCC susceptibility syndromes and 11 associated normal samples using the Illumina HumanMethylation450 BeadChip. All the HLRCC (FH mutated) and SDHB-RCC (SDHB mutated) tumors demonstrated a distinct CpG island methylator phenotype (CIMP). HLRCC tumors demonstrated an extensive and relatively uniform level of hypermethylation that showed some correlation with tumor size. SDHB-RCC demonstrated a lesser and more varied pattern of hypermethylation that overlapped in part with the HLRCC hypermethylation. Combined methylation and mRNA expression analysis of the HLRCC tumors demonstrated hypermethylation and transcription downregulation of genes associated with the HIF pathway, HIF3A and CITED4, the WNT pathway, SFRP1, and epithelial-to-mesenchymal transition and MYC expression, OVOL1. These observations were confirmed in the TCGA CIMP-RCC tumors. A selected panel of probes could identify the CIMP tumors and differentiate between HLRCC and SDHB-RCC tumors. This panel accurately detected all CIMP-RCC tumors within the TCGA RCC cohort, identifying them as HLRCC -like, and could potentially be used to create a liquid biopsy-based screening tool. The CIMP signature in these aggressive tumors could provide both a useful biomarker for diagnosis and a target for novel therapies., Competing Interests: Authors C.J.R., C.D.V., Y.W., M.J.M., P.S.M., and W.M.L. have declared that no competing interests exist. Subsequent to working on this project, author J.K.K. changed employment to work for Foundation Medicine, Inc., a commercial company that is involved in the development of diagnostic tests for cancer related therapies. All the author’s involvement with this study pre-date employment by Foundation Medicine, Inc. and so this does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Minichromosome maintenance proteins (Mcm2-7) form a hexameric complex that unwinds DNA ahead of a replicative fork. The deficiency of Mcm proteins leads to replicative stress and consequent genomic instability. Mice with a germline insertion of a Cre cassette into the 3'UTR of the Mcm2 gene (designated Mcm2 Cre ) have decreased Mcm2 expression and invariably develop precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL), due to 100-1000 kb deletions involving important tumor suppressor genes. To determine whether mice that were protected from pre-T LBL would develop non-T-cell malignancies, we used two approaches. Mice engrafted with Mcm2 Cre/Cre Lin - Sca-1 + Kit + hematopoietic stem/progenitor cells did not develop hematologic malignancy; however, these mice died of hematopoietic stem cell failure by 6 months of age. Placing the Mcm2 Cre allele onto an athymic nu/nu background completely prevented pre-T LBL and extended survival of these mice three-fold (median 296.5 vs. 80.5 days). Ultimately, most Mcm2 Cre/Cre ;nu/nu mice developed B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We identified recurrent deletions of 100-1000 kb that involved genes known or suspected to be involved in BCP-ALL, including Pax5, Nf1, Ikzf3, and Bcor. Moreover, whole-exome sequencing identified recurrent mutations of genes known to be involved in BCP-ALL progression, such as Jak1/Jak3, Ptpn11, and Kras. These findings demonstrate that an Mcm2 Cre/Cre hypomorph can induce hematopoietic dysfunction via hematopoietic stem cell failure as well as a "deletor" phenotype affecting known or suspected tumor suppressor genes., (© 2022 Federation of American Societies for Experimental Biology. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

We perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extracranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches., Competing Interests: Declaration of interests A.S.B. has advisory board relationships with Bayer, EMD Serono, and Deciphera. R.J.O. receives research support from and consults for Lentigen, a Miltenyi Biotec Company, and also consults for Umoja Biopharma. Other authors declare no competing interests., (Published by Elsevier Inc.)

Mutations in the isocitrate dehydrogenase 1 ( IDH1 ) and IDH2 genes are frequently observed in a wide variety of hematologic malignancies, including myeloid and T-cell leukemias. In this study, we generated Idh2 R140Q transgenic mice to examine the role of the Idh2 R140Q mutation in leukemia. No leukemia developed in Idh2 R140Q transgenic mice, suggesting a need for additional genetic events for leukemia development. Because myeloid cells from NUP98-HOXD13 fusion ( NHD13 ) transgenic mice frequently acquire somatic Idh mutations when they transform to acute myeloid leukemia, we generated Idh2 R140Q /NHD13 double transgenic mice. Idh2 R140Q /NHD13 transgenic mice developed an immature T-cell leukemia with an immunophenotype similar to double-negative 1 (DN1) or DN2 thymocytes. Idh2 R140Q /NHD13 leukemic cells were enriched for an early thymic precursor transcriptional signature, and the gene expression profile for Idh2 R140Q /NHD13 DN1/DN2 T-ALL closely matched that of human early/immature T-cell precursor (EITP) acute lymphoblastic leukemia (ALL). Moreover, recurrent mutations found in patients with EITP ALL, including KRAS, PTPN11, JAK3, SH2B3 , and EZH2 were also found in Idh2 R140Q /NHD13 DN1/DN2 T-ALL. In vitro treatment of Idh2 R140Q /NHD13 thymocytes with enasidenib, a selective inhibitor of mutant IDH2, led to a marked decrease in leukemic cell proliferation. These findings demonstrate that Idh2 R140Q /NHD13 mice can serve as a useful in vivo model for the study of early/immature thymocyte precursor acute lymphoblastic leukemia development and therapy. SIGNIFICANCE: T-cell leukemia induced in Idh2 R140Q /NUP98-HOXD13 mice is immunophenotypically, transcriptionally, and genetically similar to human EITP ALL, providing a model for studying disease development and treatment., (©2021 American Association for Cancer Research.)

Renal cell carcinoma (RCC) is not a single disease but is made up of several different histologically defined subtypes that are associated with distinct genetic alterations which require subtype specific management and treatment. Papillary renal cell carcinoma (pRCC) is the second most common subtype after conventional/clear cell RCC (ccRCC), representing ~20% of cases, and is subcategorized into type 1 and type 2 pRCC. It is important for preclinical studies to have cell lines that accurately represent each specific RCC subtype. This study characterizes seven cell lines derived from both primary and metastatic sites of type 1 pRCC, including the first cell line derived from a hereditary papillary renal carcinoma (HPRC)-associated tumor. Complete or partial gain of chromosome 7 was observed in all cell lines and other common gains of chromosomes 16, 17, or 20 were seen in several cell lines. Activating mutations of MET were present in three cell lines that all demonstrated increased MET phosphorylation in response to HGF and abrogation of MET phosphorylation in response to MET inhibitors. CDKN2A loss due to mutation or gene deletion, associated with poor outcomes in type 1 pRCC patients, was observed in all cell line models. Six cell lines formed tumor xenografts in athymic nude mice and thus provide in vivo models of type 1 pRCC. These type 1 pRCC cell lines provide a comprehensive representation of the genetic alterations associated with pRCC that will give insight into the biology of this disease and be ideal preclinical models for therapeutic studies., (Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Genes, Chromosomes and Cancer published by Wiley Periodicals LLC.)

Alveolar Rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with about 80% of cases characterized by either a t(1;13)(p36;q14) or t(2;13)(q35;q14), which results in the formation of the fusion oncogenes PAX7-FOXO1 and PAX3-FOXO1, respectively. Since patients with fusion-positive ARMS (FP-RMS) have a poor prognosis and are treated with an aggressive therapeutic regimen, correct classification is of clinical importance. Detection of the translocation by different molecular methods is used for diagnostics, including fluorescence in situ hybridization and RT-PCR or NGS based approaches. Since these methods are complex and time consuming, we developed specific monoclonal antibodies (mAbs) directed to the junction region on the PAX3-FOXO1 fusion protein. Two mAbs, PFM.1 and PFM.2, were developed and able to immunoprecipitate in vitro-translated PAX3-FOXO1 and cellular PAX3-FOXO1 from FP-RMS cells. Furthermore, the mAbs recognized a 105 kDa band in PAX3-FOXO1-transfected cells and in FP-RMS cell lines. The mAbs did not recognize proteins in fusion-negative embryonal rhabdomyosarcoma cell lines, nor did they recognize PAX3 or FOXO1 alone when compared to anti-PAX3 and anti-FOXO1 antibodies. We next evaluated the ability of mAb PFM.2 to detect the fusion protein by immunohistochemistry. Both PAX3-FOXO1 and PAX7-FOXO1 were detected in HEK293 cells transfected with the corresponding cDNAs. Subsequently, we stained 26 primary tumor sections and a rhabdomyosarcoma tissue array and detected both fusion proteins with a positive predictive value of 100%, negative predictive value of 98%, specificity of 100% and a sensitivity of 91%. While tumors are stained homogenously in PAX3-FOXO1 cases, the staining pattern is heterogenous with scattered positive cells only in tumors expressing PAX7-FOXO1. No staining was observed in stromal cells, embryonal rhabdomyosarcoma, and fusion-negative rhabdomyosarcoma. These results demonstrate that mAbs specific for the chimeric oncoproteins PAX3-FOXO1 and PAX7-FOXO1 can be used efficiently for simple and fast subclassification of rhabdomyosarcoma in routine diagnostics via immunohistochemical detection.

Renal cell carcinoma (RCC) is a heterogenous disease composed of several different cancer types characterized by distinct histologies and genetic alterations, including mutation of the Krebs cycle enzyme genes for fumarate hydratase and succinate dehydrogenase (SDH). This report describes a patient with multifocal renal tumors that presented with a novel, biphasic histologic morphology with one component consisting of small cells growing in a diffuse pattern occasionally forming glandular and cystic structures, reminiscent of type 1 papillary RCC, and the other component having larger cells with abundant eosinophilic and clear cytoplasm and appearing in a solid pattern of growth. Genetic analysis of multiple tumors showed that all had a somatic mutation of the IDH2 gene that created the known pathogenic, gain-of-function p.R172M alteration that results in abnormal accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). Analysis of multiple tumors demonstrated highly elevated levels of 2-HG and a CpG island methylator phenotype that is characteristic of 2-HG-related inhibition of the Ten-eleven translocation (TET) family of DNA demethylases. In combination with fumarate hydratase-deficient and succinate dehydrogenase-deficient RCCs that have increased levels of the fumarate and succinate oncometabolites, respectively, the mutation of isocitrate dehydrogenase 2 represents the third Krebs cycle enzyme alteration to be associated with oncometabolite-induced RCC tumorigenesis. This study associates the discovery of a new histologic presentation of RCC with the first report of an IDH2 gain-of-function mutation in RCC.

Long noncoding RNAs (lncRNAs) are often associated with polysomes, indicating coding potential. However, only a handful of endogenous proteins encoded by putative lncRNAs have been identified and assigned a function. Here, we report the discovery of a putative gastrointestinal-tract-specific lncRNA ( LINC00675 ) that is regulated by the pioneer transcription factor FOXA1 and encodes a conserved small protein of 79 amino acids which we termed FORCP ( FO XA1- R egulated C onserved Small P rotein). FORCP transcript is undetectable in most cell types but is abundant in well-differentiated colorectal cancer (CRC) cells where it functions to inhibit proliferation, clonogenicity, and tumorigenesis. The epitope-tagged and endogenous FORCP protein predominantly localizes to the endoplasmic reticulum (ER). In response to ER stress, FORCP depletion results in decreased apoptosis. Our findings on the initial characterization of FORCP demonstrate that FORCP is a novel, conserved small protein encoded by a mis-annotated lncRNA that regulates apoptosis and tumorigenicity in well-differentiated CRC cells., Competing Interests: XL, LP, WT, BM, MJ, SL, ED, CH, IG, QH, QS, AS, JM, BT, LJ, AR, RW, SA, RC, SS, MG, SH, CH, PM, KP, MA No competing interests declared, SD, TA is affiliated with Leidos Biomedical Research, Inc. AL Reviewing editor, eLife

CellMiner-SCLC (https://discover.nci.nih.gov/SclcCellMinerCDB/) integrates drug sensitivity and genomic data, including high-resolution methylome and transcriptome from 118 patient-derived small cell lung cancer (SCLC) cell lines, providing a resource for research into this "recalcitrant cancer." We demonstrate the reproducibility and stability of data from multiple sources and validate the SCLC consensus nomenclature on the basis of expression of master transcription factors NEUROD1, ASCL1, POU2F3, and YAP1. Our analyses reveal transcription networks linking SCLC subtypes with MYC and its paralogs and the NOTCH and HIPPO pathways. SCLC subsets express specific surface markers, providing potential opportunities for antibody-based targeted therapies. YAP1-driven SCLCs are notable for differential expression of the NOTCH pathway, epithelial-mesenchymal transition (EMT), and antigen-presenting machinery (APM) genes and sensitivity to mTOR and AKT inhibitors. These analyses provide insights into SCLC biology and a framework for future investigations into subtype-specific SCLC vulnerabilities., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

Differentiation status of tumors is correlated with metastatic potential and malignancy. FOXA1 (forkhead box A1) is a transcription factor known to regulate differentiation in certain tissues. Here, we investigate FOXA1 function in human colorectal cancer (CRC). We found that FOXA1 is robustly expressed in the normal human colon but significantly downregulated in colon adenocarcinoma. Applying FOXA1 chromatin immunoprecipitation coupled with deep sequencing and transcriptome analysis upon FOXA1 knockdown in well-differentiated CRC cells and FOXA1 overexpression in poorly differentiated CRC cells, we identified novel protein-coding and lncRNA genes regulated by FOXA1. Among the numerous novel FOXA1 targets we identified, we focused on CEACAM5 , a tumor marker and facilitator of cell adhesion. We show that FOXA1 binds to a distal enhancer downstream of CEACAM5 and strongly activates its expression. Consistent with these data, we show that FOXA1 inhibits anoikis in CRC cells. Collectively, our results uncover novel protein-coding and noncoding targets of FOXA1 and suggest a vital role of FOXA1 in enhancing CEACAM5 expression and anoikis resistance in CRC cells., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair 1-4 . Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides 5 . Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.

Renal medullary carcinoma (RMC) is a rare, aggressive disease that predominantly afflicts individuals of African or Mediterranean descent with sickle cell trait. RMC comprises 1% of all renal cell carcinoma diagnoses with a median overall survival of 13 months. Patients are typically young (median age-22) and male (male:female ratio of 2:1) and tumors are characterized by complete loss of expression of the SMARCB1 tumor suppressor protein. Due to the low incidence of RMC and the disease's aggressiveness, treatment decisions are often based on case reports. Thus, it is critical to develop preclinical models of RMC to better understand the pathogenesis of this disease and to identify effective forms of therapy. Two novel cell line models, UOK353 and UOK360, were derived from primary RMCs that both demonstrated the characteristic SMARCB1 loss. Both cell lines overexpressed EZH2 and other members of the polycomb repressive complex and EZH2 inhibition in RMC tumor spheroids resulted in decreased viability. High throughput drug screening of both cell lines revealed several additional candidate compounds, including bortezomib that had both in vitro and in vivo antitumor activity. The activity of bortezomib was shown to be partially dependent on increased oxidative stress as addition of the N-acetyl cysteine antioxidant reduced the effect on cell proliferation. Combining bortezomib and cisplatin further decreased cell viability both in vitro and in vivo that single agent bortezomib treatment. The UOK353 and UOK360 cell lines represent novel preclinical models for the development of effective forms of therapy for RMC patients., (© 2020 The Authors. Genes, Chromosomes & Cancer published by Wiley Periodicals, Inc.)

Background: Small cell lung cancer (SCLC) is an aggressive neuroendocrine lung cancer. SCLC progression and treatment resistance involve epigenetic processes. However, links between SCLC DNA methylation and drug response remain unclear. We performed an epigenome-wide study of 66 human SCLC cell lines using the Illumina Infinium MethylationEPIC BeadChip array. Correlations of SCLC DNA methylation and gene expression with in vitro response to 526 antitumor agents were examined., Results: We found multiple significant correlations between DNA methylation and chemosensitivity. A potentially important association was observed for TREX1, which encodes the 3' exonuclease I that serves as a STING antagonist in the regulation of a cytosolic DNA-sensing pathway. Increased methylation and low expression of TREX1 were associated with the sensitivity to Aurora kinase inhibitors AZD-1152, SCH-1473759, SNS-314, and TAK-901; the CDK inhibitor R-547; the Vertex ATR inhibitor Cpd 45; and the mitotic spindle disruptor vinorelbine. Compared with cell lines of other cancer types, TREX1 had low mRNA expression and increased upstream region methylation in SCLC, suggesting a possible relationship with SCLC sensitivity to Aurora kinase inhibitors. We also identified multiple additional correlations indicative of potential mechanisms of chemosensitivity. Methylation of the 3'UTR of CEP350 and MLPH, involved in centrosome machinery and microtubule tracking, respectively, was associated with response to Aurora kinase inhibitors and other agents. EPAS1 methylation was associated with response to Aurora kinase inhibitors, a PLK-1 inhibitor and a Bcl-2 inhibitor. KDM1A methylation was associated with PLK-1 inhibitors and a KSP inhibitor. Increased promoter methylation of SLFN11 was correlated with resistance to DNA damaging agents, as a result of low or no SLFN11 expression. The 5' UTR of the epigenetic modifier EZH2 was associated with response to Aurora kinase inhibitors and a FGFR inhibitor. Methylation and expression of YAP1 were correlated with response to an mTOR inhibitor. Among non-neuroendocrine markers, EPHA2 was associated with response to Aurora kinase inhibitors and a PLK-1 inhibitor and CD151 with Bcl-2 inhibitors., Conclusions: Multiple associations indicate potential epigenetic mechanisms affecting SCLC response to chemotherapy and suggest targets for combination therapies. While many correlations were not specific to SCLC lineages, several lineage markers were associated with specific agents.

Circular RNAs (circRNAs) are a class of noncoding RNAs produced by a noncanonical form of alternative splicing called back-splicing. To investigate a potential role of circRNAs in the p53 pathway, we analyzed RNA sequencing (RNA-seq) data from colorectal cancer cell lines (HCT116, RKO, and SW48) that were untreated or treated with a DNA-damaging agent. Surprisingly, unlike the strong p53-dependent induction of hundreds of p53-induced mRNAs upon DNA damage, only a few circRNAs were upregulated from p53-induced genes. circ-MDM2 , an annotated circRNA from the MDM2 locus, was one of the handful of circRNAs that originated from a p53-induced gene. Given the central role of MDM2 in suppressing p53 protein levels and p53 activity, we investigated the function of circ-MDM2 Knocking down circ-MDM2 with small interfering RNAs (siRNAs) that targeted circ-MDM2 did not alter MDM2 mRNA or MDM2 protein levels but resulted in increased basal p53 levels and growth defects in vitro and in vivo Consistent with these results, transcriptome profiling showed increased expression of several direct p53 targets, reduced retinoblastoma protein (Rb) phosphorylation, and defects in G 1 -S progression upon silencing circ-MDM2 Our results on the initial characterization of circ-MDM2 identify a new player from the MDM2 locus that suppresses p53 levels and cell cycle progression., (Copyright © 2020 American Society for Microbiology.)

Centromeric localization of CENP-A (Cse4 in Saccharomyces cerevisiae , CID in flies, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of overexpressed CENP-A contributes to aneuploidy in yeast, flies, and humans, and is proposed to promote tumorigenesis in human cancers. Hence, defining molecular mechanisms that promote or prevent mislocalization of CENP-A is an area of active investigation. In budding yeast, evolutionarily conserved histone chaperones Scm3 and chromatin assembly factor-1 (CAF-1) promote localization of Cse4 to centromeric and noncentromeric regions, respectively. Ubiquitin ligases, such as Psh1 and Slx5, and histone chaperones (HIR complex) regulate proteolysis of overexpressed Cse4 and prevent its mislocalization to noncentromeric regions. In this study, we have identified sumoylation sites lysine (K) 215/216 in the C terminus of Cse4, and shown that sumoylation of Cse4 K215/216 facilitates its genome-wide deposition into chromatin when overexpressed. Our results showed reduced levels of sumoylation of mutant Cse4 K215/216R/A [K changed to arginine (R) or alanine (A)] and reduced interaction of mutant Cse4 K215/216R/A with Scm3 and CAF-1 when compared to wild-type Cse4 Consistent with these results, levels of Cse4 K215/216R/A in the chromatin fraction and localization to centromeric and noncentromeric regions were reduced. Furthermore, in contrast to GAL- CSE4 , which exhibits Synthetic Dosage Lethality (SDL) in psh1 ∆, slx5 ∆, and hir2 ∆ strains, GAL- cse4 K215/216R does not exhibit SDL in these strains. Taken together, our results show that deposition of Cse4 into chromatin is facilitated by its C-terminal sumoylation., (Copyright © 2020 by the Genetics Society of America.)

Hairy cell leukemia (HCL) is a purine analog-responsive B-cell malignancy containing the BRAF V600E mutation, expressing CD22, CD11c, CD103, tartrate resistant acid phosphatase (TRAP) CD25, CD123, and annexin 1A. BRAF V600E and the latter 4 markers are usually absent in the more aggressive and chemoresistant variant HCLv. To evaluate differences between HCL and HCLv, expression microarrays comparing HCL with HCLv were performed for 24694 genes using 47323 probes. Microarray data from 35 HCL and 27 HCLv purified samples showed the greatest HCL-HCLv difference in the muscle-associated gene MYF6, expressed by its 2 probes 18.5- and 10.8-fold higher in HCL than HCLv (p<0.0001). By real-time quantitative PCR (RQ-PCR), 100% of 152 classic HCL samples were MYF6-positive, vs 5 (6%) of 90 blood donors. MYF6-expression was also detected in 18 (35%) of 51 with HCLv, 11 (92%) of 12 with HCL expressing unmutated IGHV4-34, 35 (73%) of 48 with chronic lymphocytic leukemia (CLL), and 1 (8%) of 12 with mantle cell lymphoma. Hypomethylation status of MYF6 supported expression in HCL more than HCLv. Posttreatment blood samples becoming negative by flow cytometry remained MYF6+ by RQ-PCR in 42 (48%) of 87 HCL patients, and MYF6 RQ-PCR could detect 1 HCL in 105 normal cells. MYF6, universally expressed in HCL and in most CLL samples, may be a useful biomarker for these leukemias. Further studies are underway to determine the role of MYF6 in HCL., Competing Interests: The authors have declared that no competing interests exist.

Restricting the localization of the histone H3 variant CENP-A (Cse4 in yeast, CID in flies) to centromeres is essential for faithful chromosome segregation. Mislocalization of CENP-A leads to chromosomal instability (CIN) in yeast, fly and human cells. Overexpression and mislocalization of CENP-A has been observed in many cancers and this correlates with increased invasiveness and poor prognosis. Yet genes that regulate CENP-A levels and localization under physiological conditions have not been defined. In this study we used a genome-wide genetic screen to identify essential genes required for Cse4 homeostasis to prevent its mislocalization for chromosomal stability. We show that two Skp, Cullin, F-box (SCF) ubiquitin ligases with the evolutionarily conserved F-box proteins Met30 and Cdc4 interact and cooperatively regulate proteolysis of endogenous Cse4 and prevent its mislocalization for faithful chromosome segregation under physiological conditions. The interaction of Met30 with Cdc4 is independent of the D domain, which is essential for their homodimerization and ubiquitination of other substrates. The requirement for both Cdc4 and Met30 for ubiquitination is specifc for Cse4; and a common substrate for Cdc4 and Met30 has not previously been described. Met30 is necessary for the interaction between Cdc4 and Cse4, and defects in this interaction lead to stabilization and mislocalization of Cse4, which in turn contributes to CIN. We provide the first direct link between Cse4 mislocalization to defects in kinetochore structure and show that SCF-mediated proteolysis of Cse4 is a major mechanism that prevents stable maintenance of Cse4 at non-centromeric regions, thus ensuring faithful chromosome segregation. In summary, we have identified essential pathways that regulate cellular levels of endogenous Cse4 and shown that proteolysis of Cse4 by SCF-Met30/Cdc4 prevents mislocalization and CIN in unperturbed cells., Competing Interests: NO authors have competing interests.

Cutaneous malignant melanoma is an aggressive cancer of melanocytes with a strong propensity to metastasize. We posit that melanoma cells acquire metastatic capability by adopting an embryonic-like phenotype, and that a lineage approach would uncover metastatic melanoma biology. Using a genetically engineered mouse model to generate a rich melanoblast transcriptome dataset, we identify melanoblast-specific genes whose expression contribute to metastatic competence and derive a 43-gene signature that predicts patient survival. We identify a melanoblast gene, KDELR3, whose loss impairs experimental metastasis. In contrast, KDELR1 deficiency enhances metastasis, providing the first example of different disease etiologies within the KDELR-family of retrograde transporters. We show that KDELR3 regulates the metastasis suppressor, KAI1, and report an interaction with the E3 ubiquitin-protein ligase gp78, a regulator of KAI1 degradation. Our work demonstrates that the melanoblast transcriptome can be mined to uncover targetable pathways for melanoma therapy.

Thrombocytopenia-absent radii (TAR) syndrome, characterized by neonatal thrombocytopenia and bilateral radial aplasia with thumbs present, is typically caused by the inheritance of a 1q21.1 deletion and a single-nucelotide polymorphism in RBM8A on the nondeleted allele. We evaluated two siblings with TAR-like dysmorphology but lacking thrombocytopenia in infancy. Family NCI-107 participated in an IRB-approved cohort study and underwent comprehensive clinical and genomic evaluations, including aCGH, whole-exome, whole-genome, and targeted sequencing. Gene expression assays and electromobility shift assays (EMSAs) were performed to evaluate the variant of interest. The previously identified TAR-associated 1q21.1 deletion was present in the affected siblings and one healthy parent. Multiple sequencing approaches did not identify previously described TAR-associated SNPs or mutations in relevant genes. We discovered rs61746197 A > G heterozygosity in the parent without the deletion and apparent hemizygosity in both siblings. rs61746197 A > G overlaps a RelA-p65 binding motif, and EMSAs indicate the A allele has higher transcription factor binding efficiency than the G allele. Stimulation of K562 cells to induce megakaryocyte differentiation abrogated the shift of both reference and alternative probes. The 1q21.1 TAR-associated deletion in combination with the G variant of rs61746197 on the nondeleted allele is associated with a TAR-like phenotype. rs61746197 G could be a functional enhancer/repressor element, but more studies are required to identify the specific factor(s) responsible. Overall, our findings suggest a role of rs61746197 A > G and human disease in the setting of a 1q21.1 deletion on the other chromosome., (Published by Cold Spring Harbor Laboratory Press.)

Mice homozygous for a hypomorphic allele of DNA replication factor minichromosome maintenance protein 2 (designated Mcm2cre/cre) develop precursor T cell lymphoblastic leukemia/lymphoma (pre-T LBL) with 4-32 small interstitial deletions per tumor. Mice that express a NUP98-HOXD13 (NHD13) transgene develop multiple types of leukemia, including myeloid and T and B lymphocyte. All Mcm2cre/cre NHD13+ mice develop pre-T LBL, and 26% develop an unrelated, concurrent B cell precursor acute lymphoblastic leukemia (BCP-ALL). Copy number alteration (CNA) analysis demonstrated that pre-T LBLs were characterized by homozygous deletions of Pten and Tcf3 and partial deletions of Notch1 leading to Notch1 activation. In contrast, BCP-ALLs were characterized by recurrent deletions involving Pax5 and Ptpn1 and copy number gain of Abl1 and Nup214 resulting in a Nup214-Abl1 fusion. We present a model in which Mcm2 deficiency leads to replicative stress, DNA double strand breaks (DSBs), and resultant CNAs due to errors in DNA DSB repair. CNAs that involve critical oncogenic pathways are then selected in vivo as malignant lymphoblasts because of a fitness advantage. Some CNAs, such as those involving Abl1 and Notch1, represent attractive targets for therapy.

Motivation: DNA copy number (CN) data are a fast-growing source of information used in basic and translational cancer research. Most CN segmentation data are presented without regard to the relationship between chromosomal regions. We offer both a toolkit to help scientists without programming experience visually explore the CN interactome and a package that constructs CN interactomes from publicly available data sets., Results: The CNVScope visualization, based on a publicly available neuroblastoma CN data set, clearly displays a distinct CN interaction in the region of the MYCN , a canonical frequent amplicon target in this cancer. Exploration of the data rapidly identified cis and trans events, including a strong anticorrelation between 11q loss and17q gain with the region of 11q loss bounded by the cell cycle regulator CCND1 ., Availability: The shiny application is readily available for use at http://cnvscope.nci.nih.gov/, and the package can be downloaded from CRAN (https://cran.r-project.org/package=CNVScope), where help pages and vignettes are located. A newer version is available on the GitHub site (https://github.com/jamesdalg/CNVScope/), which features an animated tutorial. The CNVScope package can be locally installed using instructions on the GitHub site for Windows and Macintosh systems. This CN analysis package also runs on a linux high-performance computing cluster, with options for multinode and multiprocessor analysis of CN variant data. The shiny application can be started using a single command (which will automatically install the public data package)., Competing Interests: Declaration of Conflicting Interests:The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: All authors were employees or fellows of the National Cancer Institute., (© The Author(s) 2019.)

Transgenic mice that express either a NUP98-PHF23 (NP23) or NUP98-HOXD13 (NHD13) fusion in the hematopoietic compartment develop a wide spectrum of leukemias, including myeloid, erythroid, megakaryocytic and lymphoid, at age 9-14 months. NP23-NHD13 double transgenic mice were generated by interbreeding NP23 and NHD13 mice. Remarkably, 100% of the NP23-NHD13 double transgenic mice developed acute myeloid leukemia (AML) within three months, characterized by replacement of the thymus with leukemic myeloblasts. The marked infiltration of thymus led to the intriguing hypothesis that AML generated in NP23-NHD13 mice arose in the thymus, as opposed to the bone marrow (BM). Transplantation of CD4-CD8- double negative (DN) thymocytes (which were also negative for Mac1 and Gr1) from leukemic NHD13/NP23 mice demonstrated that DN thymocytes could transmit AML, and limiting dilution studies showed that leukemia initiating cells were increased 14-fold in the thymus compared to BM. Further thymocyte fractionation demonstrated that DN1 and DN2, but not DN3 or DN4 fractions transmitted AML, and a marked expansion (100-fold) of Lineage-Sca1 + Kit + (LSK) cells in the thymus of the NP23-NHD13 mice. Taken together, these results show that the thymus of NP23-NHD13 mice acts as a reservoir for AML initiating cells and that thymic progenitors can transmit AML.

Patients who are diagnosed with osteosarcoma (OS) today receive the same therapy that patients have received over the last 4 decades. Extensive efforts to identify more effective or less toxic regimens have proved disappointing. As we enter a postgenomic era in which we now recognize OS not as a cancer of mutations but as one defined by p53 loss, chromosomal complexity, copy number alteration, and profound heterogeneity, emerging threads of discovery leave many hopeful that an improving understanding of biology will drive discoveries that improve clinical care. Under the organization of the Bone Tumor Biology Committee of the Children's Oncology Group, a team of clinicians and scientists sought to define the state of the science and to identify questions that, if answered, have the greatest potential to drive fundamental clinical advances. Having discussed these questions in a series of meetings, each led by invited experts, we distilled these conversations into a series of seven Provocative Questions. These include questions about the molecular events that trigger oncogenesis, the genomic and epigenomic drivers of disease, the biology of lung metastasis, research models that best predict clinical outcomes, and processes for translating findings into clinical trials. Here, we briefly present each Provocative Question, review the current scientific evidence, note the immediate opportunities, and speculate on the impact that answered questions might have on the field. We do so with an intent to provide a framework around which investigators can build programs and collaborations to tackle the hardest problems and to establish research priorities for those developing policies and providing funding., (© 2019 American Cancer Society.)

CellMiner (http://discover.nci.nih.gov/cellminer) and CellMinerCDB (https://discover.nci.nih.gov/cellminercdb/) are web-based applications for mining publicly available genomic, molecular, and pharmacologic datasets of human cancer cell lines including the NCI-60, Cancer Cell Line Encyclopedia, Genomics of Drug Sensitivity in Cancer, Cancer Therapeutics Response Portal, NCI/DTP small cell lung cancer, and NCI Almanac cell line sets. Here, we introduce our RNA sequencing (RNA-seq) data for the NCI-60 and their access and integration with the other databases. Correlation to transcript microarray expression levels for identical genes and identical cell lines across CellMinerCDB demonstrates the high quality of these new RNA-seq data. We provide composite and isoform transcript expression data and demonstrate diversity in isoform composition for individual cancer- and pharmacologically relevant genes, including HRAS, PTEN, EGFR, RAD51, ALKBH2, BRCA1, ERBB2, TP53, FGFR2, and CTNND1. We reveal cell-specific differences in the overall levels of isoforms and show their linkage to expression of RNA processing and splicing genes as well as resultant alterations in cancer and pharmacologic gene sets. Gene-drug pairings linked by pathways or functions show specific correlations to isoforms compared with composite gene expression, including ALKBH2-benzaldehyde, AKT3-vandetanib, BCR-imatinib, CDK1 and 20-palbociclib, CASP1-imexon, and FGFR3-pazopanib. Loss of MUC1 20 amino acid variable number tandem repeats, which is used to elicit immune response, and the presence of the androgen receptor AR-V4 and -V7 isoforms in all NCI-60 tissue of origin types demonstrate translational relevance. In summary, we introduce RNA-seq data to our CellMiner and CellMinerCDB web applications, allowing their exploration for both research and translational purposes. SIGNIFICANCE: The current study provides RNA sequencing data for the NCI-60 cell lines made accessible through both CellMiner and CellMinerCDB and is an important pharmacogenomics resource for the field., (©2019 American Association for Cancer Research.)