ABSTRACT
Lynch syndrome (LS) predisposes to cancer in adulthood and is caused by heterozygous germline variants in a mismatch repair (MMR) gene. Recent studies show an increased prevalence of LS among children with cancer, suggesting a causal relationship. For LS-spectrum (LSS) cancers, including high-grade gliomas and colorectal cancer, causality has been supported by typical MMR-related tumor characteristics, but for non-LSS cancers, causality is unclear. We characterized 20 malignant tumors of 18 children with LS, including 16 non-LSS tumors. We investigated second hits, tumor mutational load, mutational signatures and MMR protein expression. In all LSS tumors and three non-LSS tumors, we detected MMR deficiency caused by second hit somatic alterations. Furthermore, these MMR-deficient tumors carried driver variants that likely originated as a consequence of MMR deficiency. However, in 13 non-LSS tumors (81%), a second hit and MMR deficiency were absent, thus a causal link between LS and cancer development in these children is lacking. These findings demonstrate that causality of LS in children with cancer, which can be determined by molecular tumor characterization, seems to be restricted to specific tumor types. Large molecular and epidemiological studies are needed to further refine the tumor spectrum in children with LS.
Subject(s)
Brain Neoplasms , Colorectal Neoplasms, Hereditary Nonpolyposis , Colorectal Neoplasms , Neoplastic Syndromes, Hereditary , Child , Humans , Colorectal Neoplasms, Hereditary Nonpolyposis/genetics , Colorectal Neoplasms, Hereditary Nonpolyposis/pathology , Colorectal Neoplasms/pathology , Brain Neoplasms/genetics , Germ-Line Mutation , DNA Mismatch Repair/genetics , Microsatellite Instability , MutL Protein Homolog 1/geneticsABSTRACT
Somatic loss of function mutations in cohesin genes are frequently associated with various cancer types, while cohesin disruption in the germline causes cohesinopathies such as Cornelia-de-Lange syndrome (CdLS). Here, we present the discovery of a recurrent heterozygous RAD21 germline aberration at amino acid position 298 (p.P298S/A) identified in three children with lymphoblastic leukemia or lymphoma in a total dataset of 482 pediatric cancer patients. While RAD21 p.P298S/A did not disrupt the formation of the cohesin complex, it altered RAD21 gene expression, DNA damage response and primary patient fibroblasts showed increased G2/M arrest after irradiation and Mitomycin-C treatment. Subsequent single-cell RNA-sequencing analysis of healthy human bone marrow confirmed the upregulation of distinct cohesin gene patterns during hematopoiesis, highlighting the importance of RAD21 expression within proliferating B- and T-cells. Our clinical and functional data therefore suggest that RAD21 germline variants can predispose to childhood lymphoblastic leukemia or lymphoma without displaying a CdLS phenotype.
Subject(s)
Cell Cycle Proteins , DNA-Binding Proteins , Lymphoma , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Apoptosis , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Child , DNA-Binding Proteins/genetics , De Lange Syndrome/genetics , G2 Phase Cell Cycle Checkpoints , Germ Cells/metabolism , Humans , Lymphoma/genetics , Mutation , Phenotype , Precursor Cell Lymphoblastic Leukemia-Lymphoma/geneticsABSTRACT
BACKGROUND: Despite its relatively low incidence, pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths because of the aggressive growth/metastasis of the tumor, the lack of early symptoms, and the poor treatment options. Basic research to identify potential therapeutic targets for PDAC is greatly needed. METHODS: We used a negative-selection genome-wide CRISPR screen to identify essential genes in the PANC-1 human pancreatic carcinoma cell line. We validated the top hits with follow-up siRNA screens, using the HPNE, HPAF-II, AsPC-1, and Mia PaCa-2 cell lines. RESULTS: The PSMA6 gene was an identified candidate hit after the CRISPR screen, siRNA validation screen, and siRNA deconvolution screen. Spheroid formation assays and flow cytometry analysis showed that PSMA6 is critical for survival in many pancreatic ductal carcinoma cell models. Lastly, as PSMA6 protein is a proteosomal subunit of the 20S core complex, we showed that bortezomib, a proteasome inhibitor, was especially toxic in PANC-1 cells. CONCLUSIONS: Further study of PSMA6 and the proteasome subunit that it encodes, along with other hits identified in our CRISPR screens, may provide valuable insights into potential therapeutic targets for PDAC.
Subject(s)
Carcinoma, Pancreatic Ductal/genetics , Oncogenes/genetics , Pancreatic Neoplasms/genetics , Proteasome Endopeptidase Complex/genetics , Bortezomib/pharmacology , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Survival/drug effects , Cell Survival/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Genome, Human/genetics , Genomics/methods , Humans , Pancreas/pathology , Pancreatic Neoplasms/pathology , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/pharmacology , Proteasome Inhibitors/pharmacology , RNA, Small Interfering/genetics , Spheroids, CellularABSTRACT
Motivation: Large datasets containing multiple clinical and omics measurements for each subject motivate the development of new statistical methods to integrate these data to advance scientific discovery. Model: We propose bootstrap evaluation of association matrices (BEAM), which integrates multiple omics profiles with multiple clinical endpoints. BEAM associates a set omic features with clinical endpoints via regression models and then uses bootstrap resampling to determine statistical significance of the set. Unlike existing methods, BEAM uniquely accommodates an arbitrary number of omic profiles and endpoints. Results: In simulations, BEAM performed similarly to the theoretically best simple test and outperformed other integrated analysis methods. In an example pediatric leukemia application, BEAM identified several genes with biological relevance established by a CRISPR assay that had been missed by univariate screens and other integrated analysis methods. Thus, BEAM is a powerful, flexible, and robust tool to identify genes for further laboratory and/or clinical research evaluation. Availability: Source code, documentation, and a vignette for BEAM are available on GitHub at: https://github.com/annaSeffernick/BEAMR. The R package is available from CRAN at: https://cran.r-project.org/package=BEAMR. Contact: Stanley.Pounds@stjude.org. Supplementary Information: Supplementary data are available at the journal's website.
ABSTRACT
Analysis of mutational signatures can reveal underlying molecular mechanisms of the processes that have imprinted the somatic mutations found in cancer genomes. Here, we analyze single base substitutions and small insertions and deletions in pediatric cancers encompassing 785 whole-genome sequenced tumors from 27 molecularly defined cancer subtypes. We identified only a small number of mutational signatures active in pediatric cancers, compared with previously analyzed adult cancers. Further, we report a significant difference in the proportion of pediatric tumors showing homologous recombination repair defect signatures compared with previous analyses. In pediatric leukemias, we identified an indel signature, not previously reported, characterized by long insertions in nonrepeat regions, affecting mainly intronic and intergenic regions, but also exons of known cancer genes. We provide a systematic overview of COSMIC v.3 mutational signatures active across pediatric cancers, which is highly relevant for understanding tumor biology and enabling future research in defining biomarkers of treatment response.
Subject(s)
Neoplasms , Adult , Humans , Child , Mutation , Neoplasms/genetics , Oncogenes , INDEL Mutation , DNA RepairABSTRACT
Oncogenic fusions formed through chromosomal rearrangements are hallmarks of childhood cancer that define cancer subtype, predict outcome, persist through treatment, and can be ideal therapeutic targets. However, mechanistic understanding of the etiology of oncogenic fusions remains elusive. Here we report a comprehensive detection of 272 oncogenic fusion gene pairs by using tumor transcriptome sequencing data from 5190 childhood cancer patients. We identify diverse factors, including translation frame, protein domain, splicing, and gene length, that shape the formation of oncogenic fusions. Our mathematical modeling reveals a strong link between differential selection pressure and clinical outcome in CBFB-MYH11. We discover 4 oncogenic fusions, including RUNX1-RUNX1T1, TCF3-PBX1, CBFA2T3-GLIS2, and KMT2A-AFDN, with promoter-hijacking-like features that may offer alternative strategies for therapeutic targeting. We uncover extensive alternative splicing in oncogenic fusions including KMT2A-MLLT3, KMT2A-MLLT10, C11orf95-RELA, NUP98-NSD1, KMT2A-AFDN and ETV6-RUNX1. We discover neo splice sites in 18 oncogenic fusion gene pairs and demonstrate that such splice sites confer therapeutic vulnerability for etiology-based genome editing. Our study reveals general principles on the etiology of oncogenic fusions in childhood cancer and suggests profound clinical implications including etiology-based risk stratification and genome-editing-based therapeutics.
Subject(s)
Core Binding Factor Alpha 2 Subunit , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Humans , Child , Core Binding Factor Alpha 2 Subunit/genetics , Oncogene Fusion , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Transcriptome , Causality , Oncogene Proteins, Fusion/geneticsABSTRACT
BACKGROUND: The prognosis for Li-Fraumeni syndrome (LFS) patients with medulloblastoma (MB) is poor. Comprehensive clinical data for this patient group is lacking, challenging the development of novel therapeutic strategies. Here, we present clinical and molecular data on a retrospective cohort of pediatric LFS MB patients. METHODS: In this multinational, multicenter retrospective cohort study, LFS patients under 21 years with MB and class 5 or class 4 constitutional TP53 variants were included. TP53 mutation status, methylation subgroup, treatment, progression free- (PFS) and overall survival (OS), recurrence patterns, and incidence of subsequent neoplasms were evaluated. RESULTS: The study evaluated 47 LFS individuals diagnosed with MB, mainly classified as DNA methylation subgroup "SHH_3" (86%). The majority (74%) of constitutional TP53 variants represented missense variants. The 2- and 5-year (y-) PFS were 36% and 20%, and 2- and 5y-OS were 53% and 23%, respectively. Patients who received postoperative radiotherapy (RT) (2y-PFS: 44%, 2y-OS: 60%) or chemotherapy before RT (2y-PFS: 32%, 2y-OS: 48%) had significantly better clinical outcome then patients who were not treated with RT (2y-PFS: 0%, 2y-OS: 25%). Patients treated according to protocols including high-intensity chemotherapy and patients who received only maintenance-type chemotherapy showed similar outcomes (2y-PFS: 42% and 35%, 2y-OS: 68% and 53%, respectively). CONCLUSIONS: LFS MB patients have a dismal prognosis. In the presented cohort use of RT significantly increased survival rates, whereas chemotherapy intensity did not influence their clinical outcome. Prospective collection of clinical data and development of novel treatments are required to improve the outcome of LFS MB patients.
Subject(s)
Cerebellar Neoplasms , Li-Fraumeni Syndrome , Medulloblastoma , Child , Humans , Li-Fraumeni Syndrome/complications , Li-Fraumeni Syndrome/genetics , Li-Fraumeni Syndrome/therapy , Medulloblastoma/therapy , Medulloblastoma/drug therapy , Retrospective Studies , Prospective Studies , Cerebellar Neoplasms/therapy , Cerebellar Neoplasms/drug therapy , Germ-Line Mutation , Tumor Suppressor Protein p53/geneticsABSTRACT
Understanding drug resistance in cancer is paramount to improving patient outcomes, quality of life and reducing toxicities in patients receiving chemotherapy. Pharmacogenomic methods seek to understand the interaction of genomic variation and response to chemotherapeutic treatment. This chapter presents a workflow to interrogate multiple genomic inputs and individually assess their relationship with the phenotype of drug resistance using hierarchical clustering to determine the set of features that can best describe what features are associated with drug resistance. Then in a gene-centric manner regulatory features such as miRNAs, SNPs, or DNA methylation can be related back to the differential expression of genes to give understanding to the mechanism underlying resistance. In this chapter, we describe a computational method that can be adapted to a number of different diseases and phenotypes in which there are multiple genomic data types available with concordant phenotypic drug resistance information.
Subject(s)
Genomics , Quality of Life , Drug Resistance/genetics , Genomics/methods , Pharmacogenetics , PhenotypeABSTRACT
Glucocorticoids (GCs) are a mainstay of contemporary, multidrug chemotherapy in the treatment of childhood acute lymphoblastic leukemia (ALL), and resistance to GCs remains a major clinical concern. Resistance to GCs is predictive of ALL relapse and poor clinical outcome, and therefore represents a major hurdle limiting further improvements in survival rates. While advances have been made in identifying genes implicated in GC resistance, there remains an insufficient understanding of the impact of cis-regulatory disruptions in resistance. To address this, we mapped the gene regulatory response to GCs in two ALL cell lines using functional genomics and high-throughput reporter assays and identified thousands of GC-responsive changes to chromatin state, including the formation of over 250 GC-responsive super-enhancers and a depletion of AP-1 bound cis-regulatory elements implicated in cell proliferation and anti-apoptotic processes. By integrating our GC response maps with genetic and epigenetic datasets in primary ALL cells from patients, we further uncovered cis-regulatory disruptions at GC-responsive genes that impact GC resistance in childhood ALL. Overall, these data indicate that GCs initiate pervasive effects on the leukemia epigenome, and that alterations to the GC gene regulatory network contribute to GC resistance.
Subject(s)
Glucocorticoids , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Cell Line, Tumor , Chromatin , Drug Resistance, Neoplasm/genetics , Epigenomics , Glucocorticoids/pharmacology , Glucocorticoids/therapeutic use , Humans , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Receptors, Glucocorticoid/genetics , Steroids , Transcription Factor AP-1/geneticsABSTRACT
Understanding the genomic and epigenetic mechanisms of drug resistance in pediatric acute lymphoblastic leukemia (ALL) is critical for further improvements in treatment outcomes. The role of transcriptomic response in conferring resistance to l-asparaginase (LASP) is poorly understood beyond asparagine synthetase (ASNS). We defined reproducible LASP response genes in LASP-resistant and LASP-sensitive ALL cell lines as well as primary leukemia samples from newly diagnosed patients. Defining target genes of the amino acid stress response-related transcription factor activating transcription factor 4 (ATF4) in ALL cell lines using chromatin immunoprecipitation sequencing (ChIP-seq) revealed 45% of genes that changed expression after LASP treatment were direct targets of the ATF4 transcription factor, and 34% of these genes harbored LASP-responsive ATF4 promoter binding events. SLC7A11 was found to be a response gene in cell lines and patient samples as well as a direct target of ATF4. SLC7A11 was also one of only 2.4% of LASP response genes with basal level gene expression that also correlated with LASP ex vivo resistance in primary leukemia cells. Experiments using chemical inhibition of SLC7A11 with sulfasalazine, gene overexpression, and partial gene knockout recapitulated LASP resistance or sensitivity in ALL cell lines. These findings show the importance of assessing changes in gene expression following treatment with an antileukemic agent for its association with drug resistance and highlight that many response genes may not differ in their basal expression in drug-resistant leukemia cells.
Subject(s)
Aspartate-Ammonia Ligase , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Activating Transcription Factor 4/genetics , Amino Acids/therapeutic use , Asparaginase/pharmacology , Asparaginase/therapeutic use , Aspartate-Ammonia Ligase/genetics , Aspartate-Ammonia Ligase/metabolism , Cell Line, Tumor , Child , Humans , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/geneticsABSTRACT
Acute lymphoblastic leukemia (ALL) is a hematopoietic malignancy comprised of molecular subtypes largely characterized by aneuploidy or recurring chromosomal rearrangements. Despite extensive information on the ALL transcriptome and methylome, there is limited understanding of the ALL chromatin landscape. We therefore mapped accessible chromatin in 24 primary ALL cell biospecimens comprising three common molecular subtypes (DUX4/ERG, ETV6-RUNX1 and hyperdiploid) from patients treated at St. Jude Children's Research Hospital. Our findings highlight extensive chromatin reprogramming in ALL, including the identification ALL subtype-specific chromatin landscapes that are additionally modulated by genetic variation. Chromatin accessibility differences between ALL and normal B-cells implicate the activation of B-cell repressed chromatin domains and detail the disruption of normal B-cell development in ALL. Among ALL subtypes, we uncovered roles for basic helix-loop-helix, homeodomain and activator protein 1 transcription factors in promoting subtype-specific chromatin accessibility and distinct gene regulatory networks. In addition to chromatin subtype-specificity, we further identified over 3500 DNA sequence variants that alter the ALL chromatin landscape and contribute to inter-individual variability in chromatin accessibility. Collectively, our data suggest that subtype-specific chromatin landscapes and gene regulatory networks impact ALL biology and contribute to transcriptomic differences among ALL subtypes.
Subject(s)
Chromatin/genetics , Chromosome Aberrations , DNA Methylation , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Precursor Cell Lymphoblastic Leukemia-Lymphoma/classification , Transcription Factors/metabolism , Chromatin/metabolism , Epigenomics , Humans , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Transcription Factors/genetics , TranscriptomeABSTRACT
Vincristine (VCR) is one of the most widely prescribed medications for treating solid tumors and acute lymphoblastic leukemia (ALL) in children and adults. However, its major dose-limiting toxicity is peripheral neuropathy that can disrupt curative therapy. Peripheral neuropathy can also persist into adulthood, compromising quality of life of childhood cancer survivors. Reducing VCR-induced neurotoxicity without compromising its anticancer effects would be ideal. Here, we show that low expression of NHP2L1 is associated with increased sensitivity of primary leukemia cells to VCR, and that concomitant administration of VCR with inhibitors of NHP2L1 increases VCR cytotoxicity in leukemia cells, prolongs survival of ALL xenograft mice, but decreases VCR effects on human-induced pluripotent stem cell-derived neurons and mitigates neurotoxicity in mice. These findings offer a strategy for increasing VCR's antileukemic effects while reducing peripheral neuropathy in patients treated with this widely prescribed medication.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Peripheral Nervous System Diseases/prevention & control , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Ribonucleoproteins, Small Nuclear/antagonists & inhibitors , Vincristine/adverse effects , Adolescent , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cells, Cultured , Child , Drug Resistance, Neoplasm/genetics , Female , Gene Expression Regulation, Leukemic , Gene Knockdown Techniques , Humans , Induced Pluripotent Stem Cells , Male , Mice , Neurons , Peripheral Nervous System Diseases/chemically induced , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Primary Cell Culture , Ribonucleoproteins, Small Nuclear/genetics , Ribonucleoproteins, Small Nuclear/metabolism , Vincristine/therapeutic use , Xenograft Model Antitumor Assays , Young AdultABSTRACT
BACKGROUNDInterpatient differences in the accumulation of methotrexate's active polyglutamylated metabolites (MTXPGs) in leukemia cells influence its antileukemic effects.METHODSTo identify genomic and epigenomic and patient variables determining the intracellular accumulation of MTXPGs, we measured intracellular MTXPG levels in acute lymphoblastic leukemia (ALL) cells from 388 newly diagnosed patients after in vivo high-dose methotrexate (HDMTX) (1 g/m2) treatment, defined ALL subtypes, and assessed genomic and epigenomic variants influencing folate pathway genes (mRNA, miRNA, copy number alterations [CNAs], SNPs, single nucleotide variants [SNVs], CpG methylation).RESULTSWe documented greater than 100-fold differences in MTXPG levels, which influenced its antileukemic effects (P = 4 × 10-5). Three ALL subtypes had lower MTXPG levels (T cell ALL [T-ALL] and B cell ALL [B-ALL] with the TCF3-PBX1 or ETV6-RUNX1 fusions), and 2 subtypes had higher MTXPG levels (hyperdiploid and BCR-ABL like). The folate pathway genes SLC19A1, ABCC1, ABCC4, FPGS, and MTHFD1 significantly influenced intracellular MTXPG levels (P = 2.9 × 10-3 to 3.7 × 10-8). A multivariable model including the ALL subtype (P = 1.1 × 10-14), the SLC19A1/(ABCC1 + ABCC4) transporter ratio (P = 3.6 × 10-4), the MTX infusion time (P = 1.5 × 10-3), FPGS mRNA expression (P = 2.1 × 10-3), and MTX systemic clearance (P = 4.4 × 10-2) explained 42% of the variation in MTXPG accumulation (P = 1.1 × 10-38). Model simulations indicated that a longer infusion time (24 h vs. 4 h) was superior in achieving higher intracellular MTXPG levels across all subtypes if ALL.CONCLUSIONSThese findings provide insights into mechanisms underlying interpatient differences in intracellular accumulation of MTXPG in leukemia cells and its antileukemic effectsFUNDINGTHE National Cancer Institute (NCI) and the Institute of General Medical Sciences of the NIH, the Basque Government Programa Posdoctoral de Perfeccionamiento de Personal Investigador doctor, and the American Lebanese Syrian Associated Charities (ALSAC).
Subject(s)
Methotrexate/analogs & derivatives , Neoplasm Proteins , Polyglutamic Acid/analogs & derivatives , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Adolescent , Cell Line, Tumor , Child , Child, Preschool , Female , Humans , Infant , Male , Methotrexate/pharmacokinetics , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Pharmacogenomic Testing , Polyglutamic Acid/pharmacokinetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathologyABSTRACT
Identification of genomic and epigenomic determinants of drug resistance provides important insights for improving cancer treatment. Using agnostic genome-wide interrogation of mRNA and miRNA expression, DNA methylation, SNPs, CNAs and SNVs/Indels in primary human acute lymphoblastic leukemia cells, we identified 463 genomic features associated with glucocorticoid resistance. Gene-level aggregation identified 118 overlapping genes, 15 of which were confirmed by genome-wide CRISPR screen. Collectively, this identified 30 of 38 (79%) known glucocorticoid-resistance genes/miRNAs and all 38 known resistance pathways, while revealing 14 genes not previously associated with glucocorticoid-resistance. Single cell RNAseq and network-based transcriptomic modelling corroborated the top previously undiscovered gene, CELSR2. Manipulation of CELSR2 recapitulated glucocorticoid resistance in human leukemia cell lines and revealed a synergistic drug combination (prednisolone and venetoclax) that mitigated resistance in mouse xenograft models. These findings illustrate the power of an integrative genomic strategy for elucidating genes and pathways conferring drug resistance in cancer cells.