Comprehensive genetic profiling reveals frequent alterations of driver genes on the X chromosome in extranodal NK/T-cell lymphoma.

Extranodal NK/T-cell lymphoma (ENKTCL) is an Epstein-Barr virus (EBV)-related neoplasm with male dominance and a poor prognosis. A better understanding of the genetic alterations and their functional roles in ENKTCL could help improve patient stratification and treatments. Here, we performed comprehensive genetic analysis of 177 ENKTCL cases to delineate the landscape of mutations, copy number alterations (CNAs), and structural variations, identifying 34 driver genes including six previously unappreciated ones, namely HLA-B, HLA-C, ROBO1, CD58, POT1, and MAP2K1. Among them, CD274 (24%) was the most frequently altered, followed by TP53 (20%), CDKN2A (19%), ARID1A (15%), HLA-A (15%), BCOR (14%), and MSN (14%). Chromosome X (chrX) losses were the most common arm-level CNAs in females (~40%), and alterations of four X-linked driver genes (MSN, BCOR, DDX3X, and KDM6A) were more frequent in males and females harboring chrX losses. Among X-linked drivers, MSN was the most recurrently altered, and its expression was lost in approximately one-third of cases using immunohistochemical analysis. Functional studies of human cell lines demonstrated that MSN disruption promoted cell proliferation and NF-κB activation. Moreover, MSN inactivation increased sensitivity to NF-κB inhibition in vitro and in vivo. In addition, recurrent deletions were observed at the origin of replication in the EBV genome (6%). Finally, by integrating the 34 drivers and 19 significant arm-level CNAs, non-negative matrix factorization and consensus clustering identified two molecular groups with different genetic features and prognosis irrespective of clinical prognostic factors. Together, these findings could help improve diagnostic and therapeutic strategies in ENKTCL.

De novo generation of the NPM-ALK fusion recapitulates the pleiotropic phenotypes of ALK+ ALCL pathogenesis and reveals the ROR2 receptor as target for tumor cells.

BACKGROUND: Anaplastic large cell lymphoma positive for ALK (ALK+ ALCL) is a rare type of non-Hodgkin lymphoma. This lymphoma is caused by chromosomal translocations involving the anaplastic lymphoma kinase gene (ALK). In this study, we aimed to identify mechanisms of transformation and therapeutic targets by generating a model of ALK+ ALCL lymphomagenesis ab initio with the specific NPM-ALK fusion. METHODS: We performed CRISPR/Cas9-mediated genome editing of the NPM-ALK chromosomal translocation in primary human activated T lymphocytes. RESULTS: Both CD4+ and CD8+ NPM-ALK-edited T lymphocytes showed rapid and reproducible competitive advantage in culture and led to in vivo disease development with nodal and extra-nodal features. Murine tumors displayed the phenotypic diversity observed in ALK+ ALCL patients, including CD4+ and CD8+ lymphomas. Assessment of transcriptome data from models and patients revealed global activation of the WNT signaling pathway, including both canonical and non-canonical pathways, during ALK+ ALCL lymphomagenesis. Specifically, we found that the WNT signaling cell surface receptor ROR2 represented a robust and genuine marker of all ALK+ ALCL patient tumor samples. CONCLUSIONS: In this study, ab initio modeling of the ALK+ ALCL chromosomal translocation in mature T lymphocytes enabled the identification of new therapeutic targets. As ROR2 targeting approaches for other cancers are under development (including lung and ovarian tumors), our findings suggest that ALK+ ALCL cases with resistance to current therapies may also benefit from ROR2 targeting strategies.

Identification of Germline Non-coding Deletions in XIAP Gene Causing XIAP Deficiency Reveals a Key Promoter Sequence.

PURPOSE: X-linked inhibitor of apoptosis protein (XIAP) deficiency, also known as the X-linked lymphoproliferative syndrome of type 2 (XLP-2), is a rare immunodeficiency characterized by recurrent hemophagocytic lymphohistiocytosis, splenomegaly, and inflammatory bowel disease. Variants in XIAP including missense, non-sense, frameshift, and deletions of coding exons have been reported to cause XIAP deficiency. We studied three young boys with immunodeficiency displaying XLP-2-like clinical features. No genetic variation in the coding exons of XIAP was identified by whole-exome sequencing (WES), although the patients exhibited a complete loss of XIAP expression. METHODS: Targeted next-generation sequencing (NGS) of the entire locus of XIAP was performed on DNA samples from the three patients. Molecular investigations were assessed by gene reporter expression assays in HEK cells and CRISPR-Cas9 genome editing in primary T cells. RESULTS: NGS of XIAP identified three distinct non-coding deletions in the patients that were predicted to be driven by repetitive DNA sequences. These deletions share a common region of 839 bp that encompassed the first non-coding exon of XIAP and contained regulatory elements and marks specific of an active promoter. Moreover, we showed that among the 839 bp, the exon was transcriptionally active. Finally, deletion of the exon by CRISPR-Cas9 in primary cells reduced XIAP protein expression. CONCLUSIONS: These results identify a key promoter sequence contained in the first non-coding exon of XIAP. Importantly, this study highlights that sequencing of the non-coding exons that are not currently captured by WES should be considered in the genetic diagnosis when no variation is found in coding exons.

From circRNAs to fusion circRNAs in hematological malignancies.

Circular RNAs (circRNAs) represent a type of endogenous noncoding RNA generated by back-splicing events. Unlike the majority of RNAs, circRNAs are covalently closed, without a 5' end or a 3' poly(A) tail. A few circRNAs can be associated with polysomes, suggesting a protein-coding potential. CircRNAs are not degraded by RNA exonucleases or ribonuclease R and are enriched in exosomes. Recent developments in experimental methods coupled with evolving bioinformatic approaches have accelerated functional investigation of circRNAs, which exhibit a stable structure, a long half-life, and tumor specificity and can be extracted from body fluids and used as potential biological markers for tumors. Moreover, circRNAs may regulate the occurrence and development of cancers and contribute to drug resistance through a variety of molecular mechanisms. Despite the identification of a growing number of circRNAs, their effects in hematological cancers remain largely unknown. Recent studies indicate that circRNAs could also originate from fusion genes (fusion circRNAs, f-circRNAs) next to chromosomal translocations, which are considered the primary cause of various cancers, notably hematological malignancies. This Review will focus on circRNAs and f-circRNAs in hematological cancers.

Unraveling Ewing Sarcoma Tumorigenesis Originating from Patient-Derived Mesenchymal Stem Cells.

Ewing sarcoma is characterized by pathognomonic translocations, most frequently fusing EWSR1 with FLI1. An estimated 30% of Ewing sarcoma tumors also display genetic alterations in STAG2, TP53, or CDKN2A (SPC). Numerous attempts to develop relevant Ewing sarcoma models from primary human cells have been unsuccessful in faithfully recapitulating the phenotypic, transcriptomic, and epigenetic features of Ewing sarcoma. In this study, by engineering the t(11;22)(q24;q12) translocation together with a combination of SPC mutations, we generated a wide collection of immortalized cells (EWIma cells) tolerating EWSR1-FLI1 expression from primary mesenchymal stem cells (MSC) derived from a patient with Ewing sarcoma. Within this model, SPC alterations strongly favored Ewing sarcoma oncogenicity. Xenograft experiments with independent EWIma cells induced tumors and metastases in mice, which displayed bona fide features of Ewing sarcoma. EWIma cells presented balanced but also more complex translocation profiles mimicking chromoplexy, which is frequently observed in Ewing sarcoma and other cancers. Collectively, these results demonstrate that bone marrow-derived MSCs are a source of origin for Ewing sarcoma and also provide original experimental models to investigate Ewing sarcomagenesis. SIGNIFICANCE: These findings demonstrate that Ewing sarcoma can originate from human bone-marrow-derived mesenchymal stem cells and that recurrent mutations support EWSR1-FLI1 translocation-mediated transformation.

STAG2 mutations alter CTCF-anchored loop extrusion, reduce cis-regulatory interactions and EWSR1-FLI1 activity in Ewing sarcoma.

STAG2, a cohesin family gene, is among the most recurrently mutated genes in cancer. STAG2 loss of function (LOF) is associated with aggressive behavior in Ewing sarcoma, a childhood cancer driven by aberrant transcription induced by the EWSR1-FLI1 fusion oncogene. Here, using isogenic Ewing cells, we show that, while STAG2 LOF profoundly changes the transcriptome, it does not significantly impact EWSR1-FLI1, CTCF/cohesin, or acetylated H3K27 DNA binding patterns. In contrast, it strongly alters the anchored dynamic loop extrusion process at boundary CTCF sites and dramatically decreases promoter-enhancer interactions, particularly affecting the expression of genes regulated by EWSR1-FLI1 at GGAA microsatellite neo-enhancers. Down-modulation of cis-mediated EWSR1-FLI1 activity, observed in STAG2-LOF conditions, is associated with enhanced migration and invasion properties of Ewing cells previously observed in EWSR1-FLI1low cells. Our study illuminates a process whereby STAG2-LOF fine-tunes the activity of an oncogenic transcription factor through altered CTCF-anchored loop extrusion and cis-mediated enhancer mechanisms.

Repair of G1 induced DNA double-strand breaks in S-G2/M by alternative NHEJ.

The alternative non-homologous end-joining (NHEJ) pathway promotes DNA double-strand break (DSB) repair in cells deficient for NHEJ or homologous recombination, suggesting that it operates at all stages of the cell cycle. Here, we use an approach in which DNA breaks can be induced in G1 cells and their repair tracked, enabling us to show that joining of DSBs is not functional in G1-arrested XRCC4-deficient cells. Cell cycle entry into S-G2/M restores DSB repair by Pol θ-dependent and PARP1-independent alternative NHEJ with repair products bearing kilo-base long DNA end resection, micro-homologies and chromosome translocations. We identify a synthetic lethal interaction between XRCC4 and Pol θ under conditions of G1 DSBs, associated with accumulation of unresolved DNA ends in S-G2/M. Collectively, our results support the conclusion that the repair of G1 DSBs progressing to S-G2/M by alternative NHEJ drives genomic instability and represent an attractive target for future DNA repair-based cancer therapies.

Ontogenic Changes in Hematopoietic Hierarchy Determine Pediatric Specificity and Disease Phenotype in Fusion Oncogene-Driven Myeloid Leukemia.

Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specific associations between age and phenotype. We observed that fusion oncogenes, such as ETO2-GLIS2, are associated with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that ETO2-GLIS2 expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed in vivo leukemogenic potential. Chromatin accessibility and single-cell transcriptome analyses indicate ontogeny-dependent intrinsic and ETO2-GLIS2-induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes. SIGNIFICANCE: This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state.See related commentary by Cruz Hernandez and Vyas, p. 1653.This article is highlighted in the In This Issue feature, p. 1631.

STAG Mutations in Cancer.

Stromal Antigen 1 and 2 (STAG1/2) are key subunits of the cohesin complex that mediate sister chromatid cohesion, DNA repair, transcriptional regulation, and genome topology. Genetic alterations comprising any of the 11 cohesin-associated genes possibly occur in up to 26% of patients included in The Cancer Genome Atlas (TCGA) studies. STAG2 shows the highest number of putative driver truncating mutations. We provide a comprehensive review of the function of STAG1/2 in human physiology and disease and an integrative analysis of available omics data on STAG alterations in a wide array of cancers, comprising 53 691 patients and 1067 cell lines. Lastly, we discuss opportunities for therapeutic intervention.

Chromosomal Translocation Formation Is Sufficient to Produce Fusion Circular RNAs Specific to Patient Tumor Cells.

Circular RNAs constitute a unique class of RNAs whose precise functions remain to be elucidated. In particular, cancer-associated chromosomal translocations can give rise to fusion circular RNAs that play a role in leukemia progression. However, how and when fusion circular RNAs are formed and whether they are being selected in cancer cells remains unknown. Here, we used CRISPR/Cas9 to generate physiological translocation models of NPM1-ALK fusion gene. We showed that, in addition to generating fusion proteins and activating specific oncogenic pathways, chromosomal translocation induced by CRISPR/Cas9 led to the formation of de novo fusion circular RNAs. Specifically, we could recover different classes of circular RNAs composed of different circularization junctions, mainly back-spliced species. In addition, we identified fusion circular RNAs identical to those found in related patient tumor cells providing evidence that fusion circular RNAs arise early after chromosomal formation and are not just a consequence of the oncogenesis process.

Induction of Chromosomal Translocations with CRISPR-Cas9 and Other Nucleases: Understanding the Repair Mechanisms That Give Rise to Translocations.

Chromosomal translocations are associated with several tumor types, including hematopoietic malignancies, sarcomas, and solid tumors of epithelial origin, due to their activation of a proto-oncogene or generation of a novel fusion protein with oncogenic potential. In many cases, the availability of suitable human models has been lacking because of the difficulty in recapitulating precise expression of the fusion protein or other reasons. Further, understanding how translocations form mechanistically has been a goal, as it may suggest ways to prevent their occurrence. Chromosomal translocations arise when DNA ends from double-strand breaks (DSBs) on two heterologous chromosomes are improperly joined. This review provides a summary of DSB repair mechanisms and their contribution to translocation formation, the various programmable nuclease platforms that have been used to generate translocations, and the successes that have been achieved in this area.

Genomic Alterations and Complex Subclonal Architecture in Sporadic GH-Secreting Pituitary Adenomas.

Purpose: The molecular pathogenesis of growth hormone-secreting pituitary adenomas is not fully understood. Cytogenetic alterations might serve as alternative driver events in GNAS mutation-negative somatotroph tumors. Experimental Design: We performed cytogenetic profiling of pituitary adenomas obtained from 39 patients with acromegaly and four patients with sporadic gigantism by using array comparative genomic hybridization analysis. We explored intratumor DNA copy-number heterogeneity in two tumor samples by using DNA fluorescence in situ hybridization (FISH). Results: Based on copy-number profiles, we found two groups of adenomas: a low-copy-number alteration (CNA) group (<12% of genomic disruption, 63% of tumors) and a high-CNA group (24% to 45% of genomic disruption, 37% of tumors). Arm-level CNAs were the most common abnormalities. GNAS mutation-positive adenomas belonged exclusively to the low-CNA group, whereas a subgroup of GNAS mutation-negative adenomas had a high degree of genomic disruption. We detected chromothripsis-related CNA profiles in two adenoma samples from an AIP mutation-positive patient with acromegaly and a patient with sporadic gigantism. RNA sequencing of these two samples identified 17 fusion transcripts, most of which resulted from chromothripsis-related chromosomal rearrangements. DNA FISH analysis of these samples demonstrated a subclonal architecture with up to six distinct cell populations in each tumor. Conclusion: Somatotroph pituitary adenomas display substantial intertumor and intratumor DNA copy-number heterogeneity, as revealed by variable CNA profiles and complex subclonal architecture. The extensive cytogenetic burden in a subgroup of GNAS mutation-negative somatotroph adenomas points to an alternative tumorigenic pathway linked to genomic instability.

CRISPR-Cas9-guided oncogenic chromosomal translocations with conditional fusion protein expression in human mesenchymal cells.

Gene editing techniques have been extensively used to attempt to model recurrent genomic rearrangements found in tumor cells. These methods involve the induction of double-strand breaks at endogenous loci followed by the identification of breakpoint junctions within a population, which typically arise by nonhomologous end joining. The low frequency of these events, however, has hindered the cloning of cells with the desired rearrangement before oncogenic transformation. Here we present a strategy combining CRISPR-Cas9 technology and homology-directed repair to allow for the selection of human mesenchymal stem cells harboring the oncogenic translocation EWSR1-WT1 found in the aggressive desmoplastic small round cell tumor. The expression of the fusion transcript is under the control of the endogenous EWSR1 promoter and, importantly, can be conditionally expressed using Cre recombinase. This method is easily adapted to generate any cancer-relevant rearrangement.

Single-Molecule Analysis of mtDNA Replication Uncovers the Basis of the Common Deletion.

Mutations in mtDNA lead to muscular and neurological diseases and are linked to aging. The most frequent aberrancy is the "common deletion" that involves a 4,977-bp region flanked by 13-bp repeats. To investigate the basis of this deletion, we developed a single-molecule mtDNA combing method. The analysis of replicating mtDNA molecules provided in vivo evidence in support of the asymmetric mode of replication. Furthermore, we observed frequent fork stalling at the junction of the common deletion, suggesting that impaired replication triggers the formation of this toxic lesion. In parallel experiments, we employed mito-TALENs to induce breaks in distinct loci of the mitochondrial genome and found that breaks adjacent to the 5' repeat trigger the common deletion. Interestingly, this process was mediated by the mitochondrial replisome independent of canonical DSB repair. Altogether, our data underscore a unique replication-dependent repair pathway that leads to the mitochondrial common deletion.

The second European interdisciplinary Ewing sarcoma research summit--A joint effort to deconstructing the multiple layers of a complex disease.

Despite multimodal treatment, long term outcome for patients with Ewing sarcoma is still poor. The second "European interdisciplinary Ewing sarcoma research summit" assembled a large group of scientific experts in the field to discuss their latest unpublished findings on the way to the identification of novel therapeutic targets and strategies. Ewing sarcoma is characterized by a quiet genome with presence of an EWSR1-ETS gene rearrangement as the only and defining genetic aberration. RNA-sequencing of recently described Ewing-like sarcomas with variant translocations identified them as biologically distinct diseases. Various presentations adressed mechanisms of EWS-ETS fusion protein activities with a focus on EWS-FLI1. Data were presented shedding light on the molecular underpinnings of genetic permissiveness to this disease uncovering interaction of EWS-FLI1 with recently discovered susceptibility loci. Epigenetic context as a consequence of the interaction between the oncoprotein, cell type, developmental stage, and tissue microenvironment emerged as dominant theme in the discussion of the molecular pathogenesis and inter- and intra-tumor heterogeneity of Ewing sarcoma, and the difficulty to generate animal models faithfully recapitulating the human disease. The problem of preclinical development of biologically targeted therapeutics was discussed and promising perspectives were offered from the study of novel in vitro models. Finally, it was concluded that in order to facilitate rapid pre-clinical and clinical development of novel therapies in Ewing sarcoma, the community needs a platform to maintain knowledge of unpublished results, systems and models used in drug testing and to continue the open dialogue initiated at the first two Ewing sarcoma summits.

TALEN-Induced Translocations in Human Cells.

Induction of chromosomal translocations in human cells is of a great interest to study tumorigenesis and genome instability. Here, we explain in detail a method to induce translocations using the transcription activator-like effector nucleases (TALENs). We describe how to detect translocation formation by PCR, calculate translocation frequency by 96-well PCR screen, and analyze breakpoint junctions. When inducing cancer translocations, it is also possible to detect the fusion gene by FISH analysis or western blot.

Creating cancer translocations in human cells using Cas9 DSBs and nCas9 paired nicks.

Recurrent chromosomal translocations are found in numerous tumor types, often leading to the formation and expression of fusion genes with oncogenic potential. Creating chromosomal translocations at the relevant endogenous loci, rather than ectopically expressing the fusion genes, opens new possibilities for better characterizing molecular mechanisms driving tumor formation. In this chapter, we describe methods to create cancer translocations in human cells. DSBs or paired nicks generated by either wild-type Cas9 or the Cas9 nickase, respectively, are used to induce translocations at the relevant loci. Using different PCR-based methods, we also explain how to quantify translocation frequency and to analyze breakpoint junctions in the cells of interest. In addition, PCR detection of translocations is used as a very sensitive method to detect off-target effects, which has general utility.

Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining.

Breakpoint junctions of the chromosomal translocations that occur in human cancers display hallmarks of nonhomologous end-joining (NHEJ). In mouse cells, translocations are suppressed by canonical NHEJ (c-NHEJ) components, which include DNA ligase IV (LIG4), and instead arise from alternative NHEJ (alt-NHEJ). Here we used designer nucleases (ZFNs, TALENs, and CRISPR/Cas9) to introduce DSBs on two chromosomes to study translocation joining mechanisms in human cells. Remarkably, translocations were altered in cells deficient for LIG4 or its interacting protein XRCC4. Translocation junctions had significantly longer deletions and more microhomology, indicative of alt-NHEJ. Thus, unlike mouse cells, translocations in human cells are generated by c-NHEJ. Human cancer translocations induced by paired Cas9 nicks also showed a dependence on c-NHEJ, despite having distinct joining characteristics. These results demonstrate an unexpected and striking species-specific difference for common genomic rearrangements associated with tumorigenesis.

Cancer translocations in human cells induced by zinc finger and TALE nucleases.

Chromosomal translocations are signatures of numerous cancers and lead to expression of fusion genes that act as oncogenes. The wealth of genomic aberrations found in cancer, however, makes it challenging to assign a specific phenotypic change to a specific aberration. In this study, we set out to use genome editing with zinc finger (ZFN) and transcription activator-like effector (TALEN) nucleases to engineer, de novo, translocation-associated oncogenes at cognate endogenous loci in human cells. Using ZFNs and TALENs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant t(11;22)(q24;q12) and t(2;5)(p23;q35) translocations found in Ewing sarcoma and anaplastic large cell lymphoma (ALCL), respectively. We recovered both translocations with high efficiency, resulting in the expression of the EWSR1-FLI1 and NPM1-ALK fusions. Breakpoint junctions recovered after ZFN cleavage in human embryonic stem (ES) cell-derived mesenchymal precursor cells fully recapitulated the genomic characteristics found in tumor cells from Ewing sarcoma patients. This approach with tailored nucleases demonstrates that expression of fusion genes found in cancer cells can be induced from the native promoter, allowing interrogation of both the underlying mechanisms and oncogenic consequences of tumor-related translocations in human cells. With an analogous strategy, the ALCL translocation was reverted in a patient cell line to restore the integrity of the two participating chromosomes, further expanding the repertoire of genomic rearrangements that can be engineered by tailored nucleases.

DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation.

Nonhomologous end-joining (NHEJ) is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4), suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ) pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1.