Chromosomal Translocations Research Articles

Clinical and Cytogenetic Impact of Maternal Balanced Double Translocation: A Familial Case of 15q11.2 Microduplication and Microdeletion Syndromes with Genetic Counselling Implications

Clinical and Cytogenetic Impact of Maternal Balanced Double Translocation: A Familial Case of 15q11.2 Microduplication and Microdeletion Syndromes with Genetic Counselling Implications

Genome editing with the HDR-enhancing DNA-PKcs inhibitor AZD7648 causes large-scale genomic alterations.

The DNA-PKcs inhibitor AZD7648 enhances CRISPR-Cas9-directed homology-directed repair efficiencies, with potential for clinical utility, but its possible on-target consequences are unknown. We found that genome editing with AZD7648 causes frequent kilobase-scale and megabase-scale deletions, chromosome arm loss and translocations. These large-scale chromosomal alterations evade detection through typical genome editing assays, prompting caution in deploying AZD7648 and reinforcing the need to investigate multiple types of potential editing outcomes.

De Novo Balanced Translocations Disrupting the FBN1 Gene Diagnosed by Genome Sequencing: An Uncommon Cause of Marfan Syndrome Modifying Genetic Counseling.

Marfan syndrome (MFS) is a well-characterized rare genetic connective tissue disorder. The features of MFS are primarily skeletal, ocular, and cardiovascular and are mainly caused by single-nucleotide variants (SNVs) in the FBN1 gene (MIM#134797) located on chromosome 15q21.1. We describe two patients, a 26-year-old male and a 10-year-old female from unrelated distinct families, with clinically diagnosed sporadic MFS. After years of unsuccessful molecular diagnosis for genetic counseling purposes, genome sequencing was performed and revealed a balanced translocation in both patients: de novo t(9;15)(p13.3;q21.1) translocation in the male patient, and de novo t(15;16)(q21.1;p13.13) translocation in the female patient, respectively, disrupting intron 40 and 45 of FBN1. The other breakpoints were not clinically relevant. These translocations were confirmed by specific fluorescence insitu hybridization probes and conventional karyotyping. In the literature, only one family has been described, leading to four cases of MFS caused by balanced translocations. Genetic counseling for balanced translocations differs from SNVs and even interstitial deletions since it is not restricted to MFS recurrence, but also involves the risk of unbalanced gametes, leading to miscarriage or unbalanced progeny. In case of clinical certainty, MFS patients should be screened for balanced translocations to ensure appropriate genetic counseling.

Cytological mapping of a powdery mildew resistance locus PmRc1 based on wheat-Roegneria ciliaris structural rearrangement library.

A powdery mildew (Pm) resistance locus PmRc1 was identified and transferred from Roegneria ciliaris into wheat. Two compensative translocation lines carrying PmRc1 were developed. Powdery mildew (Pm), caused by the biotrophic fungal pathogen Blumeria graminis f.sp. tritici (Bgt), is a global destructive disease of bread wheat (Triticum aestivum L.). Identifying and utilizing new Pm resistance gene(s) is the most fundamental work for disease control. Roegneria ciliaris (2n = 4 x= 28, genome ScScYcYc) is a wild relative species of cultivated wheat. In this work, we evaluated wheat-R. ciliaris disomic chromosome addition lines for Pm resistance in multiple years. The introduction of R. ciliaris chromosome 1Sc into wheat enhanced resistance. The resistance locus on 1Sc was designated as PmRc1. To cytologically map PmRc1, we induced structural rearrangements using ion irradiation and increasinghomoeologous chromosomal recombination. The identified 43 1Sc translocation or deletion lines were usedto construct 1Sc cytological bin map bymarker analysis using 111 molecular markers. Based on the Pm resistance of thecharacterized structural rearrangement lines, the PmRc1 locus was cytologically mapped to bin 1ScS-8 of 1Sc short arm, flanked by markers CMH93-2 and CMH114-1. Two compensatory chromosomal translocation lines (T1ScS 1BL and T1ScS-1AS 1AL) carrying PmRc1 were developed and assessed for theiragronomic traits. Translocation chromosome T1ScS 1BL hadenhanced Pm resistance accompanied by negative effects on grain number and single plant yield. Translocation chromosome T1ScS-1AS 1AL hadenhanced Pm resistance and increased spikelet number per spike, without any obvious negativeeffect on other tested traits. Thus, T1ScS-1AS 1AL is recommended preferentiallyused in wheat breedingfor Pm resistance.

Role of MicroRNAs in Chronic Myeloid Leukemia (CML) Treatment, Biomarkers, and Resistance to Tyrosine Kinase Inhibitor: A Bioinformatics-Based Analysis

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by the reciprocal translocation of the BCR-ABL1 oncogene, which activates tyrosine kinase resulting in uncontrolled cell proliferation and apoptosis suppression. Although Imatinib (IM) is an effective treatment, IM resistance remains a significant concern. Therefore, microRNAs (miRNAs) have emerged as potential alternative therapies. These short, non-coding RNAs (20-22 nucleotides) regulate gene expression by binding to the 3' untranslated region (3'UTR) of target genes. The study aims to identify miRNAs linked to CML, determine miRNA’s target genes, construct a protein-protein interaction (PPI) network, and analyze pathways and gene ontology. A literature search using the keywords “microRNA”, and “chronic myeloid leukemia” yielded relevant papers, which were screened and categorized into three groups: 1) miRNA associated with TKI resistance, 2) miRNA as biomarkers or leukemogenesis, and 3) miRNA as therapy. Target genes for the identified miRNAs were determined using DIANA tools and miRTarBase. Gene ontology and pathway analysis were conducted using DAVID, while PPI and network visualization were performed using STRING, Cytoscape, and ClueGO. Thirteen miRNAs were selected, targeting 782 genes and forming 16 clusters. ClueGO identified clusters associated with key biological processes, including G1/S cell cycle transition, miRNA-mediated gene silencing, hematopoietic stem cell differentiation, and apoptosis regulation. Target genes are also significant in the CML pathway and other cancer pathways, such as p53, ErbB, FoxO, autophagy, apoptosis, VEGF, TNF, and microRNA. Notably, hsa-miR-16 emerged as the most promising therapeutic and biomarker candidate for CML, highlighting its role in critical pathways.

Whole genome profiling of rare pediatric thoracic tumors elucidates a YAP1::LEUTX fusion in an unclassified biphasic embryonal neoplasm

Malignant biphasic tumors of the lungs are rare, more so in the pediatric population. Here, we present the whole-genome characterization of a pleuropulmonary blastoma Type III and an unclassified biphasic thoracic embryonal neoplasm. The pleuropulmonary blastoma harbored pathogenic DICER1 germline and somatic mutations, and additional somatic variants in TP53 and BCOR. The other malignant tumor demonstrated a t(11;19) balanced translocation with a YAP1::LEUTX fusion that was confirmed by fluorescence in situ hybridization. No DICER1 germline or somatic mutation was present. YAP1 and LEUTX have been implicated in tumorigenesis of various neoplasms, and YAP1 fusion genes are an emerging oncogenic entity in a variety of malignancies. In this study we highlight the importance of whole-genome characterization of rare and unclassified tumors to identify biologic mechanisms and potential therapeutic targets.

EZH2 inhibition sensitizes retinoic acid-driven senescence in synovial sarcoma

Synovial sarcoma (SS) is driven by a unique t(18;X) chromosomal translocation resulting in expression of the SS18-SSX fusion oncoprotein, a transcriptional regulator with both activating and repressing functions. However, the manner in which SS18-SSX contributes to the development of SS is not entirely known. Here, we show that SS18-SSX drives the expression of Preferentially Expressed Antigen in Melanoma (PRAME), which is highly expressed in SS but whose function remains poorly understood. The fusion protein directly binds and activates the PRAME promoter and we found that expression of SS18-SSX and PRAME are positively correlated. We provide evidence that PRAME modulates retinoic acid (RA) signaling, forming a ternary complex with the RA receptor α (RARα) and the Enhancer of Zeste Homolog 2 (EZH2). Knockdown of PRAME suppressed the response to all-trans retinoic acid (ATRA) supporting PRAME’s role in modulating RA-signaling. Notably, we demonstrate that combined pharmacological inhibition of EZH2 and treatment with ATRA reconstituted RA signaling followed by reduced proliferation and induction of cellular senescence. In conclusion, our data provides new insights on the role of the SS18-SSX fusion protein in regulation of PRAME expression and RA signaling, highlighting the therapeutic potential of disrupting the RARα-PRAME-EZH2 complex in SS.Schematic presentation of the proposed model. A The RARα-PRAME-EZH2 ternary complex in SS. The fusion SS18-SSX oncoprotein binds to the PRAME promoter and activates its expression. PRAME in turn interacts with RARα-RXR heterodimers as well as with EZH2, and the complex binds to retinoic acid response elements (RAREs) in the DNA. This results in transcriptional repression of retinoic acid (RA) responsive genes and thus inhibition of RA-signaling, allowing tumor cell proliferation. B Therapeutic strategy. Treatment with an EZH2 inhibitor, such as GSK343, or activation of RAR receptors via all-trans retinoic acid (ATRA), disrupts the RARα-PRAME-EZH2 ternary complex and restores RA-signaling. Exposure to GSK343 or ATRA results in inhibition of cell proliferation and induction of cellular senescence, where GSK343 shows a dominant effect. The Figure was created with Biorender.com.

Abstract PR014: PVT1 fusion on extrachromosomal DNA (ecDNA) increases oncogene RNA stability and cancer cell growth

Abstract Extrachromosomal DNA (ecDNA), highly amplified circular DNA, is widespread in human cancers and serves as a primary location for oncogene amplification. Due to its dynamic structural rearrangement and asymmetric inheritance during cell division, ecDNA increases tumor genome complexity, contributing to drug resistance and shortened patient survival. Here, we show that ecDNA-mediated genomic amplification is highly associated with transcript fusion events, which are prevalent in cancer and often lead to tumor development. We discovered that transcripts with the highest fusion events originate from genes amplified on ecDNA across ecDNA(+) cell lines. Notably, PVT1 (Plasmacytoma Variant Translocation 1) long noncoding RNA, which is known to be a hotspot for chromosomal translocation, is the most frequently fused RNA species in MYC/PVT1-amplified ecDNA(+) cancer cells. Exon 1 of PVT1 is the predominant fusion partner and confers RNA stability, increasing the RNA abundance of the partner oncogene. Using a model cell line with PVT1-MYC fusion on ecDNA, we found that RNA expression of PVT1-MYC increases in vivo in an ecDNA-dependent manner, while canonical MYC does not show a prominent increase. Additionally, ectopic expression of PVT1- MYC in MYC-depleted cancer cells provides higher rescue efficiency than canonical MYC. In contrast, a PVT1-MYC mutant that is unable to enhance RNA stability fails to rescue, highlighting the functional significance of PVT1-mediated RNA stabilization in cancer. This study unveils the link between PVT1 fusion and ecDNA in cancer, providing insights for developing diagnostic and therapeutic approaches tailored to target ecDNA. Citation Format: Hyerim Yi, Shu Zhang, Matthew G Jones, Julia A Belk, Jason Swinderman, Quanming Shi, Ellis J Curtis, Vishnu P Kanakaveti, Paul S Mischel, Howard Y Chang. PVT1 fusion on extrachromosomal DNA (ecDNA) increases oncogene RNA stability and cancer cell growth [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR014.

Engineered PsCas9 enables therapeutic genome editing in mouse liver with lipid nanoparticles

Clinical implementation of therapeutic genome editing relies on efficient in vivo delivery and the safety of CRISPR-Cas tools. Previously, we identified PsCas9 as a Type II-B family enzyme capable of editing mouse liver genome upon adenoviral delivery without detectable off-targets and reduced chromosomal translocations. Yet, its efficacy remains insufficient with non-viral delivery, a common challenge for many Cas9 orthologues. Here, we sought to redesign PsCas9 for in vivo editing using lipid nanoparticles. We solve the PsCas9 ribonucleoprotein structure with cryo-EM and characterize it biochemically, providing a basis for its rational engineering. Screening over numerous guide RNA and protein variants lead us to develop engineered PsCas9 (ePsCas9) with up to 20-fold increased activity across various targets and preserved safety advantages. We apply the same design principles to boost the activity of FnCas9, an enzyme phylogenetically relevant to PsCas9. Remarkably, a single administration of mRNA encoding ePsCas9 and its guide formulated with lipid nanoparticles results in high levels of editing in the Pcsk9 gene in mouse liver, a clinically relevant target for hypercholesterolemia treatment. Collectively, our findings introduce ePsCas9 as a highly efficient, and precise tool for therapeutic genome editing, in addition to the engineering strategy applicable to other Cas9 orthologues.

'I Could Trust It': Experiences of Reciprocal Translocation Carriers and Their Partners With Prenatal Cell-Free DNA Screening for Unbalanced Translocations.

To explore the experiences of people having cfDNA screening to detect unbalanced translocations, and to understand motivations for choosing this option. We used a qualitative approach with in-depth semi-structured interviews with reciprocal translocation carriers and their partners. People who underwent cfDNA screening with translocation analysis through Victorian Clinical Genetics Services between 2015 and 2019 were invited to take part. Purposive sampling based on the participant's geographic location, requesting practitioner specialty and cfDNA screening result was used to capture a range of experiences. Interview transcripts were analysed using thematic analysis. Participants (n=13) had complex reproductive journeys associated with the translocation and opted for cfDNA screening rather than prenatal diagnosis to avoid risk to their pregnancy. Participants benefited from having a result early in pregnancy and had sufficient confidence in the result to decline a diagnostic testing procedure. Participants' experiences with cfDNA screening were intertwined with the experience of being a carrier of a reciprocal translocation. cfDNA screening with translocation analysis was perceived as an acceptable alternative to prenatal diagnosis and should be made more accessible to balanced translocation carriers. Access to specialist genetic counselling services is needed to ensure couples are provided with information about all prenatal testing options, including the benefits and limitations associated with cfDNA screening with translocation analysis.

Increased c-MYC Expression Associated with Active IGH Locus Rearrangement: An Emerging Role for c-MYC in Chronic Lymphocytic Leukemia.

This review examines the pivotal role of c-MYC in Chronic Lymphocytic Leukemia (CLL), focusing on how its overexpression leads to increased genetic instability, thereby accelerating disease progression. MYC, a major oncogene, encodes a transcription factor that regulates essential cellular processes, including cell cycle control, proliferation, and apoptosis. In CLL cases enriched with unmutated immunoglobulin heavy chain variable (IGHV) genes, MYC is significantly overexpressed and associated with active rearrangements in the IGH immunoglobulin heavy chain locus. This overexpression results in substantial DNA damage, including double-strand breaks, chromosomal translocations, and an increase in abnormal repair events. Consequently, c-MYC plays a dual role in CLL: it promotes aggressive cell proliferation while concurrently driving genomic instability through its involvement in genetic recombination. This dynamic contributes not only to CLL progression but also to the overall aggressiveness of the disease. Additionally, the review suggests that c-MYC's influence on genetic rearrangements makes it an attractive target for therapeutic strategies aimed at mitigating CLL malignancy. These findings underscore c-MYC's critical importance in advancing CLL progression, highlighting the need for further research to explore its potential as a target in future treatment approaches.

Genome-wide association for agro-morphological traits in a triploid banana population with large chromosome rearrangements

Abstract Banana breeding is hampered by the very low fertility of domesticated bananas and the lack of knowledge about the genetic determinism of agronomic traits. We analysed a breeding population of 2 723 triploid hybrids resulting from crosses between diploid and tetraploid M. acuminata parents, which was evaluated over three successive crop-cycles for 24 traits relating to yield components and plant, bunch and fruit architectures. A subset of 1 129 individuals was genotyped-by-sequencing revealing 205 612 single nucleotide polymorphisms. Most parents were heterozygous for one or several large reciprocal chromosomal translocations, which are known to impact recombination and chromosomal segregation. We applied two linear mixed models to detect associations between markers and traits: (i) a standard model with a kinship calculated using all SNPs and (ii) a model with chromosome-specific kinships that aims at recovering statistical power at alleles carried by long non-recombined haplotypic segments. For 23 of the 24 traits, we identified one to five significant quantitative trait loci (QTLs) for which the origin of favourable alleles could often be determined among the main ancestral contributors to banana cultivars. Several QTLs, located in the rearranged regions, were only detected using the second model. The resulting QTL landscape represents an important resource to support breeding programs. The proposed strategy for recovering power at SNPs carried by long non-recombined rearranged haplotypic segments is an important methodological advance for future association studies in banana and other species affected by chromosomal rearrangements.

An infant with trisomy 9 and partial trisomy 12 derived from maternal balanced translocation: A case report and literature review.

We present here, a case of a neonate with an unbalanced chromosomal translocation due to a maternal chromosomal translocation carriage that resulted in the presence of trisomy 9p combined with a partial trisomy 12p. Karyotype analysis was performed using conventional cytogenetic chromosomal analysis using the GTG-banding technique. The mother was a carrier of a balanced chromosomal translocation of 46, XX, t(9;12)(q13;p11.2), that resulted in an unbalanced translocation of the offspring, who had a karyotype 47, XX, +der(9)t(9;12)(q13;p11.2)dmat, featuring a combined trisomy of 9q13→9pter region and 12p11.2→12pter region. Clinical phenotype of the infant included cephalo-facial deformity, growth retardation, hypertonia, opisthotonus, laryngeal wheezing, foot deformity, and congenital heart disease.

Parental Balanced Translocation Carriers do not have Decreased Usable Blastulation Rates or Live Birth Rates Compared to Infertile Controls

ObjectiveTo determine if translocation carriers have a reduced number of usable blastocysts compared to infertile controls. DesignRetrospective cohort study. SubjectsAll cycles of balanced translocation carriers undergoing IVF with preimplantation genetic testing (PGT-SR) for structural rearrangements at a single infertility center compared to an age-matched control cycles of infertile patients undergoing preimplantation genetic testing for aneuploidy (PGT-A) from January 2012-August 2022. ExposureBalanced translocation carriers. Main Outcome MeasuresPrimary outcome measures were blastulation rate, usable blastulation rates and live birth rate. Secondary outcome measures were sustained implantation rate, fertilization rate, number of oocytes retrieved, number of metaphase II oocytes, total blastulation failure, number of 2 pronuclear embryos, and number of euploid embryos. Outcome measures were compared between male translocation carriers and controls, female translocation carriers and controls, and Robertsonian versus reciprocal translocation carriers. ResultsA total of 1,291 retrieval cycles from 993 patients were included, of which 255 patients were translocation carriers, while 738 were controls. Of those with translocations, 30 (11.5%) were Robertsonian and 231 (88.5%) were reciprocal carriers. There was a statistically significant difference in the blastulation rate between carriers and controls (59.5% versus 62.1%; p-value = 0.01). However, usable blastulation rates (47.2% versus 50.0%) were equivalent between groups. There were no differences in number of oocytes retrieved (18.5 versus 18.3), number of 2 pronuclear embryos (13.4 versus 12.5), sustained implantation rate (71.4% versus 75.1%) or live birth rate (63.0% versus 66.1%) between translocation carriers and controls. In both male and female translocation carriers versus controls, there were no differences in usable blastulation rates or live birth rates. When comparing Robertsonian with reciprocal translocation carriers, rates of blastulation, usable blastulation, sustained implantation, and live birth rate were equivalent. ConclusionDespite fewer euploid embryos, there were no differences in rates of usable blastulation or live birth rates in balanced translocation carriers, regardless of sex of affected partner or type of rearrangement, compared to controls. Routine karyotyping for blastulation failure may not be necessary based on these findings.

The Value of Parental Karyotyping in Recurrent Pregnancy Loss Lies in Individual Risk Assessments.

Background and Objectives: Recurrent pregnancy loss (RPL) is a multifactorial condition, encompassing genetic, anatomical, immunological, endocrine, as well as infectious and environmental factors; however, the etiology remains elusive in a substantial number of cases. Genetic factors linked to RPL include parental karyotype abnormalities (e.g., translocations, inversions, copy number variants), an increase in sperm aneuploidy, fetal microchimerism, severe skewing of X chromosome inactivation, and various gene polymorphisms. Our study aims to explore the value of routine conventional parental karyotyping in couples with RPL. Materials and Methods: A total of 213 couples (426 individuals) with a history of RPL were enrolled in this retrospective study. The peripheral blood samples included in this study were referred to the Human Genomics Laboratory of the University of Medicine and Pharmacy in Craiova, Romania, for conventional cytogenetic analysis between January 2013 and December 2023, by the Outpatient Medical Genetics Clinic of the Emergency Clinical County Hospital of Craiova. Chromosome analysis was performed using standard protocols and karyotypes were reported according to ISCN. Results: Out of 426 patients provided with conventional G-banded chromosome analysis, 410 had a normal karyotype (96.2%) and 16 had chromosome abnormalities (3.8%). The most common chromosomal abnormalities were reciprocal and Robertsonian translocations, with chromosomes 8, 11, 14, and 21 being most frequently involved. A single numerical anomaly was detected (47,XYY). One or multiple chromosomal polymorphisms were identified in 104 subjects (24.4%). In addition, we conducted a stratified analysis of the unselected group and detected chromosome abnormalities in only four cases (0.94%). Conclusions: Our results are consistent with recommendations for paternal karyotyping after an individual risk assessment in instances such as a previous live birth with congenital anomalies and/or the detection of unbalanced chromosomes or a translocation in product of conception or chorionic villi/amniotic fluid samples. In the absence of a positive history, blindly karyotyping couples may prove too expensive and labor intensive, while providing no information on fertility status or live birth rates.

Advances in Triticale (X Triticosecale) Improvement: Chromosome Manipulation and Biotechnological Approaches

Triticale (X Triticosecale) is a hybrid cereal crop with great potential for enhancing food security. It is a synthetic cereal. Meanwhile, certain genetic instabilities arising from the merging of the rye and wheat genomes have impeded the advancement of triticale, chromosome engineering advancements along with biotechnological approaches might potentially unleash the full potential of triticale. This article provides a comprehensive summary of the historical development and current status of research on conventional and molecular breeding and manipulating triticale chromosomes in order to introduce beneficial traits, correct genetic abnormalities, and accelerate breeding. Among the major strategies covered are chromosomal doubling, addition, replacement, translocation, and deletion. Beneficial genes from rye for quality of grain, yield, and disease resistance were incorporated into triticale backgrounds using addition and replacement lines. In general, using chromosome-modifying technologies within an integrated breeding framework may help with genetic stabilization, the planned evolution of triticale for greater productivity and robustness, and strategic trait integration. This study looks at the advantages, disadvantages, and potential benefits of using chromosomal engineering to enhance triticales.

Recurrent somatic mutations of FAT family cadherins induce an aggressive phenotype and poor prognosis in anaplastic large cell lymphoma

BackgroundAnaplastic Large Cell Lymphoma (ALCL) is a rare and aggressive T-cell lymphoma, classified into ALK-positive and ALK-negative subtypes, based on the presence of chromosomal translocations involving the ALK gene. The current standard of treatment for ALCL is polychemotherapy, with a high overall survival rate. However, a subset of patients does not respond to or develops resistance to these therapies, posing a serious challenge for clinicians. Recent targeted treatments such as ALK kinase inhibitors and anti-CD30 antibody-drug conjugates have shown promise but, for a fraction of patients, the prognosis is still unsatisfactory.MethodsWe investigated the genetic landscape of ALK + ALCL by whole-exome sequencing; recurring mutations were characterized in vitro and in vivo using transduced ALCL cellular models.ResultsRecurrent mutations in FAT family genes and the transcription factor RUNX1T1 were found. These mutations induced changes in ALCL cells morphology, growth, and migration, shedding light on potential factors contributing to treatment resistance. In particular, FAT4 silencing in ALCL cells activated the β-catenin and YAP1 pathways, which play crucial roles in tumor growth, and conferred resistance to chemotherapy. Furthermore, STAT1 and STAT3 were hyper-activated in these cells. Gene expression profiling showed global changes in pathways related to cell adhesion, cytoskeletal organization, and oncogenic signaling. Notably, FAT mutations associated with poor outcome in patients.ConclusionsThese findings provide novel insights into the molecular portrait of ALCL, that could help improve treatment strategies and the prognosis for ALCL patients.

Origin of Ewing sarcoma by embryonic reprogramming of neural crest to mesoderm.

Ewing sarcoma is a malignant small round blue cell tumor of bones and soft tissues caused by chromosomal translocations that generate aberrant fusion oncogenes, most frequently EWSR1::FLI1. The cell of origin and mechanisms of EWSR1::FLI1-driven transformation have remained unresolved, largely due to lack of a representative animal model. By developing a zebrafish Ewing sarcoma model, we provide evidence for a neural crest origin of this cancer. Neural crest-derived cells uniquely tolerate expression of EWSR1::FLI1 and targeted expression of EWSR1::FLI1 in these cells generates Ewing sarcomas. Single-cell analysis of tumor initiation shows that EWSR1::FLI1 reprograms neural crest-derived cells to a mesoderm-like state, strikingly resulting in ectopic fins throughout the body. By profiling chromatin accessibility and genome-wide EWSR1::FLI1 binding, we find that the fusion oncogene hijacks developmental enhancers for neural crest to mesoderm reprogramming during cancer initiation. These findings show how a single mutation profoundly alters embryonic cell fate decisions to initiate a devastating childhood cancer.

Endogenous EWSR1-FLI1 degron alleles enable control of fusion oncoprotein expression in tumor cell lines and xenografts.

Pediatric malignancies frequently harbor chromosomal translocations that induce expression of fusion oncoproteins. The EWSR1-FLI1 fusion oncoprotein acts as a neomorphic transcription factor and is the dominant genetic driver of Ewing's sarcoma. Interrogation of the mechanisms by which EWSR1-FLI1 drives tumorigenesis has been limited by a lack of model systems to precisely and selectively control its expression in patient-derived cell lines and xenografts. Here, we report the generation of a panel of patient-derived EWS cell lines in which inducible protein degrons were engineered into the endogenous EWSR1-FLI1 locus. These alleles enabled rapid and efficient depletion of EWSR1-FLI1. Complete suppression of EWSR1-FLI1 induced a reversible cell cycle arrest at the G1-S checkpoint, and we identified a core set of transcripts downstream of EWSR1-FLI1 across multiple cell lines and degron systems. Additionally, depletion of EWSR1-FLI1 potently suppressed tumor growth in xenograft models validating efforts to directly target EWSR1-FLI1 in Ewing's sarcoma.

MLL oncoprotein levels influence leukemia lineage identities

Chromosomal translocations involving the mixed-lineage leukemia (MLL) locus generate potent oncogenic fusion proteins (oncoproteins) that disrupt regulation of developmental gene expression. By profiling the oncoprotein-target sites of 36 broadly representative MLL-rearranged leukemia samples, including three samples that underwent a lymphoid-to-myeloid lineage-switching event in response to therapy, we find the genomic enrichment of the oncoprotein is highly variable between samples and subject to dynamic regulation. At high levels of expression, the oncoproteins preferentially activate either an acute lymphoblastic leukemia (ALL) program, enriched for pro-B-cell genes, or an acute myeloid leukemia (AML) program, enriched for hematopoietic-stem-cell genes. The fusion-partner-specific-binding patterns over these gene sets are highly correlated with the prevalence of each mutation in ALL versus AML. In lineage-switching samples the oncoprotein levels are reduced and the oncoproteins preferentially activate granulocyte-monocyte progenitor (GMP) genes. In a sample that lineage switched during treatment with the menin inhibitor revumenib, the oncoprotein and menin are reduced to undetectable levels, but ENL, a transcriptional cofactor of the oncoprotein, persists on numerous oncoprotein-target loci, including genes in the GMP-like lineage-switching program. We propose MLL oncoproteins promote lineage-switching events through dynamic chromatin binding at lineage-specific target genes, and may support resistance to menin inhibitors through similar changes in chromatin occupancy.