Chromosomal Translocations Research Articles

EWSR1::ATF1 Translocation: A Common Tumor Driver of Distinct Human Neoplasms

Cancer is among the leading causes of mortality in developed countries due to limited available therapeutic modalities and high rate of morbidity. Although malignancies might show individual genetic landscapes, recurring aberrations in the neoplastic genome have been identified in the wide range of transformed cells. These include translocations of frequently affected loci of the human genetic material like the Ewing sarcoma breakpoint region 1 (EWSR1) of chromosome 22 that results in malignancies with mesodermal origin. These cytogenetic defects frequently result in the genesis of fusion genes involving EWSR1 and a number of genes from partner loci. One of these chromosomal rearrangements is the reciprocal translocation between the q13 and q12 loci of chromosome 12 and 22, respectively, that is believed to initiate cancer formation by the genesis of a novel, chimeric transcription factor provoking dysregulated gene expression. Since soft-tissue neoplasms carrying t(12;22)(q13;q12) have very poor prognosis and clinical modalities specifically targeting t(12;22)(q13;q12)-harboring cells are not available to date, understanding this DNA aberration is not only timely but urgent. Here, we review our current knowledge of human malignancies carrying the specific subset of EWSR1 rearrangements that leads to the expression of the EWSR1::ATF1 tumor-driver chimeric protein.

Epigenetic Therapies.

Epigenetic therapies are emerging for pediatric cancers. Due to the relatively low mutational burden in pediatric tumors, epigenetic dysregulation and differentiation blockade is a hallmark of oncogenesis in some childhood cancers. By targeting epigenetic regulators that maintain tumor cells in a primitive developmental state, epigenetic therapies may induce differentiation. The most well-studied and clinically advanced epigenetic-targeted therapies include azacitidine and decitabine, which inhibit DNA methylation through competitive inhibition of the enzymatic activity of the DNA methyltransferase family enzymes. These DNA hypomethylating agents are Food and Drug Administration (FDA) approved for hematologic malignancies. The discovery that DNA hypermethylation occurs in patients with isocitrate dehydrogenase (IDH) mutations has led to the development and FDA approval of IDH inhibitors for hematologic and solid tumors. Epigenetic dysregulation in pediatric tumors is also driven by changes in the "histone code" that either promote oncogene expression or repress tumor suppressors. Cancers whose chromatin landscape is characterized by such aberrant histone posttranslational modifications may be amenable to targeted therapies that inhibit the chromatin-modifying enzymes that read, write, and erase these histone modifications. Small molecules that inhibit the enzymatic activity of histone deacetylases, acetyltransferases, and methyltransferases have been approved for the treatment of some adult cancers, and these agents are currently under investigation in various pediatric tumors. Chromatin regulatory complexes can be hijacked by oncogenic fusion proteins that are produced by chromosomal translocations, which are common drivers in pediatric cancer. Small molecules that disrupt oncogenic fusion protein activity and their associated chromatin complexes have demonstrated remarkable promise, and this approach has become the standard treatment for a subset of leukemias driven by the PML-RARA oncogenic fusion protein. A deeper understanding of the mechanisms that drive epigenetic dysregulation in pediatric cancer may hold the key to future success in this field, as the landscape of druggable epigenetic targets is also expanding.

The Functional Interaction Between Epstein–Barr Virus and MYC in the Pathogenesis of Burkitt Lymphoma

The Epstein–Barr virus (EBV) is associated with a wide range of diseases, malignant and non-malignant. EBV was, in fact, the first virus described with cell transformation capacity, discovered by Epstein in 1964 in lymphoma samples from African children. Since then, EBV has been associated with several human tumors including nasopharyngeal carcinoma, gastric carcinoma, T-cell lymphoma, Hodgkin lymphoma, diffuse large B cell lymphoma, and Burkitt lymphoma among others. The molecular hallmark of Burkitt lymphoma (BL) is a chromosomal translocation that involves the MYC gene and immunoglobulin loci, resulting in the deregulated expression of MYC, an oncogenic transcription factor that appears deregulated in about half of human tumors. The role of MYC in lymphoma is well established, as MYC overexpression drives B cell proliferation through multiple mechanisms, foremost, the stimulation of the cell cycle. Indeed, MYC is found overexpressed or deregulated in several non-Hodgkin lymphomas. Most endemic and many sporadic BLs are associated with EBV infection. While some mechanisms by which EBV can contribute to BL have been reported, the mechanism that links MYC translocation and EBV infection in BL is still under debate. Here, we review the main EBV-associated diseases, with a special focus on BL, and we discuss the interaction of EBV and MYC translocation during B cell malignant transformation in BL.

Uniparental disomy (UPD) exclusion in embryos following Preimplantation Genetic Testing for Structural Rearrangements (PGT-SR).

Uniparental disomy (UPD) is a genetic condition which both copies of a chromosome are inherited from a single parent, potentially leading to imprinting disorders. This study aimed to assess the integration of Short Tandem Repeat (STR) analysis into Preimplantation Genetic Testing for Structural Rearrangements (PGT-SR) to assess UPD risk and its impact on selecting euploid embryos for embryo transfer in couples with chromosomal translocations involving imprinted chromosomes. This study evaluated three couples carrying balanced chromosomal translocations: 45,XX,der(13;14)(q10;q10), 46,XX,t(10;11)(q22;q13), and 45,XY,der(14;15)(q10;q10). STR analysis was performed on trophectoderm (TE) biopsies after Whole Genome Amplification (WGA) after PGT-SR analysis using parental blood samples to assess UPD risk in euploid embryos. Haplotyping was conducted with five to six STR markers specific to each rearranged chromosome to detect UPD in euploid embryos. Of the four embryos analyzed across the three families, two couples had euploid embryos that tested negative for UPD. These embryos were successfully transferred, resulting in the birth of two healthy children. In the third family, the euploid embryo also tested negative for UPD but failed to implant after transfer, resulting in no pregnancy. Despite its rarity, UPD involving imprinted chromosomes poses significant clinical risks, as seen in disorders such as Prader-Willi syndrome and Angelman syndrome. This study highlights the importance of integrating UPD screening into PGT-SR protocols, to detect both heterodisomic and isodisomic UPD events minimizing the risk of severe genetic disorders. Integrating STR-based UPD screening within PGT-SR workflows is a reliable and cost-effective strategy that enhances embryo selection and mitigates the risk of imprinting disorders. This approach improves reproductive outcomes for families with chromosomal rearrangements, offering a practical advancement in assisted reproduction.

Transcriptional regulation of KCNA2 coding Kv1.2 by EWS::FLI1: involvement in controlling the YAP/Hippo signalling pathway and cell proliferation

BackgroundEwing sarcoma (ES), the second main pediatric bone sarcoma, is characterised by a chromosomal translocation leading to the formation of fusion proteins like EWS::FLI1. While several studies have shown that potassium channels drive the development of many tumours, limited data exist on ES. This work therefore aimed to study the transcriptional regulation of KCNA2 and define the involvement of the Kv1.2 channel encoded by KCNA2 in a key function of ES development, cell proliferation.MethodsKCNA2 expression in patients and cell lines was measured via bioinformatic analysis (RNA-Seq). The presence of a functional Kv1.2 channel was shown using patch-clamp experiments. Molecular biology approaches were used after EWS::FLI1 silencing to study the transcriptional regulation of KCNA2. Proliferation and cell count assessment were performed using cell biology approaches. KCNA2 silencing (siRNA) and RNA-Seq were performed to identify the signalling pathways involved in the ability of KCNA2 to drive cell proliferation. The regulation of the Hippo signalling pathway by KCNA2 was studied by measuring Hippo/YAP target genes expression, while YAP protein expression was studied with Western-Blot and immunofluorescence approaches.ResultsThis research identified KCNA2 (encoding for a functional Kv1.2 channel) as highly expressed in ES biopsies and cell lines. The results indicated a correlation between KCNA2 expression and patient survival. The data also demonstrated that KCNA2/Kv1.2 is a direct target of EWS::FLI1, and identified the molecular mechanisms by which this chimeric protein regulates KCNA2 gene expression at the transcriptional level. Furthermore, the results indicated that KCNA2 expression and Kv1.2 activity regulate ES cell proliferation and that KCNA2 expression drives the Hippo/YAP signalling pathway. Using the specific Kv1.2 channel inhibitor (κ-Conotoxin), the results suggested that two complementary mechanisms are involved in this process, both dependently and independently of Kv1.2 functional channels at the plasma membrane.ConclusionThis study is the first to describe the involvement of KCNA2 expression and Kv1.2 channel in cancer development. The study also unveiled the involvement of KCNA2 in the regulation of the Hippo/YAP signalling cascade. Thus, this work suggests that KCNA2/Kv1.2 could be a potential therapeutic target in ES.

ChiTaRS 8.0: the comprehensive database of chimeric transcripts and RNA-seq data with applications in liquid biopsy.

Gene fusions are nucleotide sequences formed due to errors in replication and transcription control. These errors, resulting from chromosomal translocation, transcriptional errors or trans-splicing, vary from cell to cell. The identification of fusions has become critical as key biomarkers for disease diagnosis and therapy in various cancers, significantly influencing modern medicine. Chimeric Transcripts and RNA-Sequencing database version 8.0 (ChiTaRS 8.0; http://biosrv.org/chitars) is a specialized repository for human chimeric transcripts, containing 47445 curated RNA transcripts and over 100000 chimeric sequences in humans. This updated database provides unique information on 1055 chimeric breakpoints derived from public datasets using chromosome conformation capture techniques (the Hi-C datasets). It also includes an expanded list of gene fusions that are potential drug targets, and chimeric breakpoints across 934 cell lines, positioning ChiTaRS 8.0 as a valuable resource for testing personalized cancer therapies. By utilizing text mining on a curated selection of disease-specific RNA-sequencing data from public datasets, as well as patient blood and plasma samples, we have identified novel chimeras-particularly in diseases such as oral squamous cell carcinoma and glioblastoma-now catalogued in ChiTaRS. Thus, ChiTaRS 8.0 serves as an enhanced fusion transcript repository that incorporates insights into the functional landscape of chimeras in cancers and other complex diseases, based on liquid biopsy results.

Evaluation of wheat blast resistance in the USDA hard winter wheat (Triticum aestivum L.) Northern and Southern Regional Performance Nurseries.

Wheat blast, caused by the Triticum pathotype of Pyricularia oryzae, is an emerging disease that threatens the global supply of wheat. The pathogen was first reported in Brazil and subsequently spread to the neighboring countries of Argentina, Bolivia, and Paraguay. More recently, wheat blast was reported in Asia and Africa, having been observed in Bangladesh and Zambia. The transcontinental spread of the pathogen has heightened awareness of the disease and has underscored the need to prepare for potential introductions into major wheat production regions. To this end, we have conducted greenhouse evaluations of the 2017 - 2020 hard winter wheat (Triticum aestivum L.) entries of the USDA-coordinated Northern and Southern Regional Performance Nurseries (NRPN and SRPN, respectively) of the U.S. Great Plains using an aggressive isolate of the pathogen, B-71, collected in Bolivia in 2012. Based on molecular marker analysis, all potentially resistant entries in both the NRPN and SRPN were in possession of the distal chromosomal translocation of 2NVS from Aegilops ventricosa, which has previously been associated with wheat blast resistance. No potentially new sources of resistance were evident in the 2017 - 2020 regional performance nurseries, which highlights the need for the continued use of 2NVS donor lines in Great Plains wheat breeding programs.

Comparison of chromosomal microarray and karyotyping in prenatal diagnosis using 491 amniotic fluid samples.

This study was aimed to investigate the performance of chromosomal microarray analysis (CMA) in prenatal diagnosis compared with traditional karyotyping analysis. Both CMA and karyotyping analyses were performed to detect the karyotypes in the amniotic fluid of 491 pregnant women who got prenatal diagnosis at the Center of Prenatal Diagnosis of Shangrao (China) during January 2019 to April 2021. After excluding 2 samples in the CMA analysis and 2 samples in the karyotyping analysis which were failed in detection, the remaining 487 amniotic fluid samples were detected. Both CMA and karyotyping analyses identified 22 cases of aneuploidy chromosome abnormalities, including trisomy 21 (10 cases), trisomy 18 (4 cases), sex chromosome abnormality (5 cases), and other chromosome abnormalities (3 cases). In addition, CMA and karyotyping analyses found 8 cases of fetal chromosomal imbalance. Interestingly, abnormal results were detected by CMA analysis in 10 cases whose results were normal by karyotype analysis. Furthermore, 23 cases of copy number variation (CNVs) with variation of unknown clinical significance (VOUS) were detected by CMA, which accounted for 4.68% (23/491) in all cases. However, CMA was not able to accurately identify some complex karyotypes and mixed chimeras, including 2 cases of chimeras, 4 cases of balanced translocations, 4 cases of pericentric inversions, and 8 cases of other chromosome polymorphisms, indicating karyotyping analysis was superior to detect these chromosome abnormalities compared with CMA analysis. CMA was better in detecting the fracture sites, microduplication and microdeletion with definite pathogenicity, and CNVs with VOUS compared with karyotype analysis.

Modulation of TCR stimulation and pifithrin-α improves the genomic safety profile of CRISPR-engineered human Tcells.

CRISPR-engineered chimeric antigen receptor (CAR) Tcells are at the forefront of novel cancer treatments. However, several reports describe the occurrence of CRISPR-induced chromosomal aberrations. So far, measures to increase the genomic safety of Tcell products focused mainly on the components of the CRISPR-Cas9 system and less on Tcell-intrinsic features, such as their massive expansion after Tcell receptor (TCR) stimulation. Here, we describe driving forces of indel formation in primary human Tcells. Increased Tcell activation and proliferation speed correlate with larger deletions. Editing of non-activated Tcells reduces the risk of large deletions with the downside of reduced knockout efficiencies. Alternatively, the addition of the small-molecule pifithrin-α limits large deletions, chromosomal translocations, and aneuploidy in a p53-independent manner while maintaining the functionality of CRISPR-engineered Tcells, including CAR Tcells. Controlling Tcell activation and pifithrin-α treatment are easily implementable strategies to improve the genomic integrity of CRISPR-engineered Tcells.

ТРАНСЛОКАЦИИ ХРОМОСОМ В ЛИМФОЦИТАХ ПАЦИЕНТКИ В ОТДАЛЁННЫЕ СРОКИ ПОСЛЕ АВАРИЙНОГО ЛОКАЛЬНОГО ОБЛУЧЕНИЯ

Objective: to compare the frequencies of translocations detected by one-color and three-color FISH painting in the long term (24 years) after local over-irradiation (more than 100 Gy) of a patient during radiation therapy for breast cancer. Material and methods: The material for the cytogenetical study were the cultures of venous blood lymphocytes of this patient. Cultivation of lymphocytes and preparation of chromosome preparations were carried out using variants of standard methods. Chromosomes were stained separately with one-color or three-color DNA probes for 1, 4 and 12 pairs. In both cases, 1000 metaphases were analyzed. Results: In the present study, when using the one-color FISH-method, the frequency of detected reciprocal translocations was 0.5 per 100 analyzed cells, while with tricolor painting they were 2.2 times higher (1.0 per 100 metaphases). When using the available linear-quadratic dose dependence, the average dose to the whole body was estimated at 0.28 Gy, which is a consequence of the local nature of radiation damage, the death of lymphocytes in the zone of high-dose radiation damage and the obvious impossibility of their division. Conclusion: The study of the frequency of chromosomal translocations in the long term after local overexposure during therapy in a patient for breast cancer using a three-color FISH-analysis is a more sensitive method in comparison with a one-color variant of FISH-painting.

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.