Chromosomal Translocation Events Research Articles

Non-clinical evaluation of NT-175, an autologous T cell product engineered to express an HLA-A*02:01-restricted TCR targeting TP53 R175H and resistant to TGF-b inhibition.

2560 Background: Adoptive transfer of T-cell receptor (TCR)-engineered T-cells to target shared cancer neoantigens is a promising new immunotherapy approach for patients harboring mutations in tumor suppressor genes such as TP53. TP53 is the most commonly mutated gene across all cancer types, with the R175H mutation being the most prevalent across different indications. NT-175 is an autologous engineered T-cell product expressing an HLA-A*02:01-restricted TCR that specifically targets the TP53 R175H mutation. NT-175 is additionally engineered to lack TGF-β receptor II (TGFBR2) expression, rendering T-cells insensitive to TGF-b-mediated inhibition in the tumor microenvironment. Methods: For manufacturing of NT-175, CD4 and CD8 T-cells are enriched from leukapheresis material, activated in vitro and gene-edited to knock-out both endogenous TGFBR2 and TCR β constant 1/2 (TRBC1/2) and knock-in an HLA-A*02:01-restricted TP53 R175H neoantigen-specific TCR at the TCR α constant (TRAC) locus using CRISPR-Cas9 technology. NT-175 product functionality was evaluated in vivo models and in vitro. T-cell reactivity was assessed by measuring cytotoxicity, proliferation, and cytokine production. The benefit of TGFBR2 knock-out (KO) in the presence of TGF-b was evaluated by measuring phosphorylation of SMAD2/3 proteins, the impact on cell viability and serial killing of target cells. For determination of NT-175 product safety, cross-reactivity and HLA-specificity assessments were carried out. A comprehensive analysis of potential off-target editing by CRISPR/Cas9 was performed to assess potential risk of genotoxicity in clinical grade NT-175. Results: High reactivity of the NT-175 TCR against TP53 R175H and HLA-A*02:01 expressing target cells was observed. T-cell activation and functionality was highly specific, as demonstrated by a lack of reactivity against TP53 WT, minimal cross-reactivity against antigens with up to 4 mismatches to the minimal TP53 R175H encoding epitope recognized by the NT-175 TCR, and lack of T-cell activation in the absence of HLA-A*02:01. In the presence of TGF-b, TGFBR2 KO TCR-edited T-cells displayed inhibition of SMAD2/3 phosphorylation, increased cell viability and increased cytotoxicity and proliferation in serial stimulation assays. In vivo, NT-175 T-cells were able to induce tumor clearance in two independent models. Low frequency chromosomal translocation events (<0.1%) between on-target and off-target Cas9 cleavage sites were detectable in NT-175 T-cells. However importantly, these did not result in autonomous cytokine-independent growth. Conclusions: Non-clinical studies revealed a favorable safety and efficacy profile for NT-175 and supported further clinical development of a TGF-β-resistant TCR-edited T-cell product for mutant TP53-targeted cancer immunotherapy.

Non-clinical evaluation of NT-112, an autologous T cell product engineered to express an HLA-C*08:02-restricted TCR targeting KRAS G12D and resistant to TGF-b inhibition.

e14533 Background: Adoptive transfer of T-cell receptor (TCR)-engineered T-cells to target shared cancer neoantigens is a promising new immunotherapy approach for patients harboring mutations in oncogenes such as KRAS. Activating mutations in KRAS are among the most prevalent oncogenic driver mutations in human cancers, with KRAS G12D being the most frequent KRAS mutation in colorectal carcinoma and pancreatic cancer. NT-112 is an autologous engineered T-cell product expressing an HLA-C*08:02-restricted TCR that specifically targets the KRAS G12D mutation. NT-112 is additionally engineered to lack TGF-β receptor II (TGFBR2) expression, rendering T-cells insensitive to TGF-b-mediated inhibition in the tumor microenvironment. Methods: For manufacturing of NT-112, CD4 and CD8 T-cells are enriched from leukapheresis material, activated in vitro and gene-edited to knock-out both endogenous TGFBR2 and TCR β constant 1/2 (TRBC1/2) and knock-in an HLA-C*08:02-restricted KRS G12D neoantigen-specific TCR at the TCR α constant (TRAC) locus using CRISPR-Cas9 technology. NT-112 product functionality was evaluated in both in vivo models and in vitro co-culture experiments with a panel of cell lines including those endogenously expressing the KRAS G12D mutation. T-cell reactivity was assessed by measuring cytotoxicity, proliferation, and cytokine production. For determination of NT-112 product safety, cross-reactivity and allo-reactivity assessments were carried out. A comprehensive analysis of potential off-target editing by CRISPR/Cas9 was performed to assess potential risk of genotoxicity in clinical grade NT-112. Results: High reactivity of the NT-112 TCR against KRAS G12D and HLA-C*08:02-expressing target cells was observed. T-cell activation and functionality was highly specific, as demonstrated by a lack of reactivity against KRAS WT, minimal cross-reactivity in Alanine and Glycine scans, and lack of T-cell activation in the absence of HLA-C*08:02. In vivo, NT-112 T-cells were able to induce tumor clearance in two independent models. Low frequency chromosomal translocation events (<0.1%) between on-target and off-target Cas9 cleavage sites were detectable in NT-112 T-cells. However importantly, these did not result in autonomous cytokine-independent growth. Conclusions: Non-clinical studies revealed a favorable safety and efficacy profile for NT-112 and supported further clinical development of a TGF-β-resistant TCR-edited T-cell product for mutant KRAS-targeted cancer immunotherapy.

Brd4::Nutm1 fusion gene initiates NUT carcinoma in vivo.

NUT carcinoma (NC) is an aggressive cancer with no effective treatment. About 70% of NUT carcinoma is associated with chromosome translocation events that lead to the formation of a BRD4::NUTM1 fusion gene. Because the BRD4::NUTM1 gene is unequivocally cytotoxic when ectopically expressed in cell lines, questions remain on whether the fusion gene can initiate NC. Here, we report the first genetically engineered mouse model for NUT carcinoma that recapitulates the human t(15;19) chromosome translocation in mice. We demonstrated that the mouse t(2;17) syntenic chromosome translocation, forming the Brd4::Nutm1 fusion gene, could induce aggressive carcinomas in mice. The tumors present histopathological and molecular features similar to human NC, with enrichment of undifferentiated cells. Similar to the reports of human NC incidence, Brd4::Nutm1 can induce NC from a broad range of tissues with a strong phenotypical variability. The consistent induction of poorly differentiated carcinoma demonstrated a strong reprogramming activity of BRD4::NUTM1. The new mouse model provided a critical preclinical model for NC that will lead to better understanding and therapy development for NC.

Transforming Growth Factor Beta and Alveolar Rhabdomyosarcoma: A Challenge of Tumor Differentiation and Chemotherapy Response.

Alveolar rhabdomyosarcoma (ARMS), an invasive subtype of rhabdomyosarcoma (RMS), is associated with chromosomal translocation events resulting in one of two oncogenic fusion genes, PAX3-FOXO1 or PAX7-FOXO1. ARMS patients exhibit an overexpression of the pleiotropic cytokine transforming growth factor beta (TGF-β). This overexpression of TGF-β1 causes an increased expression of a downstream transcription factor called SNAIL, which promotes epithelial to mesenchymal transition (EMT). Overexpression of TGF-β also inhibits myogenic differentiation, making ARMS patients highly resistant to chemotherapy. In this review, we first describe different types of RMS and then focus on ARMS and the impact of TGF-β in this tumor type. We next highlight current chemotherapy strategies, including a combination of the FDA-approved drugs vincristine, actinomycin D, and cyclophosphamide (VAC); cabozantinib; bortezomib; vinorelbine; AZD 1775; and cisplatin. Lastly, we discuss chemotherapy agents that target the differentiation of tumor cells in ARMS, which include all-trans retinoic acid (ATRA) and 5-Azacytidine. Improving our understanding of the role of signaling pathways, such as TGF-β1, in the development of ARMS tumor cells differentiation will help inform more tailored drug administration in the future.

Structural Aspects of the ROS1 Kinase Domain and Oncogenic Mutations

Protein kinases function as pivotal regulators in biological events, governing essential cellular processes through the transfer of phosphate groups from ATP molecules to substrates. Dysregulation of kinase activity is frequently associated with cancer, ocasionally arising from chromosomal translocation events that relocate genes encoding kinases. Fusion proteins resulting from such events, particularly those involving the proto-oncogene tyrosine-protein kinase ROS (ROS1), manifest as constitutively active kinases, emphasizing their role in oncogenesis. Notably, the chromosomal reallocation of the ros1 gene leads to fusion of proteins with the ROS1 kinase domain, implicated in various cancer types. Despite their prevalence, targeted inhibition of these fusion proteins relies on repurposed kinase inhibitors. This review comprehensively surveys experimentally determined ROS1 structures, emphasizing the pivotal role of X-ray crystallography in providing high-quality insights. We delve into the intricate interactions between ROS1 and kinase inhibitors, shedding light on the structural basis for inhibition. Additionally, we explore point mutations identified in patients, employing molecular modeling to elucidate their structural impact on the ROS1 kinase domain. By integrating structural insights with in vitro and in silico data, this review advances our understanding of ROS1 kinase in cancer, offering potential avenues for targeted therapeutic strategies.

Abstract B015: Identification of novel natural compounds that induce DNA damage and chromosomal translocations

Abstract Background: Etoposide is a well-characterized chemotherapeutic that promotes DNA double-strand breaks (DSBs) and is associated with therapy-related chromosomal translocations and leukemias. Natural compounds including bioflavonoids, which are found in food as well as in dietary supplements, have been shown by our lab to induce DSBs and promote genome rearrangements. Given the potential for bioflavonoids to cause these harmful effects, we are investigating the potential for a wide array of other natural compounds and environmental agents to also induce DNA DSBs and chromosomal translocations. The purpose of this study was to perform a high throughput screen on a National Institutes of Health (NIH) library of natural compounds to determine their ability to cause DSBs and chromosomal translocations and implicate them as risk factors for leukemias. Methods: We obtained 2 libraires from NIH containing over 2000 natural compounds. Mouse embryonic stem cells (mES) were exposed to individual compounds at 50mM for one hour. Following exposure, DNA damage was measured through γ-H2AX immunocytochemistry and microscopy to quantify the level of DNA damage induced in comparison to etoposide. Compounds identified to cause greater than or equal to 20% of the damage induced by etoposide were prioritized for study of their potential to induce chromosomal translocations. We developed a mES GFP Reporter cell line with engineered exons of GFP contained within MLL and AF9 breakpoint cluster region inserts. Cells were exposed to individual compounds at 50mM for one hour. Following exposure, cells were screened over 7 days for the generation of chromosomal translocation events as measured by appearance of GFP+ cells. Results: Exposure to the NIH natural compound library has thus far generated 22 hits with 5 - 20% the amount of damage caused by etoposide and 29 hits with greater than or equal to 20% of the damage of etoposide out of the 1,200 compounds tested thus far. Top “hits” include known inhibitors of topoisomerase I and II. Other top “hits” include natural compounds curcumin, illudin, prodigiosin, and predorine. Testing of the ability of these “hits” to cause chromosomal translocations are still underway, but compounds, such as curcumin, are showing translocation GFP+ events. Conclusions: Data from this study can be used to inform the public and doctors of natural compounds that may be believed to be beneficial for human health but that can actually cause DNA damage and potential translocations. This data could generate potential drug candidates useful in the treatment of cancer. Etoposide is known to create DSBs but it also causes frequent translocation events. This study could identify compounds that are able to cause DNA DSBs, essential to the destruction of cancer cells, with a lower likelihood of causing chromosomal translocation events in healthy cells. Citation Format: Donna Goodenow, Henry Thompson, Elizabeth Toufekoulas, Heather Derby, Christine Richardson. Identification of novel natural compounds that induce DNA damage and chromosomal translocations [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B015.

A novel neo-sex chromosome in Sylvietta brachyura (Macrosphenidae) adds to the extraordinary avian sex chromosome diversity among Sylvioidea songbirds.

We report the discovery of a novel neo-sex chromosome in an African warbler, Sylvietta brachyura (northern crombec; Macrosphenidae). This species is part of the Sylvioidea superfamily, where four separate autosome-sex chromosome translocation events have previously been discovered via comparative genomics of 11 of the 22 families in this clade. Our discovery here resulted from analyses of genomic data of single species-representatives from three additional Sylvioidea families (Macrosphenidae, Pycnonotidae and Leiothrichidae). In all three species, we confirmed the translocation of a part of chromosome 4A to the sex chromosomes, which originated basally in Sylvioidea. In S. brachyura, we found that a part of chromosome 8 has been translocated to the sex chromosomes, forming a unique neo-sex chromosome in this lineage. Furthermore, the non-recombining part of 4A in S. brachyura is smaller than in other Sylvioidea species, which suggests that recombination continued along this region after the fusion event in the Sylvioidea ancestor. These findings reveal additional sex chromosome diversity among the Sylvioidea, where five separate translocation events are now confirmed.

S5.3a Unraveling the genetic determinants of virulence in Cryptococcus neoformans

S5.3 Cellular pleomorphism and fungal virulence, September 22, 2022, 3:00 PM - 4:30 PM Cryptococcus neoformans is a human pathogenic basidiomycete yeast that can cause cryptococcal meningitis (CM), predominantly in immunocompromised individuals. The patient outcome depends on both host and pathogen-specific factors, including C. neoformans genetics. A groundbreaking 2012 study was the first to show that patient outcome is associated with genetic differences between C. neoformans isolates. Subsequent population-wide sequencing studies have revealed over 100 sequence types (ST) of C. neoformans that are associated with both geographic location and clinical outcome. All these studies have been broad, examining the severity of disease cryptococcal phenotypes in a collection of highly diverse strains. We chose a narrow focus and collected various genotypic and phenotypic data from a single ST: ST93. ST93 is a common sequence type isolated from patients globally and is the most common clinical isolate found in the sub-Saharan African country of Uganda. Previously, we performed whole genome sequencing on 38 ST93 Ugandan clinical isolates. We identified 652 unique SNPs in this ST93 population compared to the H99 reference genome. We also showed that ST93 contained two subpopulations: ST9A and ST93B. In the current study, we further characterized the genotypic, phenotypic, and virulence differences between these 38 clinical isolates. Using Illumina sequence data, we identified a pattern of linkage disequilibrium that suggested that ST93A and ST93B are evolving separately. We performed long-read sequencing on each isolate to investigate chromosomal changes and large structural variations, allowing us to identify a chromosomal translocation event wherein parts of chromosome 11 had recombined with chromosome 3. Additionally, we characterized several in vitro phenotypes for each isolate and identified three distinct phenotypic clusters based on cell wall challenge and growth experiments. Next, we infected mice with 35 isolates and observed eight different disease manifestations, including isolates that caused non-CNS infections. Overall, by working within a single sequence type, we can gain a deeper understanding of how some small genetic changes can impact strain-specific phenotypes while others have no discernable effect. Eventually, these data can be used to provide valuable information about how each clinical isolate impacts patient outcomes.

A chromosome-level reference genome of Ensete glaucum gives insight into diversity and chromosomal and repetitive sequence evolution in the Musaceae.

Background Ensete glaucum (2n = 2x = 18) is a giant herbaceous monocotyledonous plant in the small Musaceae family along with banana (Musa). A high-quality reference genome sequence assembly of E. glaucum is a resource for functional and evolutionary studies of Ensete, Musaceae, and the Zingiberales.FindingsUsing Oxford Nanopore Technologies, chromosome conformation capture (Hi-C), Illumina and RNA survey sequence, supported by molecular cytogenetics, we report a high-quality 481.5 Mb genome assembly with 9 pseudo-chromosomes and 36,836 genes. A total of 55% of the genome is composed of repetitive sequences with predominantly LTR-retroelements (37%) and DNA transposons (7%). The single 5S ribosomal DNA locus had an exceptionally long monomer length of 1,056 bp, more than twice that of the monomers at multiple loci in Musa. A tandemly repeated satellite (1.1% of the genome, with no similar sequence in Musa) was present around all centromeres, together with a few copies of a long interspersed nuclear element (LINE) retroelement. The assembly enabled us to characterize in detail the chromosomal rearrangements occurring between E. glaucum and the x = 11 species of Musa. One E. glaucum chromosome has the same gene content as Musa acuminata, while others show multiple, complex, but clearly defined evolutionary rearrangements in the change between x= 9 and 11.ConclusionsThe advance towards a Musaceae pangenome including E. glaucum, tolerant of extreme environments, makes a complete set of gene alleles, copy number variation, and a reference for structural variation available for crop breeding and understanding environmental responses. The chromosome-scale genome assembly shows the nature of chromosomal fusion and translocation events during speciation, and features of rapid repetitive DNA change in terms of copy number, sequence, and genomic location, critical to understanding its role in diversity and evolution.

Mechanism for the activation of the anaplastic lymphoma kinase receptor.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that regulates important functions in the central nervous system1,2. The ALK gene is a hotspot for chromosomal translocation events that result in several fusion proteins that cause a variety of human malignancies3. Somatic and germline gain-of-function mutations in ALK were identified in paediatric neuroblastoma4-7. ALK is composed of an extracellular region (ECR), a single transmembrane helix and an intracellular tyrosine kinase domain8,9. ALK is activated by the binding of ALKAL1 and ALKAL2 ligands10-14 to its ECR, but the lack of structural information for the ALK-ECR or for ALKAL ligands has limited our understanding of ALK activation. Here we used cryo-electron microscopy, nuclear magnetic resonance and X-ray crystallography to determine the atomic details of human ALK dimerization and activation by ALKAL1 and ALKAL2. Our data reveal a mechanism of RTK activation that allows dimerization by either dimeric (ALKAL2) or monomeric (ALKAL1) ligands. This mechanism is underpinned by an unusual architecture of the receptor-ligand complex. The ALK-ECR undergoes a pronounced ligand-induced rearrangement and adopts an orientation parallel to the membrane surface. This orientation is further stabilized by an interaction between the ligand and the membrane. Our findings highlight the diversity in RTK oligomerization and activation mechanisms.

Genome-wide identification and characterization of the Lateral Organ Boundaries Domain (LBD) gene family in polyploid wheat and related species.

BackgroundWheat (Triticum aestivum) originated from three different diploid ancestral grass species and experienced two rounds of polyploidization. Exploring how certain wheat gene subfamilies have expanded during the evolutionary process is of great importance. The Lateral Organ Boundaries Domain (LBD) gene family encodes plant-specific transcription factors that share a highly conserved LOB domain and are prime candidates for this, as they are involved in plant growth, development, secondary metabolism and stress in various species.MethodsUsing a genome-wide analysis of high-quality polyploid wheat and related species genome sequences, a total of 228 LBD members from five Triticeae species were identified, and phylogenetic relationship analysis of LBD members classified them into two main classes (classes I and II) and seven subgroups (classes I a–e, II a and II b).ResultsThe gene structure and motif composition analyses revealed that genes that had a closer phylogenetic relationship in the same subgroup also had a similar gene structure. Macrocollinearity and microcollinearity analyses of Triticeae species suggested that some LBD genes from wheat produced gene pairs across subgenomes of chromosomes 4A and 5A and that the complex evolutionary history of TaLBD4B-9 homologs was a combined result of chromosome translocation, polyploidization, gene loss and duplication events. Public RNA-seq data were used to analyze the expression patterns of wheat LBD genes in various tissues, different developmental stages and following abiotic and biotic stresses. Furthermore, qRT-PCR results suggested that some TaLBDs in class II responded to powdery mildew, regulated reproductive growth and were involved in embryo sac development in common wheat.

Abstract PO-041: Genome-wide CRISPR/Cas9 screen reveals mitochondrial gene mutation as a driver for drug resistance in Ewing sarcoma

Abstract Ewing sarcoma is a pediatric bone cancer caused by a chromosomal translocation event that generates the pathognomonic fusion protein, EWS/FLI, which acts as aberrant transcription factor. EWS/FLI is responsible for activating and repressing important genes to drive oncogenic transformation and tumorigenesis. EWS/FLI is notoriously difficult to target with small molecule inhibitors based on its lack of enzymatic activity and intrinsically disordered EWS domain, thus rational drug design is presently unfeasible. Other potential therapeutic targets are interacting partners of EWS/FLI, including epigenetic modulators, such as lysine specific demethylase-1 (LSD1). Inhibition of LSD1 by a reversible, non-competitive inhibitor, SP-2509, has been shown to cause extensive changes to gene expression and induce apoptosis in Ewing sarcoma cells. A Phase I clinical trial (NCT03600649) is currently ongoing to study the effect an SP-2509 analog, Seclidemstat (SP-2577), on patients with recurrent or refractory Ewing sarcoma tumors. Thus, understanding potential drug resistance mechanisms is vital to ensure maximal efficacy of Seclidemstat treatment for patients in the present, and in future clinical trials with this targeted therapeutic drug. Preliminary investigation using a genome-wide CRISPR/Cas9 knock-out (GeCKo) screen has revealed that the loss of genes important to mitochondrial function, specifically in the electron transport chain (ETC), play a distinct role in SP-2509 drug-resistance in Ewing sarcoma cells. We validated the GeCKo screen using cell viability assays in an Ewing sarcoma cell line (A673) via knockout (KO) of mitochondrial genes that were identified by the GeCKo screen. Consistent with the screen, we found select mitochondrial genes led to an increase in IC50 values (i.e. drug-resistance) to SP-2509, compared to WT A673 cells. We found that drug resistance could be enhanced even greater by generating monoclonal cell populations containing complete mitochondrial gene KO. We performed RNA-sequencing and metabolomics analyses to examine SP-2509 response differences between WT and our generated drug resistance cell lines. We similarly determined that treating A673 cells with mitochondrial ETC inhibitors recapitulated drug resistance. Currently, the mechanism that links mitochondrial dysfunction in Ewing sarcoma cells and resistance to the epigenetic drug SP-2509 is incompletely understood. However, our ongoing studies aim to uncover this link and ultimately provide consequential information that will help patients overcome Ewing sarcoma. Citation Format: Jack P. Tokarsky, Jesse Crow, Lillian Guenther, Emily Theisen, Kimberly Stegmaier, Stephen Lessnick. Genome-wide CRISPR/Cas9 screen reveals mitochondrial gene mutation as a driver for drug resistance in Ewing sarcoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-041.

Multiplex CRISPR/Cas9 Genome Editing Targeting the BCL11A/HBG Axis Maximizes Fetal Hemoglobin Reinduction but Generates Chromosomal Translocations Which Persist In Vivo

Sickle cell disease and β-thalassemia are among the most common monogenic disorders globally, causing significant morbidity and early mortality. The only curative option available is allogeneic hematopoietic stem cell transplantation, which is limited by a lack of matched donors and risks, including graft versus host disease and secondary malignancy. Retroviral gene transfer is being explored in clinical trials, but an alternative approach is more targeted CRISPR/Cas9 genome editing, to recapitulate naturally occurring hereditary persistence of fetal hemoglobin (HPFH) which ameliorates disease. HbF reinduction is achieved by disrupting either transcription factor binding sites within the HBG promoter, or an erythroid enhancer sequence within the HbF repressor, BCL11A. Results from preclinical studies have suggested that HbF levels may remain suboptimal when each locus is targeted individually. We thus investigated the feasibility of a dual editing approach, targeting both loci simultaneously or sequentially, to maximize HbF production. G-CSF-mobilized human peripheral blood hematopoietic stem and progenitor cells (HSPCs) underwent CRISPR/Cas9 genome editing at either HBG promoter, BCL11A erythroid enhancer, or both loci targeted either simultaneously or sequentially. Immunodeficient mice were transplanted with edited cells. HSPCs cultured in differentiation media demonstrated comparable levels of editing at each locus in the single edited arms (by TIDE).The dual editing approach did not impair editing efficiency at each site when conducted sequentially and only slightly reduced efficiency in the simultaneously-edited reactions. HbF reinduction was also greatest in sequentially double-edited reactions, with HbF/HbA ratio as high as 3.9 times that seen in mock reactions by flow cytometry and 4.1 times by HPLC. In single-edited arms, HbF reinduction was slightly greater with HBG promotor than BCL11A erythroid enhancer editing. Two separate chromosomal translocation events encompassing both loci were detected in each double-edited arm and quantified using digital droplet PCR. Both were more frequent in simultaneous (mean of 1.0% and 0.6%) than sequential reactions (means <0.2%, n=3, p=0.0420 and p=0.0296). Analysis of single BFU-E colonies grown on methocult media revealed markedly different indel patterns at the 2 target loci. Concurrent hemoglobin fraction analysis of these clonal populations by HPLC demonstrated greatest HbF proportion in double-edited colonies. Where ≥60% editing was reported at both loci mean HbF was 71.3%, compared to 47.1% with ≥60% editing at HBG only, and 29.6% with ≥60% editing at BCL11A only (p=0.0373). Following transplant into mice, engraftment and lineage differentiation were comparable among all experimental arms. At necropsy, bone marrow populations of human CD45+ cells, CD34+CD38low HSPCs and, within these, HSC-enriched CD90+CD45RA- subpopulation, were present at comparable levels indicating that the dual editing approach did not impair engraftment. Translocation events were detected, albeit at frequencies of less than 0.25%, in the bone marrows of all 4 mice transplanted with simultaneously dual-edited cells and in 5 of 6 mice transplanted with sequentially dual-edited cells. Bone marrow cells cultured ex vivo demonstrated greatest HbF from mice transplanted with sequentially double-edited cells. In summary, we present evidence of maximized HbF reinduction with sequentially applied multiplex genome editing at BCL11A erythroid enhancer and HBG promoter, with no impairment of engraftment or differentiation. However, chromosomal translocation events were consistently seen in double-edited reactions, even when edits at each locus were applied sequentially, and persisted in vivo after engraftment, thus rendering this approach inappropriate for clinical applications. However, multiplex editing and targeting both the BCL11A erythroid enhancer and HBG promoter to maximize fetal hemoglobin induction may be a promising strategy for alternative platforms such as base editors which are expected to greatly reduce or completely eliminate the occurrence of translocations. Disclosures Radtke: Forty Seven INC: Consultancy. Kiem:Enochian: Membership on an entity's Board of Directors or advisory committees; Umoja: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy; Homology Medicines: Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy; Vor Biopharma: Membership on an entity's Board of Directors or advisory committees; Rocket Pharma: Membership on an entity's Board of Directors or advisory committees.

Identification and characterization of a female gametophyte defect in sdk1-7 +/- abi3-6 +/- heterozygotes of Arabidopsis thaliana

ABSTRACT Successful reproduction in angiosperms is dependent on the highly synchronous development of their male and female gametophytes and the ensuing fusion of the gametes from these reproductive tissue types. When crossing a T-DNA insertion line sdk1-7-/-(Salk_024564), one of the S-domain receptor kinases involved in ABA responses with a fast neutron deletion line abi3-6-/-, the F1 heterozygotes (sdk1-7+/-abi3-6 +/-) displayed 50% ovule abortion suggesting a likely gametophytic defects. We identified and characterized an early stage female gametophyte developmental defect in the heterozygous mutant ovules. Recombination frequency analysis of the F2 progenies from selfed heterozygotes revealed a possible pseudo-linkage of sdk1-7 and abi3-6 suggesting a reciprocal translocation event in the heterozygote. Our study emphasizes the importance of robust analysis to distinguish gametophytic defect phenotypes caused by genetic interactions and that resulting from possible chromosomal translocation events.

High-Density Genetic Linkage Mapping of Lepidium Based on Genotyping-by-Sequencing SNPs and Segregating Contig Tag Haplotypes.

Lepidium campestre has been targeted for domestication as future oilseed and catch crop. Three hundred eighty plants comprising genotypes of L. campestre, Lepidium heterophyllum, and their interspecific F2 mapping population were genotyped using genotyping by sequencing (GBS), and the generated polymorphic markers were used for the construction of high-density genetic linkage map. TASSEL-GBS, a reference genome-based pipeline, was used for this analysis using a draft L. campestre whole genome sequence. The analysis resulted in 120,438 biallelic single-nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) above 0.01. The construction of genetic linkage map was conducted using MSTMap based on phased SNPs segregating in 1:2:1 ratio for the F2 individuals, followed by genetic mapping of segregating contig tag haplotypes as dominant markers against the linkage map. The final linkage map consisted of eight linkage groups (LGs) containing 2,330 SNP markers and spanned 881 Kosambi cM. Contigs (10,302) were genetically mapped to the eight LGs, which were assembled into pseudomolecules that covered a total of ∼120.6 Mbp. The final size of the pseudomolecules ranged from 9.4 Mbp (LG-4) to 20.4 Mpb (LG-7). The following major correspondence between the eight Lepidium LGs (LG-1 to LG-8) and the five Arabidopsis thaliana (At) chromosomes (Atx-1–Atx-5) was revealed through comparative genomics analysis: LG-1&2_Atx-1, LG-3_Atx-2&3, LG-4_Atx-2, LG-5_Atx-2&Atx-3, LG-6_Atx-4&5, LG-7_Atx-4, and LG-8_Atx-5. This analysis revealed that at least 66% of the sequences of the LGs showed high collinearity with At chromosomes. The sequence identity between the corresponding regions of the LGs and At chromosomes ranged from 80.6% (LG-6) to 86.4% (LG-8) with overall mean of 82.9%. The map positions on Lepidium LGs of the homologs of 24 genes that regulate various traits in A. thaliana were also identified. The eight LGs revealed in this study confirm the previously reported (1) haploid chromosome number of eight in L. campestre and L. heterophyllum and (2) chromosomal fusion, translocation, and inversion events during the evolution of n = 8 karyotype in ancestral species shared by Lepidium and Arabidopsis to n = 5 karyotype in A. thaliana. This study generated highly useful genomic tools and resources for Lepidium that can be used to accelerate its domestication.

Fusion protein EWS‐FLI1 partners with EWSR1 to regulate transcription in Ewing sarcoma

Chromosomal translocation events drive many types of pediatric cancers. Ewing sarcoma is a pediatric bone cancer driven by a chromosomal translocation where the N‐terminal low‐complexity domain of EWSR1 fuses to the DNA‐binding domain of the ETS transcription factor FLI1. This fusion creates the aberrant transcription factor EWS‐FLI1, which drives tumor formation by altering the transcriptional landscape. Interestingly, constitutive expression of EWSR1 is retained in Ewing sarcoma cells. The low‐complexity domain found in EWSR1 and EWS‐FLI1 is intrinsically‐disordered and can mediate protein‐protein interactions with itself and other protein partners. Moreover, this domain has the propensity to phase separate. Despite the common domain between the two proteins, very little is known about the effect of EWSR1 on EWS‐FLI1 activity. We performed RNA‐sequencing to reveal that EWSR1 and EWS‐FLI1 coregulate a large pool of genes in Ewing sarcoma cells. Loss of EWSR1 in inhibits anchorage‐independent growth, suggesting that EWSR1 plays a role in cancerous properties of Ewing sarcoma cells. We have shown EWSR1 and EWS‐FLI1 bind each other directly in vitro and in vivo. Finally, we have found the existence of EWSR1, EWS‐FLI1, and RNA Pol II in a novel granular body in Ewing sarcoma cell lines. This direct interaction of EWSR1 and EWS‐FLI1 presents a straight‐forward model that merges wild‐type and fusion properties to alter transcriptional output and drive Ewing sarcoma biology.Support or Funding InformationNational Institutes of Health, American Cancer Society

Centromere scission drives chromosome shuffling and reproductive isolation

A fundamental characteristic of eukaryotic organisms is the generation of genetic variation via sexual reproduction. Conversely, significant large-scale genome structure variations could hamper sexual reproduction, causing reproductive isolation and promoting speciation. The underlying processes behind large-scale genome rearrangements are not well understood and include chromosome translocations involving centromeres. Recent genomic studies in the Cryptococcus species complex revealed that chromosome translocations generated via centromere recombination have reshaped the genomes of different species. In this study, multiple DNA double-strand breaks (DSBs) were generated via the CRISPR/Cas9 system at centromere-specific retrotransposons in the human fungal pathogen Cryptococcus neoformans The resulting DSBs were repaired in a complex manner, leading to the formation of multiple interchromosomal rearrangements and new telomeres, similar to chromothripsis-like events. The newly generated strains harboring chromosome translocations exhibited normal vegetative growth but failed to undergo successful sexual reproduction with the parental wild-type strain. One of these strains failed to produce any spores, while another produced ∼3% viable progeny. The germinated progeny exhibited aneuploidy for multiple chromosomes and showed improved fertility with both parents. All chromosome translocation events were accompanied without any detectable change in gene sequences and thus suggest that chromosomal translocations alone may play an underappreciated role in the onset of reproductive isolation and speciation.

Myeloid-Lineage Enhancers Drive Oncogene Synergy and Represent a Novel Therapeutic Target in CSF3R-CEBPA Mutant AML

Myeloid-Lineage Enhancers Drive Oncogene Synergy and Represent a Novel Therapeutic Target in CSF3R-CEBPA Mutant AML

Biology of t(6;11) Fusion Proteins and Their Role in MLL-Rearranged Acute Leukemia Lineage Determination

Chromosomal translocations are genetic rearrangements where a chromosomal segment is transferred to a non-homologous chromosome which give rise to novel chimeras. Chromosomal rearrangements play a significant role in the development of acute leukemias (acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML)). Chromosomal translocation events occurring at 11q23 involving the KMT2A or Mixed-Lineage Leukemia (MLL) gene (n=102) can be diagnosed in about 5-10% of all acute leukemia patients (Marschalek Ann Lab Med 2016), especially prevalent in infant acute leukemias (up to 70% of cases). Different chromosomal translocation partner genes (such as AF4, AF6, AF9orENL and ELL) account for the majority of leukemia cases and have their genomic breakpoints within a major breakpoint cluster region (BCR intron 9-11; Meyer et. al. Leukemia 2018). Some rearrangements are specifically associated with particular disease phenotype e.g. the majority of ALL patients (~ 90%) are mainly caused by the following gene fusions, MLL-AF4, MLL-AF9, MLL-ENL. We are interested in a rare but yet drastic chromosomal translocation t(6;11)(q27;q23) which fuses KMT2A/MLL to Afadin (AFDN/AF6) gene. This chromosomal rearrangement has a very poor prognosis (survival-rate is ~10%) and is predominantly diagnosed in patients with high-risk AML. In this project, we investigate the molecular consequences of two different MLL-AF6 fusions and their corresponding reciprocal AF6-MLL fusions. MLL-AF6 fusions are mainly occurring within MLL intron 9 to 11 and are associated with an AML disease phenotype, while the same fusion occurring within the minor breakpoints region in MLL intron 21 until exon (ex) 24 are mainly diagnosed with T-ALL (T-cell acute lymphoblastic leukemia) disease phenotype. The molecular mechanism that determines the resulting disease phenotype is yet unknown. Therefore, we cloned all of these t(6;11) fusion proteins in order to investigate the functional consequences of the two different breakpoints (MLLex1-9::AF6ex2-30, AF6ex1::MLLex10-37; MLLex1-21::AF6ex2-30, AF6ex1::MLLex22-37). All 4 fusion genes were introduced into our inducible Sleeping Beauty system (Ivics et. al. Mobile DNA 2010; Kowarz et. al. Biotechnol J. 2015) and stably transfected reporter cell lines. Basically, these 4 fusion proteins differ only in the presence or absence of their Plant homeodomain 1-3/Bromodomain (PHD1-3/BD) domain (see Figure 1). The PHD domain regulates the epigenetic and transcriptional regulatory functions of wildtype MLL. Subsequently, we analyzed gene expression differences by the MACE-Seq (Massive Analyses of cDNA Ends). MACE data revealed fundamental differences in gene expression profiles when analyzing the two different sets of t(6;11) fusion genes. The resulting profiles have similarities to either AML or T-ALL and might give a rational explanation for the different lineages in these t(6;11) patients. Altogether, these results notably indicate that our study will provide a novel insight into this type of high-risk leukemia and subsequently will be useful for developing of novel and appropriate therapeutic strategies against acute leukemia. Disclosures No relevant conflicts of interest to declare.

Translin: A multifunctional protein involved in nucleic acid metabolism

Translin, a highly conserved, DNA/RNA binding protein, is abundantly expressed in brain, testis and in certain malignancies. It was discovered initially in the quest to find proteins that bind to alternating polypurines-polypyrimidines repeats. It has been implicated to have a role in RNA metabolism (tRNA processing, RNAi, RNA transport, etc.), transcription, DNA damage response, etc. Studies from human, mice, drosophila and yeast have revealed that it forms an octameric ring, which is important for its function. Translin is a cytoplasmic protein, but under genotoxic stress, it migrates into the nucleus, binds to the break point hot spots and therefore, thought to be involved in chromosomal translocation events as well as DNA damage related response. Its structure is known and DNA binding regions, GTP binding region and regions responsible for homotypic and heterotypic interaction are known. It forms a ball like structure with open central channel for accommodating the substrate nucleic acids. Besides this, translin protein binds to 3' and 5' UTR of certain mRNAs and probably regulates their availability for translation. It is also involved in mRNA transport and cell cycle progression. It forms a heteromeric complex with translin associated factor-X (TRAX) to form C3PO complex which is involved in RNA silencing process. Recently, it has been shown that translin is upregulated under starvation conditions in Drosophila and is involved in the integration of sleep and metabolic rate of the flies. Earlier studies classified translin as a DNA repair protein; however subsequent studies showed that it is a multifunctional protein. With this background, in this review we have summarized the translin biochemical activities, cellular function as well as structural properties of this important protein.