Mutation Frequency In Cells Research Articles

Unraveling Pathophysiology and Hematopoiesis of Vexas Syndrome By Multi-Omics Analyses and Targeted Gene Editing

Background and Rationale VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a newly described, treatment-refractory, severe, highly prevalent disease (~1:4,000 males aged >50 years) caused by somatic mutations in the UBA1 (ubiquitin-activating enzyme 1) gene of hematopoietic stem/progenitor cells (HSPCs). VEXAS is hallmarked by vacuoles in myeloid/erythroid precursors and presents with systemic inflammation. About 50% patients develop hematologic malignancies, including myelodysplasia, contributing to the poor prognosis. The pathophysiology and impact of VEXAS syndrome on human hematopoiesis remain elusive. Moreover, the lack of suitable disease models hampers investigation of disease development and preclinical drug testing. To fill these gaps, here we: i) integrated multiparametric flow cytometry and multi-omics analyses on VEXAS patients to achieve comprehensive molecular and phenotypic characterization of hematopoiesis; and ii) successfully developed in vitro and in vivo models of VEXAS syndrome by cutting-edge targeted base editing strategies. Methods Multiparametric immunophenotypic analyses and single-cell transcriptomics were performed on peripheral blood and bone marrow (BM) cells from six VEXAS patients (p.Met41>Thr; p.Met41>Val; p.Met41>Leu; c.118-1 G>C) recruited by our Unit. Circulating monocytes were analyzed by whole RNA-sequencing (RNA-seq) and metabolomics. UBA1 mutations were introduced by gene editing technologies in myeloid cell lines, primary T cells and healthy human HSPCs. Engineered HSPCs were used to reconstitute the hematopoietic compartment of immunodeficient mice to generate a humanized VEXAS model. Variant allele frequency of UBA1 mutant cells was quantified by targeted sequencing in isolated hematopoietic lineages and HSPCs. Results i) Multiparametric immunophenotypic analyses of BM cells showed unbalanced composition of the HSPC compartment in VEXAS patients compared to age-matched healthy individuals, with lower abundance of stem cells, multipotent and lymphoid progenitors and expanded myeloid progenitors. This was paralleled by a significant increase of circulating myeloid-biased HSPCs and immature myeloid cells. Transcriptomic analyses of patient-derived peripheral monocytes showed sustained VEXAS-associated inflammatory signatures and heightened glycolysis, which were confirmed by metabolomic profiling. Single-cell transcriptomics on the BM hematopoietic compartment of VEXAS patients harboring different UBA1 mutations displayed genotype-dependent gene expression patterns. Single-cell RNA-seq of peripheral blood mononuclear cells from VEXAS patients is ongoing. ii) In vitro and in vivo models of VEXAS, generated by UBA1 gene editing, faithfully recapitulated patient hematopoiesis and pathophysiology . Editing efficiency approached 90% in wild-type myeloid cell lines, primary human T cells and HSPCs. While UBA1-mutant T cells were counterselected over time in culture, myeloid ones were enriched. Colony forming unit assays of UBA1-edited HSPCs revealed an exclusive myeloid output in vitro and a significant loss of the erythroid HSPC differentiation potential compared to mock-edited controls. Transplantation of edited HSPCs in immunodeficient mice resulted in a 100-fold reduction in circulating B cells, while NK and myeloid compartments were preserved. Human BM HSPCs were 5-fold lower than in control mice, largely myeloid-biased, and displayed abnormal vacuolar morphology. Concordantly, while myeloid cells and HSPCs were mainly (>80%) UBA1 mutated, the lymphoid compartment showed predominance of wild-type alleles. Strikingly, the distribution of UBA1-mutant cells across lineages in humanized mice mirrored the composition of our VEXAS patient cohort, where UBA1-mutant cells accounted for 90% of total myeloid cells and HSPCs and for less than 5% of lymphoid lineages. Conclusions Our findingsprovide evidence thatmutations in UBA1 drive accumulation of myeloid-biased HSPCs and immature myeloid precursors. Mutant lymphoid cells are negatively selected and their peripheral myeloid counterpart displays inflammatory activation. Gene editing-based in vitro and in vivo models phenocopy most hematopoietic features of the disease and hold promise to enable preclinical testing and validation of novel therapeutics to treat VEXAS syndrome.

Characterization of POLE c.1373A > T p.(Tyr458Phe), causing high cancer risk

The cancer syndrome polymerase proofreading-associated polyposis results from germline mutations in the POLE and POLD1 genes. Mutations in the exonuclease domain of these genes are associated with hyper- and ultra-mutated tumors with a predominance of base substitutions resulting from faulty proofreading during DNA replication. When a new variant is identified by gene testing of POLE and POLD1, it is important to verify whether the variant is associated with PPAP or not, to guide genetic counseling of mutation carriers. In 2015, we reported the likely pathogenic (class 4) germline POLE c.1373A > T p.(Tyr458Phe) variant and we have now characterized this variant to verify that it is a class 5 pathogenic variant. For this purpose, we investigated (1) mutator phenotype in tumors from two carriers, (2) mutation frequency in cell-based mutagenesis assays, and (3) structural consequences based on protein modeling. Whole-exome sequencing of two tumors identified an ultra-mutator phenotype with a predominance of base substitutions, the majority of which are C > T. A SupF mutagenesis assay revealed increased mutation frequency in cells overexpressing the variant of interest as well as in isogenic cells encoding the variant. Moreover, exonuclease repair yeast-based assay supported defect in proofreading activity. Lastly, we present a homology model of human POLE to demonstrate structural consequences leading to pathogenic impact of the p.(Tyr458Phe) mutation. The three lines of evidence, taken together with updated co-segregation and previously published data, allow the germline variant POLE c.1373A > T p.(Tyr458Phe) to be reclassified as a class 5 variant. That means the variant is associated with PPAP.

Identification of Ultrasound-Sensitive Prognostic Markers of LAML and Construction of Prognostic Risk Model Based on WGCNA.

Acute myeloid leukemia (LAML) is the most widely known acute leukemia in adults. Chemotherapy is the main treatment method, but eventually many individuals who have achieved remission relapse, the disease will ultimately transform into refractory leukemia. Therefore, for the improvement of the clinical outcome of patients, it is crucial to identify novel prognostic markers. The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases were utilized to retrieve RNA-Seq information and clinical follow-up details for patients with acute myeloid leukemia, respectively, whereas samples that received or did not receive ultrasound treatment were analyzed using differential expression analysis. For consistent clustering analysis, the ConsensusClusterPlus package was utilized, while by utilizing weighted correlation network analysis (WGCNA), important modules were found and the generation of the coexpression network of hub gene was generated using Cytoscape. CIBERSORT, ESTIMATE, and xCell algorithms of the "IOBR" R package were employed for the calculation of the relative quantity of immune infiltrating cells, whereas the mutation frequency of cells was estimated by means of the "maftools" R package. The pathway enrichment score was calculated using the single sample Gene Set Enrichment Analysis (ssGSEA) algorithm of the "Gene Set Variation Analysis (GSVA)" R package. The IC50 value of the drug was predicted by utilizing the "pRRophetic." The indications linked with prognosis were selected by means of the least absolute shrinkage and selection operator (Lasso) Cox analysis. Two categories of samples were created as follows: Cluster 1 and Cluster 2 depending on the differential gene consistent clustering of ultrasound treatment. The prognosis of patients in Cluster 2 was better than that in Cluster 1, and a considerable variation was observed in the immune microenvironment of Cluster 1 and Cluster 2. Lasso analysis finally obtained an 8-gene risk model (GASK1A, LPO, LTK, PRRT4, UGT3A2, BLOCK1S1, G6PD, and UNC93B1). The model acted as an independent risk factor for the patients' prognosis, and it showed good robustness in different datasets. Considerable variations were observed in the abundance of immune cell infiltration, genome mutation, pathway enrichment score, and chemotherapeutic drug resistance between the low and high-risk groups in accordance with the risk score (RS). Additionally, model-based RSs in the immunotherapy cohort were significantly different between complete remission (CR) and other response groups. The prognosis of people with LAML can be predicted using the 8-gene signature.

Using a Humanized Animal Model to Determine the Minimum JAK2V617F Mutant Allele Burden Required to Generate MPN Pathologies

Myeloproliferative neoplasms (MPNs) are clonal hematological diseases driven by somatic mutations acquired in hematopoietic stem cells (HSCs) and characterized by the deviant, unregulated proliferation of one or more myeloid lineages. Chronic MPN can present as polycythemia vera (PV; excess erythrocytes), essential thrombocythemia (ET; excess platelets), or myelofibrosis (MF; bone marrow fibrosis). More than 300,000 patients are currently living with an MPN diagnosis in the United States. The JAK2V617F variant is an acquired somatic mutation recurrently found in MPN patients and is used as a diagnostic marker along with increased hematocrit, increased hemoglobin, and adverse thrombotic events. In MPN patients, the frequency of JAK2V617F mutant cells varies widely but is known to induce an MPN phenotype with both high (>50%) and very low (<5%) variant allelic frequency (VAF). The reason for this is currently unknown. Most JAK2-mutant animal models model a VAF which is much higher than the lower end of detectable VAFs found in MPN patients. This results in a very limited understanding of what the minimal mutant cell burden required for disease manifestation is. The understanding of genotype/phenotype relationships in patients with low JAK2V617F VAF chronic MPN is paramount to our understanding of this disease and its hematological progression. To this end, we have designed a clinically relevant, humanized mouse model wherein we employ CRISPR and homology directed repair (HDR) via a single stranded oligo donor nucleotide sequence (ssODN) to knock-in the JAK2V617Fmutation at the endogenous locus of human cells at low VAF. In addition, a synonymous JAK2V617V mutation was introduced via CRISPR/ssODN in parallel cells to serve as a control tracked by VAF alongside the VAF of JAK2V617F in their corresponding cohorts. The respective ssODNs were nucleofected into either umbilical cord blood (UCB) derived CD34+ cells or healthy adult bone marrow (BM) derived CD34+ cells to investigate the possibility that developmental age is a variable in low JAK2V617F VAF inducing a clinical MPN phenotype. After nucleofection, 20,000 CD34+ cells were intra-tibially transplanted into sub-lethally irradiated (250 rads) NSGS mice. The low cell input was utilized to limit engraftment and initial disease burden of mutant cells. Effect was measured by mutant allele burden by droplet-digital PCR (ddPCR) analysis, flow cytometry at selected timepoints to track human cell engraftment, spleen weight, and complete blood count (CBC) analysis including hematocrit, hemoglobin, white blood cells, and platelets. At 4 months post-transplant, peripheral blood hCD45+ engraftment across all cohorts averaged less than 3%. Average VAF of circulating hCD45+ cells increased in both JAK2V617F cohorts 5 months post-transplant compared to day 0 post nucleofection. Notably, overall JAK2V617F VAF in each sample remained <5% when adjusted to include both human and mouse derived peripheral blood cells. Remarkably, both hematocrit (p= .037 in UCB cohorts and p=.0096 in BM cohorts; Figure 1A) and hemoglobin (p=.046 in UCB cohorts and p=.0026 in BM cohorts; Figure 1B) were significantly elevated in both JAK2V617F cohorts compared to their respective JAK2V617V controls, while white blood cell and platelet counts remained unchanged within normal ranges. In addition, spleen weights were significantly increased in both JAK2V617F cohorts compared to their respective controls. These data demonstrate a successful clinical model of MPN which induces a classical MPN phenotype where a low VAF of JAK2V617F corresponds with a significant increase of hematocrit, hemoglobin, and spleen weight in a humanized mouse model compared to relevant controls. In addition, we demonstrate that while engraftment due to low hCD34+ cell input remains low after 4 months, the VAF of JAK2V617F within the hCD45+ sub- population increases. This successfully enables the overall VAF contributing to the MPN phenotype to remain at clinically relevant lower levels compared to other models with much higher VAF contributions. We believe this model to be of high clinical relevance into the investigation of how low JAK2V617F burden relates to disease development and progression in MPN patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-Cell Multi-Omics Defines the Cell-Type Specific Impact of SF3B1 Splicing Factor Mutations on Hematopoietic Differentiation in Human Clonal Hematopoiesis and Myelodysplastic Syndromes

Splicing factor mutations are recurrent genetic alterations in blood disorders, highlighting the importance of alternative splicing regulation in hematopoiesis. Specifically, mutations in splicing factor 3B subunit 1 (SF3B1) are implicated in the pathogenesis of myelodysplastic syndromes (MDS) and linked to a high-risk of leukemic transformation in clonal hematopoiesis (CH). SF3B1 mutations are associated with aberrant RNA splicing, leading to increased cryptic 3' splice site (ss) usage and MDS with ring sideroblasts phenotype.The study of mutant SF3B1-driven splicing aberrations in humans has been hampered by the inability to distinguish mutant and wildtype single cells in patient samples and the inadequate coverage of short-read sequencing over splice junctions. To overcome these limitations, we developed GoT-Splice by integrating Genotyping of Transcriptomes (GoT; Nam et al. 2019) with Nanopore long-read single-cell transcriptome profiling and CITE-seq (Fig. A). This allowed for the simultaneous single-cell profiling of protein and gene expression, somatic mutation status, and alternative splicing. Our method selectively enriched full-length sequencing reads with the accurate structure, enabling the capture of higher number of junctions per cell and greater coverage uniformity vs. short-read sequencing (10x Genomics; Fig. B, C).We applied GoT-Splice to CD34+ bone marrow progenitor cells from MDS (n = 15,436 cells across 3 patients; VAF: [0.38-0.4]) to study how SF3B1 mutations corrupt human hematopoiesis (Fig. D). High-resolution mapping of SF3B1 mut vs. SF3B1 wt hematopoietic progenitors revealed an increasing fitness advantage of SF3B1 mut cells towards the megakaryocytic-erythroid lineage, resulting in an expansion of SF3B1 muterythroid progenitor (EP) cells (Fig. E, F). Accordingly, SF3B1 mutEP cells displayed higher protein expression of erythroid lineage markers, CD71 and CD36, vs. SF3B1 wt cells (Fig. G). In these SF3B1 mutEP cells, we identified up-regulation of genes involved in regulation of cell cycle and checkpoint controls (e.g., CCNE1, TP53), and mRNA translation (eIFs gene family; Fig. H).Next, while SF3B1 mut cells showed the expected increase of cryptic 3' splicing vs. SF3B1 wt cells (Fig. I), they exhibited distinct cryptic 3' ss usage as a function of hematopoietic progenitor cell identity, displaying stage-specific aberrant splicing during erythroid maturation (Fig. J). In less differentiated EP cells, we observed mis-splicing of genes involved in iron homeostasis, such as the hypoxia-inducible factor HIF1A, and key regulators of erythroid cell growth, such as SEPT2. At later stages, we observed mis-splicing of BAX, a pro-apoptotic member of the Bcl-2 gene family and transcriptional target of p53, and erythroid-specific genes (e.g., PPOX). We further predicted 54% of the aberrantly spliced mRNAs to introduce premature stop codons, promoting RNA degradation through nonsense-mediated decay (NMD). In line with this notion, we observed a significant decrease in expression of NMD-inducing genes in SF3B1 mut vs . SF3B1 wt EP cells (Fig. K).Lastly, splicing factor mutations observed in CH subjects provide an opportunity to interrogate the downstream impact of SF3B1 mutations prior to development of disease. Like MDS, by applying GoT-splice to CD34+ progenitor cells from SF3B1 mut CH subjects (n = 9,007 cells across 2 subjects; VAF: [0.15-0.22]; Fig. L), we revealed increased mutant cell frequency in EP cells (Fig. M) with concomitant increased expression of genes involved in mRNA translation (Fig. N), consistent with SF3B1 mutation causing mis-splicing injury to translational machinery and ineffective erythropoiesis. Notably, CH patients already exhibited cell-type specific cryptic 3' ss usage in SF3B1 mut cells (Fig. O).In summary, we developed a novel multi-omics single-cell toolkit to examine the impact of splicing factor mutations on cellular fitness directly in human samples. With this approach, we showed that, while SF3B1 mutations arise in uncommitted HSCs, their effect on fitness increases with differentiation into committed EPs, in line with the mutant SF3B1-driven dyserythropoiesis phenotype. We revealed that SF3B1 mutations exert cell-type specific mis-splicing that leads to abnormal erythropoiesis. Finally, we demonstrated that the impact of SF3B1 mutations on EP cells begins before disease onset, as observed in CH subjects. [Display omitted] DisclosuresDai: Oxford Nanopore Technologies: Current Employment. Beaulaurier: Oxford Nanopore Technologies: Current Employment. Drong: Oxford Nanopore Technologies: Current Employment. Hickey: Oxford Nanopore Technologies: Current Employment. Juul: Oxford Nanopore Technologies: Current Employment. Wiseman: Astex: Research Funding; Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Takeda: Consultancy; StemLine: Consultancy. Harrington: Oxford Nanopore Technologies: Current Employment. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy. Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Foundation Medicine Inc: Consultancy; Merck: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Standard protocol for the PIGRET assay, a high-throughput reticulocyte Pig-a assay with an immunomagnetic separation, used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society

The PIGRET assay is one of the Pig-a assays targeting reticulocytes (RETs), an in vivo genotoxicity evaluation method using flow cytometry with endogenous reporter glycosylphosphatidylinositol anchor protein. The PIGRET assay with RETs selectively enriched with anti-CD71 antibodies has several desirable features: high-throughput assay system, low background frequency of mutant cells, and early detection of mutation. To verify the potential and usefulness of the PIGRET assay for short-term testing, an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society was conducted. The collaborating laboratories assessed the mutagenicities of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the Pig-a assay on the total red blood cell assay and the PIGRET assay. Here the standard protocol for the PIGRET assay was described in detail.

Dietary and lifestyle factors effect erythrocyte PIG-A mutant frequency in humans.

It is well understood that poor diet and lifestyle choices can increase the risk of cancer. It is also well documented that cancer is a disease of DNA mutations, with mutations in key genes driving carcinogenesis. Measuring these mutations in a minimally invasive way may be informative as to which exposures are harmful and thus allow us to introduce primary preventative measures, in a bid to reduce cancer incidences. Here, we have measured mutations in the phosphatidylinositol glycan class A (PIG-A) gene in erythrocytes from healthy volunteers (n = 156) and from non-cancer patients attending the local endoscopy department (n = 144). The X-linked PIG-A gene encodes an enzyme involved in glycosylphosphatidylinositol (GPI) anchor synthesis. A silencing mutation in which leads to the absence of GPI anchors on the extracellular surface which can be rapidly assessed using flow cytometry. The background level of PIG-A mutant erythrocytes was 2.95 (95% CI: 2.59-3.67) mutant cells (10-6). Older age increased mutant cell frequency (P < 0.001). There was no difference in mutant cell levels between males and females (P = 0.463) or smokers and non-smokers (P = 0.186). In the endoscopy group, aspirin users had lower mutant frequencies (P = 0.001). Further information on diet and exercise was available for the endoscopy patient group alone, where those with a higher health promotion index score had lower mutant frequencies (P = 0.011). Higher dietary intake of vegetables reduced mutant cell levels (P = 0.022). Participants who exercised for at least 1 h a week appeared to have reduced mutant frequencies than those who did not exercise, although this was not statistically significant (P = 0.099). This low background level of mutant erythrocytes in a population makes this assay an attractive tool to monitor exposures such as those associated with lifestyles and diet, as demonstrated here.

DOES THE USE OF SMARTPHONES CAUSE GENOTOXIC AND CYTOTOXIC EFFECTS TO ORAL EPITHELIUM? A SYSTEMATIC REVIEW WITH META-ANALYSIS

The use of the mobile device is based on radiofrequency waves, an electromagnetic wave type. To verify if cellular damage occurs, the micronucleus test was developed, aiming to identify an increase in the frequency of mutations in cells that are exposed to a varied range of agents. Objective: This study aims to answer the following question: Could the nonionizing radiation emitted by mobile telephone devices cause genotoxic and/or cytotoxic effects in the cells present in the oral mucosa, from microscopic observation by the micronucleus test? Study Design: Three stages were followed: defining the objective, tracing the search method (PICO), selecting articles, and extracting data. The databases consulted were: MEDLINE, PubMed (AllDatabases, MeSH), Scopus, LILACS, Google academic, PROSPERO, and COCHRANE using the Boolean operators Persons, Smartphone, Mutagenicity Tests, and Mouth, based on MeSH terms. Results: A total of 396 articles were found, 323 abstracts were analyzed, 312 abstracts were excluded, and w6 full texts and 6 studies were included, according to the table of scientific evidence (grade 5). Conclusion: Our study revealed that there is no occurrence of genotoxic effects; nonetheless, there is a suggestion of occurrence of cytotoxic effects caused by exposure to electromagnetic fields of radiofrequency emitted by mobile phones, especially in case of prolonged exposure.

Hmga2 Functions As an Oncogene upon the Deletion of Tet2 and Promotes the Pathogenesis of Myelodysplastic Syndrome

High Mobility Group AT-hook 2 (HMGA2) is a chromatin modifier and its overexpression has been found in a subset of patients with myelodysplastic syndrome (MDS). The high level of HMGA2 expression appears to predict poor prognosis in various tumors; however, it remains unclear how HMGA2 dysregulates expression of target genes to facilitate the transformation. To elucidate the mechanisms by which the overexpression of Hmga2 promotes the development of MDS, we generated an Hmga2-expressing Tet2-deficient (Hmga2-Tet2Δ/Δ) mouse model showing the progressive phenotype of MDS. We found that Hmga2-Tet2Δ/Δ mice had neutropenia and anemia, but variable platelet counts, accompanied by elevated frequencies of mutant cells in myeloid cells. Hmga2-Tet2Δ/Δ mice showed a similar median survival to Tet2Δ/Δ mice (274 days vs 290 days), but shorter survival than Hmga2-Tet2wt/wt mice (274 days vs undetermined). Moribund Hmga2-Tet2Δ/Δ mice showed progressive leukopenia and anemia, accompanied by the emergence of dysplastic neutrophils, myeloblasts and anisocytosis in the PB and BM and dysplastic megakaryocytes in the BM. Hmga2-Tet2Δ/Δ mice had mildly increased spleen weights, and expanded myeloid cells and HSPCs in the spleen without the deposition of fibrosis. During a 12-month observation, we found that Hmga2-Tet2Δ/Δ mice developed lethal MDS/MPN overlap disease (47%), MDS (33%), MPN (13%), and AML (7%), while 6 out of 11 Tet2Δ/Δ mice developed MPN (55%). Hmga2-Tet2wt/wt mice subsequently showed similar blood counts in PB and died without the expansion of leukemic or dysplastic blood cells. Therefore, Hmga2 overexpression did not transform wild-type HSCs but promoted the development of MDS in the absence of Tet2 in vivo. In order to elucidate the molecular mechanisms underlying the transformation of Hmga2-Tet2Δ/Δ cells, we initially performed gene expression profiling by a RNA sequencing analysis in LSK HSPCs isolated from WT, Hmga2-Tet2wt/wt, Tet2Δ/Δ, and Hmga2-Tet2Δ/Δ mice at a pre-disease stage and those isolated from two Hmga2-Tet2Δ/Δ MDS/MPN and AML mice. Hmga2-Tet2Δ/Δ leukemic cells were placed closer to one out of two Hmga2-Tet2Δ/Δ cells at the pre-disease stage, but clearly apart from the other genotype cells, indicating that Hmga2 overexpression and Tet2 loss result in the accumulation of alterations in the transcriptional program during the development of MDS.In order to clarify the mechanisms by which the overexpression of Hmga2 alters the transcriptional program in Tet2-deficient cells, we performed the ChIP-sequencing of FLAG-tagged Hmga2 in bone marrow progenitor cells isolated from WT, Hmga2-Tet2wt/wt, and Hmga2-Tet2Δ/Δ mice. The numbers of Hmga2-binding peaks were markedly lower in Tet2-deficient cells than in Hmga2-Tet2wt/wt cells (2227 peaks versus 11500 peaks). Furthermore, annotated genes adjacent to Hmga2-binding sites partially overlapped in both genotype cells, whereas 2965 out of 3843 genes identified in Tet2 wild-type cells lost the binding peaks of Hmga2 upon the deletion of Tet2. Based on the DNA-binding capacity of Hmga2, the loss of Tet2 remodeled the binding sites of Hmga2 via change in DNA methylation in Hmga2-binding flanking regions, which were not observed in the presence of Tet2, leading to significant enrichments in genes involved in cell-to-cell adhesion and cell morphogenesis in Hmga2-Tet2Δ/Δ cells. Furthermore, we found that the overexpression of Hmga2 and loss of Tet2 resulted in the activation of oncogenic pathways (e.g. TGF-b, TNF-a), but suppressed the expression of genes in the unfolded protein response. Notably, the inhibition of bile acid metabolism to reactivate the unfolded protein response markedly attenuated the proliferation of Hmga2-Tet2Δ/Δ cells. These combinatory effects on the transcriptional program and cellular functions were not redundant to those in either single mutant cell, supporting Hmga2 being a proto-oncogene because its overexpression alone was not sufficient to develop MDS in vivo. Thus, Hmga2 overexpression exerts synergistic, but also gain-of-function effects with the loss of Tet2 to target these key biological pathways and promotes the transformation of Tet2-deficient stem cells. This study also provides a new rationale for targeting the unfolded protein response in MDS cells expressing HMGA2. Disclosures No relevant conflicts of interest to declare.

Induction of in vivo Pig-a gene mutation but not micronuclei by 5-(2-chloroethyl)-2'-deoxyuridine, an antiviral pyrimidine nucleoside analogue.

5-(2-Chloroethyl)-2'-deoxyuridine (CEDU) was developed as an antiviral drug. It has been studied in a number of in vitro and in vivo genotoxicity assays and is considered an unusual nucleoside analogue owing to its potent mutagenic potential, with little to no measurable clastogenic activity. Given this atypical profile, CEDU represented an interesting compound for evaluating the in vivo Pig-a gene mutation assay, a test that is undergoing extensive validation for regulatory safety applications. The current report describes two studies with 7-week-old male Wistar Han rats, one that exposed animals to several dose levels of CEDU for 5 consecutive days, the other for 28 consecutive days. Blood samples were collected at several time points and analysed for Pig-a mutant cell frequencies via flow cytometry. These Pig-a analyses were accompanied by micronucleated reticulocyte (MN-RET) measurements performed with blood samples collected 1 day after cessation of treatment. Both studies showed robust CEDU dose-related increases in Pig-a mutant reticulocytes and mutant erythrocytes. Conversely, neither experiment showed evidence of a CEDU-related MN-RET-inducing effect. These rat haematopoietic cell results were in good agreement with those of earlier mouse studies where in vivo mutagenesis was observed, without clastogenicity/aneuploidy. Taken together, these data add further support to the concept that the Pig-a assay represents an important complement to the widely used in vivo micronucleus assay, as it expands the range of important DNA lesions that can be detected in short-term as well as protracted exposure study designs.

Suitability of Long-Term Frozen Rat Blood Samples for the Interrogation of Pig-a Gene Mutation by Flow Cytometry.

The rodent blood Pig-a assay has been undergoing international validation for use as an in vivo hematopoietic cell gene mutation assay, and given the promising results an Organization for Economic Co-operation and Development (OECD) Test Guideline is currently under development. Enthusiasm for the assay stems in part from its alignment with 3Rs principles permitting combination with other genotoxicity endpoint(s) and integration into repeat-dose toxicology studies. One logistical requirement and experimental design limitation has been that blood samples required antibody labeling and flow cytometric analysis within one week of collection. In the current report, we describe the performance of freeze-thaw reagents that enable storage and subsequent labeling and analysis of rat blood samples for at least seven months. Data generated from three laboratories are presented that demonstrate rat erythrocyte recoveries in the range of 80-90%. Despite some loss of erythrocytes, Pearson coefficients and Bland-Altman analyses based on fresh blood vs. frozen/thawed matched pairs indicate that mutant cell and reticulocyte frequencies are not significantly affected, as the measurements are highly correlated and exhibit low bias. Collectively, these data support the effectiveness and suitability of a freeze-thaw procedure that endows the assay with several new advantageous characteristics that include: flexibility in scheduling personnel/instrumentation; reliability when shipping samples from in-life facilities to analytical sites; 3Rs-friendly, as blood from positive control animals can be stored frozen to serve as analytical controls; and ability to defer a decision to generate Pig-a data until more toxicological information becomes available on a test substance. Environ. Mol. Mutagen. 60:47-55, 2019. © 2018 Wiley Periodicals, Inc.

Induction of in vivo Pig-a gene mutation but not micronuclei by 5-(2-chloroethyl)-2'-deoxyuridine, an antiviral pyrimidine nucleoside analogue.

5-(2-Chloroethyl)-2'-deoxyuridine (CEDU) was developed as an antiviral drug. It has been studied in a number of in vitro and in vivo genotoxicity assays and is considered an unusual nucleoside analogue owing to its potent mutagenic potential, with little to no measurable clastogenic activity. Given this atypical profile, CEDU represented an interesting compound for evaluating the in vivo Pig-a gene mutation assay, a test that is undergoing extensive validation for regulatory safety applications. The current report describes two studies with 7-week-old male Wistar Han rats, one that exposed animals to several dose levels of CEDU for 5 consecutive days, the other for 28 consecutive days. Blood samples were collected at several time points and analysed for Pig-a mutant cell frequencies via flow cytometry. These Pig-a analyses were accompanied by micronucleated reticulocyte (MN-RET) measurements performed with blood samples collected 1 day after cessation of treatment. Both studies showed robust CEDU dose-related increases in Pig-a mutant reticulocytes and mutant erythrocytes. Conversely, neither experiment showed evidence of a CEDU-related MN-RET-inducing effect. These rat haematopoietic cell results were in good agreement with those of earlier mouse studies where in vivo mutagenesis was observed, without clastogenicity/aneuploidy. Taken together, these data add further support to the concept that the Pig-a assay represents an important complement to the widely used in vivo micronucleus assay, as it expands the range of important DNA lesions that can be detected in short-term as well as protracted exposure study designs.

Recombinogenic Effect of the Ternary Complex of Copper(II) with Doxycycline and 1,10-Phenanthroline on Somatic Cells of Drosophila melanogaster

In the search for new anti-tumor agents, exploiting features such as the flexibility of coordination modes of metals have become an alternative strategy for synthesizing pharmaceuticals. It has been shown that the CuDP (copper(II), doxycycline, and 1,10-phenanthroline) complex cleaves DNA strands by an oxidative mechanism and by intercalating the major groove, resulting in a cytotoxic action. The objective of this study was to assess the mutagenic/recombinogenic effects of the CuDP complex in vivo using the SMART (Somatic Mutation and Recombination Test) in Drosophila melanogaster. Treatments were carried out with third instar larvae at the standard cross and high bioactivation cross using three concentrations of CuDP (6.92, 13.84 or 27.67 mM). The mutagenic doxorubicin (0.4 mM) was used as a positive control and reverse osmosis water as a negative control. For each compound, marked trans-heterozygous and balanced heterozygous individuals were analyzed to determine the mutational and recombinogenic events occurring in the cells. We found that CuDP significantly increased the frequencies of mutant cells in both standard and high bioactivation crosses, mostly by induction of recombination. These data show that CuDP is a direct recombinogenic agent that is independent of bioactivation.

Integration of liver and blood micronucleus and Pig-a gene mutation endpoints into rat 28-day repeat-treatment studies: Proof-of-principle with diethylnitrosamine

Regulatory guidance documents stress the value of assessing multiple tissues and the most appropriate endpoints when evaluating chemicals for in vivo genotoxic potential. However, conducting several independent studies to consider multiple endpoints and/or tissue compartments is resource intensive. Furthermore, conventional approaches for scoring genotoxicity endpoints are slow, tedious, and less objective than what would be considered ideal. In an effort to address these issues with current practices, we attempted to i) employ flow cytometry-based methods to score liver micronuclei, blood micronuclei, and blood Pig-a gene mutation, and ii) integrate the endpoints into a common general toxicology study design—the rat 28-day repeat dose study. A proof-of-principle experiment was performed with 6-week old male Crl:CD(SD) rats exposed to diethylnitrosamine (DEN) for 28 consecutive days. One day later blood was collected for micronucleated reticulocyte (MN-RET) and Pig-a mutation assays, and liver tissue was obtained for micronucleated hepatocyte (MNHEP) scoring. MN-RET frequencies were not affected by DEN exposure, and mean Pig-a mutant cell frequencies were only slightly elevated. On the other hand, % MNHEP showed marked, dose-related increases (2.2, 7.2, and 9.1 mean fold-increase for 5, 10, 15 mg DEN/kg/day, respectively). Concurrent with MNHEP analyses, assessments of Ki-67-positive events and the proportion of 8n nuclei provided evidence for treatment-related changes to hepatocyte proliferation. Collectively, these results reinforce the importance of evaluating chemicals’ genotoxic potential in liver in addition to hematopoietic cells, and suggest that several automated measurements can be successfully integrated into repeat-dose studies for higher efficiencies and better utilization of fewer animals.

Somatic PIK3CA Mutations are Present in Multiple Tissues of Facial Infiltrating Lipomatosis

BackgroundFacial infiltrating lipomatosis (FIL) is a congenital disorder that causes overgrowth of one side of the face. The purpose of this study was to determine whether PIK3CA mutations are present in tissues outside of the subcutaneous adipose.MethodsFIL tissues from three patients were dissected to enrich for cells from skin, subcutaneous tissue, orbicularis oris muscle, buccal fat, zygomatic bone, and mucosal neuroma. Endothelial cells within the affected tissue also were enriched using CD31 microbeads. Laser capture microdissection on formalin-fixed paraffin-embedded histologic sections was performed to collect specific cell types. DNA was extracted from each tissue and cell type, and measured for the abundance of mutant PIK3CA alleles using droplet digital PCR.ResultsWe detected mutant PIK3CA alleles in every tissue and cell type tested from each overgrown face; frequencies ranged from 1.5 to 53%. There were fewer mutant endothelial cells compared with nonendothelial cells, and the stromal cell compartment had the highest frequency of mutant cells in each tissue.ConclusionsPIK3CA mutations are not restricted to a single tissue or cell type in FIL. Overgrowth in this condition is likely due to the mutation arising in a cell that contributes to several different facial structures during embryogenesis.

Modeling Therapy Resistance in BRCA1/2-Mutant Cancers.

Although PARP inhibitors target BRCA1- or BRCA2-mutant tumor cells, drug resistance is a problem. PARP inhibitor resistance is sometimes associated with the presence of secondary or "revertant" mutations in BRCA1 or BRCA2 Whether secondary mutant tumor cells are selected for in a Darwinian fashion by treatment is unclear. Furthermore, how PARP inhibitor resistance might be therapeutically targeted is also poorly understood. Using CRISPR mutagenesis, we generated isogenic tumor cell models with secondary BRCA1 or BRCA2 mutations. Using these in heterogeneous in vitro culture or in vivo xenograft experiments in which the clonal composition of tumor cell populations in response to therapy was monitored, we established that PARP inhibitor or platinum salt exposure selects for secondary mutant clones in a Darwinian fashion, with the periodicity of PARP inhibitor administration and the pretreatment frequency of secondary mutant tumor cells influencing the eventual clonal composition of the tumor cell population. In xenograft studies, the presence of secondary mutant cells in tumors impaired the therapeutic effect of a clinical PARP inhibitor. However, we found that both PARP inhibitor-sensitive and PARP inhibitor-resistant BRCA2 mutant tumor cells were sensitive to AZD-1775, a WEE1 kinase inhibitor. In mice carrying heterogeneous tumors, AZD-1775 delivered a greater therapeutic benefit than olaparib treatment. This suggests that despite the restoration of some BRCA1 or BRCA2 gene function in "revertant" tumor cells, vulnerabilities still exist that could be therapeutically exploited. Mol Cancer Ther; 16(9); 2022-34. ©2017 AACR.

In vivo pig-a and micronucleus study of the prototypical aneugen vinblastine sulfate.

The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days -4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen. 59:30-37, 2018. © 2017 Wiley Periodicals, Inc.

Alternative Splicing of APOBEC3D Generates Functional Diversity and Its Role As a DNA Mutator

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) protein family consists of 11 members: APOBEC1, APOBEC2, seven APOBEC3s (A3s) (A/B/C/D/F/G/H), APOBEC4, and the activation-induced deaminase (AID). APOBEC1, A3s, and AID have cytidine deaminase activity and induce a cytidine (C) to thymidine (T) transition. The main function of A3s is to trigger an innate immune response to viral infection such as human immunodeficiency virus-1. Recently, it was reported that several APOBEC family proteins can induce somatic mutations into genomic DNA and thus promote cancer development. For example, AID-mediated somatic mutations contribute to B-cell lymphoma and A3B can be an enzymatic source in solid tumors such as breast cancer. However, little is known about other A3 member proteins.To determine the expression of A3 in hematopoietic cells, we performed quantitative PCR in leukemia cell lines. A3B, A3C, A3D, A3F and A3G were detected in all cell lines, but not A3A or A3H. Next, we analyzed published The Cancer Genome Atlas data from patients with acute myeloid leukemia to find somatic alterations in A3 genes using cBioPortal (http://www.cbioportal.org/). We found that each A3 was upregulated at 4-7 % of all samples and the total frequency of 23 %. Interestingly, overall survival of patients with A3upregulation was lower than those without upregulation, suggesting an important role of A3s in pathogenesis of leukemia. As it has been shown that A3A, A3B, and A3D show capacity to inflict DNA damage, we decided to investigate whether A3D can induce mutations in foreign and genomic DNA.During our attempt to generate A3D expression constructs, we detected multiple bands in leukemia and lung cancer cell lines. Based on our analysis and previous reports, we confirmed that there are at least 7 transcript variants (v1 to 7). Because A3D v3, v4 and v5 lack cytidine deaminase domains, we decided to examine A3Dv1, v2, v6 and v7.To determine whether A3D variants have foreign DNA editing activity, we performed differential DNA denaturation (3D)-PCR in HEK293T cells co-transfected with expression vectors for EGFP, UNG inhibitor, and pCAG-GS containing A3Dv1, v2, v6, v7, A3B or empty control. Total DNA was isolated 4 days after transfection. PCR products were detected at lower denaturation temperature (Td) in cells expressing A3B and all A3D variants vectors compared to control. Sequences of PCR products at lowest Td revealed that mutation frequencies in EGFP gene were 6.1-10.5 per 103 bps in cells transfected with all A3D variants and 18.7 per 103bps in cells transfected with A3B. Of note, 90 % of mutations were C/G to T/A transition in cells transfected with A3B, but in cells transfected with A3D these were 30-60 %.Next we performed 3D-PCR in HEK293T cells retrovirally infected with MigR1-IRES-EGFP containing A3Dv1, v2, v6, v7, A3B or empty to determine genomic DNA editing activity. Total DNA was isolated 4 weeks after infection. At lower Td, PCR products were detected only in cells expressing A3Dv1 and A3B. PCR products in cells expressing A3Dv2, v6, v7 were detected only at same Td as control. Sequences of the PCR products at lowest Td revealed that the mutation frequency in the EGFP gene was 22.2 per 103 bps in cells expressing A3Dv1, and 56.9 per 103 bps in cells expressing A3B. However mutation frequencies in cells expressing A3Dv6 or v7 were lower than 2 per 103bps and no mutations were detected in cells expressing A3Dv2. All of these mutations were C/G to T/A transition. Interestingly, we found 4 clones with deletion in cells expressing A3Dv1, v6 and A3B. In addition, all clones had several bases of micro homologous sequences in broken ends before joining. These results suggested that double strand breaks in genomic DNA were repaired by microhomology-mediated end joining.These DNA editing assays showed that all A3D variants had ability to induce mutations in foreign DNA, and A3Dv1 was major isoform which had ability to induce mutations in genomic DNA.Taken together, our experiments showed that A3D can be a DNA mutator and alternative splicing generates functional diversity of A3D. These findings suggest that A3D may contribute to development of hematopoietic malignancies. DisclosuresTakaori-Kondo:Mochida Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Eisai: Research Funding; Takeda Pharmaceutical: Research Funding; Chugai Pharmaceutical: Research Funding; Alexion Pharmaceuticals: Research Funding; Kyowa Kirin: Research Funding; Pfizer: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Shionogi: Research Funding; Toyama Chemical: Research Funding; Cognano: Research Funding.

Endothelial Cells from Capillary Malformations Are Enriched for Somatic GNAQ Mutations.

A somatic mutation in GNAQ (c.548G>A; p.R183Q), encoding Gαq, has been found in syndromic and sporadic capillary malformation tissue. However, the specific cell type containing the mutation is unknown. The purpose of this study was to determine which cells in capillary malformations have the GNAQ mutation. Human capillary malformation tissue was obtained from 13 patients during a clinically indicated procedure. Droplet digital polymerase chain reaction, capable of detecting mutant allelic frequencies as low as 0.1 percent, was used to quantify the abundance of GNAQ mutant cells in capillary malformation tissue. Six specimens were fractionated by fluorescence-activated cell sorting into hematopoietic, endothelial, perivascular, and stromal cells. The frequency of GNAQ mutant cells in these populations was quantified by droplet digital polymerase chain reaction. Eight capillary malformations contained GNAQ p.R183Q mutant cells, two lesions had novel GNAQ mutations (p.R183L and p.R183G), and three capillary malformations did not have a detectable GNAQ p.R183 mutation. Mutant allelic frequencies ranged from 2 to 11 percent. Following fluorescence-activated cell sorting, the GNAQ mutation was found in the endothelial but not the platelet-derived growth factor receptor-β-positive cell population; mutant allelic frequencies were 3 to 43 percent. Endothelial cells in capillary malformations are enriched for GNAQ mutations and are likely responsible for the pathophysiology underlying capillary malformation.

Creation of Mice Bearing a Partial Duplication of HPRT Gene Marked with a GFP Gene and Detection of Revertant Cells In Situ as GFP-Positive Somatic Cells.

It is becoming clear that apparently normal somatic cells accumulate mutations. Such accumulations or propagations of mutant cells are thought to be related to certain diseases such as cancer. To better understand the nature of somatic mutations, we developed a mouse model that enables in vivo detection of rare genetically altered cells via GFP positive cells. The mouse model carries a partial duplication of 3’ portion of X-chromosomal HPRT gene and a GFP gene at the end of the last exon. In addition, although HPRT gene expression was thought ubiquitous, the expression level was found insufficient in vivo to make the revertant cells detectable by GFP positivity. To overcome the problem, we replaced the natural HPRT-gene promoter with a CAG promoter. In such animals, termed HPRT-dup-GFP mouse, losing one duplicated segment by crossover between the two sister chromatids or within a single molecule of DNA reactivates gene function, producing hybrid HPRT-GFP proteins which, in turn, cause the revertant cells to be detected as GFP-positive cells in various tissues. Frequencies of green mutant cells were measured using fixed and frozen sections (liver and pancreas), fixed whole mount (small intestine), or by means of flow cytometry (unfixed splenocytes). The results showed that the frequencies varied extensively among individuals as well as among tissues. X-ray exposure (3 Gy) increased the frequency moderately (~2 times) in the liver and small intestine. Further, in two animals out of 278 examined, some solid tissues showed too many GFP-positive cells to score (termed extreme jackpot mutation). Present results illustrated a complex nature of somatic mutations occurring in vivo. While the HPRT-dup-GFP mouse may have a potential for detecting tissue-specific environmental mutagens, large inter-individual variations of mutant cell frequency cause the results unstable and hence have to be reduced. This future challenge will likely involve lowering the background mutation frequency, thus reducing inter-individual variation.