Heterozygous Cells Research Articles

SHP-1 As a Negative Regulator of FLT3 Associated Aberrant Signaling and a New Potential Target to FLT3-ITD Mutation

Background: FLT3-ITD mutation remains a great challenge in treating acute myeloid leukemia (AML) patients, for the high incidence of early relapse and drug-resistant mutations to tyrosine kinase inhibitors (TKIs). The FLT3-ITD mutation results in a significant and sustained activation of downstream signaling pathways. However, the physiological negative regulators of those aberrantly activated pathways have not been fully elucidated. In this study, we aim to identify and exploit a key molecule directly interacting with FLT3 receptor and down-regulating FLT3 associated pathways.Methods: The potential proteins interacting with FLT3 were immunoprecipitated (IP) and screened by mass spectrometry. The binding of phosphatase SHP-1 and FLT3 was verified using co-IP and in situ proximity ligation assay (PLA) in cell lines and primary samples. The phosphatase activity and phosphorylation level of SHP-1 was determined by pNPP phosphatase activity assay and western blot. Biological effects of overexpressed SHP-1 by lentiviral vectors in AML cell-lines were assessed using proliferation, apoptosis and clonal formation assays in vitro and xenografts models in vivo. Plasmids of truncated or mutated structure of SHP-1 were constructed and overexpressed in cells to identify the key domain responsible for binding with FLT3 receptor. An artificial trans-membrane peptide was designed to activate SHP-1 activity and promote apoptosis in AML cells.Results: The researchers performed immunoprecipitations (IP) followed by LC-MS/MS based screen in human leukemia cell line MV4-11 for three times, and found that the phosphatase SHP-1 binds to FLT3. In addition, the interaction between SHP-1 and FLT3-WT or FLT3-ITD was verified by co-IP and in situ proximity ligation assay (PLA) in AML cell lines, primary samples and overexpression model of 293-FT.Partial results are showed in Figure A. Furthermore, in heterozygous cell line molm13 and engineered FLT3-ITD knock-in cell line SKM-1 by crispr/cas9 techonology, combined with PLA, pNPP phosphatase activity assay and western blot, we found that SHP-1 may bind with p-FLT3 and acquired phosphatase activity(Figure B-D). In turn, p-FLT3 itself and some important proteins in the downstream pathway would be dephosphorylated and the signal transmission would be terminated. In FLT3-ITD cells, the phosphorylation level of SHP-1 significantly reduced.By using lentivirus to overexpress or knockdown shp-1 protein in FLT3-WT and FLT3-ITD cells, the biological effects were studied. Experiments on cell apoptosis, proliferation and clonal formation have shown that overexpression of SHP-1 in AML cells may promote apoptosis, inhibit proliferation and clone formation, especially in FLT3-ITD cells. On the contrary, inhibition of shp-1 could promote proliferation and increase the ability of clone formation. Furthermore, DOX-inducible overexpression cell line of SHP-1 was obtained by tet-on virus system and the western blots showed that SHP-1 may inhibit p-ERK and p-STAT5 activity. In vivo experiments of NCG mice showed that, the degree of leukemia infiltration in peripheral blood was decreased in AML transplantation mice induced by DOX to express SHP-1 when compared to control group.Next, according to the analysis of protein structure by computer simulation, we constructed truncated and mutated plasmids and overexpressed in cells, then using PLA to verify the binding , we found that FLT3 interact with N-SH2 domain of SHP-1,exactly the R30 site(Figure E). Therefore, by using specific trans-membrane peptide, the researchers found that it could activate SHP-1 and promote apoptosis in AML cells, especially FLT3-ITD ones, inhibit cell proliferation and the downstream signal pathway of FLT3.Lastly,injection of peptide in subcutaneous tumor model of nude mouse showed that the tumor was significantly smaller than that of the control group(Figure F).Conclusion: We identify a negative regulator, namely phosphatase SHP-1, which directly interacts with FLT3 receptor through the N-SH2 domain and down-regulates FLT3 downstream signaling pathways. Overexpression of SHP-1 promotes apoptosis and inhibits proliferation in cells with FLT3-ITD mutation. Artificial trans-membrane peptides activating SHP-1 can reverse FLT3 associated aberrant signaling, thus providing a new potential strategy to target FLT3-ITD in AML. The possible mechanism is showed in figure G. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Heterozygous huntingtin promotes cadmium neurotoxicity and neurodegeneration in striatal cells via altered metal transport and protein kinase C delta dependent oxidative stress and apoptosis signaling mechanisms

Huntington’s disease (HD) is functionally linked to environmental factors including cigarette use and dyshomeostasis in the levels of metals. Interestingly, one of the most abundant heavy metals in cigarettes is cadmium (Cd), which also accumulates in the striatum and causes neurotoxicity upon exposure. Thus, we hypothesized that heterozygous huntingtin (HTT), responsible for the majority of cases of HD in patients, in combination with Cd exposure would cause neurotoxicity and neurodegeneration via increased intracellular accumulation of Cd and activation of oxidative stress signaling mechanisms in a mouse striatal cell line model of HD. We report that heterozygous HTT striatal cells are significantly more susceptible to Cd-induced cytotoxicity as compared to wild-type HTT cells upon exposure for 48 h.The heterozygous HTT and Cd-induced cytotoxicity led to a NADPH oxidase (NOX) mediated oxidative stress that was attenuated by exogenous antioxidants and a NOX inhibitor, apocynin. Heterozygous HTT coupled with Cd exposure caused increased expression of protein kinase C δ (PKCδ) and other key oxidative stress proteins levels, enhanced the activation of caspase-9 and caspase-3 mediated apoptosis, and blocked the overexpression of extracellular signal-regulated kinase (ERK). We observed significantly greater intracellular accumulation of Cd and reduced expression of divalent metal transporter 1 (DMT1) protein in the heterozygous HTT striatal cells upon Cd exposure. Treatment with zinc, manganese, and iron as well as exogenous antioxidants significantly attenuated the Cd-induced cytotoxicity. Collectively, these results demonstrate that heterozygous HTT exhibits greater neurotoxic properties when coupled with Cd exposure to cause cell death via caspase mediated apoptosis, altered metal transport, and modulation of ERK and PKCδ dependent oxidative signaling mechanisms.

PPM1D Mutations Drive Clonal Hematopoiesis in Response to Cytotoxic Chemotherapy

SummaryClonal hematopoiesis (CH), in which stem cell clones dominate blood production, becomes increasingly common with age and can presage malignancy development. The conditions that promote ascendancy of particular clones are unclear. We found that mutations in PPM1D (protein phosphatase Mn2+/Mg2+-dependent 1D), a DNA damage response regulator that is frequently mutated in CH, were present in one-fifth of patients with therapy-related acute myeloid leukemia or myelodysplastic syndrome and strongly correlated with cisplatin exposure. Cell lines with hyperactive PPM1D mutations expand to outcompete normal cells after exposure to cytotoxic DNA damaging agents including cisplatin, and this effect was predominantly mediated by increased resistance to apoptosis. Moreover, heterozygous mutant Ppm1d hematopoietic cells outcompeted their wild-type counterparts in vivo after exposure to cisplatin and doxorubicin, but not during recovery from bone marrow transplantation. These findings establish the clinical relevance of PPM1D mutations in CH and the importance of studying mutation-treatment interactions.Video

Two RNase H2 Mutants with Differential rNMP Processing Activity Reveal a Threshold of Ribonucleotide Tolerance for Embryonic Development

SUMMARYRNase H2 has two distinct functions: initiation of the ribonucleotide excision repair (RER) pathway by cleaving ribonucleotides (rNMPs) incorporated during DNA replication and processing the RNA portion of an R-loop formed during transcription. An RNase H2 mutant lacking RER activity but supporting R-loop removal revealed that rNMPs in DNA initiate p53-dependent DNA damage response and early embryonic arrest in mouse. However, an RNase H2 AGS-related mutant with residual RER activity develops to birth. Estimations of the number of rNMPs in DNA in these two mutants define a ribonucleotide threshold above which p53 induces apoptosis. Below the threshold, rNMPs in DNA trigger an innate immune response. Compound heterozygous cells, containing both defective enzymes, retain rNMPs above the threshold, indicative of competition for RER substrates between active and inactive enzymes, suggesting that patients with compound heterozygous mutations in RNASEH2 genes may not reflect the properties of recombinantly expressed proteins.

Construction of a GLI3 compound heterozygous knockout human embryonic stem cell line WAe001-A-20 by CRISPR/Cas9 editing

The human GLI3 protein has a dual function as a transcriptional activator or repressor of hedgehog signaling, depending on the proteolytic processing forms of GLI3. In this study, we established a compound heterozygous GLI3 mutant human embryonic stem cell line (WAe001-A-20) through CRISPR/Cas9 editing. The WAe001-A-20 cells carried two deletions on two different alleles of exon 2 of GLI3, respectively, which resulted in a frame shift and early termination in the translation of GLI3. Moreover, WAe001-A-20 maintains a normal karyotype, parental cell morphology, pluripotent phenotype and the ability to differentiate into three germ layers.Resource tableUnlabelled TableUnique stem cell line identifierWAe001-A-20Alternative name(s) of stem cell lineH1-GLI3-KOInstitutionGuangzhou Institutes of Biomedicine and Health, Chinese Academy of SciencesContact information of distributorYin-xiong Li, li_yinxiong@gibh.ac.cnType of cell lineEmbryonic stem cell lineOriginWAe001-AAdditional origin infoAge: blastocyst stageSex: male, 46, XYCell SourceN/AClonalityClonalMethod of reprogrammingN/AGenetic ModificationYESType of ModificationDeletionAssociated diseaseGreig Cephalopolysyndactyly Syndrome, Pallister-Hall Syndrome, Postaxial PolydactylyGene/locusGLI3/7p14.1Method of modificationCRISPR/Cas9Name of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateApril 20, 2018Cell line repository/bankN/AEthical approvalCell line was used according to institutional ethical guidelines of GIBH

Mutation Clearance after Transplantation for Myelodysplastic Syndrome

BackgroundAllogeneic hematopoietic stem-cell transplantation is the only curative treatment for patients with myelodysplastic syndrome (MDS). The molecular predictors of disease progression after transplantation are unclear.MethodsWe sequenced bone marrow and skin samples from 90 adults with MDS who underwent allogeneic hematopoietic stem-cell transplantation after a myeloablative or reduced-intensity conditioning regimen. We detected mutations before transplantation using enhanced exome sequencing, and we evaluated mutation clearance by using error-corrected sequencing to genotype mutations in bone marrow samples obtained 30 days after transplantation. In this exploratory study, we evaluated the association of a mutation detected after transplantation with disease progression and survival.ResultsSequencing identified at least one validated somatic mutation before transplantation in 86 of 90 patients (96%); 32 of these patients (37%) had at least one mutation with a maximum variant allele frequency of at least 0.5% (equivalent to 1 heterozygous mutant cell in 100 cells) 30 days after transplantation. Patients with disease progression had mutations with a higher maximum variant allele frequency at 30 days than those who did not (median maximum variant allele frequency, 0.9% vs. 0%; P<0.001). The presence of at least one mutation with a variant allele frequency of at least 0.5% at day 30 was associated with a higher risk of progression (53.1% vs. 13.0%; conditioning regimen–adjusted hazard ratio, 3.86; 95% confidence interval [CI], 1.96 to 7.62; P<0.001) and a lower 1-year rate of progression-free survival than the absence of such a mutation (31.3% vs. 59.3%; conditioning regimen–adjusted hazard ratio for progression or death, 2.22; 95% CI, 1.32 to 3.73; P=0.005). The rate of progression-free survival was lower among patients who had received a reduced-intensity conditioning regimen and had at least one persistent mutation with a variant allele frequency of at least 0.5% at day 30 than among patients with other combinations of conditioning regimen and mutation status (P≤0.001). Multivariate analysis confirmed that patients who had a mutation with a variant allele frequency of at least 0.5% detected at day 30 had a higher risk of progression (hazard ratio, 4.48; 95% CI, 2.21 to 9.08; P<0.001) and a lower 1-year rate of progression-free survival than those who did not (hazard ratio for progression or death, 2.39; 95% CI, 1.40 to 4.09; P=0.002).ConclusionsThe risk of disease progression was higher among patients with MDS in whom persistent disease–associated mutations were detected in the bone marrow 30 days after transplantation than among those in whom these mutations were not detected. (Funded by the Leukemia and Lymphoma Society and others.)

The hSNM1B/Apollo variant rs11552449 is associated with cellular sensitivity towards mitomycin C and ionizing radiation

The polymorphism rs11552449 (c.181C > T, p.His61Tyr) in the hSNM1B/Apollo gene has been repeatedly shown to be associated with an increased risk for breast cancer. The aim of the current study was to investigate the association between rs11552449 and the degree of cellular sensitivity to mitomycin C (MMC) and ionizing radiation (IR). A total of 69 lymphoblastoid cell lines (LCLs) from generally healthy donors were tested for their sensitivity towards MMC and IR in growth inhibition experiments. LCLs heterozygous for rs11552449 were significantly more sensitive to MMC and IR than homozygous cells with the CC genotype (p < 0.05 and p < 0.01 for MMC and IR, respectively) and in the case of MMC also for the TT genotype (p < 0.01). Interestingly, heterozygous CT cells expressed significantly more full length hSNM1B/Apollo mRNA than cells with the homozygous CC (p < 0.0001) or TT genotypes (p < 0.00001). Thus, the observed higher sensitivity of cell lines heterozygous for rs11552449 towards MMC and IR may be a consequence of differential expression of hSNM1B/Apollo associated with rs11552449, a feature which has not been ascribed to this polymorphism before. Interestingly, relative leukocyte telomere length (rLTL) analyzed in a subset of these cells (N = 62) and in leukocytes of N = 1710 Berlin Aging Study II (BASE-II) participants was not associated with rs11552449. The results suggest that hSNM1B/Apollo is causal for the repeatedly reported association between rs11552449 and breast cancer. These results pave the way for further research regarding the clinical impact of rs11552449, e.g. on the clinical outcome of cancer therapy with DNA interstrand crosslinking agents and IR.

OR19 Integrate crispr/CAS9 for precise protein expression of HLA-B∗38:68Q

The determination of null- or low-expressed HLA alleles is clinically relevant in both hematopoietic stem cell transplantation and solid organ transplantation. Here we investigate a questionable (Q) allele HLA-B∗38:68Q, which carries a 9-nucleotide (nt) deletion at codon 230–232 on exon 4 (Fig. 1A), using CRISPR/Cas9 gene editing to study the effect of this mutation on HLA-B∗38:68Q expression. The CRISPR/Cas9, targeting gRNA and a non-PAM (Protospacer Adjacent Motif) ssDNA oligo were co-transfected to EBV-B cell line TEM665 (Fig. 1B) by Neon (Thermo Fisher Scientific) to generate homozygous deletions at codon 230–232 of HLA-B∗38:01:01. After single cell seeding and expanding, Sanger sequencing and next generation sequencing were performed to validate gene editing in single cell derived clones. Cell surface expression was determined by flow cytometric analysis using a FITC–conjugated Bw4 IgG antibody (One Lambda). The gene editing of HLA-B∗38:01:01 homozygous EBV-B cell line by CRISPR/Cas9 resulted in 25 single cell derived clones. The data showed that 21/25 (84%) clones were successfully edited. Among the edited alleles, 5 alleles were through homology directed repair (HDR) pathway and 36 alleles were through non homologous end joining (NHEJ) pathway (Fig. 1C). We acquired two precise edited clones: one was HLA-B∗38:68Q/B∗38:01:01 heterozygous and the other one was HLA-B∗38:68Q homozygous (Fig. 1D). Based on the flow cytometric analysis (Fig. 1E), the expression of HLA-B∗38:01:01 homozygous was 2.2-fold higher than HLA-B∗38:68Q/B∗38:01:01 heterozygous cells, and the HLA-B∗38:68Q/B∗38:01:01 heterozygous cells was over 2.0-fold higher than HLA-B∗38:68Q homozygous. We demonstrated that HLA-B∗38:68Q is a low-expressed allele using CRISPR/Cas9 gene editing. CRISPR/Cas9 can induce precise gene editing in HLA genes. It provides a useful tool to study the functions of insertion, deletion and intron sequences of HLA genes.Download : Download high-res image (940KB)Download : Download full-size image

Neural progenitors derived from Tuberous Sclerosis Complex patients exhibit attenuated PI3K/AKT signaling and delayed neuronal differentiation

Tuberous Sclerosis Complex (TSC) is a disease caused by autosomal dominant mutations in the TSC1 or TSC2 genes, and is characterized by tumor susceptibility, brain lesions, seizures and behavioral impairments. The TSC1 and TSC2 genes encode proteins forming a complex (TSC), which is a major regulator and suppressor of mammalian target of rapamycin complex 1 (mTORC1), a signaling complex that promotes cell growth and proliferation. TSC1/2 loss of heterozygosity (LOH) and the subsequent complete loss of TSC regulatory activity in null cells causes mTORC1 dysregulation and TSC-associated brain lesions or other tissue tumors. However, it is not clear whether TSC1/2 heterozygous brain cells are abnormal and contribute to TSC neuropathology. To investigate this issue, we generated induced pluripotent stem cells (iPSCs) from TSC patients and unaffected controls, and utilized these to obtain neural progenitor cells (NPCs) and differentiated neurons in vitro. These patient-derived TSC2 heterozygous NPCs were delayed in their ability to differentiate into neurons. Patient-derived progenitor cells also exhibited a modest activation of mTORC1 signaling downstream of TSC, and a marked attenuation of upstream PI3K/AKT signaling. We further show that pharmacologic PI3K or AKT inhibition, but not mTORC1 inhibition, causes a neuronal differentiation delay, mimicking the patient phenotype. Together these data suggest that heterozygous TSC2 mutations disrupt neuronal development, potentially contributing to the disease neuropathology, and that this defect may result from dysregulated PI3K/AKT signaling in neural progenitor cells.

A Regulatory Response to Ribosomal Protein Mutations Controls Translation, Growth, and Cell Competition

Ribosomes perform protein synthesis but are also involved in signaling processes, the full extent of which are still being uncovered. We report that phenotypes of mutating ribosomal proteins (Rps) are largely due to signaling. Using Drosophila, we discovered that a bZip-domain protein, Xrp1, becomes elevated in Rp mutant cells. Xrp1 reduces translation and growth, delays development, is responsible for gene expression changes, and causes the cell competition of Rp heterozygous cells from genetic mosaics. Without Xrp1, even cells homozygously deleted for Rp genes persist and grow. Xrp1 induction in Rp mutant cells depends on a particular Rp with regulatory effects, RpS12, and precedes overall changes in translation. Thus, effects of Rp mutations, even the reductions in translation and growth, depend on signaling through the Xrp1 pathway and are not simply consequences of reduced ribosome production limiting protein synthesis. One benefit of this system may be to eliminate Rp-mutant cells by cell competition.

Abstract 394: Elucidating tissue-specific effects of the 8q24 multicancer risk locus via CRISPR/Cas9 scarless genome editing

Abstract In the past decade, genome-wide association studies have identified a large number of genomic variants, specifically single-nucleotide polymorphisms (SNPs), associated with increased cancer risk. The molecular mechanisms underlying the vast majority of these associations remain largely unknown. Precise cellular models containing the variant of interest could be a crucial tool for establishing disease-relevant function in order to translate these discoveries into clinical application. CRISPR/Cas9 gene editing has provided a convenient and flexible method for creating isogenic cell lines via the homology-directed repair (HDR) pathway; however, methods for efficient generation and subsequent isolation of precisely edited cells have proven both expensive and time consuming. In response to this need, we have combined CRISPR/Cas9 HDR gene editing with an innovative high-throughput genotyping pipeline utilizing KASP (Kompetitive Allele-Specific PCR) technology to create scarless isogenic cell models of cancer risk variants in ~1 month without selectable markers or specialized methods such as digital droplet PCR or NGS. Utilizing this technology, we have been able to create cell lines differing by only a single base to model risk-associated SNPs located in the 8q24 risk locus in multiple cell types. The 8q24 risk locus is associated with increased risk for colorectal, breast, prostate, and, more recently, thyroid cancer. In this study, we use engineered isogenic cell lines modeling functional risk SNP rs6983267 in each of these cancer types to identify tissue-specific mechanisms of this multicancer risk locus. Rs6983267 is located within a known c-Myc enhancer and has been shown to exert risk-allele specific increase in enhancer activity. Oncogenic c-Myc is known to play a significant role in the pathogenesis of many cancers. However, how or if this mechanism varies between the different cancers this locus has been associated with remains uncertain. From preliminary studies, we do observe cell type variation in the regulatory effect of rs6983267. Not only does there seem to be a varying effect of the risk allele on c-Myc expression across the different cell types, but also risk allele enhancement of binding of the TCF7L2 transcription factor to the c-Myc enhancer rs6983267 is located within. Interestingly, rs6983267's regulatory effect seems most significant between homozygous reference cells and heterozygous cells within the same tissue type. We plan to delve further into these differences using RNA-seq and 4C-seq to assess whole transcriptomic and chromosomal interaction changes with changes in risk status. This study is one of first to compare risk mechanisms across different cancer tissue types; using precisely edited cellular models, we are able to detect cell-type specific variations in functional risk SNP effects on cancer development. Citation Format: Nicole B. Coggins, Henriette O'Geen, David J. Segal, Luis G. Carvajal-Carmona. Elucidating tissue-specific effects of the 8q24 multicancer risk locus via CRISPR/Cas9 scarless genome editing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 394.

Abstract 339: Heterozygous germline mutation in ABRAXAS causes BRCA1 mislocalization and DNA damage response defects

Abstract Whilst heterozygous germline mutations in the ABRAXAS gene have been associated with hereditary breast cancer predisposition, their initial effect on promoting tumorigenesis at the cellular level has not been explored. Here, we demonstrate in patient-derived cells that the Finnish ABRAXAS founder mutation (c.1082G&amp;gt;A, Arg361Gln), even in the heterozygous state leads to decreased BRCA1 protein levels as well as reduced nuclear localization and foci formation of BRCA1 and CtIP. This causes disturbances in basal BRCA1-A complex localization, which is reflected by a restraint in error-prone DNA double-strand break (DSB) repair pathway usage, attenuated DNA damage response, deregulated G2-M checkpoint control and apoptosis. Most importantly, mutation carrier cells display a change in their transcriptional profile, which we attribute to the reduced nuclear levels of BRCA1. The current study clearly demonstrates how the Finnish ABRAXAS founder mutation acts in a dominant-negative manner on BRCA1 to promote genome destabilisation in heterozygous carrier cells. Citation Format: Robert Winqvist, Muthiah Bose, Juliane Sachsenweger, Niina Laurila, Ann Christin Parplys, Jonas Willmann, Leila Eshraghi, Thomas W. Dunlop, Marco Groth, Katrin Rapakko, Pentti Nieminen, Thomas W. Friedl, Lisa Heiserich, Felix Meyer, Hanna Tuppurainen, Ganapathy Raman Devarajan, Hellevi Peltoketo, Heli Nevanlinna, Katri Pylkäs, Kerstin Borgmann, Lisa Wiesmuller, Helmut Pospiech. Heterozygous germline mutation in ABRAXAS causes BRCA1 mislocalization and DNA damage response defects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 339.

Abstract 5112: U2af1, a spliceosome gene commonly mutated in MDS, is required for hematopoiesis

Abstract Somatic mutations in genes encoding factors involved in pre-mRNA splicing (i.e., spliceosome genes) have been identified in more than half of myelodysplastic syndrome (MDS) patients. We are interested in the role of U2AF1, a spliceosome gene mutated in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1 have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type (WT) U2AF1 in normal hematopoiesis has not been studied. A complete understanding of the role of WT U2AF1 on hematopoiesis and RNA splicing is critical to our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of WT U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse. Homozygous embryonic deletion of U2af1 using Vav1-Cre was lethal, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wt or Mx1-Cre/U2af1wt/wt into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1 deletion, homozygous U2af1 KO mice, but not other genotypes, became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (decrease &amp;gt;1.7 fold) and multilineage cytopenias in homozygous U2af1 KO mice compared to all other genotypes (p ≤ 0.001, n=5). Deletion of U2af1 also led to rapid bone marrow failure and a reduction in bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (p ≤ 0.001, n=5). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and WT congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cells (HSCs). As early as 10 days, and up to 4 months following Mx1-Cre-induction, we observed a significant decrease in white blood cell count chimerism and complete loss of homozygous U2af1 KO HSCs, neutrophils and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). Collectively, the data indicate that normal hematopoiesis is dependent on U2af1 expression. In ongoing studies, we crossed the U2af1 KO mouse to existing transgenic mutant U2AF1-expressing mice to determine if mutant U2AF1 expression is sufficient to rescue cell survival and normal hematopoiesis when endogenous WT U2af1 is deleted. If U2AF1 mutant cells are vulnerable to loss of the WT U2AF1 allele, then selectively targeting the WT U2AF1 allele in heterozygous mutant cells could be a novel therapeutic strategy. Citation Format: Brian A. Wadugu, Amanda Heard, Joseph Bradley, Matthew Ndonwi, Matthew J. Walter. U2af1, a spliceosome gene commonly mutated in MDS, is required for hematopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5112.

PO-054 Common genetic variants at the breast cancer risk region 2Q35 map to putative IGFbp5 enhancers

IntroductionGenome-wide association studies (GWAS) have identified over 100 independently-associated regions of the genome with germline variants that modulate risk of developing breast cancer. Most signals map to non-coding regions of the genome and are thought to act at the level of gene regulation. Whilst GWAS are instrumental in highlighting regions of interest, it is important to identify the causal variant(s) at each locus and the mechanisms by which they influence risk.Material and methodsWe investigated the 2q35 breast cancer risk locus using two complementary methods. The first method involved selecting potentially causal variants based primarily on breast relevant functional data (Capture Hi-C, ChIP-seq, DNase-seq, GRO-seq) The second method involved conventional fine-mapping of case-control samples, to generate a short list of potentially causal variants that cannot be excluded on statistical grounds alone followed by intersection of the same breast-relevant functional data.Results and discussionsThe two methods identify distinct subsets of putative causal SNPs mapping to ERα/FOXA1 enhancer-like elements targeting the IGFBP5 gene promoter, a gene known to play a role in breast development. We confirmed enhancer status by performing luciferase reporter assays containing each of the regions of interest; furthermore, by introducing minor SNP alleles we identified a subset of SNPs that modulate strength of enhancer activity. ChIP-seq in heterozygous cell lines also confirms an allelic preference in enhancer binding. We are now using CRISPR/Cas9-based systems to investigate the regions of interest through perturbation studies, and generating isogenic cell lines to further investigate SNP effects on transcription factor binding, chromosome conformation and gene expression.ConclusionThe 2q35 breast cancer risk locus consists of three independent risk-associated signals. Statistical and functional approaches to variant prioritisation are complementary, identifying largely overlapping but distinct sets of candidate causal variants in putative regulatory elements (PREs). Subsequent functional investigation suggests these genetic variants may modulate the strength of these PREs; however, further investigation in isogenic cell lines is required.

Somatic clones heterozygous for recessive disease alleles of BMPR1A exhibit unexpected phenotypes in Drosophila.

The majority of mutations studied in animal models are designated as recessive based on the absence of visible phenotypes in germline heterozygotes. Accordingly, genetic studies primarily rely on homozygous loss-of-function to determine gene requirements, and a conceptually-related 'two-hit model' remains the central paradigm in cancer genetics. Here we investigate pathogenesis due to somatic mutation in epithelial tissues, a process that predominantly generates heterozygous cell clones. To study somatic mutation in Drosophila, we generated inducible alleles that mimic human Juvenile polyposis-associated BMPR1A mutations. Unexpectedly, four of these mutations had no phenotype in heterozygous carriers but exhibited clear tissue-level effects when present in somatic clones of heterozygous cells. We conclude that these alleles are indeed recessive when present in the germline, but nevertheless deleterious when present in heterozygous clones. This unforeseen effect, deleterious heteromosaicism, suggests a 'one-hit' mechanism for disease initiation that may explain some instances of pathogenesis associated with spontaneous mutation.

Reducing resistance allele formation in CRISPR gene drive

CRISPR homing gene drives can convert heterozygous cells with one copy of the drive allele into homozygotes, thereby enabling super-Mendelian inheritance. Such a mechanism could be used, for example, to rapidly disseminate a genetic payload in a population, promising effective strategies for the control of vector-borne diseases. However, all CRISPR homing gene drives studied in insects thus far have produced significant quantities of resistance alleles that would limit their spread. In this study, we provide an experimental demonstration that multiplexing of guide RNAs can both significantly increase the drive conversion efficiency and reduce germline resistance rates of a CRISPR homing gene drive in Drosophila melanogaster We further show that an autosomal drive can achieve drive conversion in the male germline, with no subsequent formation of resistance alleles in embryos through paternal carryover of Cas9. Finally, we find that the nanos promoter significantly lowers somatic Cas9 expression compared with the vasa promoter, suggesting that nanos provides a superior choice in drive strategies where gene disruption in somatic cells could have fitness costs. Comparison of drive parameters among the different constructs developed in this study and a previous study suggests that, while drive conversion and germline resistance rates are similar between different genomic targets, embryo resistance rates can vary significantly. Taken together, our results mark an important step toward developing effective gene drives capable of functioning in natural populations and provide several possible avenues for further control of resistance rates.

278 - A solution for cell therapy safety

Safety concerns such as tumourigenicity have been limiting the implementation of cell therapies to treat degenerative diseases. Here we introduce a concept and an associated genome editing strategy that addresses this issue. To maximize the expression of an inducible suicide system in dividing cells, we transcriptionally linked a suicide gene to a cell division essential locus (CDEL). Our prototype CDEL and suicide gene is CDK1, and HSV-TK, respectively. First, we generated mouse and human embryonic stem cell (ESC) lines with the described heterozygous modification. Our results showed we could eliminate proliferating cells in vitro and in vivo with dosing the prodrug ganciclovir (GCV). Elimination of these proliferating cells stopped the growth of teratomas generated by these ESCs and rendered this tissue dormant for up to one year. Despite the teratoma dormancy post-GCV treatment, we could detect GCV “escapees” among heterozygous cells, the majority of which were due to loss-of-heterozygosity (LOH). To avoid this, we generated cell lines with a homozygous CDK1 modification. Since we were unable to identify escapees in these lines in vitro, we used mathematical modelling to quantitate the risk of generating a GCV-resistant escapee. Furthermore, we demonstrated the homozygous modification of CDK1 does not compromise the differentiation capacity of the cells by evidence of viable homozygous mice and in vitro differentiation assay of modified hESCs. We also illustrated that GCV spares quiescent therapeutic cells. Lastly, we showed that our system is able to prevent tumour formation and eliminate formed tumours in a clinically relevant in vivo experiment. We are confident that our definition of safety, and the genome editing concept presented and characterized will provide a solution for cell therapy safety.

MECHANISMS OF THE IDH1/2 MUTATIONS AND ITS ASSOCIATION WITH CONTRADICTORY SURVIVAL OF GLIOBLASTOMA PATIENTS VERSUS AML PATIENTS

Isocitrate dehydrogenase 1 and 2 (IDH1/2) is mutated (IDH1/2mt) in various types of human cancer such as glioma and acute myeloid leukemia (AML), a structural alteration that leads to catalysis of α‐ketoglutarate to the oncometabolite D‐2‐hydroxyglutarate (D2HG). Heterozygous IDH1/2mt cancer cells produce less IDH1/2‐mediated NADPH, such that after exposure to ionizing radiation (IR) there were higher levels of reactive oxygen species, DNA double‐strand breaks and cell death compared to IDH1 wild‐type (IDH1wt) cells. These effects were reversed by the IDH1mt inhibitor AGI‐5198. Exposure of IDH1wt cells to D2HG was sufficient to reduce IDH‐mediated NADPH production and increase IR sensitivity. Mechanistic investigations revealed that the radiosensitivity of heterozygous cells was independent of the well‐described DNA hypermethylation phenotype in IDH1mt cancers. Thus, our results argue that altered oxidative stress responses are a plausible mechanism to understand the radiosensitivity of IDH1mt glioma. Further, they offer an explanation for the relatively longer survival of patients with IDH1mt glioma, and they imply that administration of IDH1mt inhibitors in these patients may limit irradiation efficacy in this setting.Whether this also holds true for IDH1/2mt AML was investigated using primary human IDH1mt, IDH2mt and IDH1/2wt AML cells. It was investigated whether IDH1/2 mutations cause increased DNA damage and sensitization to daunorubicin, irradiation, and the PARP inhibitors olaparib and talazoparib. IDH1/2mt inhibitors protected against these treatments. Combined treatment with a PARP inhibitor and daunorubicin had an additive effect on killing of IDH1/2mt AML cells. We provide evidence that the therapy sensitivity of IDH1/2mt cells was caused by D2HG‐mediated downregulation of expression of the DNA damage response gene ATM and not by altered redox responses due to metabolic alterations in IDH1/2mt AML cells. Thus, IDH1/2mt AML cells are sensitive to PARP inhibitors as monotherapy but especially when combined with a DNA‐damaging agent such as daunorubicin, whereas concomitant administration of IDH1/2‐mutant inhibitors during cytotoxic therapy decreases the efficacy of both agents in IDH1/2mt AML cells.Support or Funding InformationThis study was financially supported by the Dutch Cancer Society (KWF; UVA 2014‐6839; CJFVN, MK, VVVH, RJM)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Generation of human embryonic stem cell line with heterozygous RB1 deletion by CRIPSR/Cas9 nickase

The Retinoblastoma 1 (RB1) tumor suppressor, a member of the Retinoblastoma gene family, functions as a pocket protein for the functional binding of E2F transcription factors. About 1/3 of retinoblastoma patients harbor a germline RB1 mutation or deletion, leading to the development of retinoblastoma. Here, we demonstrate generation of a heterozygous deletion of the RB1 gene in the H1 human embryonic stem cell line using CRISPR/Cas9 nickase genome editing. The RB1 heterozygous knockout H1 cell line shows a normal karyotype, maintains a pluripotent state, and is capable of differentiation to the three germline layers.

BRE/BRCC45 regulates CDC25A stability by recruiting USP7 in response to DNA damage

BRCA2 is essential for maintaining genomic integrity. BRCA2-deficient primary cells are either not viable or exhibit severe proliferation defects. Yet, BRCA2 deficiency contributes to tumorigenesis. It is believed that mutations in genes such as TRP53 allow BRCA2 heterozygous cells to overcome growth arrest when they undergo loss of heterozygosity. Here, we report the use of an insertional mutagenesis screen to identify a role for BRE (Brain and Reproductive organ Expressed, also known as BRCC45), known to be a part of the BRCA1-DNA damage sensing complex, in the survival of BRCA2-deficient mouse ES cells. Cell viability by BRE overexpression is mediated by deregulation of CDC25A phosphatase, a key cell cycle regulator and an oncogene. We show that BRE facilitates deubiquitylation of CDC25A by recruiting ubiquitin-specific-processing protease 7 (USP7) in the presence of DNA damage. Additionally, we uncovered the role of CDC25A in BRCA-mediated tumorigenesis, which can have implications in cancer treatment.