Heterozygous Cells Research Articles

Functional characterization of novel ABCB6 mutations and their clinical implications in familial pseudohyperkalemia.

Isolated familial pseudohyperkalemia is a dominant red cell trait characterized by cold-induced 'passive leak' of red cell potassium ions into plasma. The causative gene of this condition is ABCB6, which encodes an erythrocyte membrane ABC transporter protein bearing the Langereis blood group antigen system. In this study analyzing three new families, we report the first functional characterization of ABCB6 mutants, including the homozygous mutation V454A, heterozygous mutation R276W, and compound heterozygous mutations R276W and R723Q (in trans). All these mutations are annotated in public databases, suggesting that familial pseudohyperkalemia could be common in the general population. Indeed, we identified variant R276W in one of 327 random blood donors (0.3%). Four weeks' storage of heterozygous R276W blood cells resulted in massive loss of potassium compared to that from healthy control red blood cells. Moreover, measurement of cation flux demonstrated greater loss of potassium or rubidium ions from HEK-293 cells expressing ABCB6 mutants than from cells expressing wild-type ABCB6. The R276W/R723Q mutations elicited greater cellular potassium ion efflux than did the other mutants tested. In conclusion, ABCB6 missense mutations in red blood cells from subjects with familial pseudohyperkalemia show elevated potassium ion efflux. The prevalence of such individuals in the blood donor population is moderate. The fact that storage of blood from these subjects leads to significantly increased levels of potassium in the plasma could have serious clinical implications for neonates and infants receiving large-volume transfusions of whole blood. Genetic tests for familial pseudohyperkalemia could be added to blood donor pre-screening. Further study of ABCB6 function and trafficking could be informative for the study of other pathologies of red blood cell hydration.

High rates of chromosome missegregation suppress tumor progression but do not inhibit tumor initiation.

Aneuploidy, an abnormal chromosome number that deviates from a multiple of the haploid, has been recognized as a common feature of cancers for >100 yr. Previously, we showed that the rate of chromosome missegregation/chromosomal instability (CIN) determines the effect of aneuploidy on tumors; whereas low rates of CIN are weakly tumor promoting, higher rates of CIN cause cell death and tumor suppression. However, whether high CIN inhibits tumor initiation or suppresses the growth and progression of already initiated tumors remained unclear. We tested this using the Apc(Min/+) mouse intestinal tumor model, in which effects on tumor initiation versus progression can be discriminated. Apc(Min/+) cells exhibit low CIN, and we generated high CIN by reducing expression of the kinesin-like mitotic motor protein CENP-E. CENP-E(+/-);Apc(Min/+) doubly heterozygous cells had higher rates of chromosome missegregation than singly heterozygous cells, resulting in increased cell death and a substantial reduction in tumor progression compared with Apc(Min/+) animals. Intestinal organoid studies confirmed that high CIN does not inhibit tumor cell initiation but does inhibit subsequent cell growth. These findings support the conclusion that increasing the rate of chromosome missegregation could serve as a successful chemotherapeutic strategy.

Endurance Training Improves Muscle Function And Metabolism In A Sickle Cell Disease Mouse Model

Sickle cell disease (SCD) is an inherited disorder resulting in the synthesis of an abnormal hemoglobin (HbS) and associated with the occurrence of painful vaso-occlusive crisis (VOC). While it has been recently suggested that oxygen supply and use might be impaired due to muscle remodeling, the corresponding metabolic effects in exercising muscle are still unknown. Considering that regular moderate-intensity exercise is known to be beneficial on muscle structure and energetics, endurance training could alleviate the muscle remodelling described in SCD and improve muscle function. PURPOSE: The aim of this study was to characterize in a mouse model of SCD i) the potential abnormalities in muscle energetics and function, and ii) the potential beneficial effects of endurance training. METHODS: Sedentary control (HbAA-SED, n=10), heterozygous (HbAS-SED, n=10) and sickle cell (HbSS-SED, n=10) mice and endurance-trained (8-weeks treadmill running) healthy (HbAA-END, n=10), heterozygous (HbAS-END, n=11) and sickle cell (HbSS-END, n=8) mice were placed in a home-built device designed for a strictly non-invasive study of posterior hindlimb muscles function and metabolism by NMR during a standardized rest - stimulation - recovery protocol. RESULTS: During the stimulation period, HbSS mice developed less force as compared to HbAS mice (p<0.01) and HbAS mice displayed a reduced total force production as compared to HbAA mice (p<0.05). Whatever the genotype, training resulted in a significantly increased force production (p<0.001). Regarding muscle energetics, the exercise-induced phosphocreatine (PCr) depletion and pH decrease were significantly (p < 0.05) larger in HbSS mice as compared to HbAA and HbAS mice. Interestingly, training resulted in a significant (p<0.05) reduction in PCr depletion and intracellular acidosis. CONCLUSIONS: We demonstrated in the present study that SCD was associated with an impaired muscle force production and a larger exercise-induced metabolic consumption. The larger acidosis, which could trigger VOC, might be explained by both an increased non-oxidative glycolytic energy production and an altered protons handling. Interestingly, endurance training improved muscle function and reduced metabolic abnormalities, including acidosis.

733. Somatically Repairing Compound Heterozygous Recessive Mutations by Chromosomal Cut-and-Paste for In Vivo Gene Therapy

Patients affected by monogenic recessive genetic disorders often carry two different mutated alleles of the same gene, which is known as compound heterozygous. Theoretically, exchanging the genetic material between the two mutated alleles will reconstitute a mutation-free allele that can be therapeutic (FigureFigure). We hypothesized that generating DNA double-stranded breaks at the same location on both mutant alleles can induce allele exchange, reconstitute a mutation-free allele, and therefore yield therapeutic benefit. We first tested this hypothesis in a targeted knock-in mouse model that carries GFPN-term-intron-tdTomatoC-term and tdTomatoN-term-intron-GFPC-term expression cassettes, respectively, at the same genomic location on each copy of Chr 11. Therefore, allele exchange at the intronic region will reconstitute the full-length GFP and tdTomato, serving as a reporter system. We injected recombinant AAV (rAAV) vectors expressing SpCas9 and sgRNA targeting the intron into adult mice by tail vein injection. Five weeks later, we observed GFP and tdTomato fluorescence in cryosections of peripheral tissues including liver and heart, whereas there was no such fluorescence observed in the tissue samples from untreated mice, demonstrating that allele exchange occurred, and that the reconstituted alleles yielded protein expression. Furthermore, we generated mice that carry two different mutations of the Aspa gene as a compound heterozygous mouse model of Canavan disease. We treated these mice with rAAV vectors expressing SpCas9 and sgRNA targeting an intron between the two mutation sites. Three weeks after treatment, we detected reconstituted, mutation-free Aspa DNA sequence by allele-specific PCR and single-molecule, high-throughput DNA sequencing in the liver. The reconstituted Aspa allele carried insertion at the predicted SpCas9 cleavage site, indicating that the DNA allele exchange was mediated by the non-homologous end joining DNA repair pathway. We also observed allele exchange in mouse liver using the SaCas9 system. Evaluation of the therapeutic benefit following Cas9/sgRNA-mediated allele exchange in various compound heterozygous mouse models and patient cell lines is underway. The gene repairing strategy described here is a novel approach to tackling a broad range of autosomal recessive genetic disorders.View Large Image | Download PowerPoint Slide

Deletion of a Yci1 Domain Protein of Candida albicans Allows Homothallic Mating in MTL Heterozygous Cells.

ABSTRACTIt has been proposed that the ancestral fungus was mating competent and homothallic. However, many mating-competent fungi were initially classified as asexual because their mating capacity was hidden behind layers of regulation. For efficient in vitro mating, the essentially obligate diploid ascomycete pathogen Candida albicans has to change its mating type locus from heterozygous MTLa/α to homozygous MTLa/a or MTLα/α and then undergo an environmentally controlled epigenetic switch to the mating-competent opaque form. These requirements greatly reduce the potential for C. albicans mating. Deletion of the Yci1 domain gene OFR1 bypasses the need for C. albicans cells to change the mating type locus from heterozygous to homozygous prior to switching to the opaque form and mating and allows homothallic mating of MTL heterozygous strains. This bypass is carbon source dependent and does not occur when cells are grown on glucose. Transcriptional profiling of ofr1 mutant cells shows that in addition to regulating cell type and mating circuitry, Ofr1 is needed for proper regulation of histone and chitin biosynthesis gene expression. It appears that OFR1 is a key regulator in C. albicans and functions in part to maintain the cryptic mating phenotype of the pathogen.

AccuCopy quantification combined with pre-amplification of long-distance PCR for fast analysis of intron 22 inversion in haemophilia A

BackgroundTo develop a digitalized intron 22 inversion (Inv22) detection in patients with severe haemophilia A. MethodsThe design included two tests: A genotyping test included two multiplex pre-amplification of LD-PCR (PLP) with two combinations of five primers to amplify wild-type and chimeric int22h alleles; a carrier mosaicism test was similar to the genotyping test except only amplification of chimeric int22h alleles by removing one primer from each of two combinations. AccuCopy detection was used to quantify PLP products. ResultsPLP product patterns in the genotyping test allowed identifying all known Inv22. Quantitative patterns accurately represented the product patterns. The results of 164 samples detected by the genotyping test were consistent with those obtained by LD-PCR detection. Limit of detection (LOD) of the carrier mosaicism test was at least 2% of heterozygous cells with Inv22. Performing the test in two obligate mothers with negative Inv22 from two sporadic pedigrees mosaic rates of blood and hair root of the mother from pedigree 1 were 8.3% and >20%, respectively and negative results were obtained in pedigree 2. ConclusionsAccuCopy quantification combined with PLP (AQ-PLP) method was confirmed to be rapid and reliable for genotyping Inv22 and highly sensitive to carrier mosaicism detection.

Mutant KRAS Copy-Number Gain Drives Metabolic Reprogramming

Abstract Homozygous and heterozygous KRAS mutant cancer cells have distinct metabolic phenotypes.

Abstract B18: Role of the tumor suppressor gene DLC-2 in ERBB2 induced mouse mammary tumorigenesis and metastasis

Abstract Introduction: Overexpression of dominant oncogenes and the loss of tumor suppressor genes are basic genetic events in acquisition of the malignant phenotype. The ERBB-2 proto-oncogene is overexpressed in 20% - 30% of human breast cancers. The tumor suppressor gene Deleted in liver cancer 2 (DLC-2, also known as STARD13) maps to chromosome 13q12.3 and is frequently downregulated by 72% in human breast cancer. It encodes a RhoGAP protein containing a START lipid binding domain. It is not known what role DLC-2 silencing plays in breast carcinogenesis. The purpose of our study is to assess the biological significance of inactivation of DLC-2 in ERBB2 induced mammary tumorigenesis mouse model. For this, we used MMTV-NIC mice, which carry an activated NEU oncogene (ERBB2) under control of the MMTV-LTR promoter that also co-expresses Cre recombinase. These mice were crossed to a conditional DLC-2 knockout mouse (floxed exon 3). Objectives: To determine if heterozygous or homozygous DLC-2 loss plays a role in mouse mammary tumor progression in MMTV-NIC mice. Results: Our preliminary results showed that heterozygous deletion of DLC-2 in the mammary epithelium of the MMTV-NIC mouse model resulted in increased tumor burden with accelerated tumor growth. Metastases to the lungs were quantified by scanning H&amp;E stained step sections (4X150uM). We observed a significant increase in the incidence of lung metastases in heterozygous DLC-2 exon3floxed/+ MMTV-NIC mice. The lung metastases showed a homogenous morphology and histopathologically similar phenotype to the MMTV-NIC strain. DLC-2 heterozygous tumor cells showed increased tumorosphere formation under low-attachment conditions with minimal growth factor supplementation. These tumor cells also showed increased expression of mesenchymal cell markers, as determined by quantitative real time RT-PCR, suggesting an Epithelial to Mesenchymal transition is occurring. Experiments with homozygous floxed DLC-2 mice are ongoing and will be presented. Conclusion: These results indicate that loss of one allele of DLC-2 is sufficient to accelerate NEU oncogene induced tumorigenesis. Citation Format: Pratima Basak, Pratima Basak, Heather Leslie, Heather Leslie, Afshin Raouf, Afshin Raouf, Michael RA Mowat, Michael RA Mowat. Role of the tumor suppressor gene DLC-2 in ERBB2 induced mouse mammary tumorigenesis and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B18.

Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities.

SummaryThe RAS/MAPK-signalling pathway is frequently deregulated in non-small cell lung cancer (NSCLC), often through KRAS activating mutations1-3. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations4-7. We recently showed that advanced lung tumours from KrasG12D/+;p53-null mice frequently exhibit KrasG12D allelic enrichment (KrasG12D/Kraswild-type>1)7, implying that mutant Kras copy gains are positively selected during progression. Through a comprehensive analysis of mutant Kras homozygous and heterozygous MEFs and lung cancer cells we now show that these genotypes are phenotypically distinct. In particular, KrasG12D/G12D cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the TCA cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous NSCLC cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of KrasG12D copy gain), but not in the corresponding early tumours (KrasG12D heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprised of two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated based on their relative mutant allelic content. We also provide the first in vivo evidence of metabolic rewiring during lung cancer malignant progression.

Establishment of a quenching probe method for detection of NPM1 mutations in acute myeloid leukemia cells.

Nucleophosmin (NPM1) mutations, generally consisting of a four base-pair insertion, are present in ~60% of all cytogenetically normal acute myeloid leukemia (AML) cases. The mutation is clinically significant as an important prognostic factor. Direct sequencing is the current standard method of mutation detection, however, it is quite costly and time consuming. The present study aimed to establish a highly sensitive quenching probe (QP) method to detect NPM1 mutations efficiently. Melting curve analysis was performed using a QP, following polymerase chain reaction for amplification of the involved region of the gene. The curve derived from the fluorescent intensity with respect to the temperature of OCI/AML3, a heterozygous NPM1 mutant AML cell line, was W-shaped with melting peaks at 61°C and 68°C. That of M-07e, the homozygous wild type cell line, was V-shaped with a melting peak at 68°C. Thus, the curve derived from the mutant allele was easily discriminated from that of the wild-type allele. The mutant allele was detected in concentrations as low as 3% as determined by a subsequent sensitivity study. With a short testing time and a high sensitivity, this assay was applicable for NPM1-mutated AML patient samples and is appropriate for screening NPM1 mutations. It does require further examination as to whether it would be useful as a detection method for other mutant alleles since NPM1 mutations may consist of 61 known types of mutant sequences. To the best of our knowledge, this is the first report describing the QP method for the detection of NPM1 mutations.

Somatic overgrowth associated with homozygous mutations in both MAN1B1 and SEC23A.

Using whole-exome sequencing, we identified homozygous mutations in two unlinked genes, SEC23A c.1200G>C (p.M400I) and MAN1B1 c.1000C>T (p.R334C), associated with congenital birth defects in two patients from a consanguineous family. Patients presented with carbohydrate-deficient transferrin, tall stature, obesity, macrocephaly, and maloccluded teeth. The parents were healthy heterozygous carriers for both mutations and an unaffected sibling with tall stature carried the heterozygous mutation in SEC23A only. Mutations in SEC23A are responsible for craniolenticosultura dysplasia (CLSD). CLSD patients are short, have late-closing fontanels, and have reduced procollagen (pro-COL1A1) secretion because of abnormal pro-COL1A1 retention in the endoplasmic reticulum (ER). The mutation we identified in MAN1B1 was previously associated with reduced MAN1B1 protein and congenital disorders of glycosylation (CDG). CDG patients are also short, are obese, and have abnormal glycan remodeling. Molecular analysis of fibroblasts from the family revealed normal levels of SEC23A in all cells and reduced levels of MAN1B1 in cells with heterozygous or homozygous mutations in SEC23A and MAN1B1. Secretion of pro-COL1A1 was increased in fibroblasts from the siblings and patients, and pro-COL1A1 was retained in Golgi of heterozygous and homozygous mutant cells, although intracellular pro-COL1A1 was increased in patient fibroblasts only. We postulate that increased pro-COL1A1 secretion is responsible for tall stature in these patients and an unaffected sibling, and not previously discovered in patients with mutations in either SEC23A or MAN1B1. The patients in this study share biochemical and cellular characteristics consistent with mutations in MAN1B1 and SEC23A, indicating a digenic disease.

Hemorrhagic Stroke: Clinical, Etiologic and Evolutive Aspects in Senegalese Children

Introduction: Hemorrhagic Stroke (HS) are uncommon among children. They have an etiological specificity and different risk factors that are different from those of adults. Prognosis depends on the etiology. Our objective was to study the characteristics of children's HS in Senegal. Methodology: Descriptive retrospective study from July 2003 to February 2014. Results: 13 patients were collected, including 9 boys. The mean age was 9.85 years. The antecedents were: rheumatic heart disease (2 patients); congenital heart disease (1 patient); arterial hypertension systolic-diastolic untreated (1 patient); heterozygous sickle cell anemia (1 patient). No history for (8 patients - 61.53%). The beginning was sudden or rapidly progressive in all patients. Clinical manifestations were hemiplegia (53.84%), headache and vomiting (46.15%), consciousness disorders (30.76%), partial motor seizures (7.69%). The topography was supratentorial (84.61%). The causes were: arteriovenous malformations (3 patients); severe thrombocytopenia (1 patient); systolic-diastolic arterial hypertension (1 patient); myeloblastic leukemia (1 patient). No etiology (7 patients). 4 patients died during the acute phase. 1 patient was transferred to pediatric oncology. 1 patient was transferred abroad for embolization. The mean follow-up was 11.8 months. Outcome was favorable in patients with ad integrum restitution. 7 patients had hemiparesis type of neurological sequelae (4 patients), motor partial epilepsy (4 patients) and aphasia (1 patient). 1 patient developed hydrocephalus. No recurrence was noted. Conclusion: This study provides a better diagnostic and etiological approach for better management.

Point-of-Care Screening for Sickle Cell Disease By a Mobile Micro-Electrophoresis Platform

In developing countries, diagnostic tests for homozygous (HbSS) or compound heterozygous (HbSC or HbS-Beta thalassemia) sickle cell disease (SCD) are not readily available at the point-of-care (POC). Very few infants are screened in Africa for SCD because of the high cost and level of skill needed to run traditional tests. Current methods are too costly and take too much time to enable equitable and timely diagnosis to save lives. The World Health Organization recognizes a crucial need for early detection of SCD in newborns, since it is estimated that 70% SCD-related deaths in Africa are preventable with early cost-effective interventions. The diagnostic barrier can be broken with affordable, POC tools that facilitate early detection immediately after birth. We have developed a mobile micro-electrophoretic device (HemeChip) through which to quickly, accurately, and affordably screen for SCD (Fig. 1A). The HemeChip uses a microfabricated platform housing cellulose acetate electrophoresis to rapidly separate hemoglobin (Hb) types. Less than 5 microliters of blood, which can be obtained through a finger stick or heel stick, is processed on a piece of cellulose paper in alkaline buffer. The HemeChip reliably identifies and discriminates amongst Hb C/A2, S, F and A0. The micro-electrophoresis results were validated against standard clinical hemoglobin screening methods, including high performance liquid chromatography (HPLC), with Pearson Correlation Coefficient (PCC) of ≥0.96 relative to HPLC for all Hb types tested. The receiver Operating-Characteristic (ROC) curves showed more than 0.89 sensitivity and 0.86 specificity for identification of hemoglobin types using the HemeChip, based on the travelling distance from the sample application point (Fig. 1B). We developed a web-based image processing application for automated and objective quantification of HemeChip results at the POC using cloud computing resources (Fig. 1C). This intensity-based mobile phone image quantitation method showed high correlation with HPLC results for tested patient blood samples (PCC=0.95). HemeChip can distinguish between different patient phenotypes, including HbSS (HbS only), transfused HbSS (HbS and HbA), and Hemoglobin SC disease (HbS and HbC). In conclusion, the HemeChip identification and quantification of hemoglobin phenotypes, as a POC technique, were comparable to standard clinical methods. This platform has clinical potential in under-served populations worldwide, in which SCD is endemic. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Dynamic Changes in the Clonal Structure of MDS and AML in Response to Epigenetic Therapy

Hematopoietic cells from patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) contain gene mutations that are variably distributed between the founding clone and daughter subclone(s). Traditional response criteria in MDS and AML are based on bone marrow morphology and may not accurately reflect antitumor activity and clinical benefit in patients treated with hypomethylating agents. We used digital sequencing of serial bone marrow samples to monitor tumor burden and to characterize the changes in the clonal structure of MDS and AML that occur during treatment with epigenetic therapy. We hypothesized that digital sequencing may provide an alternative measure of antitumor activity and identify the persistence or emergence of resistant clones during treatment which mediate disease relapse.We conducted a phase I/II study in older adults (age ≥ 60) with advanced MDS (IPSS ≥ 1.5) or AML. Subjects received a combination of decitabine 20 mg/m2 on d1-5 with the histone deacetylase inhibitor, panobinostat 10-40 mg po 3x/week every 28 days for up to 12 cycles. Serial bone marrow samples were collected for digital sequencing at baseline, after every 2 cycles of treatment and at the time of relapse.A total of 52 patients, 14 with MDS and 38 with AML were enrolled in this study. For AML patients, 10% achieved a complete remission (CR+CRi) with an additional 18% of patients achieving a morphologic leukemia-free state (mLFS) using IWG response criteria. For patients with MDS, 14% achieved a CR and 21% achieved a marrow CR. We identified 9 MDS and 16 AML patients that had banked, paired bone marrow and skin (as a source of normal DNA) samples and a somatic mutation in at least 1 of 54 recurrently mutated MDS/ AML genes. DNA was enriched for 285 genes commonly mutated in MDS and AML (n=24 patients) or whole exome probes spiked-in with the 285 genes (enhanced exome sequencing; EES) (n=7 patients), and sequenced on a HiSeq2000 instrument with 2x101bp reads. We detected an average of 4.9 SNVs and indels per patient (range 1-15) when only the 285 gene panel was used, compared to 27.4 mutations per patient (range 9-43) using EES. Ten genes were mutated in at least 3 pre-study samples. The presence of a TP53 mutation (N=8) was associated with a trend towards achieving a response (p=0.09).We then analyzed variant allele frequencies (VAF) of mutations in serial samples. We observed five distinct patterns that were associated with different clinical responses, including i) AML patients achieving a CR+CRi (n=2): mutation VAFs were undetectable by cycle 2 using standard sequencing, ii) AML with mLFS (n=2): mutation VAFs remained detectable but decreased to <10%, iii) MDS with CR/cCR+mCR (n=3): mutation VAFs decreased to <10% and were intermittently below the level of detection, iv) MDS with stable disease (n=2): mutation VAFs decreased but some remained >10%, and v) AML with treatment failure (n=5): mutation VAFs were essentially unchanged and remained >30%.We observed responding patients can have persistent measurable clonal hematopoiesis for at least one year without disease progression. Sequencing also revealed selective AML subclone clearance in a patient with treatment failure, nominating a set of mutations that may mark super-responder clones. We observed that the blast percentage decreases prior to mutation VAFs in some patients, suggesting that the differentiation of blasts could falsely underestimate tumor burden. Finally, sequencing revealed that tumor burden can be measured even in patients achieving a CR. Using an ultra-sensitive barcode sequencing approach, we sequenced 1 MDS and 1 AML patient achieving a clinical and molecular CR (based on standard sequencing). We detected extremely rare TP53 mutations months to years prior to disease relapse (VAFs = 0.23% in MDS and 0.05% in AML during a CR - equivalent to a sensitivity of 1 in 2000 heterozygous mutant cells). While patients can live with persistent clonal hematopoiesis in a CR or stable disease, ultimately we find evidence that expansion of a rare subclone drives relapse or progression from MDS to secondary AML. Digital sequencing provides an alternative measure of disease response which may augment traditional clinical response criteria and should be explored in future clinical trials. DisclosuresUy:Novartis: Research Funding. Off Label Use: Panobinostat in MDS/AML. Duncavage:Cofactor Genomics: Consultancy; DI&P Consulting: Consultancy. Jacoby:Sunesis: Research Funding; Novo Nordisk: Consultancy. Abboud:Teva Phamaceutical: Research Funding.

SETD2 Heterozygous Loss in Leukemia Leads to Chemotherapy Resistance through Attenuation of the DNA Damage Response

Relapsed leukemia is chemotherapy resistant and survival is poor. We recently found that mutations in epigenetic regulators are enriched in relapsed pediatric B-cell acute lymphoblastic leukemia (B-ALL), suggesting that they are associated with clonal survival and chemotherapy resistance. The mutations included loss of function mutations in the epigenetic regulator SETD2, which had not before been described in B-ALL. SETD2 is the only known mammalian Histone 3 Lysine 36 (H3K36) trimethyltransferase and one or two copy loss leads to a global decrease or elimination, respectively, of its chromatin mark, H3K36me3.Using CRISPR/Cas9 to engineer isogenic human and murine leukemia cells, we have demonstrated that SETD2 loss leads to resistance to DNA damaging chemotherapy used in standard ALL therapy, consistent with the gain of SETD2 mutations at relapse. Leukemia cells with SETD2 heterozygous deletion demonstrated a significant increase in IC50 to the DNA damaging anti-metabolites 6-thioguanine (6TG) and cytarabine (AraC), but not the protein synthesis inhibitor L-Asparaginase (Asp). To model the enrichment of SETD2 mutations at relapse, we competed a small population of SETD2 heterozygous cells with isogenic control leukemia cells. Within 3 weeks, treatment with 6TG and AraC, but not Asp or vehicle, led to a dramatic selection of SETD2 heterozygous cells, mimicking the selective pressure of SETD2 clones in patients.Recent reports have shown that H3K36me3is important in the DNA damage response (DDR). Consistent with this, we find that heterozygous SETD2 loss impairs the DDR signaling pathway with attenuated phosphorylation of multiple DDR components, including Chk1 and Chk2, in response to chemotherapy. Consequently, SETD2 heterozygous cells showed an abrogation of the apoptotic response to the DNA damaging chemotherapy agents 6TG and AraC, despite a normal apoptotic response to Asp.In summary, we have demonstrated that SETD2 loss leads to resistance to DNA damaging chemotherapy. The resistance is caused by a failure to trigger DDR signaling and apoptosis after chemotherapy induced DNA damage. These findings identify heterozygous SETD2 lossas a novel mechanism of chemotherapy resistance in leukemia and have implications for therapy selection and novel therapeutic interventions. DisclosuresNo relevant conflicts of interest to declare.

Measuring arterial stiffness using pOpmetre® in the African heterozygous and homozygous sickle cell disease

Measuring arterial stiffness using pOpmetre® in the African heterozygous and homozygous sickle cell disease

Radioprotection of IDH1-Mutated Cancer Cells by the IDH1-Mutant Inhibitor AGI-5198.

Isocitrate dehydrogenase 1 (IDH1) is mutated in various types of human cancer to IDH1(R132H), a structural alteration that leads to catalysis of α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate. In this study, we present evidence that small-molecule inhibitors of IDH1(R132H) that are being developed for cancer therapy may pose risks with coadministration of radiotherapy. Cancer cells heterozygous for the IDH1(R132H) mutation exhibited less IDH-mediated production of 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 with IDH1 wild-type cells. These effects were reversed by the IDH1(R132H) inhibitor AGI-5198. Exposure of IDH1 wild-type cells to D-2-hydroxyglutarate 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 IDH1-mutated cancers. Thus, our results argue that altered oxidative stress responses are a plausible mechanism to understand the radiosensitivity of IDH1-mutated cancer cells. Further, they offer an explanation for the relatively longer survival of patients with IDH1-mutated tumors, and they imply that administration of IDH1(R132H) inhibitors in these patients may limit irradiation efficacy in this setting.

Abstract 17361: Loss of Notch1 in the Secondary Heart Field Exacerbates Ascending Aortic Aneurysm and Causes Aortic Rupture in Marfan Syndrome Mice (Best of Basic Science Abstract)

Ascending aortic aneurysm (AscAA) is a life-threatening disease that affects approximately 3.5 in 100,000 people per year, but the mechanisms of AscAA remain poorly understood. We previously reported that NOTCH1 mutations are associated with familial bicuspid aortic valve (BAV), and a subset of these individuals displayed BAV aortopathy. NOTCH1 is expressed in multiple cell lineages that contribute to the aorta, and we found dysregulation of activated NOTCH1 expression in aneurysmal tissue from Marfan syndrome and BAV patients in comparison to control tissue. To investigate the role of NOTCH1 in AscAA, Notch1 haploinsufficiency was introduced into the Marfan syndrome mouse ( Fbn1 C1039G/+ ), a well-described model of AscAA. Serial echocardiographic, gross and histological characterization of the Notch1 deficient Marfan syndrome mice ( Notch1 +/- ;Fbn C1039G/+ ) demonstrated an exacerbated AscAA phenotype when compared to Fbn1 C1039G/+ mice. The compound mutant mice had increased aortic root diameter by three months of age (diameter: Notch1 +/- ;Fbn C1039G/+ = 2.11±0.11mm and Fbn1 C1039G/+ = 1.86±0.05mm, p&lt;0.05) as well as a higher rate of dilation from 1-5 months of age (rate: Notch1 +/- ;Fbn C1039G/+ =0.24±0.06mm/month and Fbn1 C1039G/+ =0.09±0.03mm/month, p&lt;0.05). Notch1 +/- and wildtype littermate mice had normal aortic diameters. Furthermore, a small subset (4/66) of the compound mutant mice succumbed to ascending aortic rupture, which was not observed in the Fbn1 C1039G/+ mice (0/63). Although endothelial Notch1 is critical for cardiovascular development, mice with heterozygous loss of Notch1 in endothelial cells that harbor the Fbn1 C1039G mutation ( Notch1 flox/+ ;Tie2-Cre +/- ;Fbn1 C1039G/+ ) did not recapitulate the phenotype of the Notch1 +/- ;Fbn C1039G/+ mice. Surprisingly, the phenotype was recapitulated with heterozygous loss of Notch1 in the lineage of secondary heart field cells ( Notch1 flox/+ ;Mef2C-AHF-Cre +/- ;Fbn1 C1039G/+ ) but not smooth muscle myosin heavy chain-expressing cells ( Notch1 flox/+ ;Myh11-Cre +/- ;Fbn1 C1039G/+ ). Our findings support a novel role for Notch1 in the non-endothelial secondary heart field and suggest that this loss of Notch1 alters a developmental process that contributes to AscAA.

Abstract 14610: Thoracic Aortic Aneurysm-causing Mutation, R258C, in Smooth Muscle Alpha-actin Inhibits Functional Effects of Wild-type Alpha-actin in Patient-derived Fibroblasts

Familial thoracic aortic aneurysms and dissections (TAAD) are most commonly caused by missense mutations in smooth muscle α-actin (SMαA), encoded by ACTA2. Although TAAD is hypothesized to result in part from a compromised ability of aortic smooth muscle cells (SMCs) to sense their mechanical environment, the specifics of how ACTA2 mutations affect cellular mechanotransduction remain to be defined. We established hTERT-immortalized lines of dermal fibroblasts from volunteers (WT) and patients with ACTA2 mutations (MT). SMαA expression was induced with adenovirus encoding MRTF-A, a potent co-activator of SM contractile genes, including ACTA2. We focus here on the ACTA2 R258C mutation, a recurrent mutation associated with early onset of TAAD. Quantification of actin expression by 2D-PAGE and western blotting showed that induced WT fibroblasts expressed ~40% β-, 30% γ- and 30% SM-α-actin, while heterozygous MT cells expressed ~40% β-, 30% γ-, 15% WT-SM-α- and 15% MT-SM-α-actin, with un-induced cells expressing &lt;5% SMαA. Cytoskeletal structure was studied by immunofluorescence and confocal microscopy, and by sedimentation for actin distribution in filamentous (F) and monomeric (G) fractions. Cell motile and contractile properties were investigated by scratch-induced cell migration, trans-well migration, and collagen matrix contraction assays. As described for other models of SMαA induction in fibroblasts, we found that MRTF-A induction of SMαA in WT cells significantly increased formation of stress fibers and focal adhesions, F/G ratio (p&lt;0.01), and matrix contraction (p&lt;0.05), and significantly decreased cell migration in scratch-induced (p&lt;0.01) and trans-well (p&lt;0.0001) migration assays. In contrast, expression of MT SMαA in heterozygous R258C MT fibroblasts abrogated the effects of SMαA on formation of stress fibers and focal adhesions, F/G ratio, matrix contraction and cell migration in comparison to induction of SMαA in WT cells. These results demonstrate that R258C MT SMαA disrupts cytoskeletal functions attributed to SMαA in WT fibroblasts and are consistent with defects in mechanotransduction, which may shed light on cellular defects due to ACTA2 mutations in both aortic SMCs and adventitial fibroblasts.

Generation of Hypertension-Associated STK39 Polymorphism Knockin Cell Lines With the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 System.

Previous genome-wide association studies identified serine threonine kinase 39 (STK39), encoding STE20/SPS1-related proline/alanine-rich kinase, as one of a limited number of hypertension susceptibility genes. A recent meta-analysis confirmed the association of STK39 intronic polymorphism rs3754777 with essential hypertension, among previously reported hypertension-associated STK39 polymorphisms. However, the biochemical function of this polymorphism in the mechanism responsible for hypertension is yet to be clarified. We generated rs3754777G>A knockin human cell lines with clustered regularly interspaced short palindromic repeats-mediated genome engineering. Homozygous (A/A) and heterozygous (G/A) knockin human embryonic kidney cell lines were generated using a double nickase, single-guide RNAs targeting STK39 intron 5 around single-nucleotide polymorphism, and a 100-bp donor single-stranded DNA oligonucleotide. Reverse transcription polymerase chain reaction with sequencing analyses revealed the identical STK39 transcripts among the wild-type and both knockin cell lines. Quantitative reverse transcription polymerase chain reaction showed increased STK39 mRNA expression, and immunoblot analysis revealed increases in total and phosphorylated STE20/SPS1-related proline/alanine-rich kinase with increased phosphorylated Na-K-Cl cotransporter isoform 1 in both knockin cell lines. The largest increases in these molecules were observed in the homozygous cell line. These findings indicated that this intronic polymorphism increases STK39 transcription, leading to activation of the STE20/SPS1-related proline/alanine-rich kinase-solute carrier family 12A signaling cascade. Increased interactions between STE20/SPS1-related proline/alanine-rich kinase and the target cation-chloride cotransporters may be responsible for hypertension susceptibility in individuals with this polymorphism.