Isogenic MDS-RS Patient-Derived iPSCs Define the Mis-Spliced Transcript Repertoire and Chromatin Landscape of SF3B1-Mutant Hematopoietic Stem/Progenitor Cells

The somatic hotspot mutation SF3B1K700E is characteristically found in myelodysplastic syndrome with ring sideroblasts (MDS-RS) and frequently occurs as an isolated mutation. However, our understanding of how this mutation drives MDS pathogenesis remains limited. To explore the downstream consequences of the SF3B1K700E mutation and its role in disease pathogenesis, we generated a panel of isogenic SF3B1K700E and SF3B1WT induced pluripotent stem cell (iPSC) lines from 3 MDS-RS patients with isolated SF3B1K700E mutation (3 SF3B1K700E and 3 SF3B1WT lines per patient, total 18). Upon hematopoietic differentiation, SF3B1K700E cells exhibited lower growth and colony-forming ability, compared to SF3B1WTcells, recapitulating hallmark phenotypes of MDS cells. To investigate the effects of the SF3B1K700E mutation on the transcriptome and chromatin landscape, we performed RNA- and ATAC- sequencing in purified CD34+/CD45+ hematopoietic stem/progenitor cells (HSPCs) derived from the panel of the 18 isogenic SF3B1K700E and SF3B1WT iPSC lines. Principal component analysis (PCA) and hierarchical clustering based on gene expression grouped the iPSC lines primarily by genotype (SF3B1K700E vs SF3B1WT) and secondarily by genetic background. To assess the impact of the SF3B1K700E mutation at the exon, transcript and gene level, we developed an analytical framework integrating differential splicing with differential transcript usage and differential gene expression analyses. We thus discovered 59 splicing events linked to 34 genes (most statistically significant events that also mapped to differentially used transcripts and differentially expressed genes). This SF3B1K700E splicing signature includes genes previously reported as mis-spliced in SF3B1K700E cells (e.g BRD9, ABCB7), as well as novel genes. We tested this signature against a published dataset of primary MDS patient samples (Pellagatti et al.). PCA based on the inclusion level of the splicing events of our signature separated SF3B1-mutated MDS patients from patients without splicing factor mutations (SF-WT) or healthy individuals. Furthermore, it identified one patient erroneously annotated as SF-WT that clustered together with the SF3B1-mutated patients, who had a, previously overlooked, 6bp in-frame deletion spanning the K700E hotspot. By comparing the chromatin accessibility profiles of SF3B1K700E and SF3B1WT iPSC-HSPCs to those defined in primary human cell types along the hematopoietic hierarchy (Corces et al.), we found that the chromatin landscape of SF3B1K700E HSPCs resembled more this of megakaryocyte-erythroid progenitor cells (MEPs) and erythroid cells, whereas that of SF3B1WT HSPCs resembled more granulocyte-monocyte progenitors (GMPs) and monocytes. This finding may underlie the more prominent involvement of the erythroid lineage in the pathology and clinical presentation of MDS-RS. To interrogate transcriptional programs in SF3B1K700E mutant cells, we performed transcription factor (TF) motif enrichment analysis. Motifs enriched in ATAC-Seq peaks more accessible in SF3B1K700E cells that were linked to genes upregulated in SF3B1K700E cells, included motifs of several TFs with known roles in hematopoiesis (GATA, ETS, STAT, AP-1). Unexpectedly, motifs of the TEAD TFs were also enriched. The TEAD family of TFs are best known as effectors of the Hippo signaling pathway, with important roles in various biological processes and malignancies, albeit no clear links to adult hematopoiesis or hematologic disease. TEAD2 and TEAD4 were upregulated in SF3B1-mutant, compared to the WT, iPSC-HSPCs and TEAD transcriptional activity, measured with a luciferase reporter construct, was higher in SF3B1K700E, compared to SF3B1WTiPSC-HSPCs. We did not find expression or activation of YAP or TAZ, which bind to DNA as a complex with TEAD upon Hippo pathway activation. These results support a Hippo-independent increase of TEAD expression and activity in SF3B1K700E cells. In summary, we generated a panel of isogenic patient-derived iPSCs that allowed us to comprehensively characterize the transcriptome and chromatin landscape of SF3B1K700E HSPCs in an isogenic system, derive a splicing signature of SF3B1K700E and identify the TEAD TF as a new transcriptional regulator of SF3B1K700Emutant HSPCs. G. Asimomitis, A.G. Deslauriers: shared 1 st authorship E. Papaemmanuil, E.P. Papapetrou: shared senior authorship Disclosures Deslauriers: Novo Nordisk A/S: Current Employment. Hellström-Lindberg: Celgene: Research Funding. Papaemmanuil: Isabl Technologies: Divested equity in a private or publicly-traded company in the past 24 months; Kyowa Hakko Kirin Pharma: Consultancy.

Novel Biphenyl Amines Inhibit Oestrogen Receptor (ER)-α in ER-Positive Mammary Carcinoma Cells

Herein, the activity of adamantanyl-tethered-biphenyl amines (ATBAs) as oestrogen receptor alpha (ERα) modulating ligands is reported. Using an ERα competitor assay it was demonstrated that ATBA compound 3-(adamantan-1-yl)-4-methoxy-N-(4-(trifluoromethyl) phenyl) aniline (AMTA) exhibited an inhibitory concentration 50% (IC50) value of 62.84 nM and demonstrated better binding affinity compared to tamoxifen (IC50 = 79.48 nM). Treatment of ERα positive (ER+) mammary carcinoma (MC) cells (Michigan Cancer Foundation-7 (MCF7)) with AMTA significantly decreased cell viability at an IC50 value of 6.4 μM. AMTA treatment of MC cell-generated three-dimensional (3D) spheroids resulted in significantly decreased cell viability. AMTA demonstrated a superior inhibitory effect compared to tamoxifen-treated MC cell spheroids. Subsequently, by use of an oestrogen response element (ERE) luciferase reporter construct, it was demonstrated that AMTA treatment significantly deceased ERE transcriptional activity in MC cells. Concordantly, AMTA treatment of MC cells also significantly decreased protein levels of oestrogen-regulated CCND1 in a dose-dependent manner. In silico molecular docking analysis suggested that AMTA compounds interact with the ligand-binding domain of ERα compared to the co-crystal ligand, 5-(4-hydroxyphenoxy)-6-(3-hydroxyphenyl)-7- methylnaphthalen-2-ol. Therefore, an analogue of AMTA may provide a structural basis to develop a newer class of ERα partial agonists.

OR07-06 The Roles of GNAQ and GNA11 in Calcium-Sensing Receptor (CaSR) Signalling

The G-protein subunits Gα 11 and Gα q, which share >90% peptide sequence identity and are encoded by the GNA11 and GNAQ genes, respectively, mediate signalling by the calcium-sensing receptor (CaSR), a class C G-protein coupled receptor (GPCR) that regulates extracellular calcium (Ca2+e) homeostasis. Germline Gα 11 inactivating and activating mutations cause familial hypocalciuric hypercalcaemia type-2 (FHH2) and autosomal dominant hypocalcaemia type-2 (ADH2), respectively, but such Gα q mutations have not been reported. We therefore investigated the DiscovEHR cohort database, which has exomes from 51,289 patients with matched phenotyping data, for such GNAQ mutations. The DiscovEHR cohort was examined for rare GNAQ variants, which were transiently expressed in CaSR-expressing HEK293A Gα q/11 knockout cells, and their effects on CaSR-mediated intracellular calcium (Ca2+i) release and MAPK activity, in response to increasing concentrations of extracellular calcium were assessed using a nuclear factor of activated T-cells response element (NFAT-RE) luciferase reporter construct and a serum response element (SRE) luciferase reporter construct, respectively. Responses were compared to those of wild-type (WT), inactivating FHH2-associated GNA11 mutations (Leu135Gln and Phe220Ser), and engineered GNAQ mutations that were equivalent to the FHH2-causing GNA11 mutations. Gα q/11 protein expression was confirmed by Western blot analysis. Six rare missense GNAQ variants (Arg19Trp, Ala110Val, Gln299His, Ala302Ser, Ala331Thr, Val344Ile) were identified in DiscovEHR individuals, all of whom had mean plasma calcium values in the normal range (8.30–10.00 mg/dL). Functional characterisation of all six Gα q variants showed no significant difference to WT Gα q responses, thereby indicating that these variants are unlikely to be disease-causing mutations. In addition, the FHH2-causing GNA11 mutations (Leu135Gln and Phe220Ser) had significantly reduced responses, compared to WT Gα 11; however, this could be compensated by WT Gα q. GNAQ Leu135Gln and Phe220Ser, in contrast to their Gα 11 counterparts, showed no differences in protein expression or signalling responses when compared to WT Gα q. Our study, which provides mechanistic insights into the differences between Gα q and Gα 11, indicates that Gα q, unlike Gα 11, does not play a major role in the pathogenesis of FHH2 or ADH2.

Benzimidazoles Downregulate Mdm2 and MdmX and Activate p53 in MdmX Overexpressing Tumor Cells

Tumor suppressor p53 is mutated in about 50% of cancers. Most malignant melanomas carry wild-type p53, but p53 activity is often inhibited due to overexpression of its negative regulators Mdm2 or MdmX. We performed high throughput screening of 2448 compounds on A375 cells carrying p53 activity luciferase reporter construct to reveal compounds that promote p53 activity in melanoma. Albendazole and fenbendazole, two approved and commonly used benzimidazole anthelmintics, stimulated p53 activity and were selected for further studies. The protein levels of p53 and p21 increased upon the treatment with albendazole and fenbendazole, indicating activation of the p53–p21 pathway, while the levels of Mdm2 and MdmX decreased in melanoma and breast cancer cells overexpressing these proteins. We also observed a reduction of cell viability and changes of cellular morphology corresponding to mitotic catastrophe, i.e., G2/M cell cycle arrest of large multinucleated cells with disrupted microtubules. In summary, we established a new tool for testing the impact of small molecule compounds on the activity of p53 and used it to identify the action of benzimidazoles in melanoma cells. The drugs promoted the stability and transcriptional activity of wild-type p53 via downregulation of its negative regulators Mdm2 and MdmX in cells overexpressing these proteins. The results indicate the potential for repurposing the benzimidazole anthelmintics for the treatment of cancers overexpressing p53 negative regulators.

Golgi Oncoprotein GOLPH3 Gene Expression Is Regulated by Functional E2F and CREB/ATF Promoter Elements

The Golgi organelle duplicates its protein and lipid content to segregate evenly between two daughter cells after mitosis. However, how Golgi biogenesis is regulated during interphase remains largely unknown. Here we show that messenger RNA (mRNA) expression of GOLPH3 and GOLGA2, two genes encoding Golgi proteins, is induced specifically in G1 phase, suggesting a link between cell cycle regulation and Golgi growth. We have examined the role of E2F transcription factors, critical regulators of G1 to S progression of the cell cycle, in the expression of Golgi proteins during interphase. We show that promoter activity for GOLPH3, a Golgi protein that is also oncogenic, is induced by E2F1-3 and repressed by E2F7. Mutation of the E2F motifs present in the GOLPH3 promoter region abrogates E2F1-mediated induction of a GOLPH3 luciferase reporter construct. Furthermore, we identify a critical CREB/ATF element in the GOLPH3 promoter that is required for its steady state and ATF2-induced expression. Interestingly, depletion of GOLPH3 with small interfering RNA (siRNA) delays the G1 to S transition in synchronized U2OS cells. Taken together, our results reveal a link between cell cycle regulation and Golgi function, and suggest that E2F-mediated regulation of Golgi genes is required for the timely progression of the cell cycle.

Mouse embryo Hox gene enhancers assayed in cell culture: Hoxb4, b8 and a7 are activated by Cdx1 protein

Mouse Hox gene enhancer elements have typically been identified and characterized using Hox/lacZ transgenic mouse embryos. Such studies have, for example, identified Cdx responsive binding motifs in the enhancers of Hoxb8 and Hoxa7. Production of transgenic mouse embryos involves issues of cost, welfare, and considerable technical skill. It would be of benefit if these studies could be performed, or advanced, in cell culture. It is shown here that Cdx1 activation of mouse Hoxb4, b8 and a7 embryo-active enhancers can be detected using a HepG2 cell culture model system. The technique employed uses co-transfection of an inducible Cdx1 expression construct together with a Hox enhancer/luciferase reporter construct. Cultures to be compared receive identical DNAs and differ only in whether or not they also receive inducer (doxycycline). Response of all three Hox enhancers to Cdx1 protein is inhibited by mutation of Cdx binding motifs which are conserved in sequence from fish or Xenopus to mammals. The magnitude of transfected chick Hoxa7 activation by Cdx1 is increased by multiple copies of its enhancer, but for maximum effect these must contain intact Cdx binding motifs. Cdx1 protein was found not to activate Hoxb4, b8 or a7 enhancers in P19 mouse pluripotential cells.

UFM1 founder mutation in the Roma population causes recessive variant of H-ABC

Objective:To identify the gene defect in patients with hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) who are negative for TUBB4A mutations.Methods:We performed homozygosity mapping and whole exome sequencing (WES) to detect the disease-causing variant. We used a Taqman assay for population screening. We developed a luciferase reporter construct to investigate the effect of the promoter mutation on expression.Results:Sixteen patients from 14 families from different countries fulfilling the MRI criteria for H-ABC exhibited a similar, severe clinical phenotype, including lack of development and a severe epileptic encephalopathy. The majority of patients had a known Roma ethnic background. Single nucleotide polymorphism array analysis in 5 patients identified one large overlapping homozygous region on chromosome 13. WES in 2 patients revealed a homozygous deletion in the promoter region of UFM1. Sanger sequencing confirmed homozygosity for this variant in all 16 patients. All patients shared a common haplotype, indicative of a founder effect. Screening of 1,000 controls from different European Roma panels demonstrated an overall carrier rate of the mutation of 3%–25%. Transfection assays showed that the deletion significantly reduced expression in specific CNS cell lines.Conclusions:UFM1 encodes ubiquitin-fold modifier 1 (UFM1), a member of the ubiquitin-like family involved in posttranslational modification of proteins. Its exact biological role is unclear. This study associates a UFM1 gene defect with a disease and sheds new light on possible UFM1 functional networks.

A comparison of cost for mycobacterial load determination in research laboratories

<p><strong>Background</strong>. Mycobacterial load determination is a critical quantitative measure needed in many clinical and research laboratory studies and selection depends on several factors including sensitivity, dynamic range and turnaround time. However, there are no data about cost, which is an important selection determinant. We therefore performed a cost analysis of five quantitative mycobacterial load assays.</p><p><strong>Methods</strong>. The costs of five mycobacterial quantification techniques were compared in a hypothetical single experiment (control and intervention) performed in triplicate. Assays evaluated were: mycobacterial colony-forming units (MCFU) using 7H10-Middlebrook solid media, automated liquid culture (BACTEC-MGIT-960), [3H]-uracil incorporation assays, luciferase-reporter construct bioluminescence, and quantitative polymerase chain reaction (PCR) (Xpert-MTB/RIF) using serial dilutions of Mycobacterium tuberculosis. Costs associated with consumables, equipment and personnel were included and expressed in 2015 South African rands and US dollars.</p><p><strong>Results</strong>. The least costly technique was the luminescence reporter construct assay (R85.68/$6.72) whereas the most expensive technique was the Xpert MTB/RIF PCR (R388.02/$30.42). The high cost of the PCR assay was mainly attributable to the costly Xpert MTB/RIF cartridges. Although the MCFU by solid culture had a similar cost compared with uracil incorporation and Xpert MTB/RIF, the purchase price of the equipment required to perform the latter assays was ~2 - 10 times higher.</p><p><strong>Conclusion</strong>. Taking into consideration the turnaround time, capital costs, discriminatory ability, the running costs (excluding staff) of the luminescence reporter assay are the lowest. Where time to result is critical, more expensive techniques such as the Xpert MTB/RIF should be used. In a clinical setting where automated culture and Xpert are routinely performed, quantitative load from time to positivity and cycle thresholds will provide extra data without additional cost.</p>

Estrogenic/antiestrogenic activity of selected selective serotonin reuptake inhibitors

Backround & Aims: Selective serotonin reuptake inhibitors (SSRIs) are one of the most prescribed classes of psychotropics. Even though the SSRI class consists of 6 molecules (citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine and sertraline), only fluoxetine was intensively studied for endocrine disruptive effects, while the other SSRIs received less attention. This study was designed to evaluate the estrogenic/antiestrogenic effect of fluoxetine, sertraline and paroxetine.Materials and methods: The in vitro (anti)estrogenic activity was assessed using a firefly luciferase reporter construct in the T47D-KBluc breast cancer cell line. These cells express nuclear estrogen receptors that can activate the transcription of the luciferase reporter gene upon binding of estrogen receptor agonists. Results: All three compounds were found to interact with the estrogen receptor. Fluoxetine had dual properties, weak estrogenic at lower concentrations and antiestrogenic effect at higher concentrations. Sertraline shared the same properties with fluoxetine, but also increased the estradiol-mediated transcriptional activity. Paroxetine presented only one type of effect, the ability to increase the estradiol-mediated transcriptional activity. Conclusions: Overall, our results indicate a possible interaction of SSRIs with the estrogen receptor. As SSRIs are being used by all categories of population, including pregnant women or children, establishing whether they can affect the endocrine mediated mechanisms should be a priority.

In vitro modulation of estrogen receptor activity by norfluoxetine

Background & Aims: Selective serotonin reuptake inhibitors (SSRIs) are antidepressants increasingly prescribed for pregnancy and postpartum depression. However, these compounds can cross the placenta and also pass into breast milk, thus reaching the fetus and infant during critical developmental stages, potentially causing adverse effects. Fluoxetine, a widely used SSRI, has been shown to affect (neuro)endocrine signaling in various organisms, including humans. This compound can also interact with estrogen receptors in vitro and cause an estrogen-dependent uterotrophic response in rodents. Consequently, the aim of the present study was to assess if the active metabolite of fluoxetine, namely norfluoxetine, shares the same capacity for estrogen receptor interaction.Materials and methods: The in vitro (anti)estrogenic activity of norfluoxetine was assessed using a firefly luciferase reporter construct in the T47D-Kbluc breast cancer cell line. These cells express nuclear estrogen receptors (ERs) that can activate the transcription of the luciferase reporter gene upon binding of ER agonists. Light emission was monitored in case of cells exposed to norfluoxetine or mixtures of norfluoxetine-estradiol. Cell viability was assessed using a resazurin-based assay.Results: During individual testing, NFLX was able to induce a significant increase in luciferase activity compared to control, but only at the highest concentration tested (10 µM). In binary mixtures with estradiol (30pM constant concentration) a significant increase in luminescence was observed at low submicromolar norfluoxetine concentrations compared to estradiol alone.Conclusion: Norfluoxetine can induce estrogenic effects in vitro and can potentiate the activity of estradiol. However, further studies are needed to clarify if these observed estrogenic effects may have detrimental consequences for human exposure.