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Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals. Neagu, van Genderen and Escudero et al. show that simultaneous inhibition of WNT and MEK signalling maintains a naive-primed intermediate pluripotency state in vitro, which displays features of the mouse embryonic rosette.

Thoracic aortic aneurysm is a potentially life-threatening disease with a strong genetic contribution. Despite identification of multiple genes involved in aneurysm formation, little is known about the specific underlying mechanisms that drive the pathological changes in the aortic wall. The aim of our study was to unravel the molecular mechanisms underlying aneurysm formation in Marfan syndrome (MFS). We collected aortic wall samples from FBN1 variant-positive MFS patients (n = 6) and healthy donor hearts (n = 5). Messenger RNA (mRNA) expression levels were measured by RNA sequencing and compared between MFS patients and controls, and between haploinsufficient (HI) and dominant negative (DN) FBN1 variants. Immunohistochemical staining, proteomics and cellular respiration experiments were used to confirm our findings. FBN1 mRNA expression levels were highly variable in MFS patients and did not significantly differ from controls. Moreover, we did not identify a distinctive TGF-β gene expression signature in MFS patients. On the contrary, differential gene and protein expression analysis, as well as vascular smooth muscle cell respiration measurements, pointed toward inflammation and mitochondrial dysfunction. Our findings confirm that inflammatory and mitochondrial pathways play important roles in the pathophysiological processes underlying MFS-related aortic disease, providing new therapeutic options.

Evasion from drug-induced apoptosis is a crucial mechanism of cancer treatment resistance. The proapoptotic protein NOXA marks an aggressive pancreatic ductal adenocarcinoma (PDAC) subtype. To identify drugs that unleash the death-inducing potential of NOXA, we performed an unbiased drug screening experiment. In NOXA-deficient isogenic cellular models, we identified an inhibitor of the transcription factor heterodimer CBFβ/RUNX1. By genetic gain and loss of function experiments, we validated that the mode of action depends on RUNX1 and NOXA. Of note is that RUNX1 expression is significantly higher in PDACs compared to normal pancreas. We show that pharmacological RUNX1 inhibition significantly blocks tumor growth in vivo and in primary patient-derived PDAC organoids. Through genome-wide analysis, we detected that RUNX1-loss reshapes the epigenetic landscape, which gains H3K27ac enrichment at the NOXA promoter. Our study demonstrates a previously unknown mechanism of NOXA-dependent cell death, which can be triggered pharmaceutically. Therefore, our data show a way to target a therapy-resistant PDAC, an unmet clinical need.

Evasion from drug-induced apoptosis is a crucial mechanism of cancer treatment resistance. The pro-apoptotic protein NOXA marks an aggressive pancreatic ductal adenocarcinoma (PDAC) subtype. To identify drugs that unleash the death-inducing potential of NOXA, we performed an unbiased drug screening experiment. In NOXA-deficient isogenic cellular models we identified an inhibitor of the transcription factor heterodimer CBFβ/RUNX1. By genetic gain and loss of function experiments we validated that the mode of action depends on RUNX1 and NOXA. Of note, RUNX1 expression is significantly higher in PDACs compared to normal pancreas. We show that pharmacological RUNX1 inhibition significantly blocks tumor growth in vivo and in primary patient-derived PDAC organoids. Through genome wide analysis, we detected that RUNX1-loss reshapes the epigenetic landscape, which gains H3K27ac enrichment at the NOXA promoter. Our study demonstrates a previously unknown mechanism of NOXA-dependent cell death, which can be triggered pharmaceutically. Therefore, our data show a novel way to target a therapy resistant PDAC, an unmet clinical need.SignificanceRecent evidence demonstrated the existence of molecular subtypes in pancreatic ductal adenocarcinoma (PDAC), which resist all current therapies. The paucity of therapeutic options, including a complete lack of targeted therapies, underscore the urgent and unmet medical need for the identification of targets and novel treatment strategies for PDAC. Our study unravels a function of the transcription factor RUNX1 in apoptosis regulation in PDAC. We show that pharmacological RUNX1 inhibition in PDAC is feasible and leads to NOXA-dependent apoptosis. The development of targeted therapies that influence the transcriptional landscape of PDAC might have great benefits for patients who are resistant to conventional therapies. RUNX1 Inhibition as a new therapeutic intervention offers an attractive strategy for future therapies.

T – B – NK + severe combined immunodeficiency (SCID) is caused by a defect in V(D)J recombination. A subset of these patients has a mutation in one of the non-homologous end joining (NHEJ) genes, most frequently the Artemis gene. Artemis is involved in opening of hairpin-sealed coding ends. The low levels of residual DH-JH junctions that could be amplified from patients' bone marrow precursor B cells showed high numbers of palindromic (P)-nucleotides. In 25% of junctions, microhomology was observed in the P-nucleotide regions, whereas this phenomenon was never observed in junctions amplified from bone marrow precursor B cells from healthy controls. We utilized this difference between Artemis-deficient cells and normal controls to develop a V(D)J recombination assay to determine hairpin-opening activity. Mutational analysis of the Artemis gene confirmed and extended the mapping of an N-terminal nuclease active site, which contains several indispensable aspartate residues. C-terminal deletion mutants did not show such severe defects in the V(D)J recombination assay using transient overexpression of (mutated) Artemis protein. However, a C-terminal deletion mutation causes T – B – NK + SCID, indicating that the Artemis C terminus is essential for V(D)J recombination at the normal Artemis expression level. The V(D)J recombination assays used in this study contribute to the diagnostic strategy for T – B – NK + SCID patients.

The intracellular uptake and retention (IUR) of imatinib is reported to be controlled by the influx transporter SLC22A1 (organic cation transporter 1). We recently hypothesized that alternative uptake and/or retention mechanisms exist that determine intracellular imatinib levels. Here, we systematically investigate the nature of these mechanisms. Imatinib uptake in cells was quantitatively determined by liquid chromatography-tandem mass spectrometry. Fluorescent microscopy was used to establish subcellular localization of imatinib. Immunoblotting, cell cycle analyses, and apoptosis assays were performed to evaluate functional consequences of imatinib sequestration. Uptake experiments revealed high intracellular imatinib concentrations in HEK293, the leukemic cell lines K562 and SD-1, and a gastrointestinal stromal tumor cell line GIST-T1. We demonstrated that imatinib IUR is time-, dose-, temperature-, and energy-dependent and provide evidence that SLC22A1 and other potential imatinib transporters do not substantially contribute to the IUR of imatinib. Prazosin, amantadine, NH4Cl, and the vacuolar ATPase inhibitor bafilomycin A1 significantly decreased the IUR of imatinib and likely interfere with lysosomal retention and accumulation of imatinib. Costaining experiments with LysoTracker Red confirmed lysosomal sequestration of imatinib. Inhibition of the lysosomal sequestration had no effect on the inhibition of c-Kit signaling and imatinib-mediated cell cycle arrest but significantly increased apoptosis in imatinib-sensitive GIST-T1 cells. We conclude that intracellular imatinib levels are primarily determined by lysosomal sequestration and do not depend on SLC22A1 expression.

Drug-induced changes in the expression or activity of ADME genes may critically affect the pharmacokinetics (PK) and the efficacy of chemotherapy as well as the incidence and severity of unwanted side effects. Drug uptake and efflux transporters are likely to be involved in the intestinal absorption, distribution and hepatic excretion of imatinib, an oral tyrosine kinase inhibitor approved for the treatment of CML and GIST. Previously, we demonstrated that imatinib is a substrate drug of both ABCB1 and ABCG2. Furthermore, we showed that prolonged imatinib exposure in Caco-2 cell cultures leads to increased expression of these drug efflux transporters. Evidently, some GIST patients chronically treated with imatinib show a time-dependent decrease in imatinib plasma levels. Altered transporter activity in the intestine and/or liver affecting the PK of imatinib may (partially) account for the observed PK resistance. We hypothesized that acquired PK resistance by decreased absorption and/or increased clearance of imatinib may be attributable to induction of intestinal and hepatic transport proteins. So, we investigated in mice whether imatinib-induced modulation of drug transporters occurs over time upon prolonged daily use of imatinib. To mimic the clinical situation we chronically treated C57/Bl6 mice, either with imatinib or with a solute control (H2O) for 4, 8 or 12 weeks. Imatinib (100 mg/kg once daily) was administered to the mice via oral gavage (∼300 μl / mice). We collected all organs involved in the absorption and elimination of imatinib including the liver and the complete GI-tract (esophagus, stomach, small and large intestine and caecum) and subsequently isolated total RNA and protein from these tissues. We performed mRNA and miRNA profiling using mouse genome 430 PM (Affymetrix) and LNA-modified oligonucleotide arrays (Exiqon), respectively, and examined the expression of >80 transporter genes using the RT2 Profiler PCR array system (SABiosciences). Prolonged imatinib treatment resulted in the modulation of various intestinal and hepatic drug transporters and clearly changed the mRNA and miRNA profiles of ADME-involved organs. Apart from the imatinib-associated transporters Abcb1a/b, Abcg2, Slc22a1 and Slco1b2, indicating proof of concept, our search identified various other potential imatinib transporter which are currently tested in uptake experiments using transporter-transfected HEK293 cells. In summary, we showed that continuous exposure of mice to daily dosages of imatinib markedly affects the expression of drug transporters and changes the mRNA and miRNA expression profiles in organs involved in uptake, distribution and elimination of imatinib. These imatinib-induced changes may critically affect the PK, efficacy and adverse events of imatinib chemotherapy. Citation Format: Herman Burger, Alexander T. den Dekker, Antonius WM Boersma, Peter de Bruijn, Jaap Verweij, Ron HJ Mathijssen, Erik AC Wiemer. Imatinib-induced modulation of gastrointestinal and hepatic membrane transporters in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 883. doi:10.1158/1538-7445.AM2013-883