ALBERT

Description: The young (~16 Myr) pre-main-sequence star in Sco–Cen 1SWASP J140747.93–394542.6, hereafter referred to as J1407, underwent a deep eclipse in 2007 April, bracketed by several shallower eclipses in the surrounding 54 d. This has been interpreted as the first detection of an eclipsing ring system circling a substellar object (dubbed J1407b). We report on a search for this companion with Sparse Aperture Mask imaging and direct imaging with both the UT4 VLT and Keck telescopes. Radial velocity measurements of J1407 provide additional constraints on J1407b and on short period companions to the central star. Follow-up photometric monitoring using the Panchromatic Robotic Optical Monitoring and Polarimetry Telescopes (PROMPT)-4 and ROAD observatories during 2012–2014 has not yielded any additional eclipses. Large regions of mass–period space are ruled out for the companion. For circular orbits the companion period is constrained to the range 3.5–13.8 yr ( a ~= 2.2–5.6 au), and stellar masses (〉80 M Jup ) are ruled out at 3 significance over these periods. The complex ring system appears to occupy more than 0.15 of its Hill radius, much larger than its Roche radius and suggesting a ring structure in transition. Further, we demonstrate that the radial velocity of J1407 is consistent with membership in the Upper Cen–Lup subgroup of the Sco–Cen association, and constraints on the rotation period and projected rotational velocity of J1407 are consistent with a stellar inclination of i * ~= 68° ± 10°.

Description: In B cell neoplasia, many transcription factors known to be involved in B cell differentiation and commitment, like E2A, EBF1 and PAX5, are frequently targeted by focal deletions, mutations or chromosomal aberrations. Recent studies have shown that the human genes BTG1 and BTG2 are commonly affected by gene alterations in different B cell malignancies, but their role in normal B cell development has not been established. BTG1 and BTG2 can act as transcriptional cofactors through recruitment of the protein arginine N-methyltransferase PRMT1, which mediates arginine methylation of transcription factors, like RUNX1, and on histone 4 arginine 3 (H4R3). Here we report that Btg1 and Btg2 display unique and overlapping functions during mouse B cell development. We observed a reduction in the fraction of B220+ progenitor cells in the bone marrow compartment of the different knockout animals, ranging from a 10% decrease in the Btg2-/-, 20% in Btg1-/- , and 40% in the Btg1-/-;Btg2-/- mice relative to wild-type controls. Deficiency for Btg1, but not Btg2, resulted in reduced outgrowth of IL-7 dependent lymphoid progenitors in methylcellulose, which correlated with a higher fraction of apoptotic cells. Btg2-/- mice showed impaired differentiation at the pre-pro-B, pro-B and pre-B cell stage, while Btg1-deficiency mainly affected later stages of B cell differentiation with reduced numbers of immature B cells. Btg1-/-;Btg2-/- mice displayed additive effects with more significant reduction of B220+ cells predominantly at the pre-B and immature B cell stage. Expression analysis revealed no reduction in the mRNA levels of master regulators E2a, Foxo1, Ebf1 and Pax5 in the absence of Btg1 and Btg2. However, higher expression levels of T cell-specific genes were observed in Btg1-/-;Btg2-/- progenitor B cells, e.g. Cd4, Ikzf2 and Tcf7 (Figure 1), some of which are known to be transcriptional repressed by Ebf1, such as Id2, Gata3, Dtx3l and Notch1. Flow cytometric analyses confirmed increased expression of CD3, CD4 and CD8 markers on CD19+ bone marrow cells lacking Btg1 and Btg2 function. Additionally, we detected enhanced levels of DC, NK and myeloid markers on Btg1-/-;Btg2-/- CD19+ BM cells, indicating that Btg1 and Btg2 repress alternative cell fates during B cell lineage specification, and are required for the maintenance of B cell identity. Biochemical studies showed evidence for a physical association between Ebf1, Btg1/Btg2 and PRMT1. We propose a model in which Btg1 and Btg2 affect the function of the critical B cell transcription factor Ebf1 by recruitment of PRMT1. Figure 1. Aberrant T-lineage expression in progenitor B cells deficient for Btg1 and Btg2. Relative expression levels of Cd4, Runx1, Ikzf2, Tcf7, Id2, Gata3, Notch1 and Dtx3l were determined on cDNA generated from B220+ BM cells of wild-type (WT), Btg1-/-, Btg2-/- and Btg1-/-;Btg2-/- mice by quantitative real-time PCR and normalized to the expression of the housekeeping gene TATA box binding protein (TBP). Data represent the mean and SEM of three independent experiments containing cDNA derived from 2 different biological samples. *, P〈 0.05, **, P〈 0.01, ***, P〈 0.001. Figure 1. Aberrant T-lineage expression in progenitor B cells deficient for Btg1 and Btg2. Relative expression levels of Cd4, Runx1, Ikzf2, Tcf7, Id2, Gata3, Notch1 and Dtx3l were determined on cDNA generated from B220+ BM cells of wild-type (WT), Btg1-/-, Btg2-/- and Btg1-/-;Btg2-/- mice by quantitative real-time PCR and normalized to the expression of the housekeeping gene TATA box binding protein (TBP). Data represent the mean and SEM of three independent experiments containing cDNA derived from 2 different biological samples. *, P〈 0.05, **, P〈 0.01, ***, P〈 0.001. Disclosures No relevant conflicts of interest to declare.

Description: Translocation t(12;21) (p13;q22), giving rise to the ETV6-RUNX1 fusion gene, is the most common genetic abnormality in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The ETV6-RUNX1 translocation arises in utero, but its expression is insufficient to induce leukemia and requires other cooperating genetic lesions for BCP-ALL to develop. Deletions affecting the transcriptional coregulator BTG1 are commonly observed in BCP-ALL (9%), but are significantly enriched in ETV6-RUNX1-positive leukemia (25%). The BTG1 protein displays no intrinsic enzymatic activity but may act by recruiting effector molecules like protein arginine methyltransferase 1 (PRMT1) to specific transcription factors. Here, we show that ETV6-RUNX1 interacts both with BTG1 and PRMT1, and this interaction is lost in c-Kit+Ter-119-Btg1-/- fetal liver (FL) derived hematopoietic progenitors (HPCs). Moreover, targeted deletion of Btg1 enhanced the proliferative capacity of ETV6-RUNX1 in FL-HPCs as measured by enhanced colony-forming and serial replating capacity (Figure 1). The combined loss of Btg1 function and ETV6-RUNX1 expression correlated with strong upregulation of the proto-oncogene Bcl6 and downregulation of BCL6 target genes, such as p19Arf and Tp53 (Figure 2). Similarly, ectopic expression of BCL6 promoted both proliferation and replating capacity of FL-derived progenitor cells in the presence of SCF, FLT3L and IL-7. This phenotype correlated with a fivefold suppression of p19Arf and a twofold suppression of Tp53 expression. Inhibition of BCL6 in a variety of human BCP-ALL cell lines by the peptide inhibitor RI-BPI resulted in decreased proliferation and induction of apoptosis as measured by Annexin-V staining. These included the ETV6-RUNX1-positive cell lines UOC-B6, AT-2 and REH, the BCR-ABL1-positive cell line SD1, as well as Nalm6. Together our results point to a novel role for BCL6 in promoting cell proliferation of primitive progenitor B cells and suggest that targeted inhibition of BCL6 may be effective in the treatment of various BCP-ALL subtypes. Figure 1. Btg1-deficiency enhances the proliferative capacity of early FL-HPCs expressing ETV6-RUNX1. FL-derived hematopoietic progenitor cells (FL-HPCs) (cKit+Ter119-) were isolated from wild-type and Btg1-/- embryos at day 13.5dpc and transduced with control and ETV6-RUNX1 virus. Control and ETV6-RUNX1 transduced FL-HPCs (1x104 cells) were added 48 hours after transduction in B cell specific methylcellulose in the presence of FLT-3L, IL-7 and SCF. Serial replating was performed under identical conditions. Mean colony counts (and SEM) were determined (〉30 cells/colony) after 6-10 days of culture. Data is a representative of 2 independent experiments. *, P〈 0.05, **, P〈 0.01. Figure 1. Btg1-deficiency enhances the proliferative capacity of early FL-HPCs expressing ETV6-RUNX1. FL-derived hematopoietic progenitor cells (FL-HPCs) (cKit+Ter119-) were isolated from wild-type and Btg1-/- embryos at day 13.5dpc and transduced with control and ETV6-RUNX1 virus. Control and ETV6-RUNX1 transduced FL-HPCs (1x104 cells) were added 48 hours after transduction in B cell specific methylcellulose in the presence of FLT-3L, IL-7 and SCF. Serial replating was performed under identical conditions. Mean colony counts (and SEM) were determined (〉30 cells/colony) after 6-10 days of culture. Data is a representative of 2 independent experiments. *, P〈 0.05, **, P〈 0.01. Figure 2. Targeted deletion of Btg1 cooperates with ETV6-RUNX1 in regulating critical effector pathways implicated in leukemia. Relative expression levels of Bcl6, Tp53 and p19arf in empty control (Ctrl) and ETV6-RUNX1 transduced wild-type and Btg1-deficient fetal liver-derived hematopoietic progenitor cells by real-time PCR and normalized to the expression of the housekeeping gene TATA box binding protein (TBP). Data represent the mean and SEM of three independent experiments. *, P〈 0.05, **, P〈 0.01, ***, P〈 0.001. Figure 2. Targeted deletion of Btg1 cooperates with ETV6-RUNX1 in regulating critical effector pathways implicated in leukemia. Relative expression levels of Bcl6, Tp53 and p19arf in empty control (Ctrl) and ETV6-RUNX1 transduced wild-type and Btg1-deficient fetal liver-derived hematopoietic progenitor cells by real-time PCR and normalized to the expression of the housekeeping gene TATA box binding protein (TBP). Data represent the mean and SEM of three independent experiments. *, P〈 0.05, **, P〈 0.01, ***, P〈 0.001. Disclosures No relevant conflicts of interest to declare.

Description: Asparaginase (ASNase) is one of the cornerstones of the multi-drug treatment protocol that is used to treat acute lymphoblastic leukemia (ALL) in pediatric and adult patients. Recent studies monitoring ASNase kinetics in patients provide evidence of a large inter-patient variability of serum ASNase concentrations and call attention to the negative effects of ASNase underexposure on treatment response and relapse risk. Despite the fact that ASNase has been used in ALL treatment protocols for decades, little is known about the biodistribution and the mechanism of ASNase turnover in patients. We used in vivo imaging to study the distribution and pharmacodynamics of ASNase in a mouse model. We injected mice with 3,000 International Units (I.U.)/kg ASNase, which was labeled with 20-25 MBq Indium-111 (In-111) and acquired micro-SPECT/CT images up 18 hours post injection. At this timepoint, serum ASNase activity has dropped to levels close to the detection limits. In addition to the expected uptake in the liver, SPECT/CT imaging revealed a rapid, strong and specific accumulation of radiolabeled ASNase in the bone marrow and spleen (Figure 1). Accumulation in these organs was confirmed by quantitative measurement of radiolabeled ASNase in the dissected organs (Figure 2). We hypothesized that macrophages which are present in high numbers in these organs, efficiently phagocytose the ASNase, thereby rapidly clearing the active enzyme from the blood. Autoradiography of spleen sections indeed showed high uptake of radiolabeled ASNase in the macrophage-rich red pulp of the spleen. Immunohistochemical stainings confirmed the presence of ASNase in cells positive for the murine macrophage marker F4/80. To provide additional evidence for the potential role of macrophages in the turnover of ASNase, we pretreated mice with a single injection of clodronate liposomes, which almost completely depletes the relevant organs from phagocytic cells. This pretreatment diminished the accumulation of ASNase in the liver, spleen and the bone marrow (Figure 2). Consistent with this notion, we found that clodronate pretreatment more than doubles the circulatory half-life of serum ASNase activity. We conclude from these experiments that ASNase is rapidly cleared from the serum by phagocytic cells. In particular, the efficient uptake of ASNase by spleen and bone marrow resident macrophages may protect leukemic cells from the nutrient depriving action of this drug and could thereby compromise therapeutic efficacy. Figure 1: SPECT/CT image of Asparaginase uptake Figure 1:. SPECT/CT image of Asparaginase uptake Lateral (A) and ventral (B) 3-dimensional volume projections of fused SPECT/CT scans of mice injected with 111Indium-labeled asparaginase (pseudocolor images with red being least intense and yellow most intense), 18 hours post injection. Numbers indicate relevant organs: 1 sternum, 2 liver, 3 spleen, 4 spine, 5, pelvis, 6 femur, 7 tibia. Figure 2: Biodistribution of Asparaginase in control and clodronate pretreated mice. Figure 2:. Biodistribution of Asparaginase in control and clodronate pretreated mice. Asparaginase uptake is depicted as percentage of the injected dose per gram of tissue (%ID/g) at 19 hours after injection in control (empty liposomes) and clodronate pretreated mice. Results are mean + standard deviation (n=5 for each group). 2-tailed t-test was used to test for significance: * p

Description: During the course of tumorigenesis and subsequent chemotherapeutic intervention, cancer cells experience various kinds of physiological stress, including hypoxia and nutrient limitation. Escaping cell death is one of the routes utilized by these malignant cells to allow continued growth and to acquire therapy resistance. B-cell Translocation Gene 1 (BTG1) is recurrently affected by genomic deletion in pediatric acute lymphoblastic leukemia (ALL) patients. Here, we define BTG1 as a mediator of the cellular stress response. When challenged with cellular stressors, such as amino acid or glucose deprivation as well as drug induced Endoplasmic Reticulum (ER) stress, mouse embryonic fibroblasts (MEFs) lacking Btg1 expression show a 20-30% increased survival rate relative to wildtype cells (Figure 1). Similarly, bone marrow B-cell progenitors isolated from Btg1 knockout mice are more resistant to Asparaginase (ASNase), a drug widely used in the treatment of ALL. Activating Transcription Factor 4 (ATF4) is the master regulator of the stress response pathway that is activated upon nutrient limitation and ER stress. Importantly, loss of ATF4 function results in an enhanced survival almost identical to the effects we measured in Btg1 knockout cells. While ATF4 protein expression itself is not different between the genotypes, gene expression analysis revealed that the induction of a subset of ATF4 target genes (Ddit3, Atf3, Trib3, Gadd34, and Ndrg1) is significantly reduced in Btg1 knockout cells. As these genes are effectors of the apoptosis machinery, increased survival in the Btg1 knockout cells may reflect an attenuation of ATF4 function. We hypothesized that BTG1 complexes with ATF4 to modify its function by recruiting Protein Arginine Methyl Transferase 1 (PRMT1). This enzyme, known to cooperate with BTG1, marks its substrate proteins with a post translational modification but has not been previously implicated in the regulation of ATF4 activity. Co-immunoprecipitation experiments indeed revealed a direct interaction between BTG1 and ATF4. We used purified proteins in an in vitro methylation assay to show that ATF4 is directly methylated by PRMT1 on arginine residue 239. Expression of the mutant ATF4 R239K, which cannot be methylated, in an ATF4 knockout background resulted in reduced transcriptional activity in response to stress relative to wildtype ATF4. In addition, we aimed to mimic the effect of BTG1 loss on the regulation ATF4 function by the addition of PRMT1 inhibitor AMI-1. Treatment of cells with this selective inhibitor faithfully recapitulates BTG1 loss by attenuating the induction of ATF4 target genes upon stress. Our findings establish the interplay of BTG1-ATF4-PRMT1 as a part of the cellular stress response. Taken together, our data indicate that BTG1 is necessary to maintain normal ATF4 function under cellular stress conditions. Loss of BTG1 expression, as it occurs during lymphoid leukemia development, may therefore provide a selective advantage for leukemic cells to survive and to resist treatment at a later stage of disease. Figure 1 Btg1 is required for survival under cellular stress. Wildtype (WT) and Btg1-/- MEFs were challenged with different treatments that cause nutrient limitation and ER stress. A MTT based assay was used to study the metabolic activity of the cells as a measure of viability. The relative cell survival as compared to untreated cells (set as 100%) is shown. Bars represent average data from four independent experiments ± SEM. 2-tailed t-test was used to test for significance: * p