The pathways regulating formation of the germinal center (GC) dark zone (DZ) and light zone (LZ) are unknown. In this study we show that FOXO1 transcription factor expression was restricted to the GC ...DZ and was required for DZ formation, since its absence in mice led to the loss of DZ gene programs and the formation of LZ-only GCs. FOXO1-negative GC B cells displayed normal somatic hypermutation but defective affinity maturation and class switch recombination. The function of FOXO1 in sustaining the DZ program involved the trans-activation of the chemokine receptor CXCR4, and cooperation with the BCL6 transcription factor in the trans-repression of genes involved in immune activation, DNA repair, and plasma cell differentiation. These results also have implications for the role of FOXO1 in lymphomagenesis because they suggest that constitutive FOXO1 activity might be required for the oncogenic activity of deregulated BCL6 expression.
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•FOXO1 expression in the germinal center is restricted to dark zone B cells•FOXO1-null mouse germinal centers lack dark zones and lose architectural polarity•FOXO1 deletion impairs affinity maturation and IgG1 class switch recombination•FOXO1 instructs the dark zone gene program directly and by licensing BCL6 activity
The factors that control germinal center polarity and cyclic reentry are unknown. Dalla-Favera and colleagues demonstrate that the transcription factor FOXO1 instructs a gene program that is required for germinal center dark zone development. Mouse germinal centers devoid of FOXO1 expression fail to support affinity maturation and class switch recombination.
Diffuse large B-cell lymphoma (DLBCL) is the most common form of human lymphoma. Although a number of structural alterations have been associated with the pathogenesis of this malignancy, the full ...spectrum of genetic lesions that are present in the DLBCL genome, and therefore the identity of dysregulated cellular pathways, remains unknown. By combining next-generation sequencing and copy number analysis, we show that the DLBCL coding genome contains, on average, more than 30 clonally represented gene alterations per case. This analysis also revealed mutations in genes not previously implicated in DLBCL pathogenesis, including those regulating chromatin methylation (MLL2; 24% of samples) and immune recognition by T cells. These results provide initial data on the complexity of the DLBCL coding genome and identify novel dysregulated pathways underlying its pathogenesis.
Inactivating mutations of the CREBBP acetyltransferase are highly frequent in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), the two most common germinal center (GC)-derived ...cancers. However, the role of CREBBP inactivation in lymphomagenesis remains unclear. Here, we show that CREBBP regulates enhancer/super-enhancer networks with central roles in GC/post-GC cell fate decisions, including genes involved in signal transduction by the B-cell receptor and CD40 receptor, transcriptional control of GC and plasma cell development, and antigen presentation. Consistently,
-deficient B cells exhibit enhanced response to mitogenic stimuli and perturbed plasma cell differentiation. Although GC-specific loss of
was insufficient to initiate malignant transformation, compound
-haploinsufficient/BCL2-transgenic mice, mimicking the genetics of FL and DLBCL, develop clonal lymphomas recapitulating the features of the human diseases. These findings establish
as a haploinsufficient tumor-suppressor gene in GC B cells and provide insights into the mechanisms by which its loss contributes to lymphomagenesis.
Loss-of-function mutations of
are common and early lesions in FL and DLBCL, suggesting a prominent role in lymphoma initiation. Our studies identify the cellular program by which reduced CREBBP dosage facilitates malignant transformation, and have direct implications for targeted lymphoma therapy based on drugs affecting CREBBP-mediated chromatin acetylation.
.
BRAF mutations in hairy-cell leukemia Tiacci, Enrico; Trifonov, Vladimir; Schiavoni, Gianluca ...
The New England journal of medicine,
06/2011, Volume:
364, Issue:
24
Journal Article
Peer reviewed
Hairy-cell leukemia (HCL) is a well-defined clinicopathological entity whose underlying genetic lesion is still obscure.
We searched for HCL-associated mutations by performing massively parallel ...sequencing of the whole exome of leukemic and matched normal cells purified from the peripheral blood of an index patient with HCL. Findings were validated by Sanger sequencing in 47 additional patients with HCL.
Whole-exome sequencing identified five missense somatic clonal mutations that were confirmed on Sanger sequencing, including a heterozygous mutation in BRAF that results in the BRAF V600E variant protein. Since BRAF V600E is oncogenic in other tumors, further analyses were focused on this genetic lesion. The same BRAF mutation was noted in all the other 47 patients with HCL who were evaluated by means of Sanger sequencing. None of the 195 patients with other peripheral B-cell lymphomas or leukemias who were evaluated carried the BRAF V600E variant, including 38 patients with splenic marginal-zone lymphomas or unclassifiable splenic lymphomas or leukemias. In immunohistologic and Western blot studies, HCL cells expressed phosphorylated MEK and ERK (the downstream targets of the BRAF kinase), indicating a constitutive activation of the RAF-MEK-ERK mitogen-activated protein kinase pathway in HCL. In vitro incubation of BRAF-mutated primary leukemic hairy cells from 5 patients with PLX-4720, a specific inhibitor of active BRAF, led to a marked decrease in phosphorylated ERK and MEK. CONCLUSIONS; The BRAF V600E mutation was present in all patients with HCL who were evaluated. This finding may have implications for the pathogenesis, diagnosis, and targeted therapy of HCL. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).
Among acute myeloid leukemia (AML) patients with a normal karyotype (CN-AML), NPM1 and CEBPA mutations define World Health Organization 2008 provisional entities accounting for approximately 60% of ...patients, but the remaining 40% are molecularly poorly characterized. Using whole-exome sequencing of one CN-AML patient lacking mutations in NPM1, CEBPA, FLT3-ITD, IDH1, and MLL-PTD, we newly identified a clonal somatic mutation in BCOR (BCL6 corepressor), a gene located on chromosome Xp11.4. Further analyses of 553 AML patients showed that BCOR mutations occurred in 3.8% of unselected CN-AML patients and represented a substantial fraction (17.1%) of CN-AML patients showing the same genotype as the AML index patient subjected to whole-exome sequencing. BCOR somatic mutations were: (1) disruptive events similar to the germline BCOR mutations causing the oculo-facio-cardio-dental genetic syndrome; (2) associated with decreased BCOR mRNA levels, absence of full-length BCOR, and absent or low expression of a truncated BCOR protein; (3) virtually mutually exclusive with NPM1 mutations; and (4) frequently associated with DNMT3A mutations, suggesting cooperativity among these genetic alterations. Finally, BCOR mutations tended to be associated with an inferior outcome in a cohort of 422 CN-AML patients (25.6% vs 56.7% overall survival at 2 years; P = .032). Our results for the first time implicate BCOR in CN-AML pathogenesis.
A significant fraction of B cell non-Hodgkin lymphomas (B-NHL) of germinal center origin carry heterozygous missense mutations in FOXO1, a member of the FOXO family of transcription factors. FOXO1 is ...a central component of the PI3K signaling cascade engaged by the B cell receptor and is essential for B cell homeostasis and survival (Dengler et al, Nat Immunol 2008; Srinivasan et al, Cell 2009; Lin et al, Nat Immunol 2010). In response to PI3K activation, AKT phosphorylates FOXO1 leading to its nuclear-cytoplasmic translocation and inactivation. Missense mutations of the FOXO1 gene are detectable in germinal center (GC)-derived B-NHL, including ~12% of Burkitt Lymphoma (BL) and ~9% of Diffuse Large B Cell Lymphoma (DLBCL) cases (Schmitz et al, Nature 2012; Trinh et al, Blood 2013; Pasqualucci et al, Cell Rep 2014). The role of FOXO1 in normal GC development as well as the contribution of its mutations to lymphomagenesis is unclear.
We show that FOXO1 expression is restricted to the dark zone of GCs, where its nuclear localization is detectable in most B cells. Mice carrying the conditional inactivation of FOXO1 in GC B cells display normal GC in number and size. However, these GCs lack phenotypically defined (CXCR4hi/CD86lo) dark zones and are entirely composed by light zone B cells (CXCR4lo/CD86hi). FOXO1-/- GC B cells express AICDA and carry a normal number of mutations in their immunonoglobulin genes, but do not undergo affinity maturation, resulting in severely impaired antigen responses.
In order to identify the biological program controlled by FOXO1 in GC B cells, we identified candidate transcriptional target genes by integrating ChIP-seq and gene expression data. These analyses showed that that the establishment of the dark zone fate relies on a FOXO1-dependent transcriptional network that is enriched for genes involved in immune signaling cascades triggered by the B cell receptor and by a variety of cytokines controlling GC polarity. Notably, a majority of these target genes are co-bound and co-regulated, in a FOXO1-dependent manner, by BCL6, a well characterized GC master regulator.
To assess the role of BL- and DLBCL-associated mutations, we first investigated the subcellular localization of FOXO1 mutant proteins by transfecting wild type and mutant GFP-tagged FOXO1 alleles into HeLa cells. As previously shown (Trinh et al, Blood 2013), this analysis showed that mutant FOXO1 proteins, but not the wild-type one, readily localize in the nucleus. Analogously, immunofluorescence analysis of BL and DLBCL samples showed the presence of nuclear FOXO1 in all tumors carrying mutations in the FOXO1 gene. However, nuclear localization was also detectable in virtually all cases carrying normal FOXO1 genes. Accordingly, in vitro experiments testing the ability of normal and mutated FOXO1 proteins to respond to various signals activating the PI3K pathway in multiple BL and DLBCL cell lines, failed to display a correlation between the presence of mutations and responsiveness to these signals. Taken together, these results suggest that other mechanisms in addition to direct gene mutation are responsible for the constitutive nuclear localization of FOXO1 in tumors. We are now examining the consequences of FOXO1 missense mutations in vivo, by reconstituting FOXO1-/- GC B cells with FOXO1 mutants using bone marrow chimeras.
No relevant conflicts of interest to declare.
Abstract 71
Acute myeloid leukemia (AML) with normal cytogenetics (CN-AML) represents about half of all adult AML. NPM1 and CEBPA mutations define WHO provisional entities accounting for ∼60% of ...CN-AML, but the remaining cases (∼40%) remain poorly characterized. To address this issue, we carried out whole-exome-sequencing (WES) of leukemic and normal cells from one patient with CN-AML that lacked mutations in NPM1, CEBPA, FLT3-ITD, and MLL-PTD. Using this approach, we identified a clonal somatic mutation of BCOR, a gene located on chromosome Xp11.4, that was present in the leukemic but not normal cells of the index AML case. The BCOR (BCL6 co-repressor) gene encodes for an ubiquitously expressed nuclear protein that is involved in repressing the activity of BCL6 and other transcriptional factors. BCOR is a key transcriptional regulator of early embryonic development, mesenchymal stem cell function and hemopoiesis. Germline mutations of BCOR are responsible for the oculo-facio-cardio-dental (OFCD) genetic syndrome that is inherited in an X-linked pattern and comprises microphtalmia, dysmorphic appearance, dental abnormalities (radiculomegaly), hammer-toe deformity and cardiac defects. WES findings in the index case were subsequently validated and further studied in a total cohort of 514 AML patients. We first performed deep-sequencing analyses of all exons of the BCOR gene in an initial set of 82 AML cases that were selected because they showed the same genetic characteristics of our index patient (i.e. normal karyotype without NPM1, CEBPA, FLT3-ITD and MLL-PTD mutations). Disruptive BCOR mutations (i.e., nonsense mutations, out-of-frame small indels, and consensus splice-site mutations) were detected in 14/82 (17.1%) of these cases. We next assessed the frequency of BCOR mutations in a series of unselected CN-AML patients (n=262) and found that they occurred in 4.2% of cases, mostly showing the typical features of BCOR-mutated cases (absence of NPM1, CEBPA, FLT3-ITD and MLL-PTD mutations). Almost mutual exclusion of BCOR and NPM1 mutations was further confirmed in a separate series of 71 NPM1-mutated only AML patients. No BCOR mutations were observed in the 89 AML cases with recurrent cytogenetic abnormalities investigated, including t(8;21)(q22;q22) (n= 29), inv(16)(p13q22) (n=40), t(15;17)(q22;q12) (n=10), and t(11q23)/MLL (n=10), and in the 10 patients with double CEBPA-mutated AML studied. BCOR mutations were: i) scattered across the whole length of the coding sequence with no hotspots identified; ii) somatic in origin and disruptive molecular events similar to germline BCOR mutations causing the OFCD genetic syndrome; iii) associated with markedly decreased BCOR mRNA levels, absence of full-length BCOR and absent or low expression of a truncated BCOR protein; iv) almost mutually exclusive with NPM1 (only 1.5% of the 197 NPM1-mutated AML investigated carried BCOR mutations); v) rarely associated with FLT3-ITD; and vi) frequently associated with DNMT3A and RUNX1 mutations, suggesting cooperation with the respective mutated pathways. Clinically, BCOR mutations correlated with poor outcome among the cohort of 160 CN-AML patients evaluated (28.0% versus 66.3% overall survival at 2 years, P=0.024). We also searched for BCOR mutations in the human AML cell lines OCI-AML2, OCI-AML3, KG1a, U937, HL-60, HL-60R, HB4, AML193, and MVP-11. Only HL-60 and HL-60R (a ATRA-resistant derivative of HL-60) carried a BCOR mutation that consisted of a hemizygous G to T transition at position 4616 in exon 10, leading to the Glu1442X nonsense mutation. Western blot analysis of HL-60 cells resulted in the absence of the full-length BCOR protein (predicted MW: 192 kDa) and presence of a low intensity 156 kDa band likely corresponding to a truncated BCOR protein. In conclusion, our results implicate for the first time BCOR in the pathogenesis of CN-AML and suggest it may act as tumor suppressor gene.
Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
Background
Patients often require a rapid sequence induction (RSI) endotracheal intubation technique during emergencies or electively to protect against aspiration, increased intracranial pressure, ...or to facilitate intubation. Traditionally succinylcholine has been the most commonly used muscle relaxant for this purpose because of its fast onset and short duration; unfortunately, it can have serious side effects. Rocuronium has been suggested as an alternative to succinylcholine for intubation. This is an update of our Cochrane review published first in 2003 and then updated in 2008 and now in 2015.
Objectives
To determine whether rocuronium creates intubating conditions comparable to those of succinylcholine during RSI intubation.
Search methods
In our initial review we searched all databases until March 2000, followed by an update to June 2007. This latest update included searching the Cochrane Central Register of Controlled Trials (CENTRAL; 2015, Issue 2), MEDLINE (1966 to February Week 2 2015), and EMBASE (1988 to February 14 2015 ) for randomized controlled trials (RCTs) or controlled clinical trials (CCTs) relating to the use of rocuronium and succinylcholine. We included foreign language journals and handsearched the references of identified studies for additional citations.
Selection criteria
We included any RCT or CCT that reported intubating conditions in comparing the use of rocuronium and succinylcholine for RSI or modified RSI in any age group or clinical setting. The dose of rocuronium was at least 0.6 mg/kg and succinylcholine was at least 1 mg/kg.
Data collection and analysis
Two authors (EN and DT) independently extracted data and assessed methodological quality for the 'Risk of bias' tables. We combined the outcomes in Review Manager 5 using a risk ratio (RR) with a random‐effects model.
Main results
The previous update (2008) had identified 53 potential studies and included 37 combined for meta‐analysis. In this latest update we identified a further 13 studies and included 11, summarizing the results of 50 trials including 4151 participants. Overall, succinylcholine was superior to rocuronium for achieving excellent intubating conditions: RR 0.86 (95% confidence interval (CI) 0.81 to 0.92; n = 4151) and clinically acceptable intubation conditions (RR 0.97, 95% CI 0.95 to 0.99; n = 3992, 48 trials). A high incidence of detection bias amongst the trials coupled with significant heterogeneity provides moderate‐quality evidence for these conclusions, which are unchanged from the previous update. Succinylcholine was more likely to produce excellent intubating conditions when using thiopental as the induction agent: RR 0.81 (95% CI: 0.73 to 0.88; n = 2302, 28 trials). In the previous update, we had concluded that propofol was the superior induction agent with succinylcholine. There were no reported incidences of severe adverse outcomes. We found no statistical difference in intubation conditions when succinylcholine was compared to 1.2 mg/kg rocuronium; however, succinylcholine was clinically superior as it has a shorter duration of action.
Authors' conclusions
Succinylcholine created superior intubation conditions to rocuronium in achieving excellent and clinically acceptable intubating conditions.
Effective delivery of protein therapeutics to the central nervous system (CNS) has been greatly restricted by the blood-brain barrier (BBB). We describe the development of a BBB transport vehicle ...(TV) comprising an engineered Fc fragment that exploits receptor-mediated transcytosis for CNS delivery of biotherapeutics by binding a highly expressed brain endothelial cell target. TVs were engineered using directed evolution to bind the apical domain of the human transferrin receptor (hTfR) without the use of amino acid insertions, deletions, or unnatural appendages. A crystal structure of the TV-TfR complex revealed the TV binding site to be away from transferrin and FcRn binding sites, which was further confirmed experimentally in vitro and in vivo. Recombinant expression of TVs fused to anti-β-secretase (BACE1) Fabs yielded antibody transport vehicle (ATV) molecules with native immunoglobulin G (IgG) structure and stability. Peripheral administration of anti-BACE1 ATVs to hTfR-engineered mice and cynomolgus monkeys resulted in substantially improved CNS uptake and sustained pharmacodynamic responses. The TV platform readily accommodates numerous additional configurations, including bispecific antibodies and protein fusions, yielding a highly modular CNS delivery platform.
Children and adolescents with juvenile idiopathic arthritis (JIA) are less physically active than their healthy peers and are at high risk of missing out on the general health benefits of physical ...activity. Wearable activity trackers are a promising option for intervening in this population with potential advantages over traditional exercise prescriptions. The objectives of this study were to: (1) determine the feasibility of a wearable activity tracker intervention in adolescents with JIA; and (2) estimate the variability in response to a wearable activity tracker intervention on the physical activity levels of adolescents with JIA.
Participants aged 12-18 years with JIA were recruited during their routine rheumatology clinic visits at a tertiary care hospital. Participants completed the 3-Day Physical Activity Recall self-reported questionnaire at baseline, 1 week and 5 week follow-up. At the 1 week follow up, participants were instructed to start wearing an activity tracker for 28 consecutive days. Participants completed a feasibility questionnaire at their end of study visit. Participant demographics, adherence rates and feasibility outcomes were summarized using descriptive statistics. The effect of wearing a tracker on moderate-to-vigorous physical activity (MVPA) and total metabolic equivalents (METs) per day were analyzed using a paired t-test.
Twenty-eight participants (74% female; median age 15.1, range 12.8-18.6) were included in the analysis. All of the participants were able to synchronize the activity tracker to a supported device, use the activity tracker correctly and complete the study measurements. On average, participants had activity logged on their smartphone application for 72% of the intervention period. The standard deviation of the change in mean METs/day was 12.148 and for mean MVPA blocks/day was 3.143 over the study period.
Wrist worn activity tracking is a feasible intervention for adolescent patients with JIA. More research is needed to examine the effect of activity tracking on physical activity levels.
Not an applicable clinical device trial as per the criteria listed on ClinicalTrials.gov as the primary objective is feasibility.
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