Challenging perspectives on the cellular origins of lymphoma.

Both B and T lymphocytes have signature traits that set them apart from other cell types. They actively and repeatedly rearrange their DNA in order to produce a unique and functional antigen receptor, they have potential for massive clonal expansion upon encountering antigen via this receptor or its precursor, and they have the capacity to be extremely long lived as 'memory' cells. All three of these traits are fundamental to their ability to function as the adaptive immune response to infectious agents, but concurrently render these cells vulnerable to transformation. Thus, it is classically considered that lymphomas arise at a relatively late stage in a lymphocyte's development during the process of modifying diversity within antigen receptors, and when the cell is capable of responding to stimulus via its receptor. Attempts to understand the aetiology of lymphoma have reinforced this notion, as the most notable advances to date have shown chronic stimulation of the antigen receptor by infectious agents or self-antigens to be key drivers of these diseases. Despite this, there is still uncertainty about the cell of origin in some lymphomas, and increasing evidence that a subset arises in a more immature cell. Specifically, a recent study indicates that T-cell lymphoma, in particular nucleophosmin-anaplastic lymphoma kinase-driven anaplastic large cell lymphoma, may originate in T-cell progenitors in the thymus.T.I.M.M. was supported by a Bloodwise Gordon Piller Studentship.This is the final version of the article. It first appeared from The Royal Society via https://doi.org/10.1098/rsob.16023

Genetic manipulation and immortalized culture of ex vivo primary human germinal center B cells.

Next-generation sequencing has transformed our knowledge of the genetics of lymphoid malignancies. However, limited experimental systems are available to model the functional effects of these genetic changes and their implications for therapy. The majority of mature B-cell malignancies arise from the germinal center (GC) stage of B-cell differentiation. Here we describe a detailed protocol for the purification and ex vivo expansion of primary, nonmalignant human GC B cells. We present methodology for the high-efficiency transduction of these cells to enable combinatorial expression of putative oncogenes. We also describe alternative approaches for CRISPR-Cas9-mediated deletion of putative tumor suppressors. Mimicking genetic changes commonly found in lymphoid malignancies leads to immortalized growth in vitro, while engraftment into immunodeficient mice generates genetically customized, synthetic models of human lymphoma. The protocol is simple and inexpensive and can be implemented in any laboratory with access to standard cell culture and animal facilities. It can be easily scaled up to enable high-throughput screening and thus provides a versatile platform for the functional interrogation of lymphoma genomic data.D.H. was personally supported by a Clinician Scientist Fellowship from the Medical Research Council (MR/M008584/1). Research in the Hodson laboratory is supported by the Kay Kendall Leukaemia Fund, The Addenbrooke’s Charitable Trust and the Evelyn Trust. The Hodson laboratory receives core funding from Wellcome (203151/Z/16/Z) and MRC to the WellcomeMRC Cambridge Stem Cell Institute and from CRUK to the CRUK Cambridge Centre (A25117

RNA binding proteins in hematopoiesis and hematological malignancy.

RNA binding proteins (RBPs) regulate fundamental processes such as differentiation and self-renewal by enabling the dynamic control of protein abundance or isoforms, or through the regulation of non-coding RNA. RBPs are increasingly appreciated as being essential for normal hematopoiesis and they are understood to play fundamental roles in hematological malignancies by acting as oncogenes or tumor suppressors. Alternative splicing has been shown to play roles in the development of specific hematopoietic lineages and sequence specific mutations in RBPs lead to dysregulated splicing in myeloid and lymphoid leukemias. RBPs that regulate translation contribute to the development and function of hematological lineages, act as nodes for the action of multiple signaling pathways and contribute to hematological malignancies. These insights broaden our mechanistic understanding of the molecular regulation of hematopoiesis and offer opportunities to develop disease biomarkers and new therapeutic modalities.We thank the Medical Research Council, The Biotechnology and Biological Sciences Research Council BBS/E/B/000C0428, Bloodwise, Cancer Research Therapeutics and Wellcome for funding research in the author’s laboratories

Molecular profiling in diffuse large B‐cell lymphoma: why so many types of subtypes?

Funder: Michael Smith Foundation for Health Research; Id: http://dx.doi.org/10.13039/501100000245Summary: The term diffuse large B‐cell lymphoma (DLBCL) includes a heterogeneous collection of biologically distinct tumours. This heterogeneity currently presents a barrier to the successful deployment of novel, biologically targeted therapies. Molecular profiling studies have recently proposed new molecular classification systems. These have the potential to resolve the biological heterogeneity of DLBCL into manageable subgroups of tumours that rely on shared oncogenic programmes. In many cases these biological programmes straddle the boundaries of our existing systems for classifying B‐cell lymphomas. Here we review the findings from these major molecular profiling studies with a specific focus on those that propose new genetic subgroups of DLBCL. We highlight the areas of consensus and discordance between these studies and discuss the implications for current clinical practice and for clinical trials. Finally, we address the outstanding challenges and solutions to the introduction of genomic subtyping and precision medicine in DLBCL

Transformational leadership: a qualitative analysis of effective leadership in women's soccer in England

This study examined perceptions of effective leadership in elite women’s soccer by team captains. Data were collected from a range of perspectives in four elite female soccer teams in England. For each of the four teams, data were collected from 6 participants (total N = 24 players). For each of the four teams, interviews were conducted with the captain and the coach as well as a focus group with 4 players regarding their perceptions of the captain’s leadership. Data were firstly deductively categorised under the four key areas of transformational leadership: idealised influence, inspirational motivation, individualised consideration and intellectual stimulation. An inductive analysis of the relevant data which did not fit into these themes revealed the importance of captains building bridges through helping to navigate the gender gap as well as to facilitate effective relationships with and between players. The implications of these findings are discussed

Application of the LymphGen classification tool to 928 clinically and genetically-characterised cases of diffuse large B cell lymphoma (DLBCL).

We recently published results of targeted sequencing applied to 928 unselected cases of DLBCL registered in the Haematological Malignancy Research Network (HMRN) registry (1). Clustering allowed us to resolve five genomic subtypes. These subtypes shared considerable overlap with those proposed in two independent genomic studies(2, 3), suggesting the potential to use genetics to stratify patients by both risk and biology. In the original studies, clustering techniques were applied to sample cohorts to reveal molecular substructure, but left open the challenge of how to classify an individual patient. This was addressed by the LymphGen classification tool (4). LymphGen assigns an individual case to one of six molecular subtypes. The tool accommodates data from exome or targeted sequencing, either with or without copy number variant (CNV) data. Separate gene expression data allows classification of a seventh, MYC-driven subtype defined by a double hit (DHL) or molecular high-grade (MHG) gene expression signature(5-7).HR was funded by a studentship from the Medical Research Council. DH was supported by a Clinician Scientist Fellowship from the Medical Research Council (MR/M008584/1). The Hodson laboratory receives core funding from Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute and core funding from the CRUK Cambridge Cancer Centre. HMRN is supported by BCUK 15037 and CRUK 18362

RNA-binding proteins ZFP36L1 and ZFP36L2 promote cell quiescence.

Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR-induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-μ at the pre-BCR checkpoint.This work was funded by the Biotechnology and Biological Sciences Research Council, a Medical Research Council CASE studentship with GSK, an MRC centenary award (A.G) and project grants from Bloodwise. DJH was supported by a Medical Research Council Clinician Scientist FellowshipThis is the author accepted manuscript. The final version is available from the American Association for the Advancement of Science via http://dx.doi.org/10.1126/science.aad597

Prolyl-4-hydroxylase 3 maintains β-cell glucose metabolism during fatty acid excess in mice

The α-ketoglutarate–dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is an HIF target that uses molecular oxygen to hydroxylate peptidyl prolyl residues. Although PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about the effects of this highly conserved enzyme in insulin-secreting β cells in vivo. Here, we show that the deletion of PHD3 specifically in β cells (βPHD3KO) was associated with impaired glucose homeostasis in mice fed a high-fat diet. In the early stages of dietary fat excess, βPHD3KO islets energetically rewired, leading to defects in the management of pyruvate fate and a shift from glycolysis to increased fatty acid oxidation (FAO). However, under more prolonged metabolic stress, this switch to preferential FAO in βPHD3KO islets was associated with impaired glucose-stimulated ATP/ADP rises, Ca(2+) fluxes, and insulin secretion. Thus, PHD3 might be a pivotal component of the β cell glucose metabolism machinery in mice by suppressing the use of fatty acids as a primary fuel source during the early phases of metabolic stress

Regulation of normal B-cell differentiation and malignant B-cell survival by OCT2.

The requirement for the B-cell transcription factor OCT2 (octamer-binding protein 2, encoded by Pou2f2) in germinal center B cells has proved controversial. Here, we report that germinal center B cells are formed normally after depletion of OCT2 in a conditional knockout mouse, but their proliferation is reduced and in vivo differentiation to antibody-secreting plasma cells is blocked. This finding led us to examine the role of OCT2 in germinal center-derived lymphomas. shRNA knockdown showed that almost all diffuse large B-cell lymphoma (DLBCL) cell lines are addicted to the expression of OCT2 and its coactivator OCA-B. Genome-wide chromatin immunoprecipitation (ChIP) analysis and gene-expression profiling revealed the broad transcriptional program regulated by OCT2 that includes the expression of STAT3, IL-10, ELL2, XBP1, MYC, TERT, and ADA. Importantly, genetic alteration of OCT2 is not a requirement for cellular addiction in DLBCL. However, we detected amplifications of the POU2F2 locus in DLBCL tumor biopsies and a recurrent mutation of threonine 223 in the DNA-binding domain of OCT2. This neomorphic mutation subtly alters the DNA-binding preference of OCT2, leading to the transactivation of noncanonical target genes including HIF1a and FCRL3 Finally, by introducing mutations designed to disrupt the OCT2-OCA-B interface, we reveal a requirement for this protein-protein interface that ultimately might be exploited therapeutically. Our findings, combined with the predominantly B-cell-restricted expression of OCT2 and the absence of a systemic phenotype in our knockout mice, suggest that an OCT2-targeted therapeutic strategy would be efficacious in both major subtypes of DLBCL while avoiding systemic toxicity.This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. DJH was supported by a Kay Kendall Leukaemia Fund Intermediate Fellowship from the UK.This is the author accepted manuscript. The final version is available from the National Academy of Sciences via http://dx.doi.org/10.1073/pnas.160055711

From dwindling ice to headwater lakes: could dams replace glaciers in the European Alps?

The potential exploitation of areas becoming ice-free in response to ongoing climate change has rarely been addressed, although it could be of interest from the water management perspective. Here we present an estimate for the potential of mitigating projected changes in seasonal water availability from melting glaciers by managing runoff through reservoirs. For the European Alps we estimate that by the end of the century, such a strategy could offset up to 65% of the expected summer-runoff changes from presently glacierized surfaces. A first-order approach suggests that the retention volume potentially available in the areas becoming deglacierized is in excess of the volume required for achieving the maximal possible mitigation by more than one order of magnitude. Obviously, however, such a strategy cannot compensate for the reduction in annual runoff caused by glacier ice depletion. Our estimates indicate that by 2070–2099, 0.73 ± 0.67 km3 a−1 of this non-renewable component of the water cycle could be missing in Alpine water supplies