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Pharmacologic inhibition of LSD1 induces molecular and morphologic differentiation of blast cells in acute myeloid leukemia (AML) patients harboring MLL gene translocations. In addition to its demethylase activity, LSD1 has a critical scaffolding function at genomic sites occupied by the SNAG domain transcription repressor GFI1. Importantly, inhibitors block both enzymatic and scaffolding activities, in the latter case by disrupting the protein:protein interaction of GFI1 with LSD1. To explore the wider consequences of LSD1 inhibition on the LSD1 protein complex we applied mass spectrometry technologies. We discovered that the interaction of the HMG-box protein HMG20B with LSD1 was also disrupted by LSD1 inhibition. Downstream investigations revealed that HMG20B is co-located on chromatin with GFI1 and LSD1 genome-wide; the strongest HMG20B binding co-locates with the strongest GFI1 and LSD1 binding. Functional assays demonstrated that HMG20B depletion induces leukemia cell differentiation and further revealed that HMG20B is required for the transcription repressor activity of GFI1 through stabilizing LSD1 on chromatin at GFI1 binding sites. Interaction of HMG20B with LSD1 is through its coiled-coil domain. Thus, HMG20B is a critical component of the GFI1:LSD1 transcription repressor complex which contributes to leukemia cell differentiation block.

Tumour-associated oncogenes induce unscheduled proliferation as well as genomic and chromosomal instability. According to current models, therapeutic strategies that block oncogene activity are likely to selectively target tumour cells. However, recent evidences have revealed that oncogenes are only essential for the proliferation of some specific tumour cell types, but not all. Indeed, the latest studies of the interactions between the oncogene and its target cell have shown that oncogenes contribute to cancer development not only by inducing proliferation but also by developmental reprogramming of the epigenome. This provides the first evidence that tumorigenesis can be initiated by stem cell reprogramming, and uncovers a new role for oncogenes in the origin of cancer. Here we analyse these evidences and propose an updated model of oncogene function that can explain the full range of genotype-phenotype associations found in human cancer. Finally, we discuss how this vision opens new avenues for developing novel anti-cancer interventions. © 2013 European Molecular Biology Organization., Research in ISG group is partially supported by FEDER and by MICINN (SAF2012‐32810), by MEC OncoBIO Consolider‐Ingenio 2010 (Ref. CSD2007‐0017), by NIH grant (R01 CA109335‐04A1), the ARIMMORA project (FP7‐ENV‐2011, European Union Seventh Framework Program) and by proyecto en red de investigación en celulas madre tumourales supported by Obra Social Kutxa y Conserjería de Sanidad de la Junta de Castilla y Leon. Research at CC's lab was partially supported by FEDER, and from an institutional grant from the Fundacion Ramon Areces. All Spanish funding is co‐sponsored by the European Union FEDER program. ISG is an API lab of the EuroSyStem project.

Understanding the cellular origin of cancer can help to improve disease prevention and therapeutics. Human plasma cell neoplasias are thought to develop from either differentiated B cells or plasma cells. However, when the expression of Maf oncogenes (associated to human plasma cell neoplasias) is targeted to mouse B cells, the resulting animals fail to reproduce the human disease. Here, to explore early cellular changes that might take place in the development of plasma cell neoplasias, we engineered transgenic mice to express MafB in haematopoietic stem/progenitor cells (HS/PCs). Unexpectedly, we show that plasma cell neoplasias arise in the MafB-transgenic mice. Beyond their clinical resemblance to human disease, these neoplasias highly express genes that are known to be upregulated in human multiple myeloma. Moreover, gene expression profiling revealed that MafB-expressing HS/PCs were more similar to B cells and tumour plasma cells than to any other subset, including wild-type HS/PCs. Consistent with this, genome-scale DNA methylation profiling revealed that MafB imposes an epigenetic program in HS/PCs, and that this program is preserved in mature B cells of MafB-transgenic mice, demonstrating a novel molecular mechanism involved in tumour initiation. Our findings suggest that, mechanistically, the haematopoietic progenitor population can be the target for transformation in MafB-associated plasma cell neoplasias.

Follicular lymphoma (FL) is incurable with conventional therapies and has a clinical course typified by multiple relapses after therapy. These tumors are genetically characterized by B-cell leukemia/lymphoma 2 (BCL2) translocation and mutation of genes involved in chromatin modification. By analyzing purified tumor cells, we identified additional novel recurrently mutated genes and confirmed mutations of one or more chromatin modifier genes within 96% of FL tumors and two or more in 76% of tumors. We defined the hierarchy of somatic mutations arising during tumor evolution by analyzing the phylogenetic relationship of somatic mutations across the coding genomes of 59 sequentially acquired biopsies from 22 patients. Among all somatically mutated genes, CREBBP mutations were most significantly enriched within the earliest inferable progenitor. These mutations were associated with a signature of decreased antigen presentation characterized by reduced transcript and protein abundance of MHC class II on tumor B cells, in line with the role of CREBBP in promoting class II transactivator (CIITA)-dependent transcriptional activation of these genes. CREBBP mutant B cells stimulated less proliferation of T cells in vitro compared with wild-type B cells from the same tumor. Transcriptional signatures of tumor-infiltrating T cells were indicative of reduced proliferation, and this corresponded to decreased frequencies of tumor-infiltrating CD4 helper T cells and CD8 memory cytotoxic T cells. These observations therefore implicate CREBBP mutation as an early event in FL evolution that contributes to immune evasion via decreased antigen presentation., This work was supported by grants from the Lymphoma Research Foundation (A.A.A.), the Leukemia and Lymphoma Society (Specialized Center of Research Excellence Program), the NIH (S10 RR02933801, R01 CA151748), the Albert and Mary Yu Gift Fund, and the Evelyn Leung Gift Fund. Research in the I.S.-G. group is supported partially by Federacion Espanola de Enfermedades Raras (FEDER) and The Ministerio de Ciencia e Innovacion (MICINN) (SAF2009-0883 and SAF2012-32810). M.R.G. is a Special Fellow of the Leukemia and Lymphoma Society. R.L. is an American Cancer Society Clinical Research Professor. A.A.A. is a Doris Duke Charitable Foundation Clinical Investigator.

PMCID: PMC3545406.-- et al., The human germinal centre-associated lymphoma gene is specifically expressed in germinal centre B-lymphocytes and germinal centre-derived B-cell lymphomas, but its function is largely unknown. Here we demonstrate that human germinal centre-associated lymphoma directly binds to Syk in B cells, increases its kinase activity on B-cell receptor stimulation and leads to enhanced activation of Syk downstream effectors. To further investigate these findings in vivo, human germinal centre-associated lymphoma transgenic mice were generated. Starting from 12 months of age these mice developed polyclonal B-cell lymphoid hyperplasia, hypergammaglobulinemia and systemic reactive amyloid A (AA) amyloidosis, leading to shortened survival. The lymphoid hyperplasia in the human germinal centre-associated lymphoma transgenic mice are likely attributable to enhanced B-cell receptor signalling as shown by increased Syk phosphorylation, ex vivo B-cell proliferation and increased RhoA activation. Overall, our study shows for the first time that the germinal centre protein human germinal centre-associated lymphoma regulates B-cell receptor signalling in B-lymphocytes which, without appropriate control, may lead to B-cell lymphoproliferation., National Institutes of Health (NIH) grants NIH CA109335 and NIH CA122105; the Dwoskin Family Foundations; NIH P01 CA34233 FEDER and by MICINN (SAF2009-08803 and SAF2012-32810 to ISG); Junta de Castilla y León (REF. CSI007A11-2 and Proyecto Biomedicina 2009-2010); MEC OncoBIO Consolider-Ingenio 2010 (Ref. CSD2007-0017); Sandra Ibarra Foundation; Group of Excellence Grant (GR15) from Junta de Castilla y Leon; the ARIMMORA project (FP7-ENV-2011, European Union Seventh Framework Programme)

Multiple myeloma (MM) is a human cancer of malignant plasma cells. Recent genomic studies have started to catalog all the genetic alterations in selected panels of MM samples, and, within a short period of time, the large majority of MM-associated genetic and transcriptional alterations will have been identified. These recent insights, on one hand, indicate that MMs can harbor extensive clonal heterogeneity, and, on the other hand, they will help to reconstruct the molecular ontogeny of the disease’s evolution (i.e., the clonal architecture of each individual MM). However, a pending challenge is to delineate the stages of hematopoietic differentiation at which the individual genetic alterations arise, or, in other words, to elucidate the cellular architecture of MM. In the classical view of the initiation and progression of MM, an initiating genetic alteration is required to immortalize a plasma cell (Fig. 1A). Such cell is then destined to acquire additional genetics hits over time. The acquisition of additional hits further deregulates the behavior of the plasma cells, thus leading to the clinically recognized features of MM, including the lytic bone lesions. In this classical model, MM is therefore a disease arising from mutations occurring in an already differentiated plasma cell. However, in the last years it has been shown that cellular heterogeneity is also a feature of human MM. Indeed, by studying the engraftment of primary MM specimens in immunodeficient mice, it was found that tumor plasma cells were unable of engraftment following intravenous injection,1 suggesting that the MM-propagating/maintaining cell was not a tumor plasma cell. Therefore, an essential gap that must be covered in order to define the cellular architecture of MM is the identification of the nature of the MM cell of origin. In fact, in spite of the assumptions of the above-mentioned classical view of MM origin, very little is still known about the cancer cell of origin from which MM arises and about how the initial genetic lesion(s) regulate this essential step. These initial genetic lesions have always been detected in differentiated tumoral plasma cells, although the presence of a human myeloma stem cell was postulated many years ago.2 It is true that the patient’s hematopoietic progenitor and stem cells (HS/PCs) seem not to show any of the initiating genetic alterations detected in tumor plasma cells; however, it is also true that these alterations would be very difficult to detect if the frequency of these putative stem cells harboring the MM-inducing genetic lesion was low. Therefore, due to this purely statistical reason, experimental results would seem to suggest that the MM cell of origin is not a stem/progenitor cell. However, until now, all the experiments in which the expression of human MM-linked oncogenes is targeted to the mouse differentiated B cell compartment have failed to reproduce the human disease in mice. Therefore, it is potentially possible that in human patients, the occurrence of MM-associated oncogenic alterations might happen in the HS/PCs compartment, and this cell of origin adopts/acquires afterwards a MM cell fate as a consequence of the oncogene activity. Figure 1. Cellular architecture and multiple myeloma (MM) development. (A) Current working model for the development of MM in humans. In the current view of the initiation and progression of MM, an initiating hit is required to immortalize a plasma ... To elucidate if MM is a stem cell-driven tissue, we initially focused on the effects of the MafB oncogene, since the MAF family of proteins seems to be implicated in 50% of human MM. In consequence, we have developed mice in which we limited MafB expression to the mouse HS/PCs Sca1+ cells.3 In human MM and in most animal models of cancer,4 the oncogenic alterations are expressed in all the cellular types that compose the tumoral tissue, from the MM stem cell to the terminal differentiated plasma cells. In our stem cell-driven Sca1-MafB model, the expression of MafB is restricted to the HS/PCs compartment, but this restricted expression pattern is nevertheless capable of generating a full-blown MM with all its differentiated cellular components, in such a way that Sca1-MafB mice share both major and minor diagnostic criteria with the human pathology. The demonstration that MM development can be established in mice by expressing MafB only in Sca1+ cells implies that MafB can impose a gene regulatory state in these stem cells that somehow persists during hematopoiesis, and that can program a tumor plasma cell phenotype symptomatic of MM. This is an observation that seems to apply to other cancer-initiating gene defects.5-10 In the oncogenic reprogramming model here presented, the reprogrammed Sca1+ population can nevertheless complete a multi-stage differentiation pathway involving an initial commitment to the B cell lineage and a subsequent differentiation to tumor plasma cells.3 This model of MM is very informative with respect to the fact that the oncogenic mutations can have different roles in MM stem cells vs. differentiated tumor plasma cells. Thus, genetically distinct MM stem cell subclones may exist on top of each MM tumor mass, revealing an even higher complex cellular composition of MM than initially thought (Fig. 1B). In order to gain even more insight into the mechanism by which MafB can induce the reprogramming of stem cells into tumor plasma cells, we have also generated in vivo genome-scale maps of DNA methylation from both stem cells and mature B cells from Sca1-MafB mice.3 We found that a substantial number of CpG islands and promoters are specifically hypermethylated or hypomethylated in the stem cells of Sca1-MafB mice, setting a pattern inherited throughout B cell development. Thus, the results presented in our study demonstrate a novel molecular mechanism involved in tumor initiation, by showing that HS/PCs can be epigenetically reprogrammed to give rise to terminally differentiated tumor plasma cells by MafB.3 To our knowledge, these results represent the most convincing evidence to date that MM development can arise and be driven by a tumor cell fate change within the stem cells. These findings represent a novel paradigm in MM biology and will revolutionize the current modeling of MM tumorigenesis and progression and are likely to have profound therapeutic implications (Fig. 1C).

Editorial.

This is an open access article., The latest scientific findings in the field of cancer research are redefining our understanding of the molecular and cellular basis of the disease, moving the emphasis toward the study of the mechanisms underlying the alteration of the normal processes of cellular differentiation. The concepts best exemplifying this new vision are those of cancer stem cells and tumoral reprogramming. The study of the biology of acute lymphoblastic leukemias (ALLs) has provided seminal experimental evidences supporting these new points of view. Furthermore, in the case of B-cells, it has been shown that all the stages of their normal development show a tremendous degree of plasticity, allowing them to be reprogrammed to other cellular types, either normal or leukemic. Here we revise the most recent discoveries in the fields of B-cell developmental plasticity and B-ALL research, and discuss their interrelationships and their implications for our understanding of the biology of the disease. © 2011 Landes Bioscience., Research in C.C. lab was partially supported by FEDER, Fondo de Investigaciones Sanitarias (PI080164), CSIC P.I.E., 200920I055 and 201120E060, from the ARIMMORA project (FP7-ENV-2011, European Union Seventh Framework Program) and from an institutional grant from the “Fundación Ramón Areces.” Research in I.S.G. group was partially supported by FEDER and by MICINN (SAF2009-08803 to I.S.G.), by Junta de Castilla y León (REF. CSI007A11-2 and Proyecto Biomedicina 2009–2010), by MEC OncoBIO Consolider-Ingenio 2010 (Ref. CSD2007-0017), by NIH grant (R01 CA109335-04A1), by Sandra Ibarra Foundation, by Group of Excellence Grant (GR15) from Junta de Castilla y Leon, and the ARIMMORA project (FP7-ENV-2011, European Union Seventh Framework Program) and by Proyecto en Red de Investigación en Celulas Madre Tumorales en Cancer de Mama, supported by Obra Social Kutxa y Conserjería de Sanidad de la Junta de Castilla y Leon. All Spanish funding is co-sponsored by the European Union FEDER program. I.S.G. is an API lab of the EuroSyStem project. E.C.S. and M.B.D. are recipients of JAE-predoc fellowships from CSIC. I.R.C. is recipient of a FPU fellowship from MICINN. E.C.S. is recipient of a “Residencia de Estudiantes” fellowship.

Abstract 438 The LMO2 is a cysteine-rich protein containing two zinc binding LIM domains indirectly regulating gene expression by mediating protein-protein interactions with other transcriptional factors (TFs), facilitating the formation of multipartite DNA-binding complexes. It is mainly expressed in endothelial and hematopoietic cells forming cell type specific complexes containing LDB1, TAL1, E2A and GATA2 proteins in endothelial cells and hematopoietic stem cells and LDB1, TAL-1, GATA1 and E proteins in the erythroid lineage. In these cells LMO2 is involved in angiogenesis and erythroid hematopoiesis. In T cells where LMO2 is only expressed in immature CD4/CD8 double-negative thymocytes, the LMO2 complex consist of LDB1, TAL1 and E2A, but may also bind to GATA3. Aberrant expression of LMO2 in T cells induces leukemogenesis. In the B cells, LMO2 is specifically up regulated in Germinal Center (GC) B cells. LMO2 is also expressed in GC-derived non-Hodgkin's lymphomas and is one of the most powerful survival predictors in DLBCL patients. However, its function in GC B cells and DLBCL is currently unknown. In the present study we aimed to characterize the LMO2 transcriptome and interactome in DLBCL cells. Gene expression arrays were performed in Rck8 cells expressing low levels of endogenous LMO2, in which LMO2 was stably overexpressed to levels observed in GCB-like DLBCL. A total of 311 differentially expressed genes (DEGs) between control cell lines transfected with mock vector and LMO2 stably transfected samples were identified at FDR of 0.05. Sixty-four genes were down-regulated by LMO2 transfection, while 247 were up-regulated. Prominent amongst these were 27 genes encoding proteins of the histone cluster 1, colocalized on chromosome 6 and consistently higher expressed in LMO2 expressing cell lines. At the same time, multiple cell-cycle-related genes were also up-regulated by LMO2 transfection, including centromere proteins (CENPE, CENPI, and CENPN), the kinetochore associated protein NDC80 and the mitotic protein CDC25C. Expression of several randomly selected genes (SPIC, LAX1, DLEU2, DOCK3, CHND2 and TNFRSF9) was validated by real time PCR and confirmed the observed gene expression changes upon LMO2 overexpression. To obtain a higher-level view of expression changes, we compared the list of DEGs to Gene Ontology (GO) categories. This revealed significant induction of genes involved in chromosome, nucleosome, and chromatin and protein-DNA complex assembly. We screened our microarray data against the Broad Institute Molecular Signatures Database, to identify TFs whose target genes were significantly up- or down-regulated by stable LMO2 transfection. This candidate list was filtered to include only those TFs whose target genes significantly overlapped with genes which had previously identified binding motifs for LMO2 complexes in their promoter sequences. Our analysis identified Sp1, NFAT, Elk1, and LEF1 as potential novel LMO2 co-factors, but not classical TAL1 and GATA partner proteins. We examined the expression of previously reported and new potential interaction partners of LMO2 in DLBCL cell lines and GC lymphocytes by Western blotting and analyzed the interaction of the expressed proteins with LMO2 by co-immunprecipitation. We identified that in DLBCL the LMO2 complex contains some of the traditional partners such as LDB1, E2A, HEB, lyl1 and ETO2, but not TAL1 or GATA proteins. Furthermore, we demonstrated LMO2 interaction also with SP1, ELK1, NFATc1 and LEF1 proteins. All the identified LMO2 interacting partners are also expressed in GC B lymphocytes, suggesting the presence of a similar protein complex in both normal and malignant B cells. LMO2 interaction with LEF protein affected its transcriptional activity, as measured by reporter assays. A Chromatin immunoprecipitation assay using promoter region of the DLEU2 gene, whose expression was up-regulated by LMO2 expression, confirmed the direct and specific LMO2 binding to a region harboring E2A and a SP1 binding sites. Overall, our studies identified an LMO2 transcriptome in DLBCL as well as its interactome, which contains novel previously unknown interacting partners. These findings suggest that LMO2 may affect unique and novel cellular functions in GC lymphocytes which may potentially contribute to DLBCL pathogenesis and are currently being investigated in our laboratory. Disclosures: No relevant conflicts of interest to declare.