Epigenetic Deregulation in Human Primary Immunodeficiencies.

Primary immunodeficiencies (PIDs) are immune disorders resulting from defects in genes involved in immune regulation, and manifesting as an increased susceptibility to infections, autoimmunity, and cancer. However, the molecular basis of some prevalent entities remains poorly understood. Epigenetic control is essential for immune functions, and epigenetic alterations have been identified in different PIDs, including syndromes such as immunodeficiency-centromeric-instability-facial-anomalies, Kabuki, or Wolf-Hirschhorn, among others. Although the epigenetic changes may differ among these PIDs, the reversibility of epigenetic modifications suggests that they might become potential therapeutic targets. Here, we review recent mechanistic advances in our understanding of epigenetic alterations associated with certain PIDs, propose that a fully epigenetically driven mechanism might underlie some PIDs, and discuss the possible prophylactic and therapeutic implications.

International meeting on Wolf-Hirschhorn syndrome: Update on the nosology and new insights on the pathogenic mechanisms for seizures and growth delay.

"An International Meeting on Wolf-Hirschhorn Syndrome (WHS)" was held at The University Hospital La Paz in Madrid, Spain (October 13-14, 2017). One hundred and twenty-five people, including physicians, scientists and affected families, attended the meeting. Parent and patient advocates from the Spanish Association of WHS opened the meeting with a panel discussion to set the stage regarding their hopes and expectations for therapeutic advances. In keeping with the theme on therapeutic development, the sessions followed a progression from description of the phenotype and definition of therapeutic endpoints, to definition of genomic changes. These proceedings will review the major points of discussion.

Novel ETV6-RUNX1 Mouse Model to Study the Role of ELF-MF in Childhood B-Acute Lymphoblastic Leukemia: a Pilot Study.

Exposure to extremely low-frequency magnetic fields (ELF-MFs) has been classified by the International Agency for Research on Cancer (IARC) as "possibly carcinogenic to humans," based on limited scientific evidence concerning childhood leukemia. This assessment emphasized the lack of appropriate animal models recapitulating the natural history of this disease. Childhood B-cell acute lymphoblastic leukemia (B-ALL) is the result of complex interactions between genetic susceptibility and exposure to exogenous agents. The most common chromosomal alteration is the ETV6-RUNX1 fusion gene, which confers a low risk of developing the malignancy by originating a preleukemic clone requiring secondary hits for full-blown disease to appear. To develop potential prophylactic interventions, we need to identify the environmental triggers of the second hit. Recently, we generated a B-ALL mouse model of the human ETV6-RUNX1+ preleukemic state. Here, we present the results from the ARIMMORA pilot study, obtained by exposing 34 Sca1-ETV6-RUNX1 mice (vs. 27 unexposed) to a 50 Hz magnetic field of 1.5 mT with both fundamental and harmonic content, with an on/off cycle of 10 min/5 min, for 20 h/day, from conception until 3 months of age. Mice were monitored until 2 years of age and peripheral blood was periodically analyzed by flow cytometry. One of the exposed mice developed B-ALL while none of the non-exposed did. Although the results are statistically non-significant due to the limited number of mice used in this pilot experiment, overall, the results show that the newly developed Sca1-ETV6-RUNX1 mouse can be successfully used for ELF-MF exposure studies about the etiology of childhood B-ALL. Bioelectromagnetics. 2019;40:343-353. © 2019 Bioelectromagnetics Society.

Tumoral reprogramming: Plasticity takes a walk on the wild side.

Cellular plasticity is the capacity that cells have to change their fate and adopt a new identity. Plasticity is essential for normal development and for tissue regeneration and, in an experimental setting, for the induction of pluripotency. All these processes involve a reprogramming of the cellular identity, mediated by signals from the environment and/or by internal changes at the transcriptional and epigenetic levels. Tumorigenesis is a process in which normal cells acquire a new malignant identity and give rise to a clonal aberrant population. This is only possible if the initiating cell has the necessary plasticity to undergo such changes, and if the oncogenic event(s) initiating cancer has the essential reprogramming capacity so as to be able to lead a change in cellular identity. The molecular mechanisms underlying tumoral reprogramming are the pathological counterparts of the normal processes regulating developmental plasticity or experimentally-induced reprogramming. In this review we will first revise the main features of non-pathological examples of reprogramming, and then we will describe the parallelisms with tumoral reprogramming, and we will also delineate how the precise knowledge of the reprogramming mechanisms offers the potential for the development of new therapeutical interventions. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.

Expression of MALT1 oncogene in hematopoietic stem/progenitor cells recapitulates the pathogenesis of human lymphoma in mice.

Chromosomal translocations involving the MALT1 gene are hallmarks of mucosa-associated lymphoid tissue (MALT) lymphoma. To date, targeting these translocations to mouse B cells has failed to reproduce human disease. Here, we induced MALT1 expression in mouse Sca1(+)Lin(-) hematopoietic stem/progenitor cells, which showed NF-κB activation and early lymphoid priming, being selectively skewed toward B-cell differentiation. These cells accumulated in extranodal tissues and gave rise to clonal tumors recapitulating the principal clinical, biological, and molecular genetic features of MALT lymphoma. Deletion of p53 gene accelerated tumor onset and induced transformation of MALT lymphoma to activated B-cell diffuse large-cell lymphoma (ABC-DLBCL). Treatment of MALT1-induced lymphomas with a specific inhibitor of MALT1 proteolytic activity decreased cell viability, indicating that endogenous Malt1 signaling was required for tumor cell survival. Our study shows that human-like lymphomas can be modeled in mice by targeting MALT1 expression to hematopoietic stem/progenitor cells, demonstrating the oncogenic role of MALT1 in lymphomagenesis. Furthermore, this work establishes a molecular link between MALT lymphoma and ABC-DLBCL, and provides mouse models to test MALT1 inhibitors. Finally, our results suggest that hematopoietic stem/progenitor cells may be involved in the pathogenesis of human mature B-cell lymphomas.

Preclinical models for prediction of immunotherapy outcomes and immune evasion mechanisms in genetically heterogeneous multiple myeloma.

The historical lack of preclinical models reflecting the genetic heterogeneity of multiple myeloma (MM) hampers the advance of therapeutic discoveries. To circumvent this limitation, we screened mice engineered to carry eight MM lesions (NF-κB, KRAS, MYC, TP53, BCL2, cyclin D1, MMSET/NSD2 and c-MAF) combinatorially activated in B lymphocytes following T cell-driven immunization. Fifteen genetically diverse models developed bone marrow (BM) tumors fulfilling MM pathogenesis. Integrative analyses of ∼500 mice and ∼1,000 patients revealed a common MAPK-MYC genetic pathway that accelerated time to progression from precursor states across genetically heterogeneous MM. MYC-dependent time to progression conditioned immune evasion mechanisms that remodeled the BM microenvironment differently. Rapid MYC-driven progressors exhibited a high number of activated/exhausted CD8+ T cells with reduced immunosuppressive regulatory T (Treg) cells, while late MYC acquisition in slow progressors was associated with lower CD8+ T cell infiltration and more abundant Treg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8+ T cells versus Treg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8+ T/Treg cell ratios underlie early progression in untreated smoldering MM, and correlated with early relapse in newly diagnosed patients with MM under Len/Dex therapy. In ICB-refractory MM models, increasing CD8+ T cell cytotoxicity or depleting Treg cells reversed immunotherapy resistance and yielded prolonged MM control. Our experimental models enable the correlation of MM genetic and immunological traits with preclinical therapy responses, which may inform the next-generation immunotherapy trials.

Cancer as a reprogramming-like disease: implications in tumor development and treatment.

Cancer is a clonal malignant disease originated in a single cell and characterized by the accumulation of partially differentiated cells that are phenotypically reminiscent of normal stages of differentiation. Given the fact that human cancer is diagnosed at later stages and cannot be monitored during its natural evolution, the origin of tumors has been a subject of continuing discussion. Animal models provide a means to determine the identity of the cell-of-origin leading to malignancy and to develop new treatments. Recent findings in mice have shown that cancer stem cells could arise through a reprogramming-like mechanism, suggesting that genetic lesions that initiate the cancer process might be dispensable for tumor progression and maintenance. This review addresses the impact of these results toward a better understanding of carcinogenesis and proposes research avenues for tackling these issues in the future.

Endogenous TAP-independent MHC-I antigen presentation: not just the ER lumen.

Altered and infected cells are eliminated by CD8+ cytotoxic T lymphocytes. This requires production of antigenic peptides mostly in the cytosol, transport to the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP), and cell surface presentation by major histocompatibility complex class I (MHC-I). Strikingly, antigen presentation occurs without TAP, although it is inefficient and associated to human pathology. TAP-independent peptides derive both from membrane and secreted proteins, as well as cytosolic ones. The efficiency of TAP-independent presentation may be impacted by the availability of receptive MHC-I, and in turn by the functional presence in the ER of the peptide-loading complex, itself anchored on TAP. Without TAP, surface expression of human leukocyte antigen (HLA)-B allotypes varies widely, with those presenting a broader peptide repertoire among the most TAP-independent. Much remains to be learned on the alternative cellular pathways for antigen presentation in the absence of TAP.

Epigenetic Priming in Immunodeficiencies.

Immunodeficiencies (IDs) are disorders of the immune system that increase susceptibility to infections and cancer, and are therefore associated with elevated morbidity and mortality. IDs can be primary (not caused by other condition or exposure) or secondary due to the exposure to different agents (infections, chemicals, aging, etc.). Most primary immunodeficiencies (PIDs) are of genetic origin, caused by mutations affecting genes with key roles in the development or function of the cells of the immune system. A large percentage of PIDs are associated with a defective development and/or function of lymphocytes and, especially, B cells, the ones in charge of generating the different types of antibodies. B-cell development is a tightly regulated process in which many different factors participate. Among the regulators of B-cell differentiation, a correct epigenetic control of cellular identity is essential for normal cell function. With the advent of next-generation sequencing (NGS) techniques, more and more alterations in different types of epigenetic regulators are being described at the root of PIDs, both in humans and in animal models. At the same time, it is becoming increasingly clear that epigenetic alterations triggered by the exposure to environmental agents have a key role in the development of secondary immunodeficiencies (SIDs). Due to their largely reversible nature, epigenetic modifications are quickly becoming key therapeutic targets in other diseases where their contribution has been known for more time, like cancer. Here, we establish a parallelism between IDs and the nowadays accepted role of epigenetics in cancer initiation and progression, and propose that epigenetics forms a "third axis" (together with genetics and external agents) to be considered in the etiology of IDs, and linking PIDs and SIDs at the molecular level. We therefore postulate that IDs arise due to a variable contribution of (i) genetic, (ii) environmental, and (iii) epigenetic causes, which in fact form a continuum landscape of all possible combinations of these factors. Additionally, this implies the possibility of a fully epigenetically triggered mechanism for some IDs. This concept would have important prophylactic and translational implications, and would also imply a more blurred frontier between primary and secondary immunodeficiencies.

Wolf-Hirschhorn Syndrome Candidate 1 Is Necessary for Correct Hematopoietic and B Cell Development.

Immunodeficiency is one of the most important causes of mortality associated with Wolf-Hirschhorn syndrome (WHS), a severe rare disease originated by a deletion in chromosome 4p. The WHS candidate 1 (WHSC1) gene has been proposed as one of the main genes responsible for many of the alterations in WHS, but its mechanism of action is still unknown. Here, we present in vivo genetic evidence showing that Whsc1 plays an important role at several points of hematopoietic development. Particularly, our results demonstrate that both differentiation and function of Whsc1-deficient B cells are impaired at several key developmental stages due to profound molecular defects affecting B cell lineage specification, commitment, fitness, and proliferation, demonstrating a causal role for WHSC1 in the immunodeficiency of WHS patients.

Homeobox NKX2-3 promotes marginal-zone lymphomagenesis by activating B-cell receptor signalling and shaping lymphocyte dynamics.

NKX2 homeobox family proteins have a role in cancer development. Here we show that NKX2-3 is overexpressed in tumour cells from a subset of patients with marginal-zone lymphomas, but not with other B-cell malignancies. While Nkx2-3-deficient mice exhibit the absence of marginal-zone B cells, transgenic mice with expression of NKX2-3 in B cells show marginal-zone expansion that leads to the development of tumours, faithfully recapitulating the principal clinical and biological features of human marginal-zone lymphomas. NKX2-3 induces B-cell receptor signalling by phosphorylating Lyn/Syk kinases, which in turn activate multiple integrins (LFA-1, VLA-4), adhesion molecules (ICAM-1, MadCAM-1) and the chemokine receptor CXCR4. These molecules enhance migration, polarization and homing of B cells to splenic and extranodal tissues, eventually driving malignant transformation through triggering NF-κB and PI3K-AKT pathways. This study implicates oncogenic NKX2-3 in lymphomagenesis, and provides a valid experimental mouse model for studying the biology and therapy of human marginal-zone B-cell lymphomas.

Lineage-specific function of Engrailed-2 in the progression of chronic myelogenous leukemia to T-cell blast crisis.

In hematopoietic malignancies, oncogenic alterations interfere with cellular differentiation and lead to tumoral development. Identification of the proteins regulating differentiation is essential to understand how they are altered in malignancies. Chronic myelogenous leukemia (CML) is a biphasic disease initiated by an alteration taking place in hematopoietic stem cells. CML progresses to a blast crisis (BC) due to a secondary differentiation block in any of the hematopoietic lineages. However, the molecular mechanisms of CML evolution to T-cell BC remain unclear. Here, we have profiled the changes in DNA methylation patterns in human samples from BC-CML, in order to identify genes whose expression is epigenetically silenced during progression to T-cell lineage-specific BC. We have found that the CpG-island of the ENGRAILED-2 (EN2) gene becomes methylated in this progression. Afterwards, we demonstrate that En2 is expressed during T-cell development in mice and humans. Finally, we further show that genetic deletion of En2 in a CML transgenic mouse model induces a T-cell lineage BC that recapitulates human disease. These results identify En2 as a new regulator of T-cell differentiation whose disruption induces a malignant T-cell fate in CML progression, and validate the strategy used to identify new developmental regulators of hematopoiesis.

Transient expression of Bcl6 is sufficient for oncogenic function and induction of mature B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma and can be separated into two subtypes based upon molecular features with similarities to germinal centre B-cells (GCB-like) or activated B-cells (ABC-like). Here we identify gain of 3q27.2 as being significantly associated with adverse outcome in DLBCL and linked with the ABC-like subtype. This lesion includes the BCL6 oncogene, but does not alter BCL6 transcript levels or target-gene repression. Separately, we identify expression of BCL6 in a subset of human haematopoietic stem/progenitor cells (HSPCs). We therefore hypothesize that BCL6 may act by 'hit-and-run' oncogenesis. We model this hit-and-run mechanism by transiently expressing Bcl6 within murine HSPCs, and find that it causes mature B-cell lymphomas that lack Bcl6 expression and target-gene repression, are transcriptionally similar to post-GCB cells, and show epigenetic changes that are conserved from HSPCs to mature B-cells. Together, these results suggest that BCL6 may function in a 'hit-and-run' role in lymphomagenesis.

Germinal centre protein HGAL promotes lymphoid hyperplasia and amyloidosis via BCR-mediated Syk activation.

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.

Acute lymphoblastic leukemia and developmental biology: a crucial interrelationship.

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 evidence 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.