Teresa Flores

Cancer induction by restriction of oncogene expression to the stem cell compartment

In human cancers, all cancerous cells carry the oncogenic genetic lesions. However, to elucidate whether cancer is a stem cell‐driven tissue, we have developed a strategy to limit oncogene expression to the stem cell compartment in a transgenic mouse setting. Here, we focus on the effects of the BCR‐ABLp210 oncogene, associated with chronic myeloid leukaemia (CML) in humans. We show that CML phenotype and biology can be established in mice by restricting BCR‐ABLp210 expression to stem cell antigen 1 (Sca1)+ cells. The course of the disease in Sca1‐BCR‐ABLp210 mice was not modified on STI571 treatment. However, BCR‐ABLp210‐induced CML is reversible through the unique elimination of the cancer stem cells (CSCs). Overall, our data show that oncogene expression in Sca1+ cells is all that is required to fully reprogramme it, giving rise to a full‐blown, oncogene‐specified tumour with all its mature cellular diversity, and that elimination of the CSCs is enough to eradicate the whole tumour.

Novel Genomic Imbalances in B-Cell Splenic Marginal Zone Lymphomas Revealed by Comparative Genomic Hybridization and Cytogenetics

Splenic marginal zone lymphoma (SMZL) has recently been recognized in the World Health Organization classification of hematological diseases as distinct type of non-Hodgkins lymphoma. In contrast to the well-established chromosomal changes associated with other B-cell non-Hodgkins lymphoma, few genetic alterations have been found associated with SMZL. The aim of our study was to analyze by comparative genomic hybridization (CGH) the chromosomal imbalances in 29 patients with SMZL and to correlate these findings with clinical and biological characteristics and patient outcome. In 21 cases, cytogenetic studies were simultaneously performed. Most of the patients (83%) displayed genomic imbalances. A total of 111 DNA copy number changes were detected with a median of four abnormalities per case (range, 1 to 12). Gains (n = 92) were more frequent than losses (n = 16), while three high-level amplifications (3q26-q29, 5p11-p15, and 17q22-q25) were observed. The most frequent gains involved 3q (31%), 5q (28%), 12q and 20q (24% each), 9q (21%), and 4q (17%). Losses were observed in 7q (14%) and 17p (10%). SMZL patients with genetic losses had a shorter survival than the remaining SMZL patients (P < 0.05). In summary, chromosomal imbalances in regions 3q, 4q, 5q, 7q, 9q, 12q, and 20q have been detected by CGH in SMZL. Patients with SMZL displaying genetic losses by CGH had a short survival.

SLUG in cancer development.

The SNAIL-related zinc-finger transcription factor, SLUG (SNAI2), is critical for the normal development of neural crest-derived cells and loss-of-function SLUG mutations have been proven to contribute to piebaldism and Waardenburg syndrome type 2 in a dose-dependent fashion. While aberrant induction of SLUG has been documented in cancer cells, relatively little is known about the consequences of SLUG overexpression in malignancy. To investigate the potential role of SLUG overexpression in development and in cancer, we generated mice carrying a tetracycline-repressible Slug transgene. These mice were morphologically normal at birth, and developed mesenchymal tumours (leukaemia and sarcomas) in almost all cases examined. Suppression of the Slug transgene did not rescue the malignant phenotype. Furthermore, the BCR–ABL oncogene, which induces Slug expression in leukaemic cells, did not induce leukaemia in Slug-deficient mice, implicating Slug in BCR–ABL leukaemogenesis in vivo. Overall, the findings indicate that while Slug overexpression is not sufficient to cause overt morphogenetic defects in mice, they demonstrate a specific and critical role for Slug in the pathogenesis of mesenchymal tumours.

Liposarcoma initiated by FUS/TLS-CHOP: the FUS/TLS domain plays a critical role in the pathogenesis of liposarcoma

The most common chromosomal translocation in liposarcomas, t(12;16)(q13;p11), creates the FUS/TLS-CHOP fusion gene. We previously developed a mouse model of liposarcoma by expressing FUS-CHOP in murine mesenchymal stem cells. In order to understand how FUS-CHOP can initiate liposarcoma, we have now generated transgenic mice expressing altered forms of the FUS-CHOP protein. Transgenic mice expressing high levels of CHOP, which lacks the FUS domain, do not develop any tumor despite its tumorigenicity in vitro and widespread activity of the EF1α promoter. These animals consistently show the accumulation of a glycoprotein material within the terminally differentiated adipocytes, a characteristic figure of liposarcomas associated with FUS-CHOP. On the contrary, transgenic mice expressing the altered form of FUS-CHOP created by the in frame fusion of the FUS domain to the carboxy end of CHOP (CHOP-FUS) developed liposarcomas. No tumors of other tissues were found in these transgenic mice despite widespread activity of the EF1α promoter. The characteristics of the liposarcomas arising in the CHOP-FUS mice were very similar to those previously observed in our FUS-CHOP transgenic mice indicating that the FUS domain is required not only for transformation but also influences the phenotype of the tumor cells. These results provide evidence that the FUS domain of FUS-CHOP plays a specific and critical role in the pathogenesis of liposarcoma.

A marginal zone pattern may be found in different varieties of non‐Hodgkin's lymphoma: the morphology and immunohistology of splenic involvement by B‐cell lymphomas simulating splenic marginal zone lymphoma

Aims: Splenic marginal zone lymphoma (SMZL) is characterized by a micronodular infiltrate of the splenic white pulp, centred on pre‐existing follicles, with a peripheral rim of ‘marginal’ zone B‐cells, always accompanied by a variable degree of red pulp infiltration. These histological features can be closely mimicked by a variety of other small B‐cell lymphomas when they involve the spleen, which makes recognition of SMZL difficult. We therefore have compared the histopathological and immunohistochemical features of other non‐Hodgkins lymphoma (NHL) types with those of SMZL.

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.

Flow cytometry immunophenotyping of fine-needle aspiration specimens: utility in the diagnosis and classification of non-Hodgkin lymphomas

Barrena S, Almeida J, García‐Macias M D C, López A, Rasillo A, Sayagués J M, Rivas R A, Gutiérrez M L, Ciudad J, Flores T, Balanzategui A, Caballero M D & Orfao A
(2011) Histopathology 58, 906–918
Flow cytometry immunophenotyping of fine‐needle aspiration specimens: utility in the diagnosis and classification of non‐Hodgkin lymphomas

A novel molecular mechanism involved in multiple myeloma development revealed by targeting MafB to haematopoietic progenitors

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.

Molecular characterization of immunoglobulin gene rearrangements in diffuse large B-cell lymphoma: antigen-driven origin and IGHV4-34 as a particular subgroup of the non-GCB subtype.

The pathogenesis of diffuse large B-cell lymphoma (DLBCL) remains partially unknown. The analysis of the B-cell receptor of the malignant cells could contribute to a better understanding of the DLBCL biology. We studied the molecular features of the immunoglobulin heavy chain (IGH) rearrangements in 165 patients diagnosed with DLBCL not otherwise specified. Clonal IGH rearrangements were amplified according to the BIOMED-2 protocol and PCR products were sequenced directly. We also analyzed the criteria for stereotyped patterns in all complete IGHV-IGHD-IGHJ (V-D-J) sequences. Complete V-D-J rearrangements were identified in 130 of 165 patients. Most cases (89%) were highly mutated, but 12 sequences were truly unmutated or minimally mutated. Three genes, IGHV4-34, IGHV3-23, and IGHV4-39, accounted for one third of the whole cohort, including an overrepresentation of IGHV4-34 (15.5% overall). Interestingly, all IGHV4-34 rearrangements and all unmutated sequences belonged to the nongerminal center B-cell-like (non-GCB) subtype. Overall, we found three cases following the current criteria for stereotyped heavy chain VH CDR3 sequences, two of them belonging to subsets previously described in CLL. IGHV gene repertoire is remarkably biased, implying an antigen-driven origin in DLBCL. The particular features in the sequence of the immunoglobulins suggest the existence of particular subgroups within the non-GCB subtype.

NIK Controls Classical and Alternative NF-κB Activation and Is Necessary for the Survival of Human T-cell Lymphoma Cells

Purpose: Peripheral T-cell lymphomas (PTCL) are a heterogeneous entity of neoplasms with poor prognosis, a lack of effective therapies, and a largely unknown molecular pathology. Deregulated NF-κB activity has been associated with several lymphoproliferative diseases, but its importance in T-cell lymphomagenesis is poorly understood. We investigated the function of the NF-κB–inducing kinase (NIK), in this pathway and its role as a potential molecular target in T-cell lymphomas. Experimental Design: We used immunohistochemistry to analyze the expression of different NF-κB members in primary human PTCL samples and to study its clinical impact. With the aim of inhibiting the pathway, we used genetic silencing of NIK in several T-cell lymphoma cell lines and observed its effect on downstream targets and cell viability. Results: We showed that the NF-κB pathway was activated in a subset of PTCLs associated with poor overall survival. NIK was overexpressed in a number of PTCL cell lines and primary samples, and a pivotal role for NIK in the survival of these tumor cells was unveiled. NIK depletion led to a dramatic induction of apoptosis in NIK-overexpressing cell lines and also showed a more pronounced effect on cell survival than inhibitor of kappa B kinase (IKK) knockdown. NIK silencing induced a blockage of both classical and alternative NF-κB activation and reduced expression of several prosurvival and antiapoptotic factors. Conclusions: The results of the present study indicate that NIK could be a promising therapeutic target in these aggressive malignancies. Clin Cancer Res; 19(9); 2319–30. ©2013 AACR.