Tongwei Mo

The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a malignancy of B cells of unknown etiology. Deletions of the chromosomal region 13q14 are commonly associated with CLL, with monoclonal B cell lymphocytosis (MBL), which occasionally precedes CLL, and with aggressive lymphoma, suggesting that this region contains a tumor-suppressor gene. Here, we demonstrate that deletion in mice of the 13q14-minimal deleted region (MDR), which encodes the DLEU2/miR-15a/16-1 cluster, causes development of indolent B cell-autonomous, clonal lymphoproliferative disorders, recapitulating the spectrum of CLL-associated phenotypes observed in humans. miR-15a/16-1-deletion accelerates the proliferation of both human and mouse B cells by modulating the expression of genes controlling cell-cycle progression. These results define the role of 13q14 deletions in the pathogenesis of CLL.

BLIMP1 is a tumor suppressor gene frequently disrupted in activated B cell like diffuse large B cell lymphoma

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease composed of at least two distinct subtypes: germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. These phenotypic subtypes segregate with largely unique genetic lesions, suggesting the involvement of different pathogenetic mechanisms. In this report we show that the BLIMP1/PRDM1 gene is inactivated by multiple mechanisms, including homozygous deletions, truncating or missense mutations, and transcriptional repression by constitutively active BCL6, in ∼53% of ABC-DLBCL. In vivo, conditional deletion of Blimp1 in mouse B cells promotes the development of lymphoproliferative disorders recapitulating critical features of the human ABC-DLBCL. These results demonstrate that BLIMP1 is a bona fide tumor-suppressor gene whose loss contributes to lymphomagenesis by blocking plasma cell differentiation.

Disruption of KMT2D perturbs germinal center B cell development and promotes lymphomagenesis

Mutations in the gene encoding the KMT2D (or MLL2) methyltransferase are highly recurrent and occur early during tumorigenesis in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). However, the functional consequences of these mutations and their role in lymphomagenesis are unknown. Here we show that FL- and DLBCL-associated KMT2D mutations impair KMT2D enzymatic activity, leading to diminished global H3K4 methylation in germinal-center (GC) B cells and DLBCL cells. Conditional deletion of Kmt2d early during B cell development, but not after initiation of the GC reaction, results in an increase in GC B cells and enhances B cell proliferation in mice. Moreover, genetic ablation of Kmt2d in mice overexpressing Bcl2 increases the incidence of GC-derived lymphomas resembling human tumors. These findings suggest that KMT2D acts as a tumor suppressor gene whose early loss facilitates lymphomagenesis by remodeling the epigenetic landscape of the cancer precursor cells. Eradication of KMT2D-deficient cells may thus represent a rational therapeutic approach for targeting early tumorigenic events.

Functional dissection of the chromosome 13q14 tumor-suppressor locus using transgenic mouse lines

Deletion of chromosomal region 13q14 represents the most common genetic aberration in B-cell chronic lymphocytic leukemia (CLL). 13q14 deletions are commonly large and heterogeneous in size and affect multiple genes. We recently found that targeted deletion in mice of the 0.11 megabase (mb)-long minimal deleted region (MDR) encompassing the DLEU2/miR-15a/16-1 cluster recapitulates the spectrum of CLL-associated lymphoproliferations in humans, including CLL, CD5(+) monoclonal B-cell lymphocytosis, and CD5(-) non-Hodgkin lymphomas. In the present study, we demonstrate that additional deletion of the 0.69-mb large genomic region telomeric to the MDR called the common deleted region (CDR) changed the spectrum of lymphoproliferations developing in CDR- versus MDR-deleted mice in that the number of CLL among B-cell lymphoproliferations was significantly elevated in the former. In addition, CDR-deleted mice seemed to succumb to their disease faster than MDR-deleted mice. Comparing HCDR3 regions of CD5(+) lymphoproliferations derived from this and published CLL mouse models, 44% (29 of 66) of junctions could be assigned to 8 sets of highly similar HCDR3 regions, demonstrating that CLL developing in mice frequently expresses almost identical, stereotypic Ag receptors. These results suggest that the size of 13q14 deletions influences the phenotype of the developing lymphoproliferations and potentially the severity of disease, suggesting a tumor-suppressor function for genetic elements in addition to DLEU2/miR-15a/16-1.

The FOXO1 Transcription Factor Instructs the Germinal Center Dark Zone Program

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.

The Crebbp Acetyltransferase is a Haploinsufficient Tumor Suppressor in B Cell Lymphoma.

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, Crebbp-deficient B cells exhibit enhanced response to mitogenic stimuli and perturbed plasma cell differentiation. Although GC-specific loss of Crebbp was insufficient to initiate malignant transformation, compound Crebbp-haploinsufficient/BCL2-transgenic mice, mimicking the genetics of FL and DLBCL, develop clonal lymphomas recapitulating the features of the human diseases. These findings establish CREBBP as a haploinsufficient tumor-suppressor gene in GC B cells and provide insights into the mechanisms by which its loss contributes to lymphomagenesis.Significance: Loss-of-function mutations of CREBBP 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. Cancer Discov; 7(3); 322-37. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 235.

Abstract B25: Disruption of KMT2D-dependent histone methylation perturbs GC B cell development and cooperates with BCL2 deregulation in lymphomagenesis

Modulation of chromatin accessibility through histone modification is a key step in the regulation of gene transcription and its disruption by genetic lesions has been implicated in malignant transformation. Indeed, a consistent theme in recent cancer genome studies has been the discovery of recurrent mutations in multiple histone/chromatin modifier genes, including methyltransferases, acetyltransferases and histone themselves. Among these, KMT2D (MLL2 or MLL4), encoding for a histone H3K4 methyltransferase, emerged as one of the most common targets of genetic lesion in B cell non-Hodgkin lymphoma, being found in ~30% of diffuse large B cell lymphoma (DLBCL) and ~90% of follicular lymphoma (FL), which together account for over 70% of all lymphoma diagnoses (Pasqualucci et al, Nat Genetics 2011; Morin et al, Nature 2011). KMT2D mutations are mostly represented by truncating events that are predicted to remove the protein C-terminal enzymatic domains, thus inactivating its function; however, missense mutations were also found in a subset of cases, suggesting selection for a functional role. These events are biallelically distributed in one third of mutated cases, while the remaining >60% harbor monoallelic mutations, consistent with a role as a tumor suppressor. Interestingly, analysis of the history of clonal evolution during FL transformation to DLBCL suggests that KMT2D mutations may be already present in a common precursor clone before divergent evolution to FL or DLBCL, suggesting an early role during B cell clonal expansion (Pasqualucci et al, Cell Rep, 2014; Green et al, Blood, 2013). To elucidate the functional consequences of KMT2D mutations, we first examined the effects of 16 DLBCL/FL-derived KMT2D missense mutant alleles on its enzymatic activity in vitro. The results showed that all 8 mutants located in the C-terminal portion of the protein were associated with significantly diminished H3K4 mono-, di- and tri-methylation activity. Consistently, a significant reduction in global methylation was observed in Kmt2d deficient murine B-cells, as well as in 4 biallelically truncated cell lines, indicating that this methyltransferase can influence all three H3K4 modifications. To gain further insights into the program regulated by KMT2D in germinal center (GC) B cells (i.e. the normal counterpart of FL/DLBCL) and the mechanism by which its loss contributes to lymphomagenesis, we crossed mice carrying a conditional Kmt2d knockout allele (Lee J et al, Elife, 2013) with either CD19Cre or Cγ1Cre deletor mice, leading to gene inactivation early during B-cell development (Kmt2dCD19KO), thus mimicking the postulated “common precursor model,” or specifically in mature, GC B-cells (Kmt2dCγ1KO). Notably, deletion of Kmt2d before, but not after initiation of the GC reaction led to a significant increase in GC B-cells and enhanced B cell proliferation. These changes were accompanied by the acquisition of distinct transcriptional signatures enriched in cell cycle regulation and apoptosis genes. A cross-species strategy combining gene expression profile analysis of murine GC B-cells and Kmt2d chromatin immunoprecipitation and sequencing of purified human GC B cells identified a core of KMT2D direct targets genes involved in biological programs with critical functions in B cells physiology, including B cell receptor signaling, lymphocyte migration, and chemokine signaling components. Finally, while loss of Kmt2d alone in Kmt2dCγ1KO was not sufficient to induce tumor development, its combination with VavP-BCL2 transgenic mice increased the incidence of GC-derived lymphomas resembling the features of the human tumors. These data support a role for KMT2D as a tumor suppressor gene whose early loss during B cell development facilitates lymphomagenesis by remodeling the epigenetic landscape of the cancer precursor cell. Citation Format: Jiyuan Zhang, David Dominguez-Sola, Shafinaz Hussein, Ji-Eun Lee, Antony B. Holmes, Mukesh Bansal, Sofija Vlasevska, Tongwei Mo, Hongyan Tang, Katia Basso, Kai Ge, Riccardo Dalla-Favera, Laura Pasqualucci. Disruption of KMT2D-dependent histone methylation perturbs GC B cell development and cooperates with BCL2 deregulation in lymphomagenesis. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B25.