Abstract B25: Disruption of KMT2D-dependent histone methylation perturbs GC B cell development and cooperates with BCL2 deregulation in lymphomagenesis
Authors: Zhang Jiyuan , Dominguez-Sola David , Hussein Shafinaz , Lee Ji-Eun , Holmes Antony B. , Bansal Mukesh , Vlasevska Sofija , Mo Tongwei , Tang Hongyan , Basso Katia , Ge Kai , Dalla-Favera Riccardo , Pasqualucci Laura 
Published: May 2016

Abstract
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 &gt;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.