PRDM11 Is a Novel Putative Tumor Suppressor In Aggressive B Cell Lymphoma
Authors: Asmar Fazila , Fog Cathrine Kolster , Jensen Klaus , Jacobsen Linda , Ralfkiær Elisabeth , Geisler Christian H. , Gr∅nbæk Kirsten , Lund Anders 
Published: October 2019

Abstract
Abstract 1002

Introduction and Aim:
Deregulation of epigenetic factors contributes together with genetic alterations to the development of cancer. The PR domain proteins (PRDMs) constitute a sub-family of the SET domain family of histone methyl transferases and consist of 17 family members. Several of the PRDMs have been characterized as tumorsuppressors, including PRDM2, PRDM3, PRDM5 and PRDM16, but the mechanisms are largely unknown. In a functional screen with overexpressed MYC, we found that shRNA mediated down regulation of PRDM11 facilitates oncogenic transformation. Given the importance of MYC in lymphoma development it is essential to understand the genetic settings that facilitate MYC-induced transformation. We thus set out to investigate the function of PRDM11 in lymphomagenesis.

Methods:
To identify PR/SET proteins collaborating with MYC in cellular transformation a retroviral vector based library of shRNA targeting 61 SET and PR domain genes from mice and humans was generated. Primary mouse embryo fibroblasts (MEFs) were transduced with the library and with the Myc overexpression vector. A conditional Prdm11 knockout (KO) mouse strain and crosses to the Eμ-Myc tumor prone strain were generated to evaluate the tumorsuppressor potential of Prdm11 in vivo. The expression levels of PRDM11 in B cell lymphoma cell lines were evaluated by RT-qPCR and immunohistochemistry staining. PRDM11 expression level in Diffuse Large B Cell Lymphoma (DLBCL) patients was assessed by immunohistochemistry staining of DLBCL tissue microarrays (TMAs) according to the H-score. In order to investigate whether the PRDM11 expression was regulated by epigenetic mechanisms we performed H3K27me3 and H3K4me3 ChIP analysis and DNA methylation specific melting curve analysis. DGGE mutation scanning was applied to analyze 17 lymphoma cell lines and 77 DLBCL patients for point mutations.

Results:
We found that overexpression of Prdm11 in MEFs diminished growth and induced apoptosis in a manner independent of p53 and the intrinsic apoptotic pathway. Furthermore, Prdm11 (KO) MEFs grew faster than their wildtype (WT) littermate controls and transformed in the presence of oncogenic Myc. Prdm11 KO mice were viable and fertile with no apparent phenotype. The Eμ-Myc mice selectively express the Myc transgene in the B-cell lineage and develop malignant lymphomas with a mean latency of 100–120 days. Importantly, we found that loss of Prdm11 potently accelerated lymphomagenesis in the Eμ-Myc mouse (p<0,0006) and induced the incidence time from 111 to 75 days. To investigate the function if PRDM11 in humans, PRDM11 expression levels were evaluated in a panel of human DLBCL and Mantle Cell Lymphoma (MCL) cell lines. Compared to normal B-cells and reactive lymph nodes, PRDM11 mRNA expression levels were significantly lower or absent in 16/19 lymphoma cell lines. PRDM11 immunohistochemistry staining of DLBCL TMAs showed lower levels compared to reactive lymph nodes. PRDM11 immunohistochemistry staining in reactive lymph nodes was mainly localized to the activated B cells in the germinal centres. Interestingly, low or absent PRDM11 expression is associated with significantly worse overall survival (p = 0,011, univariate analysis of 22 patients). We are currently in the process of investigating the prognostic significance of PRDM11 expression in another 100 patients with DLBCL. These data will be presented at the meeting. We have found an inverse correlation between the expression level of PRDM11 and the presence of the repressive chromatin mark H3K27me3 at the PRDM11 promoter by ChIP analysis. H3K27me3 is less enriched at the promoter of PRDM11 in normal B cells as well as in cell lines with EZH2 missense mutation. DNA methylation was not detected in 17 lymphoma cell lines or 77 DLBCL patients and 3 PRDM11 sequence variants were also present in the germ line.

In conclusion:
PRDM11 is a novel putative tumor suppressor in mice and men, whose downregulation may be associated with poor prognosis in DLBCL. H3K27 trimethylation of the PRDM11 promoter may be a novel target for therapy in DLBCL.

Disclosures:
No relevant conflicts of interest to declare.