SOCS1 and SOCS3 Genes Are Not Methylated In Waldenstrom Macroglobulinemia
Authors: Poulain Stephanie , Aijjou Rachid , Broucqsault Natacha , Manier Salomon , Galiégue-Zouitina Sylvie , Herbaux Charles , Tricot Sabine , Daudignon Agnes , Duthilleul Patrick , Roumier Christophe , Leleu Xavier 
Published: October 2019

Abstract
Abstract 2481

Background.
The suppressor of cytokine signalling (SOCS) 1 and 3 are inhibitors of interleukin 6 (IL6) signalling pathway and demonstrated tumour-suppressor activity. The methylation of SOCS family, resulting in transcriptional silencing, has been suggested to play an important role in development of several hematological disorders, including multiple myeloma. Waldenstrom's macroglobulinemia (WM) is a lymphoproliferative disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells that secrete monoclonal IgM immunoglobulin. Gene expression profiling has demonstrated an up-regulation in IL6 signalling in WM. IL6 also plays a critical role in cell proliferation and survival of WM cells and in the interaction between the tumour cells and their bone marrow microenvironment. We hypothesized that aberrant SOCS1 and SOCS3 expression may play a role in the pathogenesis of MW. Our study aimed to explore genetic and epigenetic aberrations of SOCS1 and SOCS3 genes.

Methods.
32 patients (pts) diagnosed with WM were included. Diagnosis was done following guidelines, and pts were not treated at time of BM collection. Approval of this protocol was obtained from the CHRU of Lille and was in accordance with the declaration of Helsinki. We studied RL and MEC1, cell lines that secrete IgM, and BCWM1 developed from a pt with WM. DNA and RNA were extracted following negative CD19 B cells selection. The levels of mRNA of SOCS1 and SOCS3 were evaluated by real-time PCR. Samples were examined at steady state and following PMA stimulation. The CpG island methylation status of SOCS1 and SOCS3 genes in the promoter region was investigated by methylation-specific (MSP) PCR. Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was used to detect both loss of heterozygosity (LOH) and copy number alteration (CNA). Paired DNA samples (tumor cells / normal T lymphocytes) were used as an intra-individual reference to distinguish germline polymorphisms from somatic abnormalities. Size, position and location of genes were referenced using UCSC HG18 assembly, LOH and CNA using genotyping console 3.02 software (Affymetrix) and Partek genomic suite.

Results.
SOCS1 and SOCS3 genes were expressed in BCWM1, RL and MEC1 cell lines but the baseline expression in cell lines was heterogeneous. PMA induced SOCS1 and SOCS3 expression in all cell lines. No methylation of SOCS1 gene was observed in these cell lines. In contrast, partial methylation of SOCS3 promoter gene was observed in RL and BCMW1. SOCS1 and SOCS3 genes were constitutively expressed at heterogeneous levels in WM pts. The expression varied for SOCS1 from 0.31 to 43 and for SOCS3 from 1.35 to 73. SOCS1 and SOCS3 expressions were enhanced by PMA treatment (n=3 pts). In order to explore genetic aberration on SOCS1 and SOCS3 genes in WM patients, SNP arrays were performed in 17 WM pts. No deletion of SOCS 1 and 3 genes were observed. In one case (5.8%), SNP array karyotyping identified an acquired uniparental disomy (UPD), a LOH without variation of copy number changes, at the SOCS3 locus (17q25). Gain of SOCS1 gene (16p13) was observed in another pt with high mRNA SOCS1 expression. Gene methylation in the promoter was studied in 25 WM pts. No methylation of CpG islands in the SOCS1 and SOCS3 gene promoters was detected in the WM pts.

Conclusion.
SOCS1 and SOCS3 genes are expressed in WM cells. Copy number aberration, UPD and epigenetic inactivation of SOCS1 and SOCS3 genes seem to be rare events in WM. Other mechanisms might explain for the heterogeneous expression levels of SOCS1 and SOCS3 genes that may contribute for IL6 signalling deregulation in WM.

Disclosures:
Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.