High-Resolution Chromatin Immunoprecipitation (ChIP) Sequencing Identifies Novel Binding Targets and Prognostic Role for SOX11 in Mantle Cell Lymphoma

Abstract 585

SOX11 belongs to the high-mobility group of transcription factors that bind to DNA and induce large conformational changes that facilitate the binding of other transcription factors. Genome-wide methylation analysis of Mantle cell lymphoma (MCL) patients using Nimblegen arrays identified SOX11 as hypomethylated and overexpressed in comparison to purified naïve B cells, which are the cells from which MCLs originate. Direct sequencing of SOX11 in MCL cell lines and patient samples failed to identify any activating mutations in SOX11, strengthening the role of aberrant promoter hypomethylation in MCL as the cause of its overexpression. SOX11 expression has been previously compared between Non-Hodgkin's lymphomas and found to be specific for MCL. The function and direct binding targets of SOX11 are unknown.

To understand the functional role of SOX11 in MCL, we used high-resolution whole-genome chromatin immunoprecipitation sequencing (ChIP-Seq) to identify the target genes directly bound by SOX11. As a model, genomic DNA from the Granta-519 MCL cell line was immunoprecipitated by SOX11 antibody and IgG and genomic enrichment sites were analyzed by Illumina sequencing. Using stringent statistical criteria (p-value<0.01, fold change≥1.5, and FDR<0.01) we identified 5682 peak positions with significant enrichment of ChIP sequencing reads corresponding to 888 unique genes. Sequencing runs from independent immunoprecipitates had more than 95% overlap in binding targets. These targets were filtered with published gene sets of differentially expressed genes following SOX11 overexpression in MCL cell lines. Pathway analysis revealed cell cycle, FOXm transcriptional network, and TGF-beta receptor signaling as the top three significantly enriched pathways by SOX11 target genes. All three pathways prominently consist of genes with anti-proliferative effects in MCL and B-NHL in general. To test our hypothesis, we transfected SOX11 by lentiviral plasmids into four MCL cell lines with varying levels of intrinsic SOX11 expression. Overexpression of SOX11 led to decreased proliferation in all four cell lines as measured by long-term tracing of living cells using a fluorescent labeled dye to track cell division. Ingenuity Pathway Analysis also identified the tumor promoting Wnt, PKC and IGFR pathways as being significantly enriched by the binding targets. We validated direct binding of SOX11 to RB1, Wnt4 and PKCA by quantitive PCR of ChIP products from Granta-519 cells. RB1 is a key regulator of entry into cell division tumor suppressor in MCL whereas Wnt signaling activates c-myc, which is oncogenic in MCL.

Previously published studies have indicated a significant negative prognostic impact of SOX11 expression on overall survival in MCL. We therefore analyzed SOX11 expression in two large independent tissue microarrays from University of Wisconsin (UW) and British Columbia Cancer Agency (BCCA), respectively. MCL patient samples were scored by experienced hematopathologists. High SOX11 expression was associated with better survival: 41 months for SOX11 positive vs. 11 months for SOX11 negative, (p=0.05) in 58 patients in the UW dataset, however the larger BCCA dataset of 140 patients did not show any statistically significant impact of SOX11 expression on survival (p=0.15). These findings suggest that the prognostic impact of SOX11 is heterogenous, and a subset of patients with high SOX11 expression may have improved survival.

In conclusion, high-resolution ChIP-Seq identifies more than 800 novel SOX11 direct binding targets. Differential activation of the diverse biological pathways affected by SOX11 target genes may help explain the differential impact of SOX11 expression on patient outcomes.