Abstract 30: SOX9 enhancer regulator may play an oncogenic role in B-cell lymphomas
Authors: Fachel Angela A. , Jiang Yanwen , Tam Wayne , Muley Ashlesha S. , Meydan Cem , Agirre Xabier , Melnick Ari M. , Richards Kristy L. 
Published: August 2018

Abstract
A majority of B-cell lymphomas, including diffuse large B-cell lymphomas (DLBCLs) and follicular lymphomas (FLs), arise from germinal center (GC) B cells. GC B cells are highly proliferative and manifest genomic instability as a byproduct of immunoglobulin affinity maturation. These characteristics make GC B cells particularly prone to malignant transformation such that DLBCLs and FLs inherit and are dependent on mutations in proteins that are also required for normal GC B cells. For example, the BCL6 transcriptional repressor is required to establish the GC phenotype in both normal B-cell development and GC B-cell malignancy through its effect on silencing expression of plasma cell differentiation and checkpoint genes. GC B cells feature a unique transcriptional program featuring upregulation of genes involved in cell proliferation and other growth pathways. Cell context-specific gene expression is largely mediated through gene enhancers. Hence we hypothesized that transcription factors that activate GC specific gene enhancers would play a critical role in formation of these cells, and would likely be required in turn to maintain the survival of GC-derived lymphoma cells.
To address this question we first mapped gene enhancers in primary human resting B cells and purified GC B cells by performing ChIP-seq to map the distribution of H3K27Acetyl, H3K4me1, and H3K4me3. This procedure allowed us to identify several thousand GC-specific enhancers. We next examined these enhancers using bioinformatic approaches to identify transcription factor binding sites and identified a list of fifteen putative GC enhancer regulators. Of these potential GC enhancer regulators, only SOX9 was highly induced in GC B cells as compared to resting B cells, pointing to SOX9 as a candidate master enhancer regulator of the GC transcriptome with potential relevance to lymphomas. Indeed, in addition to the well-known role of SOX9 as a stem cell self-renewal and cell-differentiation regulator during development, SOX9 has also been described as playing a role in colon, breast, and prostate carcinogenesis through inhibition of apoptosis, and promoting proliferation, invasion, and metastasis.
We confirmed that SOX9 is highly induced in GC cells using QPCR and Western blots. To determine whether SOX9 would indeed bind to these enhancer sites, we next performed SOX9 ChIP-seq in GC B-cell derived lymphoma cell lines. These experiments validated and showed that SOX9 binds to 1,668 upstream distal enhancer regions (-5 to -100 kb upstream from TSS) associated with 963 genes. These target genes were significantly enriched in cell cycle regulation (CCND2, CDC25B, CDK1), transcription regulation (BCOR, NCOR2), epigenetic regulation (BMI1, DNMT3A, MLL2, SUZ12, TET3), and MAPK signaling (MAP2K3, MAP3K7) and also for B-cell activation and BCL6 repressed pathways (p&lt;0.001).
We next wished to determine whether SOX9 was also expressed in GC-derived lymphoma clinical samples. Therefore we next examined RNA-seq gene expression profiles derived from cohorts of FL and DLBCL patients. We found that SOX9 is expressed at higher levels in FL patient samples, but lower levels in most DLBCLs compared to naïve B cells. These data raise the possibility that SOX9 might be differentially relevant to FL more than DLBCLs, perhaps contributing to the biologic distinction between these two GC-derived lymphomas.
To determine if SOX9 protein was also expressed in these tumors, we examined protein expression by immunohistochemistry using tissue microarrays (TMAs) containing both FLs and DLBCLs. We found that 88 of the 242 FL TMA samples (36.4%) were positive for SOX9 staining. Eleven of the 114 DLBCL TMA samples (10%) were positive for SOX9 staining, among which 80% (9/11) were GC-type DLBCL and 45% (5/11) had a prior FL history. To determine if any of the currently available GC derived lymphoma cell lines manifest high SOX9 levels, we performed a series of Western blot experiments. These studies show high levels of SOX9 protein in the GC-type DLBCL cell lines HT, Karpas422, DB, and Farage, but not in any of the eight ABC-DLBCLs subtypes.
Ongoing studies are assessing the biologic relevance of SOX9 for the GC reaction and possible contribution to lymphomagenesis using gain- and loss-of-function studies in human cells and mouse models. These data will determine whether this apparently key enhancer regulatory protein is a novel lineage factor for follicular lymphoma and potentially suitable as a therapeutic target.
Citation Format: Angela A. Fachel, Yanwen Jiang, Wayne Tam, Ashlesha S. Muley, Cem Meydan, Xabier Agirre, Ari M. Melnick, Kristy L. Richards. SOX9 enhancer regulator may play an oncogenic role in B-cell lymphomas [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 30.