Downregulation Of FOXO1 Prevents Induction Of The Tumor Suppressor BLIMP-1 In Classical Hodgkin Lymphoma

Abstract

In contrast to other B cell lymphomas, Hodgkin and Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkin lymphoma (cHL), have mainly lost their B cell identity. Recently, we reported that the transcription factor FOXO1, indispensable for B cell development and differentiation, is downregulated in HRS cells and expression of FOXO1 results in growth arrest and apoptosis in cHL cell lines.To find molecular targets of FOXO1 we performed gene expression profiling of 5 cHL cell lines expressing constitutively active FOXO1 fused to ligand binding domain of estrogen receptor (FOXO1(A3)ER).We found that FOXO1 activation led to downregulation of genes, which are normally upregulated in cHL, such as CD30/TNFRSF8 and the proto-oncogene MYC. On the other hand, FOXO1 activated expression of germinal center-specific genes including BCL6, AICDA, BACH2, and GCET2. The most surprising finding was induction of BLIMP-1/PRDM1, the master regulator of plasma cell differentiation and tumor suppressor in activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL). A positive correlation of FOXO1 and BLIMP-1 expression in microdissected HRS cells further indicated a role for FOXO1 in BLIMP-1 regulation.Of note, HRS cells do not undergo plasma cell differentiation despite constitutive expression of NF-κB, IRF4, and STAT3, known inducers of BLIMP-1. However, BLIMP-1 levels remain low in HRS cells thereby preventing terminal differentiation. The mechanisms leading to BLIMP-1 repression in cHL are poorly understood. We found that in most cHL cell lines, FOXO1 specifically upregulated BLIMP-1α representing the full-length isoform of BLIMP-1. In addition, luciferase reporter assay showed that FOXO1 activates the BLIMP-1α promoter in the cHL cell line L428. Overexpression of BLIMP-1α using a lentiviral vector strongly inhibited growth of cHL cell lines. In line with the fact that BLIMP-1 and MYC form a negative feedback loop, we found that ectopic BLIMP-1α expression resulted in downregulation of MYC protein levels, which most likely contributes to the anti-tumor effect of BLIMP-1α. On the other hand, MYC inhibition in cHL cell lines led to BLIMP-1 upregulation. Thus, our data indicate that FOXO1, BLIMP-1 and MYC constitute a negative feed-forward regulatory loop, which is controlled by FOXO1.Searching for additional mechanisms of BLIMP-1α downregulation in cHL we found that BLIMP-1α promoter was hypermethylated in two cHL cell lines but not in microdissected HRS cells. This indicates that hypermethylation is not critical for BLIMP-1α repression.Interestingly, the functionally impaired BLIMP-1 variant BLIMP-1β, which is normally not detected in normal B cells, was upregulated in cHL cell lines. BLIMP-1β is also expressed in ABC-DLBCL and has been suggested to block BLIMP-1α in a dominant-negative manner. We speculate that BLIMP-1β attenuates function of residual BLIMP-1α in cHL. However, further investigation is required.Taken together, we show for the first time that FOXO1 induces expression of BLIMP-1. We identified BLIMP-1α as a tumor suppressor which downregulates expression of the proto-oncogene MYC in cHL. Therefore, FOXO1 might exert its tumor suppressor function at least in part by upregulation of BLIMP-1α.Further work on BLIMP-1α regulation by FOXO1 and investigation of a potential role of FOXO1 in plasma cell differentiation is warranted. Disclosures:No relevant conflicts of interest to declare.