FAS Death Domain Deletions and Increased c-FLIPlong Expression Occur in Different Subtypes of Diffuse Large B-Cell Lymphoma.
Authors: Takahashi Hidenobu , Feuerhake Friedrich , Kutok Jeffery L. , Monti Stefano , Dal Cin Paola S. , Neuberg Donna , Aster Jon C. , Shipp Margaret A. 
Published: April 2020

Abstract
Diffuse large B-cell lymphomas (DLBCLs) arise from normal germinal center (GC) or post GC B cells. Negative selection of low-affinity or self-reactive normal GC B cells requires CD95 (FAS)-mediated apoptosis. Mutations of the FAS receptor that result in deletion of the cytoplastic death domain (DD) destabilize the trimeric structure and inhibit FAS-mediated apoptosis. This apoptotic pathway is also inhibited when the NFκB target, c-FLIP, interacts with the death-inducing signaling complex, assembled around the cytoplasmic FAS DD. We analyzed FAS receptor mutations and NFκB-mediated overexpression of c-FLIP as potential mechanisms for deregulating the extrinsic apoptotic pathway in DLBCL subtypes defined by gene expression profiling (GEP) -- OxPhos, BCR/proliferation and Host Response (HR) tumors (Monti et al, Blood2005; 105:1851). Study specimens included 117 newly diagnosed DLBCLs that were previously characterized for BCL2 and BCL6 translocations and assigned to the recently described consensus clusters by transcriptional profiling (40 OxPhos, 55 BCR and 22 HR tumors). The FAS coding region from each tumor was amplified by RT-PCR and sequenced. Mutations resulting in deletions of the cytoplasmic DD occurred in 8 DLBCLs. In all tumors, the resulting frameshifts or amino acid substitutions created STOP codons upstream of or at the beginning of the FAS DD. There was a significantly increased incidence of FAS DD deletions in OxPhos tumors (6 tumors, p = .05); these OxPhos tumors also had more frequent t(14;18) (p = .05), indicating that structural abnormalities of extrinsic and intrinsic apoptotic pathway components were more common in this DLBCL subtype. We previously found that HR tumors were significantly less likely to have either BCL2 or BCL6 translocations (1/22 tumors with t(14;18), no tumors with t(3;V)); these DLBCLs also had no mutations of the FAS DD. Given the brisk host inflammatory/immune cell infiltrate in HR tumors, we performed additional mixing studies and confirmed that the PCR-based assay was sufficiently sensitive to detect FAS DD mutations in HR tumor cells. HR tumors have increased expression of a large series of NFκB target genes suggesting that these tumors may be dependent upon NFκB activation (Feuerhake et al, Blood2005; 106:1392). The NFκB target gene, c-FLIPlong, inhibits FAS-induced apoptosis in normal GC B-cells and Hodgkin’s lymphomas. For these reasons, we identified c-FLIP probe sets from the long-form specific C-terminal domain and assessed c-FLIPlong transcript abundance in the DLBCL consensus clusters. The expression of c-FLIPlong was highest in HR tumors and lowest in OxPhos DLBCLs (p &lt; .0001, Kruskal-Wallis test); HR tumors also expressed significantly more abundant c-FLIPlong transcripts than normal GC B-cells (P = .03, Wilcoxon rank sum test). Therefore, NFκB-mediated upregulation of c-FLIPlong likely represents an alternative method of inhibiting FAS-mediated apoptosis in HR DLBCLs which lack structural abnormalities of the FAS receptor. These data further support the notion that GEP-defined subtypes of DLBCL differ with respect to underlying genetic lesions and suggest that these molecular signatures have therapeutic relevance.