FAS Mutations Are Associated with Therapeutic Resistance in Follicular Lymphoma

We recently reported on recurrent genomic alterations present in genome, transcriptome or exome data from 127 cases of NHL (Morin et al. Nature. 2011 Jul 27;476:298–303). Our data suggests FAS acts as a tumor suppressor in B cells based on a selection bias towards mutations that would result in truncated or altered protein, most of which were in the functional death domain. FAS is the prototypical death receptor involved in the extrinsic apoptotic pathway. Germline mutations in FAS have a dominant-negative phenotype, leading to dysfunctional FAS-mediated apoptosis, accumulation of lymphocytes and autoimmune lymphoproliferative syndrome. The role of FAS in lymphomagenesis is unclear. The FAS mutations in our study were limited to 6 cases of germinal center B lymphomas, including 3 cases of aggressive follicular lymphoma (FL). Herein we investigate the role of FAS in therapeutic resistance in FL.

One of the FL cases had treatment-refractory disease. Biopsies taken at diagnosis and after progression following 2 cycles of cyclophosphamide, vincristine, prednisone and rituximab (CVP-R) were available for transcriptome sequencing. Owing to our discovery of FAS mutations in this and other FL patients, we sequenced exon 7–9 of FAS on an extended cohort of 214 clinically-annotated FL samples to determine its potential for association with clinical outcome. To functionally validate our findings we transfected two lymphoma cell lines that are sensitive to FAS-induced apoptosis with either FAS wild-type, FAS mutant (Y232*) or empty vector control. Single clones (wild type and Y232*) with similar expression of FAS protein by flow cytometry were compared for their ability to undergo apoptosis after exposure to the FAS agonist antibody CH-11 and therapeutic levels of cyclophosphamide, adriamycin, vincristine and etoposide. We defined apoptotic cells as those expressing annexin V+/propidium iodine-negative and activated caspase 3 by flow cytometry and cells demonstrating cleaved caspase 8 by western blotting. We further determined if etoposide, chosen because it has no background fluorescence, could change the expression of FAS or FAS ligand in primary lymphocytes using multi-color flow cytometry.

We demonstrate that FAS mutations are associated with clinically-aggressive FL. In a patient with treatment-refractory FL, only 3 mutations were confirmed to be somatic and significantly enriched in the post-treatment biopsy: CTSS, RPS24 and FAS Y232* (p values <0.05), where CTSS and RPS24 have no known function in lymphocytes. In an extended cohort of FL samples, 6/214 (3%) had non-synonymous mutations in FAS predicting for a severely altered or truncated protein. Coding FAS mutations were associated with a trend towards an earlier time to progression (1 year versus 2.8 years, p=0.08) and an increased risk of histological transformation to DLBCL (n=4/6, p=0.036). Indeed, 5 of the 6 developed early resistance to primary rituximab-based chemotherapy. In vitro studies demonstrated that the FAS Y292* impairs extrinsic apoptosis induced by CH-11 (decrease in cleaved caspase 8, activated caspase 3 and Annexin V+/PI- cells). However, there was no difference in apoptosis between FAS Y232* mutants and FAS wild type cells after exposure to chemotherapy in vitro. These results led us to speculate that other factors are involved in resistance to chemotherapy. Using primary tonsilar lymphocytes in culture, we observed a significant increase in both FAS expression on primary B cells and the cognate ligand FASL expression in primary T cells following a 24-hour exposure to etoposide.

Mutations in FAS are associated with clinical therapeutic resistance in FL. FAS mutations can impair FAS-mediated apoptosis. Our results suggest that chemotherapy may potentiate an immune response in part by increasing FAS expression in B cells and FASL expression in T cells. This may enhance killing of FAS wild type but not FAS mutant expressing tumor cells. Thus the role the tumor microenvironment in chemotherapy-induced cell death may be under appreciated in FL.

No relevant conflicts of interest to declare.