FBXO11 Is Recurrently Mutated in Burkitt Lymphoma and Its Inactivation Accelerates Lymphomagenesis in Eμ-Myc mice

Abstract

INTRODUCTIONBCL6 is a key oncogene in lymphoma pathogenesis. Malignant lymphoid cells exploit several mechanisms to deregulate the expression of BCL6, including chromosomal translocations, somatic mutations in the promoter regulatory regions or inactivation of the pathway that controls its degradation. FBXO11 was recently identified as a major ubiquitin ligase involved in the degradation of BCL6 and was found to be frequently inactivated by mutations or deletion in diffuse large B cell lymphoma (DLBCL). Thus, FBXO11 acts as an oncosuppressor in DLBCL by promoting the accumulation of BCL6. Given the prominent role of FBXO11 in regulating BCL6 stability, we searched for FBXO11 mutations in BCL6-positive lymphomas, other than DLBCL, and we investigated its role in lymphoma development in vivo .METHODSWe sequenced the entire FBXO11 coding sequence by classical Sanger sequencing in 100 cases of Follicular Lymphoma (FL), 36 cases of Burkitt Lymphoma (BL), 8 BL cell lines and 8 Anaplastic Large cell lymphoma cell lines, all BCL6-positive lymphomas. Moreover, we sequenced 50 cases of Marginal Zone B cell Lymphoma (MZL), which show variable expression of BCL6.We functionally validated the FBXO11 mutations by developing mutant constructs and testing their ability to induce BCL6 and SNAIL degradation. We then applied the CRISPR/Cas9 system to disrupt the endogenous FBXO11 gene in BL cells and evaluated its effect on BCL6 stability.To dissect the in vivo role of FBXO11 in lymphomagenesis we generated conditional FBXO11 knock-out (KO) mice (FBXO11fl/fl) to delete protein expression in CD19-positive B cells. To investigate whether FBXO11 inactivation cooperates with c-myc in lymphomagenesis we crossed CD19/Cre-FBXO11fl/fl mice with Eμ-myc transgenic mice .RESULTSWe identified FBXO11 mutations in BL cases and cell lines (10/44, 22.7%), one case of FL (1/100) and one case of MZL (1/50). In FL and MZL, the mutational analysis of tissue collected by microdissection showed that the mutation was specifically acquired by the high-grade component. The frequency of FBXO11 mutation in BL was further validated on an independent cohort of 51 BL cases (6/51, 11.7%), obtained from published data of a previous whole-exome sequencing study (Love et al, Nat Genet. 2012).BL mutations were mostly missense mutations located in the functional CASH domains and also splice-site mutations that were never described before and that induced alternative splicing, as confirmed on the mRNA extracted from the tumor samples. All mutations produced a mutant FBXO11 with impaired ability to induce BCL6 and SNAIL degradation. CRISPR/Cas9 mediated KO of FBXO11 in BL cells resulted in an almost complete stabilization of BCL6, thus suggesting that FBXO11 is the main, if not unique, ubiquitin ligase that controls BCL6 stability in BL.Finally we observed an acceleration of lymphoma development in the CD19/Cre-FBXO11fl/fl mice crossed with Eμ-myc transgenic mice. The lymphomas showed histologic features of high-grade disease with a more mature B-cell phenotype, than the Eμ-myc tumors alone.CONCLUSIONSOverall our results demonstrate that FBXO11 is frequently mutated in BL. All mutants identified impair the ability of FBXO11 to regulate the degradation of BCL6. Together with our previous findings (Duan et al, Nature 2012), this study shows that FBXO11 is mostly mutated in aggressive lymphomas such as DLBCL and BL, and suggests that FBXO11 mutations could contribute to their aggressiveness. In fact, we show that FBXO11 inactivation cooperates with c-myc in accelerating lymphomagenesis. We have established a novel murine lymphoma model that resembles more closely the human BL, providing a novel promising tool for the study of lymphomagenesis and, potentially, preclinical testing of therapeutic approaches with BCL6 and/or c-myc inhibitors. DisclosuresNo relevant conflicts of interest to declare.