Epigenetic Silencing of BIM Mediates Chemotherapy Resistance of Patients with Burkitt Lymphoma That Can Be Overcome by Therapeutic Reactivation of BIM in Mouse and Human Lymphoma Models

The most critical challenge in Burkitt lymphoma is to achieve complete remission (CR) with initial high-dose chemotherapy, as most of these patients will be eventually cured. However, those not achieving CR usually die early after diagnosis. Thus, to identify the refractory cases and how to overcome initial therapeutic resistance remain unsolved issues. We have previously reported that the gene encoding the pro-apoptotic BH3-only protein Bim is frequently inactivated by genetic and epigenetic mechanisms that substantially vary among the different B-cell lymphoma subgroups. Further screening of lymphoma samples revealed that silencing of BIM by promoter hyper-methylation occurred predominantly in Burkitt lymphoma, including 22 of 47 (47%) primary biopsies and 15 of 18 (83%) cell lines, while two additional Burkitt lymphoma-derived cell lines presented BIM homozygous deletion. BIM methylation was not associated with EBV tumor status or with the presence of MYC coding mutations. Quantitative ChIP revealed that most methylated lymphomas also showed deacetylation of histone 3 in BIM promoter mediated by HDAC complexes, but Bim post-transcriptional modifications were rarely observed. In contrast to lymphomas with unmethylated BIM, methylated/deacetylated tumors did not show alterations in other BCL2-family proteins, suggesting that transcriptional repression of Bim is critical and sufficient to impair apoptosis in Burkitt lymphoma. Indeed, in a series of clinically aggressive Burkitt lymphoma/leukemia, BIM hypermethylation was associated with inferior CR rate (only 24% of methylated cases achieved CR vs. 83% of unmethylated cases, p=0.0002) and shorter overall survival (p=0.007). These data suggested that Bim silencing mediated initial therapeutic resistance in Burkitt lymphoma. To test this hypothesis, several Burkitt lymphoma cell lines were genetically manipulated by ectopic expression of BIM using retroviral vectors or by RNAi, and then incubated with various chemotherapeutic agents commonly used in Burkitt lymphoma therapy. Resistance to doxorubicin and impaired apoptosis were selectively correlated with decreased Bim expression levels. Accordingly, treatment of apoptotic-resistant cell lines with demethylating and/or histone deacetilase inhibitors alone or in combination restored Bim expression and reversed chemotherapeutic resistance in vitro. Concordant results were obtained by treating Burkitt lymphoma xenografts with different Bim expression levels that were developed in RAG2−/−IL2gc−/− immunodeficient mice. Finally, B-cell lymphomas generated in Emu-MYC transgenes crossed with BIM knock-out mice were more resistant to apoptosis after doxorubicin treatment than Emu-MYC lymphomas. In summary, our data support that

1. chemotherapy resistance in patients with Burkitt lymphoma is frequently mediated by epigenetic alteration of pro-apoptotic Bim; and

2. this resistance can be overcome by reactivating Bim expression with demethylating agents such as decitabine and/or histone deacetylase inhibitors.

These drugs, in combination with high-dose chemotherapy, may be used in the up-front therapy of patients with Burkitt lymphoma to prevent refractory disease.