High-Resolution Sequencing Identifies NOXA1 De-Methylation As a Novel Strategy to Overcome Bortezomib Resistance in Mantle Cell Lymphoma

Abstract 557

Mantle cell lymphoma (MCL) is an aggressive B-lymphoid neoplasm with frequent relapses after initial response to standard chemotherapy. Bortezomib is the first proteasome inhibitor with promising clinical efficacy in relapsed MCL, where it achieved durable responses in 30%–50% of patients. The mechanisms contributing to Bortezomib resistance remain poorly understood. Our group and others have identified aberrantly methylated genes in MCL controlling critical cellular processes like cell cycle, transcription and regulation of gene expression. We therefore hypothesized that a subset of aberrantly methylated genes may be contributing to Bortezomib resistance.

Using a massively parallel sequencing approach, HELP-tagging, we analyzed genomic methylation in MCL patients treated in a Phase II trial at the NIH. This high-resolution technique covers about 1.8 million CpGs in the human genome including gene promoters, gene bodies and CpG shores and is more sensitive and yields greater coverage of the genome as compared to array-based platforms. Unsupervised analysis of genome-wide promoter methylation of Bortezomib sensitive patients (having more than 50% reduction in absolute lymphocyte count after one cycle of Bortezomib) as compared to resistant patients revealed that methylation patterns could independently distinguish between these two groups. Similarly, three-dimensional Principal Component Analysis also clustered Bortezomib resistant patients distinctly from Bortezomib sensitive patients, confirming genomic differences in methylation between these patients. We further analyzed the differentially methylated genes using pathway analysis software, gene ontology and gene set enrichment analysis. We found that majority of differentially methylated genes were relevant to cancer biology involving metabolic pathways, regulation of transcription, cell cycle and programmed cell death. Comparing methylation profiles of patient samples taken before and 96 hours after Bortezomib treatment showed a striking genome-wide hypomethylation following Bortezomib. Western blotting of MCL cell lines showed a significant reduction in DNMT1 levels following Bortezomib treatment, which may help us understand the post-treatment hypomethylation seen in patients.

NOXA1 is a pro-apoptotic Bcl-2 family member essential for the cytotoxicity of Bortezomib. The NOXA1 gene promoter was differentially methylated in Bortezomib-sensitive MCL patients. NOXA1 was also significantly hypomethylated to a greater extent 96 hours following Bortezomib treatment in Bortezomib sensitive patients. NOXA1 hypomethylation was associated with induction of NOXA1 mRNA and protein expression in sensitive patients alone. These findings suggest a possible role for dynamic NOXA1 methylation and expression as biomarkers for Bortezomib sensitivity.

Treatment of MCL cell lines Z138 and MINO with demethylating agents Decitabine also induced NOXA1 hypomethylation and increased NOXA1 protein expression. Low dose (0.1-0.5μM) Decitabine pretreatment potentiated low (IC₂₅) dose Bortezomib causing more than 80–90% cell kill in MINO and Z138 cells. The combination of Decitabine and Bortezomib led to increased NOXA1 hypomethylation and protein expression as compared to the individual drugs. Depletion of NOXA1 by specific shRNA rescued 40–50% of the cells from the cytotoxicity of this combination. Tumor progression and chemoresistance are commonly associated with the overexpression of the prosurvival Bcl-2 family members (Bcl-2, Bcl-xL, and Mcl-1). ABT-263 is a small molecular inhibitor of the Bcl-2 family of proteins, excepting Mcl-1, which can be neutralized by an increase in NOXA1. Decitabine treatment also potently enhanced the cytotoxicity of ABT-263 in MCL cell lines.

Our data suggest that inducing hypomethylation of NOXA1 promoter can be a novel therapeutic strategy for overcoming Bortezomib resistance in MCL, and that genomic methylation patterns and specifically NOXA1 hypermethylation may be useful biomarkers for Bortezomib response. More broadly, these data demonstrate that high-resolution genomic methylation profiling can identify genes and signatures with important prognostic and therapeutic implications.