Establishment of a New Mantle Cell Lymphoma Cell Line That Has Epigenetically Silenced CD20 As a Resistance Mechanism in Vivo

Abstract 4619

Rituximab is an anti-CD20 monoclonal antibody that has significant activity in B cell lymphomas that express the cell surface protein CD20. The mechanism of its antitumor effects in vivo are not clear but include direct apoptotic effects, complement mediated cytotoxicity (CMC), and antibody dependent cellular cytotoxicity (ADCC). Previously we initiated a clinical trial of SAHA (vorinostat), cladribine, and rituximab (SCR) in patients with CD20+ B cell malignancies. In addition to its cytotoxic effects, recent evidence suggests that 2-CdA has hypomethylating properties.

A 65 year old patient with newly diagnosed blastic,leukemic, mantle cell lymphoma (MCL) was initiated on the SCR trial. He responded promptly with normalization of his blood counts and resolution of his splenomegaly. A PET/CT scan performed prior to cycle 3 was consistent with a complete metabolic remission. He developed cytopenias prior to cycle 5 that met criteria for removal from the study. Imaging and blood studies were compatible with florid relapse of his MCL including CNS disease. At post mortem analysis, cells in the CNS were CD20 positive and cells in the blood, marrow, and lymph nodes were CD20 negative by immunohistochemistry and flow cytometry. CD20 mRNA analysis by qRT-PCR demonstrated marked reduction in CD20 mRNA. A cell line was derived from the patient's blood. The cells are cyclin D1 positive and expresses Sox 11 with ∼50 times less CD20 mRNA and appropriate sized protein. This 353 cell line has been grown in continuous culture for > 1 year and remains CD20 negative, CCND1 positive and contains the t(11;14). Analysis of the CD20 promoter and coding region demonstrated no evidence of a deletion or point mutation.

Chromatin immunoprecipation (ChIP) assays were performed to access epigenetic changes at the CD20 promoter. First, we showed that the CD20 promoter in Granta MCL cells treated with cladribine contain decreased amounts of methylated histones H3K9 and K27, consistent with an epigenetic mechanism of action of cladribine involving histone methylation. Decreased DNA methylation at the CD20 promoter was also observed.

ChIP analysis of the patient's cells before and after treatment with cladribine and vorinostat in vivo demonstrated decreases in CD20 promoter DNA and histone (H3 K9 and H3K27) methylation, consistent with the known epigenetic properties of these agents.

The 353 cell line reported here will be of interest in studying epigenetic mechanisms of resistance to rituximab by silencing of CD20 gene expression. Experiments to overexpress exogenous CD20 to resensitize 353 cells to rituximab are in progress and will be reported. Future experiments will also aim at identifying a cDNA or drug that can overcome epigenetic resistance in vitro in 353 cells. These experiments could lead to improved treatments to overcome epigenetically mediated resistance to rituximab.