Identification of an alternative CD20 transcript variant in B-cell malignancies coding for a novel protein associated to rituximab resistance

Abstract

Human CD20 is a B-cell lineage-specific marker expressed by normal and leukemic B cells from the pre-B to the plasmacell stages and is a target for rituximab (RTX) immunotherapy. A CD20 reverse transcriptase-polymerase chain reaction (PCR) on B-cell lines cDNA yielded a short PCR product (⌬CD20) corresponding to a spliced mRNA transcript linking the exon 3 and exon 7 ends. We established here that this novel, alternatively spliced CD20 transcript is expressed and detectable at various levels in leukemic B cells, lymphoma B cells, in vivo tonsil-or in vitro CD40L-activated B cells, and Epstein-Barr virus (EBV)-transformed B cells, but not in resting CD19 ؉ -or CD20 ؉ -sorted B cells from peripheral blood or bone marrow of healthy donors. The truncated CD20 sequence is within the reading frame, codes a protein of 130 amino acids (ϳ 15-17 kDa) lacking large parts of the 4 transmembrane segments, suggesting that ⌬CD20 is a nonanchored membrane protein. We demonstrated the translation into a ⌬CD20 protein which is associated with the membrane CD20 protein and showed its involvement in RTX resistance. Study of patient samples before and after RTX resistance or escape confirms our in vitro findings. (Blood. 2010;115:2420-2429) Introduction CD20 (MS4A1) is a 33-to 37-kDa nonglycosylated transmembrane (TM) phosphoprotein that is widely expressed throughout B-lymphocyte ontogeny, in normal or malignant B cells. 1 The 16-kb gene encoding human CD20, consisting of 8 exons, has been mapped to chromosome 11 (11q12-q13) and belongs to the MS4A (membrane spanning 4A) gene family localized within a cluster of related genes (MS4A1 to MS4A11). 2 Its transcription leads to 3 mRNA isoforms: a dominant 2.8-kb transcript, using exons 1 to 8; a second exon 1-spliced transcript shorter by 263 bp; and a third, minor 3.4-kb transcript, 3 all encoding a full-length CD20 protein.The CD20 protein consists of cytoplasmic N-and C-termini and 4 hydrophobic regions for anchoring the molecule in the membrane. 4 A total of 3 isoforms have been identified, including a predominant 33-kDa molecule and 2 isoforms of 34.5 and 36 kDa, resulting from differential phosphorylation states (on serine and threonine residues) in relation to B-cell stimulation and proliferation. 5 CD20 appears to play a role in Ca ϩϩ conductance 6 and is also involved in cell-cycle progression by interaction with src family kinases. 7 Finally, CD20 circulating form has been identified in chronic lymphocytic leukemia (CLL), Hodgkin disease, or nonHodgkin lymphoma (NHL), and in healthy persons. 8 CD20 expression at the cell surface of malignant B cells makes it a target for monoclonal antibody (mAb) therapy. Rituximab (RTX), the first US Food and Drug Administration (FDA)-approved mAb for clinical therapy, targets the CD20 antigen 9 and leads to CD20-expressing B-cell depletion through different mechanisms 9,10 (for review, see Cartron et al 11 ). Thus, RTX is widely used against B-cell malignancies and also for autoimmune diseases such as rheumatoid arthritis, 12 steroid refract...

CD20 alternative splicing isoform generates immunogenic CD4 helper T epitopes

“…One explanation of the presence of D393-CD20 specific responses in healthy donors could be explained by the expression of D393-CD20 in EBV infected B cells. 6 Thus, the D393-CD20 specific response could be both an antiviral and an antitumor immune response. However, we have not compared the presence of this response in EBV positive and in EBV negative donors.…”

“…The two bands at position 15 to 17 kDa may correspond to different phosphorylation states of the D393-CD20 protein. 6 To assess its expression in primary lymphoma cells, we looked for D393-CD20 protein in peripheral blood lymphocytes of patient with circulating lymphoma cells. D393-CD20 protein expression was detected in five tested patient's PBMC and was absent in healthy donor's PBMC (Fig.…”

“…D393-CD20 mRNA is absent from normal resting B cells but present in various malignant or transformed B cells. [6][7][8] Moreover, high expression of D393-CD20 protein …”

“…This alternative transcript lacks 168 nucleotides within Exon 3 to 7 compared to the wild-type CD20 transcript and referred thereafter as D393-CD20. 6 D393-CD20 translation gives rise to a protein lacking the extracellular domain and the most part of the four transmembrane-spanning domains and is, therefore, cytoplasmic. D393-CD20 mRNA is absent from normal resting B cells but present in various malignant or transformed B cells.…”

“…[6][7][8] Moreover, high expression of D393-CD20 protein has been found in malignant B cells of relapsed patients previously treated with the anti-CD20 therapeutic monoclonal antibody Rituximab. 6 Therefore, we reasoned that the selective expression in leukemic B cells, as well as the expression of D393-CD20 in resistant lymphomas confers to this spliced mRNA the potential to be a tumor associated antigen. We thus investigated whether the D393-CD20 could generate epitopes recognized by T lymphocytes and eligible as new targets for immunotherapy.…”