Aberrantly Expressed Neutrophil Elastase (ELA2) Cleaves Cyclin E (CCNE) in the Nucleus and Cytoplasm of Acute Lymphocytic Leukemia Yielding Novel Leukemia-Associated Antigens.

Abstract

We have shown that donor-derived cytotoxic T lymphocytes (CTL) are specific for two HLA-A2-restricted peptides derived from CCNE (CCNE1144–152, ILLDWLMEV and CCNE2144–152, ILLDWLLEV) specifically lyse lymphoid and myeloid leukemia cells that aberrantly express CCNE. The CCNE protein is overexpressed in AML, ALL, and CML, and in solid tumors such as breast, lung, and gastric cancers. Furthermore, five low molecular weight forms (LMWFs) of CCNE, which are constitutively active to promote cell division, are found only in malignant cells, though it is not known how LMWFs are formed in leukemia. We hypothesized that the cleavage of CCNE into LMWFs occurs by enzymatic cleavage from aberrantly expressed ELA2 in leukemia, which renders the leukemia susceptible to killing by ELA2- and CCNE-specific CTL. Whole cell lysates from U937, HL60, and bone marrow from patients with B-ALL, but not from PBMC or bone marrow cells from healthy donors or ALL patients in remission, showed high expression of CCNE and LMWF by western blot (WB). Recombinant ELA2 added to B cell-derived whole cell lysates increased LMWFs, and the leukocyte elastase inhibitor Elafin prevented this cleavage. Subcellular fractions studied by coimmunoprecipitation showed that ELA2 was bound to CCNE in the nucleus, cytoplasm, and membrane-bound organelles of B-ALL, but not in normal cells. Because nuclear expression of CCNE increases during normal cell division, we studied healthy donor PBMC stimulated with anti-CD3 and anti-CD28 and found no expression of ELA2 or CCNE LMWFs by WB. We conclude that ELA2, normally expressed only in myeloid cells, is also expressed in some ALL blasts, and this data explains how CCNE LMWFs are formed in leukemia. Our findings also suggest how ELA2-mediated cleavage of the PML-RARα fusion product, required for leukemic transformation, could occur when ELA2 is aberrantly expressed in the nucleus. Finally, this work implies that overexpression of CCNE and the LMWFs that contain the immunogenic peptides could increase susceptibility of leukemia cells to CCNE-CTL lysis.