Some CLL Cells Bind Myosin-Exposed Apoptotic Cells. Exposure of Cytoplasmic Myosin Results From Transfer of Caspase-3 Dependent Cleavage Products to the Outer Cell Membrane
Authors: Cui Xiaoxuan , Zhang Lu , Magli Amanda R , Catera Rosa , Kolitz Jonathan E , Allen Steven L. , Rai Kanti R , Chiorazzi Nicholas , Chu Charles C 
Published: October 2019

Abstract
Abstract 3900
Many monoclonal antibodies (mAbs) produced by B-cell chronic lymphocytic leukemia (CLL) cells bind a subset of apoptotic cells that expose intracellular myosin on the cell surface. CLL patients with mAbs that bind these myosin-exposed apoptotic cells (MEACs) have shorter overall survival. Thus, understanding the mechanism of formation of MEACs and how CLL cells interact with MEACs may help elucidate the pathogenesis of CLL.
To test if formation of MEACs is part of general apoptotic mechanisms, apoptosis was induced in Jurkat T cells by either the intrinsic or extrinsic pathways. The intrinsic pathway was either achieved spontaneously by culturing at high cell density or induced by camptothecin (CPT) treatment. The extrinsic pathway was induced by Fas ligand (FasL) or anti-Fas mAb treatment. Apoptosis and myosin exposition were analyzed by flow cytometry. All four methods of apoptosis induction produced MEACs after prolonged incubation as detailed below. CPT, FasL or anti-Fas mAb incubation for 4 hrs induced significant apoptosis (43-58%) with a detectable fraction of MEACs (9-12%). After incubation for 16 hrs or longer, the majority of apoptotic cells were MEACs (61-89%). Similarly, spontaneous apoptosis produced more MEACs after longer incubation (20% on day 1 versus 59–69% on days 2–4). Both early apoptotic cells, which flip phosphatidylserine (PS) from the inner to outer membrane surface yet retain membrane integrity (AnnexinV+, 7-actinomycin D (7AAD)-), and late apoptotic cells, which become membrane permeable (AnnexinV+, 7AAD+), demonstrate a subpopulation of MEACs that increases with longer incubation times. In contrast, MEACs are not detectable in non-apoptotic cells (AnnexinV-, 7AAD-).
Thus, both intrinsic and extrinsic apoptotic pathways lead to MEAC formation, suggesting that a common downstream mediator may be involved. Caspase-3 activation mediates apoptotic PS exposure and membrane permeability. Therefore, we tested a caspase-3 inhibitor, Z-DEVD-FMK, and found that it significantly reduced both apoptosis and MEAC formation. For example, Z-DEVD-FMK reduced FasL induced apoptosis and MEAC formation from 74 to 14% and from 57 to 10%, respectively. In contrast, caspase-1 inhibitor, Z-YVAD-FMK, had no effect. To test if intracellular myosin is transferred from the cytoplasm to the cell membrane surface during apoptosis, cytoplasmic and membrane protein extracts were prepared, isolated by ultracentrifugation, and blotted with anti-myosin antibody. Two protein bands of the size expected for caspase-3 cleaved myosin (149 and 94 kDa) appeared in membrane extracts of apoptotic cells, but not of live cells. A protein band of the size expected for full-length myosin (250 kDa) predominated in cytoplasmic extracts of live cells. Furthermore, Z-DEVD-FMK inhibited the formation of the 149 and 94 kDa myosin bands in membrane extracts as well as the formation of caspase-3 dependant PARP cleavage products; the same treatment did not alter CD3 membrane protein or GAPDH cytoplasmic protein levels.
Taken together, these results suggest that both intrinsic and extrinsic apoptotic pathways produce MEACs at a later stage in apoptosis that involves the common downstream caspase-3 activation. In turn, myosin fragmentation occurs with subsequent exposure to the cell membrane, where the myosin fragments can serve as a potential neoantigen that may be recognized by some CLL mAbs. Because the mAbs we have used in these analyses were originally integral components of CLL surface membranes, we hypothesized that CLL cells could bind MEACs. Indeed, CLL cells binding to MEACs were visualized by confocal microscopy. To determine the functional consequences of such binding, analyses of the effects of MEAC binding on CLL cell survival in vitro are underway.

Disclosures:
No relevant conflicts of interest to declare.