Triggering of CD44 by Ultra-Low-Molecular-Weight Hyaluronan Induces Cell Death by Autophagy Via Endoplasmic Reticulum Stress in Acute Lymphoblastic Leukemia with MLL Gene Rearrangements

Abstract 1361

Acute lymphoblastic leukemia (ALL) with MLL gene rearrangements (MLL+ALL) is chemoresistant and its prognosis is still dismal. MLL+ALL shows a unique gene profile clearly distinguishable from other types of ALL and AML, and CD44 is known as one of specific molecules expressed very highly in MLL+ALL. CD44, a transmembrane glycoprotein recently attracting an attention as a cancer stem cell marker for several types of cancers, is expressed on a variety of cells, and has the specific binding sites for hyaluronic acid (HA, formally hyaluronan). High-molecular-weight (HMW)-HA which exists as extracellular matrix in tissues is incorporated into cells in a CD44-dependent manner, and then is secreted from cells as low-molecular-weight (LMW) to ultra-low-molecular-weight (ULMW)-HA in normal and pathological conditions. The purpose of this study is to investigate the biological events elicited after ligand stimulation of CD44 by using distinct molecular sizes of HA (2.5mg/ml) in MLL+ALL. When cultured in the presence of ULMW-HA (6.4kDa), but not of LMW-HA (31kDa) or HMW-HA (980kDa), thymidine uptakes of cell lines expressing CD44 at very high levels (KOPB-26 and YACL-95) were gradually inhibited in the course of culture from day3 through day5 (greater than 90% decrease at day5), although those expressing CD44 at very low levels (KOCL58, KOCL69) were largely unaffected. Cell cycle analysis of KOPB-26 and YACL-95 using propidium iodide (PI) staining after ULMW-HA stimulation revealed no increase in G0/G1 and G2/M phases and subdiploid population, suggesting that a marked inhibition of thymidine uptakes is neither due to cell cycle arrest nor induction of apoptosis. Dye exclusion test showed a marked decrease in viability at days 3–5 after ULMW-HA stimulation, and flow cytometric analysis showed a gradual increase in the low FSC/high SSC population doubly labeled by Annexin V and PI. However, cytospine smears revealed a massive cell death lacking features characteristic of apoptosis. In addition, pan-caspase inhibitor Z-VAD did not block cell death and cleaved caspase 3 was not detected on flow cytometric analysis after ULMW-HA stimulation, resulting in the conclusion that ULMW-HA-induced cell death is not elicited by induction of apoptosis. Although levels of high-mobility group protein B1 (HMGB1) usually used as a necrosis marker was progressively elevated in culture media after ULMW-HA stimulation, the necrosis inhibitor Necrostatin-1 did not block the ULMW-HA-induced cell death. Finally, electromicroscopic examination was performed and revealed a marked extension of endoplasmic reticulum (ER) and an increase in autophagsomes, and the autophagy inhibitor 3-methyladenine specifically suppressed cell death, indicating that ULMW-HA strongly induces ER stress leading to cell death by autophagy. Changes in ER stress and autophagy-associated molecules after ULMW-HA stimulation were also demonstrated. In conclusion, triggering of CD44 by ULMW-HA stimulation strongly elicits cell death by autophagy via ER stress in chemoresistant MLL+ALL. CD44-targeted therapy by ULMW-HA possibly becomes a useful strategy not only for MLL+ALL but also for CD44-high hematological and non-hematological tumors particularly for those in which the cancer stem cell population is highly positive for CD44.