Abstract
Acute lymphoblastic leukemia (ALL) with mixed lineage leukemia (MLL) gene rearrangements (MLL+ALL) has a dismal prognosis and is characterized by high surface CD44 expression. Known that CD44 has the specific binding sites for a natural ligand hyaluronan (HA), we investigated biological effects of HA with different molecular sizes on MLL+ALL cell lines, and found that the addition of ultra-low-molecular-weight (ULMW)-HA strongly suppressed their thymidine uptakes. The MLL+ALL cell line lacking surface CD44 expression established by genome editing showed no suppression of thymidine uptake. Surface CD44-high B-precursor ALL cell lines other than MLL+, but not T-ALL cell lines, were also suppressed in their thymidine uptakes. The inhibition of thymidine uptakes was because of induction of cell death, but dead cells lacked features of apoptosis on cytospin smears and flow cytometric analysis. The cell death was neither blocked by pan-caspase inhibitor nor autophagy inhibitor, but was completely blocked by necrosis inhibitor necrostatin-1. Necrotic cell death was further supported by a marked release of a high-mobility protein group B1 and morphological changes on transmission electron microscopy. Elevation of intracellular reactive oxygen species production suggested a role for inducing this necrotic cell death. ULMW-HA-triggered cell death was similarly demonstrated in surface CD44-high primary B-precursor leukemia cells. Assuming that ULMW-HA is abundantly secreted at the site of infection and inflammation, this study sheds light on understanding the mechanism of a transient inflammation-associated remission of leukemia. Further, the CD44-targeting may become an effective approach in future for the treatment of refractory B-precursor ALL by its capability of predominantly eradicating CD44-high leukemia-initiating cells.
Highlights
Mixed lineage leukemia (MLL) gene rearrangements are frequently observed in infantile acute lymphoblastic leukemia (ALL) and therapy-related second leukemia.[1,2] ALL with MLL gene rearrangements (MLL+ALL) has a unique gene profile clearly distinguishable from those of other types of ALL and acute myeloid leukemia (AML), and that the Fms-like tyrosine kinase 3 (FLT3) and CD44 genes are the most differentially expressed at very high levels.[3]
We previously reported that triggering of MLL+ALL cells by FLT3 ligand, abundantly secreted from bone marrow stromal cells, induces their cell cycle arrest showing resistance to chemotherapy, which should lead to the persistent formation of minimal residual disease accountable for the refractory nature of the disease.[4]
To address HA-mediated biological effects on MLL+ALL cells, we examined thymidine uptake after 4-day culture with or without various concentrations of ULMW-HA or HMW-HA, and found that thymidine uptake in CD44-high cell lines was markedly suppressed after culture with 2.5 mg/ml of ULMWHA (% inhibition; 78.6 ± 9.6%, mean ± S.E.), but substantially not affected (% inhibition; − 1.0 ± 5.1%) in CD44-very low cell lines
Summary
Mixed lineage leukemia (MLL) gene rearrangements are frequently observed in infantile acute lymphoblastic leukemia (ALL) and therapy-related second leukemia.[1,2] ALL with MLL gene rearrangements (MLL+ALL) has a unique gene profile clearly distinguishable from those of other types of ALL and acute myeloid leukemia (AML), and that the Fms-like tyrosine kinase 3 (FLT3) and CD44 genes are the most differentially expressed at very high levels.[3] We previously reported that triggering of MLL+ALL cells by FLT3 ligand, abundantly secreted from bone marrow stromal cells, induces their cell cycle arrest showing resistance to chemotherapy, which should lead to the persistent formation of minimal residual disease accountable for the refractory nature of the disease.[4] It should be important to characterize the role of CD44 in MLL+ALL to possibly find a new therapeutic strategy for overcoming this dismal disease. High-molecular-weight (HMW)-HA (103– 104 kD), which exists as ECM in tissues, is incorporated into cells in a CD44-dependent manner, and is secreted from them as low-molecular-weight (LMW)-HA (o500 kD) to ultraLMW (ULMW)-HA (o10 kD) under normal and pathological conditions,[12] at the site of infection and inflammation.[13,14]. The purpose of this study is to investigate the biological events elicited after ligand stimulation of CD44 by using HA with distinct molecular sizes in B-precursor ALL including MLL
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