Asparaginase therapy is a key component of chemotherapy for T-cell acute lymphoblastic leukemia (T-ALL) patients. Asparaginase depletes serum asparagine by deamination into aspartic acid. Normal hematopoietic cells can survive due to asparagine synthetase (ASNS) activity, while leukemia cells are supposed to undergo apoptosis due to silencing of the ASNS gene. Since the ASNS gene has a typical CpG island in its promoter, its methylation status in T-ALL cells may be associated with asparaginase sensitivity. Thus, we investigated the significance of ASNS methylation status in asparaginase sensitivity of T-ALL cell lines and prognosis of childhood T-ALL. Sequencing of bisulfite PCR products using next-generation sequencing technology in 22 T-ALL cell lines revealed a stepwise allele-specific methylation of the ASNS gene, in association with an aberrant methylation of a 7q21 imprinted gene cluster. T-ALL cell lines with ASNS hypermethylation status showed significantly higher in vitro l-asparaginase sensitivity in association with insufficient asparaginase-induced upregulation of ASNS gene expression and lower basal ASNS protein expression. A comprehensive analysis of diagnostic samples from childhood T-ALL patients in Japanese cohorts (n = 77) revealed that methylation of the ASNS gene was associated with an aberrant methylation of the 7q21 imprinted gene cluster. In childhood T-ALL patients in Japanese cohorts (n = 75), ASNS hypomethylation status was significantly associated with poor therapeutic outcome, and all cases with poor prognostic SPI1 fusion exclusively showed ASNS hypomethylation status. These observations demonstrate that ASNS hypomethylation status is associated with asparaginase resistance and is a poor prognostic biomarker in childhood T-ALL.

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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. Disclosures: No relevant conflicts of interest to declare.

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