Specific MLL Fusion Partners Affect the Sensitivity of Human and Murine Leukemic Cell Lines to Demethylating Agents

The CCAAT/enhancer binding protein (C/EBP)α is a transcriptional factor that plays a key role in granulocytic/monocytic differentiation. We have demonstrated that ATRA and 5-Aza-2′-deoxycytidine (5-Aza) efficiently induce over-expression of C/EBPα, leading to monocytic differentiation, cell cycle arrest and apoptosis in various AML cell lines (Fujiki A, et al. The 51st Annual ASH Meeting). In addition, we also found that THP-1 cells, which express MLL-AF9, are more sensitive to treatment with a combination of ATRA and 5-Aza than KOCL48 cells, which express MLL-AF4. To explore whether the expression of MLL fusion partners affects the degree of monocytic differentiation induced by combination treatment with ATRA and 5-Aza, we compared the expression levels of various transcriptional factors and cell surface markers in human and murine leukemic cells expressing variousMLL fusion proteins.

THP-1 and KOCL48, two human AML cell lines containing MLL rearrangements, and MLL-AF9, MLL-ENL and MLL-AF5q31, three murine leukemic cell lines expressing MLL fusion proteins, were used in this study. Thee murine cell lines were derived from murine Lin-hematopoietic progenitors transduced by retroviral vectors expressing MLL fusion proteins.

To test the effect of Aza and ATRA on cell growth, all of the cell lines were treated with 50 nM 5-Aza for two days, followed by 1 μM ATRA for an additional three or five days. Cell growth was then analyzed by nuclei counting using a Coulter counter. Cell cycle analysis was also performed by flow cytometry (FCM). In addition, an Annexin V assay was performed to measure the level of apoptosis. To assess whether monocytic differentiation was induced, the level of CD11b/Mac 1 expression was evaluated by FCM. In addition, the level of expression of different transcription factors, including C/EBPa, C/EBPe and PU.1/ Sfpi1, was analyzed by quantitative PCR (qRT-PCR) analysis.

Although both THP-1 and KOCL-48 cells were moderately resistant to ATRA (growth inhibition rate of 40–50%), the addition of 5-Aza efficiently suppressed the growth in these two cell lines (growth inhibition rate of 80%). Cell cycle analysis revealed that G1 arrest occurred at almost the same level in both cell lines. However, an Annexin V assay revealed that treatment with ATRA and 5-Aza induced 1.5 times more Annexin positive THP-1 cells than KOCL-48 cells. Morphological studies of treated THP-1 and KOCL-48 cells revealed characteristic features of apoptosis, such as an extended cytoplasm containing vacuoles, the presence of fine granules, and irregular shaped nuclei. However, FCM analysis revealed that, following treatment, CD11b was expressed at higher levels in THP-1 cells than in KOCL-48 cells. Similarly, qRT-PCR analysis demonstrated that PU.1 expression was induced to higher levels in the THP-1 cell line than in KOCL-48 cells following ATRA/5-Aza treatment (p<0.05), suggesting that these two cell lines are differentially sensitive to combination therapy. In addition, the murine cell lines expressing either MLL-AF5q31 or MLL-ENL were resistant to treatment with ATRA /5-Aza (growth inhibition rate: 10–30%). However, the murine cell line expressing MLL-AF9 was sensitive to ATRA/5-Aza treatment (growth inhibition rate: 90%). Using qRT-PCR analysis, it was found that C/EBPα and Sfpi1 expression increased in MLL-AF9-expressing murine leukemic cells but not in MLL-ENL or MLL-AF5q31-expressing murine leukemic cells.

Monocytic differentiation is more readily induced by combination treatment with ATRA and 5-Aza in MLL-AF9-expressing cells than in MLL-ENL- or MLL-AF4/AF5q31-expressing cells. The presence of specific MLL fusion partners might affect the sensitivity of cell lines to demethylating agents through inducing different degrees of DNA methylation.

No relevant conflicts of interest to declare.