ELF-153

ERG+85^High Cells Exhibit Regenerative Properties

Acute leukemia is a fast-progressing blood cancer with poor survival rates, mainly due to poorly characterized leukemia stem cells (LSCs) that resist treatments and cause relapse. Aqaqe et al. employ an ERG+85 enhancer-based fluorescent reporter system to investigate and characterize subpopulations of leukemia cells with stem cell-like properties.

To assess the stability of cellular states marked by ERG+85, they analyzed tagBFP ^dynamics in sorted tagBFP^High and tagBFP^Neg Jurkat and ELF153 cells. FACS revealed tagBFP^High cells could regenerate populations of tagBFP^High, tagBFP^Intermediate, and tagBFP^Neg cells. In contrast, tagBFP^Neg cells failed to produce tagBFP^High cells even after 30 days (Fig. 1). A similar distribution was observed when assessing ERG+85 activity in progeny from individual tagBFP^High or tagBFP^Neg cells in colony assays. ERG protein analysis showed tagBFP^High Jurkat cells had 2- to 3-fold higher ERG protein levels than tagBFP^Neg cells (Fig. 1). Notably, both cell types had similar viral integration numbers, dismissing biases or stochastic expression variations of tagBFP. The presence of varying tagBFP levels in tagBFP^High progeny suggests a gradual regulation mechanism rather than an on/off switch for ERG+85 dynamics. To compare the regenerative potential of tagBFP^High and tagBFP^Neg cells, they conducted a methycellulose colony replating assay. TagBFP^High ELF-153 cells formed more colonies than tagBFP^Neg cells across all platings, with tagBFP^Neg cells failing to form colonies by the third plating, indicating lower self-renewal potential (Fig. 2A). They assessed AML blasts' regenerative capacity using stroma-supported cultures and limiting dilution analysis (LDA). TagBFP^High cells showed 1.7- to 23-fold higher culture initiation than tagBFP^Neg cells and demonstrated significant proliferation, unlike tagBFP^Neg cells (Fig. 2B-D). In primary AML cells, tagBFP^High cells could regenerate cells with varying ERG+85 activity, while tagBFP^Neg could not produce tagBFP^High cells (Fig. 2B-D). These findings are consistent with those in reporter-expressing leukemia cell lines.

Fig. 1. Regeneration and migration/invasion potential of tagBFP^High and tagBFP^Neg fractions (Aqaqe N, Yassin M, et al., 2019).

Fig. 2. ERG+85^High cells exhibit regenerative properties (Aqaqe N, Yassin M, et al., 2019).

Aza-Caused Demethylation in Methylated CpG Sites of ELF-153 and MOLM-13 Cell Lines

Hypomethylating agents like 5-Azacytidine (Aza) are used to treat myeloid malignancies, improving survival in MDS, CMML, and AML patients. The exact molecular mechanisms behind the response to these agents remain unclear, especially since traditional treatment aims to eradicate malignant clones, yet Aza seems to stabilize clonal architecture instead.

Using next-generation sequencing (NGS), 30 myeloid leukemia-associated genes were analyzed in 15 MDS/CMML patients with excellent response to Aza. Gawliza et al. identified four main gene targets (LINE-1, EZH2, NOTCH1, RUNX1 and WT1) of Aza by methylation analysis. Then, in vitro evaluation of Aza response was conducted on ELF-153 and MOLM-13 cell lines. The 72-hour IC₅₀ for Aza was 5.8 µmol/l for ELF-153 and 2.5 µmol/l for MOLM-13. They analyzed LINE-1, a global methylation marker, and potential target genes (LINE-1, NOTCH1, RUNX1 and WT1). After 72 hours of incubation, significant demethylation of LINE-1 and all genes was observed compared to untreated controls (0 h) (Fig. 3). Genes with higher baseline methylation (e.g., EZH2) showed greater absolute methylation reduction than those with lower baseline methylation (e.g., RUNX1). EZH2 exhibited the most significant reduction: 46.1% in ELF-153 and 37.5% in MOLM-13.

Fig. 3. Decrease in CpG methylation levels of LINE-1, EZH2, NOTCH1, RUNX1 and WT1 in MOLM-13 and ELF-153 incubated with Aza for 72 h (Gawliza A L, Speith J, et al., 2019).