Paradoxical Enhancement of Leukemogenesis in Acute Myeloid Leukemia Cells with Moderately Attenuated RUNX1 Expressions

Although Runt-related transcription factor 1 (RUNX1), a member of RUNX transcription family, is known for its oncogenic role in the development of acute myeloid leukemia (AML), evidence from other groups support the oncosuppressive property of RUNX1 in leukemia cells, casting a question over the bidirectional function of RUNX1 and it is currently highly controversial. Here we report that the dual function of RUNX1 possibly arise from the total level of RUNX family expressions.

To examine the precise mechanism of RUNX1 expression in leukemogenesis, we first prepared several tetracycline-inducible short hairpin RNAs (shRNAs) which could attenuate the expressions of RUNX1 at different levels in AML cells (MV4-11 and MOLM-13 cells). Intriguingly, while AML cells transduced with shRNAs which could down-regulate RUNX1 expression below 10% at protein level (sh_Rx1_profound) deteriorated the proliferation speed of AML cells, AML cells transduced with shRNAs which could moderately down-regulate RUNX1 expression to 70% at protein level (sh_Rx1_moderate) paradoxically promoted the cell cycle progression and doubled the growth rate of AML cells. Besides, RUNX1-moderately expressing AML patient cohort exhibited the worse outcome compared to RUNX1-high or RUNX1-low expressing cohorts (n = 187), indicating an underlying mechanism that confer growth advantage to AML cells with moderately inhibited RUNX1 expressions.

To further investigate the correspondent gene in this paradoxical enhancement of oncogenesis in sh_Rx1_moderate-transduced AML cells, we performed comprehensive gene expression array and extracted genes that are highly up-regulated in RUNX1 moderate inhibition and down-regulated in AML cells transduced with sh_Rx1_profound. We hereafter focused on the top-listed gene glutathione S-transferase alpha 2 (GSTA2) and addressed the interaction of RUNX1 and GSTA2 and their functions in AML cells. Real time quantitative PCR (RT-qPCR) and immunoblotting revealed that the expression of GSTA2 was actually up-regulated in sh_Rx1_moderate-transduced AML cells and down-regulated in AML cells transduced with sh_Rx1_profound.

Interestingly, equivalent level of compensatory up-regulation of RUNX2 and RUNX3 were observed in sh_Rx1_moderate- and sh_Rx1_profound-transduced AML cells, creating an absolute gap in the expression of total amount of RUNX (RUNX1 + RUNX2 + RUNX3), which was confirmed by RT-qPCR (total amount of RUNX expressions were estimated by primers amplifying the specific sequence common to all RUNX family members). Luciferase reporter assay of GSTA2 promoter and chromatin immunoprecipitation (ChIP) assay in the proximal promoter region of GSTA2 gene proved the association of RUNX family members with this genomic region. These results indicated that total amount of RUNX family expressions modulate the expression of GSTA2 in AML cells, which might results in a paradoxical outbursts of RUNX1 moderately-inhibited AML cells.

Since GSTA2 catabolizes and scavenges free radicals such as hydrogen peroxide (H₂O₂), and decreased intracellular free radicals promote acceleration of cell cycle progression, we next measured the intracellular accumulation of H₂O₂ in RUNX1 inhibited AML cells. As we have expected, intracellular amount of H₂O₂ was decreased in sh_Rx1_moderate-transduced AML cells and increased in AML cells transduced with sh_Rx1_profound. Additive transduction of sh_RNAs targeting GSTA2 to AML cells with sh_Rx1_moderate reverted the proliferation speed to the control level, underpinning that growth advantage of moderate RUNX1 inhibition could be attributed to the GSTA2 overexpressions.

Taken together, these findings indicate that moderately attenuated RUNX1 expressions paradoxically enhance leukemogenesis in AML cells through intracellular environmental change via GSTA2, which could be a novel therapeutic target in anti-leukemia strategy.