ETV6/RUNX1 abrogates mitotic checkpoint function and targets its key player MAD2L1

Abstract

Approximately 25% of childhood B-cell precursor acute lymphoblastic leukemia have an ETV6/RUNX1 (E/R) gene fusion that results from a t(12;21). This genetic subgroup of leukemia is associated with near-triploidy, near-tetraploidy, and trisomy 21 as rather specific types of secondary changes. Here, we show that, unlike various controls, E/R-expressing Ba/F3 clones acquire a tetraploid karyotype on prolonged culture, corroborating the assumption that E/R may attenuate the mitotic checkpoint (MC). Consistent with this notion, E/Rexpressing diploid murine and human cell lines have decreased proportions of cells with 4N DNA content and a lower mitotic index when treated with spindle toxins. Moreover, both RUNX1 and E/R regulate mitotic arrestdeficient 2 L1 (MAD2L1), an essential MC component, by binding to promoter-inherent RUNX1 sites, which results in down-regulation of MAD2L1 mRNA and protein in E/R-expressing cells. Forced expression of E/R also abolishes RUNX1-induced reporter activation, whereas E/R with a mutant DNA-binding site leads to only minor effects. Our data link for the first time E/R, MC, and MAD2L1 and provide new insights into the function of the E/R fusion gene product. Although tetraploidy is an almost exclusive feature of E/R-positive leukemias, its rarity within this particular subgroup implies that further yet unknown factors are required for its manifestation.

Silencing of ETV6/RUNX1 abrogates PI3K/AKT/mTOR signaling and impairs reconstitution of leukemia in xenografts

“…Taken together with the cell cycle distribution showing a modest increase of the G0/G1 compartment in E/R-silenced cells (Figure 2d), the decelerated proliferation rate can therefore be attributed to a G1 arrest with a concomitant increase in apoptosis at the transition to S that could account for the selective loss of the S phase. A possible effect of E/R suppression on the mitotic checkpoint function 29 was addressed by phospho-histone H3 staining, but did not reveal any differences between E/R-depleted cells and controls (5 --8% positivity in two independent E/R knockdown experiments; data not shown). These findings suggest that E/R silencing alters the growth pattern of the leukemia cell lines REH and AT-2 by impeding proliferation and survival.…”

ETV6/RUNX1-positive relapses evolve from an ancestral clone and frequently acquire deletions of genes implicated in glucocorticoid signaling

“…28 Lentiviral vectors expressing shRNA sequences directed against the human (pHR-THT-sh-hBMF-SFFV-eGFP) BMF or murine (pHR-THT-sh-mBmf-SFFV-eGFP) Bmf, nontargeting the human BMF, 29 were used to transduce REH cells. Details on shRNA sequences and lentiviral supernatant production and transduction are provided in the supplemental data.…”

STAT3 mediates oncogenic addiction to TEL-AML1 in t(12;21) acute lymphoblastic leukemia

“…[20][21][22] However, there is only limited information on the signaling pathways deregulated by the fusion in these cells. [22][23][24][25] To address this question, we have used small hairpin (sh)RNA-mediated silencing to demonstrate that TEL-AML1 is responsible for inducing signal transducer and activator of transcription 3 (STAT3) activation in human t(12;21) leukemia cells. This activation is mediated via RAC1, results in transcriptional induction of MYC and is critical for the survival of TEL-AML1…”