Reciprocal activation of E/R fusion and the GNAO1 R209C mutation induces leukemogenesis. (A) RT-PCR analysis of GNAO1 upregulation in ETV6-RUNX1 (E/R)⁺ clinical ALL specimens. ACTB was used as a control. (B) WB analysis of GNAO1 expression in normal PBMCs, E/R⁺, and E/R⁻ childhood ALL specimens. (C) qRT-PCR analysis of GNAO1 mRNA expression in normal PBMCs, Reh, UoCB6, Nalm6, and THP1 leukemia cells. (D) The expression level of GNAO1 mRNA was significantly higher in E/R⁺ALL than in normal PBMCs. Expression data of GNAO1 mRNA were downloaded from the GSE79869 data set and analyzed. (E) qRT-PCR analysis of the effect of ectopic expression of the E/R fusion or an EV control on GNAO1 expression. (F) IP and WB analysis of the effects of rapamycin treatment on p300 acetylation (Ace-Lys), p300 serine/threonine phosphorylation (p-S/T), and E/R acetylation stimulated by ectopic expression of GNAO-1 WT or R209C mutation in Reh cells. (G) The K43R mutation of RUNX1 in E/R fusion inhibits E/R acetylation and association with TAF-1 stimulated by overexpression of GNAO1 WT or R209C mutant in Reh cells. (H) Treatment with the p300 inhibitor C464 reduces GNAO1 upregulation by E/R. (I) A hypothetical model showing how GNAO1 R209C mutation and E/R fusion cooperate to induce leukemia. Data are representative of 3 independent experiments with similar results. (C-E,H) Data are expressed as the mean ± SD. *P < .05; **P < .01; ***P < .001, by 2-tailed (C,E,H) or 1-tailed (D) Student t test.