CCAAT/enhancer binding protein α (C/EBPα) regulates myeloid differentiation, and its dysregulation contributes to acute myeloid leukaemia (AML) progress. Clarifying its functional implementation mechanism is of great significance for its further clinical application. In this study led by Shandong University, they show that C/EBPα regulates AML cell differentiation through liquid-liquid phase separation (LLPS), which can be disrupted by C/EBPα-p30. They used LC Sciences’ RNA Sequencing service on THP-1 and PMA-treated THP-1 cells. Considering that C/EBPα-p30 inhibits the functions of C/EBPα through the LZ region, a small peptide TAT-LZ that could instantaneously interfere with the homodimerization of C/EBPα-p42 was constructed, and dynamic inhibition of C/EBPα phase separation was observed, demonstrating the importance of C/EBPα-p42 homodimers for its LLPS. Mechanistically, homodimerization of C/EBPα-p42 mediated its phosphorylation at the novel phosphorylation site S16, which promoted LLPS and subsequent AML cell differentiation. Finally, decreasing the endogenous C/EBPα-p30/C/EBPα-p42 ratio rescued the phase separation of C/EBPα in AML cells, which provided a new insight for the treatment of the AML.

C/EBPα undergoes an abnormal particle alteration during cell differentiation

Fig. 1

a Experimental schematic for the establishment of the Dox/Ara-C (D/A) treated GFP + MLL-AF9 mouse model. b Representative photographs and weights of spleens isolated from the groups of GFP + MLL-AF9 mice at 7 days after treatment (n = 5 mice per group). A two-tailed unpaired Student’s t test was used for statistical analysis and data were presented as mean values  ±  SEM. c Flow cytometric analysis of GFP + MLL-AF9 leukaemia cells in BM of the control and D/A groups (n = 5 mice per group). A two-tailed unpaired Student’s t test was used for statistical analysis and data were presented as mean values  ±  SEM. d Immunofluorescence photograph analysis of the number of endogenous C/EBPα condensates in each GFP + MLL-AF9 leukaemia cell in the control and D/A groups. Quantification of C/EBPα condensates (n =  3 biologically independent experiments). A two-tailed unpaired Student’s t test was used for statistical analysis, and data were presented as mean values  ±  SEM. e Immunofluorescence photograph analysis of the number of endogenous C/EBPα condensates in each CD34+ primary AML cell treated with the control or D/A (Ara-C:50 nM; Dox:0.2 μg/mL) for 24 h. Quantification of C/EBPα condensates (n =  6 AML patients). A two-tailed unpaired Student’s t test was used for statistical analysis, and data were presented as mean values  ±  SEM. f Immunofluorescence photograph analysis of the number of endogenous C/EBPα condensates in each THP-1 cell stimulated with 100 ng/mL PMA for 0 h, 3 h, 6 h, 12 h, 24 h and 48 h. Quantification of C/EBPα condensates (n =  3 biologically independent experiments). A two-tailed unpaired Student’s t test was used for statistical analysis, and data were presented as mean values  ±  SEM. The experiments in (d, f) were observed at least 20 random fields in each experiment. For (e), at least 10 random fields were observed in each experiment. For (d, e), scar bars, 5 μm. For (f), scar bar, 2 μm. Source data are provided as a Source Data file.

Wang Dongmei, Sun Tao, Xia Yuan, Zhao Zhe, Sheng Xue, Li Shuying, Ma Yuechan, Li Mingying, Su Xiuhua, Zhang Fan, et. al.  (2023) Homodimer-mediated phosphorylation of C/EBPα-p42 S16 modulates acute myeloid leukaemia differentiation through liquid-liquid phase separation. Nature Communications 14(1), 6907. [article]

CCAAT/enhancer binding protein α (C/EBPα) regulates myeloid differentiation, and its dysregulation contributes to acute myeloid leukaemia (AML) progress. Clarifying its functional implementation mechanism is of great significance for its further clinical application. In this study led by Shandong University, they show that C/EBPα regulates AML cell differentiation through liquid-liquid phase separation (LLPS), which can be disrupted by C/EBPα-p30. They used LC Sciences’ RNA Sequencing service on THP-1 and PMA-treated THP-1 cells. Considering that C/EBPα-p30 inhibits the functions of C/EBPα through the LZ region, a small peptide TAT-LZ that could instantaneously interfere with the homodimerization of C/EBPα-p42 was constructed, and dynamic inhibition of C/EBPα phase separation was observed, demonstrating the importance of C/EBPα-p42 homodimers for its LLPS. Mechanistically, homodimerization of C/EBPα-p42 mediated its phosphorylation at the novel phosphorylation site S16, which promoted LLPS and subsequent AML cell differentiation. Finally, decreasing the endogenous C/EBPα-p30/C/EBPα-p42 ratio rescued the phase separation of C/EBPα in AML cells, which provided a new insight for the treatment of the AML.

C/EBPα undergoes an abnormal particle alteration during cell differentiation

Fig. 1

a Experimental schematic for the establishment of the Dox/Ara-C (D/A) treated GFP + MLL-AF9 mouse model. b Representative photographs and weights of spleens isolated from the groups of GFP + MLL-AF9 mice at 7 days after treatment (n = 5 mice per group). A two-tailed unpaired Student’s t test was used for statistical analysis and data were presented as mean values  ±  SEM. c Flow cytometric analysis of GFP + MLL-AF9 leukaemia cells in BM of the control and D/A groups (n = 5 mice per group). A two-tailed unpaired Student’s t test was used for statistical analysis and data were presented as mean values  ±  SEM. d Immunofluorescence photograph analysis of the number of endogenous C/EBPα condensates in each GFP + MLL-AF9 leukaemia cell in the control and D/A groups. Quantification of C/EBPα condensates (n =  3 biologically independent experiments). A two-tailed unpaired Student’s t test was used for statistical analysis, and data were presented as mean values  ±  SEM. e Immunofluorescence photograph analysis of the number of endogenous C/EBPα condensates in each CD34+ primary AML cell treated with the control or D/A (Ara-C:50 nM; Dox:0.2 μg/mL) for 24 h. Quantification of C/EBPα condensates (n =  6 AML patients). A two-tailed unpaired Student’s t test was used for statistical analysis, and data were presented as mean values  ±  SEM. f Immunofluorescence photograph analysis of the number of endogenous C/EBPα condensates in each THP-1 cell stimulated with 100 ng/mL PMA for 0 h, 3 h, 6 h, 12 h, 24 h and 48 h. Quantification of C/EBPα condensates (n =  3 biologically independent experiments). A two-tailed unpaired Student’s t test was used for statistical analysis, and data were presented as mean values  ±  SEM. The experiments in (d, f) were observed at least 20 random fields in each experiment. For (e), at least 10 random fields were observed in each experiment. For (d, e), scar bars, 5 μm. For (f), scar bar, 2 μm. Source data are provided as a Source Data file.

Wang Dongmei, Sun Tao, Xia Yuan, Zhao Zhe, Sheng Xue, Li Shuying, Ma Yuechan, Li Mingying, Su Xiuhua, Zhang Fan, et. al.  (2023) Homodimer-mediated phosphorylation of C/EBPα-p42 S16 modulates acute myeloid leukaemia differentiation through liquid-liquid phase separation. Nature Communications 14(1), 6907. [article]