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S. Liu et al.
microRNA networks and cell cycle inhibitors, and it also played important roles in regulating hematopoietic differ- entiation, especially myeloid and monocytic differentia- tion.15,24 Furthermore, a recent study described the role of RUNX1 in inducing intestinal goblet cell differentiation, acting through direct transcriptional activation of KLF4.25 However, the interactions between RUNX1 and KLF4 in AML and its association with leukemia development have remained largely unexplored.
In this study, we demonstrated that KLF4 was directly regulated by RUNX1 through four of seven putative bind- ing sites within the KLF4 promoter region. KLF4 expres- sion could be markedly transactivated by RUNX1 in a dose-dependent manner while leukemogenic fusion pro- tein RUNX1-ETO only had a slight effect. We expected KLF4 expression to be specifically lower in t(8;21) leukemia samples due to haploinsufficiency of RUNX1 in these cells. However, according to an analysis of a publicly available microarray gene expression profiling (GEP) dataset (containing 285 annotated AML cases and 10 healthy cases), KLF4 expression was consistently decreased in the majority of AML cases, and there was no significant difference between t(8;21) and non-t(8;21) groups.26,27 This suggested that KLF4 inactivation might be occurring in non-t(8;21) leukemia cells in distinctly differ- ent ways. One mechanism would be DNA hypermethyla- tion, which has already been confirmed by two independ- ent groups in chronic lymphocytic leukemia and adult T- cell leukemia.28,29 Other possibilities remain of great inter- est for further investigation. In addition to RUNX1, analy- sis of KLF4 promoter region also revealed putative binding sites correspond to STAT3 and NF-κB (date not shown), both of which are important transcription factors in the hematopoietic system, suggesting that RUNX1 might col- laborate with these factors to regulate KLF4 expression. Clarification of the connections between RUNX1, STAT3 and NF-κB is the subject of our ongoing study.
By analyzing RUNX1 Chip-Seq data, we identified sev- eral RUNX1 binding sites within the KLF4 promoter region. Furthermore, we found remarkable enrichment of KLF4 consensus motif within RUNX1 chip regions, which suggested the physical interaction between these two fac- tors. Supporting this possibility, both exogenous and endogenous RUNX1 and KLF4 were confirmed to co- localize and interact with each other in nuclei by co- immunoprecipitation and immunofluorescence confocal imaging assay. To the best of our knowledge, this is the first report demonstrating KLF4 both as a direct target and a binding partner of RUNX1. The physical interaction between RUNX1 and KLF4 was mediated through RHD, a critical domain of RUNX1 responsible for DNA binding and commonly involved in protein-protein interaction. RUNX1-KLF4 interaction increased KLF4 transactivation capacity on a promoter reporter driven by KLF4-response elements only (KLF4-Reporter). RUNX1-ETO was also shown to interact with KLF4; however, it had almost no co-activation effect on KLF4-Reporter. The competitive protein-protein interaction experiments showed that RUNX1-ETO disrupted RUNX1-KLF4 interaction in a competitive manner, which might block RUNX1 from co- activating KLF4 target genes. Previous studies described the role of RUNX1 in mediating the interaction between
PU.1 and NuAT and BAF families of co-activators. RUNX1-ETO displaced the co-activators from PU.1 and produced a striking switch to PU.1 interaction with co- repressors, such as Dnmt1, Sin3A, Nurd, CoRest, and B- Wich.30 Whether interaction between RUNX1-ETO and KLF4 would abolish KLF4 binding capacity with co-activa- tors or impact its DNA-binding ability are questions that need to be further addressed.
RUNX1 and KLF4 are both up-regulated genes during HDAC inhibitor-induced differentiation and apoptosis of t(8;21) leukemia cells. In this study, we performed overex- pression experiments to address their biological roles in t(8;21) leukemia cells. The results showed that both of them contributed to cell proliferation inhibition and cell apoptosis induction. Besides, KLF4 also promoted myeloid differentiation of t(8;21) leukemia cells by up-reg- ulating myeloid markers CD11b and CD15. Furthermore, we reported the role of P57, a novel target gene of KLF4 in limiting proliferation and inducing apoptosis. Thus, RUNX1, KLF4 and P57 might make up a transcriptional activation cascade in regulating t(8;21) leukemia cells sur- vival. To further investigate whether the anti-leukemic effects of the “RUNX1-KLF4-P57” pathway existed only in t(8;21) AML, we performed additional overexpression experiments of RUNX1, KLF4 and P57 in a non-RUNX1- ETO expression cell line, the HL-60 cells. The result showed that overexpression of RUNX1 also up-regulated KLF4 and P57 expression in HL-60 cells (Online Supplementary Figure S2A and B), and overexpression of either RUNX1, KLF4 or P57 (Online Supplementary Figure S2C) could inhibit the proliferation and induce apoptosis in HL-60 cells (Online Supplementary Figure S2D and F), which was similar to the results observed in Kasumi-1 cells. The above results obtained from HL-60 cells suggest- ed that the “RUNX1-KLF4-P57” pathway not only existed in RUNX1-ETO expressing cells but also in non-RUNX1- ETO expressing cells. The effect of RUNX1-ETO on the “RUNX1-KLF4-P57” pathway needs to be further clarified and this is something that we will carry forward in our future studies.
In summary, this study identified KLF4 as a target gene and a binding partner of RUNX1. KLF4 mediated prolifer- ation inhibition and apoptosis induction of t(8;21) leukemia cells through transactivating P57. Restoration of the “RUNX1-KLF4-P57” signaling pathway might be an effective therapeutic strategy for t(8;21) AML.
Acknowledgments
The authors would like to thank the staff for their kindly assis- tance, especially Wanzhu Yang, Haoyue Liang and Weichao Fu in Core Facility of flow cytometry, State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College.
Funding
This work was supported by grants from the National Natural Science Foundation of China (81570147, 81430004 and 81800153), Foundation for Innovative Research Groups of the National Natural Science Foundation of China (81421002) and CAMS Initiative Fund for Medical Sciences (2016-I2M-1-001, 2016-I2M-1-007).
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haematologica | 2019; 104(8)