Two faces of RUNX3 in myeloid transformation

•RUNX3 functions as both a tumor suppressor and an oncogene in cancers.•RUNX3 inhibits expression and function of RUNX1 in myeloid malignancies.•RUNX3 and MYC collaborate to promote myeloid transformation. RUNX3, a transcription factor, has been implicated as a tumor suppressor in various cancers, including hematological malignancies; however, recent studies revealed an oncogenic function of RUNX3 in the pathogenesis of myeloid malignancies, such as myelodysplastic syndrome and acute myeloid leukemia. In contrast to the high frequency of mutations in the RUNX1 gene, deletion of and loss-of-function mutations in RUNX3 are rarely detected in patients with hematopoietic malignancies. Although RUNX3 is expressed in normal hematopoietic stem and progenitor cells, its expression decreases with aging in humans. The loss of Runx3 did not result in the development of lethal hematological diseases in mice despite the expansion of myeloid cells. Therefore, RUNX3 does not appear to initiate the transformation of normal hematopoietic stem cells. However, the overexpression of RUNX3 inhibits the expression and transcriptional function of the RUNX1 gene, but activates the expression of key oncogenic pathways, such as MYC, resulting in the transformation of premalignant stem cells harboring a driver genetic mutation. We herein discuss the mechanisms by which RUNX3 is activated and how RUNX3 exerts oncogenic effects on the cellular function of and transcriptional program in premalignant stem cells to drive myeloid transformation. RUNX3, a transcription factor, has been implicated as a tumor suppressor in various cancers, including hematological malignancies; however, recent studies revealed an oncogenic function of RUNX3 in the pathogenesis of myeloid malignancies, such as myelodysplastic syndrome and acute myeloid leukemia. In contrast to the high frequency of mutations in the RUNX1 gene, deletion of and loss-of-function mutations in RUNX3 are rarely detected in patients with hematopoietic malignancies. Although RUNX3 is expressed in normal hematopoietic stem and progenitor cells, its expression decreases with aging in humans. The loss of Runx3 did not result in the development of lethal hematological diseases in mice despite the expansion of myeloid cells. Therefore, RUNX3 does not appear to initiate the transformation of normal hematopoietic stem cells. However, the overexpression of RUNX3 inhibits the expression and transcriptional function of the RUNX1 gene, but activates the expression of key oncogenic pathways, such as MYC, resulting in the transformation of premalignant stem cells harboring a driver genetic mutation. We herein discuss the mechanisms by which RUNX3 is activated and how RUNX3 exerts oncogenic effects on the cellular function of and transcriptional program in premalignant stem cells to drive myeloid transformation. RUNX transcription factors are critical for development and normal tissue homeostasis, and have been characterized as oncogenes or tumor suppressors in the pathogenesis of various tumors [1Speck NA Gilliland DG. Core-binding factors in haematopoiesis and leukaemia.Nat Rev Cancer. 2002; 2: 502-513Crossref PubMed Scopus (457) Google Scholar,2Ito Y. Oncogenic potential of the RUNX gene family: ‘overview’.Oncogene. 2004; 23: 4198-4208Crossref PubMed Scopus (274) Google Scholar]. RUNX family members, including RUNX1, RUNX2, and RUNX3, are highly conserved in the runt domain, which binds to the consensus DNA sequence and is involved in dimerization with the common cofactor CBFβ. Loss-of-function mutations in and deletion of RUNX1 are frequently observed in myeloid malignancies, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) [3Osato M Asou N Abdalla E et al.Biallelic and heterozygous point mutations in the runt domain of the AML1/PEBP2alphaB gene associated with myeloblastic leukemias.Blood. 1999; 93: 1817-1824Crossref PubMed Google Scholar,4Harada H Harada Y Niimi H Kyo T Kimura A Inaba T. High incidence of somatic mutations in the AML1/RUNX1 gene in myelodysplastic syndrome and low blast percentage myeloid leukemia with myelodysplasia.Blood. 2004; 103: 2316-2324Crossref PubMed Scopus (235) Google Scholar]. Furthermore, the hematopoietic cell-specific deletion of Runx1 has a minimal impact on the frequency of stem cells [5Cai X Gaudet JJ Mangan JK et al.Runx1 loss minimally impacts long-term hematopoietic stem cells.PLoS One. 2011; 6: 1-14Crossref Scopus (59) Google Scholar], but impairs the differentiation of megakaryocytes and lymphocytes, which leads to the development of myeloproliferative neoplasm (MPN)-like disease in mice after a long latency [6Ichikawa M Asai T Saito T et al.AML-1 is required for megakaryocytic maturation and lymphocytic differentiation, but not for maintenance of hematopoietic stem cells in adult hematopoiesis.Nat Med. 2004; 10: 299-304Crossref PubMed Scopus (476) Google Scholar,7Growney JD Shigematsu H Li Z et al.Loss of Runx1 perturbs adult hematopoiesis and is associated with a myeloproliferative phenotype.Blood. 2005; 106: 494-505Crossref PubMed Scopus (354) Google Scholar], indicating the tumor-suppressive function of Runx1.In contrast to the high frequency of mutations in the RUNX1 gene, the deletion and loss-of-function mutations are minimal in the RUNX3 gene in patients with hematopoietic malignancies. RUNX3 is expressed in hematopoietic stem and progenitor cells (HSPCs); however, its expression decreases with aging in humans and mice [8Balogh P Adelman ER Pluvinage JV et al.RUNX3 levels in human hematopoietic progenitors are regulated by aging and dictate erythroid–myeloid balance.Haematologica. 2020; 105: 905-913Crossref PubMed Scopus (9) Google Scholar]. This decline may contribute to the emergence of the aging phenotype in hematopoiesis, which is characterized by anemia and enhanced granulopoiesis at the expense of lymphopoiesis [9Morrison SJ Wandycz AM Akashi K Globerson A Weissman IL. The aging of hematopoietic stem cells.Nat Med. 1996; 2: 1011-1016Crossref PubMed Scopus (651) Google Scholar,10Rossi DJ Bryder D Zahn JM et al.Cell intrinsic alterations underlie hematopoietic stem cell aging.Proc Natl Acad Sci USA. 2005; 102: 9194-9199Crossref PubMed Scopus (806) Google Scholar]. The hematopoietic cell-specific deletion of murine Runx3 impaired the differentiation of erythrocytes, but retained the production of myeloid cells, resulting in development of the myeloproliferative phenotype in mice [11Wang CQ Motoda L Satake M et al.Runx3 deficiency results in myeloproliferative disorder in aged mice.Blood. 2013; 122: 562-566Crossref PubMed Scopus (24) Google Scholar]. The promoter hypermethylation of RUNX3 and RUNX1-ETO-induced transcriptional suppression of RUNX3 have been reported in myeloid leukemia cells [12Mori BN Morosetti R Lee S et al.Allelotype analysis in the evolution of chronic myelocytic leukemia.Blood. 1997; 90: 2010-2014Crossref PubMed Google Scholar, 13Est MRH Bueso-Ramos C Dinardo CD et al.RUNX3 promoter hypermethylation is frequent in leukaemia cell lines and associated with acute myeloid leukaemia inv (16) subtype.Br J Haematol. 2015; 169: 344-351Crossref PubMed Scopus (24) Google Scholar, 14Cheng CK Li L Cheng SH et al.Transcriptional repression of the RUNX3/AML2 gene by the t(8;21) and inv(16) fusion proteins in acute myeloid leukemia.Blood. 2008; 112: 3391-3402Crossref PubMed Scopus (41) Google Scholar], supporting the tumor-suppressive function of RUNX3, reductions in the expression of which may confer the fitness of normal and malignant stem cells in bone marrow during aging.Recent studies, however, revealed an oncogenic function for RUNX3 in the pathogenesis of myeloid malignancies, at least in part, caused by genomic amplifications in the 1p36 region, including the RUNX3 gene, which have been activated in MDS and AML patients [15Yoshizato T Nannya Y Atsuta Y et al.Genetic abnormalities in myelodysplasia and secondary acute myeloid leukemia: impact on outcome of stem cell transplantation.Blood. 2017; 129: 2347-2359Crossref PubMed Scopus (183) Google Scholar]. Ectopic expression of FLT3-ITD has also been found to activate the expression of Runx3 in murine Tet2-deficient AML cells, which confers a chemoresistance property and poor clinical outcomes in patients harboring the FLT3-ITD mutation [16Damdinsuren A Matsushita H Ito M et al.FLT3-ITD drives Ara-C resistance in leukemic cells via the induction of RUNX3.Leuk Res. 2014; 39: 1405-1413Crossref Scopus (12) Google Scholar,17Shih AH Jiang Y Melnick A et al.Mutational cooperativity linked to combinatorial epigenetic gain of function in acute myeloid leukemia.Cancer Cell. 2015; 27: 502-515Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar]. While the upstream regulatory mechanisms underlying the transcription of RUNX genes have been examined in various tumor cells, we recently described how RUNX3 dysregulates RUNX1 and MYC transcription factors in hematopoiesis to develop MDS. Human RUNX3-expressing Tet2-deficient murine MDS HSPCs markedly suppressed expression of the Runx1 protein due to competitive binding to the CBFβ protein and its target genes, in which RUNX3 bound to and impeded the binding of Runx1 [18Yokomizo-Nakano T Kubota S Bai J et al.Overexpression of RUNX3 represses RUNX1 to drive transformation of myelodysplastic syndrome.Cancer Res. 2020; 80: 2523-2536Crossref PubMed Scopus (11) Google Scholar]. Therefore, although RUNX3 does not appear to initiate the myeloid transformation of wild-type stem cells, the overexpression of RUNX3 promotes the development of Tet2-deficient MDS via transcription-dependent and -independent machineries. We herein discuss the mechanisms by which RUNX3 may be activated and how RUNX3 exerts oncogenic effects on the cellular function of and transcriptional program in pre-MDS stem cells in myeloid transformation.Upstream signals regulating transcription of the RUNX3 geneRUNX3 is strongly expressed in HSPCs and mature lymphoid cells in adult hematopoiesis. The level of expression of the RUNX3 protein is known to be regulated by divergent signaling pathways, such as transforming growth factor β (TGFβ), SRC, and retinoic acid [19Chuang LSH Ito Y. RUNX3 is multifunctional in carcinogenesis of multiple solid tumors.Oncogene. 2010; 29: 2605-2615Crossref PubMed Scopus (119) Google Scholar], which fine-tune the differentiation and production of mature blood cells to ensure the homeostasis of normal hematopoiesis. As a result of the alteration of these signaling pathways under stress conditions, expression of RUNX3 decreases with aging in humans and mice [8Balogh P Adelman ER Pluvinage JV et al.RUNX3 levels in human hematopoietic progenitors are regulated by aging and dictate erythroid–myeloid balance.Haematologica. 2020; 105: 905-913Crossref PubMed Scopus (9) Google Scholar], accompanied by the aging phenotype in hematopoiesis [9Morrison SJ Wandycz AM Akashi K Globerson A Weissman IL. The aging of hematopoietic stem cells.Nat Med. 1996; 2: 1011-1016Crossref PubMed Scopus (651) Google Scholar,10Rossi DJ Bryder D Zahn JM et al.Cell intrinsic alterations underlie hematopoietic stem cell aging.Proc Natl Acad Sci USA. 2005; 102: 9194-9199Crossref PubMed Scopus (806) Google Scholar]. Recent genomic sequencing analyses of the elderly revealed that somatic driver mutations in blood cells, which are shared with malignant stem cells in patients with myeloid malignancies, are frequently acquired during aging. These mutations result in clonal expansion without cytopenia and dysplasia, which are considered to be clonal hematopoiesis or so-called CHIP (clonal hematopoiesis with intermediate potential) [20Steensma DP Bejar R Jaiswal S et al.Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes.Blood. 2015; 126: 9-16Crossref PubMed Scopus (1041) Google Scholar,21Jaiswal S Ebert BL. Clonal hematopoiesis in human aging and disease.Science. 2019; 366: 4673Crossref Scopus (292) Google Scholar]. Clonal hematopoiesis has been found to increase the risk of the subsequent progression of hematological malignancies; however, the molecular mechanisms underlying the transformation of premalignant stem cells harboring a mutation, such as DNMT3A, ASXL1, and TET2, have not yet been elucidated. Runx3 knockout mice exhibited a myeloproliferative phenotype, but did not develop a lethal neoplasia [11Wang CQ Motoda L Satake M et al.Runx3 deficiency results in myeloproliferative disorder in aged mice.Blood. 2013; 122: 562-566Crossref PubMed Scopus (24) Google Scholar], which has only been detected in cells lacking Runx1 in mice because of constitutively accumulating Fanconi anemia (FA) pathway-dependent DNA damage in stem cells [22Wang CQ Krishnan V Tay LS et al.Disruption of Runx1 and Runx3 leads to bone marrow failure and leukemia predisposition due to transcriptional and DNA repair defects.Cell Rep. 2014; 8: 767-782Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar]. Therefore, if RUNX3 acts as a bona fide tumor suppressor in normal hematopoiesis, further studies are needed to clarify whether hypomorphic Runx3 gives a selective advantage to these CHIP stem cells, which compete with co-existing wild-type stem cells and initiate myeloid transformation, using an in vivo experimental model.CpG island DNA methylation is critical for regulating the function and effects of HSPCs in normal hematopoiesis [23Challen GA Sun D Jeong M et al.Dnmt3a is essential for hematopoietic stem cell differentiation.Nat Genet. 2012; 44: 23-31Crossref Scopus (768) Google Scholar,24Beerman I Bock C Garrison BS et al.Proliferation-dependent alterations of the DNA methylation landscape underlie hematopoietic stem cell aging.Cell Stem Cell. 2013; 12: 413-425Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar], and the DNA hypermethylation of RUNX3 P1 and/or P2 promoters has been reported in both AML and CML cells, as well as gastric cancer cells, suggesting that the hypermethylation of RUNX3 reduces its expression during cancer development [12Mori BN Morosetti R Lee S et al.Allelotype analysis in the evolution of chronic myelocytic leukemia.Blood. 1997; 90: 2010-2014Crossref PubMed Google Scholar, 13Est MRH Bueso-Ramos C Dinardo CD et al.RUNX3 promoter hypermethylation is frequent in leukaemia cell lines and associated with acute myeloid leukaemia inv (16) subtype.Br J Haematol. 2015; 169: 344-351Crossref PubMed Scopus (24) Google Scholar, 14Cheng CK Li L Cheng SH et al.Transcriptional repression of the RUNX3/AML2 gene by the t(8;21) and inv(16) fusion proteins in acute myeloid leukemia.Blood. 2008; 112: 3391-3402Crossref PubMed Scopus (41) Google Scholar]. However, it currently remains unclear whether DNA hypermethylation of the RUNX3 P2 promoter region initiates and/or promotes transformation by decreasing the transcription of RUNX3, or rather reflects the positive selection of tumor cells, in which the weak expression of the RUNX3 gene may only collaborate with a preferential genetic mutation, such as BCR-ABL1. In contrast to the high frequency of loss-of-function mutations in the RUNX1 gene, the deletion of and loss-of-function mutations in RUNX3 are rarely detected in patients with hematological malignancies [25Shih AH Abdel-Wahab O Patel JP Levine RL. The role of mutations in epigenetic regulators in myeloid malignancies.Nat Rev Cancer. 2012; 12: 599-612Crossref PubMed Scopus (538) Google Scholar]. A subset of patients with MDS or AML harbored the amplification of the chromosome 1p36 region and the stronger expression of RUNX3, resulting in a significantly poorer prognosis in MDS patients [15Yoshizato T Nannya Y Atsuta Y et al.Genetic abnormalities in myelodysplasia and secondary acute myeloid leukemia: impact on outcome of stem cell transplantation.Blood. 2017; 129: 2347-2359Crossref PubMed Scopus (183) Google Scholar] than in those with weaker RUNX3 expression, which is relevant to our recent finding of a progressive disease phenotype in murine RUNX3-expressing Tet2-deficient MDS. Consistent with these findings in MDS, the overexpression of RUNX3 has been correlated with a poor prognosis in patients with AML harboring the FLT3-ITD mutation [26Lacayo NJ Meshinchi S Kinnunen P et al.Gene expression profiles at diagnosis in de novo childhood AML patients identify FLT3 mutations with good clinical outcomes.Blood. 2004; 104: 2646-2655Crossref PubMed Scopus (97) Google Scholar]. FLT3-ITD activates the expression of Runx3 in murine Tet2-dificient AML cells, but also confers a chemoresistance property on AML cells [16Damdinsuren A Matsushita H Ito M et al.FLT3-ITD drives Ara-C resistance in leukemic cells via the induction of RUNX3.Leuk Res. 2014; 39: 1405-1413Crossref Scopus (12) Google Scholar,17Shih AH Jiang Y Melnick A et al.Mutational cooperativity linked to combinatorial epigenetic gain of function in acute myeloid leukemia.Cancer Cell. 2015; 27: 502-515Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar]. In addition, the Epstein–Barr virus oncoprotein has been reported to enhance the expression of RUNX3 by activating the super-enhancer of RUNX3 to promote the proliferation of transformed lymphoblastoid cells [27Zhou H Schmidt SCS Jiang S et al.Epstein–Barr virus oncoprotein super-enhancers control B cell growth.Cell Host Microbe. 2015; 17: 205-216Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar], indicating that these cell-intrinsic upstream signals directly induce the activation of RUNX3 expression in these subtypes of blood cancer. Evidence is emerging to indicate that the virus/bacterial infection-mediated propagation of hematological malignancies is due partly to the acceleration of the inflammation response pathway, which may lead to altered epigenetics and transcriptomes in stem cells [28King KY Goodell MA. Inflammatory modulation of HSCs: viewing the HSC as a foundation for the immune response.Nat Rev Immunol. 2011; 11: 685-692Crossref PubMed Scopus (366) Google Scholar,29Sur I Taipale J. The role of enhancers in cancer.Nat Rev Cancer. 2016; 16: 483-493Crossref PubMed Scopus (210) Google Scholar]. Therefore, further studies are needed to clarify whether cell-extrinsic infection and inflammatory pathways induce the activation of RUNX3 expression by altering epigenetic modifications in the regulatory region of the RUNX3 gene. RUNX3 appears to drive the development of hematological malignancies in a context-dependent manner following alterations in upstream signaling pathways and its combination with epigenetic modifications to activate transcription of the RUNX3 gene. A proposed model is provided in Figure 1. We herein describe the molecular mechanisms underlying the oncogenic effects of RUNX3 in MDS and AML.Regulation of the expression of RUNX3 isoforms in transformationRUNX1 is a master regulator of fetal and adult hematopoiesis, whereas RUNX3 is strongly expressed in HSPCs and plays important roles in the differentiation of myeloid and lymphoid cells. The deletion of Runx3 expands mature myeloid cells and increases sensitivity to the administration of granulocyte colony-stimulating factor (G-CSF) in myeloid cells in aged mice, exhibiting an MPN-like phenotype [11Wang CQ Motoda L Satake M et al.Runx3 deficiency results in myeloproliferative disorder in aged mice.Blood. 2013; 122: 562-566Crossref PubMed Scopus (24) Google Scholar]. The reduced expression of RUNX3 was recently described in aged human HSCs [8Balogh P Adelman ER Pluvinage JV et al.RUNX3 levels in human hematopoietic progenitors are regulated by aging and dictate erythroid–myeloid balance.Haematologica. 2020; 105: 905-913Crossref PubMed Scopus (9) Google Scholar], indicating a suppressive role of RUNX3 in myeloid-biased phenotype in aged human and mice. In contrast, the overexpression of RUNX3 has been described in HSPCs in patients with MDS and AML with a significantly poor prognosis in multiple independent cohorts [18Yokomizo-Nakano T Kubota S Bai J et al.Overexpression of RUNX3 represses RUNX1 to drive transformation of myelodysplastic syndrome.Cancer Res. 2020; 80: 2523-2536Crossref PubMed Scopus (11) Google Scholar]. On the basis of human cohort data, we recently determined that the ectopic expression of RUNX3 variant 2, which is driven by the P2 promoter in cells, drives the transformation of Tet2-deficient stem cells to develop MDS in mice. All RUNX genes contain two alternative promoters, a distal P1 promoter and a proximal P2 promoter, which encode the main protein isoforms that exhibit different structures and distinct expression patterns in development and differentiation. In addition, alternative splicing and noncoding RNA-mediated regulatory mechanisms may add diversity to RUNX transcripts initiated from the P1 and P2 promoters (e.g., uc002yug.2) [30Huan C Li Z Ning S Wang H Yu XF Zhang W. Long Noncoding RNA uc002yug.2 activates HIV-1 latency through regulation of mRNA levels of various RUNX1 isoforms and increased Tat expression.J Virol. 2018; 92: 1-18Crossref Scopus (26) Google Scholar], implicating the context-dependent requirements and functions of these protein isoforms [2Ito Y. Oncogenic potential of the RUNX gene family: ‘overview’.Oncogene. 2004; 23: 4198-4208Crossref PubMed Scopus (274) Google Scholar,31Mevel R Draper JE Lie-A-Ling M Kouskoff V Lacaud G RUNX transcription factors: orchestrators of development.Development. 2019; 146: 1-19Crossref Scopus (81) Google Scholar]. Although the distinct effects of RUNX1 isoforms (e.g., P1-drived RUNX1c and P2-drived RUNX1b and RUNX1a) in adult hematopoiesis have been studied [32Goyama S Schibler J Mulloy JC. Alternative translation initiation generates the N-terminal truncated form of RUNX1 that retains hematopoietic activity.Exp Hematol. 2019; 72: 27-35Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar], the physiological and pathologic roles of distinct RUNX3 isoforms in hematopoiesis remain largely unknown. The molecular mechanisms by which DNA methylation leads to changes in the proportion of RUNX3 isoforms to drive myeloid transformation will be examined in future studies.RUNX3 suppresses the expression and transcriptional function of the RUNX1 proteinRUNX transcription factors are regulated by posttranslational mechanisms through phosphorylation, methylation, acetylation, and ubiquitylation, which fine-tune the transcriptional function and stability of RUNX proteins. As there are many integrative reviews describing the posttranslational modification of RUNX proteins, such as RUNX1 [33Wang L Huang G Zhao X et al.Post-translational modifications of Runx1 regulate its activity in the cell.Blood Cells Mol Dis. 2009; 43: 30-34Crossref PubMed Scopus (28) Google Scholar], we herein briefly discuss posttranslational modifications in the RUNX3 protein. The stability of the RUNX3 protein is regulated by ubiquitylation- and phosphorylation-mediated degradation by MDM2 and SRC, respectively [34Chi XZ Kim J Lee YH et al.Runt-related transcription factor RUNX3 is a target of MDM2-mediated ubiquitination.Cancer Res. 2009; 69: 8111-8119Crossref PubMed Scopus (45) Google Scholar,35Goh YM Cinghu S Hong ETH et al.Src kinase phosphorylates RUNX3 at tyrosine residues and localizes the protein in the cytoplasm.J Biol Chem. 2010; 285: 10122-10129Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar]. The transcriptional activity of RUNX3 is regulated by its acetylation and association with p300, an epigenetic modifier that activates the transcription of target genes [36Jin YH Jeon EJ Li QL et al.Transforming growth factor-β stimulates p300-dependent RUNX3 acetylation, which inhibits ubiquitination-mediated degradation.J Biol Chem. 2004; 279: 29409-29417Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar]. In addition, RUNX proteins are stabilized by the common cofactor CBFβ [37Huang G Shigesada K Ito K Wee HJ Yokomizo T Ito Y. Dimerization with PEBP2β protects RUNX1/AML1 from ubiquitin–proteasome-mediated degradation.EMBO J. 2001; 20: 723-733Crossref PubMed Scopus (242) Google Scholar]. RUNX genes are highly conserved in the runt domain, which binds to the consensus DNA sequence and is involved in dimerization with the common cofactor CBFβ, which increases the DNA-binding affinity and stability of RUNX proteins in the complex. The functional and transcriptional interaction between Runx1 and Runx3 proteins has been reported to regulate the process of CD8+ T-cell differentiation by suppressing transcription of the CD4 gene [38Taniuchi I Osato M Egawa T et al.Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development.Cell. 2002; 111: 621-633Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar]. The mutual regulation of expression of RUNX3 and RUNX1 was also observed in Epstein–Barr virus-induced transformed lymphoid cells because RUNX3 bound to the P1 promoter of the RUNX1 gene and silenced its transcription [39Spender LC Whiteman HJ Karstegl CE Farrell PJ. Transcriptional cross-regulation of RUNX1 by RUNX3 in human B cells.Oncogene. 2005; 24: 1873-1881Crossref PubMed Scopus (75) Google Scholar,40Brady G Whiteman HJ Spender LC Farrell PJ. Downregulation of RUNX1 by RUNX3 requires the RUNX3 VWRPY sequence and is essential for Epstein–Barr virus-driven B-cell proliferation.J Virol. 2009; 83: 6909-6916Crossref PubMed Scopus (37) Google Scholar]. We previously found that ectopic expression of human RUNX3 reduces expression of the Runx1 protein by suppressing the protein stability of Runx1 and dysregulates the transcription of Runx1 target genes, such as tumor suppressor and differentiation regulator genes, leading to the development of MDS in mice [18Yokomizo-Nakano T Kubota S Bai J et al.Overexpression of RUNX3 represses RUNX1 to drive transformation of myelodysplastic syndrome.Cancer Res. 2020; 80: 2523-2536Crossref PubMed Scopus (11) Google Scholar]. The stability of the RUNX1 protein is also regulated by posttranslational modifications in RUNX1 through various modification enzymes [33Wang L Huang G Zhao X et al.Post-translational modifications of Runx1 regulate its activity in the cell.Blood Cells Mol Dis. 2009; 43: 30-34Crossref PubMed Scopus (28) Google Scholar]. For example, MLL, a methyltransferase, has been reported to increase Runx1 protein levels and activate the transcriptional function of Runx1 to activate the PU.1 gene [41Huang G Zhao X Wang L et al.The ability of MLL to bind RUNX1 and methylate H3K4 at PU.1 regulatory regions is impaired by MDS/AML-associated RUNX1/AML1 mutations.Blood. 2011; 118: 6544-6552Crossref PubMed Scopus (60) Google Scholar], whereas the MLL-fusion oncoprotein reduced Runx1 protein levels to promote the development of leukemia, which was inhibited by the ectopic expression of Runx1 [42Zhao X Chen A Yan X et al.Down-regulation of RUNX1/CBFβ by MLL fusion proteins enhances HSC self-renewal.Blood. 2014; 123: 1729-1738Crossref PubMed Scopus (27) Google Scholar]. It remains to be determined whether upstream oncogenic signals and epigenetic modifiers (e.g., p300 and MLL) may enhance the expression level and transcriptional activity of the RUNX3 protein. Nevertheless, RUNX3 reduces the expression of RUNX1 at both the mRNA and protein levels and impedes the tumor-suppressive function of RUNX1, which promotes transformation (Figure 2). Examination of whether RUNX3 suppresses expression and function of another RUNX protein, RUNX2, is warranted.Figure 2The two faces of RUNX3 in malignant transformation. Summary of the main oncogenic and tumor-suppressive functions of RUNX3 in various malignancies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)RUNX3 functions as both an oncogene and a tumor suppressor in hematopoiesisRUNX family genes have the common property of activating the expression of p19ARF to stabilize the p53 protein [43Chuang LSH Ito K Ito Y. RUNX family: Regulation and diversification of roles through interacting proteins.Int J Cancer. 2013; 132: 1260-1271Crossref PubMed Scopus (131) Google Scholar], which regulates the quiescence and self-renewal properties of adult hematopoietic stem cells [44Liu Y Elf SE Miyata Y et al.P53 regulates hematopoietic stem cell quiescence.Cell Stem Cell. 2009; 4: 37-48Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar]. As the overexpression of RUNX1 impeded the proliferation of HSPCs through the activation of myeloid differentiation [45Goyama S Schibler J Cunningham L et al.Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells.J Clin Invest. 2013; 123: 3876-3888Crossref PubMed Scopus (136) Google Scholar], the expression of Runx3 is expected to inhibit the proliferation of p53 wild-type stem cells, supporting the tumor-suppressive effects of Ruxn3 to suppress the initiation of transformation prior to the emergence of CHIP mutations, including the Trp53 gene (Figure 1). In addition, RUNX3 has been found to function as a tumor suppressor in solid tumors (e.g., liver, colon, and gastric cancers) by suppressing the transcription of genes in oncogenic pathways, such as Notch, WNT/β-catenin, and YAP [19Chuang LSH Ito Y. RUNX3 is multifunctional in carcinogenesis of multiple solid tumors.Oncogene. 2010; 29: 2605-2615Crossref PubMed Scopus (119) Google Scholar], by which the overexpression of RUNX3 may inhibit initiation of the transformation of normal HSCs. The pleiotropic effects of RUNX3 have been described in pancreatic ductal adenocarcinoma (PDA), in which the expression of RUNX3 protein was influenced by DPC4/SMAD4 gene status in a biphasic manner [46Whittle MC Izeradjene K Geetha Rani P et al.RUNX3 controls a metastatic switch in pancreatic ductal adenocarci