Overexpression of RUNX3 Represses RUNX1 to Drive Transformation of Myelodysplastic Syndrome

Author Manuscript Published OnlineFirst on April 27, 2020; DOI: 10.1158/0008-5472.CAN-19-3167 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Yokomizo-Nakano T, et al.

Overexpression of RUNX3 represses RUNX1 to drive transformation of myelodysplastic syndrome

Takako Yokomizo-Nakano1, Sho Kubota1, Jie Bai1, Ai Hamashima1, Mariko Morii1, Yuqi Sun1, Seiichiro Katagiri2,

Mihoko Iimori1, Akinori Kanai3, Daiki Tanaka1, Motohiko Oshima4, Yuka Harada5, Kazuma Ohyashiki2, Atsushi

Iwama4, Hironori Harada6, Motomi Osato7, and Goro Sashida1

1 Laboratory of Transcriptional Regulation in Leukemogenesis, International Research Center for Medical Sciences

(IRCMS), Kumamoto University, Kumamoto Japan, 2 Department of Hematology, Tokyo Medical University, Tokyo,

Japan, 3 Department of Molecular Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima

University, Hiroshima, Japan, 4 Division of Stem Cell and Molecular Medicine, Center for Stem Cell Biology and

Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo Japan, 5 Tokyo

Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo, Japan, 6 Laboratory of Oncology,

Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan, 7 Cancer Science Institute of Singapore, National

University of Singapore, Singapore.

Running title: RUNX3 represses RUNX1 and promotes MDS

Keywords: RUNX, MYC, TET2, Hematopoiesis, and Myeloproliferative neoplasm

Competing Interest: The authors declare no potential conflicts of interest to disclose.

Corresponding Author:

Goro Sashida, MD, PhD

2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan

Phone: +81-96-373-6827

Fax: +81-96-373-6869

E-mail: [email protected]

Yokomizo-Nakano T, et al.

Abstract

RUNX3, a RUNX family transcription factor, regulates normal hematopoiesis and functions as a tumor suppressor in various tumors in humans and mice. However, emerging studies have documented increased expression of

RUNX3 in hematopoietic stem/progenitor cells (HSPC) of a subset of patients with myelodysplastic syndrome

(MDS) showing a worse outcome, suggesting an oncogenic function for RUNX3 in the pathogenesis of hematological malignancies. To elucidate the oncogenic function of RUNX3 in the pathogenesis of MDS in vivo, we generated a RUNX3-expressing, Tet2-deficient mouse model with the pan-cytopenia and dysplastic blood cells characteristic of MDS in patients. RUNX3-expressing cells markedly suppressed the expression levels of Runx1, a critical regulator of hemaotpoiesis in normal and malignant cells, as well as its target genes, which included crucial tumor suppressors such as Cebpa and Csf1r. RUNX3 bound these genes and remodeled their Runx1 binding regions in Tet2-deficient cells. Overexpression of RUNX3 inhibited the transcriptional function of Runx1 and compromised hematopoiesis to facilitate the development of MDS in the absence of Tet2, indicating that

RUNX3 is an oncogene. Furthermore, overexpression of RUNX3 activated the transcription of Myc target genes and rendered cells sensitive to inhibition of Myc-Max heterodimerization. Collectively, these results reveal the mechanism by which RUNX3 overexpression exerts oncogenic effects on the cellular function of and transcriptional program in Tet2-deficient stem cells to drive the transformation of MDS.

Significance

This study defines the oncogenic effects of transcription factor RUNX3 in driving the transformation of myelodysplastic syndrome, highlighting RUNX3 as a potential target for therapeutic intervention.

Yokomizo-Nakano T, et al.

Introduction

RUNX transcription factors are critical for development and normal tissue homeostasis and have been characterized as an oncogene or tumor suppressor in the pathogenesis of various tumors (1,2). 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 co-factor CBFb. Loss-of-function mutations in and the deletion of RUNX1 are frequently observed in hematological malignancies, including myelodysplastic syndrome (MDS), in patients (3,4), and the hematopoietic cell-specific deletion of Runx1 impairs the differentiation of megakaryocytes and lymphocytes, which leads to the development of myeloproliferative neoplasm (MPN)-like disease in mice despite a long latency (5,6), indicating the tumor suppressive function of

RUNX1.

Previous studies demonstrated that the repressed expression of RUNX3 due to promoter DNA hypermethylation promoted the development of solid tumors and was associated with poor survival outcomes (e.g. colon, renal, and lung cancers) (7). RUNX3 is expressed in hematopoietic stem and progenitor cells (HSPCs); however, its expression levels decline with aging in humans and mice (8), and this may contribute to the emergence of the aging phenotype in hematopoiesis characterized by anemia and enhanced granulopoiesis (9,10).

The deletion of RUNX3 and the promoter hypermethylation and RUNX1-ETO-induced transcriptional suppression of RUNX3 have been reported in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) cells (11–13).

The deletion of Runx3 impaired the differentiation of erythrocytes, but retained the production of myeloid cells, such as granulocytes, which exhibited higher sensitivity to a G-CSF treatment in vitro, and resulted in the development of the myeloproliferative phenotype in mice (14), partially sharing the phenotype of Runx1-deficient mice.

Furthermore, the deletion of both Runx1 and Runx3 in hematopoietic cells facilitated the development of bone marrow failure and MPN diseases in mice due to alterations in the transcription-dependent and -independent functions of Runx genes (15). Therefore, the loss of Runx3 induces a compensatory function of Runx1, but also promotes the development of MPN upon the deletion of Runx1, indicating a function for RUNX3 as a tumor suppressor.

Yokomizo-Nakano T, et al.

However, emerging studies have suggested an oncogenic function for RUNX3 in the pathogenesis of hematological malignancies, including MDS, because genomic amplifications in the 1p36 region including the

RUNX3 gene have been observed in MDS patients (16). The overexpression of RUNX3 has been correlated with poor clinical outcomes in patients with AML harboring the FLT3-ITD mutation (17). FLT3-ITD has been shown to activate the expression of Runx3 in murine Tet2-dificient AML cells, but also confers a chemoresistant property to human AML cells (18,19). The Epstein-Barr virus oncoprotein enhances the expression of RUNX3 by activating the super-enhancer of RUNX3, which promotes the proliferation of transformed lymphoblastoid cells (20). Based on these findings, we herein confirmed the enhanced expression of RUNX3 mRNA and protein in a subset of MDS patients by analyzing our cohort and published datasets (21,22), and attempted to elucidate the mechanisms by which the overexpression of RUNX3 induces the transformation of MDS in vivo.

The TET2 enzyme oxidizes 5-methylcytosine (5mC) and regulates the region-specific removal of 5mC, and loss-of-function mutations in TET2 are one of the most common driver mutations in myeloid malignancies, including MDS (23,24), as well as in clonal hematopoiesis in healthy aged individuals (25,26). The loss of Tet2 promotes the self-renewal function of HSCs and results in the development of MPN-like disorders in mice, even in longer observation periods (27,28). In the present study, we generated a RUNX3-expressing Tet2-deficient MDS mouse model showing human MDS features characterized by pan-cytopenia, dysplastic myeloid cells, and impaired lymphopoiesis. We found that RUNX3-expressing Tet2-deficient HSPCs markedly suppressed the expression of the Runx1 protein and its target genes, in which RUNX3 bound to consensus DNA sequences, and remodeled the binding sites of Runx1. In addition, RUNX3 overexpression enhanced the transcription of Myc target genes, which showed higher sensitivity to the inhibition of the Myc-Max heterodimerization. The present results revealed the mechanisms by which the overexpression of RUNX3 exerts oncogenic effects on the cellular function of and transcriptional program in Tet2-deficient stem cells to initiate the transformation of MDS.

Yokomizo-Nakano T, et al.

Methods:

Mice

All mice were in the C57BL/6 background. Tet2 conditional knockout (Tet2flox/flox) mice were previously described

(27), and crossed with Rosa26:Cre-ERT2 mice (TaconicArtemis GmbH, Cologne) for conditional deletion. Two milligrams of tamoxifen (T5648, Sigma-Aldrich, St. Louis) was administered via an intraperitoneal injection for 5 consecutive days twice to completely delete the Tet2 gene. C57BL/6 mice congenic for the Ly5 locus (CD45.1) were purchased from Sankyo-Lab Service (Tokyo). All experiments using these mice were performed in accordance with our institutional guidelines for the use of laboratory animals and approved by the Review Board for

Animal Experiments of Kumamoto University (Kumamoto, Japan). All mouse experiments were performed without randomization and blinding.

MDS patient samples

All samples were obtained at Tokyo Medical University after written informed consent compliant with the

Declaration of Helsinki had been obtained from patients. Patient anonymity was ensured, and the present study was approved by the Institutional Review Committee at Tokyo Medical University (Tokyo, Japan).

Immunohistochemistry staining

Paraffin-embedded BM aspiration clots were submitted to xylene and alcohol for deparaffinization, followed by the blockade of endogenous peroxidase. Samples were incubated with an anti-RUNX3 antibody (R3-5G4, Santa

Cruz) at 4°C overnight. Envision Dual Link System-HRP (Dako) was used as the secondary antibody for 45 minutes, followed by visualization with the DAB chromogen (Dako) and counterstaining using hematoxylin.

Retrovirus vector and transduction

RUNX3 was constructed from cDNA encoding human RUNX3 and subcloned into the retrovirus vector pMYs IRES-

EGFP. Virus supernatant (VSV-G pseudotyped retroviral supernatant) was prepared by transfecting the 293GPG packaging cell line with an empty control or the FLAG-tagged RUNX3 retrovirus vector plasmid using the calcium phosphate transfection method. Virus supernatant was concentrated by centrifugation at 6,000×g for 16 hours.

The final titers of retroviral supernatants were 40,000 to 50,000 infectious units/μl, as assessed by transducing

Yokomizo-Nakano T, et al. serially diluted viral supernatants into the human Jurkat cell line. Hematopoietic cells were then incubated with the virus supernatant in 10 μg/ml protamine sulfate and 10 ng/ml RetroNectin (Takara), and infected cells were further incubated in serum-free SF-03 medium supplemented with 50 μg/ml mouse SCF and 50 μg/ml human TPO.

Cells

293T and Jurkat cell lines were obtained from RIKEN (Japan), and were cultured in DMEM or RPMI 1640 containing 10% fetal bovine serum (FBS) in a humidified incubator, respectively. 293GPG was provided by Dr. R.

Mulligan (29). PCR Mycoplasma Test Kit (Takara) was used to test for mycoplasma contamination of all cell lines.

Colony assay

Colony assays were performed using Methocult M3234 (Stem Cell Technologies) supplemented with 20 ng/ml mouse SCF, 20 ng/ml mouse IL-3, 20 ng/ml human TPO, and 2 units/ml human EPO. The number of colonies was counted on day 10 of culture. In the replating assay, colonies were scored on day 10 and 3x104 pooled cells were replated in the same medium.

Flow cytometry and antibodies

Flow cytometry and cell sorting were performed by utilizing the following anti-murine antibodies (clone and catalogue numbers): CD45.2 (104, 109820), CD45.1 (A20, 110730), Gr1 (RB6-8C5, 108404), CD11b/Mac1

(M1/70, 101208), Ter119 (116204), CD127/IL-7Rα (A7R34, 121104), B220 (RA3-6B2, 103212), CD4 (L3T4,

100526), CD8α (53-6.7, 100714), CD117/c-Kit (2B8, 105812), Sca-1 (D7, 108114), CD34 (MEC14.7, 11-0341-85),

CD71 (R27217, 113808), CD150 (TC15-12F12.2, 115924), and FcγRII-III (93, 101308). These antibodies were purchased from BioLegend, eBioscience, R&D systems, or TONBO biosciences. The lineage mixture solution contained biotin-conjugated anti-Gr1, B220, CD4, CD8a, Ter119, and IL-7Rα antibodies. Apoptotic cells were stained with an anti-Annexin V-PE antibody (BD 556422) and 7-AAD (BD 559925). In order to evaluate cell cycle progression, cells were fixed and permeabilized according to the manufacturer’s instructions, and then detected using an anti-Ki67-Alexa Fluor 647 antibody (Biolegend 350510). All flow cytometric analyses and cell sorting were performed on FACSAriaIII or FACSCantoII (BD).

ChIP sequencing

Yokomizo-Nakano T, et al.

Murine HSPCs were purified from pooled BM cells (harvested from 3-5 mice) and were fixed by 0.5 or 1.0% paraformaldehyde at 37°C for 5 min and then lysed. Cells were sonicated 15 times at an amplitude of 50% for 10 sec. Samples were incubated with anti-Runx1 antibody- (Abcam ab23980), anti-FLAG antibody- (Sigma F1804), or anti-H3K27Ac antibody-conjugated (Active motif; MABI 0309) Dynabeads protein A/G at 4°C overnight. Inputs and immunoprecipitates were incubated at 65°C for 4 h for reverse cross-linking, and DNA was purified using the

MinElute PCR Purification Kit (Qiagen). ChIP-seq libraries were generated using the ThruPLEX DNA-seq kit

(Rubicon Genomics). Bowtie2 (version 2.2.6;default parameters) was used to map the reads to the reference genome (UCSC/mm9). HOMER (version 4.9) was used for de novo motif discovery in the peaks of super- enhancers and enhancers. ChIP-seq data were deposited in the DDBJ database under the accession number

DRA008820 and DRA009681.

RNA sequencing

Total RNA was extracted using an ISOGEN (Nippon gene), and cDNA was synthesized using the SMARTer Pico

PCR cDNA Synthesis kit (Clontech). ds-cDNA was fragmented and cDNA libraries were generated using the

KAPA HyperPlus Library Preparation kit (KAPA Biosystems) and FastGene Adaptor kit (Fastgene). Sequencing was performed using NextSeq500 (Illumina) with a single-read sequencing length of 60 bp, and the CLC genomic

Workbench was used to analyze and visualize sequencing data. RNA sequencing data have been deposited in the DDBJ database under the accession number DRA008434 and DRA009681.

Quantitative-RT-PCR

Total RNA was isolated using the RNeasy Mini kit and then reverse-transcribed by the ThermoScript RT-PCR system (Invitrogen) with an oligo-dT primer. q-RT-PCR was performed on LightCycler 480 (Roche) using SYBR

Premix ExTaq (Takara) or FastStart Universal Probe Master (Roche) with a Universal Probe Library (Roche).

Expression levels were normalized to that of Gapdh. Primers for PCR are listed in Supplementary Table 1.

Immunoprecipitation and Western blotting

Whole cell lysates were used for Western blotting. Briefly, Lin-Kit+ BM cells were isolated from mice and lysed in

1 x SDS Sample buffer (50 mM Tris-HCl, pH 6.83, 1.5% SDS, 10% glycerol). In the co-immunoprecipitation

Yokomizo-Nakano T, et al. assay, 293T cells transfected with the indicated plasmids were lysed in modified RIPA buffer (50 mM Tris pH 7.5,

150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, and 1 mM EDTA). Lysates were incubated with an anti-HA antibody (3F10, Roche, 11867423001) and rotated at 4 °C overnight. Lysates were mixed with Dynabeads protein

A/G at 4°C for 1 hour, and then washed twice with IP Buffer I (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% NP40,

0.25% sodium deoxycholate) followed by IP buffer II (50 mM Tris pH 7.5, 0.1% NP40, 0.05% sodium deoxycholate). The following antibodies were used for Western blotting: FLAG (Sigma F1804), HA-Tag (CST

C29F4), RUNX3 (MBL R3-5G4), RUNX1 (Sigma R0406, Abcam ab23980), MYC (Abcam Y69), Actin (Santa Cruz

C4), H3 (Abcam ab1791), and H3K27ac (Active motif MABI 0309). Uncropped immunoblot images are available in Supplementary Figure 1.

Luciferase reporter assay

The promoter or enhancer region of Cebpa, Csf1r, and Hnrnpa0 was amplified by PCR using mouse genomic

DNA. PCR primer sequences and amplified genomic regions (mm9) were as follows: Cebpa 5’-

CTGTGGTCAACTTCTAGTGGCTTTC-3’, 5’-AGCAGAGCAACCTTAACACTCATTG-3’ and chr7:35912057+35912683; Csf1r 5’-CCCAAGTGGTGTGCCAAGTG-3’, 5’-CACAGAGGGCTGACCACACC-3’ and chr18:61250779+61250955; Hnrnpa0 5′-CCGATGAACAGCTTACAGAGCTGCG-3′, 5′-

CAGAGGGTTCCGCACGGGAG-3′ and chr13:58229640+58230427. Amplified products were cloned into the

NheI and EcoRV sites of the pGL4.23 vector (Promega). 293T cells were transiently transfected with vector plasmids and cell extracts were prepared 48 hours after transfection. Luciferase activity was assessed by performing the Promega Dual-Luciferase reporter assay system (Promega E1910).

Statistical analysis

All statistical tests were performed using Graph Pad Prism version 7 (GraphPad Software). The significance of differences was measured by an unpaired two-tailed Student’s t-test or the Mann-Whitney non-parametric test. A

P value of less than 0.05 was considered to be significant. No statistical methods were used to predetermine sample sizes for animal studies.

Accession codes:

Yokomizo-Nakano T, et al.

Sequencing data that support the results of the present study have been deposited in DDBJ under the accession numbers DRA008434, DRA008820, and DRA009681 for RNA sequencing and ChIP sequencing.

Yokomizo-Nakano T, et al.

Results:

Increased expression of RUNX3 in human MDS stem/progenitor cells

In a gene expression analysis of 183 MDS patients, as previously reported (22), RUNX3 mRNA expression levels in CD34+ HSPCs were higher in 7 out of 55 RA patients and in 4 out of 80 RAEB patients than those in the HSPCs of healthy controls (Figure 1a). We also found that 2 out of 55 MDS patients showed the increased expression of

RUNX3 in HSPCs by analyzing another cohort dataset (Figure 1b)(21). Based on the enhanced expression of

RUNX3 mRNA in HSPCs, we assessed the expression level of the RUNX3 protein in the bone marrow tissues of

MDS patients in our cohort and found that 4 out of 20 patients showed higher expression levels than those in healthy controls (Figure 1c, 1d). Detailed clinical information on our cohort was provided in Supplementary Table

2. Some RUNX3 high-expressing patients harbored high-risk -7/7q- anomalies, whereas RUNX3 low-expressing patients did not (Supplementary Figure 2). Notably, in the other cohort of 122 MDS patients (30), survival was significantly shorter in RUNX3 high-expressing patients than in RUNX3 low-expressing patients (median survival

702.5 days versus 1533 days, p=0.0287) (Figure 1e). By utilizing the dataset on gene expression in HSPCs

(21,22), we found similar TET2 mRNA expression levels in RUNX3 high- and low-expressing patients in these cohorts; however, RUNX3 high-expressing HSPCs showed significant enrichments in the expression of up- and down-regulated genes defined in murine RUNX3-Tet2 Δ/Δ MDS HSPCs (as described below) (Supplementary Figure

3). In addition, RUNX3-high-expressing HSPCs showed markedly positive enrichments in the TNF-a and TGF-b signaling pathways over RUNX3-low-expressing HSPCs (Figure 1f), implying that RUNX3 overexpression contributes to activating the expression of genes in these key oncogenic pathways in MDS. Thus, RUNX3 appears to function as an oncogene in the development of MDS in patients showing a worse outcome.

RUNX3 overexpression drives the development of MDS in the absence of Tet2

In order to establish whether RUNX3 promotes the development of MDS in vivo, we generated a hematopoietic cell-specific RUNX3-expressing Tet2-deficient mouse model because the loss-of-function mutation in TET2 is one of the most common genetic mutations observed in clonal hematopoiesis in aged individuals (23,24), and in

Yokomizo-Nakano T, et al. patients with MDS showing stronger RUNX3 expression (30). We initially deleted Tet2 by activating Cre recombinase via intraperitoneal injections of tamoxifen in Cre-ERT2 and Tet2flox/flox;Cre-ERT2 mice. Two months after the tamoxifen injection, hematopoietic stem cells (HSCs) purified from mice were infected with an empty control or FLAG-tagged RUNX3 retrovirus vector under liquid culture conditions supplemented with SCF and TPO showing comparable transduction efficacies (Supplementary Figure 4), and were transplanted into lethally- irradiated CD45.1+ wild-type recipient mice together with freshly harvested CD45.1+ wild-type bone marrow cells

(Figure 2a). We hereafter refer to recipient mice reconstituted with control, RUNX3-expressing, Tet2Δ/Δ, and

RUNX3-expressing Tet2Δ/Δ HSCs as WT, RUNX3-Tet2 wt/wt, Tet2Δ/Δ, and RUNX3-Tet2 Δ/Δ mice, respectively. We confirmed the overexpression of the RUNX3 protein in c-Kit+ cells (Figure 2b), and the decreased expression of

Tet2 mRNA in Lineage-c-Kit+Sca-1+ (LSK) HSPCs isolated from these mice (Figure 2c).

Complete blood count (CBC) analyses revealed no significant changes between these genotypes at 2 months post transplantation (Supplementary Figure 5). While neither RUNX3-Tet2 wt/wt mice nor Tet2 Δ/Δ mice showed significant changes in CBC at 9 months post transplantation, platelet counts in RUNX3-Tet2 Δ/Δ mice gradually decreased from 7 months and these mice developed thrombocytopenia at 9 months post transplantation

(Figure 2d). Moribund RUNX3-Tet2 Δ/Δ mice showed lower frequencies of CD45.2+GFP+ cells in B and T cells but higher frequency in mature myeloid cells in PB than RUNX3-Tet2 Δ/Δ mice at the pre-disease stage (Figure 2e).

Although survival times were not shorter in RUNX3-Tet2 Δ/Δ mice than in Tet2 Δ/Δ mice (median survival 455 days versus 351.5 days, p=0.4862), which develop MDS/MPN showing leukocytosis as previously reported, moribund

RUNX3-Tet2 Δ/Δ mice had severe neutropenia (Figure 2f), anemia, and thrombocytopenia (Figure 2d), accompanied by dysplastic neutrophils, anisocytosis, and giant platelets in PB and dysplastic megakaryocytes in BM and the spleen (Figure 2g), which are characteristic features of MDS in patients. While RUNX3-Tet2 Δ/Δ mice showed similar numbers of BM cells to mice with other genotypes (Figure 2h), spleen weights were variable in RUNX3-

Tet2 Δ/Δ MDS mice (Figure 2i) and the expansion of Lineage-Sca-1+c-Kit+ (LSK) HSPCs was observed in the spleen

(Supplementary Figure 6), indicating extramedullary hematopoiesis in RUNX3-Tet2 Δ/Δ MDS mice. We found that

RUNX3-Tet2 wt/wt mice and Tet2Δ/Δ mice developed MDS and MDS/MPN according to the criteria for murine MDS

Yokomizo-Nakano T, et al. and the MDS/MPN subcategories (31), whereas RUNX3-Tet2 Δ/Δ stem cells dominantly induced the development of

MDS in mice showing dysplasia and cytopenia, but lacking leukocytosis in PB (Figure 2j). Thus, the overexpression of RUNX3 compromises normal hematopoiesis and drives the development of MDS in the absence of Tet2 in vivo.

RUNX3-expressing Tet2-deficient HSPCs maintain the repopulating capacity but impair the differentiation

We then performed a detailed phenotypic analysis of the BM cells of all genotype mice 2 months post transplantation and moribund RUNX3-Tet2 Δ/Δ MDS mice. Pre-MDS RUNX3-Tet2 Δ/Δ mice showed similar frequencies of CD45.2+GFP+ cells in LSK HSPCs and granulocyte/macrophage progenitor cells (GMPs) in BM, while RUNX3-Tet2 Δ/Δ MDS mice subsequently showed larger numbers of these cells in HSPCs and GMPs (Figure

3a). Based on the increased proportion of RUNX3-Tet2 Δ/Δ cells in HSPCs, we performed Ki67 and Annexin V staining by flow cytometry and found that RUNX3-Tet2 Δ/Δ mice showed a slightly higher frequency of Ki67-positive cells (Figure 3b), but less apoptosis in HSPCs in BM (Figure 3c). The results obtained also showed that RUNX3-

Tet2 Δ/Δ HSPCs maintained larger numbers of in vitro re-plating colonies than Tet2Δ/Δ HSPCs showing monocyte colonies in the third plating and also generated mixed-lineage colonies (e.g. GEMM and GM) (Figure 3d). To further assess the proliferative capacity of RUNX3-Tet2Δ/Δ cells in vivo, we performed the competitive transplantation of CD45.2+GFP+ HSPCs purified from primary transplanted mice with freshly-harvested wild-type

CD45.1+ BM cells into lethally-irradiated recipient mice (Figure 3e). WT HSPCs did not repopulate in this experimental setting based on the low frequencies of CD45.2+GFP+ cells in PB (Figure 3f) and HSPCs in BM at 6 months post transplantation (Figure 3g), while RUNX3-Tet2 Δ/Δ mice showed a higher frequency of CD45.2+GFP+ cells in myeloid cells, but negligible amounts in lymphoid cells in PB (Figure 3f), and showed the up-regulated expression of the RUNX3 protein in c-Kit+ cells in BM (Figure 3h). RUNX3-Tet2 Δ/Δ mice showed a similar

CD45.2+GFP+ frequency in HSPCs, CMPs and GMPs to those in single mutant mice in BM (Figure 3g), indicating that RUNX3 overexpression and/or a Tet2 deficiency sustained the repopulating capacity of HSPCs and myeloid progenitor cells in vivo. In contrast, RUNX3-Tet2 Δ/Δ mice showed a smaller frequency of CD45.2+GFP+ in

Yokomizo-Nakano T, et al. megakaryocyte/erythroid progenitor cells (MEPs) than that in Tet2 Δ/Δ mice (Figure 3g), which may have contributed to thrombocytopenia in primary transplanted mice (Figure 2d). Thus, although RUNX3-Tet2 Δ/Δ HSPCs maintain their repopulating capacity, their differentiation is impaired, leading to the development of MDS.

RUNX3 overexpression reduces the expression of Runx1 protein and dysregulates the transcription of

Runx1 target genes

In order to elucidate the mechanisms by which RUNX3-Tet2 Δ/Δ HSPCs develop MDS in vivo, we performed gene expression analyses using RNA sequencing on HSPCs isolated from WT, RUNX3-Tet2wt/wt, Tet2Δ/Δ, and RUNX3-

Tet2 Δ/Δ mice at 2 months post transplantation, one Tet2 Δ/Δ MDS/MPN mouse, two Tet2 Δ/Δ MDS mice, and two

RUNX3-Tet2 Δ/Δ MDS mice. As expected, a principal component analysis (PCA) revealed that RUNX3-Tet2 Δ/Δ cells at the pre-MDS and MDS stages clustered closer, but separately from WT, Tet2 Δ/Δ and Tet2Δ/Δ MDS cells (Figure

4a). We observed 68, 258, and 151 up-regulated genes and 279, 615, and 415 down-regulated genes in RUNX3-

Tet2 wt/wt, Tet2Δ/Δ, and RUNX3-Tet2 Δ/Δ cells, respectively, at the pre-MDS stage from those in WT cells (Figure 4b and Supplementary Data 1). We also found that RUNX3-Tet2 Δ/Δ MDS HSPCs shared approximately 50% up- and down-regulated genes in RUNX3-Tet2 Δ/Δ HSPCs at the pre-MDS stage (Figure 4c), but shared fewer dysregulated genes with Tet2 Δ/Δ MDS and MDS/MPN cells (Supplementary Figure 7), indicating an altered transcriptional program in HSPCs during the development of Tet2-deficient MDS due to the overexpression of RUNX3. We performed gene ontology (GO) enrichment analyses and found that RUNX3-Tet2 Δ/Δ MDS cells showed positive enrichments in genes involved in TGF-b, TNF-a, and inflammatory response pathways, as expected from the result showing that human MDS cells express RUNX3 (Figure 1e), but negative enrichments in genes in immune responses and leukocyte differentiation (Figure 4d and Supplementary Data 2). RUNX3-Tet2 Δ/Δ MDS cells showed a positive enrichment in the expression of RUNX1-ETO target genes, but negative enrichments in other acute leukemia-related fusion genes, such as MLL-AF9 and BCR-ABL1 (Figure 4d), indicating that RUNX3-Tet2 Δ/Δ cells suppress the transcriptional function of Runx1, which has been shown to be repressed by RUNX1-ETO

(32,33).

Yokomizo-Nakano T, et al.

We attempted to clarify whether the overexpression of RUNX3 inhibits the expression and/or function of

Runx1. While RUNX3-expressing HSPCs showed similar expression levels of Runx1 mRNA (Figure 4e), RUNX3-

Tet2 wt/wt and RUNX3-Tet2 Δ/Δ c-Kit+ progenitor cells both had markedly decreased expression levels of the Runx1 protein (Figure 4f). Since the stability of the RUNX1 protein is enhanced by binding to CBFb (34), we transiently transfected RUNX1, RUNX3, and CBFb vectors in human cells and performed an IP-WB experiment to assess binding between RUNX1, RUNX3, and CBFb. We found that RUNX3 inhibited the binding of RUNX1 to CBFb

(Supplementary Figure 8), which supports the overexpression of the RUNX3 protein decreasing Runx1 protein levels due to their competitive binding to CBFb. Consistent with the results obtained in the GO analysis, the Gene

Set Enrichment Analysis (GSEA) revealed that RUNX3-expressing cells had significantly decreased or increased expression levels of canonical Runx1 target genes (Figure 4g), which were up- or down-regulated in Runx1- knockout cells defined by a microarray (GSE40155) (Supplementary Data 3). RUNX3-Tet2 Δ/Δ MDS HSPCs showed significantly lower or higher expression levels of Runx1 target genes than Tet2 Δ/Δ MDS HSPCs (Figure 4g).

Among Runx1 target genes, we performed quantitative RT-PCR (q-PCR) using these HSPCs and confirmed that

RUNX3-Tet2 Δ/Δ MDS cells showed the significantly weaker expression of Runx1 target tumor suppressor genes, such as Cebpa and Csf1r (Figure 4h), the expression levels of which were slightly decreased in these HSPCs in

RNA sequencing. Therefore, RUNX3 overexpression reduces the expression of the Runx1 protein in cells and dysregulates the transcription of Runx1 target genes.

RUNX3 overexpression remodels the binding regions of and impedes the transcriptional function of Runx1

Based on the dysregulated expression of Runx1 target genes, in order to clarify whether RUNX3 impeded the DNA binding of and transcriptional function of RUNX1, we first performed Runx1-ChIP sequencing (ChIP-seq) on WT,

RUNX3-Tet2 wt/wt, and RUNX3-Tet2 Δ/Δ cells and FLAG-tagged RUNX3-ChIP-seq on RUNX3-Tet2 wt/wt and RUNX3-

Tet2 Δ/Δ cells after expanding HSPCs in an in vitro culture. To assess the level of active histone modifications in

Runx-binding sites, we also performed H3K27ac-ChIP-seq on HSPCs isolated from mice of all genotypes. As expected, RUNX3- and Runx1-binding peaks were enriched at the promoter to transcription start site (TSS) and

Yokomizo-Nakano T, et al. intron regions in these cells (Figure 5a and Supplementary Figure 9). Motif enrichment analyses of RUNX3- and

Runx1-ChIP-seq revealed robust enrichments in consensus Runx-binding sequences, supporting the accuracy of these ChIP-seq data (Table 1 and Supplementary Data 4). While RUNX3-binding peaks mostly overlapped between RUNX3-Tet2 wt/wt and RUNX3-Tet2 Δ/Δ cells, we found that Runx1-binding peaks showed a large variation between WT, RUNX3-Tet2 wt/wt and RUNX3-Tet2 Δ/Δ cells (Figure 5b). The Runx1-binding sites identified in WT cells overlapped with those in RUNX3-Tet2 Δ/Δ cells, which were partly shared by RUNX3-binding sites in RUNX3-Tet2 Δ/Δ cells (Figure 5c), indicating that the overexpression of RUNX3 and loss of Tet2 remodeled the binding regions of

Runx1. Among these Runx1 target genes, by visualizing ChIP-seq data, we found that RUNX3-Tet2 Δ/Δ cells showed RUNX3-binding peaks in the promoter regions of the Cebpa, Csf1r, and Hnrnpa0 genes (Figure 5d and

Supplementary Figure 10).

While we did not detect any changes in H3K27ac expression levels in c-Kit+ progenitor cells among these different genotypes (Figure 5e), Runx1 target genes showed lower levels of the H3K27ac modification in

RUNX3-expressing cells than in control cells (Figure 5f, 5g), supporting the inhibition by RUNX3 of the transcription of Runx1 target genes (Figure 4h). To determine whether RUNX3 competed with RUNX1 to activate the promoter activity of their target genes, we performed a luciferase assay in which activity was regulated by the promoter/enhancer region of either Cebpa, Csf1r or Hnrnpa0 containing RUNX-binding-motif sequences (Figure

5h). In this experimental setting, the transfection of RUNX3 expression suppressed luciferase activity driven by

RUNX1 expression in a dose-dependent manner (Figure 5i). Therefore, RUNX3 overexpression remodels the binding regions of Runx1 with the loss of Tet2, but also impedes the transcriptional function of Runx1 accompanied by the reduction of H3K27ac modification in the region-dependent manner.

RUNX3 overexpression enhances the transcription of Myc target genes and sensitivity to the inhibition of

Myc

As we further analyzed gene expression profiles in HSPCs in an unsupervised manner, we found that the overexpression of RUNX3 and/or loss of Tet2 resulted in positive enrichments in the expression of the MYC

Yokomizo-Nakano T, et al. hallmark and TGF-b signaling pathways (Figure 6a). Given the importance of these biological pathways in the pathogenesis of MDS, we found that RUNX3-Tet2 Δ/Δ MDS cells showed the stronger expression of genes in the

MYC hallmark and TGF-b signaling pathways than RUNX3-Tet2 Δ/Δ pre-MDS cells (Figure 6b). RUNX3-Tet2 Δ/Δ

MDS HSPCs did not show a significant change in the expression of Myc hallmark genes or Myc-RUNX3-common target genes (as defined below) from Tet2 Δ/Δ MDS HSPCs (Figure 6a), implying that the loss of Tet2 exerted a dominant effect on the transcription of Myc target genes. These genotype cells showed similar mRNA and protein expression levels of Myc (Figure 6c and 6d), in which blood-specific enhancers harbored similar levels of the

H3K27ac modification (Supplementary Figure 11), implying that the overexpression of RUNX3 and loss of Tet2 promote the transcriptional function of Myc rather than increasing the expression of Myc. A motif enrichment analysis of RUNX3-ChIP-seq revealed significant enrichments in E-box and Myc/Max-binding motif sequences in

RUNX3-Tet2 Δ/Δ cells (Table 1 and Supplementary Data 4); therefore, we found that RUNX3-binding sites were shared by Myc-binding sites, which were identified by Myc-ChIP-seq in RUNX3-Tet2 Δ/Δ cells (Figure 6e). Mutually,

Myc-ChIP-seq revealed a significant enrichment in the Runx-binding motif sequence in RUNX3-Tet2 Δ/Δ cells (Table

1). In fact, Myc-RUNX3 common target genes showed a positive enrichment in RUNX3-Tet2 Δ/Δ HSPCs rather than in either of the single mutant HSPCs (Figure 6a).

To determine whether Myc was critical for the cell growth capacity of RUNX3-expressing and/or Tet2- deficient HSPCs, we treated these HSPCs with the Myc inhibitor, 10058-F4, which blocks its heterodimerization with Max under in vitro culture conditions. Although Tet2 Δ/Δ cells showed the increased expression of Myc hallmark genes (Figure 6a), they showed a similar cell growth capacity after the treatment with 10058-F4 (Figure

6f), suggesting that the loss of Tet2 increased the expression of Myc target genes in a Myc-Max-independent manner. The number of RUNX3-expressing cells was significantly decreased by the treatment with 10058-F4

(Figure 6f), indicating the transcriptional function of Myc was critical for the cell growth property of RUNX3-Tet2Δ/Δ cells. Although further studies are needed to assess the in vivo effects of Myc inhibition, the overexpression of

RUNX3 promoted the transcriptional function of Myc and sensitivity to the inhibition of Myc-Max heterodimerization, at least under this in vitro condition.

Yokomizo-Nakano T, et al.

Discussion

The expression levels of RUNX transcription factors are fine-tuned by signaling pathways, transcription factors, and epigenetic modifiers at the promoter and enhancer regions of RUNX in a cell type-specific manner in development and adult tissue homeostasis, while the dysregulation of these processes initiates tumorigenesis (1). RUNX3 is known to function as a tumor suppressor in various tumors including MPN (7), and we demonstrated the enhanced expression and oncogenic function of RUNX3 in the pathogenesis of MDS in patients and mice. Although it currently remains unclear how RUNX3 is activated in MDS HSPCs in patients, in the present study, the ectopic expression of RUNX3 clearly facilitated the initiation of Tet2-deficient MDS in mice following alterations in the transcriptional program involved in Runx1-mediated hematopoiesis and cancer-related biological pathways (e.g.

MYC and TGF-b) (a model is shown in Figure 6g), which were partly shared in RUNX3-high-expressing MDS

HSPCs in patients, thereby supporting our mouse model recapitulating the pathogenesis of human MDS.

Runx1 is expressed in HSPCs and its loss impairs normal hematopoiesis, leading to the development of

MPN-like disease in vivo, a similar phenotype to that observed in Runx3-deficient mice (5,14), suggesting a functional redundancy between RUNX1 and RUNX3 for regulating the expression of their common target genes.

A Runx1 deficiency is compensated for by Runx3 in terms the proliferative capacity of HSPCs being maintained and the deletion of both genes leading to bone marrow failure disease in mice (15). In contrast, direct transcriptional inhibition between RUNX genes has been implicated in pathogenesis because RUNX3 directly binds to the promoter of RUNX1 in order to repress the transcription of RUNX1 and promote the development of EBV- mediated B-cell malignancies, which markedly enhance the expression of RUNX3 due to its activated super- enhancer (20,35,36). In the present study, RUNX3-expressing MDS cells suppressed the transcription of canonical Runx1 target genes following the remodeling of Runx1 binding sites via RUNX3 accompanied by a reduced level of the H3K27ac modification. Among these common peaks, we found that RUNX3-Tet2 Δ/Δ MDS cells markedly suppressed the expression of Runx1 target genes, such as Cebpa and Csf1r, which regulate differentiation and play tumor-suppressive roles in myeloid transformation including MDS (37–39), indicating that the region-specific competition of RUNX proteins and recruitment of repressor complexes drive the suppression of

Yokomizo-Nakano T, et al. target genes. Although the functional roles of these repressed genes in RUNX3-Tet2 Δ/Δ MDS cells currently remain unclear, RUNX3-Tet2 Δ/Δ HSPCs impeded the differentiation and production of mature myeloid cells and platelets and MDS rather than MDS/MPN subsequently developed in mice. These results indicate that the overexpression of RUNX3 drives MDS clones and/or suppresses MPN clones in Tet2-deficient stem cells; however, further studies are needed to elucidate the mechanisms underlying competition between MDS clones and MPN clones in vivo.

Another interesting result was reduced Runx1 protein levels in spite of similar mRNA levels in RUNX3- over-expressing cells. The co-factor CBFb enhances Runx1 stability by preventing its ubiquitin-mediated degradation (34). The stability of the Runx1 protein is tightly regulated by post-translational modifications in

Runx1 through various modification enzymes (40) because wild-type MLL increases, whereas the MLL-fusion oncoprotein reduces Runx1 protein levels in order to promote the development of leukemia, which was inhibited by the ectopic expression of Runx1 (41). The present results showed that the RUNX3 protein competed with the

RUNX1 protein for binding to CBFb, whereas the RUNX1 protein did not inhibit RUNX3 protein binding to CBFb in an ex vivo setting, supporting the oncogenic property of RUNX3, the overexpression of which reduces RUNX1 protein levels to promote transformation in vivo.

MYC is critical for the development of various tumors through its regulation of cancer-related biological pathways (e.g. cell cycle, apoptosis, and translation). MYC and RUNX family genes have been shown to collaborate in order to initiate and propagate tumorigenesis, for example, MYC and RUNX2 collaborate to promote the development of blastic plasmacytoid dendritic cell neoplasms (BPDCN) as well as T-cell lymphoma, at least in part, due to the suppression of MYC-dependent apoptosis (42,43). RUNX3 has also been shown to function as an oncogene in natural killer/T-cell lymphoma and is transcriptionally activated by MYC (44). In the present study, we demonstrated that RUNX3-Tet2 Δ/Δ cells enhanced the expression of Myc target genes in order to activate cancer-related biological pathways, which were partly bound by RUNX3, as identified in ChIP-seq and motif enrichment analyses. Although the mechanisms by which the loss of Tet2 or overexpression of RUNX3 activates the transcriptional function of Myc currently remain unclear, RUNX3 directly binds to Myc target genes and

Yokomizo-Nakano T, et al. facilitates the transcriptional function of Myc, resulting in the development of MDS. Indeed, we found that RUNX3- expressing cells showed greater vulnerability to the functional inhibition of Myc by 10058-F4 than control cells, at least, in an in vitro setting. We also applied this result to human cells and demonstrated that human AML cells strongly expressing RUNX3 with activated histone modifications at the enhancer showed higher sensitivity to

10058-F4 and JQ1, a BRD4 inhibitor that inhibits the function of super-enhancers (45,46), following the moderate reduction of RUNX3 and MYC expression. Further studies are needed to examine the combinatorial inhibition of

MYC and activated enhancers of target genes, including the region of the RUNX3 gene in MDS cells in vivo.

In the present study, we generated a murine model for Tet2-deficient MDS expressing RUNX3 and demonstrated that the overexpression of RUNX3 exerts oncogenic effects on the cellular function of and the transcriptional program in MDS stem cells. Several MDS patient-derived xenograft murine models have recently been reported using humanized cytokine knock-in mice and supplementing patient-derived mesenchymal stroma cells with human MDS stem cells (47–49). These xenograft models for MDS remain challenging, but may provide a more detailed understanding of the pathogenesis of MDS. An integrated study on genetically engineered mice and these xenograft models is warranted in the future for the development of novel therapeutics that target MDS stem cells.

Acknowledgments

The authors thank the members of the Sashida Laboratory for their discussions during the preparation of this manuscript and Mr. Shinji Kudoh and Dr. Takaaki Ito for their technical help. This work was supported in part by a grant from the Uehara Memorial Foundation (G.S.), the Princess Takamatsu Cancer Research Fund (G.S.), the

Japanese Society of Hematology (G.S.), the Naito Foundation (T.YN.), the Takeda Science Foundation (T.YN.), and

Grants-in-Aid for Scientific Research (16KT0113, 16K19579, 18H02842, 19K22640 and 19K08842) from the

Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

Yokomizo-Nakano T, et al.

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Table:

Table 1. Motif enrichments in sequences bound by Runx1 or RUNX3 in RUNX3-Tet2wt/wt and RUNX3-Tet2Δ/Δ cells and Myc in RUNX3-Tet2Δ/Δ cells

IP: Runx1 IP: FLAG-RUNX3 IP: Myc

Name RUNX3-Tet2 wt/wt RUNX3-Tet2 Δ/Δ RUNX3-Tet2 wt/wt RUNX3-Tet2 Δ/Δ RUNX3-Tet2 Δ/Δ

cell cell cell cell cell

RUNX 1e-1153 1e-596 1e-2073 1e-1147 1e-5

E-box 1e-42 1e-32 1e-30 1e-22 1e-3

Max 1e-15 1e-12 1e-24 1e-18 1e-41

Myc 1e-14 1e-4 1e-11 1e-8 1e-36

Yokomizo-Nakano T, et al.

Figure legends:

Figure 1. Increased expression of RUNX3 in BM cells of MDS patients a, b) Stronger expression of RUNX3 mRNA in the CD34+ HSPCs of a subset of MDS patients than in healthy controls in GSE19429 (a) and GSE4619 (b). c) Increased expression of the RUNX3 protein in the BM cells of MDS patients (n=20). d) Representative histology of BM cells in MDS patients observed by hematoxylin staining and anti-RUNX3 staining. e) Shorter survival of RUNX3 high-expressing patients (n=12, red line) than RUNX3 low-expressing patients (n=12, black line) in a cohort of 122 MDS patients (GSE58831). The Log-rank test was used for the P value. f) GSEA for TGF-b and TNF-a hallmark pathway genes in the HSPCs of MDS patients showing stronger RUNX3 expression than in those showing weaker RUNX3 expression, as shown in Figure 1a (GSE19429). a, b, c) Bars show means ± SD.

Figure 2. RUNX3 overexpression induces the development of MDS in the absence of Tet2 a) Experimental scheme utilizing the RUNX3 vector and Tet2 conditional KO mice for transplantation assays. b) Successful transduction of the RUNX3 protein in Lineage-c-Kit+ BM cells. Levels of actin were used as loading controls. c) Expression level of Tet2 mRNA examined by q-PCR in HSPCs in BM (n=3-10). d) Complete blood cell counts in the PB of WT, RUNX3-Tet2 wt/wt, Tet2Δ/Δ, and RUNX3-Tet2 Δ/Δ mice (n=9-18) at 9 months post transplantation, moribund Tet2Δ/Δ mice (MDS, n=8, opened circle; MDS/MPN, n=10, axis) and moribund RUNX3-Tet2 Δ/Δ MDS mice (n=14). e, f) Proportions of CD45.2+GFP+ cells in Gr-1+/Mac-1+ myeloid cells, B220+ B-cells, and CD4+/CD8+ T-cells in the

PB (e) and absolute Gr-1+/Mac-1+ myeloid cell counts in the PB (f) of WT, RUNX3-Tet2wt/wt, Tet2Δ/Δ, and RUNX3-

Tet2 Δ/Δ mice (n=4-5) at 2 months post transplantation, moribund Tet2 Δ/Δ mice (n=15) and moribund RUNX3-Tet2 Δ/Δ

MDS mice (n=7). Bars show means ± SEM.

Yokomizo-Nakano T, et al. g) Dysplastic blood cells in the PB and BM of RUNX3-Tet 2 Δ/Δ mice observed by May-Grünwald Giemsa staining.

Representative histology of the BM and spleen of RUNX3-Tet2 Δ/Δ mice observed by hematoxylin-eosin staining.

Scar bars show 10 µm or 50 µm. h, i) Bone marrow cell counts isolated from 1 femur and 1 tibia (h) and spleen weights (i) of WT, RUNX3-Tet2 wt/wt,

Tet2 Δ/Δ, and RUNX3-Tet2 Δ/Δ mice (n=5-12) at 6 months post transplantation, moribund Tet2Δ/Δ mice (MDS, n=4, opened circle; MDS/MPN, n=8, axis) and moribund RUNX3-Tet2 Δ/Δ MDS mice (n=13). j) Disease types of hematological malignancies identified in RUNX3-Tet2 wt/wt (n=9), Tet2Δ/Δ (n=18), and RUNX3-

Tet2 Δ/Δ mice (n=15). Data are combined from 5 independent experiments. c, d, h, i) Bars show means ± SD, *p<0.05, **p<0.01, and ***p<0.001 by the Student’s t-test.

Figure 3. RUNX3-expressing Tet2-deficient HSPCs maintain the repopulating capacity but impair the differentiation a) Strategy of FACS gating boundaries for HSPCs and myeloid progenitors and proportions of CD45.2+GFP+ cells in LSK cells and GMP cells in the BM of WT, RUNX3-Tet 2 wt/wt, Tet2Δ/Δ, and RUNX3-Tet2 Δ/Δ mice (n=4-5) at 2 months post transplantation and moribund RUNX3-Tet2 Δ/Δ MDS mice (n=4). b) Frequencies of Ki67+ cells in CD45.2+GFP+LSK HSPCs in the BM of WT, RUNX3-Tet2 wt/wt, Tet2Δ/Δ, and RUNX3-

Tet2 Δ/Δ mice (n=3-6). c) Frequencies of Annexin V+ apoptotic cells in CD45.2+GFP+LSK HSPCs in the BM of WT, RUNX3-Tet2wt/wt,

Tet2 Δ/Δ, and RUNX3-Tet2 Δ/Δ mice (n=7-10). d) Serial colony formation capacities of HSPCs isolated from WT, RUNX3-Tet2 wt/wt, Tet2Δ/Δ, and RUNX3-Tet2 Δ/Δ mice (n=3). e) Experimental scheme utilizing HSPCs for the secondary competitive transplantation assay. f) Proportions of CD45.2+GFP+ cells in Gr-1+/Mac-1+ myeloid cells and B220+ B-cells in the PB of WT, RUNX3-

Tet2 wt/wt, Tet2Δ/Δ, and RUNX3-Tet2 Δ/Δ mice (n=8-9) at 6 months post transplantation. g) Proportions of CD45.2+GFP+ cells in LSK, CMP, GMP, and MEP cells in the BM of WT, RUNX3-Tet2 wt/wt, Tet2Δ/Δ,

Yokomizo-Nakano T, et al. and RUNX3-Tet2 Δ/Δ mice (n=8-9) at 6 months post transplantation. h) Overexpression of the RUNX3 protein in Lineage-c-Kit+ BM cells. b, c, d) Bars show the mean ± SD, *p<0.05 by the Student’s t-test. a, f, g) Bars show the mean ± SEM, *p<0.05, and **p<0.01 by the Mann-Whitney U test.

Figure 4. RUNX3-expressing Tet2-deficient HSPCs accumulate an altered transcriptional program a) Principal component analysis based on the gene expression of HSPCs isolated from WT, RUNX3-Tet2wt/wt,

Tet2 Δ/Δ, and RUNX3-Tet2 Δ/Δ mice at the pre-disease stage, two Tet2 Δ/Δ MDS mice, one Tet2Δ/Δ MDS/MPN mouse, and two RUNX3-Tet2 Δ/Δ MDS mice. b) Venn diagrams of overlaps in up- and down-regulated genes in HSPCs isolated from RUNX3-Tet2wt/wt (yellow),

Tet2 Δ/Δ (green), and RUNX3-Tet2 Δ/Δ (purple) mice at the pre-disease stage from those isolated from WT mice. c) Venn diagrams of overlaps in up- and down-regulated genes in HSPCs isolated from RUNX3-Tet2 Δ/Δ mice at the pre-disease stage and two RUNX3-Tet2 Δ/Δ disease mice from those in WT cells. d) Gene ontology (GO) analysis for up- and down-regulated genes in HSPCs detected in Figure 4c. e,f) Expression levels of Runx1 mRNA examined by q-PCR in HSPCs (n=7-10), and the Runx1 protein in Lineage- c-Kit+ BM cells. Levels of actin were used as loading controls. g) GSEA for canonical Runx1 target genes defined by Runx1-ChIP-seq and either up- or down-regulated genes in

Runx1-knockout cells in HSPCs isolated from WT, RUNX3-Tet2 wt/wt, Tet2Δ/Δ, and RUNX3-Tet2 Δ/Δ mice at the pre- disease stage, moribund Tet2Δ/Δ MDS and MDS/MPN mice, and moribund RUNX3-Tet2 Δ/Δ MDS mice. Depicted genotype HSPCs were compared with either WT, Tet2Δ/Δ or Tet2 Δ/Δ-MDS HSPCs. h) Expression levels of Cebpa, Csf1r and Hnrnpa0 mRNA examined by q-PCR in HSPCs WT, RUNX3-Tet2wt/wt,

Tet2 Δ/Δ, and RUNX3-Tet2 Δ/Δ mice at the pre-disease stage (n=5-7), moribund Tet2Δ/Δ mice (n=3), and moribund

RUNX3-Tet2 Δ/Δ mice (n=5). e, h) Bars show means ± SD, *p<0.05, and **p<0.01 by the Student’s t-test.

Yokomizo-Nakano T, et al.

Figure 5. RUNX3 remodels Runx1-binding sites and inhibits its transcriptional function a) Pie charts showing the binding regions of Runx1 and RUNX3 in RUNX3-Tet2 Δ/Δ cells. b) Venn diagram showing the significant overlap of Runx1-binding sites between WT (blue circle), RUNX3-Tet2wt/wt

(yellow circle), and RUNX3-Tet2 Δ/Δ (purple circle) cells. c) Venn diagram showing overlaps between Runx1-binding sites in WT cells (blue circle), Runx1-binding sites in

RUNX3-Tet2 Δ/Δ cells (green circle), and RUNX3-binding sites in RUNX3-Tet2 Δ/Δ cells (red circle). d) Representative Runx1-binding and RUNX3-binding peaks in the promoter region of Cebpa, Csf1r and Hnrnpa0 genes. e) Similar expression of the H3K27ac modification in Lineage-c-Kit+ BM cells. Levels of histone H3 were used as loading controls. f) H3K27ac modification levels in the canonical Runx1 target region examined in WT, RUNX3-Tet2 wt/wt, Tet2Δ/Δ, and

RUNX3-Tet2 Δ/Δ cells. g) Representative H3K27ac peaks in the promoter region of Cebpa and Hnrnpa0 genes. h) Experimental scheme of the luciferase reporter assay. i) Luciferase assay showing the RUNX3-dependent inhibition of RUNX1 activating the reporter activity of Cebpa,

Csf1r and Hnrnpa0, of which the promoter/enhancer regions contained consensus RUNX-binding sequences. i) Bars show means ± SD, *p<0.05, **p<0.01, and ***p<0.001 by the Student’s t-test.

Figure 6. RUNX3 overexpression enhances the expression of Myc target genes and sensitivity to the inhibition of Myc a) GSEA for Myc hallmark genes, TGF-b hallmark pathway genes, and RUNX3-Myc common target genes in

HSPCs isolated from RUNX3-Tet2 wt/wt, Tet2Δ/Δ, and RUNX3-Tet2 Δ/Δ mice at the pre-disease stage, Tet2Δ/Δ MDS and

MDS/MPN mice, and RUNX3-Tet2 Δ/Δ MDS mice relative to that of WT cells or that of Tet2 Δ/Δ MDS cells. b) GSEA for Myc hallmark genes in HSPCs isolated from RUNX3-Tet2 Δ/Δ MDS mice relative to that of RUNX3-

Tet2 Δ/Δ MDS mice at the pre-disease stage.

Yokomizo-Nakano T, et al. c, d) Expression levels of Myc mRNA in HSPCs (n=6-10) and the Myc protein in Lineage-c-Kit+ BM cells. Levels of actin were used as loading controls. e) Venn diagram showing the overlap of the target genes of RUNX3 in RUNX3-Tet2 wt/wt cells and RUNX3-Tet2 Δ/Δ cells and Myc identified in RUNX3-Tet2 Δ/Δ cells. f) Reduced cell growth capacity of 10058-F4-treated RUNX3-expressing HSPCs under in vitro culture conditions from that of control DMSO-treated RUNX3-expressing HSPCs (n=3). g) Model of the pathogenesis of RUNX3-expressing Tet2-deficient MDS. c, f) Bars show the mean ± SD, *p<0.05, **p<0.01, and ***p<0.001 by the Student’s t-test.

a GSE1942194299 b GSEGSE46146199 c RUNRUNX3 X3IHC 1500 1000 30

800 1000 20 600 RU NX3 RU NX3 400 h 500 h 10

RMA intensity RMA intensity 200 NX3 positive cells positive RU NX3 0 0 0 % h % RUNX3 positive cells (%) RA MDS MDS Healthy Healthy Healthy RAEB1/2 d SurvivalSurvival prpropoportions:ortions: SurvivalSurvival ofof TOP16TOP16e RURUNNX3_HighX3 High 100100 RUNX3_High SurvivalRU prNXop3_Lortions:ow Survivalp=0.028 of7 TOP12 RURUNNX3_LX3ow Low p=0.0085 100 p=0.0085 Control 50 50 ent survival erc ent P ent survival erc ent P 0 50 0 1000 2000 3000 0 Days Percent survival Percent 0 1000 2000 30Percent survival 00 Days 0 MDS 0 1000 2000 3000 Days

f TNFα-Signaling via NFκB TGF-β Signaling NES 3.53 NES 2.50 p value 0.0 p value 0.0 FDR q-value 0.0 FDR q-value 0.0

RUNX3 High RUNX3 Low RUNX3 High RUNX3 Low

a BM cells Lin-Kit+ Lin-Kit+Sca1- LSK GMP LSK GMP 100 100 CD45.2+GFP+ 75 Lin MEP 75 CD45.2+GFP- c-Kit % CD45.1+

CD16/32 50 LK CMP 50 c-Kit Sca-1 CD34 25 25 0 0 /Δ /Δ Δ Δ/Δ Δ/Δ Δ Δ/Δ Δ/Δ

WTWT/WT WTWT/WT Tet2 MDS Tet2 MDS b Ki67 posiKi6t7+ive cells c AApoppopttosisosis 100 12 * RUNX3-Tet2RUNX3-Tet2 RUNX3-Tet2RUNX3-Tet2 * RUNX3-Tet2 RUNX3-Tet2 75 9 e WT CD45.2+

% % 50 6 WT/WT + nn exinV+ RUNX3-Tet2 GFP Δ/Δ 25 3 Tet2 Primary LSK cells % A % RUNX3-Tet2Δ/Δ transplanted 0 0 mice /Δ Δ Δ Δ/Δ / TE ΔTE TRΔ/Δ WTWE WR TR WE WR WT/WT WTWT/WT Lethally-irradiated Tet2 Tet2 recipient mice 120 CD45.1+ (Ly5.1+) 5 RUNX3-Tet2 RUNX3-Tet2 WT mice 2x10 RUNX3-Tet2 RUNX3-Tet2 (Ly5.1+) BM cells Myeloid cells d 90 Myeloid cells BB cellcellss 120 f * GM * GMPCD45.2+GFP+ CD45.2+GFP+ 100 100 CD45.2+GFP- CD45.2+GFP- 90 M CD45.1+ CD45.1+ GEMM 60 75 75 CD45.2+GFP+ 100 CD45.2+GFP-

GEMM 60 % 50 50 CD45.1+ 25 75 25

Colony number Colony 30 Colony number Colony

Colony number 30 0 50 0 Δ / /Δ Δ Δ/Δ Δ Δ/Δ 0 WT -RUNX3 WT WT/WT 25 WT/WT -RUNX3 WT-EmptyWT-RUNX3 Tet2 WT-EmptyWT-RUNX3 et2 KO-Empty Tet2 Plating 1 2 3 1 2 3 1 2 3 1 2 3 T et2 KO et2 KO-Empty 3_3rd T T et2 KO ty_2nd ty_3rd 3_2nd 0 y_2nd ty_3rd 3_2nd 0 T WT-Empty et2-Empty Δ/Δ WT-RUNX3 T et2-RUNX3 WT -RUNRUNX3-X Tet2 T RUNX3- WT-Emp et2-Emp WT-Emp WT WT-RUNWT/WTX T et2-RUNX Δ/Δ RUNX3-Tet2 RUNX3-Tet2 Tet2 Tet2-Empt T Tet2Tet2-RUNX3_3rd RUNX3-Tet2GMP 3_3rd RUNX3-Tet2 ty_2nd ty_3rd y_2nd ty_3-RUrdNX3 3_2nd WT-EmptyWT-RUNX3 g WT-Empty GMP MEPet2-Empty LSKLSK CMCMPP GMPWT-RUNX3 MEPT et2 KO-EmptyetCD45.2+GFP+2 KO et2-RUNX3 ** T T T ** et2-Emp *WT-Emp WT-Emp 100 WT-RUNX * CD45.2+GFP- et2-RUNX ** * * WT-RUNX3_2nd Tet2-EmptT Tet2 KO-RUNX3_MDS Tet2-RUNX3_3rd CD45.1+ T 100 100 100 CD45.2+GFP+ 75 100 CD45.2+GFP+ CD45.2+GFP+ CD45.2+GFP+ CD45.2+GFP- CD45.2+GFP- CD45.2+GFP- 75 75 75 75 CD45.2+GFP- CD45.1+ 50 CD45.1+ CD45.1+ CD45.1+ h 50 50 50 50 Δ/Δ 25 25 25 25 25 0 0 0 0 0 /Δ /Δ /Δ /Δ Δ Δ/Δ Δ Δ/Δ Δ Δ/Δ Δ Δ/Δ kDa WT RUNX3-Tet2 WT WT/WT WT WT/WT WT WT/WT WT WT/WT -RUNX3 -RUNX3 -RUNX3 -RUNX3 -RUNX3 50 anti-RUNX3 WT-Empty Tet2 WT-EmptyWT-RUNX3Tet2 WT-EmptyWT-RUNX3Tet2 WT-EmptyWT-RUNX3Tet2 WT-RUNX3 WT-EmptyWT-RUNX3 et2 KO-Empty et2 KO-Empty et2 KO-Empty et2 KO-Empty et2 KO T et2 KO T et2 KO T et2 KO T T T T et2 KO-Emptyet2 KO T anti-actin T T 35 RUNX3-Tet2 RUNX3-Tet2 RUNX3-Tet2 RUNX3-Tet2 Tet2 KO-RUNX3_MDS RUNX3-Tet2 RUNX3-Tet2 RUNX3-Tet2 RUNX3-Tet2

15 regulated-genes regulated genes RUNX3-Tet2Δ/Δ 62 10 WT 31 RUNX3-Tet2WT/WT PC2 5 Tet2Δ/Δ 17 5 _MDS/MPN 10 83 103 Tet2Δ/Δ 0 36 217 371 Δ/Δ 26 Δ/Δ -5 Tet2 79 RUNX3-Tet2 _MDS 119 110 -10 RUNX3-Tet2Δ/Δ_MDS -15 -20 -10 0 10 20 30 PC1 c UP DOWN d UP-regulated in RUNX3-Tet2GO_Runx3-Tet2KO_MDS_UPΔ/Δ_MDS regulated genes regulated genes TNFA signaling via NFKB Inflammatory response Targets of RUNX1-RUNX1T1 fusion RUNX3-Tet2Δ/Δ 61 90 83 126 289 276 TGFB1 targets RUNX3-Tet2Δ/Δ_MDS Targets of EWSR1-FLI1 fusion Targets of PAX-FOXO1 fusion_DN 0 20 40 60 80 e Runx1 f -log (P value)

3 WT/WT Δ/Δ DOWN-regulated in RUNX3-Tet2GO_Runx3-Tet2KO_MDS_DNΔ/Δ_MDS

/Δ 2 Δ Immune response Targets of MLL-AF9 fusion kDa WT RUNX3-Tet2Tet2 RUNX3-Tet2 Targets of HOXA9 and MEIS1_DN 1 50 anti-Runx1 Targets of NUP98-HOXA9 fusion_DN 1.00 0.29 2.00 0.31 Runx1/actin ratio Leukocyte differentiation Relative expression Relative 50 0 Targets of BCR-ABL1 fusion Δ anti-actin / Δ/Δ Δ/Δ WT Δ 35 0 50 100 150 WTRunx3WT/WT et2KO T -RUNX3 Tet2 MDS -log (P value) et2KO T

RUNX3-Tet2Tet2KO-RUNX3_MDSRUNX3-Tet2 RUNX3-Tet2 g FDR NES Enriched in h CebpCebpaa CCsf1rsf1r HnrnpaHnrnpa00 (shade) (area) Mutant WT <0.05 >1.5 4 * 6 * 3 * * * ** <0.0005 >2.5 <0.25 >1.0 3 4 2 <0.005 >2.0 >0.25 <1.0 2

Δ/Δ 2 1 vs vs Tet2 1 vs WT Tet2Δ/Δ_MDS Relative expression Relative Relative expression Relative 0 0 expression Relative 0 Runx1 CHIP /Δ Δ Δ Δ Δ/Δ Δ/Δ / Δ/Δ Δ/Δ / Δ/Δ Δ/Δ WT WT Δ WT Δ _MDS WTWT/WTRunx3et2KO _MDS WTRunx3et2KO WT Runx3et2KO _MDS Up-regulated T WT/WTT -RUNX3 WT/WT T _MDS-RUNX3 MDS _MDS MDS _MDS-RUNX3 MDS Tet2Δ/Δ Tet2et2KΔ/Δ O Tet2 et2KO T Δ/Δet2KO in Runx1 KO T et2KO et2KO T et2KO Down-regulated T T T Tet2 Tet2 Tet2KO-RUNX3_MDS Tet2KO-RUNX3_MDS Tet2 Tet2KO-RUNX3_MDS in Runx1 KO RUNX3-Tet2RUNX3-Tet2 RUNX3-Tet2RUNX3-Tet2 RUNX3-Tet2RUNX3-Tet2 /Δ Δ Δ/Δ Δ/Δ RUNX3-Tet2 RUNX3-Tet2 RUNX3-Tet2 WT/WT _MDS _MDS Tet2 /Δ _MDS Δ/Δ Δ/Δ Δ

Tet2 RUNX3-Tet2 RUNX3-Tet2 RUNX3-Tet2 RUNX3-Tet2 RUNX3-Tet2

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 27, 2020; DOI: 10.1158/0008-5472.CAN-19-3167 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Figure 5 a b c IP: Runx1 IP: FLAG-RUNX3 IP: Runx1 IP: Runx1 IP: FLAG-RUNX3 IP: Runx1 RUNX3-Tet2Δ/Δ 10646 WT 20Run,662x3-IP _TR-Cpeaks Run20x1-Run,IP711x1-_TR-CIP _TR-Cpeaks 1565 2072 1284 1800 7591 promoter-TSS promoter-TSSpromoter-TSS Cebpa 25.9% exon 28.0% exonexon 5806 9553 5' UTR 5' UTR5' UTR 7360 13302 14391 4560 Hnrnpa0 27.4% promoter 28.1% promoter CpG CpGCpG 554 6759 Intergenic -TSS Intergeniintronc -TSS intronintron 777 TTS TTS TTS 4781 3' UTR 3' UTR3' UTR Csf1r 34.7% 33.4% Intergenic 8491 Intergenic Intergenic non-coding intron noinn-codingtron non-coding WT/WT Δ/Δ WT RUNX3-Tet2 RUNX3-Tet2 IP: FLAG-RUNX3 Total=20662 Total=20711 Total=20711 RUNX3-Tet2Δ/Δ d Runx1-IP_TR-CWT WT WT

Runx1 promoter-TSSWT/WT Runx1 WT/WT Runx1 WT/WT RUNX3-Tet2exon RUNX3-Tet2 RUNX3-Tet2 ChIP-seq 5' UTR ChIP-seq ChIP-seq Δ/Δ Δ/Δ RUNX3-Tet2CpG Δ/Δ RUNX3-Tet2 intron RUNX3-Tet2 TTSWT/WT WT/WT WT/WT RUNX3-Tet23' UTR RUNX3-Tet2 RUNX3-Tet2 RUNX3 Intergenic RUNX3 RUNX3 Δ/Δ Δ/Δ ChIP-seq RUNX3-Tet2non-coding ChIP-seq RUNX3-Tet2Δ/Δ ChIP-seq RUNX3-Tet2 Total=20711

Cebpa Hnrnpa0 Csf1r g e f H3K27Ac ChIP-seq WT/WT Δ/Δ H3K27Ac peak WT WT Tet2Δ/Δ Δ/Δ RUNX3-Tet2WT/WT RUNX3-Tet2WT/WT RUNX3-Tet2Δ/Δ kDa WT RUNX3-Tet2Tet2 RUNX3-Tet2 anti- WT Tet2Δ/Δ Tet2Δ/Δ WT/WT 15 H3K27Ac RUNX3-Tet2 RUNX3-Tet2Δ/Δ RUNX3-Tet2Δ/Δ 15 anti-H3

Runx1 target region Cebpa Hnrnpa0 h i Csf1r-C_M_2 Cebpa(7)_F3_3 Hnrnpa0_3 Luc2 Cebpa Hnrnpa0 Csf1r 30030000 4000005' 2000 ** Runx binding ** 4x10 2000 promoter site * RUNX1 RUNX3 * * *** 30003x10005' 15015000 20020000

2000005' 1000 Transfection Lumine sc enc e 2x10 1000 Ratio Ratio Ratio 293T 10010000 cells 10001x10005' 505000

48hrs Rela tive 00 00 00 RUNX1 0 1 1 1 1 (μg) 0 1 1 1 1 (μg) 0 1 1 1 1 (μg) 1:0.2 1:0.5 Assay Luciferase activity Empty 1:0.2 1:0.5 RUNX3 0 0 0.2 0.5 1 ( g) Empty0 0 0.2 0.5 1 ( g) 0 0 1:00.2.2 1:00.5.5 1 ( g) 1:RX3=1:0 1:RX3=1:1 μ μ Empty μ 1:RX3= 1:RX3= 1:RX3=1:0 1:RX3=1:1 RX RX 1:RX3= 1:RX3= 1:RX3=1:0 1:RX3=1:1 RX RX RX RX 1:RX3= 1:RX3= Empty 2 1 0.8 0.5 0 (μg) 2RX 1 RX0.8 0.5 0 (μg) RX2 1 0.8RX0.5 0 (μg) RX RX

a vs b vs WT Tet2Δ/Δ_MDS NES 1.66 p value 0.0 MYC targets version2 FDR NES Enriched in MYC targets (shade) (area) Mutant WT version2 FDR q-value 0.0056 Myc-RUNX3 targets <0.0005 >2.5 TGFβ Signaling <0.005 >2.0 MDS pre-MDS Δ/Δ /Δ <0.05 >1.5 RUNX3-Tet2 Δ Δ/Δ WT/WT <0.25 >1.0 NES 1.51 /Δ _MDS _MDS _MDS Tet2Δ Δ/Δ Δ/Δ >0,25 <1.0 TGFβ p value 0.033 Tet2 Signaling FDR q-value 0.016 RUNX3-Tet2 RUNX3-Tet2 RUNX3-Tet2RUNX3-Tet2 MDS pre-MDS Δ/Δ c Myc d e RUNX3-Tet2 IP: Myc 2.5 WT/WT Δ/Δ RUNX3-Tet2Δ/Δ 2.0 IP: FLAG-RUNX3 WT/WT /Δ RUNX3-Tet2 1.5 Δ 188 147 14271 445 1.0 kDa WT RUNX3-Tet2Tet2 RUNX3-Tet2 75 0.5 anti-Myc 12828 Relative expression Relative 0.0 50 7303 /Δ 1.00 0.82 0.88 1.07 Δ Δ/Δ Δ/Δ Myc/actin ratio WT IP: FLAG-RUNX3 WTWT/WTRunx3 et2KO Tet2KO-TRU-RUNNX3X3 Δ/Δ Tet2 anti-actin RUNX3-Tet2 et2KO MDS 35 T

Tet2KO-RUNX3_MDS RUNX3-Tet2RUNX3-Tet2 8000 RUNX3-Tet2 DMSO MDS stem cell 6000 f 10058-F4 5uM g

WT/WT RUNX3 MYC TET2 loss 4000 WT-EmptyWT RUNX3-Tet2WT-RUNX3 ** 12000 4000 2000 DMSO 9000 ** 30DMSO00 10058-F4 5uMRUNX1 and MYC *** 10058-F4 5uM 0 6000 2000 RUNX1 targets targets 0 2 4 6 Cell number number Cell 3000 number Cell 1000 Time in days *** *** 0 0 ** 0 2 4 6 0 2 4 6 Differentiation Tumorigenesis Time in days Time in days block

Tet2KO-EmptyTet2Δ/Δ RUNX3-Tet2Tet2KO-RUNX3Δ/Δ 20000 8000 DMSO 15000 DMSO 10058-F4 5uM 6000 10058-F4 5uM 10000 4000 ** Cell number number Cell Cell number number Cell 5000 2000 * ** 0 0 *** 0 2 4 6 0 2 4 6 Time in days Time in days

Overexpression of RUNX3 represses RUNX1 to drive transformation of myelodysplastic syndrome

Takako Yokomizo-Nakano, Sho Kubota, Jie Bai, et al.

Cancer Res Published OnlineFirst April 27, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-3167

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2020/04/25/0008-5472.CAN-19-3167.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.

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