B7-H4 enhances the differentiation of murine leukemia-initiating cells via the PTEN/AKT/RCOR2/RUNX1 pathways

Leukemia-initiating cells (LICs) are believed to be responsible for the initiation, development and relapse of leukemia.1 To effectively eliminate LICs, new molecular targets and therapeutic strategies for targeting either extracellular or intracellular signaling are needed. Studies from our and other groups have suggested that targeting specific surface immune molecules of LICs, including LILRB2,2 CD123,3 CD474 and CD93,5 may be a promising strategy to block leukemogenesis. To identify more such surface immune molecules, we screened approximately 30 cell surface proteins expressed in immune systems, and found that several immune molecules, including IREM-1, BTLA, CD244, JAM3, B7-H1 and B7-H4, were highly expressed on MLL-AF9-induced human acute myeloid leukemia (AML) cells.6 Interestingly, one of the identified candidates B7-H4 was also expressed on one fraction of human LIC-enriched CD34+ AML cells as determined by flow cytometric analysis (Supplementary Figure 1a). More importantly, in silico analysis using data extracted from the curated database Leukemia Gene Atlas (http://www.leukemia-gene-atlas.org) also showed that B7-H4 expression level was positively correlated with the overall survival of AML patients (Supplementary Figure 1b), indicating that B7-H4 may serve as a tumor suppressor during leukemia development.

For secondary and tertiary transplantation, a total of 1x10 4 of YFP + BM leukemia cells from primary and secondary recipients were sorted using flow cytometry followed by transplantation into recipients. For the rescue experiments, the lentiviral plasmid pGIPZ-shRcor2-GFP (targeting murine Rcor2, sTable 1) and packaging plasmids pSPAX2 and pMD2G (4:3:1) were transfected in 293T cells using a calcium phosphate transfection method. The resulting lentiviral supernatant was harvested for the spin infection with B7-H4-null bone marrow (BM) leukemia cells followed by retro-orbital injection into the recipients. For each group, at least 5 recipient mice (usually 8-10-week old) were randomly and blindly divided into 2-3 groups and used 2 for the transplantation. All the experiments were repeated at least 3 times. In another experiment, Rcor2 was silenced in C1498 cells, an AML cell line, using Rcor2-targeting shRNA followed by the analysis of RUNX1 protein level using western blotting.

Western blotting and co-immunoprecipitation
Nuclear and cytoplasmic fractions were extracted using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology), and total proteins were isolated by Radio Immune Precipitation Assay (RIPA) buffer (Beyotime Biotechnology) according to the manufacture's protocols. A combination of different plasmids of MSCV-HA-AKT-IRES-mCherry, pBabe-PTEN-3XFlag, pLVX-strepII-B7-H4-IRES-GFP and pLVX-strepII-IRES-GFP (empty vector) were transfected into 293T cells followed by co-immunoprecipitation (co-IP) for the analysis of their interactions. Whole cell lysates or cytoplasmic and nuclear fractions were electrophoresed on 8-10% sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% non-fat milk/TBS and

RNA sequencing
Total RNA was extracted from B7-H4 WT and B7-H4-null YFP + Mac-1 + c-Kit + LICs for RNA-sequencing library construction using a standard TruSeq RNA sample preparation v2 protocol (Illumina). The samples were subsequently sequenced using the Illumina HiSeq2500 platform. Gene expression levels are represented as fragments per kilobase per million mapped reads (FPKM). The RNA-sequencing data were deposited in the Gene Expression Omnibus (GEO) repository under accession number GSE89161.

Immunofluorescence staining, Wright-Giemsa staining and hematoxylin and esosin staining
To determine the localization of B7-H4, immunofluorescence staining was performed in B7-H4-overexpression C1498 cells or primary mouse leukemia cells by using Anti-HA (Sigma-Aldrich, catalog number: H9658, for detecting ectopically expressed B7-H4) or anti-B7-H4 (Proteintech, catalog number: 12080-I-AP, for detecting endogenous B7-H4) antibodies, respectively. Wright-Giemsa staining was performed with BM leukemia cells of secondary recipient mice and B7-H4-overexpression JOSK-I cells, a human acute monocytic leukemia cell line (Cancer Res., 1986, 3067-3074) and the frequencies of blast cells were calculated according to their typical morphologies. Liver and spleen tissues were fixed in 4% paraformaldehyde 4 and embedded in paraffin. Sections were stained with hematoxylin and esosin for the analysis of the infiltration of leukemia cells.

Analysis for luciferase activities
To evaluate the transcriptional regulation of RUNX1 by B7-H4 or RCOR2, and PTEN

Chromatin immunoprecipitation assays
Chromatin immunoprecipitation (ChIP) assays were performed as previously described (Cell Stem Cell, 2010, 380-390). The 293T cells were transfected with the pLX304-Rcor2-V5 plasmids and collected for 24 h later for the cross-linking with formaldehyde at a final concentration of 1%. The samples were sonicated with a Q700 sonicator six times (5 sec on and 10 sec off for each round of sonication).

RCOR2
was immunoprecipitated with anti-V5 antibodies (Biodragon Immunotechnologies Co., Ltd， B1005) and protein A/G beads (Beyotime). DNA fragments were purified using a Qiagen PCR purification kit and quantified by semi-quantitative PCR with primers for the Runx1 promoter as listed in sTable 1.

Statistical analysis
The data are expressed as the means± SEM. The data were analyzed using Student's t test (two-sided), and statistical significance was set at p<0.05. The survival rates of the two groups were analyzed using the Log-rank Test. If the mice are dead due to other unexpected reasons, but not because of the leukemia development, they will not be included for the calculation of the survival ratio. All the experiment shown was replicated 3-5 times or more.  sFigure 4. B7-H4 suppresses leukemogenesis by down-regulating the RCOR2 level. a) WT or B7-H4-null YFP + Mac-1 + c-Kit + LICs of primary recipients were subjected to mRNA-sequencing. Gene ontology (GO) analysis of biological process is shown, and the candidate changes in "transcription, chromosome organization or chromatin organization" are highlighted in red. b) Heat-map of significantly changed genes from mRNA-sequencing analysis in panel a. c) mRNA-sequencing results for the potential candidate genes related to transcription factors, myeloid differentiation and self-renewal. d) RCOR2 levels were validated in WT cells, B7-H4-null cells and Rcor2-knockdown B7-H4-null cells from the rescue experiment.