SUV39H1 regulates the progression of MLL-AF9-induced acute myeloid leukemia

Epigenetic regulations play crucial roles in leukemogenesis and leukemia progression. SUV39H1 is the dominant H3K9 methyltransferase in the hematopoietic system, and its expression declines with aging. However, the role of SUV39H1 via its-mediated repressive modification H3K9me3 in leukemogenesis/leukemia progression remains to be explored. We found that SUV39H1 was down-regulated in a variety of leukemias, including MLL-r AML, as compared with normal individuals. Decreased levels of Suv39h1 expression and genomic H3K9me3 occupancy were observed in LSCs from MLL-r-induced AML mouse models in comparison with that of hematopoietic stem/progenitor cells. Suv39h1 overexpression increased leukemia latency and decreased the frequency of LSCs in MLL-r AML mouse models, while Suv39h1 knockdown accelerated disease progression with increased number of LSCs. Increased Suv39h1 expression led to the inactivation of Hoxb13 and Six1, as well as reversion of Hoxa9/Meis1 downstream target genes, which in turn decelerated leukemia progression. Interestingly, Hoxb13 expression is up-regulated in MLL-AF9-induced AML cells, while knockdown of Hoxb13 in MLL-AF9 leukemic cells significantly prolonged the survival of leukemic mice with reduced LSC frequencies. Our data revealed that SUV39H1 functions as a tumor suppressor in MLL-AF9-induced AML progression. These findings provide the direct link of SUV39H1 to AML development and progression.


Immuno-blotting analysis
Leukemic bone marrow cells or c-Kit + cells were lysed with 1× SDS sample buffer for 30 min at room temperature, boiled at 100°C for 10 min, and stored at -20°C for use. Samples of equal amount of protein were separated by SDS-PAGE, transferred on a PVDF membrane (Millipore), blocked with fat-free milk, and then incubated with primary antibodies. After washing, blots were incubated with HRP-conjugated secondary antibody for 1h. Signals were detected with the Immobilon Western Chemiluminescent HRP substrate (Millipore). Densitometry was performed and calculated with ImageJ (1.49v, NIH). The antibodies used for immune-blotting are listed in Supplementary Table 1.

Cell line culture and CCK8 analysis
Human cell lines HEK293T, THP-1, Molm13 and Nomo-1 cells were obtained from cell bank of SKLEH (purchased from ATCC and maintained by cell bank). The cells were tested for mycoplasma contamination monthly using a mycoplasma detection kit (InvivoGen). Cell lines were validated for authentication using the short tandem repeat method (Beijing Microread Corporation Limited). THP-1, Molm13 and Nomo-1 were cultured at 4-6×10 5 cells/ml in RPMI-1640 GlutaMAX containing 10% FBS, 25 mM HEPES, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were transduced with lentiviruses expressing SUV39H1-eGFP, and 48h after infection, GFP + cells were sorted with flow cytometry and collected. Cells were then seeded in 96-well plates at a density of 5000 cells/well in 100 µl volume. Ten µl of Cell Counting Kit-8 (Dojindo) were added to each well and allowed to react with cells for 4h. The absorbance was read at 450 nm by Synergy H4 Microplate Reader (Biotek).

Colony forming capacity (CFC) assays for mouse cells
Leukemic cells (eGFP + for SUV39H1 overexpression) were sorted, and cultured in methylcellulose-based medium (3231, STEMCELL Technologies) supplied with 10 ng/mL mIL-6, 10 ng/mL mIL-3, 50 ng/mL mSCF and 10 ng/mL mGM-CSF (Peprotech) and 100U/mL Penicillin-Streptomycin. The cells were plated in 24-well plates in a 0.5 ml volume at 400 cells per well. CFCs were scored under an inverted microscope after 7-10 days' culture. In the replating CFC assays, 500 cells were plated in each well in the first-round CFC. For replating assay, colony cells were collected and replated in methylcellulose every 6 days with 500 cells as input for each passage (number of colonies at the 3 rd plating were calculated). and Hoxb13 (mature RNA: ATCATGACAACTAGTACTG) was synthesized by BGI tech company, annealed and then inserted into SF-LV-shRNA-BFP plasmid.

Apoptosis assay
The apoptosis assay was performed as previously described 1 . Briefly, leukemic mouse BM or spleen cells were labeled with surface antibodies. Cells were washed with 1 × PBS, resuspended in 1 × Binding Buffer (BD Bioscience, Carlsbad, CA, USA) and stained with Annexin V and 7-AAD (BD Bioscience, Carlsbad, CA, USA) according to the manufacture manual. Cells were analyzed using a CANTO II flow cytometer (BD Bioscience, USA).

Cell cycle analysis
For cell cycle analyses, leukemic mouse BM or spleen cells were labeled with surface antibodies, fixed and permeabilized with BD IntraSure Kit (BD Bioscience, San Jose, CA, USA). Cells were intracellularly stained with antibodies against Ki67 (BD Bioscience, Carlsbad, CA, USA) and Hoechst 33342 (Sigma-Aldrich), and measured using the CANTO II flow cytometer (BD Bioscience, USA). BrdU incorporation assay was used to analyze cell cycle. Cells were incubated with BrdU (1 mM) for 1-2 hours before detecting. Cells were fixed, permeabilized and stained with anti-BrdU antibody and 7-AAD by using BD Pharmingen™ BrdU Flow Kits (BD Bioscience, San Jose, CA, USA). Cells were measured by the CANTO II flow cytometer (BD Bioscience, USA).

Lentivirus preparations and bone marrow transplantation
All procedures were performed as described previously 1 . EGFP + , mCherry + or BFP + cells were sorted and transplanted into sub-lethally (4.5 Gy) irradiated mouse recipients.

Droplet digital PCR (ddPCR)
The QX200 droplet digital PCR System (Bio-Rad) was used to detect the target genes in a duplex reaction. Evagreen PCR reaction mixture was assembled from a 2×ddPCR

BrdU incorporation assay
Mice were intraperitoneal injected with 1 mg of BrdU 2h before they were sacrificed. BM cells were harvested and stained with fluorescent-conjugated antibodies. Labeled BM cells were fixed, permeabilized, and treated with DNase to expose the incorporated BrdU using a BrdU Flow Kit (BD Biosciences). Cells were then stained with an APC-labeled anti-BrdU and analyzed using a CANTO II flow cytometer.

RNA-seq and ChIP-seq analysis
RNA-seq libraries were generated using the Illumina TruSeq RNA sample preparation kit v2 following manufacturer's protocol. The libraries were sequenced on an Illumina Hiseq 2000 platform using a 150-bp paired-end sequencing strategy. Read alignment against mouse genome reference (mm10) was performed using TopHat2 2 .
The FPKM (fragments per kilobase of transcript per million mapped reads) value of transcripts were calculated by the Cufflink algorithm 3 representing the gene expression level. Differentially enriched genes were enriched with DESeq2. ChIPed DNA was subjected to library construction according to Illumina's TruSeq ® ChIP Sample Preparation according to the protocol. 50-cycle single-read sequencing was performed using an Illumina HiSeq 2500. (c) The densitometry of relative Suv39h1 and H3K9me3 levels in SUV OE groups (P1 and P2) was normalized to β-tubulin and H3, respectively. Densitometry was calculated with ImageJ, n =3 for each group.