Supplementalexperimental Methods

SupplementalExperimental Methods

In vivo mouse model

Purified Lin- cells from B6-Ly5.2 mice were infected with a retrovirus carrying MSCV-MLL-AF9-IRES-GFP as previously described. After 2-3 days of infection, approximately 1×106 unpurified cells were injected into lethally irradiated (9.5 Gy) B6-Ly5.1 recipients. At the end-stage of leukemia (~60 days), the mice were sacrificed, and primary leukemic cells (P0) were harvested from the bone marrow. Secondary transplantation of the leukemic cells does not require the irradiation of the recipients. We harvested a sufficient number of leukemic cells from the bone marrow of the 4th recipients (P3) to transplant 1×106 P3 leukemic cells into immunocompetent B6-Ly5.1 recipients for the in vivo experiments.

Sample preparation

Aliquots of normal human BM mononuclear cells (BMMNCs) were obtained from allogeneic transplant donors at the State Key Institute of Hematology. AML and ITP samples were obtained from untreated patients. Sample acquisition was approved by the Institutional Review Boards (IRB) at the State Key Institute of Hematology. MNCs were isolated from umbilical cord blood using Ficoll separation. c-Kit+ and CD34+ cells were isolated using a positive magnetic bead selection protocol (Miltenyi, Auburn, CA, USA). All donors signed informed consent forms.

Murine niche cells from bone were harvested from control and leukemic mice as previously reported1.

Small-molecule compounds

The small-molecule compounds that were used in this study were purchased as described below: BX471 (Sigma-Aldrich), A1B1 (AxonMedchem), Maraviroc (Selleck), J113863 (R&D)2-5.

Flow cytometry

A mixture of anti-CD3, anti-CD4, anti-CD8, anti-CD45R, anti-CD11b, anti-Gr-1, and anti-Ter-119 antibodies was used for the Lineage mix (Lin). The following antibodies were used for HSC and HPC analysis: Lin mixture, anti-c-Kit, anti-Sca1, anti-CD34, anti-CD16/32 (FcγR), anti-CD135 (Flt3/Flk2), anti-CD45.1 and anti-CD45.2 (BD Bioscience). The anti-Ter-119 and anti-CD71 antibodies were used for erythroblast analysis. Anti-CD41 was used for megakaryocyte analysis. DAPI was used to exclude dead cells. For cell cycle analysis, the cells were first labeled with surface markers, fixed and permeabilized with the Intrasure Kit (BD Biosciences), then washed and stained with anti-Ki67 (BD Bioscience) for 30 minutes at room temperature. Hoechst 33342 was added prior to flow cytometry analysis. For apoptosis analysis, the cells were stained with candidate surface markers and then labeled with the anti-Annexin-V antibody and 7-AAD in binding buffer according to the manufacturer’s instructions (BD Biosciences).

CD34+ cord blood CFC

Human CD34+ cells were collected from cord blood. For clonogenic assays, 500 CD34+ cells were plated in methylcellulosemediumH4435 (StemCell Technologies) with 1%penicillin/streptomycin(Gibco) per well. AML patients’ serum was added in H4435 at a concentration of 80 μl/ml. CFCs were scored after 14 days of incubation.

Leukemia CFC assay

EGFP+ leukemic cells were sorted and cultured in methylcellulose-based medium (3231, Stemcell Technologies), which included 10 ng/mL mIL-6, 10 ng/mL mIL-3, 50 ng/mL mSCF and 10 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF). The cells were plated in 24-well plates in a 0.5 ml volume at a density of 400 cells per well. The colonies were counted and collected after 7days of incubation. Colonies of types A, B, and C were characterized as described in reference The colonies were counted and collected after 7-14 days of incubation. Colonies of types A, B, and C were characterized as described in reference6.

In vitro liquid culture

For erythroid differentiation, HSPCs were cultured in SFEM (StemCell Technologies) medium containing 50 ng/ml rmSCF, 10 ng/ml rmTPO, and 5 U/ml rhEPO. For HSPC maintenance, HSPCs were cultured in SFEM containing 100 ng/ml rmSCF and 10 ng/ml TPO (PeproTech).

Single cell colony assays

For the single cell colony assays, LT-HSCs, CMPs, and MEPs were sorted individually into the central 60 wells of 96-well plates containing 200 µl of SFEM (StemCellTechnologies) supplemented with 2 mM L-glutamine, 50 µM β-mercaptoethanol, 10% FBS, 50 ng/ml rmSCF, 10 ng/ml rmTPO, 10 ng/ml rmL-3, 10 ng/ml rmGM-CSF and 1 U/ml hEPO (PeproTech). LT-HSC and CMPs/MEPs colonies were analyzed individually on day 14 and 8, respectively. Colonies were imaged using a ZEISS microscope at a 5× magnification.

Western blotting (WB) analysis

One million cells were lysed with 50 μl of 2× SDS sample buffer, sonicated for 10 s and then boiled at 95°C for 5 min. These extractions were resolved by SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore). The following primary antibodies were used: anti-P38 (Cell Signaling Technology, 1:2,000, #9212), anti-Phospho-p38 (Thr180/Tyr182) (Cell Signaling Technology, 1:1,000, #9211), anti-ERK (Millipore, 1:2,000, #ABS44), anti-phos ERK (Cell Signaling Technology, 1:1,000, #5013), anti-AKT (Cell Signaling Technology, 1:1,000, #4691), anti-AKT S473 (Cell Signaling Technology, 1:1,000, #9271), anti-GATA1(Abcam, 1:1,000, ab28839),anti-Tublin (Cell Signaling Technology, 1:5,000, #2144) and anti-β-actin (Cell Signaling Technology, 1:4,000, #4967). Horseradish peroxidase–conjugated antibodies to rabbit (Cell Signaling Technology, 1:4,000, #7074) and mouse (Cell Signaling Technology, 1:2,000, #7076) IgG were used as the secondary antibodies. The Luminescent Image Analyzer (ImageQuant LAS 4000) was used for detection.

Immunofluorescence staining

Cells were cytospun on slides, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X100l, and blocked with 5% BSA for 45 min at room temperature. Then, the cells were incubated with primary antibodies overnight at 4°C. The cells were washed in PBS and incubated with Alexa Fluor 488 goat anti-rabbit IgG or Alexa Fluor 561 goat anti-rabbit IgG (H+L) for 1 h at room temperature. Slides were mounted with cover glasses with the Prolong Gold Antifade Reagent (Invitrogen) and viewed with an Olympus confocal microscope.

qRT-PCR

Total RNA was isolated from candidate cells with the Qiagen RNeasy mini kit according to the manufacturer’s instructions. Quantitative RT–PCR was performed on a StepOne real-time PCR system (Applied Biosystems).Primers used for experiments were shown in Table S6.

References

1.Nakamura Y, Arai F, Iwasaki H, Hosokawa K, Kobayashi I, Gomei Y et al. Isolation and characterization of endosteal niche cell populations that regulate hematopoietic stem cells. Blood 2010; 116: 1422-1432.

2.Liang M, Mallari C, Rosser M, Ng HP, May K, Monahan S et al. Identification and characterization of a potent, selective, and orally active antagonist of the CC chemokine receptor-1. The Journal of biological chemistry 2000; 275: 19000-19008.

3.Revesz L, Bollbuck B, Buhl T, Eder J, Esser R, Feifel R et al. Novel CCR1 antagonists with oral activity in the mouse collagen induced arthritis. Bioorganic & medicinal chemistry letters 2005; 15: 5160-5164.

4.Marciniak E, Faivre E, Dutar P, Alves Pires C, Demeyer D, Caillierez R et al. The Chemokine MIP-1alpha/CCL3 impairs mouse hippocampal synaptic transmission, plasticity and memory. Scientific reports 2015; 5: 15862.

5.Amat M, Benjamim CF, Williams LM, Prats N, Terricabras E, Beleta J et al. Pharmacological blockade of CCR1 ameliorates murine arthritis and alters cytokine networks in vivo. British journal of pharmacology 2006; 149: 666-675.

6.Johnson JJ, Chen W, Hudson W, Yao Q, Taylor M, Rabbitts TH et al. Prenatal and postnatal myeloid cells demonstrate stepwise progression in the pathogenesis of MLL fusion gene leukemia. Blood 2003; 101: 3229-3235.

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