PRMT1 Promotes Leukemia Cell Transformation.

A. 6133 cells were stained with E84, then FACS was sorted based on E84 intensity. 3×105 sorted cells were intravenously transferred to sublethally irradiated mice.

B. Leukemia progression in recipient mice was shown on Kaplan-Meier curves.

C. Leukemia cell manifestation in the bone marrow and peripheral blood of recipient mice was measured by flow cytometry. For the E84-low group, the bottom five dots represent five recipient mice sacrificed on day 90 post transfer. Closed symbols represent moribund mice, and open symbols represent non-terminally ill, inhibitor-treated mice sacrificed on day 88.

D. PRMT1 expression renders 6133 cells’ cytokine-independent growth. 6133 cells and 6133/PRMT1 cells were cultured with or without mouse stem cell factor (SCF). Cell viabilities were measured daily.

E. Schematic for inducing leukemia by injecting 6133 or 6133/PRMT1 cells intravenously into sub-lethally irradiated recipient mice (n=7).

F. Leukemia progression in recipient mice was documented on Kaplan-Meier curves.

PRMT1 Inhibitor MS023 Blocks Leukemia Progression.

A. Survival of 6133/PRMT1 cell is highly sensitive to treatment of MS023. 6133 and 6133/PRMT1 cells were treated with MS023 for 48 hours and cell viability was determined by counting.

B. 6133/PRMT1 cells were intravenously transferred into sub-lethally irradiated Mice. Recipient mice were intraperitoneally injected with PRMT1 inhibitor or vehicle for 15 doses every other day.

C. Leukemia progression shown on Kaplan-Meier curves. n=5.

D. Leukemia cells in recipient mice (n=6) were measured by flow cytometry. The right panel indicates the weights of recipient mice spleens. Closed symbols represent moribund mice, and open symbols represent non-terminally ill, inhibitor-treated mice sacrificed on day 40 and 120.

E. Peripheral blood was collected from non-terminally ill, inhibitor-treated mice at 40 and 120 days post cell transfer.

F. MS203 treatment of in vitro cultured 6133 and 6133/cMPLW515L cells. n=3.

G. Schematic of leukemia induced by 6133 c-mplW515L transplantation.

H. Kaplan-Meier curves for MS023 treated leukemia mice induced by 6133 c-mplW515L cells. n=7.

PRMT1 Promotes Glycolysis.

A. Extracellular acidification rates (ECAR) in 6133 and 6133/PRMT1 cells were measured by Seahorse assays. 150,000 cells were seeded in special 24-well plates for the Seahorse Xf-24 analyzer. Oligomycin, FCCP, and antimycin were sequentially injected into wells as indicated.

B. Extracellular acidification rates in PRMT1 inhibitor treated 6133 cells. Cells were pretreated with PRMT1 inhibitor MS023 overnight prior to Seahorse analysis.

C. Protein levels of lactate dehydrogenase A (LDHA) and p-Y10-LDHA in 6133 and 6133-PRMT1 cells by Western blotting. Cells were seeded in the same density and extracts were harvested after overnight culture.

D. Intracellular and extracellular lactate levels were measured by an L-lactate kit. Cells were seeded at 1×107 cells/ml and cultured for 24 hours, followed by centrifugation. Both medium/supernatant (extracellular) and cell pellet (intracellular) were collected for analysis. The results of triplicates are plotted. * p < 0.05

PRMT1 Changes Oxygen Consumption and Redox Status.

A. Oxygen consumption rates (OCR) in 6133 and 6133/PRMT1 cells were measured by Seahorse assays. 150,000 cells were seeded in special 24-well plates for Seahorse Xf-24 analyzer. Oligomycin, FCCP and antimycin were sequentially injected into wells as indicated.

B. Oxygen consumption rates in PRMT1 inhibitor-treated 6133 cells. Cells were pretreated with PRMT1 inhibitor MS023 overnight prior to Seahorse analysis.

C. Mitotracker Deep Red FM stain was used to assess the mitochondria mass in 6133 and 6133-PRMT1 cells.

D. Mitochondrial DNA amount was measured by quantitative PCR of mito-specific gene cytochrome B.

E. TMRE staining was performed to measure mitochondrial membrane potential. Cells were seeded at 1.1×105 cells/ml, and 0.1 volume of 5mM TMRE was added to the culture to reach a final concentration of 500nM. After 20 minutes, cells were collected and used for FACS analysis.

F. Intracellular ROS level was measured by H2-DCFDA staining. 1×105 cells were incubated in a warm staining solution containing 10 uM of H2-DCFDA for 30 minutes and then washed and subjected to analysis.

G. Mitochondrial ROS was measured by MitoSOX staining. 5×105 cells were incubated with a warm staining solution containing 2.5 uM of MitoSOX for 10 minutes and then washed and subjected to analysis.

H. Intracellular levels of GSH/GSSG and NADP/NADPH ratio were measured. In each assay, 5×105 cells were used for extract preparation.

Metabolomic analysis of PRMT1-regulated metabolism in 6133 cells. 6133 cells expressing doxycycline-inducible PRMT1 were induced to express PRMT1. The activated group (labelled as A): metabolites collected after PRMT1 was induced. Control group (labelled as C): metabolites collected before PRMT1 was induced.

A: principle component analysis (PCA) for the metabolites. The C group and A group were clustered at different positions in PCA analysis.

B: heatmap of metabolites differentially expressed in these two groups.

C: volcano plots for metabolites. 204 metabolites are changed more than 2 folds with P <0.5.

D: Metabolic pathways are mostly affected by PRMT1 overexpression in 6133 cells. The metabolite data were analyzed by a web-based program called MetaboAnalystR6.0.

PRMT1 Causes Leukemia Cells’ Heavy Dependency on Glucose consumption.

A. Glucose colorimetric assay of 6133 and 6133/PRMT1 cells. 1.25× 105 cells were seeded per well and cultured overnight. After centrifugation, supernatant/medium was collected and used for the assay.

B. Cell viability of 6133 and 6133/PRMT1 cells under glucose-free conditions. Cells were seeded in a 96-well plate with fresh regular or glucose-minus RPMI1640 medium. Cell viability was measured by CellTiter-Glo kit. Ratios were normalized to the wells with regular RPMI-1640 medium.

C. Cell viability of 6133 and 6133/PRMT1 cells with 2-DG treatment. Cells were seeded in 96-well plates supplemented with 2-DG. Cell viability was measured by CellTiter-Glo kit. The growth of cells without 2-DG addition was used for normalization.

D. 6133/PRMT1 cells were intravenously transferred into sub-lethally irradiated mice. Starting on day 4 post-transfer, recipient mice were intraperitoneally injected with saline or 0.25g/Kg body weight of 2-DG every other day. The survival of recipient mice is shown as Kaplan-Meier curves.

E. Percentages of leukemia cells in bone marrow and peripheral blood of recipient mice were measured by flow cytometry. Closed symbols represent mice transplanted with 6133/PRMT1 cell-induced leukemia. The open square is a control wild type mouse without leukemia.

PRMT1 Alters Fatty Acid Oxidation in Leukemia Cells.

A. mRNA level of CPT1a and PPARα in 6133 and 6133-PRMT1 cells. Cell pellets were harvested in Trizol before RNA extraction, followed by cDNA synthesis and qPCR analysis.

B. Western blotting of 6133 and 6133-PRMT1 cell lines.

C. BODIPY Staining of 6133 cells. Cells were incubated with 200nM of BODIPY at 37oC for 15 minutes before being washed with medium and sent for FACS analysis.

D. Cell viability of 6133 and 6133-PRMT1 cells with Etomoxir treatment. 6133 and 6133-PRMT1 cells were seeded in a 96-well plate, supplemented with Etomoxir. Cell viability was measured by CellTiter-Glo Kit. Ratios were normalized to day 0.

E. Cell viability of 6133 and 6133-PRMT1 cells with Orlistat treatment.

F. Viability of 6133 and 6133-PRMT1 cells with a supplement of short-chain fatty acid (triacetin/ tripropoinin/tributyrin) under glucose-free conditions. Cells were cultured with RPMI with or without glucose, supplemented with 100nM of triacetin/tripropionin/tributyrin, respectively. Cell viability was measured after 72 hours. Fold change of viability is normalized to glucose+ cells.

G. Growth curves of 6133-PRMT1 cells transduced with CPT1A, CPT1A/H473A and CPT1A/G710E.

Expression of metabolic regulated genes in cells with differential PRMT1 Expression levels.

A. Two populations of 6133 cells were FACS-sorted based on E84 staining intensity, as in Figure 1. Sorted cells were harvested and subjected to western blotting.

B. RNA was extracted from sorted cells and used for quantitative real-time PCR.

RBM15-MKL1 (OTT-MAL) Protein Stability is not Affected by PRMT1 Activity.

A. HEK293T cells were transfected with constructs expressing HA-tagged RBM15-MKL1 with or without PRMT1. Extracts were harvested at 24 hours post transfection and then used for western blotting. Endogenous RBM15 is indicated.

B. NB4 cells were treated with PRMT1 inhibitor MS023 for 24 hours. Extracts were harvested for western blotting.

C. Global arginine di-methylation (Di-me-R) of bone marrow cells from mice injected with PRMT1 inhibitor MS023. Mice were injected with 80 mg/kg body weight of MS023 or vehicle every other day, for 30 days. Bone marrow cells were collected and extracts were prepared for western blotting.

Responsiveness to Glucose Restriction of Cells with Differential PRMT1 Expression levels. FACS-sorted E84-high and E84-low 6133 cells were washed with fresh medium and then seeded in glucose-free RPMI1640 medium for continuing culture. The viability of E84-high/low cells was measured by CellTiter-Glo assay.