Figure 5.
Biosynthetic failure in AML impairs cell cycle progression and drives differentiation. (A) Representative Cytospin and Hema3 stains of NrasG12D; MLL-AF9 clones transduced with the indicated shRNAs after 4 days of doxycycline treatment (n = 9 per condition). Scale bar, 10 μm. (B-C) Gene set enrichment analysis (GSEA) after OGDH depletion. NrasG12D; MLL-AF9 AML clones (B) or p53R172H AML cells (C) transduced with retroviruses encoding doxycycline-inducible shControl or shOgdh (2 independent shRNAs sequenced separately and pooled during analysis) were subjected to RNA sequencing after 4 days of treatment with doxycycline. GSEA identified pathways of interest that were upregulated or downregulated. (D) Transcript levels of Gata2 (left) and Gypa (right) in p53R172H AML cells treated with doxycycline for 4 days and sorted for BFP. BFP+ indicates shRNA on; BFP– indicates shRNA off. P values for shOgdh off (BFP–) vs on (BFP+) indicated (unpaired t test; n = 4 per condition) (E-F) Cell cycle analysis by 5-ethynyl-2′-deoxyuridine (EdU) tracing. NrasG12D; MLL-AF9 (E) or p53R172H (F) AML cells treated with doxycycline for 4 days and sorted for GFP or BFP, respectively, were labeled with EdU for 1 hour. They were then fixed, permeabilized, stained with propidium iodide (PI), and subjected to cell cycle analysis by flow cytometry. Left lower gate represents G1 phase; upper gate, S phase; right lower gate, G2 phase/mitosis (M). Percentages of cells falling within each gate are indicated. (G) Extracellular aspartate levels in media were measured at 48 hours after knockdown of p53R172H AML cells with the indicated shRNAs. (H) Baseline transcript levels of Slc1a3 and Slc1a5 in p53R172H AML cells, MLL-fusion AML cells, and normal murine cKit-positive progenitor cells. (I) Relative number (% of control) of BFP+ cells after 4 days of induction of the indicated shRNA in p53R172H AML cells transduced with an empty vector or an exogenous Asp transporter (+SLC1A3). Media containing fetal bovine serum (FBS) was either unmodified (RPMI) or supplemented with 5 mM L-aspartate (L-Asp) or 10 mM L-glutamine (L-Gln) as indicated. P values are indicated (1-way ANOVA with either Dunn or Sidak multiple comparisons test as appropriate). (J) Median fluorescent intensity (MFI) of Cd11b after 4 days of induction of the indicated shRNAs in p53R172H AML cells transduced with an empty vector or vector for expression of an exogenous aspartate transporter (+SLC1A3). Media containing FBS was supplemented with 5 mM L-Asp. P values are indicated (1-way ANOVA with Sidak multiple comparisons test). FDR, false discovery rate; LSC, leukemia stem cell; NES, normalized enrichment score; ns, not significant; TPM, transcripts per million.

Biosynthetic failure in AML impairs cell cycle progression and drives differentiation. (A) Representative Cytospin and Hema3 stains of NrasG12D; MLL-AF9 clones transduced with the indicated shRNAs after 4 days of doxycycline treatment (n = 9 per condition). Scale bar, 10 μm. (B-C) Gene set enrichment analysis (GSEA) after OGDH depletion. NrasG12D; MLL-AF9 AML clones (B) or p53R172H AML cells (C) transduced with retroviruses encoding doxycycline-inducible shControl or shOgdh (2 independent shRNAs sequenced separately and pooled during analysis) were subjected to RNA sequencing after 4 days of treatment with doxycycline. GSEA identified pathways of interest that were upregulated or downregulated. (D) Transcript levels of Gata2 (left) and Gypa (right) in p53R172H AML cells treated with doxycycline for 4 days and sorted for BFP. BFP+ indicates shRNA on; BFP– indicates shRNA off. P values for shOgdh off (BFP–) vs on (BFP+) indicated (unpaired t test; n = 4 per condition) (E-F) Cell cycle analysis by 5-ethynyl-2′-deoxyuridine (EdU) tracing. NrasG12D; MLL-AF9 (E) or p53R172H (F) AML cells treated with doxycycline for 4 days and sorted for GFP or BFP, respectively, were labeled with EdU for 1 hour. They were then fixed, permeabilized, stained with propidium iodide (PI), and subjected to cell cycle analysis by flow cytometry. Left lower gate represents G1 phase; upper gate, S phase; right lower gate, G2 phase/mitosis (M). Percentages of cells falling within each gate are indicated. (G) Extracellular aspartate levels in media were measured at 48 hours after knockdown of p53R172H AML cells with the indicated shRNAs. (H) Baseline transcript levels of Slc1a3 and Slc1a5 in p53R172H AML cells, MLL-fusion AML cells, and normal murine cKit-positive progenitor cells. (I) Relative number (% of control) of BFP+ cells after 4 days of induction of the indicated shRNA in p53R172H AML cells transduced with an empty vector or an exogenous Asp transporter (+SLC1A3). Media containing fetal bovine serum (FBS) was either unmodified (RPMI) or supplemented with 5 mM L-aspartate (L-Asp) or 10 mM L-glutamine (L-Gln) as indicated. P values are indicated (1-way ANOVA with either Dunn or Sidak multiple comparisons test as appropriate). (J) Median fluorescent intensity (MFI) of Cd11b after 4 days of induction of the indicated shRNAs in p53R172H AML cells transduced with an empty vector or vector for expression of an exogenous aspartate transporter (+SLC1A3). Media containing FBS was supplemented with 5 mM L-Asp. P values are indicated (1-way ANOVA with Sidak multiple comparisons test). FDR, false discovery rate; LSC, leukemia stem cell; NES, normalized enrichment score; ns, not significant; TPM, transcripts per million.

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