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Mutant CEBPA directly drives the expression of the targetable tumor-promoting factor CD73 in AML

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Science Advances  10 Jul 2019:
Vol. 5, no. 7, eaaw4304
DOI: 10.1126/sciadv.aaw4304
  • Fig. 1 Distinct enhancer binding of CEBPA in WT GMPs versus p30 L-GMPs.

    (A) Representative examples of CEBPA, H3K4me1, and H3K27Ac ChIP-seq tracks in WT GMPs and Lp30 L-GMPs, showing L-GMP p30-specific CEBPA binding. (B) Distribution of p42-specific, p30-specific, and common CEBPA-bound regions. (C) Normalized intensity tags per million (TPM) of CEBPA (left), H3K4me1 (center), and H3K27ac (right) at midpoint-centered enhancers [rows, ±5000 base pairs (bp)] across the three classes of regions in either WT GMPs or Lp30 L-GMPs. (D) Normalized intensity (TPM) for either H3K4me1 (purple) or H3K27ac (green) across the three classes of regions (midpoint-centered ±500 bp). “Random” represents all regions shuffled randomly across the genome. (E) Distribution of the three classes of regions into LSK-, preGM-, GMP-, and granulocyte-specific enhancers. (F) Subdivision of the three enhancer categories based on whether their nearest-neighbor genes are up- or down-regulated in Lp30 L-GMPs versus WT GMPs (log2 fold change > 0.58, P < 0.05 by edgeR, see Materials and Methods). Statistics are two-sided Fisher’s exact test between groups. ns, not significant.

  • Fig. 2 Motif enrichment analysis of p42, common, and p30 CEBPA-bound regions.

    (A) TF motif enrichment analysis, combining results from three TF motif databases. The set of common regions is used as reference, and the relative enrichment z scores for p42 and p30 regions (compared to common) are displayed as shades of orange (enriched) or blue (depleted). (B) Percentages of CEBPA-bound regions overlapping with regions bound by ERG, FLI1, and PU1 in the HPC-7 cell line. (C) Proteomics quantification of TFs with enriched motifs, comparing FACS isolated WT GMPs and Lp30 L-GMPs (Limma multifactorial analysis, Benjamini-Hochberg corrected P value. *P ≤ 0.05; **P ≤ 0.01). (D) RNA-seq quantification of TF gene expression with enriched motifs, comparing WT GMPs and Lp30 L-GMPs (RNA-seq, *FDR ≤ 0.05; ****FDR ≤ 0.0001). (E) RT-qPCR quantification of Cebpa expression normalized to Actg1 (WT GMPs, n = 3; Lp30 L-GMPs, n = 7, Student’s t test. ***P < 0.001). (F) CEBPA Western blot using WT GMPs and Lp30 L-GMPs. The CEBPA and Histone H3 bands were obtained from the same blot incubated with different Abs. (G) All CEBPA motifs found in p42, common, and p30 region sets tested for their match to the CEBPA consensus (Jasper DB. One-tailed Wilcoxon test. **P ≤ 0.001). (H) Frequencies of bins of CEBPA-consensus matching motifs in each region set.

  • Fig. 3 Gene expression analysis of murine and human CEBPA mutant AML.

    (A) RNA-seq assessment of total up- and down-regulated transcripts in L-GMPs sorted from human AML samples with biallelic CEBPA mutations versus WT GMPs (log2 fold change > 0.58, P < 0.05 by edgeR, see Materials and Methods). (B) Overrepresented GO categories (David online) of deregulated genes in L-GMPs versus normal GMPs. Included are representative, nonredundant categories found among the top 12 (up) or 64 (down) enriched in either species. “Negative regulation of cell proliferation” was included in AML-up for comparison to the down-regulated genes “cell cycle” category enrichment. “Transcription” was included for AML-down as it was enriched above the shared “DNA damage” in humans. Shared categories are colored; species specific are outlined in gray. See data file S4 for full list. (C) Stringently assessed deregulated mRNAs (FDR ≤ 0.01) in leukemic GMPs versus normal GMPs from human (teal) and murine (orange) BM. Lists of deregulated mRNAs were reduced to genes for which orthologs could be found in both species. Observed overlap (orthologs with shared directionality) (top) and expected random overlap (bottom) between the two species are indicated. (D) Quantified differential expression of the 102 overlapping genes shown in (C). Left: log2 fold change for each species. Right: Relative expression levels normalized for each species individually. (E) CD73 and ITGAX protein levels assessed by mass spectrometry (MS) (Limma multifactorial analysis, Benjamini-Hochberg corrected P value. *P ≤ 0.05).

  • Fig. 4 CEBPA-p30 mediated activation of Nt5e expression.

    (A) RT-qPCR quantification of Nt5e expression normalized to Actg1 (WT GMPs, n = 3; Lp30 L-GMPs, n = 4; mean ± SD, t test). (B) CD73 expression on the surface of WT GMPs and Lp30 L-GMPs as determined by flow cytometry (left) and the associated quantification CD73 expression GMPs (right; WT GMPs, n = 2; Lp30 L-GMPs, n = 3; mean ± SD, t test). (C) Top: ChIP-seq analysis on WT GMPs and Lp30 L-GMPs, showing a putative p30-specific enhancer located 40 kb upstream of the murine Nt5e gene. Middle: Assessment of direct interaction between the −40-kb enhancer and the Nt5e TSS by 3C-qPCR. Primer localization is depicted by open circles. qPCR (n = 3 to 5) was normalized to cKit–enriched BM cells (multiple t test, Holm-Sidak). Bottom: CRISPRi-mediated knockdown of Nt5e in Lp30 L-GMPs using gRNAs targeting the positions indicated by open circles. Nt5e expression was quantified by RT-qPCR, and expression levels were normalized to Actg1 and to cells transduced with gRNA targeting position −112 kb [n = 3, mean ± SD, one-way analysis of variance (ANOVA) with Dunnett’s correction, testing expression reductions between targeting enhancer or TSS positions versus the −112-kb control position]. (D) Dual luciferase assay assessing p42- or p30-mediated trans-activation of the −40-kb enhancer cloned upstream of a minimal promoter compared to an empty vector control (n = 3, mean ± SEM, t test). For all panels, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

  • Fig. 5 Up-regulation of NT5E in human AML samples with biallelic CEBPA mutations.

    (A) RT-qPCR quantification of CEBPA mRNA in sorted GMPs from healthy individuals as well as patients with AML with monoallelic and biallelic CEBPA mutations. Normalized to POL2 and H6PD (n = 5, 4, and 4, mean ± SEM). (B) RT-qPCR quantification of NT5E expression levels in sorted GMPs from healthy individuals and patients with AML with the indicated mutational status. Normalized to POL2 and H6PD (n = 8, 3, 4, 6, 6, 9, 3, 3, 4, 4, 1, 6, 22, 7, 8, and 5, mean ± SEM). Statistical significance was assessed using one-way ANOVA with Dunnett’s correction for (A) and (B), where the mean of each group was compared to the WT-GMP group. (C) Normalized expression of CEBPA and NT5E in datasets extracted from the cBioPortal (TCGA data, RNA-seq) and Leukemia Gene Atlas (expression array) databases (mean ± SD). For Kohlmann and Verhaak datasets, the monoallelic and biallelic CEBPA-mutated subtypes are binned as one group, and for TCGA data, the biallelic subtype is separate. (D) Top: ChIP-seq analysis on GMPs from healthy individuals and L-GMPs from patients with AML with monoallelic and biallelic CEBPA mutations showing a putative enhancer located 48 kb upstream of the human NT5E gene. Center: ChIP-seq tracks from CD34+ hematopoietic progenitor cells. Bottom: Publicly available Hi-C data in black and red bars (30) and ChIP-seq tracks (31) from the same region in CD8 T cells. (E) Dual luciferase assay assessing p42- or p30-mediated trans-activation of the −48-kb enhancer cloned upstream of a minimal promoter compared to an empty vector control (n = 3, mean ± SEM, Student’s t test). For all panels, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

  • Fig. 6 Effect of Nt5e KD on in vivo AML progression and identification of CD73 tumor-protective adenosinergic signaling as a potential therapeutic target.

    (A) Nt5e knockdown efficiency; RT-qPCR quantification of Nt5e normalized to Actg1. GFP+ sorted Lp30 cells transduced with shNt5e or scrambled control (n = 3, mean ± SD). (B) Schematic outline of competitive BM transplant experiment. (C) Representative FACSs of Lp30 cells transduced with shRNA targeting Nt5e or scrambled control at the input and output time points. (D) Target-to-competitor (GFP/YFP) ratio of Lp30 cells transduced with shRNA targeting Nt5e or scrambled control after BMT and 4 weeks of leukemia progression. Ratio was normalized to input cells. (n = 4, mean ± SD). (E) Survival of recipient mice transplanted with 2.5 × 104 GFP+-sorted Lp30 cells transduced with shNt5e or scrambled control (n = 8). (F) Survival of recipient mice transplanted with 5 × 103 cells double positive for KRAB-dCas9-mCherry and sgRNA-GFP targeting Nt5e TSS, a putative enhancer (−40 kb), or a negative upstream region (−100 kb) (n = 7 and 8). (G) RT-qPCR quantification of Adora1, Adora2a, Adora2b, and Adora3 in Lp30 L-GMP and WT-GMP cells, displayed as ratios to levels in whole BM cells. Normalized for each cell type to Actg1 (wBM, n = 3; GMPs, n = 5 to 6). (H) Signatures enriched in both Nt5e knockdown groups in vivo compared to scrambled control based on RNA-seq (Scr, n = 3; shNt5e #1, n = 3; shNt5e #2, n = 2). Color indicates log2 fold change (red) and P value (purple) for each signature. See data file S6 for full list. (I) Plot and statistical values from GSEA testing genes of the negative A2A signature in biallelic CEBPA-mutated AML versus all other AML subtypes; TCGA AML expression dataset. (J) Survival of recipient mice transplanted with 100 freshly harvested Lp30 cells. Mice were injected intraperitoneally with mock, A2ARi (SCH-58261, 20 μg), anti-CD73 (2C5-IgG2a, 100 μg), and control-Ig (NIP228-IgG2a, 100 μg) in combinations as indicated. Treatments were twice weekly from day 1 until the first mouse was euthanized, as specified on the graph. (K) Model of Nt5e differential leukemia-specific expression as a consequence of elevated CEBPA-p30, leading to engagement of the Nt5e AML enhancer. Blue ovals represent CEBPA-p42, and red ovals represent CEBPA-p30 monomers. Bottom: Half blue ovals represent CEBPA-p42 mutated in the DNA-binding domain, often present in human biallelic CEBPA-mutated AML. Statistics were determined by Student’s two-tailed t test (two groups) or one-way ANOVA corrected for multiple comparisons (Dunnett’s correction) between control and treatment groups (three or more groups) or log-rank test (survival). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaaw4304/DC1

    Supplementary Materials and Methods

    Fig. S1. Distinct enhancer-binding of CEBPA in WT GMPs versus p30 L-GMPs, related to Fig. 1.

    Fig. S2. Motif enrichment analysis of p42, common and p30 CEBPA-bound regions, related to Fig. 2.

    Fig. S3. Gene expression analysis of murine and human CEBPA mutant AML, related to Fig. 3.

    Fig. S4. CEBPA-p30 directly activates the expression of Nt5e, related to Fig. 4.

    Fig. S5. CD73-positive GMPs in human AML and CBF-MYH binding of the NT5E promoter in INV(16) AML, related to Fig. 5.

    Fig. S6. A2AR-dependent adenosinergic autocrine survival signaling promoted by CD73, related to Fig. 6.

    Data File S1. CEBPA-peak coordinates, enriched motifs in CEBPA regions, related to Fig. 1.

    Data File S2. RNA-seq data, related to Figs. 2 and 3.

    Data File S3. MS data, related to Figs. 2 and 3.

    Data File S4. Gene signatures, related to Fig. 3.

    Data File S5. RNA-seq data, related to Fig. 6.

    Data File S6. Gene signatures, related to Fig. 6.

    Data File S7. Materials lists, related to Materials and Methods.

    References (5273).

  • Supplementary Materials

    The PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. Distinct enhancer-binding of CEBPA in WT GMPs versus p30 L-GMPs, related to Fig. 1.
    • Fig. S2. Motif enrichment analysis of p42, common and p30 CEBPA-bound regions, related to Fig. 2.
    • Fig. S3. Gene expression analysis of murine and human CEBPA mutant AML, related to Fig. 3.
    • Fig. S4. CEBPA-p30 directly activates the expression of Nt5e, related to Fig. 4.
    • Fig. S5. CD73-positive GMPs in human AML and CBF-MYH binding of the NT5E promoter in INV(16) AML, related to Fig. 5.
    • Fig. S6. A2AR-dependent adenosinergic autocrine survival signaling promoted by CD73, related to Fig. 6.
    • Legends for data files S1 to S7.
    • References (5273).

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). CEBPA-peak coordinates, enriched motifs in CEBPA regions, related to Fig. 1.
    • Data file S2 (Microsoft Excel format). RNA-seq data, related to Figs. 2 and 3.
    • Data file S3 (Microsoft Excel format). MS data, related to Figs. 2 and 3.
    • Data file S4 (Microsoft Excel format). Gene signatures, related to Fig. 3.
    • Data file S5 (Microsoft Excel format). RNA-seq data, related to Fig. 6.
    • Data file S6 (Microsoft Excel format). Gene signatures, related to Fig. 6.
    • Data file S7 (Microsoft Excel format). Materials lists, related to Materials and Methods.

    Files in this Data Supplement:

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