Research ArticleLEUKEMIA

MAFB enhances oncogenic Notch signaling in T cell acute lymphoblastic leukemia

See allHide authors and affiliations

Science Signaling  14 Nov 2017:
Vol. 10, Issue 505, eaam6846
DOI: 10.1126/scisignal.aam6846
  • Fig. 1 Screen to identify novel enhancers of Notch signaling.

    (A) Schematic of the methodology used in the cDNA gain-of-function screen. (B) List of candidate genes potentiating NOTCH1 LPΔP activity. MAML1control is the MAML1 positive control. Black dots indicate candidates that were independently verified in reporter assays. (C) Luciferase induction using the Notch-responsive TP1 reporter and LPΔP to validate candidates identified in the screen. Data were quantified relative to the empty vector (EV) control. (D) Luciferase assay using the TP1 reporter (40 ng per well) and LPΔP (40 ng per well) and assaying dose dependency of Notch activation by ETS2 (40, 80, and 120 ng per well) or MAFB (40 and 80 ng per well). (E) Luciferase TP1 reporter assay with LPΔP when MAFB and ETS2 were singly or cotransfected (at 40 ng each). To normalize the amount of plasmid per well, we added EV so that the total amount of DNA was 80 ng per well (not including reporters). Data are from three independent experiments. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.0001 by unpaired t tests. NS, not significant.

  • Fig. 2 MAFB enhances T-ALL onset of a weak Notch gain-of-function allele.

    (A) Schematic representation of experimental design for retroviral transduction of 5-fluorouracil (5-FU)–treated BM progenitors. When only a single cDNA was expressed (MAFB-only, ICN-only, or LPΔP-only), an equivalent dose of the retroviral vector (either MigR1 or MigR1-NGFR) was used to normalize the total retroviral titers. (B) Peripheral blood analysis of the four cohorts of transplanted mice (ICN, n = 8; MAFB n = 9; LPΔP, n = 14; and LPΔP+MAFB, n = 15) 4 weeks after BMT. Representative populations shown were gated for live, singlet, and Lin cells. NGFR is the surrogate marker for MAFB transduction, and GFP is the surrogate marker for Notch-mutant transduction. (C) Kaplan-Meier plot indicating percent T-ALL–free mice (that is, insert space between free and mice) after BMT. Additional animals that succumbed to non–T-ALL conditions, such as irradiation poisoning (MAFB-only, n = 1; LPΔP-only, n = 1) and BM failure or anemia (LPΔP-only, n = 1), were excluded from the analysis. (D) WBC counts in the peripheral blood of the mice in each of the experimental cohorts. A diagnostic threshold for tumor onset of 40 million/ml of WBCs and circulating double-positive (DP) T cells in the peripheral blood was used as a benchmark for T-ALL, as previously described (8, 25). ***P ≤ 0.0005 by Mantel-Cox test (C) or unpaired t test (D).

  • Fig. 3 MafB-deficient T-ALL cells down-regulate Notch targets and perform poorly in competitive culture conditions.

    (A) MafB expression measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in T6E cells 48 hours after treatment with indicated siRNAs relative to that in cells cultured with transfection reagent (untreated). *P ≤ 0.05, **P ≤ 0.005 by unpaired t test. (B) Relative expression analysis by qRT-PCR of Notch signaling and Myc signaling (CAD) targets and expression controls (GAPDH) in MafB-siRNA#1–treated cells relative to each in Scrmb-siRNA–treated cells. (C) ChIP assay measuring the occupancy of P300 at the Hes1 promoter and at the NDME in MafB-shRNA#3–treated T6E cells. Genomic DNA sequence of GAPDH is used as the nonspecific control. ***P < 0.0005 by unpaired t test. (D) Pie chart representation of Notch-dependent gene expression stratified on the basis of differential H3K27Ac abundance measured by ChIP-seq analysis of T6E cells transfected with shScrmb or shMafB#3. Of the 59 Notch-dependent transcripts, as determined by a GSI-washout experiment, 37 have nearby MafB-dependent H3K27Ac regions, which display a fold change of ≤−1.5 (dark gray portion of the pie chart). (E) Genome tracts of individual examples of MafB-dependent H3K27Ac for Notch target genes (Dtx1, top; Myc, bottom) in T6E cells transfected with shScrmb or shMafB#3. kb, kilobase. (F) Schematic representation of the experimental design for MafB shRNA–treated T-ALL cells. The cells were sorted for their respective markers and subsequently cultured in equal numbers directly afterward. Every 3 days, the percentage of the cellular population bearing each marker was determined in an aliquot of the competition culture. FACS, fluorescence-activated cell sorting. (G) Left: Competition assay of T6E cells treated with GFP+ or NGFR+ shScrmb plated in equal numbers (2.5 × 105) at day 0 (no significant difference; n = 3). Middle and right: Competition assay of T6E cells treated with GFP+ shScrmb or one of two NGFR+ MafB shRNAs plated in equal numbers (2.5 × 105) at day 0 (n = 3). P = 0.02 and 0.03, respectively.

  • Fig. 4 Ectopic MAFB expression in T-ALL cells enhances Notch target gene expression and limits the effect of GSI treatment.

    (A) Western blotting for MafB (with anti-MafB antibody; Abcam) and loading control (GAPDH) in whole-cell lysates (10 μg) from T6E cells expressing Flag-MAFB or HA-MAFB. MigR1, empty vector. (B and C) Hes1 (B) and Myc (C) expression measured by qRT-PCR in T6E cells overexpressing MAFB. EF1a, elongation factor 1α. (D and E) Hes1 (D) and Myc (E) expression measured by qRT-PCR in T6E cells treated with dimethyl sulfoxide (DMSO) or GSI (0.1 μM) for 24 hours and subsequently washed out for 6 hours. Black bars, control cells; gray bars, MAFB-overexpressing cells. (A to E) n = 3 to 6 experiments. (F) Proliferation of MigR1-transfected control or MAFB-overexpressing T6E cells (2.5 × 105) plated in a medium containing DMSO or GSI (10 nM). n = 2 experiments with three technical replicates per experiment. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.00001 by unpaired t test.

  • Fig. 5 MAFB interacts with ETS proteins and recruits cofactors to Notch/RBPj binding sites.

    (A) Schematic representation of the experimental design for the OIP assay in U2OS cells. A biotin-tagged oligo from the TP1 Notch reporter was used as the bait to immunoprecipitate proteins interacting at the CSL or EBS regions. (B) Representative results of OIP assay in U2OS cells transfected with MigR1, ETS2, Flag-MAFB, or ETS2 + Flag-MAFB and probed with ETS2 and Flag antibodies after OIP. Ten percent loading of nuclear lysates indicates protein abundance; GAPDH is the loading control. (C) Schematic representation of the tagged MafB and MafB ΔbZIP mutant. Right: Western blotting (WB) on mixed lysates of HA-MAFB and HA–MAFB ΔbZIP immunoprecipitated from doubly transduced T6E cells to show relative protein size and HA-pulldown efficiency. (D) Representative results of HA- or FLAG-IP assessing MAFB interactions with ETS1, ETS2, P300, and PCAF in T6E T-ALL cells. (E) Representative HA-IP in T6E cells transduced with MigR1, HA-MAFB, or HA–MAFB ΔbZIP and probed for HA, ETS2, PCAF, and P300. The background bands in the HA 10% loading control likely result from the blot being probed and restriped multiple times. (F) Representative HA-IP in T6E cells transduced with HA-MafB and probed for cleaved-Notch1 (Val1744). (G) Representative FLAG-IP in T6E cells transduced with MigR1, FLAG–MAFB ΔbZIP, or FLAG-MAFB and probed for RBPJ and FLAG. (H) Notch TP1 reporter assay in T6E cells transfected with LPΔP and the indicated plasmids. EV is the vector control. **P ≤ 0.005 by unpaired t test; n = 3. (I) Kaplan-Meier plot comparing percent of T-ALL–free survival after BMT with progenitors transduced with LPΔP-only, LPΔP+MAFB, or LPΔP + MAFB ΔbZIP domain deletion mutant. LPΔP, n = 6; LPΔP+MAFB, n = 12; LPΔP+MAFB ΔbZIP, n = 10. Animals that succumbed to non–T-ALL conditions, such as BM failure/anemia (LPΔP-only, n = 1), were excluded from the analysis. ***P ≤ 0.0001 by Mantel-Cox test.

  • Fig. 6 Loss of MAFB suppresses Notch target gene expression and cell proliferation in human T-ALL.

    (A) MAFB mRNA expression in human T-ALL cell lines (U2OS cells used as negative control). (B) MAFB mRNA expression after MAFB knockdown in KOPT-K1 cells by each of the two shRNAs. (C) Notch target gene expression in sorted KOPT-K1 cells 72 hours after transduction with shRNA against MAFB relative to KOPT-K1 cells treated with Scrmb-shRNA. (D) Proliferation of sorted KOPT-K1 cells after transduction with shRNAs against MAFB. Growth is compared to cells treated with Scrmb-shRNA (n = 3). (E) Representative Western blotting for ETS1 and ETS2 in human T-ALL cell lines. GAPDH is the loading control. (F) Representative FLAG-IP in KOPT-K1 cells transduced with MigR1 or FLAG-MAFB and probed for FLAG, ETS2, and RBPJ. Ten percent loading of lysates indicates RBPJ and ETS2 abundance. GAPDH is the loading control. **P ≤ 0.005, ***P ≤ 0.001.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/505/eaam6846/DC1

    Fig. S1. Validation of Notch1 gain-of-function screen and MAFB expression in murine and human T-ALL.

    Fig. S2. Analysis of spleen and peripheral blood after BMT.

    Fig. S3. Validation of MafB siRNA and shRNA reagents.

    Fig. S4. Ectopic MAFB expression enhances T6E cell growth.

    Fig. S5. MAFB interacts with DNA through ETS factors, and the loss of ETS1 delays onset of T-ALL.

    Fig. S6. Zmiz1 expression in T6E cells is not affected by the loss of MafB.

    Table S1. Notch signaling enhancement of Notch-mutant LPΔP by candidate genes from the cDNA library.

    Table S2. MafB suppression affects a subset of Notch signaling gene targets in T6E cells.

    Table S3. List of oligo sequences and primer sets for qPCR and OIP.

  • Supplementary Materials for:

    MAFB enhances oncogenic Notch signaling in T cell acute lymphoblastic leukemia

    Kostandin V. Pajcini,* Lanwei Xu, Lijian Shao, Jelena Petrovic, Karol Palasiewicz, Yumi Ohtani, Will Bailis, Curtis Lee, Gerald B. Wertheim, Rajeswaran Mani, Natarajan Musuthamy, Yunlei Li, Jules P. P. Meijerink, Stephen C. Blacklow, Robert B. Faryabi, Sara Cherry, Warren S. Pear*

    *Corresponding author. Email: kvp{at}uic.edu (K.V.P.); wpear{at}mail.med.upenn.edu (W.S.P.)

    This PDF file includes:

    • Fig. S1. Validation of Notch1 gain-of-function screen and MAFB expression in murine and human T-ALL.
    • Fig. S2. Analysis of spleen and peripheral blood after BMT.
    • Fig. S3. Validation of MafB siRNA and shRNA reagents.
    • Fig. S4. Ectopic MAFB expression enhances T6E cell growth.
    • Fig. S5. MAFB interacts with DNA through ETS factors, and the loss of ETS1 delays onset of T-ALL.
    • Fig. S6. Zmiz1 expression in T6E cells is not affected by the loss of MafB.
    • Legends for tables S1 and S2
    • Table S3. List of oligo sequences and primer sets for qPCR and OIP.

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.88 MB

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Notch signaling enhancement of Notch mutant LPΔP by candidate genes from the cDNA library.
    • Table S2 (Microsoft Excel format). MafB suppression affects a subset of Notch signaling gene targets in T6E cells.

    [Download Tables S1 and S2]


    Citation: K. V. Pajcini, L. Xu, L. Shao, J. Petrovic, K. Palasiewicz, Y. Ohtani, W. Bailis, C. Lee, G. B. Wertheim, R. Mani, N. Musuthamy, Y. Li, J. P. P. Meijerink, S. C. Blacklow, R. B. Faryabi, S. Cherry, W. S. Pear, MAFB enhances oncogenic Notch signaling in T cell acute lymphoblastic leukemia. Sci. Signal. 10, eaam6846 (2017).

    © 2017 American Association for the Advancement of Science

Stay Connected to Science Signaling

Navigate This Article