Research ArticleCancer

Dual inhibition of MDMX and MDM2 as a therapeutic strategy in leukemia

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Science Translational Medicine  11 Apr 2018:
Vol. 10, Issue 436, eaao3003
DOI: 10.1126/scitranslmed.aao3003
  • Fig. 1 MDMX is targetable in acute myeloid leukemia using an α-helical stapled peptide (ALRN-6924).

    (A) mRNA expression analysis of MDMX in human cancers using The Cancer Genome Atlas (TCGA) data sets and (B) MDMX and MDM2 mRNA expression in hematopoietic stem (HSC) and granulocyte-monocytic progenitor–enriched (GMP) cells from acute myeloid leukemia (AML) patients (n = 14) and healthy controls (n = 6) (data shown as mean ± SD, where **P < 0.01, ***P < 0.001; n.s., not significant). (C) Fluorescence polarization (mP) of a fixed concentration of ALRN-3618 was measured in the presence of varying concentrations (log M) of mouse double minute 2 homolog (MDM2) and MDMX (top panels) (n = 2). Graphs display varying concentrations of ALRN-6924 used to displace ALRN-3618 at its EC80 from each target protein (bottom panels). (D) IC50 and Ki values (n = 2) for each target protein, as determined from a nonlinear four-parameter curve obtained from plotting fluorescence polarization versus drug concentration in (C) (bottom). (E) Sensorgrams for the binding of ALRN-6924 (blue) and RG7388 (red) to surfaces with immobilized MDM2 (top) and MDMX (bottom) (n = 2). (F) Enzyme-linked immunosorbent assay demonstrating the expression of human p21 protein (left) (21 hours after treatment with ALRN-6924) and cellular caspase activity (right) (48 hours after treatment with ALRN-6924) determined using Caspase-Glo 3/7 assay reagent in SJASA-1 cells (n = 2; data shown as mean ± SD). ACC, adrenocortical carcinoma; DLBC, diffuse large B cell lymphoma; PCPG, pheochromocytoma and paraganglioma; RCC, renal cell carcinoma.

  • Fig. 2 ALRN-6924 rapidly increases transcription at the p21 locus and affects its bursting dynamics.

    (A) Schematic representation of the engineered p21 gene locus containing MS2 stem-loops (p21-MS2). The transcription start site is shown as an arrow, exons are shown as blue rectangles, introns are shown as angled lines, and 5′ and 3′ untranslated regions (UTRs) are in gray. Scale bar, 1 kb. (B) Live-cell imaging of U2OS p21-MS2 cells in untreated medium. Top panel: Images of cells containing p21-MS2 (maximum intensity projections of z-dimension stacks). Scale bar, 10 μm; the green box outlines the transcription site (TS). Bottom panel: Quantification of the fluorescence intensity at the TS marked in the top panel. (C) Live-cell imaging of U2OS p21-MS2 cells in ALRN-6924–treated medium as described in (B). (D) Proportion of cells containing an active TS (≥1.5-fold the background). For each time point and condition, the proportion of active TS in a cell population was calculated using a rolling average of 30 min before and after each time point [85 untreated cells (top) and 54 cells treated with 5 μM ALRN-6924 (bottom)]. The pink area represents the 95% confidence interval. (E) Average cumulative transcription plot of p21-MS2 gene in untreated (green broken lines) versus ALRN-6924–treated cells (red solid lines). MCP-GFP, green fluorescent protein–tagged MS2 capsid protein; a.u., arbitrary units.

  • Fig. 3 ALRN-6924 blocks MDM2- and MDMX-p53 protein-protein interactions and activates the p53 pathway in AML.

    (A) Intracellular staining of FAM-labeled ALRN-6924 and p53-APC 3 hours after treatment with vehicle or ALRN-6924 at the indicated doses [a representative histogram is shown, black arrow denotes rightward shift (increase) in mean fluorescence intensity (MFI)]. (B) High-resolution fluorescence microscopy of MOLM13 cells treated with FAM-labeled ALRN-6924 3 hours after treatment with the indicated doses (shown is a representative image). (C) Coimmunoprecipitation with anti-MDM2 (left), anti-p53 (left) and anti-MDMX (right) antibodies or immunoglobulin G (IgG) isotype control using MOLM13 cellular extracts followed by Western blot analysis with anti-p53, anti-actin, anti-MDM2, or anti-MDMX antibodies. IP, immunoprecipitation. (D) Western blot analysis showing expression of p53 and p53-target proteins (p21 and MDM2) in primary patient AML cells (representative blots are shown). (E) Hierarchical clustering of genes differentially expressed in MOLM13 cells after exposure to 1 μM ALRN-6924 for 6 hours compared to vehicle-treated cells (n = 3). (F) Gene set enrichment analysis (GSEA) showing the top gene signatures differentially expressed in response to ALRN-6924. (G) Targeted gene expression analysis by quantitative reverse transcription polymerase chain reaction (qRT-PCR) of p53 target genes [CDKN1A (p21), MDM2, BAX, PUMA, and GADD45a] in response to increasing concentrations of ALRN-6924 in MOLM13 cells (n = 3, shown as the mean ± SD; **P < 0.01). KEGG, Kyoto Encyclopedia of Genes and Genomes; PID, Pathway Interaction Database; NES, normalized enrichement score; NT, no treatment.

  • Fig. 4 ALRN-6924 inhibits cellular proliferation and clonogenic capacity, and induces cell cycle arrest and apoptosis in AML cell lines.

    (A) Cellular proliferation measured by trypan blue exclusion in AML cell lines 24 hours after treatment with increasing concentrations of ALRN-6924 (n = 3). (B) Time-dependent changes in proliferation in AML cell lines treated with 1 μM ALRN-6924 (n = 3). (C) Fluorescence-activated cell sorting (FACS) analysis of cellular viability, apoptosis, and necrosis measured by annexin V and 4′,6-diamidino-2-phenylindole (DAPI) staining in AML cell lines 24 hours after treatment with increasing concentration of ALRN-6924 (n = 3). (D) Colony-forming capacity (CFC) assay of AML cell lines that were treated with vehicle, 0.5 μM RG7388, or 0.5 μM ALRN-6924 and grown in cytokine-free methylcellulose semisolid medium for 10 days (n = 3). (E) Western blot analysis of p53 in cell lines expressing an empty vector or one of two different p53 short hairpin RNAs (shRNAs) (a representative blot is shown). (F) Cellular proliferation measured by trypan blue exclusion in isogeneic p53 shRNA AML cell lines treated with ALRN-6924 (n = 3). (G) Apoptosis analysis by annexin V and DAPI staining in isogeneic p53 shRNA AML cell lines treated with ALRN-6924 (n = 3). Data are shown as the mean ± SD, and *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 ALRN-6924 shows robust antileukemic activity in primary AML cells and in vivo.

    (A) Dose- and time-dependent effects on cellular proliferation measured by trypan blue exclusion in primary AML cells treated with increasing concentrations of ALRN-6924 (data shown as mean ± SD of technical triplicates). (B) Dose-dependent effects of ALRN-6924 on clonogenic capacity of primary AML cells (n = 4) compared to healthy peripheral blood mononuclear cells (PBMNCs) isolated from cord blood (n = 3) and bone marrow mononuclear cells (BMMNCs) isolated from one AML patient in clinical remission (data shown as mean ± SD; P statistics determined using unpaired two-tailed Student’s t test). (C) FACS analysis of cellular viability (left panel) and apoptosis (right panel, plotted as fold change relative to baseline) measured by annexin V and DAPI staining in leukemia stem cell–enriched (CD34+CD38) primary bone marrow cells from AML patients (n = 3) and healthy donors (n = 3) 48 hours after treatment with 5 μM ALRN-6924 (data shown as mean ± SD, unpaired one-tailed Student’s t test). (D) Serial replating capacity of AML cell lines treated with either 0.5 μM ALRN-6924 or 0.5 μM RG7388, measured by CFC assay. (E) Human AML cellular engraftment analysis by FACS of mouse bone marrow (BM) from experimental animals 5 weeks after the start of treatment. Human AML cells were identified by costaining mouse BM cells with human CD45 and mouse CD45.1 antibodies [BIW, two times per week (n = 10); TIW, three times per week (n = 5)]. (F) Kaplan-Meier curve showing overall survival of mice xenotransplanted with MOLM13 AML cells and treated with ALRN-6924 or vehicle. (G) Median survival times for the curves shown in (F). (H) FACS analysis of mouse hematopoietic stem and progenitor frequencies after treatment with ALRN-6924 (20 mg/kg) TIW for 4 weeks (HSC = LSK CD150+CD48, LSK = LinSca+Kit+, and LK = LinKit+ progenitors). (I) Neutrophil and platelet counts assessed by Hemavet analysis of peripheral blood from mice (n = 10) on treatment with ALRN-6924 (20 mg/kg) TIW for 4 weeks. Data shown as the mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 6 ALRN-6924 selectively activates p53 in leukemia cells of an MDS/AML patient in vivo.

    (A) mRNA expression analysis by qRT-PCR of TP53, MDMX-FL, MDMX-S, and MDM2 in leukemic cells isolated from peripheral blood of a patient with myelodysplastic syndrome (MDS)/AML. (B) Cell viability assay using patient-derived leukemic PBMNCs evaluated by trypan blue exclusion. PBMNCs were treated in liquid culture with vehicle, 10 μM ALRN-6924, or 10 μM RG7388 and monitored over time. (C) CFC assay using patient-derived leukemic cells treated with vehicle or 0.5 μM ALRN-6924 as in Fig. 5B. Cells were plated in methylcellulose medium containing either vehicle or 1 μM ALRN-6924 for 10 days. (D) Intracellular FACS analysis of PBMNCs isolated from the MDS/AML patient treated with ALRN-6924 at the indicated time points after infusion. Total PBMNCs were isolated and stained with hematopoietic stem cell and progenitor markers CD34 and CD38 and pan-lymphocyte markers followed by intracellular staining of p53 and p21. FACS analysis of p53 activity in AML cells was performed by gating on lymphocyte marker–negative (CD3, CD4, CD8, CD10, and CD19), CD34+CD38 cells. The MFI of p53 and p21 in CD34+CD38 cells compared to the MFI of p53 and p21 in healthy lymphocytes at the indicated times after infusion with ALRN-6924 is shown. (E) Pathological report summarizing the percentages of peripheral blood blasts at the indicated time points after infusion with ALRN-6924. (F) Peripheral blood analysis of the patient’s platelets and neutrophils at the indicated times after infusion with ALRN-6924. Error bars represent the average of technical replicates (n = 3) ± SD; *P < 0.05 unpaired one-tailed Student’s t test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/436/eaao3003/DC1

    Materials and Methods

    Fig. S1. MDMX is overexpressed in cancers of hematopoietic and lymphoid origin.

    Fig. S2. MS2-SL repeats were inserted into a single allele of the p21 gene.

    Fig. S3. ALRN-6924 disrupts the p53-MDM2 and p53-MDMX protein-protein interactions and activates p53-dependent pathways.

    Fig. S4. MDMX-FL is differentially expressed in p53 WT AML cell lines sensitive to ALRN-6924.

    Fig. S5. In vivo xenograft models of leukemia development are sensitive to ALRN-6924.

    Fig. S6. Peripheral blood leukemic blasts from an MDS/AML patient treated with ALRN-6924 were analyzed by flow cytometry.

    Table S1. ALRN-6924 binding kinetics to MDMX and MDM2 compared to RG7388, a small-molecule MDM2 inhibitor.

    Table S2. Sensitivity of leukemia cell lines expressing WT and mutant p53 to ALRN-6924.

    Movie S1. Video showing transcriptional bursting of the p21 gene over time in vehicle-treated cells.

    Movie S2. Video showing transcriptional bursting of the p21 gene over time in ALRN-6924–treated cells.

    References (8490)

  • Supplementary Material for:

    Dual inhibition of MDMX and MDM2 as a therapeutic strategy in leukemia

    Luis A. Carvajal, Daniela Ben Neriah, Adrien Senecal, Lumie Benard, Victor Thiruthuvanathan, Tatyana Yatsenko, Swathi-Rao Narayanagari, Justin C. Wheat, Tihomira I. Todorova, Kelly Mitchell, Charles Kenworthy, Vincent Guerlavais, D. Allen Annis, Boris Bartholdy, Britta Will, Jesus D. Anampa, Ioannis Mantzaris, Manuel Aivado, Robert H. Singer, Robert A. Coleman, Amit Verma, Ulrich Steidl*

    *Corresponding author. Email: ulrich.steidl{at}einstein.yu.edu

    Published 11 April 2018, Sci. Transl. Med. 10, eaao3003 (2018)
    DOI: 10.1126/scitranslmed.aao3003

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. MDMX is overexpressed in cancers of hematopoietic and lymphoid origin.
    • Fig. S2. MS2-SL repeats were inserted into a single allele of the p21 gene.
    • Fig. S3. ALRN-6924 disrupts the p53-MDM2 and p53-MDMX protein-protein interactions and activates p53-dependent pathways.
    • Fig. S4. MDMX-FL is differentially expressed in p53 WT AML cell lines sensitive to ALRN-6924.
    • Fig. S5. In vivo xenograft models of leukemia development are sensitive to ALRN-6924.
    • Fig. S6. Peripheral blood leukemic blasts from an MDS/AML patient treated with ALRN-6924 were analyzed by flow cytometry.
    • Table S1. ALRN-6924 binding kinetics to MDMX and MDM2 compared to RG7388, a small-molecule MDM2 inhibitor.
    • Table S2. Sensitivity of leukemia cell lines expressing WT and mutant p53 to ALRN-6924.
    • Legends for movies S1 and S2
    • References (8490)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Video showing transcriptional bursting of the p21 gene over time in vehicle-treated cells.
    • Movie S2 (.avi format). Video showing transcriptional bursting of the p21 gene over time in ALRN-6924–treated cells.

    [Download Movies S1 and S2]

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