Human models of NUP98-KDM5A megakaryocytic leukemia in mice contribute to uncovering new biomarkers and therapeutic vulnerabilities

Abstract

Acute megakaryoblastic leukemia (AMKL) represents ∼10% of pediatric acute myeloid leukemia cases and typically affects young children (<3 years of age). It remains plagued with extremely poor treatment outcomes (<40% cure rates), mostly due to primary chemotherapy refractory disease and/or early relapse. Recurrent and mutually exclusive chimeric fusion oncogenes have been detected in 60% to 70% of cases and include nucleoporin 98 (NUP98) gene rearrangements, most commonly NUP98-KDM5A. Human models of NUP98-KDM5A-driven AMKL capable of faithfully recapitulating the disease have been lacking, and patient samples are rare, further limiting biomarkers and drug discovery. To overcome these impediments, we overexpressed NUP98-KDM5A in human cord blood hematopoietic stem and progenitor cells using a lentiviral-based approach to create physiopathologically relevant disease models. The NUP98-KDM5A fusion oncogene was a potent inducer of maturation arrest, sustaining long-term proliferative and progenitor capacities of engineered cells in optimized culture conditions. Adoptive transfer of NUP98-KDM5A-transformed cells into immunodeficient mice led to multiple subtypes of leukemia, including AMKL, that phenocopy human disease phenotypically and molecularly. The integrative molecular characterization of synthetic and patient NUP98-KDM5A AMKL samples revealed SELP, MPIG6B, and NEO1 as distinctive and novel disease biomarkers. Transcriptomic and proteomic analyses pointed to upregulation of the JAK-STAT signaling pathway in the model AMKL. Both synthetic models and patient-derived xenografts of NUP98-rearranged AMKL showed in vitro therapeutic vulnerability to ruxolitinib, a clinically approved JAK2 inhibitor. Overall, synthetic human AMKL models contribute to defining functional dependencies of rare genotypes of high-fatality pediatric leukemia, which lack effective and rationally designed treatments.

Graphical abstract

Figure 1.

Overexpression of NUP98-KDM5A efficiently induces maturation block and sustains the proliferative and progenitor capacities of CB-CD34⁺cells. (A) Experimental procedures used to establish in vitro models of N5A-driven leukemia. CD34⁺ cells isolated from single-donor CB were seeded in 96-well plates and infected with lentiviral particles carrying the chimeric NUP98-KDM5A oncogene. The lentiviral vector encodes FLAG-tagged NUP98-KDM5A and a GFP reporter gene, driven by MNDU3 and PGK promoters, respectively. Independent cell lines derived from each well were grown for 3 to 5 days in optimized culture conditions before GT evaluation and further in vitro expansion (20% of the cells from each well). (B) CD34⁺GFP⁺ enrichment in long-term cultures of CB-CD34⁺ cells transduced with a control (CTL, n = 4) or NUP98-KDM5A (N5A, n = 12) vector. (C) Short-term proliferation kinetic of transduced cells in independent cultures of CB-CD34⁺ cells transduced with N5A or control lentiviral vector. Cultures were initiated from 2 independent CBs (eg, CB1 and CB2) transduced with control (n = 6 per CB) or N5A (n = 14 per CB) lentiviral vector, as indicated. (D) Fluorescence-activated cell sorting profiles showing the time course of GFP and CD34 expression in 2 independent samples transduced with control (eg, CTL_C) or N5A lentiviral vector (eg, N5A_A). Transduced CB-CD34⁺ cells were derived from a single donor. (E) Giemsa-stained cytospins showing immature cellular morphology of an N5A-expressing cell line (N5A_C, bottom) at day 80 and differentiation of matched-CTL cells at day 59. Original magnification ×1000. (F) Acquisition by flow cytometry showing differentiation of control cells (GFP⁺CD34⁻ C-KIT^hi) and a maturation arrest of N5A-transduced cells (GFP⁺CD34⁺ C-KIT^low). (G) Graph showing the percentage of GFP⁺KIT^low immature cells in each indicated culture, defined as median fluorescence intensity <1.5 × 10⁴ for KIT^low cells; n = 3 independent experiments, n = 4 CB units, n = 43 cultures of N5A cells, and n = 19 cultures of CTL-cells. (H) Clonogenic progenitor frequency for freshly isolated (day 0, n = 2) and CTL or N5A-transduced CB-CD34⁺ cells, plated at days 8 and 88 of culture (n = 2 for CTL; n = 4 for N5A; mean ± standard error of the mean [SEM]). Phenotypic classification of clonogenic progenitors is presented in supplemental Figure 1. (I) Representative image of a typical long-term colony generated from the N5A_C cell line at day 60 of culture. Top: bright field microscopy; bottom: epifluorescence microscopy. Original magnification ×10. **** P < .0001.

Figure 2.

Overexpression of N5A fusion in CB-CD34⁺cells induces acute megakaryoblastic leukemia and multilineage leukemia subtypes in xenograft models. (A) Representation of the experimental procedures used to establish in vivo models of N5A-driven leukemia. Human CD34⁺ cells were isolated from CB and transduced as described in Figure 1A. After GT evaluation, 70% of the cells from each well were injected into a primary recipient mouse. Leukemia xenograft cells were collected and characterized phenotypically, molecularly, and functionally. (B) Distribution of generated xenograft models classified by leukemia subtypes based on molecular markers and cytology analyses (supplemental Table 3). Models originated from 6 experimental groups initiated from 7 independent CB samples (supplemental Table 2). (C) Detection of N5A fusion transcript expression by RT-PCR with RNA isolated from BM or spleen (Sp) cells of leukemic mice, as indicated. Normal nontransduced CB-CD34⁺ sample was used as negative control (CTL). (D-J) AMKL xenograft models. (D) Brittle white bones and mild splenomegaly were observed in AMKL xenograft models (xAMKL-3, N5A vector) compared with control xenograft-recipient mice (CTL, empty vector). (E) Representative hematoxylin-phloxine-saffron–stained longitudinal sections of tibia BM harvested from xCTL and xAMKL-3 recipient mice. (F) Blasts infiltration percentage (hCD45^lowCD41⁺/CD61⁺ cells) in BM and spleen of primary AMKL (n = 6). (G) Giemsa-stained peripheral blood (PBL) smear and BM cytospin from an AMKL primary (1^ary) recipient mouse (xAMKL-3) highlighting the presence of leukemic blasts (48 weeks after transplantation). (H) Fluorescence-activated cell sorting (FACS) profiles revealing typical hCD45^lowCD34⁻CD41⁺ megakaryoblasts in the BM of a primary recipient mouse (xAMKL-3), along with CD45^hiCD3⁺-activated T cells. The spleen is infiltrated by CD45^hiCD3⁺ activated T cells with hCD45^lowCD34⁻CD41⁺ barely detectable. Characterization of an additional mouse xenograft model (xAMKL-1) with detailed T-cell immunophenotype is shown in supplemental Figure 2. (I) Giemsa-stained BM cytospin and spleen touch preparations showing leukemic blasts derived from a secondary (2^ary) recipient mouse with a 2.2 × 10⁶ xAMKL-3 BM–cell transplant (33 weeks after transplantation). (J) FACS profiles revealing hCD45^lowCD34⁻CD41⁺GFP⁺ megakaryoblasts in the BM and spleen of the secondary recipient mouse. *P < .05. HSC, hemopoietic stem cell; LSC, leukemic stem cell.

Figure 3.

Distinct expression profiles of NUP98-KDM5A leukemia subtypes compared with normal CB CD34⁺cells. (A) Hierarchical clustering heat map of the top 100 genes differentially expressed between the 3 leukemia subtypes observed in N5A xenograft models (AMKL xenograft [xAMKL], n = 4; B-ALL xenograft [xB-ALL], n = 4; AML-O xenograft [xAML-O], n = 3) and normal CB CD34⁺ cells (CB-CD34⁺ cells, n = 4). Read count data were converted into log₂ values and represented according to a blue-yellow-red–colored gradient scale. The size factor values, reflecting the correction factor for normalization of the relative depth of sample libraries, are represented by a gradient of green tones according to the size factor scale bar. Sample conditions are described by a binary (pink, C/control; blue, E/experimental leukemia) color code. (B) Principal component analysis plots of the first and second principal components of NUP98-KDM5A models, patients, and controls, calculated using the 500 most variable genes between all conditions.

Figure 4.

Molecular characterization of N5A AMKL. (A) Scatterplots showing pairwise correlation of gene expression values (log FPKM, RNAseq) between N5A AMKL derived from pediatric patients and xenograft models (N5A pAMKL and xAMKL, respectively). For each scatterplot the Pearson correlation coefficients (r) are indicated in mirroring cells. The red diagonal line represents a perfect correlation (r = 1.0). (B) Top 30 genes differentially expressed by at least 10-fold in a sampling of BM cells derived from 2 patients and 4 xenograft models presenting N5A AMKL, as compared with normal CB-CD34⁺ cells (n = 4). Genes with expression values of ≥10 FPKM for all N5A AMKL samples and fold changes ≥10 compared with CB-CD34⁺ samples are displayed (see supplemental Figure 7 for the complete list). The indicated FPKM values are represented by a logarithmic color scale (log₁₀). (C) Expression of HOXA/HOXB cluster and MEIS genes in N5A AMKL samples derived from patients (n = 2) and xenograft models (n = 4) compared with control CB-CD34⁺ cells (n = 4). Values are presented as the mean ± SEM. (D) Top 10 ranked gene sets in the GSEA of genes upregulated in N5A AMKL patients and xenograft models compared with CB-CD34⁺ samples. Enrichment plots for selected gene sets (in red) are depicted in panel E and in supplemental Figure 6.

Figure 5.

Specific biomarkers of AMKL. (A) Heat map of expression values in FPKM (RNAseq) of 7 top-ranked genes encoding cell surface proteins that are differentially expressed by a least 10-fold in leukemic BM cells derived from patients (pAMKL) or mouse xenograft models (xAMKL) in the middle panel, as compared with normal CB-CD34⁺ cells in the left panel, and expressed at low levels (≤5 FPKM) in CB-CD34⁺ cells. Genes with expression values of ≥5 FPKM in all N5A AMKL samples, fold change ≥10 compared with CB-CD34⁺ samples, and low expression levels (≤5 FPKM) in CB-CD34⁺ cells are listed in supplemental Table 8. Expression of the selected genes in leukemic BM cells derived from patients presenting other genetic subtypes of AMKL (non-N5A pAMKL) or non-AMKL leukemia subtype involving NUP98 rearrangement (NUPr pAML) are also indicated. Expression of ITGA2B/CD41, ITGB3/CD61, and NCAM1 is indicated in red for comparison. Expression of the selected genes in the validation cohort are shown in the right panel, represented as mean expression per indicated genetic group. (B) Distribution of selected gene expression values (FPKM) in BM-derived pediatric AML cells classified according to the FAB nomenclature (M0-M7); n = 284 pediatric AML cases from the National Cancer Institute (NCI) TARGET database. Horizontal lines represent median values. Pairwise gene expression comparisons between M7 and other FAB categories were performed with a Mann-Whitney rank sum test with the Benjamini-Hochberg correction (shown below graphs). M7 leukemia (n = 11) involved the following exclusive genetic lesions: NUP98-KDM5A (n = 1), CBFA2T3-GLIS2 (n = 4), KMT2A-MLLT10 (n = 1), and RBM15-MKL1 (n = 1). (C) Pairwise scatterplot representations showing correlative expression of the indicated genes in a pediatric AML (NCI, TARGET database). Representations were created with the bioinformatic tool MiSTIC. AML classified as FAB M7 or M6 are indicated in red and blue, respectively. (D) Selection of specimens expressing the highest levels of RHAG, NEO1, GP9, or ITGB3/CD61 combined with ITGA2B/CD41 significantly enriches for FAB M7 AML (eg, AMKL). M7: 8 of 9 selected P = 6.1e-11. See data set and bioinformatic tool in panel C. (E) SELP expression, as assessed by flow cytometry in freshly isolated CB-CD34⁺ cells and in AMKL BM cells from an N5A mouse xenograft model. (F) Expression of NEO1 detected by RT-PCR using RNA derived from the BM of leukemic xenograft models or from CB-CD34⁺ cells. KDM5B expression was used as the endogenous control. (G) Expression of NEO1 detected by RT-PCR using RNA derived from the BM of an infant with NUP98-BPTF AMKL. RNA was isolated at diagnosis (NUP98r pAMKL-3D) and after 2 cycles of chemotherapy when disease burden was ∼2% by cytology (NUP98r pAMKL-3 MRD). Human placental RNA was used as the nontumor control. ns/not significant P > .05; *P < .05; **P < .01; ***P < .001; ****P < .0001.

Figure 6.

Primary NUP98r xenograft cells are vulnerable to JAK-STAT signaling inhibitors. (A) Schematic overview of the procedures used to perform the cell surface proteomic analysis of CD41⁺ AMKL cells isolated from the spleen of NUP98r (NUP98-BPTF) PDX mice. Cell surface proteins were biotinylated and isolated using streptavidin pulldown and stringent washes. Analysis by liquid chromatography-mass spectrometry allowed for identification of 411 cell surface proteins, including AMKL-specific biomarkers (supplemental Table 9). (B) Top 10 hallmark gene sets enriched in the analysis of the cell surface proteins detected on NUP98r AMKL primary xenograft using Metascape (see supplemental Methods for details). The complete analysis is provided in supplemental Table 10. (C) Dose-response curves and half maximal inhibitory concentrations (IC₅₀) determined for each indicated cell type submitted to a viability assay in presence of an inhibitor or DMSO vehicle (Cell-Titer Glo, 6-day incubation). Experiments were conducted with 4 replicates. CMK, N5A xAMKL (E771, G662), and CB-CD34⁺ (no. 5/6 and 7); n = 1 experiment. M07e, ML-2, 2^ary NTF AMKL PDX (I603, I604); n = 2 experiments.