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Acute myeloid leukemia (AML) is a molecularly diverse malignancy with a poor prognosis, whose largest subgroup is characterized by somatic mutations in NPM1, the gene for Nucleophosmin1. These mutations, termed NPM1c, result in cytoplasmic dislocation of Nucleophosmin1 and are associated with distinctive transcriptional signatures2, yet their role in leukemogenesis remains obscure. Here we report that activation of a humanized Npm1c knock-in allele in murine hemopoietic stem cells causes Hox overexpression, enhanced self-renewal and expanded myelopoiesis. One third of mice developed delayed-onset AML, suggesting a requirement for cooperating mutations. We identified such mutations using a Sleeping Beauty3-4 transposon which caused rapid-onset AML in 80% of Npm1c+ mice, associated with mutually exclusive integrations in Csf2, Flt3 or Rasgrp1 in 55 of 70 leukemias. Recurrent integrations were also identified in known and novel leukemia genes including Nf1, Bach2, Dleu2 and Nup98. Our results give new pathogenetic insights and identify therapeutic targets in NPM1c+ AML.
Nucleophosmin, has roles in several cellular processes including ribosome biogenesis and centrosome duplication5-6, for which it relies on its ability to shuttle between the nucleolus, nucleus and cytoplasm using subcellular localization signals7. This ability is impaired in 35% of AMLs, as a result of NPM1c mutations1 which disrupt the N-terminal nucleolar localization signal of Nucleophosmin and generate a nuclear export signal in its place1,8. NPM1c mutations are mutually exclusive of fusion genes found in other types of AML9, but frequently co-occur with activating mutations in FLT31 or RAS10.
NPM1c is known to bind to and alter the subcellular distribution of several proteins including HEXIM1, Fbw7γ, p19Arf and NF-kappaB8, however the relevance of these interactions to AML is unknown8. Selected transgenic mouse lines overexpressing NPM1c in myeloid progenitors display an increased incidence of mild myeloproliferative syndromes, but the significance of this observation is unclear as these mice do not develop AML11.
To study the hemopoietic effects of NPM1c, we generated a conditional knock-in mouse model of the commonest form of NPM1c mutation, type A1. We confirmed that the human (NPM1cA) and “humanized” mouse (Npm1cA) type A mutant proteins (Supplementary figure 1) displayed the same sub-cellular localization (Figure 1a) and proceeded to modify the Npm1 locus in mouse embryonic stem (ES) cells. The conditional allele, Npm1flox-cA, was designed to minimize interference with the native locus, as this could itself be leukemogenic12, yet switch to the mutant allele, Npm1cA, after Cre-loxP recombination (Figure 1b). We confirmed that after Cre-loxP recombination Npm1flox-cA/+ ES cells expressed the Npm1cA mutant mRNA and protein (Figure 1c,d), and established the Npm1flox-cA/+ allele in mice which were born at Mendelian ratios. However, the Npm1cA allele was incompatible with normal embryonic development as crosses between Npm1flox-cA/flox-cA mice and mice heterozygous for Stella-Cre, which mediates Cre-loxP recombination in the early embryo (PL, unpublished), gave no double transgenic live offspring (0/80) or embryos at E8 (n=11), E10 (n=10) and E12 (n=23). By contrast, the offspring of Npm1flox-cA/flox-cA − Mx1-Cre+ crosses were born at Mendelian ratios (Figure 1e).
To study the hemopoietic effects of Npm1cA, 5- to 8-week-old Npm1+/+ and Npm1flox-cA/+ mice (hereafter collectively referred to as Npm1WT) and Npm1flox-cA/+; Mx1-Cre+ mice (hereafter referred to as Npm1cA/+), were treated with Polyinosinic-Polycytidylic acid (pIpC) and analyzed 8 weeks later. Cre-loxP recombination was seen in >90% of bone-marrow-derived hemopoietic colonies from Npm1cA/+ mice (Supplementary Figure 2), reflecting efficient recombination in hemopoietic stem cells (HSCs) and Npm1cA protein was detectable in Npm1cA/+ hemopoietic tissues (Figure 2a). Gene expression profiling of Npm1cA/+ compared to Npm1WT lineage negative marrow progenitors (Lin−), showed differential overexpression of HoxA5, HoxA7, HoxA9, HoxA10 and HoxB5 genes (Figure 2b and Supplementary Table 1). Similar gene expression changes were seen with total bone marrow nucleated cells (BMNC) and B220+ cells, but not Gr1+/Mac1+ cells (Supplementary tables 2-4). For Npm1cA/+ BMNC HoxA9, HoxA7 and HoxA5 were the 3 most significantly overexpressed mRNAs in the mouse genome and expression of several lymphoid-specific genes was reduced (Supplementary table 2). Total Npm1 mRNA expression was unaltered (Supplementary tables 1-4 and Supplementary figure 3).
Npm1cA/+ and Npm1WT blood counts did not differ (Figure 2c); although Npm1cA/+ mice had increased mean red cell (MCV) and platelet (MPV) volumes (Figure 2d). Bone marrow histo-morphological examination revealed no detectable abnormalities/differences (Supplementary Figure 4a). Flow cytometric analysis of marrow cells showed no significant differences in stem or progenitor cell compartment sizes (Figure 2e-f and Supplementary Figure 4b). However, Npm1cA/+ mice had increased numbers of Gr1+/Mac1+ myeloid cells and decreased numbers of late B-cells (B220+/CD19+) (Figure 2g-i). Additionally, Npm1cA/+ cells showed increased serial re-plating ability in methylcellulose, an in vitro surrogate for self-renewing potential13 (Figure 2j). There were no differences in levels of Gr1+/Mac1+ cell apoptosis or DNA damage (Supplementary Figure 4c-e).
To study the leukemogenicity of Npm1cA, we aged 43 Npm1cA/+ and 44 Npm1WT mice. Npm1cA/+ mice had a shorter overall survival compared to Npm1WT animals (617 vs 769 days, p=0.018) as a result of excess deaths due to AML (13 vs 0, p=0.0001) (Figure 2k-l). Morphologically AMLs showed maturation (“myeloid leukemia with maturation”14) and all 5 AMLs tested were Gr1+/Mac1+. Of 13 Npm1cA/+ mice with AML, 6 were found to have coincidental non-extensive B-lymphoid tumors. Compared to Npm1cA/+ mice dying of non-hematological causes, mice with AML had enlarged livers (2.6g vs 1.8g, p<0.001) and spleens (1.3g vs 0.3g, p<0.001) and higher blood leukocyte counts (77.1 vs 7.0 ×106/l, p=0.006)(Supplementary table 5). Also 2/2 AMLs tested were transplantable into sublethally irradiated syngeneic mice (Supplementary table 6). Finally, Npm1cA/+ and Npm1WT mice developed lymphoid tumors at the expected rate for their age/strain15 and had similar rates of non-hematological mortality (Figure 2k and Supplementary table 5).
The above data show that Npm1c can initiate AML, however the long latency reflects the need for additional mutations. To identify cooperating mutations, we subjected Npm1cA/+ mice to insertional mutagenesis with Sleeping Beauty (SB)3. We generated transgenic mice carrying approximately 80 copies of GrOnc, a novel bi-functional PiggyBac/SB transposon16 capable of both gene activation and disruption (Figure 3a-b and Supplementary Figure 5). GrOnc harbors the Graffi1.4 murine leukemia virus LTR, which preferential promotes myeloid rather than lymphoid leukemia in predisposed backgrounds, an effect attributed to the LTR17.
We crossed Npm1flox-cA/+, GrOnc, Mx1-Cre and conditional Rosa26flox-SB transposase (Supplementary Figure 6) mouse lines as per Supplementary Figure 7a to generate 125 Npm1flox-cA/+; Rosaflox-SB/+; GrOnc+; Mx1-Cre+ (Npm1cA/+ mutagenesis cohort), and 45 Npm1+/+; Rosaflox-SB/+; GrOnc+; Mx1-Cre+ (Npm1+/+ mutagenesis cohort). All mutagenised mice developed aggressive leukemia/lymphoma within one year of pIpC induction (Figure 3c). However, mean survival was significantly shorter in the Npm1cA/+ compared to Npm1WT mice (99 vs. 150 days, p=0.001) (Figure 3c). The first 121 mice (87 Npm1cA/+ and 34 Npm1+/+) were studied in detail and tumors classified by histological type (Supplementary Table 6), confirmed by flow cytometry in 18 leukemias. Compared to Npm1+/+, Npm1cA/+ mice had a significantly higher fraction of AMLs (80.5% vs. 26.5%, p=0.00000002) and no T-cell leukemias (0% vs. 17.6%, p=0.00006) (Figure 3d-f). These findings indicate strong synergy between Npm1cA and GrOnc in promoting myeloid leukemogenesis. In some mice a coincidental second tumor was identified (leukemia/lymphoma in 19.8% and angiosarcoma in 3.3%) (Supplementary Table 7).
In order to identify the genetic mutations collaborating with Npm1c to cause AML, we mapped the transposon integration sites in hemopoietic tumors from 87 Npm1cA/+ and 34 Npm1+/+ mice using barcoded splinkerette PCR and 454-sequencing. A total of 219,556 high-quality reads mapped evenly throughout the genome although, as expected for SB3-4, we observed local hopping around the GrOnc donor site at Megabase (Mb) 30 on chromosome 19 (Supplementary Figure 8). CISs for Npm1cA/+ and Npm1+/+ tumors were identified using the kernel convolution method18 and those mapping within 2Mb of the donor site discarded. The only other CIS on chromosome 19 involved Pten located at 32.8Mb, which makes its significance uncertain. The CIS sets in Npm1cA/+ and Npm1+/+ tumors included several known and many putative novel leukemia genes (Figure 4a, Supplementary Figures 8 and 9). The two sets overlapped but were clearly different, indicating that distinct molecular pathways operated in the two groups.
In the Npm1cA group, the most striking finding was the identification of activating integrations upstream of Csf2, the gene for Gm-csf, in 48.3% of leukemias, associated with LunSD-Csf2 fusion transcripts, Csf2 mRNA overexpression (Figure 4b-d) and increased levels of Gm-csf in leukemic cell supernatants (p=0.0007, Supplementary Table 8). We studied transposon integrations in single-cell derived methylcellulose colonies from three Csf2+ AMLs and found that over 90% of CFU colonies (11/12, 12/12 and 19/20) carried Csf2 insertions. We went on to map transposon integrations in 3 such colonies from the AML 36 and found that individual colonies carried 67-90 independent integrations each, yet only two integrations were shared between them, those involving Csf2 and the myeloid oncogene Myst4 (Moz) (Figure 4e). This is the first study of the subclonal make-up of transposon-derived malignancies and shows that, if the transposase remains active, transposons are continuously re-mobilized generating a highly heterogeneous population; bar for a small number of key insertions, possibly the leukemia “drivers”. We went on to study transposon integrations in leukemias developing in irradiated recipients transplanted with AMLs 26 and 38. We found that Csf2 insertions persisted in all 5 recipient mice studied (Supplementary Table 5), whilst the vast majority (>98%) of other insertions were lost. These data strongly indicate that Csf2 insertions are critical to the growth of these leukemias and thus persist during leukemic evolution/propagation, whilst “passenger” insertions are lost. Csf2 can behave as a myeloid oncogene in mice when activated by endogenous retroviruses19, whilst autocrine Gm-csf production has been reported to drive the growth of human AML blasts in-vitro 20-21 and this is usually, albeit not always, inhibited by anti-GM-CSF antibodies.
Other important CISs included Flt3 (Figure 4f), a gene frequently co-mutated in human NPM1c+ AML and Nup98 (Figure 4g), a key component of the nuclear pore and a leukemogenic fusion gene partner not been previously identified as a retroviral or transposon CIS. NUP98 fusion proteins can directly interfere with nucleo-cytoplasmic transport of NPM1c22, raising the possibility that Nup98 insertions may operate in a similar way hence their specificity to Npm1cA/+ AMLs. Other CISs included inactivating GrOnc insertions in signal transduction inhibitors such as Nf1, Ptpn1 and Ptptn2 and insertions in transcription factors including Bach2, Cnot1, Pax5 and Zfp521 (Figure 5a and Supplementary figure 10).
Co-occurrence analysis of CIS genes from Npm1cA/+ AMLs, showed that Csf2 integrations were mutually exclusive of integrations in Flt3 (p=0.002) and Rasgrp1 (p=0.008) (Figure 5a and Supplementary figure 11). This was also true for Kras but did not reach statistical significance (p=0.12). These observations suggest that these genes provide alternative proliferation signals that complement Npm1c in promoting cellular transformation. Interestingly Rasgrp1, previously associated with T-lymphoblastic leukemia in retroviral mutagenesis screens23, was recently identified as a drug resistance gene in another mouse model of AML24. Hox overexpression persisted in Npm1c/+ transposon-derived AMLs, irrespective of transposon insertions, suggesting that the molecular effects of Npm1c mutations remained operative in leukemic cells (Figure 5b). HoxA7 and HoxA9 are frequently targeted by retroviruses in BXH2 myeloid leukemias25 and mediate leukemic transformation by MLL oncoproteins26. Also, in line with our findings, HoxA9−/− mice exhibit defects in maturing myeloid and B-lymphoid cells, but not early progenitors27. Overall, this makes it highly plausible that Hox overexpression mediates the leukemogenic effects of NPM1c, particularly as expression levels seen in our mice are comparable to those in human AMLs.
Taken together, our data demonstrate that Npm1c are AML-initiating mutations which cause Hox overexpression, impart increased self-renewal to and prime hemopoietic stem/progenitor cells to leukemic transformation by activation of a narrow set of pro-proliferative molecules/pathways, usually in combination with mutations in transcriptional regulators (Figure 5c, Supplementary Tables 1-3). Observations from whole genome sequencing studies are generating data in support of such a model in this and other subgroups of AML with a normal karyotype10,28. Also, our data explain other important features of human NPM1c+ AML, including a consistent negativity for the primitive marker CD341 (effects of Npm1cA most noticeable on later progenitors) and a failure to observe NPM1c mutations in the human germline (embryonic lethality). Finally, our approach to define the effects of a mutation in isolation and proceed to map its collaborative oncogenic pathways provides a model for the study of other human cancer-associated mutations and can be used to complement and help decipher recent and impending whole cancer genome sequencing studies.
For all sequences see Supplementary table 9
Amino-terminal GFP-NPM1/GFP-Npm1 fusion constructs were generated and K562 cells photographed 24hrs after electroporation.
A 9.8kb was retrieved from BAC bMQ-282D14 using oligonucleotide recombineering29 and modified to the Npm1flox-cA targeting construct (duplicated region: NCBIM37-chr11:33051153-33053141). Npm1cA (Post-Cre) ES cell clones were derived under FIAU selection30. Npm1flox-cA/+ and Npm1flox-cA/flox-cA mice were crossed with Stella-Cre for embryonic viability and Mx1-Cre31 mice for hemopoietic studies. For Mx1-Cre activation 500μg pIpC x6 was used.
Generated by WW and PL by insertion of IRES-Cre into the 3'UTR of Stella, they show 100% Cre-loxP recombination at several loci in embryos and germline.
Generated by pronuclear injection of linearized GrOnc DNA.
Generated as per Supplementary Figure 6.
We used PCR assays with primers P1-P16 (e.g. Supplementary Figures 2, 6, 7).
Mouse femurs and spleens were processed as described32. For BM lineage analysis we stained with Gr1-PE and CD11b(Mac1)-FITC, or B220-APC-Alexa750 and CD19-PE. For HSC/progenitor analyses, we depleted lineage-positive cells using MACS columns and stained with cKit-APC, ScaI-PB, CD34-FITC and either Flt3-PE (HSC) or FcRIII-PE (progenitors). Antibodies from BD Pharmingen except CD11b-FITC (Caltag). We used a CyAn-ADP analyzer and FlowJo. B220+ and Gr1+/Mac1+ cells were sorted using a MoFlo sorter. For leukemia phenotyping we used combinations of the above.
Global profiling was done using Illumina mouse-6 expression beadchip version 2. Data were quantile normalised33 and analyzed using the bioconductor (http://www.bioconductor.org/), lumi (http://www.bioconductor.org/packages/2.0/bioc/html/lumi.html) and limma34 packages, then p-value adjusted for multiple testing35. Microarray data and description of experimental design were deposited to Array Express with accession number E-MEXP-3113.
BM or spleen cells were plated in M3434 (Stem Cell Technologies) as described33. Colonies were counted/picked after 10-12 days. For re-plating assays 50,000 BM cells from 6xNpm1cA/+ and 6xNpm1WT mice were plated and counted after 8 days with 30,000 cells re-plated etc.
Npm1 targeting: PstI-digested ES cell DNA was hybridized with a PCR-generated-probe (Figure 1b). GrOnc copy number and clonal integrations: SacI-digested tail DNA or BamHI-digested leukemic DNA respectively, were hybridized with the XbaI-SacII GrOnc fragment (Supplementary Figure 5).
For Npm1+ and Npm1cA cDNAs we used allele-specific primers R1-R3 and for GrOnc-Csf2 primers Graffi1.4-LTR_F and Csf2_exon3_R.
For GrOnc copy number En2-SA and β-Actin were quantified from tail DNA. En2-SA/β-Actin ratio was normalized against wild-type mice (2 copies of En2-SA). For Csf2 qRT-PCR we designed specific primers/probe and normalized against Gapdh. For Hox genes, we used standardized assays (Applied Biosystems), normalized against Gapdh and expressed values relative to Npm1+/+.
Anti-Npm1cA-specific rabbit antisera, were generated and affinity-purified using peptide Hydrazine-IQDLCLAVEEVSLRK and used at 1/100. Mouse monoclonal anti-Actin (Sigma) was used at 1/5000.
The location of the 80-copy GrOnc donor site, was identified using Texas Red-labeled GrOnc DNA on blood metaphases.
Blood counts were performed on a VetABC analyzer (Horiba ABX).
These were done as described36 using SB-specific primers and 454-sequenced.
ssaha2 was used to map GrOnc insertions to mouse genome. Sequences were filtered to contain the SBcommon-Sp2-454R primer then the end of the SB repeat, followed by genomic sequence starting with TA and ending at GATC (MboI restriction site). Sequences <17 nucleotides or with <85% maximal genomic identity were discarded. For sequences matching several loci, we calculated a normalized score difference (NSD) comparing the best hit to the second best hit, whereby NSD= ((score of best hit) − (score of second-best hit)) / query-length*100. We mapped 5000 random mouse genomic fragments to the genome and found that 96.5% of correctly and only 1.5% of wrongly mapped reads had NSD ≥4 and removed reads with NSD <4 from analysis. Finally, redundant sequences from the same tumor and same location were “collapsed” to one integration. To identify CISs, non-redundant insertions were analyzed using a Gaussian Kernel Convolution-based framework37 for 10Kb, 30Kb, 60Kb and 120Kb windows and all windows merged to compile CIS lists38. To identify previously described CISs we searched the retroviral tagged cancer gene database (RTCGD, http://rtcgd.ncifcrf.gov)36 and recent insertional mutagenesis publications3-4,39-40.
Formalin-fixed, paraffin-embedded sections were stained with H&E and anti-CD3, anti-B220 and anti-myeloperoxidase, detected by immunoperoxidase. All material was examined by two experienced histopathologists (PW and MA) blinded to mouse genotypes. A primary diagnosis was established and hemopoietic tissues examined for additional malignancies.
Cryopreserved AML cells were plated in duplicate at 100,000 per 100ul RPMI/ 10%FCS. Supernatants were collected after 2 days and assayed (EMGMCSF, Thermo).
Syngeneic mice were sublethally irradiated (500rad) and injected with 1×106 cryopreserved AML cells into the tail vein. Mice were culled when unwell or after 180 days.
We used Fisher's exact test for 2×2 comparisons and mutual exclusivity testing, χ2-test for nominal data and t-test for continuous data unless normality test failed, when Mann-Whitney Rank Sum tests were used (Sigma Stat). SEMs are represented in error bars.
We acknowledge the use of the Research Support Facility at the WTSI, the Department of Pathology Tissue Bank and the Cambridge NIHR Biomedical Research Centre, University of Cambridge. We thank F. Law and J. Gadiot for assistance in generating the Npm1flox-CA and Rosaflox-SB mice, F. Foyer and B. Graham for help with fluorescent microscopy, B. Ling, W. Cheng, R. Macintyre and P. Chan for help with Flow Cytometry; R. Bautista for help with gene expression images; C, Hale and A. Nyzhnyk for help with ELISAs, B. Huntly, D. Adams, J. Cadinanos, H. Prosser, N. Conte, K. Yusa and Q. Liang, for helpful discussions during the project; P. Campbell and A. Green for critical reading of the manuscript. This work was supported by a Clinician Scientist Fellowship from Cancer Research UK (G.V.).
Database Accession numbers: Microarray data and description of experimental design were deposited to Array Express with accession number E-MEXP-3113.
Author Contributions. G.V. and A.B. designed the study; G.V. generated Npm1flox-cA mice, GrOnc mice and GFP-NPM1 constructs, managed mouse colonies, designed and validated polyclonal anti-Npm1c sera and carried out western blots; J.L.C. and G.V. performed mouse genotyping, tumor processing/banking and K562 transfections; G.V., J.C., R.R. and L.R. performed mouse necropsies; G.V. J.L.C. and J.C. performed hemopoietic analyses; G.V. and C.G. performed qPCR; G.V., P.E. and R.A. performed gene expression analysis studies; S.R, G.V. and R.R. performed mapping and analysis of transposon integration sites; G.V. and R.B. performed fluorescence-in-situ hybridization; W.W. and P.L. generated the Stella-Cre mice; A.U. generated the Rosaflox-SB mice; P.W. and M.A. performed histological analyses; A.B. supervised the study; all authors contributed to the writing of the manuscript.
The authors have no competing financial interests to declare