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J.N. Fisher et al.
row (BM).3-6 In cases in which these fusions do not con- tain the KMT2A-SET (suppressor of variegation 3–9, enhancer of zeste, and trithorax) domain, they acquire H3K79 or H4R3 histone methyltransferase- or acetyl- transferase activity through interactions with several co- factors.5,6 The interaction between chromatin and KMT2A fusions, mediated by the N-terminal menin- and the LEDGF (lens epithelium-derived growth factor) bind- ing domain, has been shown to be crucial for mainte- nance of the leukemic phenotype.7-10 Exploration of the KMT2A-menin-LEDGF interaction triad has led to the development of a series of promising small molecules with potent antileukemic activity.11,12 More recent studies have proposed physical interactions between NUP98, and NUP98 fusion proteins, with KMT2A and non-specific lethal histone-modifying protein complexes. Parallel genetic studies using mouse models suggested that NUP98-fusion gene driven leukemogenesis might be dependent on KMT2A function.13-15
Inv(11)(p15q23) has been reported as the sole chromo- somal abnormality in patients with several hematologic malignancies including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML);16-20 however, to date NUP98-KMT2A fusion expression has only been reported in two patients with AML.19 Using fluorescent in situ hybridization and reverse transcription quantitative polymerase chain reaction (PCR), Kaltenbach et al. found that inv(11)(p15q23) leads to fusion of the NUP98-FG- repeats to almost the entire KMT2A open reading frame (ORF).19 In this case, exon 1 encoding for the N-terminal menin-LEDGF interaction domain is lost. In contrast to other KMT2A- or NUP98-fusion associated diseases, NUP98-KMT2A+ leukemic blasts did not express known KMT2A targets such as the HOXA-gene cluster (HOXA5, HOXA7, HOXA9, or HOXA10) suggesting alternative mechanisms of transformation. As the size of the NUP98- KMT2A fusion ORF (>12 kb) limits the ability to test its transforming activity by retroviral expression in BM cells, we generated an inducible transgenic mouse model. We found that iNUP98-KMT2A expression led to a sympto- matic21 hematologic disease mimicking human MDS or AML that, as in patients, was not associated with elevat- ed expression of the Hox-A-B-C gene cluster.19 Thus, our work formally proves that a fusion, in which the N-termi- nus of KMT2A is replaced by the FG-repeats of NUP98, is a leukemogenic oncogene.
Methods
Primary induction of iNUP98-KMT2A expression
Adult transgenic mice were provided with doxycycline- impregnated chow pellets (400 ppm Doxycycline Diet, Harlan- Teklad) ad libitum from 6-8 weeks of age until analysis. All exper- iments were conducted in compliance with Swiss animal wel- fare laws and were approved by the Swiss Cantonal Veterinary Office of Basel Stadt.
Flow cytometry, colony-forming assays and cell culture
Total BM cells were isolated from wildtype (WT) C57BL/6 and iNUP98-KMT2A mice and processed with the Direct Lineage Cell Depletion kit (Miltenyi Biotec, Bergisch Gladbach, Germany). For immunophenotypic analysis, cells were incubat- ed with antibodies recognizing the mouse lineage markers: CD11b (Mac-1), Ly-6G (Gr-1), CD117 (c-Kit), FcgRII/III, Ter119,
CD71, B220, CD3, and CD34. For lineage marker-negative Sca- 1+ c-Kit+ (LSK) characterization, lineage marker negative (Lin-) BM cells were stained with Ly-6A/E (Sca-1), c-Kit, CD150 (SLAM1) and CD48, as well as CD45.1 and CD45.2 antibodies.
For proliferation experiments, 1x105 Lin- BM cells were cul- tured in liquid media containing murine stem cell factor (100 ng/mL), murine interleukin 3 (6 ng/mL), human interleukin 6 (10 ng/mL) and doxycycline (1 μg/mL). For colony-forming assays, 5x103 Lin- cells were plated in 2 mL of methylcellulose (MethoCult M3434, StemCell Technologies, Vancouver, Canada) and counted after 8-10 days. For cell cycle analysis, cells were fixed for 16 h at 4°C in a 4% paraformaldehyde solution (Thermo Scientific, Monza, Italy) then stained with Hoechst 33342 (Invitrogen, Waltham, USA) and pyronin Y (Sigma, St. Louis, USA) for 40 min at room temperature, flowed for 15 min on ice, before washing in FACS buffer and analysis. For in vitro experiments, unless indicated otherwise, doxycycline was used at a concentration of 1 μg/mL.
PO-PRO-1 and 7-aminoactinomycin D staining of apop- totic cells
Apoptotic cells were quantified using the PO-PRO-1 and 7- aminoactinomycin D staining kit (Invitrogen, Waltham, USA) in accordance with the kit protocol.
Expression analysis of senescence-related genes in iNUP98-KMT2A mouse embryonic fibroblasts
Mouse embryonic fibroblasts (MEF) were generated by isola- tion of E14.5 embryos from iNUP98-KMT2A mice and the geno- type was checked by PCR. To investigate senescence, WT and iNUP98-KMT2A MEF were grown in Dulbecco modified Eagle medium with doxycycline and serially passaged into 100 mm plates when 90% confluence was reached. β-Galactosidase staining was performed using the Senescence β-Galactosidase Staining kit (Cell Signaling, Leiden, the Netherlands) when WT MEF started showing signs of senescence. RNA was extracted from MEF cell pellets at early and late passages and senescence- related gene expression was analyzed by quantitative real-time PCR using a commercially available kit (RT2 Profiler PCR Array, QIAGEN AG, Hombrechtikon, Switzerland) (Online Supplementary Table S2) as well as by manual quantitative PCR using specific primers. Gene expression levels on the RT2 Profiler PCR Array were normalized to an internal panel of housekeep- ing genes, whereas individual quantitative PCR data were nor- malized to Gapdh.
Exposure of leukemic blasts to menin and BET inhibitors
iNUP98-KMT2A and KMT2A-AF9 leukemic blasts were iso- lated and treated for 48 h in vitro with 0-500 nM of the BRD4 inhibitor JQ1/vehicle (dimethylsulfoxide, DMSO) or 0-12 μM of the menin-interaction inhibitor MI-2-2/vehicle (DMSO). Cell cycle analysis was performed at the 48 h time-point after stain- ing with Hoechst 33342 and pyronin Y as described above.
Results
Establishing iNUP98-KMT2A transgenic mice
To address the transforming potential of NUP98- KMT2A, we cloned a full-length human fusion ORF into the p2LOX targeting-vector to integrate it into the Hprt gene locus on the X-chromosome under the control of a doxycycline-responsive element in embryonic stem cells. A reverse Tet transactivator (rtTA) stably integrated in the
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