Mice

C57BL/6 J mice were purchased from SLC Japan and maintained in a specific pathogen-free environment at the University of Tokyo animal center. All animal experiments were performed according to the institutional guidelines and protocol (PA21-67) approved by the Laboratory Animal Research Center of the Institute of Medical Science at the University of Tokyo.

Patient samples

The pediatric patients with NPM1::MLF1 fusion or NPM1::CCDC28A were reported by Shiba et al. [3]. The patients’ leukemic samples were obtained from the bone marrow at the time of diagnosis. RNA extraction and cDNA synthesis were performed as previously described [3]. NPM1::MLF1 and NPM1::CCDC28A were amplified by polymerase chain reaction (PCR) using the following primers:

NPM1 Fw: GCTCGAGGCCACCATGGACTACAAGGACGACGATGACAAAGAAGATTC

GATGGACATG,

NPM1::MLF1 Rv: GCGCGGCCGCCAGTTATTTTTTGTTGCTTTTCAC,

NPM1::CCDC28A Rv: TGTCAATGCCAAAAAAAATGCCATTCCAGTGAG.

PCR cycling conditions were as follows: 2 min at 94 °C for one cycle, followed by 25 cycles of 10 s at 98 °C, 30 s at 55 °C, 1 min 30 s at 68 °C, followed by a final extension of 10 min at 72 °C.

Plasmids and retrovirus infection

Flag-tagged forms of the NPM1::MLF1 fusion and NPM1::CCDC28A, wild-type NPM1, NPM1c, MLL::ENL were cloned into the pMYs-IRES-GFP retroviral vector. We generated ectopic retrovirus using Plat-E packaging cells [21] with the calcium-phosphate coprecipitation method. The mouse bone marrow cells were incubated with ectopic retrovirus for 48 h using retronectin (Takara Bio Inc., Otsu, Shiga, Japan)).

Western blotting

HEK293T cells were transiently transfected with the indicated plasmids using polyethyleneimine (PEI). Forty-eight hours after transfection, cells were lysed with cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA; #9803). Lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Bio-Rad). The blot was incubated with anti-Flag (Sigma-Aldrich, catalog F1804, clone M2, 1:200) and anti-GAPDH (Cell Signaling Technology, catalog #5174, clone D16H11, 1:1000) antibodies. Signals were detected with ECL Western Blotting Substrate (Promega, Madison, WI, USA) and visualized with Amersham Imager 600 (GE Healthcare).

Immunostaining

HEK293T cells and K562 cells were transduced with the indicated plasmids using PEI. Twenty-four hours after transduction, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were then permeabilized with 0.2% Triton X-100 for 5 min and blocked with 5% goat serum for 1 h. The HEK293T cells and K562 cells were then stained with anti-flag antibody (Fujifilm, catalog 014-22383), followed by AlexaFluor633-mouse antibody (Invitrogen, catalog A-21052. Cell nuclei were stained with DAPI (BioLegend, catalog 422801). Fluorescence images were captured with an N-SIM super-resolution microscope (Nikon) and were analyzed with NIS-Elements software (Nikon).

Colony replating assay and bone marrow transplantation assay

Eight to ten weeks old mice were treated with 5FU (150 mg/kg, intraperitoneal administration). Bone marrow cells were harvested 4 days after 5FU injection, and were transduced with pMY-IRES-GFP vector, NPM1::MLF1 or NPM1::CCDC28A in RPMI supplemented with 10% fetal bovine serum, mouse SCF 50 ng/μl (R& D, catalog 455-MC), human TPO 50 ng/μl (R& D, catalog 288-TP), mouse FLT3 50 ng/μl (R& D, catalog 427-FL) for 3 days.

For the colony replating assay, 1 × 104 GFP-positive cells were sorted by FACS Aria III (BD Biosciences, San Jose, CA, USA) and plated in M3234 (STEMCELL Technologies) methylcellulose containing 10 ng/mL mouse SCF (R& D, catalog 455-MC), 10 ng/mL mouse IL-3 (R& D, catalog 403-ML), mouse IL-6 (R& D, catalog 406-ML), and 10 ng/μl GM-CSF (R& D, catalog 415-ML). For each round of plating, 1 × 104 cells were plated. Colonies were counted and replated every 7 days. A colony was defined as a cluster of at least 50 cells.

For the bone marrow transplantation assay, 1 × 106 GFP+ cells were injected into 8–12 weeks old C57BL/6 J mice after sublethal irradiation (4.5 Gy). GFP+ leukemia cells were harvested from bone marrows of moribund mice, and were serially transplanted into sublethally irradiated secondary recipient mice by intravenous injection.

Flow cytometry analysis

Bone marrow cells and peripheral blood cells were obtained from leukemic mice. After removal of red blood cells using RBC lysis buffer, the cells were stained with the following antibodies: CD11b-PE-cy7 (BioLegend, 101215), B220-APC-cy7 (BioLegend, 103223), CD3-APC (BioLegend, 100235) and c-kit-PE-Cy7 (BioLegend, 105814). For analysis of mouse lineage- c-Kit+ (LK) cells, the cells were incubated with a cocktail of biotinylated monoclonal antibodies against lineage markers (CD5, B220, CD11b, Gr-1, and Ter119). Cells were then stained with c-kit-PE-Cy7 (BioLegend, 105814), Sca1-APC (BioLegend, 108112), and Streptavidin-Brilliant Violet 605 (BioLegend, 405229) CD34-Alexa flora647 (BioLegend, 119314), CD16/32-PE (BioLegend, 101307). Propidium iodide (PI) and 4’, 6-diamidino-2-phenylindole (DAPI) were used to exclude dead cells. All data were collected using FACS Verse or FACS AriaIII (BD Biosciences, San Jose, CA, USA) and were analyzed with FlowJo software.

Morphological analysis

Cytospin preparations were stained with May-Giemsa staining solution. Images were captured using a BX51 microscope and a DP12 camera (Olympus).

RNA Isolation and RNA sequence

Murine bone marrow cells were transduced with vector, NPM1::MLF1 or NPM1::CCDC28A, and were transplanted into sub-lethally irradiated mice. Ten weeks after transplantation, bone marrow cells were collected from mice and GFP+ cells were sorted by FACS AriaIII (BD Biosciences, San Jose, CA, USA). Total RNA was extracted from GFP+ cells using the RNeasy Mini kit (QIAGEN, 74004), and mRNA was purified from total RNA using poly‐T oligo‐attached magnetic beads. Pair‐end sequencing FASTQ files were aligned to the mouse reference genome (mm10) using HISAT2 30 on the Galaxy platform (https://usegalaxy.org). Raw gene counts were obtained from the read alignments using Subread 31 (v2.4.3) and converted to counts per million (CPM) by edgeR 32 (v3.32.1). After filtering out low‐expressed genes with CPM less than 1, all CPM values were log2 transformed to generate unsupervised clustering dendrograms and heat maps. Differential expression was analyzed with the linear model using limma 33 (v3.46.0). Genes with false discovery rate (FDR) < 0.05 adjusted by the Benjamini‐Hochberg method were considered significant differentially expressed genes (DEGs). For MSigDB gene pathway overlap analysis, DEGs with FDR < 0.05 were examined at http://www.gsea‐msigdb.org/gsea/msigdb/annotate.jsp.

CUT&RUN assay

1 × 105NPM1::CCDC28A cells were collected per sample to perform the CUT&RUN assay using CUT&RUN kit (Cell Signaling, #86652). The CUT&RUN assay was performed according to the protocol provided by Cell Signaling (https://www.cellsignal.com/learn-and-support/protocols/cut-and-run-protocol) with an anti-flag antibody (FUJIFILM catalog 014-22383) and Rabbit (DA1E) mAb IgG XP® Isotype control antibody (Cell Signaling#66362) as a negative control.

Quantitative RT-PCR

Total RNA was extracted using the RNeasy Mini kit (QIAGEN) and reverse transcribed (RT) using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, 4368814). The cDNA was then subjected to quantitative RT-PCR using a SYBR Select Master Mix (Applied Biosystems). The sequences of the 5ʹ to 3ʹ primers used for RT-PCR in this study, from 5ʹ to 3ʹ are as follows:

mouse Hoxa3 Forward CTCATTTTGCGGTCGCTATCC

mouse Hoxa3 Reverse ATCCATGCCATTGTAGCCGTA

mouse Hoxa9 Forward ACAATGCCGAGAATGAGAGC

mouse Hoxa9 Reverse GTTCCAGCGTCTGGTGTTTT

mouse Hoxa10 Forward CAGCCCCTTCAGAAAACAGT

mouse Hoxa10 Reverse TCTTTGCTGTGAGCCAGTTG

mouse Hoxa11 Forward GGCCACACTGAGGACAAGG

mouse Hoxa11 Reverse GAACTCTCGCTCCAGCTCTC

mouse Gapdh Forward TTGATGGCAACAATCTCCAC,

mouse Gapdh Reverse CGTCCCGTAGACAAAATGGT

ChIP-qPCR

Chromatin immunoprecipitation (ChIP) was performed using Simple chip kit (Cell Signaling Technology, #9002) with antibodies against Flag (Sigma-Aldrich, catalog F1804, clone M2, 1:200) according to the manufacturer’s protocol. The purified DNA was then used for qPCR using a SYBR Select Master Mix (Applied Biosystems). The sequences of the 5‘ to 3‘ primers used for ChIP-qPCR in this study are as follows:

mouse Hoxb9 Forward CCACCGACTGGCTTCCTCGC

mouse Hoxb9 Reverse CCAGGGGTCACACCTCCCCA

mouse Hoxa9 Forward AGGAGTCGCTGCTTTCTGTT

mouse Hoxa9 Reverse ATTAGAACGGGGAGGGGTAA

mouse Hoxa10 Forward TAGATGCTTGCAGAAGGAAAGG),

mouse Hoxa10 Reverse CCATATGGCAAGAGGCAAAGA

Dual luciferase Assay

HEK293T cells were seeded in 24-well plates and co-transfected with 560 ng of each plasmid (pMYs-IG vector, NPM1::MLF1, NPM1::CCDC28A, wild-type NPM1, NPM1c or MLL::ENL), 3.8 ng pGL4.74 vector and 190 ng HoxA9 reporter [22] using PEI. Forty-eight hours after transfection, cells were lysed and firefly and renilla luciferase activities were measured with the Dual-Luciferase Assay System (Promega) according to the manufacturer’s protocol on a FLUOstar Optima.

Selinexor treatment

Selinexor (KPT-330) was purchased from Selleckchem (catalog# S7252). For the colony forming assay, 1 × 104NPM1::MLF1 or NPM1::CCDC28A expressing AML cells were cultured in M3234 (STEMCELL Technologies) methylcellulose containing 10 ng/μl mouse SCF (R& D, catalog 455-MC), 10 ng/μl mouse IL-3(R& D, catalog 403-ML), 10 ng/μl mouse GM-CSF (R& D, catalog 415-ML) together with the indicated concentrations of selinexor (10 nM, 25 nM, 50 nM, and 100 nM). The number of colonies was counted after seven days of treatment with selinexor.

For the luciferase assay, HEK293T cells were seeded in 24-well plates and co-transfected with 560 ng of each plasmid (pMYs-IG vector, NPM1::MLF1, NPM1::CCDC28A, wild-type NPM1, NPM1c), 3.8 ng pGL4.74 vector and 190 ng HoxA9 reporter using PEI. Selinexor (100 nM) was added to some cultures 24 h after transfection. Reporter activity was measured 48 h after transfection.

Cell viability assay

Cells were plated into flat-bottomed 96-well plates at a density of 5 × 104 for NPM1::CCDC28A cells, 1 × 105 for cord blood CD34+ cells, or 1 × 105 for mouse c-kit+ bone marrow cells per well. Cells were incubated for 72 h with Selinexor (Selleckchem catalog#S7252) at 0, 0.025, 0.05, 0.1, 0.25, 0.5, and 1 μM, or VTP50469 (MedChemExpress catalog# HY-114162) at 0, 0.1, 0.3, 1, 3, 10, and 30 μM. The growth inhibitory effect of the drugs was evaluated both individually and in combination. Following the treatment, 8 µL of Cell Counting Kit 8 (Dojindo Molecular Technologies) was added to each well, and the plates were incubated at 37 °C for an additional 1 h. The absorbance of each sample was then measured using a CLARIOstar Plus Microplate Reader (BMG LABTECH) at 450 nm.

Statistical analysis

GraphPad Prism 9 was used for statistical analysis. Two-tailed unpaired t test was used for pairwise comparisons, and one-way ANOVA was used for multiple comparisons of significance. The p value (<0.05, <0.01, <0.001) is indicated by one to three asterisks (*, **, ***). p values higher than 0.05 were considered not significant (ns).

Ethics approval and consent to participate

The present study was conducted in accordance with the Declaration of Helsinki and approved by the institutional review boards of Gunma Children’s Medical Center and the participating institutes and the ethical review board of the JPLSG AML-05 trial. Written informed consent was provided by all patients or their parents/guardians.