Stabilization of NF-κB-Inducing Kinase Suppresses MLL-AF9-Induced Acute Myeloid Leukemia

SUMMARY Canonical NF-κB signaling is constitutively activated in acute myeloid leukemia (AML) stem cells and is required for maintenance of the self-renewal of leukemia stem cells (LSCs). However, any potential role for NF-κB non-canonical signaling in AML has been largely overlooked. Here, we report that stabilization of NF-κB-inducing kinase (NIK) suppresses AML. Mechanistically, stabilization of NIK activates NF-κB non-canonical signaling and represses NF-κB canonical signaling. In addition, stabilization of NIK-induced activation of NF-κB non-canonical signaling upregulates Dnmt3a and downregulates Mef2c, which suppresses and promotes AML development, respectively. Importantly, by querying the connectivity MAP using up- and downregulated genes that are present exclusively in NIK-stabilized LSCs, we discovered that verteporfin has anti-AML effects, suggesting that repurposing verteporfin to target myeloid leukemia is worth testing clinically. Our data provide a scientific rationale for developing small molecules to stabilize NIK specifically in myeloid leukemias as an attractive therapeutic option.


Supplemental Experimental Procedures
Mice NIKΔT3 flSTOP mice have been described previously (Sasaki et al., 2008). Vav-Cre mice (Stock Number: 008610) were obtained from the Jackson Laboratory and the ROSA-CreER T2 mice were kindly provided by Dr. Yiying Zhang (Guo et al., 2007). Both strains are in a C57BL6 background and were used at 8-12 weeks old. All the mice used throughout our experiments were NIKΔT3 flSTOP heterozygous, therefore only one allele of NIKΔT3 was activated. Transplant recipients (C57BL/Ka CD45.1) were 8-12 weeks old. The Institutional Animal Care and Use Committee of the University of Iowa approved all animal experiments.

Human Samples
De-identified human AML samples were obtained from the flow cytometry laboratory at the University of Iowa (Institutional IRB Approval #201508729). Mononuclear cells were purified by Ficoll-density gradient centrifugation and enriched for CD34 + or lineage − leukemic stem/progenitor cells by positive or negative selection using the EasySep™ Human CD34 Positive Selection Kit or the Human Progenitor Cell Enrichment Kit, respectively (StemCell Technologies), depending on CD34 positivity of the blasts.

In Vivo Leukemogenesis Assays and Verteporfin Treatment
FACS-sorted lineage − c-kit + Sca-1 + or lineage − c-kit + BM cells from control, ROSACreER T2 , or NIKERT2 mice were cultured in Dulbecco Modified Eagle Medium supplemented with 10% FBS, 50 ng/ml SCF, 50 ng/ml TPO, 50 ng/ml FLT3 ligand, and 10 ng/ml IL-3 (all from Peprotech, Rocky Hill, NJ) overnight. Then the cells were plated on virus-loaded retronectin-coated plates for 24 hours and spin-infected with viral supernatant supplemented with polybrene (4 mg/ml) at 1000 g for 90 min at room temperature and transplanted retro-orbitally into lethally irradiated (950 cGy, single dose) CD45.1 recipients (45-100K/recipient) along with 2-3 × 10 5 rescue cells. Tamoxifen (5 mg/mouse/day, Sigma, St. Louis, MO) was administered daily by oral gavage for 4-5 days at indicated time points post-BM transplantation. For secondary BM transplantations, bulk leukemia cells (300,000/recipient) or sorted leukemia stem cells (500/recipient) from spleens or BM of primary recipient mice were transplanted into sublethally (6 Gy) irradiated CD45.1 recipients. For therapeutic treatments of the primary recipients, verteporfin (Sigma) was given at 140 mg/kg body weight by intraperitoneal injection 1 week after transplantation for 12 days and then observed daily for signs of morbidity.

Cell Proliferation Analysis
For Ki67 staining, leukemia cells from BM or spleen were first stained with surface markers to define the LSC population, followed by fixation and permeabilization with Cytofix/Cytoperm (BD Biosciences) and intracellular staining with Ki67-PE. For drug treatment, leukemia cells were cultured in RPMI with 20% fetal bovine serum (FBS) and 10 ng/ml IL-3 with dimethyl sulfoxide (DMSO) vehicle alone or different small molecules at the indicated concentrations. Cell viability was measured by CellTiter-Blue ® Cell Viability Assay (Promega).

RNA-Seq and Transcriptome Analysis
Total RNA was extracted from sorted LSCs with or without stabilized NIK (GFP ± lin − ckit + Sca1 − CD16 + CD34 + ) from tamoxifen-treated or control leukemic mice, and three biological replicates, each pooled from two control or tamoxifen-treated leukemic mice, were obtained for each genotype. cDNA synthesis and amplification were performed using the SMARTer Ultra Low Input RNA Kit (Clontech) starting with 20 ng of total RNA per sample, following the manufacturer's instructions. cDNA was fragmented with Q800R sonicator (Qsonica) and used as input for NEBNext Ultra DNA Library Preparation Kit (NEB). Libraries were sequenced on Illumina's HiSeq2000 in single-read mode with read length of 50 nucleotides producing 60-70 million reads per sample. Sequence data in fastq format were generated using the CASAVA 1.8.2 processing pipeline from Illumina. We considered genes differentially expressed between two groups of samples when the DESeq2 analysis resulted in an adjusted p-value of <0.01 and the fold-change in gene expression was 1.5 or higher. Heat maps were generated with normalized data of RNA-seq analyses.

Connectivity Map Analysis
The gene expression profiles of control HSPCs and LSCs were compared to those of NIK-stabilized HSPCs and LSCs. Significantly (p £ 0.01 and log2-fold change ³1.5) upand down-regulated genes exclusively present in NIK-stabilized LSCs were used to query