. 2019 Apr 24;5(4):eaau8389.

doi: 10.1126/sciadv.aau8389. eCollection 2019 Apr.

RUNX represses Pmp22 to drive neurofibromagenesis

Ashley Hall¹, Kwangmin Choi¹, Wei Liu¹, Jonathan Rose¹, Chuntao Zhao¹, Yanan Yu^{1

2}, Youjin Na¹, Yuqi Cai¹, Robert A Coover¹, Yi Lin¹, Eva Dombi³, MiOk Kim⁴, Ditsa Levanon⁵, Yoram Groner⁵, Elisa Boscolo^{1

6}, Dao Pan^{1

6}, P Paul Liu⁷, Q Richard Lu^{1

6}, Nancy Ratner^{1

6}, Gang Huang^{1

6}, Jianqiang Wu^{1

6}

Affiliations

¹ Cincinnati Children's Hospital Medical Center, Division of Experimental Hematology and Cancer Biology, Cancer and Blood Diseases Institute, University of Cincinnati, 3333 Burnet Ave., Cincinnati, OH 45229, USA.
² Department of Cancer and Cell Biology, College of Medicine, University of Cincinnati, Cincinnati, OH 45267, USA.
³ Pediatric Oncology Branch, National Cancer Institute, Bethesda, MD 20892, USA.
⁴ Department of Epidemiology and Biostatistics, UCSF, Box 0128, 1450 3rd St. Suite 285, San Francisco, CA 94143, USA.
⁵ Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
⁶ Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.
⁷ National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

PMID: 31032403
PMCID: PMC6482019
DOI: 10.1126/sciadv.aau8389

RUNX represses Pmp22 to drive neurofibromagenesis

Ashley Hall et al. Sci Adv. 2019.

. 2019 Apr 24;5(4):eaau8389.

doi: 10.1126/sciadv.aau8389. eCollection 2019 Apr.

Authors

Affiliations

¹ Cincinnati Children's Hospital Medical Center, Division of Experimental Hematology and Cancer Biology, Cancer and Blood Diseases Institute, University of Cincinnati, 3333 Burnet Ave., Cincinnati, OH 45229, USA.
² Department of Cancer and Cell Biology, College of Medicine, University of Cincinnati, Cincinnati, OH 45267, USA.
³ Pediatric Oncology Branch, National Cancer Institute, Bethesda, MD 20892, USA.
⁴ Department of Epidemiology and Biostatistics, UCSF, Box 0128, 1450 3rd St. Suite 285, San Francisco, CA 94143, USA.
⁵ Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
⁶ Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.
⁷ National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

PMID: 31032403
PMCID: PMC6482019
DOI: 10.1126/sciadv.aau8389

Abstract

Patients with neurofibromatosis type 1 (NF1) are predisposed to develop neurofibromas, but the underlying molecular mechanisms of neurofibromagenesis are not fully understood. We showed dual genetic deletion of Runx1 and Runx3 in Schwann cells (SCs) and SC precursors delayed neurofibromagenesis and prolonged mouse survival. We identified peripheral myelin protein 22 (Pmp22/Gas3) related to neurofibroma initiation. Knockdown of Pmp22 with short hairpin RNAs increased Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre tumor-derived sphere numbers and enabled significantly more neurofibroma-like microlesions on transplantation. Conversely, overexpression of Pmp22 in mouse neurofibroma SCs decreased cell proliferation. Mechanistically, RUNX1/3 regulated alternative promoter usage and induced levels of protein expression of Pmp22 to control SC growth. Last, pharmacological inhibition of RUNX/core-binding factor β (CBFB) activity significantly reduced neurofibroma volume in vivo. Thus, we identified a signaling pathway involving RUNX1/3 suppression of Pmp22 in neurofibroma initiation and/or maintenance. Targeting disruption of RUNX/CBFB interaction might provide a novel therapy for patients with neurofibroma.

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Figures

**Fig. 1. Dual deletion of Runx1/3 prolongs survival time and decreases tumor number and size in the *Nf1^fl/fl;DhhCre* neurofibroma mouse.**
(A) Kaplan-Meier survival curve. Purple, *Runx1^fll+;Runx3 ^fl/+;Nf1^fllfl;DhhCre;* green, Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre; red, Runx1^fl/fl;Runx3^fl/fl;Nf1^fl+;*DhhCre*. (B) Each mouse volume at 7 and 12 months in controls (*Runx1^fll+;Runx3 ^fl/+;Nf1^fllfl;DhhCre* mice) (left) and Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre mice (right). Dashed lines indicate all the control mice volume >40 mm³ at 7 months, while the double-knockout mice volume varied. Mouse tumor volume statistical analysis: Mixed model analysis. Mixed-effects analysis of tumor volume showed P < 0.001 between *Runx1^fll+;Runx3 ^fl/+;Nf1^fllfl;DhhCre* mice and Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre mice at 7 and 12 months, respectively. (C) Representative gross dissections of thoracic paraspinal neurofibromas and nerve roots in 12 months of age-matched WT (left), *Nf1^fl/fl*;*DhhCre* mice (second from the left), Runx1^fl/fl;Nf1^fl/fl;DhhCre mice (third from the left), and *Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre* mice (right). White arrows point to tumors. Ruler showed 1-mm markings. (Photo credit: Ashley Hall and Jianqiang Wu). (D) Representative photographs of toluidine blue–stained semithin sections from age-matched WT (left), *Nf1^fl/fl*;*DhhCre* (middle), and *Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre* (right) mouse DRGs. Top-left corner insertions are high magnification of imagings to each corresponding genotype. (E) Tumor diameter in the *Runx1^fll+;Runx3 ^fl/+;Nf1^fllfl;DhhCre* mice (circles; n = 3 mice with 20 tumors) and Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre mice (squares; n = 4 mice with nine tumors) at 12 months. (F) Average tumor number per mouse at 12 months in the *Runx1^fll+;Runx3^fl/+;Nf1^fllfl;DhhCre* mice (black bar; n = 3) and Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre mice (white bar; n = 4). (G) Cell proliferation shown as percentage of BrdU⁺ cells in *Runx1^fll+;Runx3^fl/+;Nf1^fllfl;DhhCre* mice (black bar; n = 5) and Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre mice (white bar; n = 5). (H) Cell death shown as percentage of CC3⁺ cells in *Runx1^fll+;Runx3^fl/+;Nf1^fllfl;DhhCre* mice (black bar; n = 5) and Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre mice (white bar; n = 5). (D to G) Unpaired Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001. *R1^fl/fl;R3^fl/fl;Nf1^fl/fl;DhhCre* = *Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre*.

**Fig. 2. Combined transcriptome profiling, ChIP-seq, and ATAC-seq analysis identifies 14 highly plausible direct targets of Runx.**
(A) Heat maps showed the antibody-binding pattern around the TSSs region. Each panel represents 3 kb upstream and downstream of the TSSs. Left, IgG control; right, RUNX1 antibody. (B) Circle chart depicted RUNX1-binding site distribution in relation to the nearest annotated TSSs. Numbers represented distance from TSS (kb), and percentages represented percentage of bound regions. (C) Venn diagram showed the overlap of genes among RNA-seq DEGs, ChIP-seq peak, and ATAC-seq peak. For ChIP-seq and ATAC-seq, we consider differential peaks detected within ±20 kb around TSS. (D) Heat map showed the 14 commonly shared genes from *Nf1^fl/fl;DhhCre* and Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre tumor RNA-seq. (E) qRT-PCR confirmed the relative mRNA expression of the 14 targets. (F) Representative two genes (Rnf157: up-regulated gene; Ccnd1: down-regulated gene) showed their RNA-seq gene expression (blue: down-regulation; red: up-regulation), RUNX1-binding ChiP-seq peak, and ATAC-seq peak. *P < 0.05, **P < 0.01.

**Fig. 3. Loss of *Nf1* mediated PMP22 functions as tumor suppressor to contribute to neurofibroma initiation.**
(A) Venn diagram showed the overlap of the 14 RUNX1/3 target genes with human neurofibroma-initiating cell microarray DEGs. (B) Western blot of PMP22 in WT mouse sciatic nerves (1 to 3), *Nf1^fl/fl;DhhCre* tumors (4 to 6), and *Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre* tumors (7 to 9). β-Tubulin was used as a loading control. (C) *ShPmp22* partially rescued numbers *of Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre* spheres. **P < 0.01. (D) Western blot showed the levels of *Pmp22* knockdown by *shPmp22*. Numbers indicated the percentage of knockdown. (E) Neurofibroma-like lesions formed after subcutaneous injection of *shNT*- or *shPmp22*-transduced *Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre* DRG/neurofibroma-derived sphere cells. Statistics: Fisher’s exact test. P < 0.05. (F) A representative gross photograph of a lesion (dashed black circle) under reflected skin in a *nu/nu* mouse injected with *shPmp22*-transduced *Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre* DRG/neurofibroma-derived sphere cells. Ruler showed 1-mm markings. (Photo credit: Ashley Hall and Jianqiang Wu). (G) H&E-stained section of the tumor from (G) showed spindle cells (white arrows). (H) IHC showing S100β⁺ cells (brown; white arrows) in tumor. Blue is H&E counterstain. (I) Toluidine blue staining showed mast cells (black arrows). (J) Electronic micrograph showed lesions containing SCs, identified by continuous basal lamina in high magnification (white arrows).

**Fig. 4. *Pmp22* inhibits mouse neurofibroma SC proliferation, and overexpression of *Pmp22* induces cell senescence.**
(A) Western blot showing PMP22 expression on lentivirus control (Ctrl) or PMP22 overexpression (OE) lentivirus-transduced mouse neurofibroma SCs. β-Tubulin was used as a loading control. (B) MTS assay showing significantly decreased cell growth in Pmp22 overexpressing mouse neurofibroma SCs compared with lentivirus control cells starting at day 3 and stopping cell growth at day 5. OD, optical density. (C) Representative EdU (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) images on control (top) and Pmp22 overexpressing (bottom) mouse neurofibroma SCs. (D) Quantification of percentage of EdU⁺ cells on control (black bar) and Pmp22 overexpressing cells (white bar). (E) qRT-PCR of the relative mRNA expression of Pmp22 on Δ*Runx* cells normalized to control (n = 3 per group). (F) Representative Western blot of Pmp22 on three independent controls and three independent ΔRunx cells. (G) Representative bright-field images of mouse neurofibroma SCs. Top (control): Cells were transduced by lentivirus, but the RUNX-*Pmp22*–binding sites remained intact. Bottom (Δ*Runx*-*Pmp22*): Homozygous deletion of five putative RUNX-binding sites in *Pmp22* gene by CRISPR-Cas9 approach (Δ*Runx*). (H) Representative EdU (green) and DAPI (blue) images on control (top) and Δ*Runx* (bottom) mouse neurofibroma SCs. (I) Quantification of percentage of EdU⁺ cells on control (white bar; n = 3) and Δ*Runx* cells (light gray bar; n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001.

**Fig. 5. *Runx1/3* regulates the alternative promoter usage and affects posttranslational modification of *Pmp22*.**
(A) Upper two bands: RUNX1 ChIP-seq showed the binding peak near *Pmp22* (blue box). IgG was used as control. Lower two bands: ATAC-seq on *Nf1^fl/fl;DhhCre* and *Runx*1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre showed more *Pmp22* P2 open chromatin in *Nf1^fl/fl;DhhCre* SCs (blue box). Top-right corner arrow pointed to TSS. Underneath, red bars indicated that *Pmp22* mRNA expression was up-regulated in *Runx*1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre versus *Nf1^fl/fl;DhhCre* mice. Ab, antibody. (B) Ratio of normalized *Pmp22* P1 (white bar) and P2 (light gray bar) transcript abundance in *Runx*1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre (R1R3Nf1Ko) versus *Nf1^fl/fl;DhhCre* (Nf1Ko) from bulk tumor RNA-seq data. (C) qRT-PCR showed *Pmp22* P1 (white bar) and P2 (light gray bar) relative mRNA expression in *Runx1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre* (R1R3Nf1Ko) over *Nf1^fl/fl;DhhCre* (Nf1Ko) from FACS-sorted EGFP⁺ cells. n.s., not significant. (D) Diagram showed the cloning strategy of Pmp22 P1 5′-UTR or P2 5′-UTR with CMV into pGL2 luciferase reporter assay vector. Only CMV was used as control. (E) Luciferase reporter assay showed that P2 5′-UTR (CMV-P2) had less posttranscriptional modification activity compared with P1 5′-UTR (CMV-P1) in immortalized WT mouse SCs. (F) Representative immunofluorescence images of GM130 (green) and PMP22 (red) in WT (top), *Nf1^fl/fl;DhhCre* (middle), and *Runx*1^fl/fl;Runx3^fl/fl;Nf1^fl/fl;DhhCre (bottom) mouse neurofibroma/DRG SCs. White arrows pointed to GM130 (Golgi body) and PMP22 colocalization. *P < 0.05 and **P < 0.01.

**Fig. 6. RUNX/CBFB interaction inhibitor, Ro5-3335, significantly decreases mouse neurofibroma growth in vivo.**
(A) Waterfall plot showed mouse tumor volume change. Data from each mouse were shown as a single bar. Change in tumor volume was quantified between 7 and 9 months for each individual mouse treated with vehicle control (control) (black bars; n = 13) or Ro5-3335 (gray bars; n = 10) for 8 weeks. (B and C) Ro5-3335–inhibited neurofibroma cell proliferation readout by the percentage of Ki67⁺ cells. (D and E) Ro5-3335–induced neurofibroma cell apoptosis as assessed by the percentage of CC3⁺ cells. (F and G) Relative mRNA expression of Ro5-3335–treated mouse neurofibromas compared with vehicle controls on up- (F) and down-regulated (G) genes. (H) Western blot of PMP22 in *Nf1^fl/fl;DhhCre* tumors (1 and 2) and Ro5-3335–treated *Nf1^fl/fl;DhhCre* tumors (3 and 4). β-Actin was used as loading control. *P < 0.05 and **P < 0.01.

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RUNX represses Pmp22 to drive neurofibromagenesis

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RUNX represses Pmp22 to drive neurofibromagenesis

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