RUNX represses Pmp22 to drive neurofibromagenesis

RUNX 1 and RUNX3 drive Nf1 neurofibromagenesis by mediating the promoter usage and inducing levels of protein expression of PMP22.


INTRODUCTION
Neurofibromatosis type 1 (NF1) is a common inherited human disorder, with a frequency of 1:2500 worldwide (1). About 95% of patients with NF1 develop dermal and/or plexiform neurofibromas, for which no effective cure exists. Often, surgery removal is impossible because of the integration of tumor into critical peripheral nerves (1). The cytostatic MAPK (mitogen-activated protein kinase) kinase inhibitor selumetinib has shown promising efficacy, but tumors regrow after stopping the treatment (2,3). New therapeutic strategies and targets for the treatment of neurofibromas are urgently needed.
The drivers of neurofibromagenesis have not been fully identified. NF1 encodes a RAS guanosine triphosphatase-activating protein that activates downstream RAS pathways. Therefore, loss of NF1 is considered a potential major driver of neurofibromagenesis (4,5). Other genes, such as STAT3 and EGFR, also contribute to neurofibroma formation (6,7). However, none of them can eliminate tumor formation, suggesting that neurofibroma formation involves multiple drivers.
Three RUNT-related transcription factor (RUNX1, RUNX2, and RUNX3) genes are present in human and in mouse. Their gene products share many structural similarities but have distinct biological activities. RUNX1 (also called AML1) is required for the maturation of megakaryocytes and differentiation of T and B cells. RUNX2 is critical for skeletal morphogenesis (8). RUNX3 is important for neu-rogenesis of proprioceptive neurons in the dorsal root ganglia (DRG) and for hematopoiesis (9,10). All three RUNX proteins bind to the core-binding factor  (CBFB) via the same protein motif. CBFB lacks a DNA binding domain but, when bound to RUNX, substantially increases the Runt domain DNA binding affinity, thereby enhancing RUNX transcriptional activities (11). RUNX can function as either tumor suppressors or oncogenes. RUNX1 has been implicated as a tumor suppressor in solid tumors, including breast cancer, esophageal adenocarcinoma, colon cancer, and possibly, prostate cancer and as an oncogene in skin cancer, endometrial cancer, and epithelial cancer (12). RUNX2 has been implicated in metastasis including bone metastasis. RUNX3 acts as a tumor suppressor in gastric cancer but functions as an oncogene in ovarian cancers (12). Extracellular signal-regulated kinase (ERK) signaling (downstream of NF1/RAS/MAPK) phosphorylates RUNX1 at S276/S293 or arginine methylation of R206 and R210 (13). ERK phosphorylation caused by loss of NF1 increases RUNX1 activity and contributes to neurofibroma formation (14).
Schwann cells (SCs) develop from neural crests, and Nf1 −/− SCs and/or their precursors are cells of origin for neurofibromas (15,16). There are both myelinating and nonmyelinating SCs within a neurofibroma, and neurofibromas harbor increased Remak bundleassociated (nonmyelinating) SCs. One of the SC's functions is to form a myelin sheath containing the peripheral myelin protein 22 (PMP22). PMP22 is mainly expressed in myelinating SCs (17,18) and neurons (19). Its expression is regulated via transcriptional regulation and posttranscriptional modification. Pmp22 has two major different mRNAs that differ only in their 5′-untranslated regions (5′-UTRs). This difference causes alternative usage of two promoters located upstream of the exons 1A (P1) and 1B (P2). Both P1 and P2 are developmentally regulated in SCs and contribute to Pmp22 levels in mature SCs. P1 is SC specific, and P2 is more often used in other tissues where Pmp22 is expressed at a lower level (17,18). Increased PMP22 in gene dosage results in the inherited demyelinating peripheral neuropathies Charcot-Marie-Tooth disease type 1A (20), while decreases cause hereditary neuropathy with liability to pressure palsies (21).
The PMP22 gene expression has been detected within and outside the nervous system and in several cancers, and a number of studies have reported either up-or down-regulation of the gene/ protein in different models. Amplification of chromosome 17p11.2 region and the PMP22 protein expression have been associated with human osteosarcoma and glioblastoma (22). The PMP22 gene/protein expression is markedly up-regulated in gastric cancer-derived tumorspheric cells and contributes to cell proliferation, tumorsphere formation, and chemoresistance to cisplatin (23). Conversely, the PMP22 gene expression is diminished in metastatic carcinoma cells compared with primary carcinoma cells, suggesting that it might serve as a prognostic marker (24). Here, we showed that dual deletion of mouse Runx1/3 in SCs and SC precursors (SCPs) significantly delayed neurofibromagenesis and prolonged mouse survival. We showed that RUNX1/3 regulated Pmp22 expression by switching alternative promoter usage and markedly induced levels of protein expression of Pmp22 to drive neurofibromagenesis. We also showed that pharmacological inhibition of RUNX/CBFB activity significantly reduced mouse neurofibroma growth in vivo, implicating a novel signaling pathway involving RUNX1/3 repression of Pmp22 in neurofibroma initiation and/or maintenance.

Conditional knockout of Runx1 transiently delays neurofibroma growth and induces compensatory overexpression of Runx3 in the Runx1 fl/fll ;Nf1 fl/fl ;DhhCre mouse model
We have previously shown that targeted genetic deletion of Runx1 in SCs and SCPs decreases neurofibroma formation at 4 months (14), but we found that there was no difference in tumor volume at 7 months of age (P = 0.38) (fig. S1A). Because all three RUNX proteins (with CBFB) bind to the same DNA motif to exert their effects, it is possible that phenotypes observed upon conditional inactivation of Runx1 were attenuated by compensation of Runx2 and/or Runx3. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) on Runx1 fl/fl ;Nf1 fl/fl ;DhhCre mouse tumors/DRGs and age-matched Nf1 fl/fl ;DhhCre tumors indicated that Runx3 expression displayed a pronounced time-dependent, increase (fig. S1B). Immunohistochemistry (IHC) on 7-month-old Runx1 fl/fl ;Nf1 fl/fl ;DhhCre mouse DRG/tumors verified stronger Runx3 expression compared with age-matched Nf1 fl/fl ;DhhCre tumors (fig. S1C), suggesting induced compensation of Runx3 upon conditional knockout of Runx1.
To determine whether Runx3 affects Runx1 knockout SCPs, we used shRunx3 to transduce Runx1 fl/fll ;Nf1 fl/fl ;DhhCre DRG/tumor-derived mouse neurofibroma spheres. We found a significant decrease in the numbers of neurofibroma spheres in all three tested clones compared with shnon-target control (shNT; fig. S1D), indicating that Runx3 compensated for the tumor growth upon conditional inactivation of Runx1 in the Nf1 fl/fl ;DhhCre cells.

Runx1/3 drives neurofibromagenesis by activating oncogenic pathways and reprogramming the neuronal and immune systems
We performed RNA sequencing (RNA-seq) to investigate whether Runx1/3 mediates changes in gene regulation involved in neurofibromagenesis. To avoid the complexity of heterozygous knockout of Runx1/3, we compared RNA-seq gene expression profiles of Nf1 fl/fl ;DhhCre tumors and Runx1 fl/fl ;Runx3 fl/fl ;Nf1 fl/fl ;DhhCre mouse DRG/tumors using a false discovery rate (FDR) P value of <0.05 and >2-fold change as the significant threshold. We identified 2887 differentially expressed RUNX1/3-responsive genes, of which 1677 genes were up-regulated and 1210 genes were down-regulated in immune-related pattering ( fig. S2C). In contrast, extensive activation of pathways associated with SC development and myelination or apoptosis, including neuronal system, axon guidance, diacylglycerol (DAG). and inositol 1,4,5-trisphosphate signaling, oxidative phosphorylation, and ion channel transportation, was remarkably upregulated ( fig. S2D).
RUNX mediates changes in chromatin structure (25). We performed assay for transposase-accessible chromatin sequencing (ATAC-seq) to determine whether RUNX is associated with changes in chromatin accessibility in our system. To avoid the complexity of cell type heterogeneity within a tumor, we FACS  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.

Pmp22 is a direct target of RUNX1/3 in neurofibroma
We previously showed that P75 + /EGFR + SCs are potential neurofibromainitiating cells (26) and that they showed differential gene expression profile compared with WT P75 + /EGFR − SCs. To identify genes that might be related to neurofibroma initiation, we compared these 14 genes to those overexpressed in a neurofibroma tumor-initiating cell microarray dataset GSE122773 (14). We identified Pmp22 as the only common gene (Fig. 3A). The Pmp22 expression was confirmed by qRT-PCR (Fig. 2E). Western blots showed that PMP22 protein was expressed in WT mouse sciatic nerves but was almost undetectable in Nf1 fl/fl ;DhhCre mouse neurofibromas, while robust expression in Runx1 fl/fl ;Runx3 fl/fl ;Nf1 fl/fl ;DhhCre mouse DRG/tumors was observed (Fig. 3B). We analyzed the existing transcriptomic data (www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE41747 for mouse and www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE14038 for human). We found decreased gene expression on the SC differentiation/myelination markers (PMP22, MPZ, and MBP) in both mouse and human plexiform neurofibromas versus WT nerve controls. On the contrary, RUNX family gene (RUNX1, RUNX2, and RUNX3) expression was increased in plexiform neurofibromas ( fig. S4, A and B), suggesting that RUNX factors might drive the dedifferentiation/myelination of SCs in our model.
Reports show that Pmp22 is also regulated by other genes such as Sox10, Egr2, and Hippo regulators (27). To determine whether these regulators are related to Pmp22 expression in our system, we searched the gene expression change on the Runx1 fl/fl ;Runx3 fl/fl ;Nf1 fl/fl ;DhhCre versus Nf1 fl/fl ;DhhCre tumor RNA-seq data. We found that there was no significant differential expression among these genes ( fig. S5), suggesting that more sample replicates might be needed or these regulators might be less relevant to Pmp22 expression upon loss of Nf1.

Loss of Nf1 mediated PMP22 functions as tumor suppressor to contribute to neurofibroma initiation
We used neurofibroma sphere culture, a system enabling detection of growth and self-renewal of nervous system stem cells/progenitors, to determine whether Pmp22 was related to SCP growth upon loss of Nf1. We knocked down Pmp22 from Runx1 fl/fl ;Runx3 fl/fl ;Nf1 fl/fl ;DhhCre mouse DRG/neurofibroma-derived spheres, which has high Pmp22 expression using shPmp22s or shNT. Sphere numbers were significantly increased 4 days after shPmp22 transduction versus shNT controls (Fig. 3D). The overall average sphere size was similar (not shown). We confirmed decreased Pmp22 by Western blot (Fig. 3E). Thus, loss of Runx1/3 increased Pmp22 expression in Runx1 fl/fl ;Runx3 fl/fl ;Nf1 fl/fl ;DhhCre, while decreasing Pmp22 expression increased Runx1 fl/fl ;Runx3 fl/fl ;Nf1 fl/fl ;DhhCre SCP's self-renewal in vitro.
In many cancers, self-renewing stem/progenitor-like cells contribute to tumorigenesis. To test whether tumor formation was affected by Pmp22 reduction, we transplanted the above dissociated shPmp22 or shNT control sphere cells into nude mice (nu/nu). Eight weeks after transplantation, neurofibroma-like microlesions were detected in 8 of 10 nu/nu mice, which were transplanted subcutaneously with shPmp22 lentivirus-transduced Runx1 fl/fl ;Runx3 fl/fl ;Nf1 fl/fl ;DhhCre mouse DRG/tumor-derived sphere cells, while fewer lesions (3 of 10) were detected in shNT controls (P < 0.05) (Fig. 3, E and F). H&E staining of tissue sections showed features of neurofibroma-like lesion with spindle shape cells (Fig. 3G), anti-S100 + SCs (Fig. 3H), and toluidine blue-positive mast cells (Fig. 3I). On electron micrographs, these lesions contained SCs, identified by their wrapping of axons and continuous basal lamina (Fig. 3J). These in vitro and in vivo genetic results supported the conclusion that Pmp22 was an important target of Runx1/3-mediated neurofibromagenesis.

Overexpression of Pmp22 in mouse neurofibroma SCs decreases cell proliferation
We overexpressed Pmp22 in mouse neurofibroma SCs to further determine the role of Pmp22 on SC growth. Western blot confirmed Pmp22 expression on these selected cells (Fig. 4A). MTS assay showed that cell growth decreased significantly by passage 4 cells (day 3), and almost no cell growth was detected from days 5 to 7 (Fig. 4B). 5-ethynyl-2′-deoxyuridine (EdU) assays showed significantly decreased cell proliferation (21 days after puromycin selection) (Fig.4, C and D) but no difference in apoptosis as detected by CC3.

Deletion of RUNX-binding sites in Pmp22 gene decreases cell proliferation and increases Pmp22 expression
ChIP-seq identified five putative RUNX-binding sites (TGTGG) in the Pmp22 gene ( fig. S6A). To determine the importance of these RUNX-Pmp22-binding sites, we used a CRISPR-Cas9 approach to delete this sequence in mouse neurofibroma SCs. We identified four independent homozygous deletion clones (clones #12, #65, #79, and #85; designated Runx). We confirmed the deletion by sequencing ( fig. S6, B and C). Consistent with the in vivo genetic results, qRT-PCR showed increased expression of PMP22 at both RNA and protein levels on the analyzed homozygous clones compared with controls ( Fig. 4, E and F). We noted that the in vitro Pmp22 RNA expression change was similar to the genetic data (~2-folds), but the induction of protein levels was much less marked compared with the in vivo genetic data, and we also detected PMP22 protein expression in WT controls in Western blot. One of the possible explanations is that these cells have been cultured in vitro for a long time (~4 months) in the presence of forskolin, which can increase both SC PMP22 mRNA and protein expression in vitro. The Runx cells showed decreased cell growth on bright microscopy (Fig. 4G), EdU + proliferating cells decreased significantly as compared with cells transduced by lentivirus, but the five binding sites remained intact (Fig. 4, H and I). No difference was detected on the number of apoptotic cells (not shown). The PMP22 expression increased in Runx cells in immunofluorescence staining. However, the PMP22 protein expression was retained in perinuclear-reticular structures, especially in Golgi body (not shown).

RUNX1/3 regulates Pmp22 promoter usage
Pmp22 has four protein-coding transcript variants [ENSMUST00000108702 (encoding using P1 promoter), ENSMUST0000001836 (encoding using P2 promoter), ENSMUST00000108700, and ENSMUST00000108701] (www.ensembl.org). Although all four transcripts can produce the same protein product, the preferential usage of each transcript depends on tissue types and developmental context. Analysis of chromatin accessibility by ATAC-seq revealed a preponderance of open chromatin accessibility at P2 region in FACS-sorted Nf1 fl/fl ;DhhCre SCs compared with Runx1 fl/fl ;Runx3 fl/fl ;Nf1 fl/fl ;DhhCre tumor SCs (Fig. 5A). Notably, Pmp22 mRNA relative expression fold change (Fig. 2E) was lower than bulk tumor RNA-seq analysis (2.3×; table S1). One of the possibilities is that different transcripts (promoters) might contribute to this difference because RNA-seq only identified statistically significant expressed transcripts, while the primers we used in the bulk tumor qRT-PCR recognized all four transcripts. To determine whether that was the case, we checked the abundance of each transcript from bulk tumor RNA-seq data using Kallisto. We found that the abundances of ENSMUST00000108700 and ENSMUST00000108701 were negligible. Only ENSMUST00000108702 and ENSMUST0000001836 were abundant. Runx1 fl/fl ; Runx3 fl/fl ; Nf1 fl/fl ;DhhCre P1 transcript was 2.3-fold up-regulated compared with Nf1 fl/fl ;DhhCre (P < 0.05; Fig. 5B). The P2 transcript was ~2× more abundant than the P1 transcript in both tumors, but no statistically significant differential expression was detected between; therefore, P2 transcript's effect on the gene-level differential change could be small in the bulk tumor RNA-seq. To exclude the influence of Pmp22 RNAs from WT SCs and possible other tissues in bulk tumors, we checked the above two transcript variants' relative mRNA expression levels on FACS-sorted EGFP + SCs using special primers that only recognized each transcript by qRT-PCR. Consistent with the RNA-seq data, we found that only P1 transcript showed statistically significant differential expression in Runx1 fl/fl ;Runx3 fl/fl ;Nf1 fl/fl ;DhhCre versus Nf1 fl/fl ;DhhCre, with a 2.7-fold up-regulation (P < 0.05; Fig. 5C). These results indicated that P1 transcript was the actual contributor on Pmp22 gene expression in SCs in both genotypes, while in Nf1 fl/fl ;DhhCre tumor cells, P2 might have been used to drive Pmp22 expression in other tissues.
It is previously reported that PMP22 protein expression might be partially influenced by the conserved alternative 5′-UTR (28). RegRNA2 search (http://regrna2.mbc.nctu.edu.tw) showed that there was a significant difference in the motif compositions of P1 and P2 5′-UTR regions; therefore, they might affect posttranscriptional control and translation efficiency. Thus, we adapted a luciferase reporter assay system to test whether the 5′-UTRs of the Pmp22 P1 and P2 transcripts affect the PMP22 translation in our system. The EGFP luciferase was activated by two components: a cytomegalovirus (CMV) promoter and the P1 5′-UTR or P2 5′-UTR (Fig. 5D). CMV alone caused low luciferase transcriptional activity but was markedly increased by P1 5′-UTR or P2 5′-UTR (Fig. 5E). Significantly higher luciferase activity was driven by the P1 5′-UTR promoter vector compared with the P2 5′-UTR promoter (Fig. 5E), suggesting that the P1 5′-UTR had stronger activity in modifying posttranscriptional Pmp22 mRNA compared with the P2 5′-UTR.
In WT SCs, PMP22 is rapidly degraded in the ER, so that only a small portion of the total PMP22 accumulates in the Golgi body, to be translocated to the SC membrane when axonal contact and myelination occur (29). We performed immunofluorescence staining of PMP22 together with a cis-Golgi marker, GM-130, to visualize the PMP22 localization. The PMP22 expression was high in WT mouse SCs, both on the cell membrane and in the Golgi body ( fig. S5F), but was almost undetectable in Nf1 fl/fl ;DhhCre tumor cells. High expression of PMP22 was detected in the Runx1 fl/fl ;Runx3 fl/fl ;Nf1 fl/fl ;DhhCre tumor cells in intracellular structures, including Golgi body, suggesting that RUNX1/3 changes the intracellular trafficking of PMP22. Together, we concluded that the combined outcome of the Runx1/ 3-mediated Pmp22 promoter usage and protein metabolism leads to alternative diminished Pmp22 expression in Nf1 −/− SCs and SCPs and neurofibromagenesis.

Ro5-3335 decreases neurofibroma growth by inhibiting SC proliferation and inducing SC apoptosis
Last, we tested in vivo the effect of inhibition of RUNX/Cbf- activity using the small molecular inhibitor, Ro5-3335, which inactivates RUNX and Cbf- functions by sandwich-like binding to RUNX and CBF-B, thereby interrupting the association between them (30). Because Ro5-3335 can be toxic, we first determined the maximum tolerated dose (MTD) and dose de-escalation. We defined the MTD as 100 mg/kg twice daily from Mondays to Fridays and once daily on Saturdays and Sundays. We then treated the Nf1 fl/fl ;DhhCre mice by oral gavage Ro5-3335 (n = 12) or vehicle control (n = 13) at MTD for 8 weeks. A few mice lost <10% weight during the first 2 weeks, but all regained normal weight thereafter. Two drug-treated mice were removed from the cohort because of severe dermatitis and were excluded from the statistical analysis. All other mice survived, and there were no obvious side effects apart from drowsiness due to Ro5-3335 benzodiazepine derivative effects (30). Volumetric measurement showed a significant decrease in tumor volume in Ro5-3335-treated mice compared with vehicle controls (Fig. 6A).
Ro5-3335 significantly reduced the number of Ki67 + proliferating cells (Fig. 6, B and C) and induced CC3 + apoptotic cells (Fig. 6, D and E). The relative mRNA expression changes on four selected genes from RNA-seq (two up-regulated and two downregulated in genetic results) were consistent with the genetic results (Fig. 6, F and G). Western blot verified that the PMP22 protein expression increased in these Ro5-3335-treated mouse neurofibromas compared with vehicle controls (Fig. 6H).

DISCUSSION
In this study, we performed in-depth investigation into the cooperative oncogenic roles of Runx1/3 in Nf1 neurofibromagenesis. We showed that either genetic deletion or pharmacological inhibition of Runx1/3 function decreased mouse neurofibroma burden in vivo. We further showed that the underlying mechanism was that Runx1/3 regulated alternative promoter usage and markedly induced levels of protein expression of PMP22, a direct target of RUNX1/3, to drive neurofibromagenesis. The RUNX family of genes can play pivotal roles in the initiation and/or maintenance of tumor cells via transcriptional misregulation, DNA repair defects, and genomic instability and promotes the development of cancers (12). They often compensate each other upon conditional inactivation of one Runx gene. We showed that Runx3 compensated Runx1 to contribute to neurofibromagenesis but that dual deletion of Runx1/3 in the Nf1 fl/fl ;DhhCre mice overcame the gene dose compensation and exhibited more durable effects on tumor initiation and/or maintenance. Notably, the Runx1/3 double-knockout mice were still dying, although the mouse survival time was prolonged (Fig. 1A). It is unlikely that Runx2 compensated the Runx1/3 activities because RNA-seq results showed that the Runx2 expression was 2.8-fold down in Runx1 fl/fl ;Runx3 fl/fl ;Nf1 fl/fl ; DhhCre versus Nf1 fl/fl ;DhhCre mouse tumors (not shown). Further experiments are needed to determine the genes/pathways that might be involved in the remaining tumors.
The RUNX transcription factors play important roles during the development in both the central and peripheral nervous systems. In the peripheral nervous system, RUNX1 and RUNX3 orchestrate dynamic gene expression change during the differentiation and maturation of DRG neurons (31). RUNX1 promotes adult mouse neurosphere proliferation and neuronal differentiation (32). We found that overexpressed Runx1/3 represses Pmp22 to contribute to neurofibroma initiation and maintenance. PMP22 is highly expressed in myelinating SCs and contributes to membrane organization in compact myelin to maintain the integrity of myelinated peripheral nerves (19). We show that Pmp22 expression decreases significantly in Nf1 −/− neurofibroma SCs and increases after Runx1/3 knockout or RUNX1/3-Pmp22-binding site deletion in mouse neurofibroma SCs. This might contribute to the increase in neurofibroma nonmyelinating SCs, as compared with Runx1/3 knockout nerves in electronic micrographs (not shown).
PMP22 expression has also been detected outside of myelinating SCs and can play important functions in cell proliferation, differentiation, and death. PMP22 protein is expressed in rat DRG SCs at embryonic day 16 (E16). These PMP22-expressing progenitors are multipotent and can generate non-neural cells (such as smooth muscle cells) in response to factors of the TGF- family (33). We showed that the expression of Pmp22 was increased in Runx1 fl/fl ;Runx3 fl/fl ; Nf1 fl/fl ;DhhCre mouse DRG/tumor spheres and that decreasing Pmp22 expression elevated precursor number, as evidenced by increased sphere numbers (Fig. 3C). It is plausible that overexpression of Runx1/3 leads to low levels of PMP22 protein expression; therefore, P2 is used more to drive PMP22 protein expression in other tissues during neurofibroma formation. This hypothesis is strongly supported by ATAC-seq data on FACS-sorted EGFP + tumor SCs, in which there is more open chromatin access in neuronal P2 promoter in Nf1 fl/fl ;DhhCre cells (Fig. 5A), RNA-seq data on bulk tumor (Fig. 5B), and qRT-PCR results on FACS-sorted EGFP + tumor SCs (Fig. 5C).
Our results also suggest that the reduction of PMP22 proteins in Nf1 fl/fl ;DhhCre mouse neurofibromas may be attributed not only to reduced transcriptional regulation but also possibly to loss of RNA stability and diminished rate of posttranscriptional and/or translational modification in neurofibroma SCs, and further experiments are needed to determine whether and how these posttranscriptional steps are involved in regulating PMP22 protein expression. PMP22 expression in neurofibroma may result in alternative availability of the glial PMP22 protein during the cell cycle, leading to aberrant regulation of cell growth control during neurofibromagenesis. On the other hand, Pmp22 mRNA levels are not always consistent with PMP22 protein expression. The varied expression of PMP22 in different tumor types indicates the role of PMP22 in growth arrest, and differentiation may be cell and tissue specific. PMP22 mRNA levels in colon cancer are not altered compared with normal tissue, yet protein levels, as measured by IHC and Western blot analysis, are significantly reduced in colon carcinoma samples compared with normal tissue (34). However, the rather subtle induction of Pmp22 RNA and use of alternative promoters would not explain the markedly induced levels of PMP22 protein upon elimination of RUNX factors in our system. There are several possible explanations: (i) Different promoters might be used during neurofibroma development; (ii) transcription and posttranscriptional modification might be involved; (iii) translation might play an important role; and (iv) it might be PMP22 protein accumulation due to increased myelin sheaths (Fig. 1D), which can stabilize PMP22 protein, in Runx1 fl/fl ;Runx3 fl/fl ;Nf1 fl/fl ;DhhCre. All these possibilities highlight the need for dynamic PMP22 protein analysis in neurofibroma SCs.
In normal SCs, TEAD1 and its coactivators are required for PMP22 expression and for the expression of early growth response protein 2 (EGR2) (27). Enhancers that regulate PMP22 expression are regulated by the SRY sex-determining region Y-box 10 (SOX10) and EGR2/KROX20 transcription factors (27). EGR2 and SOX10 activity mediates the developmental induction of Pmp22 expression by histone modification (35). Contrary to activation, we show that RUNX1/3 represses Pmp22 in Nf1 −/− SCs. Although the predicted SOX10-binding motif [consensus: AATTCATG, using the CIS-BP (Catalog of Inferred Sequence Binding Preferences) database] is located in chr11:63115220-63115227 and the locations between RUNX1/3 peaks and SOX10 probably overlap, no SOX10-binding motifs have been identified between our RUNX1/3 peak and the first exon of Pmp22. SOX10 may compete with RUNX1/3 for the same binding site because protein-protein interaction analysis shows that RUNX1/3 and SOX10 are not direct interacting partners. SOX10 expression is down-regulated in NF1 −/− neurofibromas (36), and it is possible that RUNX1/3 binds to this TF-binding motif in our system to drive neurofibromagenesis. However, the effect on both Pmp22 and Mbp suggests that perhaps common regulators of Mbp and Pmp22 may be involved during tumor initiation and/or progression. Notably, the RUNX1binding peak is located 17.9 kb upstream of Pmp22 TSS (Fig. 5A). This suggests that a chromatin looping structure might be involved in the binding process. Chromosome conformation capture experiments will help to better understand the mechanisms. Besides Pmp22, it is highly possible that other RUNX1/3 targets, such as SDC4, ERN1, and/or MBP, might be involved in tumor formation.
Targeting transcription factors is becoming a realistic option with increased understanding of transcription factor biology and technological advances. Increased expression of other RUNX family member compensation for the antitumor effect elicited by single RUNX member silencing suggests that simultaneous attenuation of all RUNX family members might lead to much stronger antitumor effects than suppression of individual RUNX members. Morita et al. (37) show that targeting RUNX clustering using pyrrole-imidazole polyamides bind to RUNX-binding consensus sites (5′-TGTGGT-3′ and 5′-TGCGGT-3′) is effectively against AML. During Ro5-3335 treatment, RUNX/CBFB-binding sites are blocked, and none of the RUNX can bind to CBFB to achieve their transcriptional activities. It is different from the Runx1 genetically conditional knockout, in which RUNX1 has no function but CBFB function is undisturbed, and the compensated Runx3 gene produces RUNX3 that can bind to CBFB to achieve RUNX transcriptional activities and contribute to neurofibroma formation.
In summary, we show that the SC myelinating gene Pmp22 serves as a tumor suppressor to contribute to neurofibromagenesis. Overexpression of PMP22 in mouse neurofibroma SCs decreases cell proliferation. Loss of Nf1 activates RUNX1/3 to enable tumor initiation by repressing Pmp22 through alternative promoter usage and other mechanisms to induce protein level expression of PMP22. The efficacy of Ro5-3335 on mouse neurofibroma growth suggests that disruption of RUNX/CBFB interaction or targeting RUNX gene cluster might provide a novel therapeutic strategy for patients with neurofibroma.

Animals
Mice were housed in temperature-and humidity-controlled facilities on 12-hour dark-light cycles with free access to food and water. The animal care and use committees of the Cincinnati Children's Hospital Medical Center approved all animal procedures. Institutional Animal Care and Use Committee guidelines were followed with animal subjects. To study the specific function of SCs, we used Dhh-Cre transgenic mouse, where Cre-mediated recombination activity would result in deletion of the floxed Nf1 allele in SC/SCPs of the developing peripheral nerves at E12.5. We bred the Runx1 fl/fl ;Runx3 fl/fl mice (38,39) (16).

Western blots
Western blots were performed using antibodies recognizing PMP22, -tubulin, and -actin (Cell Signaling Technology, Danvers, MA). At least three different tumor/cell lysates were analyzed per antigen.

Tumorigenesis assay in nude mice
We subcutaneously injected 5 × 10 5 mouse sphere cells per injection into athymic nude mice (males and females, Harlan, Indianapolis, IN). After 2 months, we dissected tumors and fixed them overnight in 4% paraformaldehyde for paraffin embedding or in Karnovsky fixative for electron microscopy.

Lentiviral transduction
We transduced secondary Nf1 fl/fl ;DhhCre or Runx1 fl/fl ;Runx3 fl/fl ; Nf1 fl/fl ;DhhCre DRG/neurofibroma spheres with purified shRNAs or nontarget control lentivirus (Sigma-Aldrich, St. Louis, MO). We incubated lentiviral particles with neurofibroma spheres at multiplicity of infection of 1:10 to 1:50 for 4 days and counted sphere numbers. For in vivo transplantation, Runx1 fl/fl ;Runx3 fl/fl ;Nf1 fl/fl ;DhhCre DRG/ neurofibroma spheres were transduced with lentivirus in the presence of Polybrene (8 g/ml; Sigma-Aldrich) for 16 to 20 hours, followed by culturing for 4 days. Spheres were collected and dissociated into single cells for transplantation. For overexpression studies, we used puromycin at 1 g/ml to eliminate untransduced cells.
For microarray, tumor initiating-like cells were FACS-sorted from neurofibroma tumors, and total mRNA was extracted using the Qiagen Kit. Affymetrix Human Genome U133 Plus 2.0 array was used for transcriptome analysis. CEL files were preprocessed using Bioconductor's affy package with Robust Multi-array Average normalization method. DEGs were predicted using Bioconductor's limma package. The |fold change| > 2× and FDR P < 0.05 were used as cutoffs to detect DEGs.

Statistics
We used a Gehan-Breslow-Wilcoxon log-rank test for Kaplan-Meier analysis. Neurofibroma growth was modeled by mixed-effects model analysis. P values were generated with a random-effects model analysis on log-transformed tumor volume data using the SAS mixed procedure (44). Fisher's exact test was used on transplanted nude mice tumor formation analysis. We used unpaired two-tailed Student's t tests to analyze the significance of cell proliferation and cell death quantification in tissue sections when two samples were compared. Data were reported as means ± SEM. P < 0.05 was considered significant.