Activation of Receptor Tyrosine Kinases Mediates Acquired Resistance to MEK Inhibition in Malignant Peripheral Nerve Sheath Tumors.

Malignant peripheral nerve sheath tumors often arise in patients with neurofibromatosis type 1 and are among the most treatment-refractory types of sarcoma. Overall survival in patients with relapsed disease remains poor, and thus novel therapeutic approaches are needed. NF1 is essential for negative regulation of RAS activity and is altered in about 90% of malignant peripheral nerve sheath tumors (MPNST). A complex interplay of upstream signaling and parallel RAS-driven pathways characterizes NF1-driven tumorigenesis, and inhibiting more than one RAS effector pathway is therefore necessary. To devise potential combination therapeutic strategies, we identified actionable alterations in signaling that underlie adaptive and acquired resistance to MEK inhibitor (MEKi). Using a series of proteomic, biochemical, and genetic approaches in an in vitro model of MEKi resistance provided a rationale for combination therapies. HGF/MET signaling was elevated in the MEKi-resistant model. HGF overexpression conferred resistance to MEKi in parental cells. Depletion of HGF or MET restored sensitivity of MEKi-resistant cells to MEKi. Finally, a combination of MEK and MET inhibition demonstrated activity in models of MPNST and may therefore be effective in patients with MPNST harboring genetic alterations in NF1. SIGNIFICANCE: This study demonstrates that MEKi plus MET inhibitor may delay or prevent a novel mechanism of acquired MEKi resistance, with clinical implications for MPNST patients harboring NF1 alterations.

suz12 inactivation in p53- and nf1-deficient zebrafish accelerates the onset of malignant peripheral nerve sheath tumors and expands the spectrum of tumor types.

Polycomb repressive complex 2 (PRC2) is an epigenetic regulator of gene expression that possesses histone methyltransferase activity. PRC2 trimethylates lysine 27 of histone H3 proteins (H3K27me3) as a chromatin modification associated with repressed transcription of genes frequently involved in cell proliferation or self-renewal. Loss-of-function mutations in the PRC2 core subunit SUZ12 have been identified in a variety of tumors, including malignant peripheral nerve sheath tumors (MPNSTs). To determine the consequences of SUZ12 loss in the pathogenesis of MPNST and other cancers, we used CRISPR-Cas9 to disrupt the open reading frame of each of two orthologous suz12 genes in zebrafish: suz12a and suz12b We generated these knockout alleles in the germline of our previously described p53 (also known as tp53)- and nf1-deficient zebrafish model of MPNSTs. Loss of suz12 significantly accelerated the onset and increased the penetrance of MPNSTs compared to that in control zebrafish. Moreover, in suz12-deficient zebrafish, we detected additional types of tumors besides MPNSTs, including leukemia with histological characteristics of lymphoid malignancies, soft tissue sarcoma and pancreatic adenocarcinoma, which were not detected in p53/nf1-deficient control fish, and are also contained in the human spectrum of SUZ12-deficient malignancies identified in the AACR Genie database. The suz12-knockout tumors displayed reduced or abolished H3K27me3 epigenetic marks and upregulation of gene sets reported to be targeted by PRC2. Thus, these zebrafish lines with inactivation of suz12 in combination with loss of p53/nf1 provide a model of human MPNSTs and multiple other tumor types, which will be useful for mechanistic studies of molecular pathogenesis and targeted therapy with small molecule inhibitors.

Genetic disruption of the small GTPase RAC1 prevents plexiform neurofibroma formation in mice with neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome caused by mutations in the NF1 tumor suppressor gene. NF1 encodes neurofibromin, a GTPase-activating protein for RAS proto-oncogene GTPase (RAS). Plexiform neurofibromas are a hallmark of NF1 and result from loss of heterozygosity of NF1 in Schwann cells, leading to constitutively activated p21RAS. Given the inability to target p21RAS directly, here we performed an shRNA library screen of all human kinases and Rho-GTPases in a patient-derived NF1-/- Schwann cell line to identify novel therapeutic targets to disrupt PN formation and progression. Rho family members, including Rac family small GTPase 1 (RAC1), were identified as candidates. Corroborating these findings, we observed that shRNA-mediated knockdown of RAC1 reduces cell proliferation and phosphorylation of extracellular signal-regulated kinase (ERK) in NF1-/- Schwann cells. Genetically engineered Nf1flox/flox;PostnCre+ mice, which develop multiple PNs, also exhibited increased RAC1-GTP and phospho-ERK levels compared with Nf1flox/flox;PostnCre- littermates. Notably, mice in which both Nf1 and Rac1 loci were disrupted (Nf1flox/floxRac1flox/flox;PostnCre+) were completely free of tumors and had normal phospho-ERK activity compared with Nf1flox/flox ;PostnCre+ mice. We conclude that the RAC1-GTPase is a key downstream node of RAS and that genetic disruption of the Rac1 allele completely prevents PN tumor formation in vivo in mice.

Ketotifen Modulates Mast Cell Chemotaxis to Kit-Ligand, but Does Not Impact Mast Cell Numbers, Degranulation, or Tumor Behavior in Neurofibromas of Nf1-Deficient Mice.

Neurofibromatosis Type 1 (NF1) is one of the most common genetic tumor predisposition syndromes in humans. Mutant NF1 results in dysregulated RAS allowing neoplasms throughout the neuroaxis. Plexiform neurofibromas (pNF) afflict up to 50% of patients with NF1. They are complex tumors of the peripheral nerve that cause major morbidity via nerve dysregulation and mortality via conversion to malignant sarcoma. Genetically engineered mouse models (GEMM) of NF1 provide valuable insights for the identification of therapies that have utility in people with pNF. Preclinical studies in GEMMs implicate mast cells and the c-Kit/Kit ligand pathway in pNF tumorigenesis. Kit ligand is a potent chemokine secreted by tumorigenic, Nf1-deficient Schwann cells. Ketotifen is an FDA-approved drug for the treatment of allergic conjunctivitis and asthma that promotes mast cell stabilization and has been used in prior case studies to treat or prevent pNFs. This study investigated the effect of ketotifen on mast cell infiltration and degranulation in the presence and absence of Kit ligand provocation and the effect of ketotifen on shrinking or preventing pNF formation in the Nf1flox/flox ;PostnCre + GEMM. Ketotifen decreased mast cell infiltration in response to exogenous Kit ligand administration, but did not affect mast cell degranulation. Importantly, ketotifen did not reduce mast cells numbers or activity in pNF and did not prevent pNF formation or decrease the volume of established pNF despite administration of pharmacologically active doses. These findings suggest that ketotifen has limited use as monotherapy to prevent or reduce pNF burden in the setting of Nf1 mutations.

Intrinsic Resistance to MEK Inhibition through BET Protein-Mediated Kinome Reprogramming in NF1-Deficient Ovarian Cancer.

Mutation or deletion of Neurofibromin 1 (NF1), an inhibitor of RAS signaling, frequently occurs in epithelial ovarian cancer (EOC), supporting therapies that target downstream RAS effectors, such as the RAF-MEK-ERK pathway. However, no comprehensive studies have been carried out testing the efficacy of MEK inhibition in NF1-deficient EOC. Here, we performed a detailed characterization of MEK inhibition in NF1-deficient EOC cell lines using kinome profiling and RNA sequencing. Our studies showed MEK inhibitors (MEKi) were ineffective at providing durable growth inhibition in NF1-deficient cells due to kinome reprogramming. MEKi-mediated destabilization of FOSL1 resulted in induced expression of receptor tyrosine kinases (RTK) and their downstream RAF and PI3K signaling, thus overcoming MEKi therapy. MEKi synthetic enhancement screens identified BRD2 and BRD4 as integral mediators of the MEKi-induced RTK signatures. Inhibition of bromo and extra terminal (BET) proteins using BET bromodomain inhibitors blocked MEKi-induced RTK reprogramming, indicating that BRD2 and BRD4 represent promising therapeutic targets in combination with MEKi to block resistance due to kinome reprogramming in NF1-deficient EOC. IMPLICATIONS: Our findings suggest MEK inhibitors will likely not be effective as single-agent therapies in NF1-deficient EOC due to kinome reprogramming. Cotargeting BET proteins in combination with MEKis to block reprogramming at the transcriptional level may provide an epigenetic strategy to overcome MEKi resistance in NF1-deficient EOC.

Loss of atrx cooperates with p53-deficiency to promote the development of sarcomas and other malignancies.

The SWI/SNF-family chromatin remodeling protein ATRX is a tumor suppressor in sarcomas, gliomas and other malignancies. Its loss of function facilitates the alternative lengthening of telomeres (ALT) pathway in tumor cells, while it also affects Polycomb repressive complex 2 (PRC2) silencing of its target genes. To further define the role of inactivating ATRX mutations in carcinogenesis, we knocked out atrx in our previously reported p53/nf1-deficient zebrafish line that develops malignant peripheral nerve sheath tumors and gliomas. Complete inactivation of atrx using CRISPR/Cas9 was lethal in developing fish and resulted in an alpha-thalassemia-like phenotype including reduced alpha-globin expression. In p53/nf1-deficient zebrafish neither peripheral nerve sheath tumors nor gliomas showed accelerated onset in atrx+/- fish, but these fish developed various tumors that were not observed in their atrx+/+ siblings, including epithelioid sarcoma, angiosarcoma, undifferentiated pleomorphic sarcoma and rare types of carcinoma. These cancer types are included in the AACR Genie database of human tumors associated with mutant ATRX, indicating that our zebrafish model reliably mimics a role for ATRX-loss in the early pathogenesis of these human cancer types. RNA-seq of p53/nf1- and p53/nf1/atrx-deficient tumors revealed that down-regulation of telomerase accompanied ALT-mediated lengthening of the telomeres in atrx-mutant samples. Moreover, inactivating mutations in atrx disturbed PRC2-target gene silencing, indicating a connection between ATRX loss and PRC2 dysfunction in cancer development.

Tonic ATP-mediated growth suppression in peripheral nerve glia requires arrestin-PP2 and is evaded in NF1.

Normal Schwann cells (SCs) are quiescent in adult nerves, when ATP is released from the nerve in an activity dependent manner. We find that suppressing nerve activity in adult nerves causes SC to enter the cell cycle. In vitro, ATP activates the SC G-protein coupled receptor (GPCR) P2Y2. Downstream of P2Y2, ß-arrestin-mediated signaling results in PP2-mediated de-phosphorylation of AKT, and PP2 activity is required for SC growth suppression. NF1 deficient SC show reduced growth suppression by ATP, and are resistant to the effects of ß-arrestin-mediated signaling, including PP2-mediated de-phosphorylation of AKT. In patients with the disorder Neurofibromatosis type 1, NF1 mutant SCs proliferate and form SC tumors called neurofibromas. Elevating ATP levels in vivo reduced neurofibroma cell proliferation. Thus, the low proliferation characteristic of differentiated adult peripheral nerve may require ongoing, nerve activity-dependent, ATP. Additionally, we identify a mechanism through which NF1 SCs may evade growth suppression in nerve tumors.

Neurofibromin Deficiency Induces Endothelial Cell Proliferation and Retinal Neovascularization.

Purpose: Neurofibromatosis type 1 (NF1) is the result of inherited mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin. Eye manifestations are common in NF1 with recent reports describing a vascular dysplasia in the retina and choroid. Common features of NF1 retinopathy include tortuous and dilated feeder vessels that terminate in capillary tufts, increased endothelial permeability, and neovascularization. Given the retinal vascular phenotype observed in persons with NF1, we hypothesize that preserving neurofibromin may be a novel strategy to control pathologic retinal neovascularization. Methods: Nf1 expression in human endothelial cells (EC) was reduced using small hairpin (sh) RNA and EC proliferation, migration, and capacity to form vessel-like networks were assessed in response to VEGF and hypoxia. Wild-type (WT), Nf1 heterozygous (Nf1+/-), and Nf1flox/+;Tie2cre pups were subjected to hyperoxia/hypoxia using the oxygen-induced retinopathy model. Retinas were analyzed quantitatively for extent of retinal vessel dropout, neovascularization, and capillary branching. Results: Neurofibromin expression was suppressed in response to VEGF, which corresponded with activation of Mek-Erk and PI3-K-Akt signaling. Neurofibromin-deficient EC exhibited enhanced proliferation and network formation in response to VEGF and hypoxia via an Akt-dependent mechanism. In response to hyperoxia/hypoxia, Nf1+/- retinas exhibited increased vessel dropout and neovascularization when compared with WT retinas. Neovascularization was similar between Nf1+/- and Nf1flox/+;Tie2cre retinas, but capillary drop out in Nf1flox/+;Tie2cre retinas was significantly reduced when compared with Nf1+/- retinas. Conclusions: These data suggest that neurofibromin expression is essential for controlling endothelial cell proliferation and retinal neovascularization and therapies targeting neurofibromin-deficient EC may be beneficial.

p53 and NF 1 loss plays distinct but complementary roles in glioma initiation and progression.

Malignant glioma is one of the deadliest types of cancer. Understanding how the cell of origin progressively evolves toward malignancy in greater detail could provide mechanistic insights and lead to novel concepts for tumor prevention and therapy. Previously we have identified oligodendrocyte precursor cell (OPC) as the cell of origin for glioma following the concurrent deletion of p53 and NF1 using a mouse genetic mosaic system that can reveal mutant cells prior to malignancy. In the current study, we set out to deconstruct the gliomagenic process in two aspects. First, we determined how the individual loss of p53 or NF1 contributes to aberrant behaviors of OPCs. Second, we determined how signaling aberrations in OPCs progressively change from pre-malignant to transformed stages. We found that while the deletion of NF1 leads to mutant OPC expansion through increased proliferation and decreased differentiation, the deletion of p53 impairs OPC senescence. Signaling analysis showed that, while PI3K and MEK pathways go through stepwise over-activation, mTOR signaling remains at the basal level in pre-transforming mutant OPCs but is abruptly up-regulated in tumor OPCs. Finally, inhibiting mTOR via pharmacological or genetic methods, led to a significant blockade of gliomagenesis but had little impact on pre-transforming mutant OPCs, suggesting that mTOR is necessary for final transformation but not early progression. In summary, our findings show that deconstructing the tumorigenic process reveals specific aberrations caused by individual gene mutations and altered signaling events at precise timing during tumor progression, which may shed light on tumor-prevention strategies.

Cooperative p16 and p21 action protects female astrocytes from transformation.

Mechanisms underlying sex differences in cancer incidence are not defined but likely involve dimorphism (s) in tumor suppressor function at the cellular and organismal levels. As an example, sexual dimorphism in retinoblastoma protein (Rb) activity was shown to block transformation of female, but not male, murine astrocytes in which neurofibromin and p53 function was abrogated (GBM astrocytes). Correlated sex differences in gene expression in the murine GBM astrocytes were found to be highly concordant with sex differences in gene expression in male and female GBM patients, including in the expression of components of the Rb and p53 pathways. To define the basis of this phenomenon, we examined the functions of the cyclin dependent kinase (CDK) inhibitors, p16, p21 and p27 in murine GBM astrocytes under conditions that promote Rb-dependent growth arrest. We found that upon serum deprivation or etoposide-induced DNA damage, female, but not male GBM astrocytes, respond with increased p16 and p21 activity, and cell cycle arrest. In contrast, male GBM astrocytes continue to proliferate, accumulate chromosomal aberrations, exhibit enhanced clonogenic cell activity and in vivo tumorigenesis; all manifestations of broad sex differences in cell cycle regulation and DNA repair. Differences in tumorigenesis disappeared when female GBM astrocytes are also rendered null for p16 and p21. These data elucidate mechanisms underlying sex differences in cancer incidence and demonstrate sex-specific effects of cytotoxic and targeted therapeutics. This has critical implications for lab and clinical research.

Human stem cell modeling in neurofibromatosis type 1 (NF1).

The future of precision medicine is heavily reliant on the use of human tissues to identify the key determinants that account for differences between individuals with the same disorder. This need is exemplified by the neurofibromatosis type 1 (NF1) neurogenetic condition. As such, individuals with NF1 are born with a germline mutation in the NF1 gene, but may develop numerous distinct neurological problems, ranging from autism and attention deficit to brain and peripheral nerve sheath tumors. Coupled with accurate preclinical mouse models, the availability of NF1 patient-derived induced pluripotent stem cells (iPSCs) provides new opportunities to define the critical factors that underlie NF1-associated nervous system disease pathogenesis and progression. In this review, we discuss the generation and potential applications of iPSC technology to the study of NF1.

The promise of signal transduction in genetically driven sarcomas of the nerve.

Neurofibromatosis type 1 (NF1) is an autosomal dominant tumor predisposition syndrome. Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas arising from peripheral nerve sheaths, and the most commonly lethal feature associated with NF1. The hallmark of NF1 and NF1-related MPNST is the loss of neurofibromin expression. Loss of neurofibromin is considered a tumor-promoting event, and leads to constitutive activation of RAS and its downstream effectors. However, RAS activation alone is not sufficient for MPNST formation, and additional tumor suppressors and signaling pathways have been implicated in tumorigenesis of MPNST. Taking advantage of the rapid development of novel therapeutics targeting key molecular pathways across all cancer types, the best-in-class modulators of these pathways can be assessed in pre-clinical models and translated into clinical trials for patients with MPNST. Here, we describe the genetic changes and molecular pathways that drive MPNST formation and highlight the promise of signal transduction to identify therapies that may treat these tumors more effectively.

Neurofibromatosis type 1 and optic pathway glioma: Molecular interplay and therapeutic insights.

Children with neurofibromatosis type 1 (NF1) are predisposed to develop central nervous system neoplasms, the most common of which are low-grade gliomas (LGGs). The absence of human NF1 associated LGG-derived cell lines, coupled with an inability to generate patient-derived xenograft models, represents barriers to profile molecularly targeted therapies for these tumors. Thus, genetically engineered mouse models have been identified to evaluate the interplay between Nf1-deficient tumor cells and nonneoplastic stromal cells to evaluate potential therapies for these neoplasms. Future treatments might also consider targeting the nonneoplastic cells in NF1-LGGs to reduce tumor growth and neurologic morbidity in affected children.

Lipoamide Inhibits NF1 Deficiency-induced Epithelial-Mesenchymal Transition in Murine Schwann Cells.

BACKGROUND AND AIMS: Neurofibromatosis type I (NF1) is one of the most common neurocutaneous syndromes characterized by development of adult neurofibromas which is mainly made up of Schwann cells. The disease is generally accepted to be caused by inactivation mutation of Nf1 gene. And Nf1 deficiency had been reported to lead to ROS overproduction and epithelial-mesenchymal transition (EMT) phenotype. This study was designed to investigate whether excessive ROS conferred to Nf1 deficiency-induced EMT in Schwann cells. METHODS: Colony formation, wound healing assay and transwell assay was used to evaluate the effects of stable Nf1 knockdown in SW10 Schwann cells. Western blot and ROS assay was conducted to explore the molecular mechanisms of Nf1 inactivation in tumorigenesis. Animal experiments were performed to assess the inhibitory effects of lipoamide, which is the neutral amide of α-lipoic acid and functions as a potent antioxidant to scavenge ROS, on Nf1-deficiency tumor growth in vivo. RESULTS: Nf1 knockdown enhanced the cellular capacities of proliferation, migration and invasion, promoted ROS generation, decreased the expression of epithelial surface marker E-cadherin, and up-regulated several EMT-associated molecules in Schwann cells. Moreover, lipoamide dose-dependently inhibited not only Nf1 deficiency-induced EMT but also spontaneous EMT. Furthermore, lipoamide markedly suppresses tumor growth in a mouse model of NF1-associated neurofibroma. CONCLUSIONS: Our results clearly reveal that ROS overproduction is responsible for Nf1 deficiency-induced EMT and plays a crucial role in NF1 tumor growth. The findings presented herein shed light on the potential of antioxidant therapy to prevent the progression of NF1-associated neurofibroma.

Targeted Disruption of NF1 in Osteocytes Increases FGF23 and Osteoid With Osteomalacia-like Bone Phenotype.

Neurofibromatosis type 1 (NF1, OMIM 162200), caused by NF1 gene mutations, exhibits multi-system abnormalities, including skeletal deformities in humans. Osteocytes play critical roles in controlling bone modeling and remodeling. However, the role of neurofibromin, the protein product of the NF1 gene, in osteocytes is largely unknown. This study investigated the role of neurofibromin in osteocytes by disrupting Nf1 under the Dmp1-promoter. The conditional knockout (Nf1 cKO) mice displayed serum profile of a metabolic bone disorder with an osteomalacia-like bone phenotype. Serum FGF23 levels were 4 times increased in cKO mice compared with age-matched controls. In addition, calcium-phosphorus metabolism was significantly altered (calcium reduced; phosphorus reduced; parathyroid hormone [PTH] increased; 1,25(OH)2 D decreased). Bone histomorphometry showed dramatically increased osteoid parameters, including osteoid volume, surface, and thickness. Dynamic bone histomorphometry revealed reduced bone formation rate and mineral apposition rate in the cKO mice. TRAP staining showed a reduced osteoclast number. Micro-CT demonstrated thinner and porous cortical bones in the cKO mice, in which osteocyte dendrites were disorganized as assessed by electron microscopy. Interestingly, the cKO mice exhibited spontaneous fractures in long bones, as found in NF1 patients. Mechanical testing of femora revealed significantly reduced maximum force and stiffness. Immunohistochemistry showed significantly increased FGF23 protein in the cKO bones. Moreover, primary osteocytes from cKO femora showed about eightfold increase in FGF23 mRNA levels compared with control cells. The upregulation of FGF23 was specifically and significantly inhibited by PI3K inhibitor Ly294002, indicating upregulation of FGF23 through PI3K in Nf1-deficient osteocytes. Taken together, these results indicate that Nf1 deficiency in osteocytes dramatically increases FGF23 production and causes a mineralization defect (ie, hyperosteoidosis) via the alteration of calcium-phosphorus metabolism. This study demonstrates critical roles of neurofibromin in osteocytes for osteoid mineralization. © 2017 American Society for Bone and Mineral Research.

An inflammatory gene signature distinguishes neurofibroma Schwann cells and macrophages from cells in the normal peripheral nervous system.

Neurofibromas are benign peripheral nerve tumors driven by NF1 loss in Schwann cells (SCs). Macrophages are abundant in neurofibromas, and macrophage targeted interventions may have therapeutic potential in these tumors. We generated gene expression data from fluorescence-activated cell sorted (FACS) SCs and macrophages from wild-type and mutant nerve and neurofibroma to identify candidate pathways involved in SC-macrophage cross-talk. While in 1-month-old Nf1 mutant nerve neither SCs nor macrophages significantly differed from their normal counterparts, both macrophages and SCs showed significantly altered cytokine gene expression in neurofibromas. Computationally reconstructed SC-macrophage molecular networks were enriched for inflammation-associated pathways. We verified that neurofibroma SC conditioned medium contains macrophage chemo-attractants including colony stimulation factor 1 (CSF1). Network analysis confirmed previously implicated pathways and predict novel paracrine and autocrine loops involving cytokines, chemokines, and growth factors. Network analysis also predicted a central role for decreased type-I interferon signaling. We validated type-I interferon expression in neurofibroma by protein profiling, and show that treatment of neurofibroma-bearing mice with polyethylene glycolyated (PEGylated) type-I interferon-α2b reduces the expression of many cytokines overexpressed in neurofibroma. These studies reveal numerous potential targetable interactions between Nf1 mutant SCs and macrophages for further analyses.

Pharmacologically targeting beta-catenin for NF1 associated deficiencies in fracture repair.

Patients with Neurofibromatosis type 1 display delayed fracture healing and the increased deposition of fibrous tissue at the fracture site. Severe cases can lead to non-union and even congenital pseudarthrosis. Neurofibromatosis type 1 is caused by a mutation in the NF1 gene and mice lacking the Nf1 gene show a fracture repair phenotype similar to that seen in patients. Tissue from the fracture site of patients with Neurofibromatosis type 1 and from mice deficient in the Nf1 gene both show elevated levels of ß-catenin protein and activation of ß-catenin mediated signaling. Constitutively elevated ß-catenin leads to a delayed and fibrous fracture repair process, and (RS)-5-methyl-1-phenyl-1,3,4,6-tetrahydro-2,5-benzoxazocine (Nefopam, a centrally-acting, non-narcotic analgesic agent) inhibits ß-catenin mediated signaling during skin wound repair. Here we investigate Nefopam's potential as a modulator of bone repair in mice deficient in Nf1. Mice were treated with Nefopam and investigated for bone fracture repair. Bone marrow stromal cells flushed from the long bones of unfractured mice were treated with Nefopam and investigated for osteogenic potential. Treatment with Nefopam was able to lower the ß-catenin level and the Axin2 transcript level in the fracture calluses of Nf1 deficient mice. Cultures from the bone marrow of Nf1-/- mice had significantly lower osteoblastic colonies and mineralized nodules, which was increased when cells were cultured in the presence of Nefopam. Fracture calluses were harvested and analyzed 14days and 21days after injury. Nf1-/- calluses had less bone, less cartilage, and higher fibrous tissue content than control calluses. Treatment with Nefopam increased the bone and cartilage content and decreased the fibrous tissue content in Nf1-/- calluses. These findings present a potential treatment for patients with Neurofibromatosis 1 in the context of bone repair. Since Nefopam is already in use in patient care, it could be rapidly translated to the clinical setting.

Absence of Neurofibromin Induces an Oncogenic Metabolic Switch via Mitochondrial ERK-Mediated Phosphorylation of the Chaperone TRAP1.

Mutations in neurofibromin, a Ras GTPase-activating protein, lead to the tumor predisposition syndrome neurofibromatosis type 1. Here, we report that cells lacking neurofibromin exhibit enhanced glycolysis and decreased respiration in a Ras/ERK-dependent way. In the mitochondrial matrix of neurofibromin-deficient cells, a fraction of active ERK1/2 associates with succinate dehydrogenase (SDH) and TRAP1, a chaperone that promotes the accumulation of the oncometabolite succinate by inhibiting SDH. ERK1/2 enhances both formation of this multimeric complex and SDH inhibition. ERK1/2 kinase activity is favored by the interaction with TRAP1, and TRAP1 is, in turn, phosphorylated in an ERK1/2-dependent way. TRAP1 silencing or mutagenesis at the serine residues targeted by ERK1/2 abrogates tumorigenicity, a phenotype that is reverted by addition of a cell-permeable succinate analog. Our findings reveal that Ras/ERK signaling controls the metabolic changes orchestrated by TRAP1 that have a key role in tumor growth and are a promising target for anti-neoplastic strategies.

The primacy of NF1 loss as the driver of tumorigenesis in neurofibromatosis type 1-associated plexiform neurofibromas.

Neurofibromatosis type 1 (NF1) is a common tumor-predisposition disorder due to germline mutations in the tumor suppressor gene NF1. A virtually pathognomonic finding of NF1 is the plexiform neurofibroma (PN), a benign, likely congenital tumor that arises from bi-allelic inactivation of NF1. PN can undergo transformation to a malignant peripheral nerve sheath tumor, an aggressive soft-tissue sarcoma. To better understand the non-NF1 genetic contributions to PN pathogenesis, we performed whole-exome sequencing, RNASeq profiling and genome-wide copy-number determination for 23 low-passage Schwann cell cultures established from surgical PN material with matching germline DNA. All resected tumors were derived from routine debulking surgeries. None of the tumors were considered at risk for malignant transformation at the time; for example, there was no pain or rapid growth. Deep (~500X) NF1 exon sequencing was also conducted on tumor DNA. Non-NF1 somatic mutation verification was performed using the Ampliseq/IonTorrent platform. We identified 100% of the germline NF1 mutations and found somatic NF1 inactivation in 74% of the PN. One individual with three PNs had different NF1 somatic mutations in each tumor. The median number of somatic mutations per sample, including NF1, was one (range 0-8). NF1 was the only gene that was recurrently somatically inactivated in multiple tumors. Gene Set Enrichment Analysis of transcriptome-wide tumor RNA sequencing identified five significant (FDR<0.01) and seven trending (0.01⩽FDR<0.02) gene sets related to DNA replication, telomere maintenance and elongation, cell cycle progression, signal transduction and cell proliferation. We found no recurrent non-NF1 locus copy-number variation in PN. This is the first multi-sample whole-exome and whole-transcriptome sequencing study of NF1-associated PN. Taken together with concurrent copy-number data, our comprehensive genetic analysis reveals the primacy of NF1 loss as the driver of PN tumorigenesis.

Stat5 is critical for the development and maintenance of myeloproliferative neoplasm initiated by Nf1 deficiency.

Juvenile myelomonocytic leukemia is a rare myeloproliferative neoplasm characterized by hyperactive RAS signaling. Neurofibromin1 (encoded by the NF1 gene) is a negative regulator of RAS activation. Patients with neurofibromatosis type 1 harbor loss-of-function mutations in NF1 and have a 200- to 500-fold increased risk of juvenile myelomonocytic leukemia. Leukemia cells from patients with juvenile myelomonocytic leukemia display hypersensitivity to certain cytokines, such as granulocyte-macrophage colony-stimulating factor. The granulocyte-macrophage colony-stimulating factor receptor utilizes pre-associated JAK2 to initiate signals after ligand binding. JAK2 subsequently activates STAT5, among other downstream effectors. Although STAT5 is gaining recognition as an important mediator of growth factor signaling in myeloid leukemias, the contribution of STAT5 to the development of hyperactive RAS-initiated myeloproliferative disease has not been well described. In this study, we investigated the consequence of STAT5 attenuation via genetic and pharmacological approaches in Nf1-deficient murine models of juvenile myelomonocytic leukemia. We found that homozygous Stat5 deficiency extended the lifespan of Nf1-deficient mice and eliminated the development of myeloproliferative neoplasm associated with Nf1 gene loss. Likewise, we found that JAK inhibition with ruxolitinib attenuated myeloproliferative neoplasm in Nf1-deficient mice. Finally, we found that primary cells from a patient with KRAS-mutant juvenile myelomonocytic leukemia displayed reduced colony formation in response to JAK2 inhibition. Our findings establish a central role for STAT5 activation in the pathogenesis of juvenile myelomonocytic leukemia and suggest that targeting this pathway may be of clinical utility in these patients.