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Plexiform neurofibromas are benign nerve sheath Schwann cell tumors characterized by biallelic mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene. Atypical neurofibromas show additional frequent loss of CDKN2A/Ink4a/Arf and may be precursor lesions of aggressive malignant peripheral nerve sheath tumors (MPNST). Here we combined loss of Nf1 in developing Schwann cells with global Ink4a/Arf loss and identified paraspinal plexiform neurofibromas and atypical neurofibromas. Upon transplantation, atypical neurofibromas generated genetically engineered mice (GEM)-PNST similar to human MPNST, and tumors showed reduced p16INK4a protein and reduced senescence markers, confirming susceptibility to transformation. Superficial GEM-PNST contained regions of nerve-associated plexiform neurofibromas or atypical neurofibromas and grew rapidly on transplantation. Transcriptome analyses showed similarities to corresponding human tumors. Thus, we recapitulated nerve tumor progression in NF1 and provided preclinical platforms for testing therapies at each tumor grade. These results support a tumor progression model in which loss of NF1 in Schwann cells drives plexiform neurofibromas formation, additional loss of Ink4a/Arf contributes to atypical neurofibromas formation, and further changes underlie transformation to MPNST.Significance:New mouse models recapitulate the stepwise progression of NF1 tumors and will be useful to define effective treatments that halt tumor growth and tumor progression in NF1.

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Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft-tissue sarcomas that cause significant mortality in adults with Neurofibromatosis Type 1 (NF1). We compared gene expression of growth factors in normal human nerves to MPNST and normal human Schwann cells (NHSC) to MPNST cell lines. We identified WNT5A as the most significantly upregulated ligand-coding gene and verified its protein expression in MPNST cell lines and tumors. In many contexts WNT5A acts as an oncogene. However, inhibiting WNT5A expression using shRNA did not alter MPNST cell proliferation, invasion, migration, or survival in vitro. Rather, shWNT5A-treated MPNST cells upregulated mRNAs associated with the remodeling of extracellular matrix and with immune cell communication. In addition, these cells secreted increased amounts of the proinflammatory cytokines CXCL1, CCL2, IL-6, CXCL8, and ICAM1. Versus controls, shWNT5A-expressing MPNST cells formed larger tumors in vivo. Grafted tumors contained elevated macrophage/stromal cells, larger and more numerous blood vessels, and increased levels of Mmp9, Cxcl13, Lipocalin-1, and Ccl12. In some MPNST settings, these effects were mimicked by targeting the WNT5A receptor ROR2. These data suggest that the non-canonical Wnt ligand WNT5A inhibits MPNST tumor formation by modulating the MPNST microenvironment, so that blocking WNT5A accelerates tumor growth in vivo.

Purpose Plexiform neurofibromas (NF) develop in children with Neurofibromatosis Type 1 (NF1) and can be associated with several skeletal co-morbidities. Preclinical mouse studies revealed Nf1 deficiency in osteoprogenitor cells disrupts, in a MEK-dependent manner, pyrophosphate (PPi) homeostasis and skeletal mineralization. The etiology of NF-associated skeletal manifestations remains unknown. Methods We used mouse models of NF1 neurofibromas to assess bone mineralization of skeletal structures adjacent to tumors. Expression of genes involved in pyrophosphate homeostasis was assessed in mouse and human NF tumors and Schwann cell cultures. We used Dual-energy X-ray Absorptiometry (DXA) to assess tumor-associated changes in bone mineral density (BMD) in an individual with NF1 following treatment with the MEK inhibitor selumetinib. Results We detected increased non-mineralized bone surfaces adjacent to tumors in mouse models of NF1 neurofibromas. Expression of Enpp1, a PPi-generating ectophosphatase, and ANKH, a PPi transporter, was increased in mouse and human neurofibroma-derived tissues and Schwann cells, respectively. In one patient, tumor-associated reductions in BMD were partially rescued following therapy with selumetinib. Conclusion Results indicate that NF-associated skeletal pathologies in NF1 are associated with dysregulated pyrophosphate homeostasis in adjacent NF tumors and suggest that treatment of NFs with MEK inhibitors may improve skeletal manifestations of the disease.

Neurofibromatosis type 1 (NF1) is a genetic disorder characterized by nerve tumors called neurofibromas, in which Schwann cells (SCs) lack NF1 and show deregulated RAS signaling. NF1 is also implicated in regulation of cAMP. Gene expression profiling and protein expression identified P2RY14 in SCs and SC precursors (SCPs) implicating P2RY14 as a candidate upstream regulator of cAMP in EGF-dependent SCP. We found that SCP self-renewal was reduced by genetic or pharmacological inhibition of P2RY14. In NF1 deficient SCs and malignant peripheral nerve sheath tumor (MPNST) cells, P2RY14 inhibition decreased EGFR-driven phospho-Akt and increased cAMP signaling. In a neurofibroma mouse model, genetic deletion of P2RY14 increased mouse survival, delayed neurofibroma initiation and rescued cAMP signaling. Conversely, elevation of cAMP diminished SCP number in vitro and diminished SC proliferation in neurofibroma bearing mice in vivo. These studies identify the purinergic receptor P2RY14 as a critical G-protein-coupled receptor (GPCR) in NF1 mutant SCPs and SCs and suggest roles for EGFR-GPCR crosstalk in facilitating SCP self-renewal and neurofibroma initiation via cAMP and EGFR-driven phospho-Akt.

Neurofibromatosis type 1 (NF1) is characterized by nerve tumors called neurofibromas, in which Schwann cells (SCs) show deregulated RAS signaling. NF1 is also implicated in regulation of cAMP. We identified the G-protein-coupled receptor (GPCR) P2ry14 in human neurofibromas, neurofibroma-derived SC precursors (SCPs), mature SCs, and mouse SCPs. Mouse Nf1-/- SCP self-renewal was reduced by genetic or pharmacological inhibition of P2ry14. In a mouse model of NF1, genetic deletion of P2ry14 rescued low cAMP signaling, increased mouse survival, delayed neurofibroma initiation, and improved SC Remak bundles. P2ry14 signals via Gi to increase intracellular cAMP, implicating P2ry14 as a key upstream regulator of cAMP. We found that elevation of cAMP by either blocking the degradation of cAMP or by using a P2ry14 inhibitor diminished NF1-/- SCP self-renewal in vitro and neurofibroma SC proliferation in in vivo. These studies identify P2ry14 as a critical regulator of SCP self-renewal, SC proliferation, and neurofibroma initiation.

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, β-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 β-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. Electronic supplementary material The online version of this article (10.1186/s40478-018-0635-9) contains supplementary material, which is available to authorized users.

// Mark A. Currier 1 , Les Sprague 1 , Tilat A. Rizvi 2 , Brooke Nartker 1 , Chun-Yu Chen 1 , Pin-Yi Wang 1 , Brian J. Hutzen 1 , Meghan R. Franczek 1 , Ami V. Patel 2 , Katherine E. Chaney 2 , Keri A. Streby 1,3 , Jeffrey A. Ecsedy 4 , Joe Conner 5 , Nancy Ratner 2 and Timothy P. Cripe 1,3 1 Center for Childhood Cancer and Blood Diseases, Nationwide Children’s Hospital, The Ohio State University, Columbus, Ohio, USA 2 Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center; Cincinnati, Ohio, USA 3 Division of Hematology/Oncology/Blood and Marrow Transplantation, Nationwide Children’s Hospital, The Ohio State University, Columbus, Ohio, USA 4 Takeda Pharmaceuticals International Co, Cambridge, MA, USA 5 Virttu Biologics, Ltd, Biocity, Scotland, Newhouse, United Kingdom Correspondence to: Timothy P. Cripe, email: // Keywords : oHSV, Aurora A kinase, neuroblastoma, MPNST Received : November 07, 2016 Accepted : January 17, 2017 Published : February 02, 2017 Abstract Malignant peripheral nerve sheath tumor (MPNST) and neuroblastoma models respond to the investigational small molecule Aurora A kinase inhibitor, alisertib. We previously reported that MPNST and neuroblastomas are also susceptible to oncolytic herpes virus (oHSV) therapy. Herein, we show that combination of alisertib and HSV1716, a virus derived from HSV-1 and attenuated by deletion of RL1, exhibits significantly increased antitumor efficacy compared to either monotherapy. Alisertib and HSV1716 reduced tumor growth and increased survival in two xenograft models of MPNST and neuroblastoma. We found the enhanced antitumor effect was due to multiple mechanisms that likely each contribute to the combination effect. First, oncolytic herpes virus increased the sensitivity of uninfected cells to alisertib cytotoxicity, a process we term virus-induced therapeutic adjuvant (VITA). Second, alisertib increased peak virus production and slowed virus clearance from tumors, both likely a consequence of it preventing virus-mediated increase of intratumoral NK cells. We also found that alisertib inhibited virus-induced accumulation of intratumoral myeloid derived suppressor cells, which normally are protumorigenic. Our data suggest that clinical trials of the combination of oHSV and alisertib are warranted in patients with neuroblastoma or MPNST.

Neurofibromatosis type 1 (NF1) and Neurofibromatosis type 2 (NF2) are two dominantly inherited disorders that cause tumors in Schwann cells. NF1 patients have a high risk for malignant peripheral nerve sheath tumors (MPNST), which are often inoperable and do not respond well to current chemotherapies or radiation. NF2 patients have a high risk for schwannomas. To identify potential therapeutic targets in these two tumors, we screened the NF1 MPNST cell line, ST88-14, and the NF2 schwannoma cell line, HEI-193, against ~2000 drugs of known mechanisms of action (including ~600 cancer relevant drugs), and also screened the cell lines against an siRNA library targeting most protein kinases. Both the drug screen and the siRNA screen identified Polo-like kinase 1 (PLK1) among the most potent hits in both cell lines. Since PLK1 acts on the cell cycle primarily at the G2/M transition, the same stage where aurora kinase (AURKA) acts, we explored PLK1 and its relationship to aurora kinase in MPNST. Quantitative profiling of PLK1 inhibitors against a panel of 10 neurofibromatosis cell lines found that they were potent inhibitors and, unlike AURKA inhibitors, were not more selective for NF1 over NF2 tumor cells. Furthermore, one PLK1 inhibitor, BI6727 stabilized tumor volume in MPNST xenografts. We conclude that PLK1 is a therapeutic target for MPNSTs and schwannomas, but inhibitors may have a narrow therapeutic index that limits their use as a single agent.

Purpose: In neurofibromatosis type 1 (NF1) and in highly aggressive malignant peripheral nerve sheath tumors (MPNSTs), constitutively active RAS-GTP and increased MAPK signaling are important in tumorigenesis. Dual specificity phosphatases (DUSPs) are negative regulators of MAPK signaling that dephosphorylate p38, JNK, and ERK in different settings. Although often acting as tumor suppressors, DUSPs may also act as oncogenes, helping tumor cells adapt to high levels of MAPK signaling. We hypothesized that inhibiting DUSPs might be selectively toxic to cells from NF1-driven tumors. Experimental Design: We examined DUSP gene and protein expression in neurofibroma and MPNSTs. We used small hairpin RNA (shRNA) to knock down DUSP1 and DUSP6 to evaluate cell growth, downstream MAPK signaling, and mechanisms of action. We evaluated the DUSP inhibitor, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), in MPNST cell lines and in cell-line and patient-derived MPNST xenografts. Results: DUSP1 and DUSP6 are expressed in NF1-deleted tumors. Knockdown of DUSP1 and DUSP6, alone or in combination, reduced MPNST cell growth and led to ERK and JNK hyperactivation increasing downstream TP53 and p-ATM. The DUSP inhibitor, BCI, diminished the survival of NF1-deleted Schwann cells and MPNST cell lines through activation of JNK. In vivo, treatment of an established cell-line xenograft or a novel patient-derived xenograft (PDX) of MPNSTs with BCI increased ERK and JNK activation, caused tumor necrosis and fibrosis, and reduced tumor volume in one model. Conclusions: Targeting DUSP1 and DUSP6 genetically or with BCI effectively inhibits MPNST cell growth and promotes cell death, in vitro and in xenograft models. The data support further investigation of DUSP inhibition in MPNSTs.

Malignant peripheral nerve sheath tumors (MPNST) are rare soft tissue sarcomas that are a major source of mortality in neurofibromatosis type 1 (NF1) patients. To identify MPNST driver genes, we performed a lentiviral short hairpin (sh) RNA screen, targeting all 130 genes up-regulated in neurofibroma and MPNSTs versus normal human nerve Schwann cells. NF1 mutant cells show activation of RAS/MAPK signaling, so a counter-screen in RAS mutant carcinoma cells was performed to exclude common RAS-pathway driven genes. We identified 7 genes specific for survival of MPSNT cells, including MEIS1. MEIS1 was frequently amplified or hypomethylated in human MPSNTs, correlating with elevated MEIS1 gene expression. In MPNST cells and in a genetically engineered mouse model, MEIS1 expression in developing nerve glial cells was necessary for MPNST growth. Mechanistically, MEIS1 drives MPNST cell growth via the transcription factor ID1, thereby suppressing expression of the cell cycle inhibitor p27Kip and maintaining cell survival., Highlights • Targeting over-expressed genes facilitates identification of sarcoma driver genes. • We identify MEIS1 as a MPNST oncogene. • MEIS1 suppresses p27Kip enabling MPNST survival. We identify MEIS1 as a sarcoma oncogene, and identify an additional 7 genes specific for survival of malignant peripheral nerve sheath cells. MEIS1 was frequently amplified or hypomethylated in human tumors, correlating with elevated MEIS1 gene and protein expression. MEIS1 enables cell cycle progression in these tumor cells through downregulation of expression of a pro-cell death protein p27Kip. Thus, inhibitors targeting cell cycle checkpoints and/or upregulating p27Kip may have therapeutic value for these patients, and perhaps for other tumor types in which MEIS1 is an oncogene.

// Jed J. Kendall 1 , Katherine E. Chaney 1 , Ami V. Patel 1 , Tilat A. Rizvi 1 , David A. Largaespada 2 , Nancy Ratner 1 1 Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital, Department of Pediatrics, University of Cincinnati, Cincinnati, OH 45229, USA 2 Department of Pediatrics, Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA Correspondence to: Nancy Ratner, email: Nancy.Ratner@cchmc.org Keywords: MPNST, CK2, G2 arrest, NF1, β-catenin Received: March 10, 2016 Accepted: June 07, 2016 Published: July 18, 2016 ABSTRACT Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that are a major cause of mortality of Neurofibromatosis type 1 (NF1) patients. MPNST patients have few therapeutic options available and only complete surgical resection can be curative. MPNST formation and survival are dependent on activated β-catenin signaling. The goal of this study was to determine if inhibition of the CK2 enzyme can be therapeutically exploited in MPNSTs, given CK2’s role in mainta ining oncogenic phenotypes including stabilization of β-catenin. We found that CK2α is over-expressed in MPNSTs and is critical for maintaining cell survival, as the CK2 inhibitor, CX-4945 (Silmitasertib), and shRNA targeting CK2α each significantly reduce MPNST cell viability. These effects were preceded by loss of critical signaling pathways in MPNSTs, including destabilization of β-catenin and TCF8. CX-4945 administration in vivo slowed tumor growth and extends survival time. We conclude that CK2 inhibition is a promising approach to blocking β-catenin in MPNST cells, although combinatorial therapies may be required for maximal efficacy.

We previously reported xenograft models of neuroblastoma and malignant peripheral nerve sheath tumor (MPNST) are responsive to direct intratumoral injection of oncolytic Herpes Simplex Viruses (Parikh et al., Ped Blood Can 44:469, 2005; Mahller et al., Ped Blood Can 46:745, 2006; Mahller et al., Can Res 68:1170, 2008). We have confirmed these findings using an oncolytic HSV, Seprehvir (HSV1716), which is in clinical trials that include pediatric patients (NCT00931931, NCT02031965). The models showed wide variability, however, with some models having maintained complete responses and others showing only delayed growth. Because DNA viruses are highly dependent on normal nuclear machinery for DNA synthesis/replication and virus packaging/assembly, we postulated that increased transit time in mitosis may be beneficial to an oncolytic DNA virus. Aurora A Kinase is an enzyme critical for formation of centrioles and mitotic spindles during mitosis and is often dysregulated in cancer. The selective AAK inhibitor, Alisertib (MLN8237), arrests cells in G2/M and has significant activity in models of neuroblastoma (data from the Pediatric Preclinical Testing Program) and in MPNST (data from our prior study: Patel et al., Clin Can Res 18:5020, 2012). We found tumor responses in xenograft models using the combination of Seprehvir and Alisertib was more than additive. Immunohistochemical staining for HSV antigens revealed a marked difference in the extent and spread of virus as early as 24 hrs after virus injection. Our preliminary data assessing kinetics of virus infection suggest that Alisertib pretreatment enhances virus uptake and spread, accounting for the combinatorial effects. These data strongly suggest alisertib pretreatment “primes” tumor cells, making them highly susceptible to virus infection and spread and greatly accelerates the virolytic effects. Overall, our findings support clinical testing of Alisertib combined with Seprehvir in patients with refractory neurogenic tumors such as neuroblastoma and MPNST. Citation Format: Mark Currier, Tilat Rizvi, Brooke Nartker, Duo Chen, Chun-Yu Chen, Pin-Yi Wang, Les Sprague, Meghan Franczek, Ami Patel, Katherine Chaney, Keri Streby, Jeff Ecsedy, Joe Conner, Nancy Ratner, Timothy Cripe. Enhanced antitumor efficacy by combining oncolytic herpes simplex virus and Aurora A kinase inhibition in models of neuroblastoma and malignant peripheral nerve sheath tumor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B35.