Malignant Peripheral Nerve Sheath Tumors Pathogenesis Research Articles

Huge Malignant Peripheral Nerve Sheath Tumors Originating In Neurofibromatosis Type 1

Malignant peripheral nerve sheath tumors (MPNST) are malignant soft tissue neoplasms which account for 2% of all soft tissue sarcomas. These tumors are associated with poor prognosis, propensity to metastasize and high recurrence rates. About half of the MPNST arise from pre-existing neurofibroma and are associated with Neurofibromatosis type I (NF1), 10% are radiotherapy induced others are sporadic. Pathogenesis of MPNST is not fully understood yet. MPNST are common in extremities followed by trunk but are less common in head and neck area. MPNST show limited sensitivity to radiotherapy and chemotherapy and wide surgical resection is mainstay of treatment. We present a series of 3 cases of MPNST of head and neck region originating in patients having NF1.

In Silico Identification of Therapeutic Targets and Novel Drug Candidates for Malignant Peripheral Nerve Sheath Tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are an aggressive form of sarcomas with a poor prognosis and limited treatment options. Therefore, new therapeutic targets are urgently needed to identify novel drugs. Based on the Gene Expression Omnibus database, an integrated analysis was performed to identify differentially expressed genes (DEGs) in MPNSTs compared to neurofibromas (NFs). Then functional enrichment analyses, protein-protein interaction (PPI) network construction, and hub gene selection were conducted. We explored DEG-guided repurposable drugs to treat MPNST based on the Library of Integrated Network-Based Cellular Signatures (LINCS) database. Furthermore, the binding affinity between predicted drug candidates and the MPNST-associated hub gene was calculated using molecular docking. We identified 89 DEGs in common with all three MPNSTs datasets. In the PPI networks, twist family bHLH transcription factor 1 (Twist1) with higher node degrees was further evaluated as a therapeutic target. Cytochalasin-d, cabozantinib, everolimus, refametinib, and BGT-226 were extracted from the LINCS database, which showed lower normalized connectivity scores (-1.88, -1.81, -1.78, -1.76, and -1.72, respectively) and was considered as drug candidates. In addition, the results of molecular docking between the five drugs and Twist1 showed a binding affinity of -6.61, -7.03, -7.73, -3.94, and -7.07 kcal/mol, respectively. Overall, our results describe the importance of Twist1 in MPNST pathogenesis. Everolimus was also found to be a potential therapeutic drug for MPNSTs.

Endoglin, a Novel Biomarker and Therapeutical Target to Prevent Malignant Peripheral Nerve Sheath Tumor Growth and Metastasis.

Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive soft-tissue sarcomas that lack effective treatments, underscoring the urgent need to uncover novel mediators of MPNST pathogenesis that may serve as potential therapeutic targets. Tumor angiogenesis is considered a critical event in MPNST transformation and progression. Here, we have investigated whether endoglin (ENG), a TGFβ coreceptor with a crucial role in angiogenesis, could be a novel therapeutic target in MPNSTs. ENG expression was evaluated in human peripheral nerve sheath tumor tissues and plasma samples. Effects of tumor cell-specific ENG expression on gene expression, signaling pathway activation and in vivo MPNST growth and metastasis, were investigated. The efficacy of ENG targeting in monotherapy or in combination with MEK inhibition was analyzed in xenograft models. ENG expression was found to be upregulated in both human MPNST tumor tissues and plasma-circulating small extracellular vesicles. We demonstrated that ENG modulates Smad1/5 and MAPK/ERK pathway activation and pro-angiogenic and pro-metastatic gene expression in MPNST cells and plays an active role in tumor growth and metastasis in vivo. Targeting with ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST growth and metastasis in xenograft models by reducing tumor cell proliferation and angiogenesis. Moreover, combination of anti-ENG therapy with MEK inhibition effectively reduced tumor cell growth and angiogenesis. Our data unveil a tumor-promoting function of ENG in MPNSTs and support the use of this protein as a novel biomarker and a promising therapeutic target for this disease.

Establishment and genomic characterization of a sporadic malignant peripheral nerve sheath tumor cell line

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive Schwann cell-derived neoplasms that occur sporadically or in patients with neurofibromatosis type 1 (NF1). Preclinical research on sporadic MPNSTs has been limited as few cell lines exist. We generated and characterized a new sporadic MPNST cell line, 2XSB, which shares the molecular and genomic features of the parent tumor. These cells have a highly complex karyotype with extensive chromothripsis. 2XSB cells show robust invasive 3-dimensional and clonogenic culture capability and form solid tumors when xenografted into immunodeficient mice. High-density single nucleotide polymorphism array and whole exome sequencing analyses indicate that, unlike NF1-associated MPNSTs, 2XSB cells have intact, functional NF1 alleles with no evidence of mutations in genes encoding components of Polycomb Repressor Complex 2. However, mutations in other genes implicated in MPNST pathogenesis were identified in 2XSB cells including homozygous deletion of CDKN2A and mutations in TP53 and PTEN. We also identified mutations in genes not previously associated with MPNSTs but associated with the pathogenesis of other human cancers. These include DNMT1, NUMA1, NTRK1, PDE11A, CSMD3, LRP5 and ACTL9. This sporadic MPNST-derived cell line provides a useful tool for investigating the biology and potential treatment regimens for sporadic MPNSTs.

Efficacy of MEK inhibition in a recurrent malignant peripheral nerve sheath tumor

The prognosis of recurrent malignant peripheral nerve sheath tumors (MPNST) is dismal, with surgical resection being the only definitive salvage therapy. Treatment with chemoradiation approaches has not significantly improved patient outcomes. Similarly, trials of therapies targeting MPNST genomic drivers have thus far been unsuccessful. Improved understanding of the molecular pathogenesis of MPNST indicates frequent activation of the mitogen-activated protein kinase (MAPK) cell signaling pathway. MEK inhibitors have shown activity in preclinical studies; however, their clinical efficacy has not been reported to date. We describe here a case of sustained complete response to MEK inhibition in an adolescent patient with a recurrent metastatic MPNST with multiple alterations in the MAPK pathway, guided by a precision oncology approach.

CSIG-02. R-RAS SUBFAMILY PROTEINS ELICIT DISTINCT PHYSIOLOGIC EFFECTS AND PHOSPHOPROTEOME ALTERATIONS IN NEUROFIBROMIN-NULL MPNST CELLS

Abstract Loss of the Ras GTPase-activating protein neurofibromin promotes the development of aggressive spindle cell neoplasms known as Malignant Peripheral Nerve Sheath Tumors (MPNSTs) in patients with the genetic disorder neurofibromatosis type 1 (NF1). Currently, the available chemotherapeutic regimens and radiotherapy are ineffective against MPNSTs, so the prognosis for patients with these neoplasms is poor. Neurofibromin loss dysregulates multiple Ras proteins in the classic (H-Ras, N-Ras, K-Ras) and R-Ras (R-Ras, R-Ras2/TC21, R-Ras3/M-Ras) subfamilies. Consequently, it is unclear which Ras proteins or pathways regulated by these Ras proteins should be therapeutically targeted in MPNSTs. We have previously shown that classic Ras proteins drive MPNST cell proliferation and survival. However, the role(s) of the R-Ras subfamily of proteins in MPNSTs have not been elucidated. To determine how R-Ras proteins contribute to the pathogenesis of neurofibromin-null MPNSTs, we introduced dominant negative (DN) R-Ras mutants, which are pan-inhibitors of the R-Ras subfamily, into MPNST cells and assessed the impact of R-Ras subfamily inhibition on mitogenesis, migration and the phosphoproteome. A panel of MPNST cell lines (STS-26T, YST-1, ST88-14, 90–8, NMS2, NMS-PC, S462, T265-2c) was used. Methodologies utilized include immunoblotting, PCR, Transwell migration, 3H-thymidine incorporation, and mass spectrometric analysis of phosphoprotein-enriched specimens. We found that R-Ras and R-Ras2 are widely expressed and can be activated in neurofibromin-null MPNST cells. In contrast to classic Ras proteins, we found that R-Ras proteins drive MPNST both mitogenesis and migration. Using mass spectrometry-based phosphoproteomics, we identified thirteen protein networks that were regulated by DN R-Ras, including networks affecting cellular movement via effects on microtubules. We chose to further study changes in ROCK1 phosphorylation and found that R-Ras subfamily proteins function, at least in some part, through the same pathways as ROCK1. We conclude that R-Ras proteins promote tumorigenesis by regulating distinct signaling pathways that regulate MPNST mitogenesis and migration.

RABL6A Is an Essential Driver of MPNSTs that Negatively Regulates the RB1 Pathway and Sensitizes Tumor Cells to CDK4/6 Inhibitors.

Malignant peripheral nerve sheath tumors (MPNST) are deadly sarcomas that lack effective therapies. In most MPNSTs, the retinoblastoma (RB1) tumor suppressor is disabled by hyperactivation of cyclin-dependent kinases (CDK), commonly through loss of CDK-inhibitory proteins such as p27(Kip1). RABL6A is an inhibitor of RB1 whose role in MPNSTs is unknown. To gain insight into MPNST development and establish new treatment options, we investigated RABL6A-RB1 signaling and CDK inhibitor-based therapy in MPNSTs. We examined patient-matched MPNSTs and precursor lesions by RNA sequencing (RNA-Seq) and IHC. Molecular and biological effects of silencing RABL6A and/or p27 in MPNST lines and normal human Schwann cells were determined. Tumor-suppressive effects of CDK inhibitors were measured in MPNST cells and orthotopic tumors. RABL6A was dramatically upregulated in human MPNSTs compared with precursor lesions, which correlated inversely with p27 levels. Silencing RABL6A caused MPNST cell death and G1 arrest that coincided with p27 upregulation, CDK downregulation, and RB1 activation. The growth-suppressive effects of RABL6A loss, and its regulation of RB1, were largely rescued by p27 depletion. Importantly, reactivation of RB1 using a CDK4/6 inhibitor (palbociclib) killed MPNST cells in vitro in an RABL6A-dependent manner and suppressed MPNST growth in vivo. Low-dose combination of drugs targeting multiple RB1 kinases (CDK4/6, CDK2) had enhanced antitumorigenic activity associated with potential MPNST cell redifferentiation. RABL6A is a new driver of MPNST pathogenesis that acts in part through p27-RB1 inactivation. Our results suggest RB1 targeted therapy with multiple pathway drugs may effectively treat MPNSTs.

Suz12 inactivation in p53- and nf1-deficient zebrafish accelerates the onset of malignant peripheral nerve sheath tumors and expands the spectrum of tumor types.

ABSTRACTPolycomb 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.

ErbB4 promotes malignant peripheral nerve sheath tumor pathogenesis via Ras-independent mechanisms

BackgroundWe have found that erbB receptor tyrosine kinases drive Ras hyperactivation and growth in NF1-null malignant peripheral nerve sheath tumors (MPNSTs). However, MPNSTs variably express multiple erbB receptors with distinct functional characteristics and it is not clear which of these receptors drive MPNST pathogenesis. Here, we test the hypothesis that altered erbB4 expression promotes MPNST pathogenesis by uniquely activating key cytoplasmic signaling cascades.MethodsErbB4 expression was assessed using immunohistochemistry, immunocytochemistry, immunoblotting and real-time PCR. To define erbB4 functions, we generated mice that develop MPNSTs with floxed Erbb4 alleles (P0-GGFβ3;Trp53+/−;Erbb4flox/flox mice) and ablated Erbb4 in these tumors. MPNST cell proliferation and survival was assessed using 3H-thymidine incorporation, MTT assays, Real-Time Glo and cell count assays. Control and Erbb4-null MPNST cells were orthotopically xenografted in immunodeficient mice and the growth, proliferation (Ki67 labeling), apoptosis (TUNEL labeling) and angiogenesis of these grafts was analyzed. Antibody arrays querying cytoplasmic kinases were used to identify erbB4-responsive kinases. Pharmacologic or genetic inhibition was used to identify erbB4-responsive kinases that drive proliferation.ResultsAberrant erbB4 expression was evident in 25/30 surgically resected human MPNSTs and in MPNSTs from genetically engineered mouse models (P0-GGFβ3 and P0-GGFβ3;Trp53+/− mice); multiple erbB4 splice variants that differ in their ability to activate PI3 kinase and nuclear signaling were present in MPNST-derived cell lines. Erbb4-null MPNST cells demonstrated decreased proliferation and survival and altered morphology relative to non-ablated controls. Orthotopic allografts of Erbb4-null cells were significantly smaller than controls, with reduced proliferation, survival and vascularization. ERBB4 knockdown in human MPNST cells similarly inhibited DNA synthesis and viability. Although we have previously shown that broad-spectrum erbB inhibitors inhibit Ras activation, Erbb4 ablation did not affect Ras activation, suggesting that erbB4 drives neoplasia via non-Ras dependent pathways. An analysis of 43 candidate kinases identified multiple NRG1β-responsive and erbB4-dependent signaling cascades including the PI3K, WNK1, STAT3, STAT5 and phospholipase-Cγ pathways. Although WNK1 inhibition did not alter proliferation, inhibition of STAT3, STAT5 and phospholipase-Cγ markedly reduced proliferation.ConclusionsErbB4 promotes MPNST growth by activating key non-Ras dependent signaling cascades including the STAT3, STAT5 and phospholipase-Cγ pathways. ErbB4 and its effector pathways are thus potentially useful therapeutic targets in MPNSTs.

TYK2 promotes malignant peripheral nerve sheath tumor progression through inhibition of cell death.

BackgroundMalignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas that arise most commonly in the setting of the Neurofibromatosis Type 1 (NF1) cancer predisposition syndrome. Despite aggressive multimodality therapy, outcomes are dismal and most patients die within 5 years of diagnosis. Prior genomic studies in our laboratory identified tyrosine kinase 2 (TYK2) as a frequently mutated gene in MPNST. Herein, we explored the function of TYK2 in MPNST pathogenesis.MethodsImmunohistochemistry was utilized to examine expression of TYK2 in MPNSTs and other sarcomas. To establish a role for TYK2 in MPNST pathogenesis, murine and human TYK2 knockdown and knockout cells were established using shRNA and CRISPR/Cas9 systems, respectively.ResultsWe have demonstrated that TYK2 was highly expressed in the majority of human MPNSTs examined. Additionally, we demonstrated that knockdown of Tyk2/TYK2 in murine and human MPNST cells significantly increased cell death in vitro. These effects were accompanied by a decrease in the levels of activated Stats and Bcl‐2 as well as an increase in the levels of Cleaved Caspase‐3. In addition, Tyk2‐KD cells demonstrated impaired growth in subcutaneous and metastasis models in vivo.ConclusionTaken together, these data illustrate the importance of TYK2 in MPNST pathogenesis and suggest that the TYK2 pathway may be a potential therapeutic target for these deadly cancers.

Therapeutic Targets for Malignant Peripheral nerve Sheath Tumors

Malignant peripheral nerve sheath tumors (MPNSTs) are rare and aggressive soft-tissue sarcomas with dismal prognosis. Complete resection, which is the only known definitive therapy, is not feasible with every tumor, and local recurrence after surgery is another challenge to successful treatment. Treatments used with other sarcoma types have not proven beneficial to MPNST patients. Targeted therapies blocking several signaling pathways known to drive MPNST pathogenesis have also not improved patient outcomes in clinical trials. This review discusses existing therapies and targeted chemotherapeutic options currently being tested clinically, and potential therapeutic avenues identified in preclinical studies that include targeting signaling pathways such as the HIPPO-YAP pathway and epigenetic mechanisms as well as multi-agent strategies.

Programming of Schwann Cells by Lats1/2-TAZ/YAP Signaling Drives Malignant Peripheral Nerve Sheath Tumorigenesis

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive Schwann cell (SC)-lineage-derived sarcomas. Molecular events driving SC-to-MPNST transformation are incompletely understood. Here, we show that human MPNSTs exhibit elevated HIPPO-TAZ/YAP expression, and that TAZ/YAP hyperactivity in SCs caused by Lats1/2 loss potently induces high-grade nerve-associated tumors with full penetrance. Lats1/2 deficiency reprograms SCs to a cancerous, progenitor-like phenotype and promotes hyperproliferation. Conversely, disruption of TAZ/YAP activity alleviates tumor burden in Lats1/2-deficient mice and inhibits human MPNST cell proliferation. Moreover, genome-wide profiling reveals that TAZ/YAP-TEAD1 directly activates oncogenic programs, including platelet-derived growth factor receptor (PDGFR) signaling. Co-targeting TAZ/YAP and PDGFR pathways inhibits tumor growth. Thus, our findings establish a previously unrecognized convergence between Lats1/2-TAZ/YAP signaling and MPNST pathogenesis, revealing potential therapeutic targets in these untreatable tumors.

Expression and inhibition of BRD4, EZH2 and TOP2A in neurofibromas and malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNST) are rare, highly aggressive sarcomas that can occur spontaneously or from pre-existing plexiform neurofibromas in neurofibromatosis type1 (NF1) patients. MPNSTs have high local recurrence rates, metastasize easily, are generally resistant to therapeutic intervention and frequently fatal for the patient. Novel targeted therapeutic strategies are urgently needed. Standard treatment for patients presenting with advanced disease is doxorubicin based chemotherapy which inhibits the actions of the enzyme topoisomerase IIα (TOP2A). Recent molecular studies using murine models and cell lines identified the bromodomain containing protein 4 (BRD4) and enhancer of zeste homolog 2 (EZH2) as novel targets for MPNST treatment. We investigated the expression and potential use of BRD4, EZH2 and TOP2A as therapeutic targets in human NF1-derived MPNSTs. The transcript levels of BRD4, EZH2 and TOP2A were determined in paired formalin-fixed paraffin-embedded (FFPE) neurofibroma/MPNST samples derived from the same NF1 patient and in a set of plexiform neurofibromas, atypical neurofibromas and MPNST. We further examined the effect on cell viability of genetic or pharmacological inhibition of BRD4, EZH2 and TOP2A in an MPNST cell line panel. Our results indicated that in MPNST samples BRD4 mRNA levels were not upregulated and that MPNST cell lines were relatively insensitive to the bromodomain inhibitor JQ1. We corroborated that EZH2 mRNA expression is increased in MPNST but failed to confirm its reported pivotal role in MPNST pathogenesis as EZH2 knockdown by siRNA did not interfere with cellular proliferation and viability. Finally, the relation between TOP2A levels and sensitivity for doxorubicin was examined, confirming reports that TOP2A mRNA levels were overexpressed in MPNST and showing that MPNST cell lines exhibited relatively high TOP2A protein levels and sensitivity to doxorubicin. We tentatively conclude that the potential for effective therapeutic intervention in MPNST by targeting BRD4, EZH2 and TOP2A individually, may be limited. Clinical studies are necessary to ultimately prove the relevance of BRD4 and EZH2 inhibition as novel therapeutic strategies for MPNST.

Abstract 4433: Deregulated microRNAs in NF1 derived malignant peripheral nerve sheath tumors (MPNST) and their role in carcinogenesis

Abstract Background and aim: MPNSTs are rare and highly aggressive soft tissue sarcomas that can occur spontaneously (sporadic MPNST) or from pre-existing plexiform neurofibromas in neurofibromatosis type 1 (NF1) patients. MPNSTs are usually diagnosed relatively late, metastasize easily, are resistant to therapeutic intervention and frequently fatal for the patient. Diagnostic biomarkers that detect early malignant transformation in the NF1 setting as well identification of putative targets for treatment are urgently needed. In this context we investigated the miRNA expression profiles in NF1-derived MPNST. Materials and Methods: Total RNA was isolated from sections of formalin fixed paraffin embedded (FFPE) tumor samples. miRNA expression was determined using a RT-PCR platform (TaqMan® Low Density Array Human MicroRNA Cards; Applied Biosystems). The expression of selected miRNAs was transiently modulated in neurofibroma and MPNST cell lines by transfection with miRNA mimics (Dharmacon) or LNA™ based anti-miRs (Exiqon) and effects on cell proliferation, invasion/migration and Wnt/β-catenin signaling were studied. Results: In order to identify miRNAs that are specifically deregulated in MPNST we analyzed miRNA expression in a unique set paired tumor samples (n=9) of plexiform neurofibroma and MPNST which were derived from the same NF1 patient. At least 90 miRNAs were found to be differentially expressed (FDR <10%; p<0.025) between neurofibromas and MPNST; the vast majority (82/90) of miRNAs being downregulated in MPNST. Interestingly, and in contrast to reports in the literature, sporadic MPNST samples (n=10) could be easily discriminated from NF1-derived MPNST by their miRNA expression profiles. On the basis of their statistical significance, fold difference and expression level, we selected miRNAs to examine their role in MPNST pathogenesis. MiRNA levels were modulated in MPNST (ST88-14, sNF96.2, 90-8TL) and neurofibroma (Hs53.T) cell lines using miRNA mimics for let-7b, miR-16, miR-26a, miR-29a, miR-139-5p, miR-146a and antisense inhibitors for miR-889, miR-135b. The effects of miRNA expression levels on cellular proliferation, invasion and migration were determined. As Wnt/β-catenin signaling is considered a cancer related driver pathway in MPNST we also investigated whether differentially expressed miRNAs could regulate this pathway focusing on miRNA previously linked to Wnt regulation or predicted to target Wnt signaling components. Using TOP/FOP luciferase reporter assays we analyzed the effects of miR-135b, miR-30a, miR-139-5p and miR-146a. Conclusion: miRNAs specifically deregulated in MPNST may fulfil key oncogenic roles and may be used as biomarker or for therapeutic purposes. Citation Format: Azadeh Amirnasr, Patricia F. van Kuijk, Robert M. Verdijk, Walter Taal, Stefan Sleijfer, Erik A. C. Wiemer. Deregulated microRNAs in NF1 derived malignant peripheral nerve sheath tumors (MPNST) and their role in carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4433. doi:10.1158/1538-7445.AM2017-4433

Spatially- and temporally-controlled postnatal p53 knockdown cooperates with embryonic Schwann cell precursor Nf1 gene loss to promote malignant peripheral nerve sheath tumor formation.

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that arise sporadically or in association with the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome. In individuals with NF1, MPNSTs are hypothesized to arise from Nf1-deficient Schwann cell precursor cells following the somatic acquisition of secondary cooperating genetic mutations (e.g., p53 loss). To model this sequential genetic cooperativity, we coupled somatic lentivirus-mediated p53 knockdown in the adult right sciatic nerve with embryonic Schwann cell precursor Nf1 gene inactivation in two different Nf1 conditional knockout mouse strains. Using this approach, ∼60% of mice with Periostin-Cre-mediated Nf1 gene inactivation (Periostin-Cre; Nf1flox/flox mice) developed tumors classified as low-grade MPNSTs following p53 knockdown (mean, 6 months). Similarly, ∼70% of Nf1+/− mice with GFAP-Cre-mediated Nf1 gene inactivation (GFAP-Cre; Nf1flox/null mice) developed low-grade MPNSTs following p53 knockdown (mean, 3 months). In addition, wild-type and Nf1+/− mice with GFAP-Cre-mediated Nf1 loss develop MPNSTs following somatic p53 knockout with different latencies, suggesting potential influences of Nf1+/− stromal cells in MPNST pathogenesis. Collectively, this new MPNST model system permits the analysis of somatically-acquired events as well as tumor microenvironment signals that potentially cooperate with Nf1 loss in the development and progression of this deadly malignancy.

Whole Exome Sequencing Reveals the Order of Genetic Changes during Malignant Transformation and Metastasis in a Single Patient with NF1-plexiform Neurofibroma.

Malignant peripheral nerve sheath tumors (MPNST) occur at increased frequency in individuals with neurofibromatosis type 1 (NF1), where they likely arise from benign plexiform neurofibroma precursors. While previous studies have used a variety of discovery approaches to discover genes associated with MPNST pathogenesis, it is currently unclear what molecular events are associated with the evolution of MPNST from plexiform neurofibroma. Whole-exome sequencing was performed on biopsy materials representing plexiform neurofibroma (n = 3), MPNST, and metastasis from a single individual with NF1 over a 14-year period. Additional validation cases were used to assess candidate genes involved in malignant progression, while a murine MPNST model was used for functional analysis. There was an increasing proportion of cells with a somatic NF1 gene mutation as the tumors progressed from benign to malignant, suggesting a clonal process in MPNST development. Copy number variations, including loss of one copy of the TP53 gene, were identified in the primary tumor and the metastatic lesion, but not in benign precursor lesions. A limited number of genes with nonsynonymous somatic mutations (βIII-spectrin and ZNF208) were discovered, several of which were validated in additional primary and metastatic MPNST samples. Finally, increased βIII-spectrin expression was observed in the majority of MPNSTs, and shRNA-mediated knockdown reduced murine MPNST growth in vivo. Collectively, the ability to track the molecular evolution of MPNST in a single individual with NF1 offers new insights into the sequence of genetic events important for disease pathogenesis and progression for future mechanistic study.

Combinatorial therapy with tamoxifen and trifluoperazine effectively inhibits malignant peripheral nerve sheath tumor growth by targeting complementary signaling cascades.

Chemotherapeutic agents effective against malignant peripheral nerve sheath tumors (MPNSTs) are urgently needed. We recently found that tamoxifen potently impedes xenograft growth. In vitro, tamoxifen inhibits MPNST proliferation and survival in an estrogen receptor-independent manner; these effects are phenocopied by the calmodulin inhibitor trifluoperazine. The present study was performed to establish the mechanism of action of tamoxifen in vivo and optimize its therapeutic effectiveness. To determine if tamoxifen has estrogen receptor-dependent effects in vivo, we grafted MPNST cells in castrated and ovariectomized mice; xenograft growth was unaffected by reductions in sex hormones. To establish whether tamoxifen and trifluoperazine additively or synergistically impede MPNST growth, mice xenografted with neurofibromatosis type 1-associated or sporadic MPNST cells were treated with tamoxifen, trifluoperazine, or both drugs for 30 days. Both monotherapies inhibited graft growth by 50%, whereas combinatorial treatment maximally reduced graft mass by 90% and enhanced decreases in proliferation and survival. Kinomic analyses showed that tamoxifen and trifluoperazine have both shared and distinct targets in MPNSTs. In addition, trifluoperazine prevented tamoxifen-induced increases in serum/glucocorticoid regulated kinase 1, a protein linked to tamoxifen resistance. These findings suggest that combinatorial therapy with tamoxifen and trifluoperazine is effective against MPNSTs because these agents target complementary pathways that are essential for MPNST pathogenesis.

PRC2 is recurrently inactivated through EED or SUZ12 loss in malignant peripheral nerve sheath tumors.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) represent a group of highly aggressive soft tissue sarcomas that may occur sporadically, in association with neurofibromatosis type I (NF1-), or after radiotherapy1–3. Using comprehensive genomic approaches, we identified loss-of-function (LOF) somatic alterations of the Polycomb repressive complex 2 (PRC2) core components, EED or SUZ12, in 92% of sporadic, 70% of NF1-associated and 90% of radiotherapy-associated MPNSTs. MPNSTs with PRC2 loss showed complete loss of H3K27me3 and aberrant transcriptional activation of multiple PRC2-repressed homeobox master regulators and their regulated developmental pathways. Introduction of the PRC2 component in a PRC2-deficient MPNST cell line restored H3K27me3 and decreased cell growth. Additionally, we identified frequent somatic alterations of CDKN2A (81% of all MPNSTs) and NF1 (72% of non-NF1-associated MPNSTs), and they significantly co-occur with PRC2 alterations. The highly recurrent and specific inactivation of PRC2, NF1, CDKN2A posits their critical and potentially cooperative roles in MPNST pathogenesis.

TAGLN expression is upregulated in NF1-associated malignant peripheral nerve sheath tumors by hypomethylation in its promoter and subpromoter regions.

Neurofibromatosis type 1 (NF1) caused by NF1 gene mutation is a commonly inherited autosomal dominant disorder. Malignant peripheral nerve sheath tumors (MPNSTs), a type of aggressive sarcoma, are a major cause of mortality in NF1 patients. The malignant transformation of benign plexiform neurofibromas (PNs) to MPNSTs is a marked peculiarity in NF1 patients, yet the pathogenesis remains poorly understood. We found that an actin-associated protein transgelin (SM22) was highly expressed in NF1-deficient MPNST tissues compared to NF1-deficient PN tissues using immunohistological staining and primary cultured MPNST cells in western blot analysis. We further found that this transgelin upregulation was caused by increased transcriptional expression of the TAGLN gene encoding transgelin. Comparison of DNA methylation values in the promoter and subpromoter regions of the TAGLN gene in three types of NF1-deficient primary-cultured cells, derived from an NF1 patient’s normal phenotype, a benign PN and MPNST tissues, revealed that the TAGLN gene was hypomethylated in the MPNST cells. Next, to determine the functional role of transgelin in MPNST pathogenesis, we manipulated the TAGLN gene expression and investigated the alteration of the RAS-mitogen-activated protein kinase (MAPK) signaling pathway in the normal-phenotypic and malignant tumor cells. The downregulation of TAGLN expression in NF1-deficient MPNST tumor cells through the treatment of the small interfering RNA resulted in a decrease in the RAS activation (GTP-RAS) and the downstream ERK1/2 activation (phosphorylated ERK1/2), while the overexpression of TAGLN in normal-phenotypic NF1-deficient cells caused an increase in RAS and ERK1/2 activation. These results indicate that upregulation of transgelin caused by hypomethylation of the TAGLN gene is closely involved in tumor progression in NF1.

BET Bromodomain Inhibition Triggers Apoptosis of NF1-Associated Malignant Peripheral Nerve Sheath Tumors through Bim Induction

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that develop sporadically or in neurofibromatosis type 1 (NF1) patients. There is no effective treatment for MPNSTs and they are typically fatal. To gain insights into MPNST pathogenesis, we utilized an MPNST mouse model that allowed us to study the evolution of these tumors at the transcriptome level. Strikingly, in MPNSTs we found upregulation of a chromatin regulator, Brd4, and show that BRD4 inhibition profoundly suppresses both growth and tumorigenesis. Our findings reveal roles for BET bromodomains in MPNST development and report a mechanism by which bromodomain inhibition induces apoptosis through induction of proapoptotic Bim, which may representa paradigm shift in therapy for MPNST patients.Moreover, these findings indicate epigenetic mechanisms underlying the balance of anti- and proapoptotic molecules and that bromodomain inhibitioncan shift this balance in favor of cancer cell apoptosis.