Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

LPAR1 and aberrantly expressed LPAR3 differentially promote the migration and proliferation of malignant peripheral nerve sheath tumor cells.

Schwann cell-derived neoplasms known as malignant peripheral nerve sheath tumors (MPNSTs) are the most common malignancy and the leading cause of death in individuals with neurofibromatosis Type 1. Using genome-scale shRNA screens, we have previously found evidence suggesting that lysophosphatidic acid receptors (LPARs) are essential for MPNST proliferation and/or survival. Here, we examine the expression and mutational status of all six LPA receptors in MPNSTs, assess the role that individual LPA receptors play in MPNST physiology and examine their ability to activate key neurofibromin-regulated signaling cascades. We found that human Schwann cells express LPAR1 and LPAR6, while MPNST cells express predominantly LPAR1 and LPAR3. Whole exome sequencing of 16 MPNST cell lines showed no evidence of mutations in any LPAR genes or ENPP2, a gene encoding a major LPA biosynthetic enzyme. Oleoyl-LPA, an LPA variant with an unsaturated side chain, promoted MPNST cell proliferation and migration. LPAR1 knockdown ablated the promigratory effect of LPA, while LPAR3 knockdown decreased proliferation. Inhibition of R-Ras signaling with a doxycycline-inducible dominant negative (DN) R-Ras mutant, which inhibits both R-Ras and R-Ras2, blocked LPA's promigratory effect. In contrast, DN R-Ras did not affect migration induced by neuregulin-1ß (NRG1ß), suggesting that LPA and NRG1ß promote MPNST migration via distinct pathways. LPA-induced migration was also inhibited by Y27632, an inhibitor of the ROCK1/2 kinases that mediate R-Ras effects in MPNSTs. Thus, LPAR1 and aberrantly expressed LPAR3 mediate distinct effects in MPNSTs. These receptors and the signaling pathways that they regulate are potentially useful therapeutic targets in MPNSTs.

The RASopathies: Biology, genetics and therapeutic options.

The RASopathies are a group of genetic diseases in which the Ras/MAPK signaling pathway is inappropriately activated as a result of mutations in genes encoding proteins within this pathway. As their causative mutations have been identified, this group of diseases has expanded to include neurofibromatosis type 1 (NF1), Legius syndrome, Noonan syndrome, CBL syndrome, Noonan syndrome-like disorder with loose anagen hair, Noonan syndrome with multiple lentigines, Costello syndrome, cardiofaciocutaneous syndrome, gingival fibromatosis and capillary malformation-arteriovenous malformation syndrome. Many of these genetic disorders share clinical features in common such as abnormal facies, short stature, varying degrees of cognitive impairment, cardiovascular abnormalities, skeletal abnormalities and a predisposition to develop benign and malignant neoplasms. Others are more dissimilar, even though their mutations are in the same gene that is mutated in a different RASopathy. Here, we describe the clinical features of each RASopathy and contrast them with the other RASopathies. We discuss the genetics of these disorders, including the causative mutations for each RASopathy, the impact that these mutations have on the function of an individual protein and how this dysregulates the Ras/MAPK signaling pathway. As several of these individual disorders are genetically heterogeneous, we also consider the different genes that can be mutated to produce disease with the same phenotype. We also discuss how our growing understanding of dysregulated Ras/MAPK signaling had led to the development of new therapeutic agents and what work will be critically important in the future to improve the lives of patients with RASopathies.

R-Ras subfamily proteins elicit distinct physiologic effects and phosphoproteome alterations in neurofibromin-null MPNST cells.

BACKGROUND: Loss of the Ras GTPase-activating protein neurofibromin promotes nervous system tumor pathogenesis in patients with neurofibromatosis type 1 (NF1). Neurofibromin loss potentially hyperactivates classic Ras (H-Ras, N-Ras, K-Ras), M-Ras, and R-Ras (R-Ras, R-Ras2/TC21) subfamily proteins. We have shown that classic Ras proteins promote proliferation and survival, but not migration, in malignant peripheral nerve sheath tumor (MPNST) cells. However, it is unclear whether R-Ras, R-Ras2 and M-Ras are expressed and hyperactivated in MPNSTs and, if so, whether they contribute to MPNST pathogenesis. We assessed the expression and activation of these proteins in MPNST cells and inhibited them to determine the effect this had on proliferation, migration, invasion, survival and the phosphoproteome. METHODS: NF1-associated (ST88-14, 90-8, NMS2, NMS-PC, S462, T265-2c) and sporadic (STS-26T, YST-1) MPNST lines were used. Cells were transfected with doxycycline-inducible vectors expressing either a pan-inhibitor of the R-Ras subfamily [dominant negative (DN) R-Ras] or enhanced green fluorescent protein (eGFP). Methodologies used included immunoblotting, immunocytochemistry, PCR, Transwell migration, 3H-thymidine incorporation, calcein cleavage assays and shRNA knockdowns. Proteins in cells with or without DN R-Ras expression were differentially labeled with SILAC and mass spectrometry was used to identify phosphoproteins and determine their relative quantities in the presence and absence of DN R-Ras. Validation of R-Ras and R-Ras2 action and R-Ras regulated networks was performed using genetic and/or pharmacologic approaches. RESULTS: R-Ras2 was uniformly expressed in MPNST cells, with R-Ras present in a major subset. Both proteins were activated in neurofibromin-null MPNST cells. Consistent with classical Ras inhibition, DN R-Ras and R-Ras2 knockdown inhibited proliferation. However, DN R-Ras inhibition impaired migration and invasion but not survival. Mass spectrometry-based phosphoproteomics identified thirteen protein networks distinctly regulated by DN R-Ras, including multiple networks regulating cellular movement and morphology. ROCK1 was a prominent mediator in these networks. DN R-Ras expression and RRAS and RRAS2 knockdown inhibited migration and ROCK1 phosphorylation; ROCK1 inhibition similarly impaired migration and invasion, altered cellular morphology and triggered the accumulation of large intracellular vesicles. CONCLUSIONS: R-Ras proteins function distinctly from classic Ras proteins by regulating distinct signaling pathways that promote MPNST tumorigenesis by mediating migration and invasion. Mutations of the NF1 gene potentially results in the activation of multiple Ras proteins, which are key regulators of many biologic effects. The protein encoded by the NF1 gene, neurofibromin, acts as an inhibitor of both classic Ras and R-Ras proteins; loss of neurofibromin could cause these Ras proteins to become persistently active, leading to the development of cancer. We have previously shown that three related Ras proteins (the classic Ras proteins) are highly activated in malignant peripheral nerve sheath tumor (MPNST) cells with neurofibromin loss and that they drive cancer cell proliferation and survival by activating multiple cellular signaling pathways. Here, we examined the expression, activation and action of R-Ras proteins in MPNST cells that have lost neurofibromin. Both R-Ras and R-Ras2 are expressed in MPNST cells and activated. Inhibition of R-Ras action inhibited proliferation, migration and invasion but not survival. We examined the activation of cytoplasmic signaling pathways in the presence and absence of R-Ras signaling and found that R-Ras proteins regulated 13 signaling pathways distinct from those regulated by classic Ras proteins. Closer study of an R-Ras regulated pathway containing the signaling protein ROCK1 showed that inhibition of either R-Ras, R-Ras2 or ROCK1 similarly impaired cellular migration and invasion and altered cellular morphology. Inhibition of R-Ras/R-Ras2 and ROCK1 signaling also triggered the accumulation of abnormal intracellular vesicles, indicating that these signaling molecules regulate the movement of proteins and other molecules in the cellular interior. Video Abstract.

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells.

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed. This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

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.

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

BACKGROUND: We 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. METHODS: ErbB4 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-GGFß3;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. RESULTS: Aberrant erbB4 expression was evident in 25/30 surgically resected human MPNSTs and in MPNSTs from genetically engineered mouse models (P0-GGFß3 and P0-GGFß3;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 NRG1ß-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. CONCLUSIONS: ErbB4 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.

Classic Ras Proteins Promote Proliferation and Survival via Distinct Phosphoproteome Alterations in Neurofibromin-Null Malignant Peripheral Nerve Sheath Tumor Cells.

Neurofibromin, the tumor suppressor encoded by the neurofibromatosis type 1 (NF1) gene, potentially suppresses the activation of H-Ras, N-Ras, and K-Ras. However, it is not known whether these classic Ras proteins are hyperactivated in NF1-null nerve sheath tumors, how they contribute to tumorigenesis, and what signaling pathways mediate their effects. Here we show that H-Ras, N-Ras, and K-Ras are coexpressed with their activators (guanine nucleotide exchange factors) in neurofibromin-null malignant peripheral nerve sheath tumor (MPNST) cells, and that all 3 Ras proteins are activated. Dominant negative (DN) H-Ras, a pan-inhibitor of the classic Ras family, inhibited MPNST proliferation and survival, but not migration. However, NF1-null MPNST cells were variably dependent on individual Ras proteins. In some lines, ablation of H-Ras, N-Ras, and/or K-Ras inhibited mitogenesis. In others, ablation of a single Ras protein had no effect on proliferation; in these lines, ablation of a single Ras protein resulted in compensatory increases in the activation and/or expression of other Ras proteins. Using mass spectrometry-based phosphoproteomics, we identified 7 signaling networks affecting morphology, proliferation, and survival that are regulated by DN H-Ras. Thus, neurofibromin loss activates multiple classic Ras proteins that promote proliferation and survival by regulating several distinct signaling cascades.

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.