A heat shock protein 90 inhibitor reduces oncoprotein expression and induces cell death in heterogeneous glioblastoma cells with EGFR, PDGFRA, CDK4, and NF1 aberrations.

AIMS: Glioblastoma (GBM) is a highly malignant brain tumor. After treatment with the first-line drug temozolomide, only 50% of patients are responsive. Recent literature shows that the difficulty in treating GBM is mainly due to the heterogeneity of its four major cellular states, which are characterized by differences in EGFR, PDGFRA, CDK4, and NF1. Therefore, development of a multitarget drug is a potential strategy for treating heterogeneous GBM. MAIN METHODS: In this study, the antitumor ability of a potent heat shock protein 90 inhibitor, NVP-AUY922 (AUY922), was evaluated in GBM cell lines (U-87 MG and T98G cells) and patient-derived GBM cell lines [P#5 and P#5 temozolomide-resistant (TMZ-R) cells]. KEY FINDINGS: We found that AUY922 significantly reduced cell viability and colony formation in four GBM cell lines. AUY922 also significantly induced apoptosis by increasing PARP1 cleavage and the number of annexin V-positive cells. The autophagy indicators as MAP1LC3B cleavage and MAP1LC3B puncta were increased after AUY922 treatment. AUY922-induced cell death could be partially reversed by pharmacological inhibition of either apoptotic inhibitor or autophagy inhibitor. Moreover, AUY922 reduced the mRNA and protein expressions of EGFR, PDGFRA, CDK4, and NF1, which contribute to the four cellular state subtypes in GBM cells. In addition, the downstream signaling proteins of these four proteins, AKT/p-AKT, MAPK/p-MAPK, and BRAF, were downregulated after AUY922 treatment. SIGNIFICANCE: Taken together, AUY922 led to GBM cell death via apoptosis and autophagy, and reduced the mRNA and protein expression of EGFR, PDGFRA, CDK4, and NF1in heterogeneous GBM cells.

Inactivation of NF1 Promotes Resistance to EGFR Inhibition in KRAS/NRAS/BRAFV600 -Wild-Type Colorectal Cancer.

Through the use of an unbiased, genome-scale CRISPR modifier screen, we identified NF1 suppression as a mechanism of resistance to EGFR inhibition in NRAS/KRAS/BRAFV600 -wild-type colorectal cancer cells. Reduced NF1 expression permitted sustained signaling through the MAPK pathway to promote cell proliferation in the presence of EGFR inhibition. Targeting of MEK in combination with EGFR inhibition leads to synergistic antiproliferative activity. Human KRAS/NRAS/BRAFV600 -wild-type colorectal cancer cell lines with NF1 mutations displayed reduced NF1 mRNA or protein expression and were resistant to EGFR blockade by gefitinib or cetuximab. Cooccurring loss-of-function mutations in PTEN were associated with resistance to dual EGFR/MEK inhibition but cotreatment with a PI3K inhibitor further suppressed proliferation. Loss of NF1 may be a useful biomarker to identify patients that are less likely to benefit from single-agent anti-EGFR therapy in colorectal cancer and may direct potential combination strategies. IMPLICATIONS: This study suggests that further clinical validation of NF1 status as predictor of response to anti-EGFR targeting antibodies in patients with colorectal cancer with KRAS/NRAS/BRAFV600 -wild-type tumors is warranted.

Genomic Profiling of Childhood Tumor Patient-Derived Xenograft Models to Enable Rational Clinical Trial Design.

Accelerating cures for children with cancer remains an immediate challenge as a result of extensive oncogenic heterogeneity between and within histologies, distinct molecular mechanisms evolving between diagnosis and relapsed disease, and limited therapeutic options. To systematically prioritize and rationally test novel agents in preclinical murine models, researchers within the Pediatric Preclinical Testing Consortium are continuously developing patient-derived xenografts (PDXs)-many of which are refractory to current standard-of-care treatments-from high-risk childhood cancers. Here, we genomically characterize 261 PDX models from 37 unique pediatric cancers; demonstrate faithful recapitulation of histologies and subtypes; and refine our understanding of relapsed disease. In addition, we use expression signatures to classify tumors for TP53 and NF1 pathway inactivation. We anticipate that these data will serve as a resource for pediatric oncology drug development and will guide rational clinical trial design for children with cancer.

mTORC1 signaling is essential for neurofibromatosis type I gene modulated osteogenic differentiation of BMSCs.

Neurofibromatosis type I (NF1), which is caused by mutations in the NF1 gene, is a common autosomal dominant genetic disease leading to skeletal abnormalities. Both NF1 gene and mammalian target of rapamycin complex 1 (mTORC1) signaling are associated with the osteogenic differentiation of bone marrow stem cells (BMSCs). In this study, we hypothesized that mTORC1 signaling is involved in NF1-modulated osteoblast differentiation of BMSCs. Human BMSCs were cultured in an osteogenic induction medium. The expression of NF1 was either inhibited or overexpressed by transfecting NF1 with a specific small interfering RNA (siRNA) or pcDNA3.0 plasmid, respectively. In addition, an mTORC1 signaling inhibitor and agonist were used to investigate the effects of mTORC1 on NF1-modulated osteogenic differentiation of BMSCs. The results indicated that inhibiting the expression of NF1 with siRNA significantly decreased the mRNA levels of NF1, whereas overexpressing the expression of NF1 with pcDNA3.0 plasmid significantly increased the mRNA levels of NF1 at days 3, 7, 14 and 21 after culture. We observed reduced osteogenic differentiation and cell proliferation in the NF1-siRNA group and enhanced osteogenic differentiation and cell proliferation of BMSCs in the NF1-pcDNA3.0 group. The activity of mTORC1 signaling (p-mTORC1, p-S6K1, and p-4EBP1) was significantly upregulated in the NF1-siRNA group and significantly inhibited in the NF1-pcDNA3.0 group, 7 and 14 days after culture. The effects of NF1-siRNA and NF1-pcDNA3.0 on osteogenic differentiation of BMSCs and cell proliferation were reversed by mTORC1 inhibitor and agonist, respectively. In conclusion, NF1 modulates osteogenic differentiation and cell proliferation of human BMSCs and mTORC1 signaling is essential for this process.

Genomic Profiling in Patients With Malignant Peripheral Nerve Sheath Tumors Reveals Multiple Pathways With Targetable Mutations.

Background: The aim of this study was to determine the frequency of alterations in BRAF and other RAS/RAF genes, as well as other targetable pathways in malignant peripheral nerve sheath tumors (MPNSTs). Patients and Methods: Pathology specimens were available for 2 cohorts: (1) patients with MPNST at Swedish Cancer Institute (n=17) from 2004 through 2016, and (2) patients with MPNST evaluated for >300 genomic alterations at Foundation Medicine from 2014 through 2016 (n=186; including 2 Swedish patients with BRAF-mutated MPNST). Results: Of 201 MPNSTs, 13 (6.5%) demonstrated BRAF alterations. In the Foundation Medicine cohort, 10 of 84 tumors (11.9%) with no NF1 alterations had BRAF mutations (5 were V600E, 5 other), as did 3 of 102 (2.9%) tumors with NF1 alterations (1 V600E, 2 other). In the Foundation Medicine cohort, 47% of patients had an alteration in at least one other gene in the RAS/RAF pathway (not including NF1 or BRAF); 46% had alterations in the PI3 pathway, with 70% having alterations in at least 1 of the 2 pathways; 57% had a CDKN2A alteration (80% in BRAF-mutated and 71% in NF1-altered patients); and 70% had an alteration in DNA repair genes. MPNST, both NF1 wild-type and NF1-mutated, often harbor alterations in the RAS/RAF pathway as well as changes related to DNA repair and CDKN2A/B V600E and other mutations occur in BRAF, suggesting the need for second-generation activating BRAF inhibitors. The concurrence of BRAF and/or NF1 alterations with CDKN2A/B mutations, in particular, may be significant in the transformation of neurologic tumors from benign to malignant. Conclusions: All MPNSTs would benefit from a comprehensive genomic analysis. Treatments targeted to RAS/RAF, DNA repair, and CDKN2A/B pathways should be used and/or developed to treat this uncommon tumor.

LukS-PV-Regulated MicroRNA-125a-3p Promotes THP-1 Macrophages Differentiation and Apoptosis by Down-Regulating NF1 and Bcl-2.

BACKGROUND/AIMS: LukS-PV is a component of Panton-Valentine leukocidin (PVL). We have previously demonstrated that LukS-PV potently promoted differentiation and induced apoptosis in THP-1 cells. However, the precise mechanisms of these actions remain unknown. MicroRNAs (miRs) play important roles in cellular differentiation and apoptosis. This study aimed to investigate the role of miR-125a-3p in LukS-PV-regulated differentiation and apoptosis and its underlying mechanism in THP-1 cells. METHODS: MicroRNA profiling analyses were conducted to determine differential miRNA expression levels in THP-1 cells treated with LukS-PV. Cell differentiation and apoptosis were measured in THP-1 cells by gain-of-function and loss-of-function experiments. Bioinformatics analysis and luciferase reporter assays were used to confirm the targets of miR-125a-3p. The effects of the miR-125a-3p targets on cellular differentiation were determined by knocking them down. RESULTS: MiR-125a-3p was up-regulated after treating the human monocytic leukaemia cell line THP-1 with LukS-PV. In addition, miR-125a-3p positively regulated apoptosis and differentiation in THP-1 cells treated with LukS-PV. Concordantly, luciferase reporter assays confirmed that neurofibromatosis type 1 (NF1) and B-cell lymphoma 2 (Bcl-2) were direct target genes of miR-125a-3p. Moreover, NF1 knockdown in THP-1 cells significantly promoted differentiation in vitro. Finally, the extracellular signal-regulated kinase (ERK) pathway, a downstream target of NF1, was activated after NF1 knockdown. CONCLUSIONS: These findings confirm that miR-125a-3p is involved in LukS-PV-mediated cell differentiation and apoptosis in THP-1 cells.

Ets Factors Regulate Neural Stem Cell Depletion and Gliogenesis in Ras Pathway Glioma.

As the list of putative driver mutations in glioma grows, we are just beginning to elucidate the effects of dysregulated developmental signaling pathways on the transformation of neural cells. We have employed a postnatal, mosaic, autochthonous glioma model that captures the first hours and days of gliomagenesis in more resolution than conventional genetically engineered mouse models of cancer. We provide evidence that disruption of the Nf1-Ras pathway in the ventricular zone at multiple signaling nodes uniformly results in rapid neural stem cell depletion, progenitor hyperproliferation, and gliogenic lineage restriction. Abolishing Ets subfamily activity, which is upregulated downstream of Ras, rescues these phenotypes and blocks glioma initiation. Thus, the Nf1-Ras-Ets axis might be one of the select molecular pathways that are perturbed for initiation and maintenance in glioma.

Neurofibromin interacts with the cytoplasmic Dynein Heavy Chain 1 in melanosomes of human melanocytes.

Neurofibromin (NF1) is encoded by the NF1 tumour suppressor gene. Mutations result in a disorder known as Neurofibromatosis Type 1 (NF-1), and patients are often diagnosed due to the presence of unusual pigmentary patterns that include Café au lait macules (CALMs). Little is known about how loss of NF1 results in pigmentary defects in melanocytes. We sought to identify novel NF1 interacting proteins and elucidate the molecular mechanisms underlying the pigmentary defects. The cytoplasmic Dynein Heavy Chain 1 (DHC) was found to interact with NF1 along microtubules in vesicular structures identified to be melanosomes. Our studies suggest that NF1 is involved in melanosomal localization, and that disruptions in NF1-DHC interactions may contribute to the abnormal pigmentary features commonly associated with this debilitating syndrome.

Sensitivity of malignant peripheral nerve sheath tumor cells to TRAIL is augmented by loss of NF1 through modulation of MYC/MAD and is potentiated by curcumin through induction of ROS.

Malignant peripheral nerve sheath tumor (MPNST) is a rare aggressive form of sarcoma often associated with the tumor syndrome neurofibromatosis type 1 (NF1). We investigated the effects of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) on NF1 associated MPNST and determinants of TRAIL sensitivity. MPNST cell lines with complete neurofibromin deficiency were sensitive to apoptotic cell death induced by TRAIL whereas MPNST cells with retained neurofibromin expression or normal human Schwann cells were resistant. Increased sensitivity to TRAIL was associated with overexpression of death receptors, especially DR5. Re-expression of the GAP related domain of neurofibromin (NF1-GRD) suppressed DR5 expression and decreased sensitivity to TRAIL. We show that death receptor expression and TRAIL sensitivity critically depend on c-MYC and that c-MYC amounts are increased by MEK/ERK and PI3K/AKT signalling pathways which are suppressed by neurofibromin. Furthermore PI3K/AKT signalling strongly suppresses the MYC-antagonist MAD1 which significantly contributes to TRAIL sensitivity. Re-expression of the NF1-GRD decreased c-MYC and increased MAD1 amounts suggesting that neurofibromin influences TRAIL sensitivity at least in part by modulating the MYC/MAX/MAD network. The phytochemical curcumin further increased the sensitivity of neurofibromin deficient MPNST cells to TRAIL. This was presumably mediated by ROS, as it correlated with increased ROS production, was blocked by N-acetylcysteine and mimicked by exogenous ROS.

Targeting oncogenic Ras signaling in hematologic malignancies.

Ras proteins are critical nodes in cellular signaling that integrate inputs from activated cell surface receptors and other stimuli to modulate cell fate through a complex network of effector pathways. Oncogenic RAS mutations are found in ∼25% of human cancers and are highly prevalent in hematopoietic malignancies. Because of their structural and biochemical properties, oncogenic Ras proteins are exceedingly difficult targets for rational drug discovery, and no mechanism-based therapies exist for cancers with RAS mutations. This article reviews the properties of normal and oncogenic Ras proteins, the prevalence and likely pathogenic role of NRAS, KRAS, and NF1 mutations in hematopoietic malignancies, relevant animal models of these cancers, and implications for drug discovery. Because hematologic malignancies are experimentally tractable, they are especially valuable platforms for addressing the fundamental question of how to reverse the adverse biochemical output of oncogenic Ras in cancer.

The receptor tyrosine kinase Alk controls neurofibromin functions in Drosophila growth and learning.

Anaplastic Lymphoma Kinase (Alk) is a Receptor Tyrosine Kinase (RTK) activated in several cancers, but with largely unknown physiological functions. We report two unexpected roles for the Drosophila ortholog dAlk, in body size determination and associative learning. Remarkably, reducing neuronal dAlk activity increased body size and enhanced associative learning, suggesting that its activation is inhibitory in both processes. Consistently, dAlk activation reduced body size and caused learning deficits resembling phenotypes of null mutations in dNf1, the Ras GTPase Activating Protein-encoding conserved ortholog of the Neurofibromatosis type 1 (NF1) disease gene. We show that dAlk and dNf1 co-localize extensively and interact functionally in the nervous system. Importantly, genetic or pharmacological inhibition of dAlk rescued the reduced body size, adult learning deficits, and Extracellular-Regulated-Kinase (ERK) overactivation dNf1 mutant phenotypes. These results identify dAlk as an upstream activator of dNf1-regulated Ras signaling responsible for several dNf1 defects, and they implicate human Alk as a potential therapeutic target in NF1.

Proteasomal and genetic inactivation of the NF1 tumor suppressor in gliomagenesis.

Loss-of-function mutations in the NF1 tumor suppressor result in deregulated Ras signaling and drive tumorigenesis in the familial cancer syndrome neurofibromatosis type I. However, the extent to which NF1 inactivation promotes sporadic tumorigenesis is unknown. Here we report that NF1 is inactivated in sporadic gliomas via two mechanisms: excessive proteasomal degradation and genetic loss. NF1 protein destabilization is triggered by the hyperactivation of protein kinase C (PKC) and confers sensitivity to PKC inhibitors. However, complete genetic loss, which only occurs when p53 is inactivated, mediates sensitivity to mTOR inhibitors. These studies reveal an expanding role for NF1 inactivation in sporadic gliomagenesis and illustrate how different mechanisms of inactivation are utilized in genetically distinct tumors, which consequently impacts therapeutic sensitivity.

Neurofibromatosis type 1 (NF1) tumor suppressor, neurofibromin, regulates the neuronal differentiation of PC12 cells via its associating protein, CRMP-2.

Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, a negative regulator of Ras. Neurofibromin is implicated in the neuronal abnormality of NF1 patients; however, the precise cellular function of neurofibromin has yet to be clarified. Using proteomic strategies, we identified a set of neurofibromin-associating cellular proteins, including axon regulator CRMP-2 (Collapsin response mediator protein-2). CRMP-2 directly bound to the C-terminal domain of neurofibromin, and this association was regulated by the manner of CRMP-2 phosphorylation. In nerve growth factor-stimulated PC12 cells, neurofibromin and CRMP-2 co-localized particularly on the distal tips and branches of extended neurites. Suppression of neurofibromin using NF1 small interfering RNA significantly inhibited this neurite outgrowth and up-regulated a series of CRMP-2 phosphorylations by kinases identified as CDK5, GSK-3b, and Rho kinase. Overexpression of the NF1-RAS-GAP-related domain rescued these NF1 small interfering RNA-induced events. Our results suggest that neurofibromin regulates neuronal differentiation by performing one or more complementary roles. First, neurofibromin directly regulates CRMP-2 phosphorylation accessibility through the complex formation. Also, neurofibromin appears to indirectly regulate CRMP-2 activity by suppressing CRMP-2-phosphorylating kinase cascades via its Ras-GAP function. Our study demonstrates that the functional association of neurofibromin and CRMP-2 is essential for neuronal cell differentiation and that lack of expression or abnormal regulation of neurofibromin can result in impaired function of neuronal cells, which is likely a factor in NF1-related pathogenesis.

Psoriatic skin expresses the transcription factor Gli1: possible contribution of decreased neurofibromin expression.

BACKGROUND: Psoriasis is a chronic inflammatory disorder of skin characterized by hyperproliferation of keratinocytes. Intracellular signalling pathways inducing the hyperproliferation of keratinocytes remain to be elucidated. An inhibitor of Hedgehog (Hh) signalling, cyclopamine, was recently reported to clear psoriatic skin lesions, suggesting involvement of the Hh signalling pathway in the hyperproliferation of lesional keratinocytes. We have previously observed activation of the Hh signalling pathway in Schwann cells of plexiform neurofibroma in neurofibromatosis type 1 (NF1), which results from functional loss of the NF1 encoding protein, neurofibromin. In psoriasis, deficiency of neurofibromin expression has been observed in lesional keratinocytes. OBJECTIVES: To investigate whether the Hh signalling pathway would be activated in psoriasis and whether inhibition of neurofibromin expression would enhance the activation of the Hh signalling pathway. METHODS: Activation of the Hh signalling pathway was examined by protein expression of one of the target genes, GLI1, coding for the transcription factor Gli1. Immunohistochemical studies were performed on seven psoriatic skin samples and seven control normal skin samples with a standard immunoperoxidase technique. mRNA expression of GLI1 was analysed by reverse transcriptase-polymerase chain reaction in HaCaT cells transfected with double-strand small interfering RNA for NF1. RESULTS: Our results showed Gli1 expression in psoriatic skin but not in control normal skin. Inhibition of neurofibromin expression in HaCaT cells upregulated mRNA expression of GLI1. CONCLUSIONS: Our findings indicate that the Hh signalling pathway is activated in psoriasis and that neurofibromin deficiency may upregulate the pathway.

The neurofibromatosis type 1 gene product neurofibromin enhances cell motility by regulating actin filament dynamics via the Rho-ROCK-LIMK2-cofilin pathway.

Neurofibromin is a neurofibromatosis type 1 (NF1) tumor suppressor gene product with a domain that acts as a GTPase-activating protein and functions, in part, as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation, predisposing to a variety of tumors of the peripheral and central nervous systems. We show here, using the small interfering RNA (siRNA) technique, that neurofibromin dynamically regulates actin cytoskeletal reorganization, followed by enhanced cell motility and gross cell aggregation in Matrigel matrix. NF1 siRNA induces characteristic morphological changes, such as excessive actin stress fiber formation, with elevated negative phosphorylation levels of cofilin, which regulates actin cytoskeletal reorganization by depolymerizing and severing actin filaments. We found that the elevated phosphorylation of cofilin in neurofibromin-depleted cells is promoted by activation of a Rho-ROCK-LIMK2 pathway, which requires Ras activation but is not transduced through three major Ras-mediated downstream pathways via Raf, phosphatidylinositol 3-kinase, and RalGEF. In addition, the exogenous expression of the NF1-GTPase-activating protein-related domain suppressed the NF1 siRNA-induced phenotypes. Neurofibromin was demonstrated to play a significant role in the machinery regulating cell proliferation and in actin cytoskeletal reorganization, which affects cell motility and adhesion. These findings may explain, in part, the mechanism of multiple neurofibroma formation in NF1 patients.