Neurofibromatosis Type 1 Mutations Research Articles

Density and function of actin-microdomains in healthy and NF1 deficient osteoclasts revealed by the combined use of atomic force and stimulated emission depletion microscopy

Actin and myosins (IIA, IIB, and X) generate mechanical forces in osteoclasts that drive functions such as migration and membrane trafficking. In neurofibromatosis, these processes are perturbed due to a mutation in neurofibromatosis type 1 (NF1) gene. This mutation leads to generation of hyperactive bone-resorbing osteoclasts that increases incidence of skeletal dysplasia e.g. early-onset osteoporosis in patients suffering from neurofibromatosis. To study the density and function of actin clusters in mutated cells we introduce a new approach for combined use of a stimulated emission depletion (STED) microscope with an atomic force microscope (AFM). We resolved actin-cores within actin-microdomains at four typical structures (podosome-belt, podosome raft, actin patches, and sealing zone) for osteoclasts cultured on bone as well as on glass. Densities of actin-cores in these structures were higher on bone than on glass, and the nearest neighbor distances were shortest in sealing zones, where also an accumulation of vesicular material was observed at their center. In NF1 deficient osteoclasts, the clustering was tighter and there was also more vesicular material accumulated inside the sealing zone. Using the STED-AFM system, we measured the condensation of the actin structures in real-time after a bone-coated cantilever was placed in contact with a differentiated osteoclast and found that the condensation of actin was initiated at 40 min, after sufficient local actin concentration was reached. A functional implication of the less dense clustering in NF1 deficient cells was that the adhesion of these cells was less specific for bone. The data and new methodologies presented here build a foundation for establishing novel actomyosin dependent mechanisms during osteoclast migration and resorption.

Prevalence, Type, and Molecular Spectrum of NF1 Mutations in Patients with Neurofibromatosis Type 1 and Congenital Heart Disease.

The aim of this study was to assess the prevalence and type of congenital heart disease (CHD) and the associated mutation spectrum in a large series of patients with neurofibromatosis type 1 (NF1), and correlate the mutation type with the presence and subgroups of cardiac defects. The study cohort included 493 individuals with molecularly confirmed diagnosis of NF1 for whom cardiac evaluation data were available. CHD was reported in 62/493 (12.6%) patients. Among these patients, 23/62 (37.1%) had pulmonary valve stenosis/dysplasia, 20/62 (32.3%) had mitral valve anomalies, and 10/62 (16.1%) had septal defects. Other defects occurred as rare events. In this NF1 subcohort, three subjects carried a whole-gene deletion, while 59 were heterozygous for an intragenic mutation. A significantly increased prevalence of non-truncating intragenic mutations was either observed in individuals with CHD (22/59, 37.3%) or with pulmonary valve stenosis (13/20, 65.0%), when compared to individuals without CHD (89/420, 21.2%) (p = 0.038) or pulmonary valve stenosis (98/459, 21.4%) (p = 0.002). Similarly, patients with non-truncating NF1 mutations displayed two- and six-fold higher risk of developing CHD (odds ratio = 1.9713, 95% confidence interval (CI): 1.1162–3.4814, p = 0.0193) and pulmonary valve stenosis (odds ratio = 6.8411, 95% CI: 2.6574–17.6114, p = 0.0001), respectively. Noteworthy, all but one patient (19/20, 95.0%) with pulmonary valve stenosis, and 18/35 (51.4%) patients with other CHDs displayed Noonan syndrome (NS)-like features. Present data confirm the significant frequency of CHD in patients with NF1, and provide further evidence for a higher than expected prevalence of NF1 in-frame variants and NS-like characteristics in NF1 patients with CHD, particularly with pulmonary valve stenosis.

Assessment of nociception and related quality-of-life measures in a porcine model of neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder resulting from germline mutations in the NF1 gene, which encodes neurofibromin. Patients experience a variety of symptoms, but pain in the context of NF1 remains largely underrecognized. Here, we characterize nociceptive signaling and pain behaviors in a miniswine harboring a disruptive NF1 mutation (exon 42 deletion). We present the first characterization of pain-related behaviors in a pig model of NF1, identifying unchanged agitation scores, lower tactile thresholds (allodynia), and decreased response latencies to thermal laser stimulation (hyperalgesia) in NF1 (females only) pigs. Male NF1 pigs with tumors showed reduced sleep quality and increased resting, 2 health-related quality-of-life symptoms found to be comorbid in people with NF1 pain. We explore these phenotypes in relationship to suppression of the increased activity of the N-type voltage-gated calcium (CaV2.2) channel by pharmacological antagonism of phosphorylation of a regulatory protein-the collapsin response mediator protein 2 (CRMP2), a known interactor of neurofibromin, and by targeting the interface between the α subunit of CaV2.2 and the accessory β-subunits with small molecules. Our data support the use of NF1 pigs as a large animal model for studying NF1-associated pain and for understanding the pathophysiology of NF1. Our findings demonstrate the translational potential of 2 small molecules in reversing ion channel remodeling seen in NF1. Interfering with CaV2.2, a clinically validated target for pain management, might also be a promising therapeutic strategy for NF1-related pain management.

Abstract 5311: Establishment of neurofibroma cells and dedifferentiated fat (DFAT) cells from tumor tissues from patients diagnosed with NF1 (Neurofibromatosis type 1)

Abstract Background: The NF1 tumor suppressor gene encodes neurofibromin and is a functional Ras GTPase-activating protein (RasGAP) that negatively regulates the Ras signaling pathway by accelerating the conversion of activated Ras-GTP to inactive Ras-GDP. NF1 gene germline mutations cause various Neurofibromatosis type 1 (NF1) symptoms, including neurofibroma development. We are developing in vitro models that recapitulate the pathological and clinical properties of neurofibromas with the aim of developing therapeutic strategies to treat patients with NF1 gene-deficient tumors. Methods: Neurofibroma cells and dedifferentiated fat (DFAT) cells were established from NF1 patient tumors. Tissue samples were obtained during tumor resection at our hospital from patients who met NIH clinical diagnostic criteria for NF1. Whole-blood specimens were also obtained for gene analysis. All patients provided written informed consent. The institutional review board at our university approved this aspect of our study. Tumor tissues were dissociated in DMEM containing collagenase. The neurofibroma cells at the bottom of the tube and the floating stromal adipocytes were collected separately after centrifugation. To establish DFAT cells, the stromal adipocytes were placed in a culture flask filled with 20% FBS-DMEM, and then the flask was inverted and incubated at 37 °C in a humidified atmosphere of 5% CO2. The stromal adipocytes floated up through the medium and adhered to the ceiling of the flask. After 1 week, the cells were firmly attached to the ceiling and had dedifferentiated. The DFAT cells as well as the neurofibroma cells can be passaged. The DFAT cells exhibited multipotent differentiation abilities into a variety of cell types. Results: We established neurofibroma cells and DFAT cells from NF1-associated neurofibromas. We performed flow cytometry analysis and found that the cells derived from NF1 patients expressed SOX10, S100, and CD90, all of which are expressed in Schwann cells. We identified the NF1 mutations in patients by next-generation sequencing. Peripheral blood specimens from patients 1 and 2 were positive for c.1466A>G, p.Tyr489Cys and c.3213_3214delAA, p.Ser1072Hisfs*16 mutations of NF1, respectively. We also identified NF1 mutations in the cells that we had established from tumors. In the tumor specimen of patient 1, we identified an additional somatic mutation, c.6772C>T, p.Arg2258X of NF1 gene. Conclusions: We established NF1 gene-deficient neurofibroma cells and NF1 gene-deficient DFAT cells from the tumor tissues from NF1 patients with NF1 gene mutations. These cells may well be useful in studying the pathophysiology of NF1 gene-deficient tumors as well as cell-based drug screening to facilitate the development of new treatments. Note: This abstract was not presented at the meeting. Citation Format: Yoshimi Arima, Hiroyuki Nobusue, Shigeki Sakai, Kazuo Kishi, Toshiki Takenouchi, Kenjiro Kosaki, Hideyuki Saya. Establishment of neurofibroma cells and dedifferentiated fat (DFAT) cells from tumor tissues from patients diagnosed with NF1 (Neurofibromatosis type 1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5311.

Neurofibromatosis type 1‐related pseudarthrosis: Beyond the pseudarthrosis site

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting approximately 1 in 2,000 newborns. Up to 5% of NF1 patients suffer from pseudarthrosis of a long bone (NF1-PA). Current treatments are often unsatisfactory, potentially leading to amputation. To gain more insight into the pathogenesis we cultured cells from PA tissue and normal-appearing periosteum of the affected bone for NF1 mutation analysis. PA cells were available from 13 individuals with NF1. Biallelic NF1 inactivation was identified in all investigated PA cells obtained during the first surgery. Three of five cases sampled during a later intervention showed biallelic NF1 inactivation. Also, in three individuals, we examined periosteum-derived cells from normal-appearing periosteum proximal and distal to the PA. We identified the same biallelic NF1 inactivation in the periosteal cells outside the PA region. These results indicate that NF1 inactivation is required but not sufficient for the development of NF1-PA. We observed that late-onset NF1-PA occurs and is not always preceded by congenital bowing. Furthermore, the failure to identify biallelic inactivation in two of five later interventions and one reintervention with a known somatic mutation indicates that NF1-PA can persist after the removal of most NF1 negative cells.

Loss of GTPase activating protein neurofibromin stimulates paracrine cell communication via macropinocytosis

Neurofibromin, the protein product of the neurofibromatosis type 1 (NF1) tumor suppressor gene, is a negative regulator of Ras signaling. Patients with mutations in NF1 have a strong predisposition for cardiovascular disease, which contributes to their early mortality. Nf1 heterozygous (Nf1+/−) bone marrow to wild type chimeras and mice with heterozygous recombination of Nf1 in myeloid cells recapitulate many of the vascular phenotypes observed in Nf1+/− mutants. Although these results suggest that macrophages play a central role in NF1 vasculopathy, the underlying mechanisms are currently unknown. In the present study, we employed macrophages isolated from either Nf1+/− or Lysm Cre+/Nf1f/f mice to test the hypothesis that loss of Nf1 stimulates macropinocytosis in macrophages. Scanning electron microscopy and flow cytometry analysis of FITC-dextran internalization demonstrated that loss of Nf1 in macrophages stimulates macropinocytosis. We next utilized various cellular and molecular approaches, pharmacological inhibitors and genetically modified mice to identify the signaling mechanisms mediating macropinocytosis in Nf1-deficient macrophages. Our results indicate that loss of Nf1 stimulates PKCδ-mediated p47phox phosphorylation via RAS activation, leading to increased NADPH oxidase 2 activity, reactive oxygen species generation, membrane ruffling and macropinocytosis. Interestingly, we also found that Nf1-deficient macrophages internalize exosomes derived from angiotensin II-treated endothelial cells via macropinocytosis in vitro and in the peritoneal cavity in vivo. As a result of exosome internalization, Nf1-deficient macrophages polarized toward an inflammatory M1 phenotype and secreted increased levels of proinflammatory cytokines compared to controls. In conclusion, the findings of the present study demonstrate that loss of Nf1 stimulates paracrine endothelial to myeloid cell communication via macropinocytosis, leading to proinflammatory changes in recipient macrophages.

Trametinib for aggressive gliomas in adults with neurofibromatosis type 1.

e13562 Background: Neurofibromatosis type 1 (NF1) is caused by mutations of the tumor suppressor gene NF1 resulting in decreased levels of neurofibromin and dysregulation of the RAS pathway, including MEK/ERK upregulation. NF1 is characterized by multiple benign and malignant tumors. Central nervous system tumors have an overall standardized incidence ratio of 37.5 in population studies with high rates of aggressive gliomas in adults with NF1. The response to standard glioma therapies is mixed. MEK1/2 inhibitors have shown activity in NF1 driven plexiform neurofibromas and low grade gliomas, however, data is lacking about the optimal dose or sequencing of MEK1/2 inhibitors for adults with NF1 associated aggressive gliomas. We present recent experiences with the MEK1/2 inhibitor trametinib in adults with NF1 and aggressive gliomas. Methods: A retrospective chart review was conducted on all adult patients with NF1 and astrocytoma treated with trametinib at the Johns Hopkins Brain Cancer Program between 2016-2018. Results: Three patients met selection criteria. Two received trametinib as initial therapy (2 mg daily). All developed an acneiform rash; one had transient transaminitis requiring temporary discontinuation and subsequent dose reductions. Clinical benefit was achieved associated with two partial radiographic responses. Conclusions: Astrocytomas in adults with NF1 often have a more aggressive course than suggested by histology. The cases presented here demonstrate clinical benefit from single agent MEK inhibition as well as possible synergistic effects with conventional therapies for gliomas in this patient population. These observations highlight the need for further research in this area. [Table: see text]

A novel pathogenic mutation in neurofibromatosis type 1

A novel pathogenic mutation in neurofibromatosis type 1

Neurofibromatosis type 2 gene mutation and progesterone receptor messenger RNA expression in the pathogenesis of sporadic orbitocranial meningioma.

To investigate neurofibromatosis type 2 (NF2) gene mutation at mRNA levels in sporadic orbitocranial meningioma and its association with progesterone receptor (PR) mRNA expression. This was a case-control study. Thirty-four sporadic meningioma patients with no familial NF2-related meningioma history were recruited. They were interviewed for their obstetric, gynecologic, and contraception history. PR investigation was performed with real-time polymerase chain reaction (PCR). NF2 mutation was investigated using Qbiomarker Somatic Mutation PCR Assay at NF2 mRNA level after its cDNA extraction (four mRNA mutation cytoband coordinates for nucleotide change: c.634C>T/p.Q212, c.655G>A/p.V219M, c.784C>T/p.R262 and c.1228C>T/p. Q410). After mutation analysis at mRNA level, NF2 gene mutation was found in 35.29% patients. Non-mutation group was strongly associated with exogenous hormonal exposure (non-mutation vs mutation: 95.5% vs 83.3%, P<0.001). PR mRNA was found significantly lower in non-mutation group (P=0.033) which presumed as long term exogenous progesterone exposure. However, mutation group was associated with higher rate of progression to grade II (mutation vs non-mutation, 18.2% vs 5%, P<0.001) and was associated more in fibrous and anaplastic tumor tissue. NF2 mutation-meningioma is associated with higher grade of meningioma. Non NF2 mutation-meningioma is strongly associated with exogenous progesterone exposure and lower PR expression.

SAT-324 An Unusual Association between Pheochromocytoma and Hyperparathyroidism in a Patient with Neurofibromatosis Type 1: A Possible Co-Occurrence of Two Hereditary Endocrine Tumor Syndromes

Background: Patients with neurofibromatosis type 1 (NF1) have increased risk of developing various types of endocrine tumors including pheochromocytoma; however, an association with hyperparathyroidism is a rare entity. We present an unusual combination of pheochromocytoma and hyperparathyroidism in a NF-1 patient; a double mutation or digenic inheritance in hereditary endocrine syndromes is proposed. Clinical case: A 66-year-old woman with clinical features of NF1 presented with alteration of consciousness. Physical examination was unremarkable except a large lower abdominal mass and normotension. Severe hypercalcemia (15.02 mg/dL) with markedly elevated parathyroid hormone (PTH 1,550.6 pg/mL; 15-68.3) levels was revealed. A neck ultrasonography showed a 1.6-cm well-defined heterogeneous hypoechoic lesion at postero-inferior aspect of right thyroid lobe corresponding to the result from Tc99m MIBI parathyroid scintigraphy suggestive of parathyroid adenoma. CT whole abdomen revealed a huge uterine myoma and an accidental finding of a 2.5-cm well-defined enhancing nodule at right adrenal gland. Pheochromocytoma was diagnosed by an elevated of 24-h urine metanephrine (771.1 ug; < 276.1) with a normal level of 24 h-urine normetanephrine (273.7 ug; < 668.7). Genetic study showed a heterogeneous mutation at Gly681Ser in exon 11 of RET gene. The co-existence of NF1 and multiple endocrine neoplasia type 2A (MEN2A) was proposed. Laparoscopic right adrenalectomy was performed and its pathological findings confirmed the diagnosis of pheochromocytoma. Subsequently, total parathyroidectomy with thyroidectomy was done with its pathological study showing a parathyroid adenoma, multinodular goiter without the evidence of medullary thyroid carcinoma. The patient refused to perform the treatment for her myoma uteri. During the follow-up period, she has remained asymptomatic with calcium/vitamin D and thyroid hormone replacement without an evidence of pheochromocytoma or hyperparathyroidism recurrence. Conclusion: Our patient has the distinctive features of pheochromocytoma and primary hyperparathyroidism in the patient carrying the double mutations in NF1 and RET gene. This gene-gene interaction may explain the phenotype variability among the affected patients with the hereditary endocrine tumor syndromes.

MON-381 A Missed Case of Pheochromocytoma in NF1 Patient Presenting as Pre-Eclampsia

Background Neurofibromatosis type 1 (NF1) is a rare autosomal dominant disorder manifesting with skin lesions and neurofibromas. An increased risk of pheochromocytoma and paraganglioma (PPGL) is known with an estimated incidence of 0.1-5.7%1. While some data suggested screening for PPGL in NF1 patients2, this has not been emphasized on guidelines or in practice. Herein, we report a case of pheochromocytoma diagnosed in a patient with NF1 shortly after a pre-eclampsia complicated pregnancy. Clinical case A 25 year old woman with NF1 had poorly controlled hypertension, palpitations, and anxiety during her first pregnancy attributed to pre-eclampsia. She presented to the ED shortly post-partum with episodes of palpitations, tachycardia, hypotension, and GI symptoms. A CT scan of the abdomen and pelvis identified a heterogeneous, well-defined, 35 HU, left adrenal mass measuring 4.9 x 4.5 x 4.2 cm. Biochemical data noted elevated plasma metanephrines (2.42 nmol/L, n<0.49 nmol/L) and normetanephrines (15.85 nmol/L, n<0.89 nmol/L). 24 hour urinary metanephrines (1292 ug/g, n<300 ug/g), normetanephrines (4769 ug/g, n<44 ug/g), norepinephrine (731 ug/g, n<45 ug/g), and epinephrine (77 ug/g, n<20 ug/g) were elevated. She was started on an alpha-adrenergic receptor blockade (in addition to her beta and calcium channel blockers) 3 weeks, and metyrosine 3 days before she underwent a left laparoscopic adrenalectomy. A few weeks after surgery, she was able to stop all blood pressure and anxiety medications. She asked about PPGL screening in her daughter as she carries the NF1 mutation. Clinical Lesson PPGL occur in a small but meaningful number of patients with NF1, and can be associated with significant morbidity and mortality if not detected. The current guidelines suggests screening for PPGL need not be considered unless indicated by signs or symptoms. This case emphasize the significance of biochemical screening for PPGL in NF1 patients prior to elective surgical procedures and conception to avoid fatal complications.

MicroRNA‑92a promotes cell proliferation, migration and survival by directly targeting the tumor suppressor gene NF2 in colorectal and lung cancer cells.

Inactivation of the tumor suppressor protein Merlin leads to the development of benign nervous system tumors in neurofibromatosis type 2 (NF2). Documented causes of Merlin inactivation include deleterious mutations in the encoding neurofibromin 2 gene (NF2) and aberrant Merlin phosphorylation leading to proteasomal degradation. Rare somatic NF2 mutations have also been detected in common human malignancies not associated with NF2, including colorectal and lung cancer. Furthermore, tumors without NF2 mutations and with unaltered NF2 transcript levels, but with low Merlin expression, have been reported. The present study demonstrated that NF2 is also regulated by microRNAs (miRNAs) through direct interaction with evolutionarily conserved miRNA response elements (MREs) within its 3′-untranslated region (3′UTR). Dual-Luciferase assays in human colorectal carcinoma (HCT116) and lung adenocarcinoma (A549) cells revealed downregulation of NF2 by miR-92a-3p via its wild-type 3′UTR, but not NF2−3′UTR with mutated miR-92a-3p MRE. HCT116 cells overexpressing miR-92a-3p exhibited significant downregulation of endogenous NF2 mRNA and protein levels, which was rescued by co-transfection of a target protector oligonucleotide specific for the miR-92a-3p binding site within NF2−3′UTR. miR-92a-3p overexpression in HCT116 and A549 cells promoted migration, proliferation and resistance to apoptosis, as well as altered F-actin organization compared with controls. Knockdown of NF2 by siRNA phenocopied the oncogenic effects of miR-92a overexpression on HCT116 and A549 cells. Collectively, the findings of the present study provide functional proof of the unappreciated role of miRNAs in NF2 regulation and tumor progression, leading to enhanced oncogenicity.

Reprogramming Captures the Genetic and Tumorigenic Properties of Neurofibromatosis Type 1 Plexiform Neurofibromas.

SummaryNeurofibromatosis type 1 (NF1) is a tumor predisposition genetic disease caused by mutations in the NF1 tumor suppressor gene. Plexiform neurofibromas (PNFs) are benign Schwann cell (SC) tumors of the peripheral nerve sheath that develop through NF1 inactivation and can progress toward a malignant soft tissue sarcoma. There is a lack of non-perishable model systems to investigate PNF development. We reprogrammed PNF-derived NF1(−/−) cells, descendants from the tumor originating cell. These NF1(−/−)-induced pluripotent stem cells (iPSCs) captured the genomic status of PNFs and were able to differentiate toward neural crest stem cells and further to SCs. iPSC-derived NF1(−/−) SCs exhibited a continuous high proliferation rate, poor myelination ability, and a tendency to form 3D spheres that expressed the same markers as their PNF-derived primary SC counterparts. They represent a valuable model to study and treat PNFs. PNF-derived iPSC lines were banked for making them available.

Preclinical assessment of MEK1/2 inhibitors for neurofibromatosis type 2-associated schwannomas reveals differences in efficacy and drug resistance development.

Neurofibromatosis type 2 (NF2) is a genetic tumor-predisposition disorder caused by NF2/merlin tumor suppressor gene inactivation. The hallmark of NF2 is formation of bilateral vestibular schwannomas (VS). Because merlin modulates activity of the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, we investigated repurposing drugs targeting MEK1 and/or MEK2 as a treatment for NF2-associated schwannomas. Mouse and human merlin-deficient Schwann cell lines (MD-MSC/HSC) were screened against 6 MEK1/2 inhibitors. Efficacious drugs were tested in orthotopic allograft and NF2 transgenic mouse models. Pathway and proteome analyses were conducted. Drug efficacy was examined in primary human VS cells with NF2 mutations and correlated with DNA methylation patterns. Trametinib, PD0325901, and cobimetinib were most effective in reducing MD-MSC/HSC viability. Each decreased phosphorylated pERK1/2 and cyclin D1, increased p27, and induced caspase-3 cleavage in MD-MSCs. Proteomic analysis confirmed cell cycle arrest and activation of pro-apoptotic pathways in trametinib-treated MD-MSCs. The 3 inhibitors slowed allograft growth; however, decreased pERK1/2, cyclin D1, and Ki-67 levels were observed only in PD0325901 and cobimetinib-treated grafts. Tumor burden and average tumor size were reduced in trametinib-treated NF2 transgenic mice; however, tumors did not exhibit reduced pERK1/2 levels. Trametinib and PD0325901 modestly reduced viability of several primary human VS cell cultures with NF2 mutations. DNA methylation analysis of PD0325901-resistant versus -susceptible VS identified genes that could contribute to drug resistance. MEK inhibitors exhibited differences in antitumor efficacy resistance in schwannoma models with possible emergence of trametinib resistance. The results support further investigation of MEK inhibitors in combination with other targeted drugs for NF2 schwannomas.

RAS-MPAK Pathway Signaling in Health and Disease

The p21ras (Ras) family of signal switch molecules is an essential component of proliferative responses to many extracellular stimuli including most hematopoietic growth factors and cytokines. Phosphorylation of tyrosine residues on activated cytokine/growth factor receptors triggers multiple signaling pathways including JAK/STAT, Ras and PI3K. Activated Ras proteins further activate downstream effectors such as RAF/MEK/ERK, PI3K and RalGDS. Point mutations of Ras proteins are commonly found in human cancers and are estimated to occur in about 30% of cancer cases. These mutations lock Ras in the constitutively active conformation. In addition to point mutations of RAS, other genetic lesions such as the BCR-ABL fusion, PTPN11 mutations, FLT3 internal tandem duplications, CBL mutations and NF1 inactivation all lead to hyperactive Ras signaling. Besides cancers, RAS and Ras-activating mutations have been found in RASopathies, such as Neurofibromatosis type 1 (NF1), Noonan Syndrome, Costello syndrome, and Cardiofaciocutaneous syndrome (CFC).RAS mutations are highly prevalent in hematologic malignancies including chronic and juvenile myelomonocytic leukemia (CMML and JMML), acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL) and multiple myeloma (MM). In contrast to epithelial cancers where KRAS mutations are highly prevalent, most mutations found in hematologic malignancies alter NRAS. Human genetic data and results from genetic mouse models support the idea that deregulated Ras signaling is initiating event in myeloproliferative neoplasms (MPNs) and MPN/MDS overlapping diseases. RAS mutations are detected among the initiating mutations in many cases of CMML and JMML, and can persist in patients who have achieved remission either spontaneously or after treatment. Furthermore, mutations of Nras and Kras, as well as leukemia-associated mutations that result in hyperactive Ras such as BCR-ABL, loss of NF1, PTPN11 or CBL mutations all induce MPN or MPN/MDS overlapping disease in mouse models. Although in adult de novo AMLs, human genetic data support a model in which RAS and Ras-activation mutations are likely secondary event, recent large scale next generation sequencing studies show that RAS and Ras-activating mutations are much more commonly found in pediatric than adult AML cases, and pediatric AMLs in general lack “adult pre-leukemic mutation' such as TET2, DNMAT3 or IDH1/2. These results suggest a possibility that RAS mutations can be initiating mutations in pediatric AMLs. In addition, even in AMLs where RAS mutations are secondary event, hyperactive Ras promotes leukemic stem cell self-renewal and is critical for leukemia maintenance, underscoring the importance of targeting Ras for leukemia treatment.Targeting Ras proteins remains to be challenging and significant ongoing effort focuses on developing direct inhibitors of Ras. In recent years, small molecules that bind to Ras proteins to prevent GEF-mediated GDP/GTP exchange, or target the RasG12C mutant allele, showed promising pre-clinical results. Besides, targeting Ras effectors, such as MEK/ERK, individually or in combination with other pathways, remains as a valid approach to halt Ras-induced malignant proliferation. Better understanding of the subcellular localization, feedback regulation and redundancy of Ras-activated signaling network under physiological and pathological contexts will shed light on developing new strategies to effectively attack Ras-driven cancers. DisclosuresNo relevant conflicts of interest to declare.

A proteasome-resistant fragment of NIK mediates oncogenic NF-κB signaling in schwannomas.

Schwannomas are common, highly morbid and medically untreatable tumors that can arise in patients with germ line as well as somatic mutations in neurofibromatosis type 2 (NF2). These mutations most commonly result in the loss of function of the NF2-encoded protein, Merlin. Little is known about how Merlin functions endogenously as a tumor suppressor and how its loss leads to oncogenic transformation in Schwann cells (SCs). Here, we identify nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase (NIK) as a potential drug target driving NF-κB signaling and Merlin-deficient schwannoma genesis. Using a genomic approach to profile aberrant tumor signaling pathways, we describe multiple upregulated NF-κB signaling elements in human and murine schwannomas, leading us to identify a caspase-cleaved, proteasome-resistant NIK kinase domain fragment that amplifies pathogenic NF-κB signaling. Lentiviral-mediated transduction of this NIK fragment into normal SCs promotes proliferation, survival, and adhesion while inducing schwannoma formation in a novel in vivo orthotopic transplant model. Furthermore, we describe an NF-κB-potentiated hepatocyte growth factor (HGF) to MET proto-oncogene receptor tyrosine kinase (c-Met) autocrine feed-forward loop promoting SC proliferation. These innovative studies identify a novel signaling axis underlying schwannoma formation, revealing new and potentially druggable schwannoma vulnerabilities with future therapeutic potential.

Phenotypic expression of a spectrum of Neurofibromatosis Type 1 (NF1) mutations identified through NGS and MLPA

Neurofibromatosis Type 1 (NF1) is caused by mutations of the NF1 gene. The aim of this study was to identify the genetic causes underlying the disease, attempt possible phenotype/genotype correlations and add to the NF1 mutation spectrum. A screening protocol based on genomic DNA was established in 168 patients, encompassing sequencing of all coding exons and adjoining introns using a custom targeted next generation sequencing protocol and subsequent confirmation of findings with Sanger sequencing. MLPA was used to detect deletions/duplications and positive findings were confirmed by RNA analysis. All novel findings were evaluated according to ACMG Standards and guidelines for the interpretation of sequence variants with the aid of in-silico bioinformatic tools and family segregation analysis. A germline variant was identified in 145 patients (86%). In total 49 known and 70 novel variants in coding and non-coding regions were identified. Seven patients carried whole or partial gene deletions. NF1 patients, present with high phenotypic variability even in cases where the same germline disease causing variant has been identified. Our findings will contribute to a better knowledge of the genetic causes and the phenotypic expression related to the disease.

Germline and Somatic NF1 Alterations Are Linked to Increased HER2 Expression in Breast Cancer.

NF1 germline mutation predisposes to breast cancer. NF1 mutations have also been proposed as oncogenic drivers in sporadic breast cancers. To understand the genomic and histologic characteristics of these breast cancers, we analyzed the tumors with NF1 germline mutations and also examined the genomic and proteomic profiles of unselected tumors. Among 14 breast cancer specimens from 13 women affected with neurofibromatosis type 1 (NF1), 9 samples (NF + BrCa) underwent genomic copy number (CN) and targeted sequencing analysis. Mutations of NF1 were identified in two samples and TP53 were in three. No mutation was detected in ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, and STK11 HER2 (ErbB2) overexpression was detected by IHC in 69.2% (9/13) of the tumors. CN gain/amplification of ERBB2 was detected in 4 of 9 with DNA analysis. By evaluating HER2 expression and NF1 alterations in unselected invasive breast cancers in TCGA datasets, we discovered that among samples with ERBB2 CN gain/amplification, the HER2 mRNA and protein expression were much more pronounced in NF1-mutated/deleted samples in comparison with NF1-unaltered samples. This finding suggests a synergistic interplay between these two genes, potentially driving the development of breast cancer harboring NF1 mutation and ERBB2 CN gain/amplification. NF1 gene loss of heterozygosity was observed in 4 of 9 NF + BrCa samples. CDK4 appeared to have more CN gain in NF + BrCa and exhibited increased mRNA expression in TCGA NF1--altered samples. Cancer Prev Res; 11(10); 655-64. ©2018 AACR.

Recent Advances in the Diagnosis and Pathogenesis of Neurofibromatosis Type 1 (NF1)-associated Peripheral Nervous System Neoplasms.

The diagnosis of a neurofibroma or a malignant peripheral nerve sheath tumor (MPNST) often raises the question of whether the patient has the genetic disorder neurofibromatosis type 1 (NF1) as well as how this will impact the patient's outcome, what their risk is for developing additional neoplasms and whether treatment options differ for NF1-associated and sporadic peripheral nerve sheath tumors. Establishing a diagnosis of NF1 is challenging as this disorder has numerous neoplastic and non-neoplastic manifestations which are variably present in individual patients. Further, other genetic diseases affecting the Ras signaling cascade (RASopathies) mimic many of the clinical features of NF1. Here, we review the clinical manifestations of NF1 and compare and contrast them with those of the RASopathies. We also consider current approaches to genetic testing for germline NF1 mutations. We then focus on NF1-associated neurofibromas, considering first the complicated clinical behavior and pathology of these neoplasms and then discussing our current understanding of the genomic abnormalities that drive their pathogenesis, including the mutations encountered in atypical neurofibromas. As several neurofibroma subtypes are capable of undergoing malignant transformation to become MPNSTs, we compare and contrast patient outcomes in sporadic, NF1-associated and radiation-induced MPNSTs, and review the challenging pathology of these lesions. The mutations involved in neurofibroma-MPNST progression, including the recent identification of mutations affecting epigenetic regulators, are then considered. Finally, we explore how our current understanding of neurofibroma and MPNST pathogenesis is informing the design of new therapies for these neoplasms.

Novel and Recurring Disease-Causing NF1 Variants in Two Chinese Families with Neurofibromatosis Type 1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder primarily characterized by multiple café-au-lait macules, peripheral neurofibromas, skinfold freckling, and Lisch nodules. The causative genetic factor is the neurofibromin 1 gene (NF1), which encodes a Ras GTPase-activating protein called neurofibromin. NF1 variants may lead to loss of neurofibromin function and activation of downstream cell growth. This study aims to discover the disease-causing variants responsible for NF1 in two Han Chinese families by using exome sequencing combined with Sanger sequencing. A recurrent missense variant c.269T>C (p.Leu90Pro) and a novel nonsense variant c.2993dupA (p.Tyr998*) in the NF1 gene were identified. These variants co-segregated with the disorder in the pedigrees and were absent in the normal controls. The results broaden the NF1 mutation spectrum responsible for NF1. This may be helpful in genetic counseling, clinical management, and gene-targeted therapies for NF1.