Loss Of Neurofibromatosis Type 1 Research Articles

Is vestibular schwannoma really a benign tumor? Case report and review

Introduction: Vestibular schwannoma (VS) is a benign tumor that develops from Schwann cells of the eighth cranial pair, mainly in the cerebellopontine angle. Case Presentation: We report the case of a 30-year-old female patient who developed left otalgia associated with neglected tinnitus, the evolution of which was marked by the development of a static cerebellar syndrome and a behavioral disorder, whose brain MRI revealed a locally advanced process in the cerebellopontine angle at the expense of the vestibulocochlear nerve, in favor of a VS, complicated by involvement of the tonsils, which unfortunately led to the patient’s death. Discussion: VS, formerly known as acoustic neuroma, is an extra-axial intracranial tumor that accounts for over 80% of pontocerebellar angle tumors, and is secondary in the majority of cases to inactivation of the neurofibromatosis type 2 (NF2) tumor suppressor gene, either by mutation of the NF2 gene or loss of chromosome 22q. In the majority of cases, it is unilateral and solitary, but in almost 8% of cases, it is associated with NF2. Cerebral MRI is the examination of choice for the detection, characterization, and diagnosis of VS without the need for biopsy, mainly with T1-weighted sequences before and after gadolinium injection. Treatment is based essentially on surgery or radiosurgery, depending on the size, impact, and expertise of the treatment team. Conclusion: VS remains an important intracranial tumor entity, which can be life-threatening in cases of advanced local invasion.

A Call for Discovery and Therapeutic Development for Cutaneous Neurofibromas

A Call for Discovery and Therapeutic Development for Cutaneous Neurofibromas

Loss of NF1 in Drosophila Larvae Causes Tactile Hypersensitivity and Impaired Synaptic Transmission at the Neuromuscular Junction.

Autism spectrum disorder (ASD) is a neurodevelopmental condition in which the mechanisms underlying its core symptomatology are largely unknown. Studying animal models of monogenic syndromes associated with ASD, such as neurofibromatosis type 1 (NF1), can offer insights into its etiology. Here, we show that loss of function of the Drosophila NF1 ortholog results in tactile hypersensitivity following brief mechanical stimulation in the larva (mixed sexes), paralleling the sensory abnormalities observed in individuals with ASD. Mutant larvae also exhibit synaptic transmission deficits at the glutamatergic neuromuscular junction (NMJ), with increased spontaneous but reduced evoked release. While the latter is homeostatically compensated for by a postsynaptic increase in input resistance, the former is consistent with neuronal hyperexcitability. Indeed, diminished expression of NF1 specifically within central cholinergic neurons induces both excessive neuronal firing and tactile hypersensitivity, suggesting the two may be linked. Furthermore, both impaired synaptic transmission and behavioral deficits are fully rescued via knock-down of Ras proteins. These findings validate NF1 -/- Drosophila as a tractable model of ASD with the potential to elucidate important pathophysiological mechanisms.SIGNIFICANCE STATEMENT Autism spectrum disorder (ASD) affects 1-2% of the overall population and can considerably impact an individual's quality of life. However, there are currently no treatments available for its core symptoms, largely because of a poor understanding of the underlying mechanisms involved. Examining how loss of function of the ASD-associated NF1 gene affects behavior and physiology in Drosophila may shed light on this. In this study, we identify a novel, ASD-relevant behavioral phenotype in NF1 -/- larvae, namely an enhanced response to mechanical stimulation, which is associated with Ras-dependent synaptic transmission deficits indicative of neuronal hyperexcitability. Such insights support the use of Drosophila neurofibromatosis type 1 (NF1) models in ASD research and may provide outputs for genetic or pharmacological screens in future studies.

Abstract 1615: Developing a novel model of neurofibromatosis type 1 associated malignant peripheral nerve sheath tumors using induced pluripotent stem cells

Abstract The genetic tumor predisposition syndrome Neurofibromatosis type 1 (NF1) results from the inheritance of a mutant copy of NF1, a RAS-GAP tumor suppressor gene. Subsequent loss of the remaining wild-type NF1 allele in Schwann lineage cells of the peripheral nervous system leads to complete functional loss of the encoded protein, neurofibromin. Schwann cells lacking neurofibromin exhibit hyperactive RAS signaling, and contribute to the formation of benign plexiform neurofibromas (PNFs). Through additional mutations in tumor suppressor genes CDKN2A/CDKN2B, these PNFs may escape senescence and progress to atypical neurofibromatous neoplasms of uncertain biological potential (ANNUBP). Furthermore, loss of function mutations in Polycomb Repressive Complex 2 (PRC2) have been strongly implicated in the progression of ANNUBPs to lethal malignant peripheral nerve sheath tumors (MPNSTs). Although these alterations have been associated with MPNST formation, their temporal dependence during Schwann cell development and contribution to malignant transformation using a human cell model has not been studied. We hypothesize that sequential loss of NF1, CDKN2A, and finally SUZ12 in Schwann cells transforms them into MPNST, and this process is possible to study using a human induced pluripotent stem cell (iPSC) model system. To test our hypothesis, we generated Schwann lineage cells from commercially available iPSCs. Cells were subjected to targeted mutations using CRISPR/Cas9 ribonucleoproteins at different stages of development to mimic potential tumorigenesis. Our preliminary results show that multiple iPSC donor lines can reliably be differentiated to Neural Crest Cells (NCCs), and further to mature Schwann cells. We have edited multiple target loci at high levels (>70%) throughout Schwann cell development. NCCs lacking neurofibromin showed an increase in proliferation and migration. While iPSC-derived Schwann cells (iSCs) lacking NF1 and CDKN2A with or without SUZ12 loss became senescent after several passages. These mutant iSCs were unable to form tumors when subcutaneously injected into the flank of NRG mice, while commonly utilized immortalized human Schwann cell lines lacking NF1 were able to form large subcutaneous tumors. Further refinement of this induced-MPNST (iMPNST) model through targeting of CDKN2B, ATRX, or TP53 may allow for better understanding of the order and timing at which these transforming mutations are sufficient to result in MPNST formation. Additionally, the application of single-cell RNA sequencing technology to this system could uncover potential heterogeneity that could harbor a clone primed for malignant transformation. This model could aid in uncovering novel genetic mutations and pathways which contribute to transformation of benign PNFs to MPNSTs, providing additional targets for future therapeutic intervention. Citation Format: Garrett M. Draper, Daniel Panken, Mahathi Patchava, Wendy Hudson, Kyle B. Williams, David A. Largaespada. Developing a novel model of neurofibromatosis type 1 associated malignant peripheral nerve sheath tumors using induced pluripotent stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1615.

Integrated Drug Mining Reveals Actionable Strategies Inhibiting Plexiform Neurofibromas.

Neurofibromatosis Type 1 (NF1) is one of the most common genetic tumor predisposition syndromes, affecting up to 1 in 2500 individuals. Up to half of patients with NF1 develop benign nerve sheath tumors called plexiform neurofibromas (PNs), characterized by biallelic NF1 loss. PNs can grow to immense sizes, cause extensive morbidity, and harbor a 15% lifetime risk of malignant transformation. Increasingly, molecular sequencing and drug screening data from various preclinical murine and human PN cell lines, murine models, and human PN tissues are available to help identify salient treatments for PNs. Despite this, Selumetinib, a MEK inhibitor, is the only currently FDA-approved pharmacotherapy for symptomatic and inoperable PNs in pediatric NF1 patients. The discovery of alternative and additional treatments has been hampered by the rarity of the disease, which makes prioritizing drugs to be tested in future clinical trials immensely important. Here, we propose a gene regulatory network-based integrated analysis to mine high-throughput cell line-based drug data combined with transcriptomes from resected human PN tumors. Conserved network modules were characterized and served as drug fingerprints reflecting the biological connections among drug effects and the inherent properties of PN cell lines and tissue. Drug candidates were ranked, and the therapeutic potential of drug combinations was evaluated via computational predication. Auspicious therapeutic agents and drug combinations were proposed for further investigation in preclinical and clinical trials.

Tumor and Constitutional Sequencing for Neurofibromatosis Type 1.

PURPOSENF1 variants in tumors are important to recognize, as multiple mechanisms may give rise to biallelic variants. Both deletions and copy-neutral loss of heterozygosity (LOH) are potential mechanisms of NF1 loss, distinct from point mutations, and additional genes altered may drive different tumor types. This study investigates whether tumors from individuals with neurofibromatosis type 1 (NF1) demonstrate additional gene variants and detects NF1 second hits using paired germline and somatic sequencing. In addition, rare tumor types in NF1 may also be characterized by tumor sequencing.MATERIALS AND METHODSSequences of 529 cancer driver genes were analyzed in 6,381 tumors, yielding 391 NF1-mutated tumors in which NF1 LOH analysis was performed. Driver genes were evaluated by tumor type including malignant peripheral nerve sheath tumors and gliomas.RESULTSNF1 LOH was seen in 133 of 391 tumor samples in the cohort. Individuals with NF1 had more prevalent copy-neutral LOH (P < .0001), suggesting somatic intrachromosomal recombination. Osteosarcoma in NF1 also had NF1 LOH and additional p53 alteration. NF1 second hit data from tumors were informative for inferring deleteriousness of missense variants that were conflicting in ClinVar, potentially helping to add to NF1 annotation. Although criteria for evaluating germline and somatic variants are different, deleterious effects on NF1 function may be shared.CONCLUSIONSequencing of NF1-associated tumors demonstrated a spectrum of second hits in NF1 and the prevalence of copy-neutral LOH. Future work may be aimed at further understanding of LOH mechanisms and strategies to mitigate tumor risk.

Neurofibromin and suppression of tumorigenesis: beyond the GAP.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disease and one of the most common inherited tumor predisposition syndromes, affecting 1 in 3000 individuals worldwide. The NF1 gene encodes neurofibromin, a large protein with RAS GTP-ase activating (RAS-GAP) activity, and loss of NF1 results in increased RAS signaling. Neurofibromin contains many other domains, and there is considerable evidence that these domains play a role in some manifestations of NF1. Investigating the role of these domains as well as the various signaling pathways that neurofibromin regulates and interacts with will provide a better understanding of how neurofibromin acts to suppress tumor development and potentially open new therapeutic avenues. In this review, we discuss what is known about the structure of neurofibromin, its interactions with other proteins and signaling pathways, its role in development and differentiation, and its function as a tumor suppressor. Finally, we discuss the latest research on potential therapeutics for neurofibromin-deficient neoplasms.

Neurofibromatosis Type 1 Gene Alterations Define Specific Features of a Subset of Glioblastomas.

Neurofibromatosis type 1 (NF1) gene mutations or alterations occur within neurofibromatosis type 1 as well as in many different malignant tumours on the somatic level. In glioblastoma, NF1 loss of function plays a major role in inducing the mesenchymal (MES) subtype and, therefore defining the most aggressive glioblastoma. This is associated with an immune signature and mediated via the NF1–MAPK–FOSL1 axis. Specifically, increased invasion seems to be regulated via mutations in the leucine-rich domain (LRD) of the NF1 gene product neurofibromin. Novel targets for therapy may arise from neurofibromin deficiency-associated cellular mechanisms that are summarised in this review.

Multiplatform Genomic Profiling and Immunohistochemistry Identify Prognostic and Predictive Signatures in Malignant Peripheral Nerve Sheath Tumors (MPNSTs)

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive neoplasms arising either sporadically or in patients with neurofibromatosis type 1 (NF1). To elucidate markers of prognosis and treatment response, we retrospectively identified MPNST tumor resection specimens at a single institution and performed histopathologic evaluation, immunohistochemistry (IHC), DNA methylation profiling, and whole exome sequencing (WES).We identified a total of 54 consecutive patients treated at one institution between 1990 and 2019 with tumors meeting diagnostic criteria for MPNST and had sufficient tissue available for comprehensive histopathological evaluation (n = 54), DNA methylation profiling (n = 45) and whole exome sequencing (n = 35, n = 15 with paired normal). Overall survival (OS), metastasis free survival (MFS), and locoregional failure free rate (LFFR), were estimated using the Kaplan-Meier method. Log-rank test, Cox Proportional Hazards regression, and hierarchical clustering were performed in R.The median follow up was 22 months with no significant differences in LFFR, MFS, or OS between NF1 associated (n = 32) and sporadic MPNSTs (n = 22). Radiotherapy significantly improved LFFR (P = 0.05) but not OS. With regard to histopathologic and IHC analyses, increasing tumor grade using a two-tiered system (P = 0.01) and Ki-67 index (P = 0.01) were associated with worse OS. Unsupervised hierarchical clustering of DNA methylation profiles identified two groups with significant differences in chromosome instability and tumor purity but not clinical outcome. Recurrently identified copy number variants (CNVs) included chromosome 22q loss, 11q loss, 10 loss, 9p loss, 8q gain, and 1q loss. Chromosome 9p loss (P = 0.02) and 8q gain (P = 0.04) were significantly associated with worse OS. On WES analysis, recurrently mutated genes included NF1, EED, SUZ12, TP53, and CDKNA2A. Of these alterations, only CKND2A loss (on Chr9p) was associated with worse OS (P = 0.01). With regard to treatment response, MPNSTs exhibiting NF1 and H3K27 trimethylation loss demonstrated greater LFFR benefit from radiation therapy (P = 0.004) compared to NF1 and H3K27 trimethylation intact tumors (P = 0.83) with clinical NF1 diagnosis as the only significantly different baseline parameter between these two subgroups.MPNSTs are a common cause of death in adult patients with NF1. Radiotherapy improves LFFR in all patients, and integrated IHC and genomic analysis further identifies putative prognostic and predictive markers, including the identification of a patient subset in whom the benefit of radiotherapy is most pronounced. Taken together, these data support radiation therapy in management of MPNSTs and suggest molecular characterization may be important for counseling patients with MPNST or potentially informing clinical trial design. Prospective and multi-institutional analyses are needed to validate these findings.

Neurofibromin Deficiency and Extracellular Matrix Cooperate to Increase Transforming Potential through FAK-Dependent Signaling.

Plexiform neurofibromas (Pnfs) are benign peripheral nerve sheath tumors that are major features of the human genetic syndrome, neurofibromatosis type 1 (NF1). Pnfs are derived from Schwann cells (SCs) undergoing loss of heterozygosity (LOH) at the NF1 locus in an NF1+/- milieu and thus are variably lacking in the key Ras-controlling protein, neurofibromin (Nfn). As these SCs are embedded in a dense desmoplastic milieu of stromal cells and abnormal extracellular matrix (ECM), cell-cell cooperativity (CCC) and the molecular microenvironment play essential roles in Pnf progression towards a malignant peripheral nerve sheath tumor (MPNST). The complexity of Pnf biology makes treatment challenging. The only approved drug, the MEK inhibitor Selumetinib, displays a variable and partial therapeutic response. Here, we explored ECM contributions to the growth of cells lacking Nfn. In a 3D in vitro culture, NF1 loss sensitizes cells to signals from a Pnf-mimicking ECM through focal adhesion kinase (FAK) hyperactivation. This hyperactivation correlated with phosphorylation of the downstream effectors, Src, ERK, and AKT, and with colony formation. Expression of the GAP-related domain of Nfn only partially decreased activation of this signaling pathway and only slowed down 3D colony growth of cells lacking Nfn. However, combinatorial treatment with both the FAK inhibitor Defactinib (VS-6063) and Selumetinib (AZD6244) fully suppressed colony growth. These observations pave the way for a new combined therapeutic strategy simultaneously interfering with both intracellular signals and the interplay between the various tumor cells and the ECM.

Fluorescence in situ hybridization detection of chromosome 22 monosomy in pleural effusion cytology for the diagnosis of mesothelioma.

Malignant pleural mesothelioma (MPM) is characterized by mutations in several genes, including cyclin-dependent kinase-inhibitor 2A/p16 in the 9p21 locus, BRCA1-associated protein 1 (BAP1), and neurofibromatosis type 2 (NF2) in the 22q12 locus. Recent studies indicate that fluorescence in situ hybridization (FISH) detects hemizygous loss of NF2 in tissue specimens of MPM. The authors investigated whether NF2 FISH, either alone or in combination with other diagnostic assays (9p21 FISH, methylthioadenosine phosphorylase [MTAP] immunohistochemistry [IHC], and BAP1 IHC), effectively distinguishes MPM cells from reactive mesothelial cells (RMCs) in cell blocks prepared from pleural effusions. FISH assays were used to examine the deletion status of NF2 and 9p21, and IHC was used to determine the expression of MTAP and BAP1 in cell blocks from 54 cases with MPM and 18 cases with RMCs. Hemizygous NF2 loss (chromosome 22 monosomy or hemizygous deletion) showed 51.9% sensitivity (48.1% for chromosome 22 monosomy and 3.7% for hemizygous deletion) and 100% specificity in differentiating MPM cells from RMCs. Combinations of NF2 FISH, 9p21 FISH, and BAP1 IHC assays yielded greater sensitivity (98.1%) than any assay alone (9p21 FISH, 61.1%; MTAP IHC, 52.8%; or BAP1 IHC, 60.4%). The level of hemizygous NF2 loss in cell blocks positively correlated with that in corresponding tissues. Furthermore, to overcome cytologic specimen-specific challenges, FISH combined with cytokeratin AE1/AE3 immunofluorescence was necessary in 25.9% of MPM cases for FISH assessment of predominantly scattered MPM cells. NF2 FISH alone or in combination with other diagnostic assays effectively differentiates MPM cells from RMCs in cell blocks prepared from pleural effusions.

Humanized neurofibroma model from induced pluripotent stem cells delineates tumor pathogenesis and developmental origins.

Neurofibromatosis type 1 (NF1) is a common tumor predisposition syndrome caused by NF1 gene mutation, in which affected patients develop Schwann cell lineage peripheral nerve sheath tumors (neurofibromas). To investigate human neurofibroma pathogenesis, we differentiated a series of isogenic, patient-specific NF1-mutant human induced pluripotent stem cells (hiPSCs) into Schwannian lineage cells (SLCs). We found that, although WT and heterozygous NF1-mutant hiPSCs-SLCs did not form tumors following mouse sciatic nerve implantation, NF1-null SLCs formed bona fide neurofibromas with high levels of SOX10 expression. To confirm that SOX10+ SLCs contained the cells of origin for neurofibromas, both Nf1 alleles were inactivated in mouse Sox10+ cells, leading to classic nodular cutaneous and plexiform neurofibroma formation that completely recapitulated their human counterparts. Moreover, we discovered that NF1 loss impaired Schwann cell differentiation by inducing a persistent stem-like state to expand the pool of progenitors required to initiate tumor formation, indicating that, in addition to regulating MAPK-mediated cell growth, NF1 loss also altered Schwann cell differentiation to promote neurofibroma development. Taken together, we established a complementary humanized neurofibroma explant and, to our knowledge, first-in-kind genetically engineered nodular cutaneous neurofibroma mouse models that delineate neurofibroma pathogenesis amenable to future therapeutic target discovery and evaluation.

Cdkn2a Loss in a Model of Neurofibroma Demonstrates Stepwise Tumor Progression to Atypical Neurofibroma and MPNST.

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

Abstract 1087: NF1 regulates the RAS-related GTPases, RRAS and RRAS2, independent of RAS activity

Abstract Neurofibromin, which is encoded by the neurofibromatosis type 1 (NF1) gene, is a tumor suppressor that acts as a RAS-GTPase activating protein (RAS-GAP) to stimulate the intrinsic GTPase activity of RAS as well as the closely related RAS subfamily members, RRAS, RRAS2, and MRAS. This results in the conversion of the active GTP-bound form of RAS into the inactive GDP-bound state leading to the downregulation of several RAS downstream effector pathways, most notably MAPK signaling. While the region of NF1 that regulates RAS activity represents only a small fraction of the entire protein, a large extent of the NF1 structural domains and their corresponding mechanistic functions remain uncharacterized despite the fact there is a high frequency of NF1 mutations in several different types of cancer. Thus, we wanted to elucidate the underlying biochemical and signaling functions of NF1 that are unrelated to the regulation of RAS and how loss of these functions contributes to the pathogenesis of cancer. To accomplish this objective, we used CRISPR-Cas9 methods to knockout NF1 in an isogenic “RASless” MEF model system, which is devoid of the major oncogenic RAS isoforms (HRAS, KRAS, and NRAS) and reconstituted with the KRAS4b wild-type or mutant KRASG12C or KRASG12D isoform. Loss of NF1 led to elevated RAS-GTP levels, however, this increase was not as profound as the levels in KRAS-mutated cells or provided a proliferative advantage. Although ablation of NF1 resulted in sustained activation of MAPK signaling, it also unexpectedly, resulted in a robust increase in AKT phosphorylation compared to KRAS-mutated cells. Surprisingly, loss of NF1 in KRAS4b wild-type and KRAS-mutated cells potently suppressed the RAS-related GTPases, RRAS and RRAS2, with modest effects on MRAS, at both the transcript and protein levels. A Clariom™D transcriptome microarray analysis revealed a significant downregulation in the NF-κB target genes, insulin-like growth factor binding protein 2 (IGFBP2), argininosuccinate synthetase 1 (ASS1), and DUSP1, in both the NF1 knockout KRAS4b wild-type and KRAS-mutated cells. Moreover, NF1Null melanoma cells also displayed a potent suppression of RRAS and RRAS2 as well as these NF-κB transcription factors. Since RRAS and RRAS2 both contain the same NF-κB transcription factor binding sites, we hypothesize that IGFBP2, ASS1, and/or DUSP1 may contribute to the NF1-mediated regulation of these RAS-related GTPases. More importantly, this study provides the first evidence of at least one novel RAS-independent function of NF1 to regulate the RAS-related subfamily members, RRAS and RRAS2, in a manner exclusive of its RAS-GTPase activity and this may provide insight into new potential biomarkers and molecular targets for treating patients with mutations in NF1. Citation Format: Jillian M. Silva, Lizzeth Canche, Frank McCormick. NF1 regulates the RAS-related GTPases, RRAS and RRAS2, independent of RAS activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1087.

New Model Systems and the Development of Targeted Therapies for the Treatment of Neurofibromatosis Type 1-Associated Malignant Peripheral Nerve Sheath Tumors.

Neurofibromatosis Type 1 (NF1) is a common genetic disorder and cancer predisposition syndrome (1:3000 births) caused by mutations in the tumor suppressor gene NF1. NF1 encodes neurofibromin, a negative regulator of the Ras signaling pathway. Individuals with NF1 often develop benign tumors of the peripheral nervous system (neurofibromas), originating from the Schwann cell linage, some of which progress further to malignant peripheral nerve sheath tumors (MPNSTs). Treatment options for neurofibromas and MPNSTs are extremely limited, relying largely on surgical resection and cytotoxic chemotherapy. Identification of novel therapeutic targets in both benign neurofibromas and MPNSTs is critical for improved patient outcomes and quality of life. Recent clinical trials conducted in patients with NF1 for the treatment of symptomatic plexiform neurofibromas using inhibitors of the mitogen-activated protein kinase (MEK) have shown very promising results. However, MEK inhibitors do not work in all patients and have significant side effects. In addition, preliminary evidence suggests single agent use of MEK inhibitors for MPNST treatment will fail. Here, we describe the preclinical efforts that led to the identification of MEK inhibitors as promising therapeutics for the treatment of NF1-related neoplasia and possible reasons they lack single agent efficacy in the treatment of MPNSTs. In addition, we describe work to find targets other than MEK for treatment of MPNST. These have come from studies of RAS biochemistry, in vitro drug screening, forward genetic screens for Schwann cell tumors, and synthetic lethal screens in cells with oncogenic RAS gene mutations. Lastly, we discuss new approaches to exploit drug screening and synthetic lethality with NF1 loss of function mutations in human Schwann cells using CRISPR/Cas9 technology.

Combination therapy with mTOR kinase inhibitor and dasatinib as a novel therapeutic strategy for vestibular schwannoma

Neurofibromatosis type 2 (NF2) is an inherited disorder characterized by bilateral vestibular schwannomas (VS) that arise from neoplastic Schwann cells (SCs). NF2-associated VSs are often accompanied by meningioma (MN), and the majority of NF2 patients show loss of the NF2 tumor suppressor. mTORC1 and mTORC2-specific serum/glucocorticoid-regulated kinase 1 (SGK1) are constitutively activated in MN with loss of NF2. In a recent high-throughput kinome screen in NF2-null human arachnoidal and meningioma cells, we showed activation of EPH RTKs, c-KIT, and SFK members independent of mTORC1/2 activation. Subsequently, we demonstrated in vitro and in vivo efficacy of combination therapy with the dual mTORC1/2 inhibitor AZD2014 and the multi-kinase inhibitor dasatinib. For these reasons, we investigated activated mTORC1/2 and EPH receptor-mediated signaling in sporadic and NF2-associated VS. Using primary human VS cells and a mouse allograft model of schwannoma, we evaluated the dual mTORC1/2 inhibitor AZD2014 and the tyrosine kinase inhibitor dasatinib as monotherapies and in combination. Escalating dose-response experiments on primary VS cells grown from 15 human tumors show that combination therapy with AZD2014 and dasatinib is more effective at reducing metabolic activity than either drug alone and exhibits a therapeutic effect at a physiologically reasonable concentration (~0.1 µM). In vivo, while AZD2014 and dasatinib each inhibit tumor growth alone, the effect of combination therapy exceeds that of either drug. Co-targeting the mTOR and EPH receptor pathways with these or similar compounds may constitute a novel therapeutic strategy for VS, a condition for which there is no FDA-approved pharmacotherapy.

The Role of RUNX1 in NF1-Related Tumors and Blood Disorders.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder. NF1 patients are predisposed to formation of several type solid tumors as well as to juvenile myelomonocytic leukemia. Loss of NF1 results in dysregulation of MAPK, PI3K and other signaling cascades, to promote cell proliferation and to inhibit cell apoptosis. The RUNX1 gene is associated with stem cell function in many tissues, and plays a key role in the fate of stem cells. Aberrant RUNX1 expression leads to context-dependent tumor development, in which RUNX1 may serve as a tumor suppressor or an oncogene in specific tissue contexts. The co-occurrence of mutation of NF1 and RUNX1 is detected rarely in several cancers and signaling downstream of RAS-MAPK can alter RUNX1 function. Whether aberrant RUNX1 expression contributes to NF1-related tumorigenesis is not fully understood. This review focuses on the role of RUNX1 in NF1-related tumors and blood disorders, and in sporadic cancers.

Hemizygous loss of NF2 detected by fluorescence in situ hybridization is useful for the diagnosis of malignant pleural mesothelioma.

Neurofibromatosis type 2 (NF2) gene, a tumor suppressor gene located on chromosome 22q12.2, is frequently abnormal in mesothelioma. Recent studies have revealed the effectiveness of diagnostic assays for differentiating malignant pleural mesothelioma from reactive mesothelial hyperplasia. These include detection of homozygous deletion of the 9p21 locus by fluorescence in situ hybridization (FISH) (9p21 FISH), loss of expression of BAP1 as detected by immunohistochemistry, and loss of expression of methylthioadenosine phosphorylase (MTAP) as detected by immunohistochemistry. However, the application of FISH detection of NF2 gene deletion (NF2 FISH) in differentiation of malignant pleural mesothelioma from reactive mesothelial hyperplasia has not been fully evaluated. In this study, we investigated whether NF2 FISH, either alone or in a combination with other diagnostic assays (9p21 FISH, MTAP immunohistochemistry, and BAP1 immunohistochemistry), is effective for distinguishing malignant pleural mesothelioma from reactive mesothelial hyperplasia. This study cohort included malignant pleural mesothelioma (n = 47) and reactive mesothelial hyperplasia cases (n = 27) from a period between 2001 and 2017. We used FISH to examine deletion status of NF2 and 9p21 and immunohistochemistry to examine expression of MTAP and BAP1 in malignant pleural mesothelioma and in reactive mesothelial hyperplasia. Hemizygous NF2 loss (chromosome 22 monosomy or hemizygous deletion) was detected in 25 of 47 (53.2%) mesothelioma cases. None of the mesothelioma cases showed homozygous NF2 deletion. Hemizygous NF2 loss showed 53.2% sensitivity and 100% specificity in differentiating malignant pleural mesothelioma from reactive mesothelial hyperplasia. A combination of NF2 FISH, 9p21 FISH, and BAP1 immunohistochemistry yielded greater sensitivity (100%) than that detected for either diagnostic assay alone (53.2% for NF2 FISH, 78.7% for 9p21 FISH, 70.2% for MTAP immunohistochemistry, or 57.4% for BAP1 immunohistochemistry). Thus, NF2 FISH in combination with other diagnostic assays is effective for distinguishing malignant pleural mesothelioma from reactive mesothelial hyperplasia.

363 NF1 heterozygosity fosters de novo tumorigenesis but impairs malignant transformation

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder. Patients with NF1 have mono-allelic loss of the tumor suppressor gene NF1 in their germline, which predisposes them to develop a wide array of benign lesions. Intriguingly, recent sequencing efforts revealed that the NF1 gene is frequently mutated in multiple malignant tumors not typically associated with NF1 patients, suggesting that NF1 heterozygosity is refractory to at least some cancer types. In two orthogonal mouse models representing tumors associated with NF1 (neurofibroma and malignant peripheral nerve sheath tumor) and non-NF1-related tumors (papilloma and squamous cell carcinoma), we serendipitously discover that an NF1heterozygosity microenvironment accelerates the formation of benign tumors but impairs further progression to malignancy. Analysis of benign and malignant tumors commonly associated with NF1 patients, as well as those with high NF1 gene mutation frequency, reveals an antagonistic role for NF1 heterozygosity in tumor initiation and malignant transformation and helps to reconciliate the role of the NF1 gene in both NF1 and non-NF1 patient contexts. Our novel framework to study the role of NF1 in tumorigenesis integrates the concept of NF1 as a tumor predisposition gene and we have uncovered mechanisms underlying the impairment of cancer progression by tumor microenvironment cells harboring tumor suppressor genes in the haploinsufficient state, such as NF1 heterozygosity, may translate into novel therapeutic strategies.

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.