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Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft-tissue sarcomas with a poor survival rate, presenting either sporadically or in the context of neurofibromatosis type 1 (NF1). The histological diagnosis of MPNSTs can be challenging, with different tumors exhibiting great histological and marker expression overlap. This heterogeneity could be partly responsible for the observed disparity in treatment response due to the inherent diversity of the preclinical models used. For several years, our group has been generating a large patient-derived orthotopic xenograft (PDOX) MPNST platform for identifying new precision medicine treatments. Herein, we describe the expansion of this platform using six primary tumors clinically diagnosed as MPNSTs, from which we obtained six additional PDOX mouse models and three cell lines, thus generating three pairs of in vitro-in vivo models. We extensively characterized these tumors and derived preclinical models, including genomic, epigenomic, and histological analyses. Tumors were reclassified after these analyses: three remained as MPNSTs (two being classic MPNSTs), one was a melanoma, another was a neurotrophic tyrosine receptor kinase (NTRK)-rearranged spindle cell neoplasm, and, finally, the last was an unclassifiable tumor bearing neurofibromin-2 (NF2) inactivation, a neuroblastoma RAS viral oncogene homolog (NRAS) oncogenic mutation, and a SWI/SNF-related matrix-associated actin-dependent regulator of chromatin (SMARCA4) heterozygous truncated variant. New cell lines and PDOXs faithfully recapitulated histology, marker expression, and genomic characteristics of the primary tumors. The diversity in tumor identity and their specific associated genomic alterations impacted treatment responses obtained when we used the new cell lines for testing compounds against known altered pathways in MPNSTs. In summary, we present here an extension of our MPNST precision medicine platform, with new PDOXs and cell lines, including tumor entities confounded as MPNSTs in a real clinical scenario. This platform may constitute a useful tool for obtaining correct preclinical information to guide MPNST clinical trials., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Cutaneous neurofibromas (cNFs) are benign Schwann cell (SC) tumors arising from subepidermal glia. Individuals with neurofibromatosis type 1 (NF1) may develop thousands of cNFs, which greatly affect their quality of life. cNF growth is driven by the proliferation of NF1-/- SCs and their interaction with the NF1+/- microenvironment. We analyzed the crosstalk between human cNF-derived SCs and fibroblasts (FBs), identifying an expression signature specific to the SC-FB interaction. We validated the secretion of proteins involved in immune cell migration, suggesting a role of SC-FB crosstalk in immune cell recruitment. The signature also captured components of developmental signaling pathways, including the cAMP elevator G protein-coupled receptor 68 (GPR68). Activation of Gpr68 by ogerin in combination with the MEK inhibitor (MEKi) selumetinib reduced viability and induced differentiation and death of human cNF-derived primary SCs, a result corroborated using an induced pluripotent stem cell-derived 3D neurofibromasphere model. Similar results were obtained using other Gpr68 activators or cAMP analogs/adenylyl cyclase activators in combination with selumetinib. Interestingly, whereas primary SC cultures restarted their proliferation after treatment with selumetinib alone was stopped, the combination of ogerin-selumetinib elicited a permanent halt on SC expansion that persisted after drug removal. These results indicate that unbalancing the Ras and cAMP pathways by combining MEKi and cAMP elevators could be used as a potential treatment for cNFs.

Neurofibromas are benign peripheral nervous system tumors associated with neurofibromatosis type 1, which originate from NF1(-/-) Schwann cell precursors. We describe a protocol to generate neurofibromaspheres by differentiating NF1(-/-) Schwann cells from induced pluripotent stem cells and combining them with neurofibroma primary fibroblasts. We also describe the development of neurofibroma-like tumors when neurofibromaspheres are engrafted in the sciatic nerve of nude mice. This model constitutes a versatile platform for drug screening and the study of neurofibroma biology. For complete details on the use and execution of this protocol, please refer to Mazuelas et al. (2022). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Malignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas of the peripheral nervous system that develop either sporadically or in the context of neurofibromatosis type 1 (NF1). MPNST diagnosis can be challenging and treatment outcomes are poor. We present here a resource consisting of the genomic characterization of 9 widely used human MPNST cell lines for their use in translational research. NF1-related cell lines recapitulated primary MPNST copy number profiles, exhibited NF1 , CDKN2A , and SUZ12/EED tumor suppressor gene (TSG) inactivation, and presented no gain-of-function mutations. In contrast, sporadic cell lines collectively displayed different TSG inactivation patterns and presented kinase-activating mutations, fusion genes, altered mutational frequencies and COSMIC signatures, and different methylome-based classifications. Cell lines re-classified as melanomas and other sarcomas exhibited a different drug-treatment response. Deep genomic analysis, methylome-based classification, and cell-identity marker expression, challenged the identity of common MPNST cell lines, opening an opportunity to revise MPNST differential diagnosis., Competing Interests: The authors declare that they have no competing interests., (© 2023 The Author(s).)

Plexiform neurofibromas (pNFs) are developmental tumors that appear in neurofibromatosis type 1 individuals, constituting a major source of morbidity and potentially transforming into a highly metastatic sarcoma (MPNST). pNFs arise after NF1 inactivation in a cell of the neural crest (NC)-Schwann cell (SC) lineage. Here, we develop an iPSC-based NC-SC in vitro differentiation system and construct a lineage expression roadmap for the analysis of different 2D and 3D NF models. The best model consists of generating heterotypic spheroids (neurofibromaspheres) composed of iPSC-derived differentiating NF1(-/-) SCs and NF1(+/-) pNF-derived fibroblasts (Fbs). Neurofibromaspheres form by maintaining highly proliferative NF1(-/-) cells committed to the NC-SC axis due to SC-SC and SC-Fb interactions, resulting in SC linage cells at different maturation points. Upon engraftment on the mouse sciatic nerve, neurofibromaspheres consistently generate human NF-like tumors. Analysis of expression roadmap genes in human pNF single-cell RNA-seq data uncovers the presence of SC subpopulations at distinct differentiation states., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neurofibromatosis Type 1 (NF1) is a genetic condition affecting approximately 1:3500 persons worldwide. The NF1 gene codes for neurofibromin protein, a GTPase activating protein (GAP) and a negative regulator of RAS. The NF1 gene undergoes alternative splicing of exon 23a (E23a) that codes for 21 amino acids placed at the center of the GAP related domain (GRD). E23a-containing type II neurofibromin exhibits a weaker Ras-GAP activity compared to E23a-less type I isoform. Exon E23a has been related with the cognitive impairment present in NF1 individuals. We designed antisense Phosphorodiamidate Morpholino Oligomers (PMOs) to modulate E23a alternative splicing at physiological conditions of gene expression and tested their impact during PC12 cell line neuronal differentiation. Results show that any dynamic modification of the natural ratio between type I and type II isoforms disturbed neuronal differentiation, altering the proper formation of neurites and deregulating both the MAPK/ERK and cAMP/PKA signaling pathways. Our results suggest an opposite regulation of these pathways by neurofibromin and the possible existence of a feedback loop sensing neurofibromin-related signaling. The present work illustrates the utility of PMOs to study alternative splicing that could be applied to other alternatively spliced genes in vitro and in vivo.

Plexiform neurofibromas (pNFs) are benign tumors of the peripheral nervous system (PNS) that can progress towards a deadly soft tissue sarcoma termed malignant peripheral nerve sheath tumor (MPNST). pNFs appear during development in the context of the genetic disease Neurofibromatosis type 1 (NF1) due to the complete loss of the NF1 tumor suppressor gene in a cell of the neural crest (NC) - Schwann cell (SC) axis of differentiation. NF1(-/-) cells from pNFs can be reprogrammed into induced pluripotent stem cells (iPSCs) that exhibit an increased proliferation rate and maintain full iPSC properties. Efficient protocols for iPSC differentiation towards NC and SC exist and thus NC cells can be efficiently obtained from NF1(-/-) iPSCs and further differentiated towards SCs. In this review, we will focus on the iPSC modeling of pNFs, including the reprogramming of primary pNF-derived cells, the properties of pNF-derived iPSCs, the capacity to differentiate towards the NC-SC lineage, and how well iPSC-derived NF1(-/-) SC spheroids recapitulate pNF-derived primary SCs. The potential uses of NF1(-/-) iPSCs in pNF modeling and a future outlook are discussed., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Malignant peripheral nerve sheath tumor (MPNST) constitutes the leading cause of neurofibromatosis type 1-related mortality. MPNSTs contain highly rearranged hyperploid genomes and exhibit a high division rate and aggressiveness. We have studied in vitro whether the mitotic kinesins KIF11, KIF15, and KIF23 have a functional role in maintaining MPNST cell survival and can represent potential therapeutic vulnerabilities., Methods: We studied the expression of kinesin mRNAs and proteins in tumors and cell lines and used several in vitro functional assays to analyze the impact of kinesin genetic suppression (KIF15, KIF23) and drug inhibition (KIF11) in MPNST cells. We also performed in vitro combined treatments targeting KIF11 together with other described MPNST targets., Results: The studied kinesins were overexpressed in MPNST samples. KIF15 and KIF23 were required for the survival of MPNST cell lines, which were also more sensitive than benign control fibroblasts to the KIF11 inhibitors ispinesib and ARRY-520. Co-targeting KIF11 and BRD4 with ARRY-520 and JQ1 reduced MPNST cell viability, synergistically killing a much higher proportion of MPNST cells than control fibroblasts. In addition, genetic suppression of KIF15 conferred an increased sensitivity to KIF11 inhibitors alone or in combination with JQ1., Conclusions: The mitotic spindle kinesins KIF11 and KIF15 and the cytokinetic kinesin KIF23 play a clear role in maintaining MPNST cell survival and may represent potential therapeutic vulnerabilities. Although further in vivo evidences are still mandatory, we propose a simultaneous suppression of KIF11, KIF15, and BRD4 as a potential therapy for MPNSTs., (© The Author(s) 2020. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)