Deep genomic analysis of malignant peripheral nerve sheath tumor cell lines challenges current malignant peripheral nerve sheath tumor diagnosis

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 suppres-sor 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 muta-tional frequencies and COSMIC signatures, and different methylome-based clas-sifications. 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

Endoglin, a novel biomarker and therapeutical target to prevent malignant peripheral nerve sheath tumor growth and metastasis.

PURPOSE Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft-tissue sarcomas that lack effective treatments, underscoring the urgent need to uncover novel mediators of MPNST pathogenesis that may serve as potential therapeutic targets. Tumor angiogenesis is considered a critical event in MPNST transformation and progression. Here, we have investigated whether endoglin (ENG), a TGF-β coreceptor with a crucial role in angiogenesis, could be a novel therapeutic target in MPNSTs. EXPERIMENTAL DESIGN ENG expression was evaluated in human peripheral nerve sheath tumor tissues and plasma samples. Effects of tumor cell-specific ENG expression on gene expression, signaling pathway activation and in vivo MPNST growth and metastasis were investigated. The efficacy of ENG targeting in monotherapy or in combination with MEK inhibition was analyzed in xenograft models. RESULTS ENG expression was found to be upregulated in both human MPNST tumor tissues and plasma circulating small extracellular vesicles. We demonstrated that ENG modulates Smad1/5 and MAPK/ERK pathway activation and pro-angiogenic and pro-metastatic gene expression in MPNST cells and plays an active role in tumor growth and metastasis in vivo. Targeting with ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST growth and metastasis in xenograft models by reducing tumor cell proliferation and angiogenesis. Moreover, combination of anti-ENG therapy with MEK inhibition effectively reduced tumor cell growth and angiogenesis. CONCLUSIONS Our data unveil a tumor-promoting function of ENG in MPNSTs and support the use of this protein as a novel biomarker and a promising therapeutic target for this disease.We apologize to those authors whose work could not be cited due to size limitations. We thank Dr. Eduard Serra, Dr. Conxi Lázaro and Dr. David Lyden for their support in the project. We also thank Héctor Tejero for his help in analyzing RNA-seq data. Dr. Peinado laboratory is funded by US Department of Defense (W81XWH-16-1-0131), Agencia Estatal de Investigación/Ministerio de Ciencia e Innovación (AEI/MCIN) (PID2020-118558RB-I00/AEI/10.13039/501100011033), Fundación Proyecto Neurofibromatosis, European Union’s Horizon 2020 research and innovation programme “proEVLifeCycle” under the Marie Skłodowska-Curie grant agreement No 860303, and Fundación Científica AECC. We are also grateful for the support of the Ministerio de Universidades (Programa de Formación de Profesorado Universitario (FPU)) for the fellowship FPU016/05356 awarded to T. González-Muñoz and to the Translational NeTwork for the CLinical application of Extracellular VesicleS (TeNTaCLES) RED2018-102411-T(AEI/10.13039/501100011033). A. Di Giannatale was supported during this work by a research gran Nuovo-Soldati Foundation. The CNIO, certified as Severo Ochoa Excellence Centre, is supported by the Spanish Government through the Instituto de Salud Carlos III.N

Expansion of a Malignant Peripheral Nerve Sheath Tumor preclinical platform and its application in a high-throughput screening strategy to identify novel drug treatments

Programa de Doctorat en Genètica / Tesi realitzada a l'Institut d’investigació Biomèdica de Bellvitge (IDIBELL) i a l'Institut Català d'Oncologia (ICO)[eng] Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are aggressive soft-tissue sarcomas. They usually appear in the clinical context of Neurofibromatosis type 1 (NF1), however, they also occur at a low frequency in the general population. There is a medical need to develop new therapies to treat MPNSTs as they are the leading cause of death in NF1 patients, and the five-year survival rate is low. Additionally, the intrinsic heterogeneity of MPNSTs complicates their histological classification, particularly in sporadic cases. For several years, our group has been collecting patient MPNSTs to develop a preclinical platform of cell lines and patient-derived orthotopic xenograft (PDOX) mouse models to study MPNST biology, as well as to use them to conduct preclinical trials and personalized medicine strategies. Through using this platform, this PhD thesis centers on two main objectives: the identification of new synergistic drug combinations to treat MPNSTs from a high-throughput screening (HTS) of a large library of almost 2,000 drugs and the expansion of the platform by comprehensively characterizing six primary tumors diagnosed as MPNSTs and their derived PDOX and cell line models. HTS identified a total of 113 synergistic pairwise drug combinations, from which we selected 21 according to two synergy models (Bliss Independence and Highest Single Agent) and their targeted pathways. We tested these combinations to replicate the observed synergy in vitro using several tumor-derived cell lines, some of them derived by our group. The combination of MK-1775 (a WEE1 inhibitor) with Doxorubicin (a DNA Topoisomerase II inhibitor) is the only one presenting synergy in most cell lines, especially in those with gene TP53 mutated, significantly reducing the tumor growth rate in PDOX models. Regarding the second objective, the three cell models and six PDOXs generated from the six primary tumors clinically diagnosed as MPNSTs faithfully recapitulate the patients’ main genomic and histological features. Notably, we have generated three pairs of in vitro-in vivo models derived from the same primary tumors for the preclinical platform. The study of the six primary tumors was performed at different levels: 1) genetically, by analyzing the mutational status of the most recurrently mutated tumor suppressor genes in MPNSTs (NF1, CDKN2A, and SUZ12/EED from PRC2), and the mutational burden and signatures; 2) epigenetically, by analyzing the methylome profile of the three cell lines for molecular tumor classification; and 3) histologically, by analyzing the expression of markers clinically relevant for MPNST (SOX10, S100B, H3K27me3). The results illuminated the clinical reality of MPNSTs, finding that three of the tumors (all sporadic cases) presented features incompatible with their initial clinical classification as MPNSTs and were thus reclassified as distinct tumor entities. In conclusion, our preclinical platform is a bona fide tool for the study of MPNSTs since it is representative of the clinical diversity of tumors diagnosed as MPNSTs, as well as being an excellent tool to test and identify novel therapeutic approaches.[spa] Los tumores malignos de la vaina del nervio periférico (Malignant Peripheral Nerve Sheath Tumors, MPNSTs) son sarcomas de tejidos blandos agresivos. Suelen aparecer en el contexto clínico de la Neurofibromatosis tipo 1 (NF1), aunque también se dan en baja frecuencia en la población general. Existe una elevada urgencia médica de desarrollar nuevas terapias para tratar los MPNSTs, ya que son la principal causa de muerte en pacientes con NF1, y la tasa de supervivencia a cinco años es pobre. Además, la heterogeneidad intrínseca de los MPNSTs complica su clasificación histológica, especialmente en los casos esporádicos. Durante varios años, nuestro grupo ha estado reuniendo MPNSTs de pacientes para desarrollar una plataforma preclínica de líneas celulares y modelos murinos de xenoinjerto ortotópico derivados de paciente (Patient-derived orthotopic xenograft, PDOX) para estudiar la biología de los MPNST, así como para llevar a cabo ensayos preclínicos y estrategias de medicina personalizada. Usando esta plataforma, la tesis doctoral se centra en dos objetivos principales: la identificación de nuevas combinaciones sinérgicas de fármacos para tratar los MPNSTs a partir de un cribado de alto rendimiento (High-throughput screening, HTS) de una extensa librería de casi 2.000 fármacos y la ampliación de la plataforma de MPNSTs mediante la caracterización histológica y molecular de seis tumores primarios diagnosticados como MPNSTs y las líneas celulares y PDOX generados a partir de éstos. El HTS identificó un total de 113 combinaciones sinérgicas de fármacos, de las que seleccionamos 21 según dos modelos de sinergia (Bliss Independence y Highest Single Agent) y sus dianas moleculares. Estas combinaciones fueron testadas por nuestro grupo para replicar la sinergia observada en el HTS, utilizando varias líneas celulares derivadas de tumores, algunas de ellas derivadas por nuestro grupo. La combinación de MK-1775 (un inhibidor de WEE1) con Doxorrubicina (un inhibidor de la Topoisomerasa II del ADN) es la única que presenta sinergia en la mayoría de las líneas celulares, especialmente en aquellas con el gen TP53 mutado, y reduce significativamente la tasa de crecimiento tumoral en modelos PDOX. En cuanto al segundo objetivo, los tres modelos celulares y los seis PDOX generados a partir de los seis tumores primarios diagnosticados clínicamente como MPNSTs recapitulan fielmente las principales características genómicas e histológicas de los pacientes. En particular, hemos generado tres parejas de modelos in vitro-in vivo derivados de los mismos tumores primarios. El estudio de los seis tumores primarios se realizó a diferentes niveles: 1) genético, mediante el análisis del estado mutacional de los genes supresores tumorales más recurrentemente inactivados en los MPNST (NF1, CDKN2A y SUZ12/EED del PRC2), y de la carga y las firmas mutacionales; 2) epigenético, mediante el análisis del perfil del metiloma de las tres líneas celulares para la clasificación molecular de los tumores; y 3) histológicamente, analizando la expresión de marcadores clínicamente relevantes para los MPNSTs (SOX10, S100B, H3K27me3). Los resultados arrojaron luz sobre la realidad clínica de los MPNSTs, descubriéndose que tres de los tumores (todos ellos casos esporádicos) presentaban características incompatibles con su clasificación clínica como MPNST, por lo que fueron reclasificados como otras entidades tumorales. En conclusión, nuestra plataforma preclínica es una útil herramienta para el estudio de los MPNSTs ya que es representativa de la diversidad clínica de los tumores diagnosticados como MPNST, además de ser una excelente herramienta para probar e identificar nuevas aproximaciones terapéuticas

Expansion of a Malignant Peripheral Nerve Sheath Tumor preclinical platform and its application in a high-throughput screening strategy to identify novel drug treatments

[eng] Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are aggressive soft-tissue sarcomas. They usually appear in the clinical context of Neurofibromatosis type 1 (NF1), however, they also occur at a low frequency in the general population. There is a medical need to develop new therapies to treat MPNSTs as they are the leading cause of death in NF1 patients, and the five-year survival rate is low. Additionally, the intrinsic heterogeneity of MPNSTs complicates their histological classification, particularly in sporadic cases. For several years, our group has been collecting patient MPNSTs to develop a preclinical platform of cell lines and patient-derived orthotopic xenograft (PDOX) mouse models to study MPNST biology, as well as to use them to conduct preclinical trials and personalized medicine strategies. Through using this platform, this PhD thesis centers on two main objectives: the identification of new synergistic drug combinations to treat MPNSTs from a high-throughput screening (HTS) of a large library of almost 2,000 drugs and the expansion of the platform by comprehensively characterizing six primary tumors diagnosed as MPNSTs and their derived PDOX and cell line models. HTS identified a total of 113 synergistic pairwise drug combinations, from which we selected 21 according to two synergy models (Bliss Independence and Highest Single Agent) and their targeted pathways. We tested these combinations to replicate the observed synergy in vitro using several tumor-derived cell lines, some of them derived by our group. The combination of MK-1775 (a WEE1 inhibitor) with Doxorubicin (a DNA Topoisomerase II inhibitor) is the only one presenting synergy in most cell lines, especially in those with gene TP53 mutated, significantly reducing the tumor growth rate in PDOX models. Regarding the second objective, the three cell models and six PDOXs generated from the six primary tumors clinically diagnosed as MPNSTs faithfully recapitulate the patients’ main genomic and histological features. Notably, we have generated three pairs of in vitro-in vivo models derived from the same primary tumors for the preclinical platform. The study of the six primary tumors was performed at different levels: 1) genetically, by analyzing the mutational status of the most recurrently mutated tumor suppressor genes in MPNSTs (NF1, CDKN2A, and SUZ12/EED from PRC2), and the mutational burden and signatures; 2) epigenetically, by analyzing the methylome profile of the three cell lines for molecular tumor classification; and 3) histologically, by analyzing the expression of markers clinically relevant for MPNST (SOX10, S100B, H3K27me3). The results illuminated the clinical reality of MPNSTs, finding that three of the tumors (all sporadic cases) presented features incompatible with their initial clinical classification as MPNSTs and were thus reclassified as distinct tumor entities. In conclusion, our preclinical platform is a bona fide tool for the study of MPNSTs since it is representative of the clinical diversity of tumors diagnosed as MPNSTs, as well as being an excellent tool to test and identify novel therapeutic approaches.[spa] Los tumores malignos de la vaina del nervio periférico (Malignant Peripheral Nerve Sheath Tumors, MPNSTs) son sarcomas de tejidos blandos agresivos. Suelen aparecer en el contexto clínico de la Neurofibromatosis tipo 1 (NF1), aunque también se dan en baja frecuencia en la población general. Existe una elevada urgencia médica de desarrollar nuevas terapias para tratar los MPNSTs, ya que son la principal causa de muerte en pacientes con NF1, y la tasa de supervivencia a cinco años es pobre. Además, la heterogeneidad intrínseca de los MPNSTs complica su clasificación histológica, especialmente en los casos esporádicos. Durante varios años, nuestro grupo ha estado reuniendo MPNSTs de pacientes para desarrollar una plataforma preclínica de líneas celulares y modelos murinos de xenoinjerto ortotópico derivados de paciente (Patient-derived orthotopic xenograft, PDOX) para estudiar la biología de los MPNST, así como para llevar a cabo ensayos preclínicos y estrategias de medicina personalizada. Usando esta plataforma, la tesis doctoral se centra en dos objetivos principales: la identificación de nuevas combinaciones sinérgicas de fármacos para tratar los MPNSTs a partir de un cribado de alto rendimiento (High-throughput screening, HTS) de una extensa librería de casi 2.000 fármacos y la ampliación de la plataforma de MPNSTs mediante la caracterización histológica y molecular de seis tumores primarios diagnosticados como MPNSTs y las líneas celulares y PDOX generados a partir de éstos. El HTS identificó un total de 113 combinaciones sinérgicas de fármacos, de las que seleccionamos 21 según dos modelos de sinergia (Bliss Independence y Highest Single Agent) y sus dianas moleculares. Estas combinaciones fueron testadas por nuestro grupo para replicar la sinergia observada en el HTS, utilizando varias líneas celulares derivadas de tumores, algunas de ellas derivadas por nuestro grupo. La combinación de MK-1775 (un inhibidor de WEE1) con Doxorrubicina (un inhibidor de la Topoisomerasa II del ADN) es la única que presenta sinergia en la mayoría de las líneas celulares, especialmente en aquellas con el gen TP53 mutado, y reduce significativamente la tasa de crecimiento tumoral en modelos PDOX. En cuanto al segundo objetivo, los tres modelos celulares y los seis PDOX generados a partir de los seis tumores primarios diagnosticados clínicamente como MPNSTs recapitulan fielmente las principales características genómicas e histológicas de los pacientes. En particular, hemos generado tres parejas de modelos in vitro-in vivo derivados de los mismos tumores primarios. El estudio de los seis tumores primarios se realizó a diferentes niveles: 1) genético, mediante el análisis del estado mutacional de los genes supresores tumorales más recurrentemente inactivados en los MPNST (NF1, CDKN2A y SUZ12/EED del PRC2), y de la carga y las firmas mutacionales; 2) epigenético, mediante el análisis del perfil del metiloma de las tres líneas celulares para la clasificación molecular de los tumores; y 3) histológicamente, analizando la expresión de marcadores clínicamente relevantes para los MPNSTs (SOX10, S100B, H3K27me3). Los resultados arrojaron luz sobre la realidad clínica de los MPNSTs, descubriéndose que tres de los tumores (todos ellos casos esporádicos) presentaban características incompatibles con su clasificación clínica como MPNST, por lo que fueron reclasificados como otras entidades tumorales. En conclusión, nuestra plataforma preclínica es una útil herramienta para el estudio de los MPNSTs ya que es representativa de la diversidad clínica de los tumores diagnosticados como MPNST, además de ser una excelente herramienta para probar e identificar nuevas aproximaciones terapéuticas

Generation of two compound heterozygous HGSNAT-mutated lines from healthy induced pluripotent stem cells using CRISPR/Cas9 to model Sanfilippo C syndrome

Sanfilippo C syndrome (Mucopolysaccharidosis IIIC) is a rare lysosomal storage disorder caused by mutations in the HGSNAT gene. It is characterized by a progressive and severe neurodegeneration, for which there is no treatment available. Here, we report the generation of two HGSNAT-mutated cell lines from a healthy human induced pluripotent stem cell (hiPSC) line using CRISPR/Cas9 editing. These novel cell lines have a normal karyotype, express pluripotency specific markers and have the capability to differentiate into all three germ layers in vitro. These hiPSC lines will be useful for the generation of in vitro models of Sanfilippo C syndrome

Generation of two compound heterozygous HGSNAT-mutated lines from healthy induced pluripotent stem cells using CRISPR/Cas9 to model Sanfilippo C syndrome

Sanfilippo C syndrome (Mucopolysaccharidosis IIIC) is a rare lysosomal storage disorder caused by mutations in the HGSNAT gene. It is characterized by a progressive and severe neurodegeneration, for which there is no treatment available. Here, we report the generation of two HGSNAT-mutated cell lines from a healthy human induced pluripotent stem cell (hiPSC) line using CRISPR/Cas9 editing. These novel cell lines have a normal karyotype, express pluripotency specific markers and have the capability to differentiate into all three germ layers in vitro. These hiPSC lines will be useful for the generation of in vitro models of Sanfilippo C syndrome

Use of patient derived orthotopic xenograft models for real-time therapy guidance in a pediatric sporadic malignant peripheral nerve sheath tumor

Background: The aim of this study was to test the feasibility and utility of developing patient-derived orthotopic xenograft (PDOX) models for patients with malignant peripheral nerve sheath tumors (MPNSTs) to aid therapeutic interventions in real time. Patient & methods: A sporadic relapsed MPNST developed in a 14-year-old boy was engrafted in mice, generating a PDOX model for use in co-clinical trials after informed consent. SNP-array and exome sequencing was performed on the relapsed tumor. Genomics, drug availability, and published literature guided PDOX treatments. Results: A MPNST PDOX model was generated and expanded. Analysis of the patient's relapsed tumor revealed mutations in the MAPK1, EED, and CDK2NA/B genes. First, the PDOX model was treated with the same therapeutic regimen as received by the patient (everolimus and trametinib); after observing partial response, tumors were left to regrow. Regrown tumors were treated based on mutations (palbociclib and JQ1), drug availability, and published literature (nab-paclitaxel; bevacizumab; sorafenib plus doxorubicin; and gemcitabine plus docetaxel). The patient had a lung metastatic relapse and was treated according to PDOX results, first with nab-paclitaxel, second with sorafenib plus doxorubicin after progression, although a complete response was not achieved and multiple metastasectomies were performed. The patient is currently disease free 46 months after first relapse. Conclusion: Our results indicate the feasibility of generating MPNST-PDOX and genomic characterization to guide treatment in real time. Although the treatment responses observed in our model did not fully recapitulate the patient's response, this pilot study identify key aspects to improve our co-clinical testing approach in real time