Sox2 protein stability is enhanced by BRafV600E and Pten deletion in adult neural stem/progenitor cells

Sox2 protein stability is enhanced by BRafV600E and Pten deletion in adult neural stem/progenitor cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Sox2 protein stability is enhanced by BRafV600E and Pten deletion in adult neural stem/progenitor cells Eugenia Guida, Ambra Colopi, Valeriana Cesarini, Marco Pieraccioli, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7515762/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Apr, 2026 Read the published version in npj Precision Oncology → Version 1 posted 8 You are reading this latest preprint version Abstract BRAF mutations are oncogenic drivers present in about 7% of human cancers, with the V600E substitution being the most frequent. In the nervous system, BRAFV600E has been identified in both low- and high-grade gliomas (LGG and HGG) and in tumors of the peripheral nervous system. To investigate the mechanisms underlying BRafV600E-driven tumorigenesis, we generated a mouse model in which the BRafV600E mutation and Pten deletion can be induced through the Sox2-CreERT2 system. This inducible deleter is active in adult neural stem/progenitor cells (aNSPCs) and Schwann cell precursors (SCPs). In this model, BRafV600E-mutated/Pten-deleted telencephalic aNSPCs give rise to diffuse LGG with oligodendroglioma-like features resembling the human diffuse LGG, MAPK pathway–altered subtype. In contrast, mutated SCPs develop schwannomas, cutaneous neurofibromas, and malignant peripheral nerve sheath tumors (MPNSTs). Sox2 deletion in BRafV600E/Ptendel mice markedly reduces tumor formation, indicating that aNSPCs act as tumor cells of origin. In vitro analyses show increased proliferation of BRafV600E/Ptendel aNSPCs and preferential differentiation toward oligodendroglia-like tumor cells. BRaf-mutant aNSPCs display increased Sox2 protein, but not mRNA levels, suggesting post-transcriptional regulation. Sox2 phosphorylation at T118 is promoted by BRafV600E, potentially stabilizing the protein during aNSPC transformation, although the involvement of downstream kinases cannot be excluded. Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Molecular biology Biological sciences/Neuroscience Health sciences/Oncology BRafV600E Mapk Pten Sox2 PLNTY schwannoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The RAS/BRAF/MEK/ERK pathway is highly mutated in cancer 1 , with mutations of the BRAF gene occurring in about 7% of cases 2 . The most frequently observed BRAF mutation is the substitution of valine 600 with glutamic acid (V600E), which induces persistent, RAS-independent activation of BRAF, inhibiting its inactivation and leading to a continuous stimulation of MEK and ERK 3 . Such mutation is recurrent in melanomas, hairy cell leukemias, thyroid and colon carcinomas, and acts as a tumor driver in animal models for these tumors 4-7 . In the nervous system, MAPK-altered tumors are frequently found in pediatric low-grade glioneural tumors (GNTs), with FGFR1 or BRAF being the predominant mutated genes 8 . BRAF alterations account for a restricted number of tumors and can occur as BRAF fusions ( KIAA1549-BRAF ) or substitutions (BRAFV600E). Specifically, the BRAFV600E allele has been identified in many central nervous system (CNS) tumors ranging from GNTs (such as gangliogliomas and dysembryoplastic neuroepithelial tumors) 7 , to astrocytic tumors (such as pleomorphic xanthoastrocytomas 9,10 and pilocytic astrocytomas 11 ), papillary craniopharyngioma 12 , oligodendroglial tumors (such as oligodendroglioma-like tumors of pediatric origin 13 , polymorphous low-grade neuroepithelial tumor of the young, PLNTY 14 ), and pediatric or adult glioblastoma multiforme (GBM) 15 . Exploiting genetic mouse models (GEMMs) carrying cell-specific promoter/enhancer constructs to direct oncogene activation or tumor suppressor deletion in vivo, it has been shown that embryonic and adult neural stem/progenitor cells (NSPCs), heterogeneous and specialized stem cells that generate specific types of neurons and glia 16 , are the cells of origin of many high-grade gliomas (HGGs) 17 . On the contrary, peripheral nerve sheath (PNS) tumors have been shown to arise from Schwann cell precursors (SCPs; also defined as skin cell precursors, SKPs), a cell population considered as multipotent as NSPCs. 18 These cells are found along outgrowing peripheral nerves 19 and hair bulbs 18 , or derive from PLP1-expressing Schwann cells 20 . Mouse models of low-grade gliomas (LGGs), in particular the GNT subtypes, have primarily been obtained by in utero brain injections of oncogenes, resulting in massive transformation of fetal neural precursors and leaving open the question of whether adult NSPCs (aNSPCs) are competent to give origin to GNTs. We previously showed that Sex determining region Y-box 2 (Sox2) expression is transcriptionally regulated by BRafV600E in a melanoma mouse model 21 . Sox2 is a transcriptional regulator essential during early embryonic development for orchestrating stem cell pluripotency and neural commitment 22 . Aside from its physiological role in neural development, Sox2 plays an oncogenic role in brain tumors 23 , and its expression can be regulated at multiple levels through transcriptional, translational, and post-translational mechanisms 24,25 . Recent findings have shown that Akt-mediated phosphorylation on threonine 118 (T118) prevents the neighbor lysine (K119) ubiquitylation in murine embryonic stem cells (ESCs) 25 and that Sox2 protein stability is enhanced by regulating its ubiquitylation in NSPCs 26 . In the present work, we investigated the mechanism/s involved in aNSPCs transformation operated by BRafV600E alone or in combination with Pten deletion, both in vivo and in vitro. We found that symptomatic GNTs resembling PLNTY lesions and schwannomas develop and rapidly progress after oncogenic induction in BRafV600E/Pte n del young adult mice in vivo, while aNSPCs shift to high proliferative activity following transduction, in vitro. We demonstrate that the BRafV600E mutation, alone or in combination with Pten deletion, enhances Sox2 protein stability by decreasing Sox2 ubiquitylation, highlighting the essential role of concomitantly overactivated Mapk and Akt pathways in sustaining the oncogenic role of Sox2 in GNT formation. Results Sox2-CreERT2 promoter is active in telencephalic aNSPCs and in SCPs To activate the BR af V600E mutation and Pten deletion specifically in aNSPCs, we employed the Sox2-CreERT2 deleter, proved to be active in aNSPCs from the sub-ventricular zone (SVZ) of the lateral ventricles and in the sub-granular zone (SGZ) of the hippocampus 27 . To limit the occurrence of the oncogenic mutations only to a few numbers of aNSPCs, we induced Cre-mediated recombination by a single injection of tamoxifen in one-month-old Sox2-CreERT2 ; R26R-LacZ reporters (Figure 1A). Cre activation was monitored by tracing b- galactosidase ( b- gal ) activity in the brain, spinal cord, and dermis at 10 and 30 days after tamoxifen administration. On day 10, we found b-gal-positive cells in the SVZ and SGZ neurogenic niches, as expected, in the corpus callosum and the cerebral cortex (Supplementary Figure S1A), and their numbers increased one-month post-tamoxifen injection, extending also into the caudoputamen (Figure 1B-C). We also found scattered b-gal-positive cells in adult dorsal root ganglia (DRG) and in hair bulbs from skin sections, indicating that the Sox2 telencephalic promoter was also active in SCP/SKPs (Figure 1D-E). Sox2-CreERT2 activity at embryonic stage E13.5 was identified in the anterior telencephalic region (Figure 1F), as previously observed for Sox2-CreERT2; R26R-LacZ embryos 27 and within the developing DRG 31 . BRafV600E and Pten deletion in Sox2-expressing aNSPCs and SCPs induce the formation of brain and nerve tumors in adult mice To understand if Mapk overactivation might impact cell transformation and/or aberrant cell differentiation, we activated the BRafV600E mutation (thereafter defined B) along with Pten deletion (thereafter defined P) in aNSPCs by means of the Sox2-CreERT2 deleter . To trace Sox2-CreERT2 edited cells , Sox2-CreERT2; R26R-Eyfp mice were crossed to BRafV600E and Pten flox/flox mice to obtain the genotypes shown in Figure 2A . One-month-old mice were primed with a single intraperitoneal (i.p.) injection of tamoxifen and constantly observed. Tremor was usually the first symptom appearing only in BbPP and BBPP mutants at about 30 days after tamoxifen administration and was then accompanied by weight loss, apathy, sporadic epilepsy, kyphosis, and hindlimb retraction, indicating a neurological condition (Supplementary Video). Control (Wt) mice did not show any phenotype. BbPP mice required euthanasia to prevent respiratory paralysis, with a median survival time of 60 days post-injection (dpi). Mice carrying the BBPP alleles exhibited a similar median survival time (60 dpi) compared to BbPP mice (Figure 2B). In contrast, Wt mice, as well as BBpp, bbPP, and most BbPp and BBPp mice, did not develop symptoms when followed up to 150 dpi (Figure 2B and Supplementary Figure S1B). Three out of eight BbPp and three out of eight BBPp mice developed a neurological phenotype at around 70 and 60 dpi, respectively, suggesting that loss of Pten heterozygosity occurred (Supplementary Figure S1B). At autopsy, freshly isolated brains from euthanized BbPP and BBPP mice did not show gross cortical anomalies. We then evaluated the brain/body weight ratio in 6 out of 13 BbPP and 6 out of 12 BBPP mice, observing a two-fold increase in brain weight in the mutated genotypes compared to Wt (Figure 2C and Supplementary Figure S1C). In 100% of the affected mice from both genotypes (13/13 and 12/12, respectively), hematoxylin and eosin (H&E) brain sections showed tumor masses in the telencephalic vesicles that, in some cases, induced enlargement of lateral ventricles (Figure 2D), suggesting obstructive hydrocephalus. All the tamoxifen-injected BbPP and BBPP mice showed a mass on the ventral surface of the brainstem associated with the trigeminal nerve (cranial nerve, CN V) (Supplementary Figure S1D). All the inspected spinal cords showed enlarged, often bilateral, DRG masses at many vertebral levels, also invading the intervertebral foramina to extend in the peri-vertebral space (Figure 2E and Supplementary Figure S1E). Such masses showed larger dimensions in BBPP compared to BbPP genotypes and signs of compression on the spinal cord, which led to animal death (Figure 2E). We found that the entire cohort of BbPP and BBPP mice developed DRG tumors (100%) that were accompanied by brain tumors (100%), and occasionally by skin masses (23% in BbPP and 33% in BBPP) (Figure 2A and F). BBpp or bbPP brains and DRGs did not exhibit areas of hyperproliferation when animals were sacrificed approximately 150 days after tamoxifen injection (not shown). To further prove that aNSPCs were the tumor cells of origin, we specifically deleted Sox2 in these cells by crossing BbPP mice with conditional Sox2 flox/flox mice and generating the Sox2 flox/flox BbPP mice (Supplementary Figure S1F). Cre recombination was induced by tamoxifen in five BbPP and five Sox2 flox/flox BbPP mice. None of the injected Sox2 -deleted ( Sox2 de l ) conditional mice showed symptoms 30 days after tamoxifen administration, while Sox2 wt BbPP mice were symptomatic and were sacrificed between 60 and 75 days after tamoxifen induction (Supplementary Figure S1G). Sox2 del mice were followed for an additional 4 months and then sacrificed. Isolated brains and spinal cords revealed that Sox2 del BbPP mice did not develop brain tumors, nor schwannomas (data not shown). Collectively, these findings demonstrate that BRafV600E expression combined with Pten loss can give rise to brain tumor formation in Sox2-expressing aNSPCs, and DRG and/or skin tumors in Sox2-expressing SCPs. Moreover, our experiments confirm that Sox2 expression is a prerequisite for aNSPCs/SCPs transformation. BRafV600E / Pten -deleted aNSPCs are prone to transform into neuroglial tumor subtypes. The telencephalic tumors from both genotypes (BbPP and BBPP) were identified as heterogeneous glioneural lesions forming neurocytic rosettes characterized by proliferating cells with rounded nuclei that were Olig2-positive and NeuN-negative with scattered neuropil islands (Figure 3A and Supplementary Figure S2A), while no gross histological alterations in BBpp, or bbPP brains were found (data not shown). All lesions observed showed areas of uniformly round nuclei with a perinuclear halo, conferring an “oligo-like” appearance (Figure 3A and Supplementary Figure S2A), a feature frequently described in diffuse glioneuronal tumors with oligodendroglioma-like features such as PLNTY 32 . Immunohistochemical (IHC) analysis showed Olig2-positive rounded neuroepithelial cells with scant cytoplasm and without evidence of cytoplasmic processes, intermingled with giant astrocytic cells showing intense Gfap and Nestin positivity, corresponding to reactive astrocytes (Figure 3A and Supplementary S2B-C). Reactive astrocytes associated with the tumor tissue had the typical stellate morphology and long, thick cytoplasmic processes 33 that differed morphologically from small Gfap-positive cells within the tumor, which were excluded from the oligodendroglioma-like areas (Figure 3A and Supplementary S2B-C). BbPP tumors were characterized by a low Ki67 index (5.6% ± 3.1% Ki67-positive cells) while BBPP tumors showed a higher proliferation activity with a Ki67 index reaching 15.5% ± 5.5% (Figure 3B). Tumors from both BbPP and BBPP genotypes always contained Olig2, Gfap, as well as Sox2-positive cells (Figure 3A and Supplementary Figure S2D), while Nestin and Tubb3 immunoreactivity was in the non-tumoral compartment (Supplementary Figure S2D). Indeed, normal NeuN-positive neurons were engulfed by neoplastic cells (Figure 3A and Supplementary Figure S2D). In some cases, we observed that BBPP lesions presented with rhythmic palisades with a spongioblastic pattern (Supplementary Figure S2A), a feature described in a wide range of CNS neoplasms, including pilocytic astrocytoma and oligodendroglioma 34 . Staining combinations with Olig2-Sox2, Olig2-Gfap, and Sox2-Gfap antibodies showed that the majority of Olig2-positive tumor cells were Sox2- but not Gfap-positive, while Gfap-Sox2 positivity mainly co-localized in reactive astrocytes (Figure 3C). We observed a few Gfap-Eyfp-positive cells within the tumor, suggesting that a minority of tumor cells could be of astrocytic nature (Figure 3D). Pten loss and BRafV600E expression were confirmed in serial sections of Eyfp-positive BbPP tumors by immunofluorescence (IF) and by IHC, and, accordingly, pErk1,2 immunoreactivity was also observed (Figure 3E and Supplementary Figure S2E). BBpp, but not bbPP brains, showed a few scattered BRafV600E-positive cells in the subventricular region, which were not sufficient to induce tumor formation (Supplementary Figure S2F). Altogether, our results suggest that BRafV600E expression in a Pten -deleted background affects aNSPCs proliferation and differentiation, inducing the formation of LGG tumors that are more proliferative in the presence of a homozygous BRafV600E- mutated allele. Human PLNTY lesions express SOX2 within the oligodendroglioma-like tumor area. To investigate whether BRAFV600E-positive PLNTY lesions express SOX2, we identified two PLNTY cases diagnosed in a 16-year-old male and a 29-year-old female patient. Consistent with the literature, H&E staining revealed lesions with a diffuse growth pattern and oligodendroglioma-like cellular components that were strongly positive for OLIG2 (Figure 4 A-B). The Ki-67 labeling indices were approximately 10% and 5%, respectively. Notably, within the oligodendroglioma-like areas, tumor cells showed high levels of SOX2 and strong BRAFV600E immunoreactivity, and were negative for IDH1R132H, supporting the diagnosis of PLNTY. In contrast to the mouse lesions, GFAP staining was diffusely distributed throughout the tumor areas. We also probed the lesions for PTEN and pAKT expression as readout of PI3K pathway overactivation, and pERK1,2 to assess MAPK pathway activation (Figure 4 A-B). These results suggest a correlation between BRAFV600E and SOX2 expression, in agreement with previous work on colon cancer (CRC) and recurrent ameloblastoma tumor cells 35,36 . BRafV600E and Pten -deleted SCPs transform into paraspinal schwannomas Histological examination of brainstem-associated masses involving CN V and enlarged DRGs revealed whorls of Schwann cells, consistent with cellular schwannomas, a tumor variant characterized by high cellularity, predominantly composed of Antoni A areas, and lacking Verocay bodies 37 . These tumors displayed neuronal cell bodies and axons entrapped by spindle-shaped neoplastic cells, which also invaded the spinal nerve roots. Immunohistochemical characterization showed diffuse S100b, Sox2, and Sox10 positivity in both BbPP and BBPP genotypes (Figure 5A–B and Supplementary Figure S2G). Ki67 staining indicated moderate proliferative activity, with labeling indices of 9.2% ± 2.5% and 11.2% ± 3.7% in BbPP and BBPP tumors, respectively (Figure 5A–C and Supplementary Figure S2G). Both genotypes also developed skin masses (Figure 2F and Figure 5D), spanning a spectrum from benign cutaneous neurofibromas (cNFs) to high-grade malignant peripheral nerve sheath tumors (MPNSTs), as determined by histological and immunohistochemical analyses (Figure 5D and E). cNFs consisted of spindle-shaped Schwann cells positive for S100b and Sox10, admixed with perineurial cells and fibroblasts (Figure 5D). In contrast, MPNSTs, corresponding phenotypically to sciatic nerve masses in both genotypes (one case in BbPP and one in BBPP), were composed of densely packed spindle cells with elongated, wavy nuclei and nuclear atypia. These tumors showed strong immunoreactivity for S100b, Sox2, and Sox10 (Figure 5E) while Ki67 indices showed significant differences between the two tumor genotypes (14.4% ± 1.1% vs 20.4% ± 2.9%, BbPP and BBPP, respectively; Figure 5F). Schwann cell transformation of the cranial and spinal DRG resulted in overgrowth and invasion of the posterior cranial fossa and the vertebral canal, respectively, leading to animal death. BRafV600E-mutated aNSPCs show increased proliferation in vitro Attempts to establish cell cultures from brain or DRG tumors in vitro from both BbPP and BBPP genotypes were unsuccessful; thus, we obtained in vitro deleted aNSPCs isolated from the brains of the different genotypes. To this end, SVZ cells were collected from the brains of one-month-old Wt, BbPP, and BBPP mice to obtain aNSPCs. To better dissect the independent contribution of Mapk or Akt overactivated pathways during cell transformation, we also included BBpp and bbPP aNSPCs. aNSPCs grown as neurospheres were treated with 2μM 4-hydroxytamoxifen (4HT) to induce Cre-mediated mutation. Single cell suspensions were then FACS-sorted to obtain a homogeneous Eyfp-positive cell population, genotyped to verify Pten deletion (Supplementary Figure S3A), and/or stained with BRafV600E-specific antibody for the inclusion of V600E mutation (Supplementary Figure S3B). To assess BRafV600E levels in BbPP and BBPP, compared to Wt neurospheres, we quantified total BRaf and BRafV600E expression in IF experiments (Supplementary Figure S3B-C). This analysis revealed increased BRafV600E fluorescence intensity in the BBPP compared with BbPP cells, consistent with successful gene editing. 5-bromo-2’-deoxyuridine (BrdU) incorporation rates in the presence of canonical Growth Factors (GFs) showed that aNSPCs from all the genotypes were more proliferative compared to Wt cells (BbPP 33.3% ± 1.7%, BBPP 37.3% ± 0.8%, BBpp 31.4% ± 3.1&, bbPP 31.3% ± 2.3%; Figure 6A) in vitro. BRafV600E inhibition by 0.5 or 1mM PLX-4032 (PLX) affected BRaf-mutated aNSPCs growth, and BrdU incorporation was strongly decreased in BbPP compared to Wt cells (Supplementary Figure S3D). Since GFs independence is considered a hallmark of malignancy secondary to the development of autocrine GF loops in cancer cells 38 , we evaluated whether mutated aNSPCs were able to proliferate in low concentrations of GFs or in their absence. Cells from all genotypes but not Wt were able to proliferate in the absence of GFs after 5 days of culture and formed large colonies in complete GFs deprivation; however, BRafV600E-containing genotypes showed a consistently higher proliferation activity compared to Pten -only deleted aNSPCs (Figure 6B and Supplementary Figure S3E). Braf-mutated aNSPCs preferentially differentiate into oligodendrocytes in vitro It is well established that NSPCs can differentiate along the three neural lineages when exposed to serum in vitro 39 . To evaluate the impact of the BRafV600E mutation and/or Pten deletion on the developmental fate of aNSPCs, we performed pseudo-differentiation experiments on cultured cells from all the genotypes in 1% FBS for 10 days. Wt aNSPCs preferentially differentiated along the astrocytic lineage with 61.37% ± 9.29% of Gfap-positive cells, compared to the neuronal lineage or oligodendrocyte lineage (30.68% ± 8.25% Tubb3-positive cells and 7.95% ± 1.05% MBP-positive cells, respectively). On the contrary, the number of Gfap-positive cells was significantly decreased in BBpp (9.54% ± 4.53%), BbPP (23.74% ± 12.9%), or BBPP (15.01% ± 8.26%) aNSPCs, in favor of MBP-positive cells, that accounted for 63.17% ± 10.47%, 31.93% ± 3.59% and 57.96% ± 5.5%, respectively. Tubb3-positive cell numbers were not significantly different in the BRafV600E-mutated genotypes (BBpp 24.39% ± 5.04%, BbPP 33.84% ± 10.31%, and BBPP 20.0% ± 0.68%) compared to the control genotype (Figure 6C). bbPP aNSPCs differentiated preferentially toward the neuronal pathway (Tubb3 positivity: 85% ± 1.04%) as previously described 40,41 . The remaining cells were positive for Gfap (13.4% ± 2.04%), but none differentiated along the oligodendrocyte lineage. Wt or bbPP aNSPCs cultures did not retain any proliferating undifferentiated cells, while in all the BRafV600E genotypes they were still present, with numbers ranging from 2.9% ± 1.3% in BBpp to 10.5% ± 2.0% in BbPP and 7.0% ± 3.0% in BBPP genotypes, respectively (Figure 6C). Treatment with PLX completely abrogated BrdU-incorporating undifferentiated cells in all the BRafV600E genotypes, while Wt or bbPP were not affected (Supplementary Figure S3F). Overall, these results suggest that, also in differentiating conditions, BRafV600E -mutant aNSPCs retain their ability to proliferate, while preferentially differentiating toward the oligodendrocyte lineage at the expense of the astrocyte and neuron lineages. BRafV600E mutation and Pten deletion both enhance Sox2 stability in aNSPCs Sox2 is required to maintain aNSPCs and cancer stem cells 27,42 . It also acts as a glioma-reprogramming factor 23 , and its deletion in PDGF-B-transformed oligodendroglioma prevents cancer cells from generating secondary tumors when orthotopically transplanted 42 . By IF and western blot (WB) analysis, we found that BbPP and BBPP aNSPCs showed higher levels of Sox2 protein expression compared to Wt cells; however, quantitative RT-PCR analysis did not show significant differences in Sox2 mRNA levels (Figure 7A-B and Supplementary Figure S4A-B). On the contrary, Gfap mRNA levels were dramatically decreased in BbPP aNSPCs, as well as in all the other mutant genotypes (Supplementary Figure S4B). Treatment with PLX or PD98059 (PD) for 6 h, to inhibit Mapk activation, showed a strong reduction of Sox2 protein levels in BbPP aNSPCs (Figure 7C and Supplementary Figure S4C). It has been shown that Akt activation decreases Sox2 ubiquitylation, enhancing its stability in ESCs and esophageal cancer cells 25,43 , whereas Sox2 ubiquitylation is increased upon neuronal differentiation in aNSPC 26 . Thus, to determine whether protein synthesis or degradation rates could be affected by overactivated Mapk/Akt pathways, we treated Wt, BbPP, BBpp, and bbPP aNSPCs for 5 h with the proteasome inhibitor MG132 (MG) or the protein synthesis inhibitor cycloheximide (CHX). Sox2 protein levels were upregulated by proteasome inhibition and downregulated by protein synthesis inhibition in Wt aNSPCs (Figure 7D and Supplementary Figure S4D). As expected, Sox2 levels were increased both in BbPP or BBpp aNSPCs compared to Wt cells, and its levels slightly changed following MG or CHX treatments, suggesting that BRafV600E promotes Sox2 protein stability by affecting the ubiquitylation process (Figure 7D and Supplementary Figure S4D). We also found that Sox2 mRNA levels were upregulated in Akt-deregulated bbPP aNSPCs (Supplementary Figure 4B) and, importantly, that proteasome inhibition but not CHX treatment increased Sox2 protein levels by an Akt-dependent post-translational mechanism (Figure 7D and Supplementary Figure S4D), as previously shown in ESCs and esophageal cancer cells 25,43 . We then monitored Sox2 ubiquitylation by BRafV600E following transfection of HA-Ubiquitin, Sox2, and FLAG-BRafV600E- or FLAG-empty-expressing plasmids in HEK293T cells. Notably, input extracts showed that Sox2 levels were increased following 5 h MG treatment compared to mock-treated controls in FLAG-BRafV600E/Sox2/HA-Ubiquitin cells, but not in FLAG-empty/Sox2/HA-Ubiquitin cells. We observed that Sox2 levels were higher in FLAG-BRafV600E/Sox2 co-transfected cells compared to FLAG-Sox2-transfected cells, due to a transcriptional effect of Mek1 overactivation on CMV-promoter driven expression plasmids 44 . Ubiquitylated immunoprecipitated Sox2 levels, monitored by HA expression, were increased in Sox2-only transfected cells following MG treatment (2-fold), while they were decreased both in control and in MG-treated (about 0.4-fold) FLAG-BRafV600E/Sox2 transfected cells, indicating that BRafV600E expression prevented Sox2 ubiquitylation (Figure 7E and Supplementary S4E). These results corroborated the finding that inhibition of ubiquitylation by MG was a major mechanism for Sox2 stabilization either in BBpp or in BbPP aNSPCs and suggested a role for the Mapk-activated pathway in preventing Sox2 degradation. It is known that phosphorylation of threonine 118 (pT118) mediated by Akt prevents Sox2 lysine 119 methylation and ubiquitylation, increasing Sox2 stability in ESCs 25,26 . We found that T118 was a target of the BRaf overactivated pathway, as mutant T118A-Sox2 was not phosphorylated in BRafV600E-transfected HEK293T cells (Figure 7F and Supplementary Figure S4F). We found that FLAG-BRafV600E was present in the complex obtained from Sox2 immunoprecipitation (Figure 7E), and conversely, while Wt-Sox2 co-immunoprecipitated, T118A-Sox2 was not present in the complex obtained from FLAG-BRafV600E immunoprecipitation in HEK293T transfected cells (Figure 7G). To assess if the T118A mutation affected Sox2 stability in FLAG-BRafV600E overexpressing cells, we transiently expressed Wt- or T118A-Sox2 in HEK293T cells that were treated after 72 h from transfection for 1, 3, or 6 h with CHX. While Wt-Sox2 levels did not change throughout the treatment time, we found that T118A-Sox2 levels were significantly decreased after 6 h of treatment (Figure 7H and Supplementary Figure S4G). The role of BRafV600E in phosphorylating and stabilizing Sox2 levels was further confirmed in aNSPCs cell extracts that showed increased total- and pT118-Sox2 levels in all BRafV600E mutated genotypes (BbPP, BBPP, BBpp) (Figure 7I). As in ESCs 25 , T118 was a target of Akt-mediated phosphorylation, as shown in bbPP cells, confirming that the Akt-activated pathway promotes T118 phosphorylation also in aNSPCs (Figure 7I and Supplementary Figure S4H). We next interrogated the TCGA PanCancer Atlas dataset to determine whether human high-grade tumors exhibit differential SOX2 mRNA expression according to BRAF mutational status. Interestingly, we found no statistically significant differences between BRAF-mutated or non-mutated samples in SOX2 expression levels in the LGG and GBM cohorts, although the mutated cohort in the dataset was too small compared to the non-mutated. Similar results were obtained by analyzing skin cutaneous melanomas (SKCM) (in which the two cohorts were similar in size, Supplementary Figure S4I). On the contrary, BRAF-mutated colorectal adenocarcinomas (COADs) showed significantly higher SOX2 levels, compared to non-mutated tumors, despite the mutated cohort representing about 1/6th of the non-mutated group (Supplementary Figure S4I). These results suggest that BRAFV600 mutations may differentially affect tumor cells depending on their cell of origin, as observed in neural precursors that give origin to GBM or to SKCM, compared with endoderm-derived epithelial cells that give origin to COAD. Altogether, these results suggest that the BRafV600E-activated pathway promotes Sox2 T118 phosphorylation in mutated aNSPCs, thereby preventing Sox2 degradation and thus enhancing its stability. As previously identified in ESCs, our results also highlight the role of the Akt-activated pathway in mediating T118-Sox2 phosphorylation in Pten -deleted aNSPCs. Discussion MAPK is one of the most affected pathways in diffuse LGGs. A WHO category of diffuse LGG with MAPK pathway alterations has been recently described, in which BRAF alterations represent more than 80% of cases 45 , the majority being of paediatric age 10,46 . While susceptibility of the cell-of-origin and deregulation of signaling pathway/s have emerged as the most critical determinants in GBM, it has not been clearly demonstrated whether these factors can also be responsible for LGGs. We tested the hypothesis that MAPK overactivated signaling, operated by BRafV600E mutation, plays a transforming role in mouse aNSPCs, introducing the homonymous BRafV600E mutation, through CreERT2 recombineering specifically in Sox2-expressing aNSPCs. The stemness nature of the Sox2 promoter was validated by analysis of b-galactosidase (b-gal?) expression in the brain, DRGs, and skin of adult mice following tamoxifen induction. While no overt pathological phenotypes were elicited in BRafV600E -only or Pten -only deleted animals during the observation period, as also previously reported 40,47 , both mutations were required for tumor transformation of aNSPCs in the lateral ventricles and in the DRGs, leading animals to death very early from the recombination events. Brain tumors were identified as low-grade neuroepithelial tumors with oligodendroglioma-like features and peripheral nerve tumors as schwannomas (WHO, 2021), highlighting the role of concomitant MAPK and PI3K/AKT overactivated pathways in directing LGG and schwannoma tumorigenesis. BRafV600E has been described as an oncogenic driver when oncosuppressors such as Tp53 or Ink4/Arf were concomitantly deleted in fetal brains, promoting the development of malignant ganglioglioma or astrocytoma, respectively 48,49 . Here we demonstrate that, in the presence of concomitant Pten deletion, BRafV600E expression specifically in aNSPCs and SCPs promotes cell transformation leading to low-grade oligodendroglioma-like lesions and to schwannomas. Indeed, GNTs and PLNTY with oligodendrocyte-like differentiation are driven by aberrant members of the MAPK-activated pathway, including genetic anomalies of BRAF 14,32,50 . Consistent with these findings, oligodendroglial-like tumor cells from PLNTY lesions harboring the BRAFV600E mutation express both OLIG2 and SOX2. Notably, an association between BRAFV600E and SOX2 expression has been reported in two other human tumors, CRC and ameloblastoma, in which inhibition of MAPK signaling led to SOX2 downregulation 35,36 . Consistent with this, in our brain tumor model Olig2-positive tumor cells were Sox2- but not Gfap- positive, and Gfap-Sox2-co-staining was observed mainly in reactive astrocytes. While GFAP staining was diffusely positive throughout the human tumor areas, mouse lesions exhibited only scant Gfap expression within tumor cells, with strong labeling restricted to the reactive astroglial component. This discrepancy may reflect differences in tumor kinetics and in the timing of tissue collection and analysis between mouse models and human samples. Whereas mouse tumors were collected at an early stage following the initial transformation event induced by CreERT2 activation, human lesions were obtained after an unknown and likely prolonged period of tumor evolution. This temporal difference may have allowed the accumulation of downstream events associated with increased GFAP expression in the human samples. In agreement, another mouse model resembling PLNTY lesions reported scant GFAP expression in tumor cells 48 . Biallelic BRafV600E mutation significantly increased the Ki67 index compared to the monoallelic condition in the brain tumors, indicating that reinforced MAPK pathway activation enhances tumor proliferative activity, as it has been recently reported for biallelic RasG12V-induced hepatocarcinomas 51 . Despite a higher Ki67 index in homozygous compared with heterozygous BRafV600E tumors, we did not observe a significant difference in median survival time. Such discrepancy was likely due to the rapid worsening of the symptomatology in both genotypes induced by the DRG tumors that compressed the spinal cord, requiring euthanasia for ethical concerns at very close intervals, in a matter of days. It is well established that Pten -deleted aNSPCs gain enhanced self-renewal, proliferative ability, and decreased GF dependency 52 . As expected, in vitro-cultured Pten -deleted aNSPCs were more proliferative than Wt cells 40 , but, most interestingly, the BRafV600E mutation alone also increased proliferative ability in aNSPCs, strongly enhancing GF independence. While it has previously been shown that in vivo homozygous deletion of Pten in aNSPCs does not result in tumor formation 40 , BRafV600E overexpression in fetal neural cells guided by a strong viral promoter leads to circumscribed lesions composed of oligoid cells 48 . We did not detect any brain anomalies in BBpp mice at the time of sacrifice; however, we cannot exclude the possibility that limited, asymptomatic lesions may have developed at later time points. These results suggest that, in vivo, two BRafV600E mutant alleles are insufficient to drive tumor formation unless an additional genetic hit, such as Pten inactivation, occurs. In agreement with tumor histology, Mapk overactivation in vitro primed aNSPCs toward oligodendrocyte-like differentiation, as shown by MBP positivity at the expense of the astrocytic fate. Forced Erk1,2 activation during developmental myelination of the CNS and PNS promotes oligodendrocyte precursor cells expansion and myelin production from both oligodendrocyte and Schwann cells in animal models 53 . Since the BRafV600E mutation has been identified in 10% of sporadic schwannomas and in MPNSTs not associated with NF1/2 mutations 54,55 , our results further demonstrate that schwannomas or MPNSTs can originate from postnatal SCPs following BRafV600E mutation and concomitant Pten inactivation. In many brain tumors, Sox2 is expressed at high levels and is required for GBM stem cell propagation 56 . A recently described mouse model for skin tumors has clearly shown that BRafV600E expression in skin progenitor cells, along with Mapk activation, induces upregulation of Sox2 expression and tumor formation, making these cells permissive to rapid transformation 57 . Consistently, we found that Sox2 deletion in BbPP mice abrogated tumor formation, indicating that Sox2 acts as a stemness factor essential for aNSPC survival and transformation. We found that aNSPCs obtained from all the BRafV600E genotypes showed enhanced Sox2 expression at the protein but not mRNA levels, while bbPP aNSPCs mediated Sox2 expression mainly at the transcriptional level. Sox2 increase mediated by BRafV600E, instead, was not linked to increased protein synthesis, but rather to the decrease of Sox2 ubiquitylation, as revealed by proteasomal inhibition experiments. In agreement, BRafV600E expression reduced Sox2 ubiquitylation in HEK293T transfected cells, corroborating the results obtained in aNSPCs. A mechanism that involves an ubiquitylation switch mediated by Cul4a/Det1/Cop1 has been shown to regulate Sox2 levels during aNSPCs development, while a phosphorylation/methylation/ubiquitylation switch in residues T118/K119 of Sox2, mediated by Akt1/Set7/WWP2, has been shown to be involved in the control of ESC stemness 25 . Moreover, phosphorylated levels of Sox2-T118A were not increased following BRAFV600E transfection in HEK293T cells, supporting the role of this residue as a substrate for BRaf-induced phosphorylation. Our results indicate that PI3K/AKT signaling promotes both Sox2 transcription and protein stabilization, while Mapk activation reinforces Sox2 expression by enhancing protein stability via T118 phosphorylation, highlighting a cooperative interplay between the two pathways in aNSPCs. Even if none of the tested downstream members of the MAPK pathway, such as pMek1,2, pErk1,2, Rsk1, and Rsk2, were found to interact with Sox2 in immunoprecipitation (IP) experiments (not shown), we cannot exclude that these downstream members or other unknown BRaf-interacting kinase/s could mediate Sox2 T118 phosphorylation and stabilization. In summary, this study identifies the BRafV600E and MAPK-activated pathways as essential events together with Pten loss for aNSPC/SCP transformation that lead to LGG and schwannoma formation, as outlined in the model presented in Figure 8. Although the direct phosphorylation of Sox2 by BRafV600E was not demonstrated, our results uncover a novel downstream event initiated by BRafV600E that leads to increased Sox2 protein stabilization through T118 phosphorylation and impaired Sox2 ubiquitylation in mutated aNSPCs. Our findings suggest that this mechanism may represent a parallel oncogenic process downstream of MAPK/PI3K signaling, contributing to cellular transformation. Methods Reagents All reagents used throughout the experiments are listed in Supplementary Table 1. Mice and Tamoxifen treatment Mice carrying the Sox2-CreERT2 transgene 27 were crossed with transgenic mice carrying conditional BRafV600E , Pten flox/flox alleles, and the R2R6-Eyfp reporter or R26R-LacZ (Jackson Laboratories, Bar Harbour, ME, USA ) and with Sox2 flox/flox 27 . Animals were backcrossed into the C57BL/6 strain. Experimental animals carried the Sox2-CreERT2 in heterozygosity. Transgenic 1-month-old mice were injected i.p. with 225mg/kg tamoxifen dissolved in a 9:1 corn oil:ethanol mixture, once. For oral gavage, 11.5 days post coitum (dpc) pregnant Sox2-CreERT2; R26R-LacZ animals were treated with 100μL of 5mg/ml tamoxifen for two consecutive days and sacrificed at 13.5 dpc to obtain recombined embryos. Experiments were carried out on both male and female mice, in similar ratios. Animals were individually identified and monitored daily using a predefined clinical scoring system to assess disease progression. Scoring was performed by trained personnel blinded to group allocation. Phenotypic signs were recorded at each observation. Ruffled fur was assigned a score of 1, and kyphotic posture a score of 2. Body weight was measured regularly, with weight loss <5% assigned a score of 1 and weight loss <10% a score of 3. Clinical signs included respiratory distress (score 2) and paralysis (score 5). Behavioral alterations were also assessed, with reduced mobility and ataxia each assigned a score of 3. Animals exhibiting severe clinical signs or reaching a threshold score of 5 were humanely euthanized in accordance with the guidelines of the Stazione per la Tecnologia Animale – University of Rome Tor Vergata and with protocol no. 700-PR/2017 from the Italian Ministry of Health. PCR and genotyping DNA was extracted from tails or aNSPCs with Proteinase K. Cre alleles and Cre-mediated recombined alleles were verified by animal genotyping. PCRs were performed using the primers listed in Supplementary Table 2. Histology, tumor analysis, immunohistochemistry, and immunofluorescence Human tumor specimens were obtained for routine diagnosis. Patients’ data were collected anonymously, and written informed consent, as part of the diagnostic routine and treatment procedures, was obtained from patients or their guardians in accordance with the Declaration of Helsinki, and the study adhered to Good Clinical Practice guidelines. Samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and processed. PLNTY cases were independently and blindly validated by the two pathologists (M.M. and M.G.) based on histomorphology and immunohistochemistry (IHC). For molecular analyses, two serial sections (10 µm thick) were obtained from formalin-fixed paraffin-embedded (FFPE) tissue blocks. Tumor tissue represents greater than 50% of the sample. After deparaffinization, genomic DNA was extracted from microdissected FFPE tissue using the QIAamp DNA FFPE Tissue Kit on the QIAcube automated platform, according to the manufacturer’s instructions. DNA concentration and quality were assessed using the QIAxpert system. The mutational status of the BRAF gene was assessed using the BRAF Codon 600 Mutation Analysis Kit II, a diagnostic assay for qualitative detection of mutations at codon 600 in exon 15. The assay specifically identifies five clinically relevant mutations: V600E, V600K, V600D, V600R, and V600M. Interpretation of the mutational status was performed according to the manufacturer’s recommendations. BRAF mutation (c.1799T>A; p.V600E) was detected. Mouse tumor specimens were obtained following animal perfusion and post-fixed with 4% paraformaldehyde. Five mm sections from tumor samples were processed for H&E, IHC, and IF stainings. For IHC and IF analysis, antigen retrieval on tissue sections was performed using citrate buffer pH 6.0. For IF analysis of cultured aNSPCs, cells were fixed in 4% PFA and permeabilized with 0.1% Triton X-100. For β-galactosidase staining, embryos and tissues were processed as previously described 28 . All antibodies used, and relative dilutions are listed in Supplementary Table 3. Ki-67 assessment was obtained as the ratio of the number of tumor cells stained by Ki-67 antibodies to the total number of tumor cells counted in at least 5 non-overlapping microscopic fields at 400x magnification. The fluorescence intensity measurements were performed using LAS-X software (Leica Microsystems, Wetzlar, Germany). aNSPCs culture and assays aNSPCs were isolated as previously described by Donegà et al. 29 aNSPCs were obtained from pools of 3 adult (1-month-old) brains cultured in NeuroCult Basal Medium, Pen/Strep, 20 ng/ml EGF, 15 ng/ml bFGF, and 0.0002% Heparin. Experiments were run utilizing at least 3 batches of aNSPCs between passage 5 and 20. All aNSPCs cultures were tested negative for Mycoplasma spp. In vitro, tamoxifen was administered by adding 0.2 μM 4HT for 10 consecutive days to cell cultures. Sox2-CreERT2 activation was reported by Eyfp expression in cells. FACS sorting Single cell suspension of Cre-activated aNSPCs was loaded onto BD FACS Aria III cell sorter (BD Biosciences, Franklin Lakes, NJ, USA) and gated for Eyfp fluorescence to obtain Eyfp-positive cell population. To increase the sorting stringency, a purity criterion was set. Eyfp-negative cells were discarded. In vitro assays For the differentiation assays, 12000 cells/cm 2 were seeded on plates pre-coated with 1:200 Geltrex and cultivated in Neurobasal+ Medium with B27+, 0.5 mM GlutaMAX, 1% FBS, Pen/Strep for 10 days. For the proliferation assay, single cells were incubated with 100 μM BrdU for 30 minutes. Cells were fixed and immunostained with anti-BrdU antibody. For the clonogenic assay, 3000 cells/cm 2 were plated and allowed to form secondary spheres for 10 days. Western blot and immunoprecipitation Cell extraction was performed in RIPA buffer. Primary antibodies listed in Supplementary Table 3 were all used at a 1:1000 dilution. For IP, cells were homogenized in lysis buffer, and 1 mg of cell extract was incubated with 3 μg of mouse anti-Sox2 antibody, mouse anti-FLAG, or mouse immunoglobulin G (IgG) (negative control) and Dynabeads Protein G in 1 ml, overnight at 4°C under constant rotation. All samples were resuspended in 4x SDS sample buffer and processed for WB analysis. RNA extraction and qPCR RNA was collected from aNSPCs and extracted using the RNeasy Kit. cDNA was synthesized from 1 mg of total RNA with SuperScript III Reverse Transcriptase and random primers. Actb was used as a control for mRNA normalization. The relative amounts of each substrate were calculated by the 2 −ΔΔCT method, and expression levels were represented as fold increases relative to the control sample, which was set to 1. All primers were provided by Sigma-Aldrich and are listed in Supplementary Table 2. Plasmids pMT2-HAubq 30 was a gift from Dr. A. Peschiaroli (National Research Council, CNR, Rome), p3XFLAG-BRAFV600E was purchased from Addgene, and p3XFLAG-CMV-14 from Sigma-Aldrich. Full-length Sox2 was PCR-amplified, subcloned into the TOPO TA cloning kit, and then into pcDNA3 using XhoI/EcoRI restriction enzymes (see Supplementary Table 1-2). T118A mutation was inserted by PCR-directed mutagenesis from pcDNA3-Sox2 as starting template, subcloned in TOPO TA cloning kit, and then into pcDNA3 using XhoI/EcoRI restriction enzymes. Plasmids were Sanger-sequenced to verify the DNA sequence. Statistical and bioinformatic analysis All statistical analyses were conducted using GraphPad Prism version 10 software. Data are presented as means and standard deviations; tests were considered significant for relative values with p < 0.05. All experiments were performed on at least three biological replicates. For the bioinformatic analysis, SOX2 gene expression and BRAF somatic mutation data of Colorectal Adenocarcinoma (TGCA, PanCancer Atlas), Brain Lower Grade Glioma (TGCA, PanCancer Atlas), Glioblastoma Multiforme (TGCA, PanCancer Atlas), and Skin Cutaneous Melanoma (TGCA, PanCancer Atlas) were retrieved from the cBioPortal database (https://www.cbioportal.org/). Boxplot visualization and statistical analyses were conducted using R software (version 4.1.1). p-values were calculated by the Wilcoxon test with Benjamini-Hochberg (BH) adjustment using the R package ggpubr 0.4.0. Declarations DATA AVAILABILITY All the data reported in this paper will be shared by the lead contact upon request. ACKNOWLEDGEMENTS Authors thank Dr A. Peschiaroli (Istituto di Farmacologia Traslazionale - (IFT); CNR- Rome, Italy) for sharing pMT2-HAubq plasmid. This paper was supported by grants from Italian Ministry of University and Research Prin2022CE79J_004 to SD; Prin 2022AWB8T4 and P2022SE38P_004, Mnesys NRRP PE0000006 to EAJ; Prin 2022X4FWZ5 to MS. Author Contribution: Conceptualization: E.G. and S.D. wrote the main manuscript text Methodology: E.G. (Figs 1-7), A.C. (Figs 1,2,3,4,6,7) V.C. (Phenotype analysis, Fig.1), L.M. and M.P. (Fig.5), S.D.C (FACS analysis and sorting) M.M. and M.G. (Fig.4) Resources: S.K.N., S.D. 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E.A.J. is or has been a consultant and/or paid speaker for Bayer, FQM, Ibsa, Kanna, Menarini, Merck, Otsuka, Pfizer, Recordati, Shionogi and Viatris. The other authors declare no competing interests. Supplementary Files Supplementarymaterial.pdf Cite Share Download PDF Status: Published Journal Publication published 20 Apr, 2026 Read the published version in npj Precision Oncology → Version 1 posted Editorial decision: Accepted 08 Apr, 2026 Reviews received at journal 05 Apr, 2026 Reviewers agreed at journal 05 Apr, 2026 Reviews received at journal 03 Apr, 2026 Reviewers agreed at journal 03 Apr, 2026 Reviewers invited by journal 03 Apr, 2026 Submission checks completed at journal 01 Apr, 2026 First submitted to journal 01 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7515762","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":604369846,"identity":"431c121b-feb1-4173-8d75-c629049bb9b2","order_by":0,"name":"Eugenia Guida","email":"","orcid":"","institution":"University of Rome Tor Vergata","correspondingAuthor":false,"prefix":"","firstName":"Eugenia","middleName":"","lastName":"Guida","suffix":""},{"id":604369847,"identity":"1fc4eddb-9f15-413d-b300-4fc7828ca2e4","order_by":1,"name":"Ambra Colopi","email":"","orcid":"","institution":"University of Rome Tor Vergata","correspondingAuthor":false,"prefix":"","firstName":"Ambra","middleName":"","lastName":"Colopi","suffix":""},{"id":604369848,"identity":"0018330f-5c31-4d69-83fe-572d30e93ad4","order_by":2,"name":"Valeriana Cesarini","email":"","orcid":"","institution":"Humanitas University","correspondingAuthor":false,"prefix":"","firstName":"Valeriana","middleName":"","lastName":"Cesarini","suffix":""},{"id":604369849,"identity":"b77d6bb0-82d3-49dd-8f14-67c5f0c942e7","order_by":3,"name":"Marco Pieraccioli","email":"","orcid":"","institution":"Catholic University of the Sacred Heart","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Pieraccioli","suffix":""},{"id":604369850,"identity":"e180ba87-8cb5-4999-9bd9-a3e61a567d7f","order_by":4,"name":"Luca Mignini","email":"","orcid":"","institution":"Bambino Gesù Children's 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Heart","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Gessi","suffix":""},{"id":604369854,"identity":"8c074f17-b0d5-4bbc-bd84-bf9af8c30c5d","order_by":8,"name":"Maurizio Salvati","email":"","orcid":"","institution":"University of Rome Tor Vergata","correspondingAuthor":false,"prefix":"","firstName":"Maurizio","middleName":"","lastName":"Salvati","suffix":""},{"id":604369855,"identity":"ba869336-2af0-4522-a219-50184ec08a65","order_by":9,"name":"Silvia Kirsten Nicolis","email":"","orcid":"","institution":"University of Milan Bicocca","correspondingAuthor":false,"prefix":"","firstName":"Silvia","middleName":"Kirsten","lastName":"Nicolis","suffix":""},{"id":604369856,"identity":"617e1063-0b3c-4b7e-b088-57ad4c2351b7","order_by":10,"name":"Emmanuele Angelo Jannini","email":"","orcid":"","institution":"University of Rome Tor Vergata","correspondingAuthor":false,"prefix":"","firstName":"Emmanuele","middleName":"Angelo","lastName":"Jannini","suffix":""},{"id":604369857,"identity":"8696b410-c9ca-40f0-8a9e-d59f710c3d22","order_by":11,"name":"Susanna Dolci","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYDCCA8wNDAkVDAxsYF4FAw8RWhiBWs7AtJxh4CGsB6SFsQ3KATEIauE73tj44eG8w3l80oePbvjBxiBjT0iL5JmDzRKJ2w4Xs/Glpd3s4SHCYQY3EtsYgFoS23h4zG7wSBCtZQ5Ey80/BkRraYBouc2TQIQWsF8SjqUDtbCl3ZY5IMHDc4CAFr7jzQc//qixTpzfw3zs5tt/NvbsDYSsQQMSJKofBaNgFIyCUYAVAACIfD565PhafgAAAABJRU5ErkJggg==","orcid":"","institution":"University of Rome Tor Vergata","correspondingAuthor":true,"prefix":"","firstName":"Susanna","middleName":"","lastName":"Dolci","suffix":""}],"badges":[],"createdAt":"2025-09-02 09:08:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7515762/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7515762/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41698-026-01439-5","type":"published","date":"2026-04-20T15:59:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":104780478,"identity":"e2b3803b-00ee-422b-9ec7-4f44e4954f68","added_by":"auto","created_at":"2026-03-17 07:53:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12638324,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSox2-CreERT2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e transgene is active in adult and embryonic aNSPCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of \u003cem\u003eSox2-CreERT2\u003c/em\u003e \u003cem\u003eR26R-LacZ\u003c/em\u003e transgene construct. \u003cstrong\u003e(B-C)\u003c/strong\u003e β-gal staining on adult brain section 30 days post tamoxifen administration\u003cstrong\u003e. \u003c/strong\u003eβ-gal activity was present in the cerebral cortex (Ctx), caudoputamen (CP), sub-ventricular zone (SVZ) and sub-granular zone (SGZ). Scale bar: 2mm (B) and 50 µm (C). \u003cstrong\u003e(D)\u003c/strong\u003e Whole mount β-gal staining of spinal cord showing positive cells within DRGs in adult mice 30 days post tamoxifen administration. Scale bars: 1mm.\u003cstrong\u003e \u0026nbsp;(E)\u003c/strong\u003e β-gal staining of adult mouse skin sections, showing positivity in hair bulbs. Scale bars: 25 µm.\u003cstrong\u003e (F)\u003c/strong\u003e Representative images of whole mount β-gal staining on \u003cem\u003eSox2-CreERT2\u003c/em\u003e \u003cem\u003eR26R-LacZ\u003c/em\u003e 13.5 embryo. β-gal-positive cells were found in the telencephalon and in the developing DRG. Scale bars: 100 µm.\u003c/p\u003e","description":"","filename":"Figure1rev.png","url":"https://assets-eu.researchsquare.com/files/rs-7515762/v1/7b93604c14d07dc431358c69.png"},{"id":104780299,"identity":"64d8ec5a-9581-4fdd-aafb-b644749cdb57","added_by":"auto","created_at":"2026-03-17 07:52:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":14299316,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBRafV600E\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emutation and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePten\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deletion in aNSPCs induce brain and nerve tumors in adult mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of \u003cem\u003eSox2-CreERT2\u003c/em\u003e, \u003cem\u003eBRafV600E, Pten\u003c/em\u003e\u003csup\u003e\u003cem\u003efloxf/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e \u003cem\u003eR26R-Eyfp\u003c/em\u003e alleles (left panel) and a summarizing table of the different genotypes, cohort size, median survival, and occurrence of tumors right panel. \u003csup\u003e1 \u003c/sup\u003eN=3 mice also showed skin masses; \u003csup\u003e2 \u003c/sup\u003eN=4 mice also showed skin masses. \u003cstrong\u003e(B)\u003c/strong\u003e Kaplan-Meier survival curve for BbPP (N=13) and BBPP (N=12) compared to Wt mice after tamoxifen injection (N=12). 0 corresponds to tamoxifen administration in 1-month-old mice. \u003cstrong\u003e(C)\u003c/strong\u003e Stereomicroscopic inspection of BbPP and BBPP brains. Scale bar: 500 µm.\u003cstrong\u003e (D)\u003c/strong\u003e H\u0026amp;E-stained sections of BbPP and BBPP affected brains, showing brain mass and enlarged lateral ventricles. Scale bar: 500 µm. \u003cstrong\u003e(E)\u003c/strong\u003e Representative images of spinal cord and DRG in Wt, BbPP and BBPP mice. Arrowheads point to DRG masses that were larger in BBPP compared to BbPP mice. Scale bar: 500 µm.\u003cstrong\u003e (F)\u003c/strong\u003e Representative images of cutaneous tumors in BbPP and BBPP mice\u003cstrong\u003e \u003c/strong\u003eafter tamoxifen induction.\u003c/p\u003e","description":"","filename":"Figure2rev.png","url":"https://assets-eu.researchsquare.com/files/rs-7515762/v1/48c857a70b5b8056256d9fab.png"},{"id":104469232,"identity":"a457a7a8-6096-442f-92b4-0c0b1a22c6d4","added_by":"auto","created_at":"2026-03-12 07:05:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31608293,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBRafV600E and\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Pten-\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edeleted\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eNSPCs induce the formation of LGG tumors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e H\u0026amp;E and IHC analyses on BbPP (M#10 adult male sample and F#2 adult female sample) or BBPP (M#4 adult male sample and F#4 adult female sample) brain tumor sections probed for Sox2, Olig2, Ki67, Gfap, and NeuN. Scale bar: 50 µm.\u003cstrong\u003e (B)\u003c/strong\u003eQuantification of the percentage of Ki67-positive cells in BbPP or BBPP brain tumors compared to Wt brain. Mean ± s.d. ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 (One-way ANOVA with Dunnett’s multiple comparisons test). Ki67 quantifications were obtained from 5 consecutive tissue slices at 40x magnification. The percentage of cells was evaluated as the ratio of positively stained cells to total nuclei.\u003cstrong\u003e (C-E)\u003c/strong\u003eIF co-staining analysis on BbPP or BBPP brain tumors for Olig2, Sox2, Gfap, BRafV600E, Pten, Eyfp. Nuclei were counterstained with DAPI. Scale bar: 50 µm. N=3.\u003c/p\u003e","description":"","filename":"Figure3rev.png","url":"https://assets-eu.researchsquare.com/files/rs-7515762/v1/ed8df19c4b3d57e6a9c9ba1b.png"},{"id":104469233,"identity":"633fd120-59a4-4dcc-a07a-f96d40bc0b9e","added_by":"auto","created_at":"2026-03-12 07:05:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":40986264,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePLNTY lesions express SOX2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E and IHC analyses on two PLNTY lesions isolated from a 16-year-old male patient and a 29-year-old female patient, respectively. IHC staining for BRAFV600E, IDH1R132H expression, OLIG2, GFAP, SOX2, Ki-67, pERK1,2, pAKT and PTEN. Normal brain tissue (NBT); tumor (T). Scale bar: 50 µm.\u003c/p\u003e","description":"","filename":"Figure4rev.png","url":"https://assets-eu.researchsquare.com/files/rs-7515762/v1/08bf53128222a6d31e9239d5.png"},{"id":104469234,"identity":"ae280c47-6e14-4ff6-bb3c-9a921ea8e4a6","added_by":"auto","created_at":"2026-03-12 07:05:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":31481942,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBRafV600E/\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePten\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003edel\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e peripheral SCPs transform into paraspinal schwannomas\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e H\u0026amp;E of brainstem associated mass of CN V (left upper panel) from BbPP mice, scale bar: 2mm. H\u0026amp;E, 40x magnification (upper right panel) of CN V mass and IHC (remaining panels) for Ki67, S100b, Sox2, and Sox10. Scale bar: 50mm. Schwannoma (SCW); Cortex (Ctx); Antoni A areas (A). \u003cstrong\u003e(B)\u003c/strong\u003e H\u0026amp;E-stained sections of spinal cords within the vertebral canal vertebral bone (VB); spinal cord (SC); schwannoma (SCW) from BbPP (left panel, first row) or BBPP (left panel, second row) mice. Scale bars: 100 mm. Right panels represent 40x magnifications. Arrowhead indicated neuronal cell bodies entrapped by spindled neoplastic cells. Scale bars: 50mm. IHC (remaining panels) for Ki67, S100b, Sox2, and Sox10. Scale bar: 50mm. \u003cstrong\u003e(C) \u003c/strong\u003eQuantification of the percentage of Ki67-positive cells in schwannomas from BbPP or BBPP compared to Wt. Mean ± s.d ****p \u0026lt; 0.0001, ns=non-significant (Student’s \u003cem\u003et\u003c/em\u003e-test, two-tailed). Ki67 cell quantifications were obtained from 5 consecutive tissue slices at 40x magnification. The percentage of cells was evaluated as the ratio of positively stained cells on total nuclei. \u003cstrong\u003e(D) \u003c/strong\u003eH\u0026amp;E of cNF (left panel, first row) and IHC for S100b and Sox10 (left panel, second and third row respectively). Scale bar:\u003cstrong\u003e \u003c/strong\u003e50mm\u003cstrong\u003e (E) \u003c/strong\u003eH\u0026amp;E of MPNST showing tightly packed spindled cells (left panel, upper row scale bar: 50mm) and nuclear atypia (, right panel, upper row scale bar: 10mm). Middle and lower panels show IHC for Ki67, S100b, Sox2, and Sox10. Scale bar:50mm. \u003cstrong\u003e(F)\u003c/strong\u003e Quantification of the percentage of Ki67-positive cells in MPNSTs from BbPP or BBPP compared to Wt. Mean ± s.d ***p \u0026lt; 0.0001, ****p \u0026lt; 0.0001 (Student’s \u003cem\u003et\u003c/em\u003e-test, two-tailed). Ki67 cell quantifications were obtained from 5 consecutive tissue slices at 40x magnification. The percentage of cells was evaluated as the ratio of positively stained cells to total nuclei.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure5rev.png","url":"https://assets-eu.researchsquare.com/files/rs-7515762/v1/129ed1d13c92b1329052ea0e.png"},{"id":104469230,"identity":"9fe747f5-f07e-4c28-92ad-9e6e10f89494","added_by":"auto","created_at":"2026-03-12 07:05:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8233996,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBRafV600E maintains aNSPCs proliferation under differentiating conditions and favors their differentiation toward the oligodendrocyte fate in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Representative IF images of BrdU incorporation assay performed on Wt and mutated aNSPCs and relative quantification of the percentage of BrdU-positive cells. BbPP, BBPP, BBpp showed statistically significant increase of BrdU-incorporating cells compared to Wt. *p \u0026lt; 0.05 (Student’s \u003cem\u003et\u003c/em\u003e-test, two-tailed). Scale bar: 50 mm. N=3\u003cstrong\u003e. \u003c/strong\u003eQuantification of BrdU-positive cells was obtained from 5 consecutive tissue slices at 40x magnification. The percentage of cells was evaluated as the ratio of positively stained cells to total nuclei. \u003cstrong\u003e(B) \u003c/strong\u003eRepresentative merged images (light/epifluorescence) of Wt and mutated aNSPCs cultured in the absence of growth factors (GF) and relative quantification of the number of living BbPP, BBPP, BBpp cells compared to Wt. *p \u0026lt; 0.05, **p \u0026lt; 0.01 (Student’s \u003cem\u003et\u003c/em\u003e-test, two-tailed). Scale bar: 50 mm. N=3.\u003cstrong\u003e (C)\u003c/strong\u003e IF images of aNSPCs differentiation assay and quantification of percentage of differentiated cells in the mutated aNSPCs compared to Wt (right panel). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ns= non-significant (Student’s \u003cem\u003et\u003c/em\u003e-test, two-tailed). Scale bar: 50 mm. N=3\u003cstrong\u003e. \u003c/strong\u003eGfap, Tubb3, MBP, and BrdU cell quantifications were obtained from 5 consecutive tissue slices at 40x magnification. The percentage of cells was evaluated as the ratio of positively stained cells to total nuclei.\u003c/p\u003e","description":"","filename":"Figure6rev.png","url":"https://assets-eu.researchsquare.com/files/rs-7515762/v1/e2b69504adbd0c40e13a6abf.png"},{"id":104469228,"identity":"d3ec0523-39fc-48f3-9784-ae2542727f4c","added_by":"auto","created_at":"2026-03-12 07:05:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5413089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBRafV600E and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePten-\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edeletion\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003epromote Sox2 protein stabilization in aNSPCs by preventing its proteasomal degradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eRepresentative images of Sox2 expression in Wt, BbPP and BBPP cells and relative quantification Sox2-positive cells. BbPP and BBPP aNSPCs showed statistically significant increase of Sox2-positive cells compared to Wt. ***p \u0026lt; 0.001 ****p \u0026lt; 0.0001 (Student’s \u003cem\u003et\u003c/em\u003e-test, two-tailed). Scale bar: 50 µm.\u003cstrong\u003e \u003c/strong\u003eN=3\u003cstrong\u003e. \u003c/strong\u003eSox2 cell quantifications were obtained from 5 consecutive tissue slices at 40x magnification. The percentage of cells was evaluated as the ratio of positively stained cells to total nuclei.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eWB analysis on Wt and BbPP aNSPCs extracts probed for Sox2, pAKT, Akt, pErk1,2 or Erk2. β-Tubulin is the reference gene. (n=3)\u003cstrong\u003e (C)\u003c/strong\u003e WB analysis on cell extracts from BbPP aNSPCs treated for 6h with PD98059 (PD, 1 µM), \u0026nbsp;or PLX-4032 (PLX, 1 µM), and probed for pErk1,2, Erk2, Sox2 or β-Tubulin, versus control (Ctr). N=3. \u003cstrong\u003e(D) \u003c/strong\u003eWB analysis for Sox2 expression on Wt, BbPP, BBPP, BBpp and bbPP aNSPCs treated for 5h with MG132 (MG, 20µM), or Cycloheximide (CHX, 10 µg/ml), versus control (Ctr). N=3. \u003cstrong\u003e(E)\u003c/strong\u003e WB analysis on anti-Sox2 immunoprecipitated cell extracts obtained from HEK293T cells transfected with HA-Ubiquitin, Sox2, and FLAG-BRafV600E or HA-Ubiquitin, Sox2, and FLAG expressing plasmids, in the presence or absence of MG. Immunoprecipitated protein extracts were probed for Sox2, HA, pT118-Sox2, FLAG, or β-Tubulin. Input samples represent 1/80 of immunoprecipitated protein extracts. N=3. \u003cstrong\u003e(F) \u003c/strong\u003eWB analysis on cell extracts from HEK293T cells transfected with FLAG-BRafV600E Wt-Sox2 or FLAG-BRafV600E T118A-Sox2 expressing plasmids, in the presence or absence of MG. Protein extracts were probed for FLAG, Sox2, pT118-Sox2, or β-Tubulin. N=3. \u003cstrong\u003e(G)\u003c/strong\u003e WB analysis on anti-FLAG immunoprecipitated cell extracts obtained from HEK293T cells transfected with FLAG-BRafV600E in the presence of Wt- or T118A-Sox2 expressing plasmids.\u003cstrong\u003e \u003c/strong\u003eImmunoprecipitated protein extracts were probed for FLAG, Sox2, or β-Tubulin. N=3.\u003cstrong\u003e (H) \u003c/strong\u003eWB analysis on cell extracts from HEK293T cells transfected with Wt- or T118A-Sox2 expressing plasmids. 72h after transfection cells were treated with CHX for 1, 3, or 6h and cells extract probed for FLAG, Sox2 or β-Tubulin.\u003cstrong\u003e (I) \u003c/strong\u003eWB analysis on cell extracts from Wt, BbPP, BBPP, BBpp or bbPP aNSPCs probed for pAkt, pErk1,2, Sox2, pT118-Sox2, or β-Tubulin. N=3.\u003c/p\u003e","description":"","filename":"Figure7rev.png","url":"https://assets-eu.researchsquare.com/files/rs-7515762/v1/fa44a90896479fddb7ecf93d.png"},{"id":104469226,"identity":"ffe5199d-b429-476e-8f1c-e24dc9cf15b0","added_by":"auto","created_at":"2026-03-12 07:05:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1657494,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBRafV600E and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePten\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deletion promote Sox2 stabilization in aNSPCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematic representation of the proposed signaling cascade in which MAPK/PI3K pathway activation, driven by BRafV600E overactivation and \u003cem\u003ePten\u003c/em\u003e deletion, induces Sox2 phosphorylation at the T118 residue, leading to protein stabilization in aNSPCs. Dashed arrows indicate potential kinases responsible for Sox2 phosphorylation at T118. Question marks indicate potential intermediate mediators.\u003c/p\u003e","description":"","filename":"Figure8REV.png","url":"https://assets-eu.researchsquare.com/files/rs-7515762/v1/13d510accfae04ba712d5553.png"},{"id":107929514,"identity":"87cf58e0-bee5-4920-ada0-58088135fa76","added_by":"auto","created_at":"2026-04-27 16:17:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":229034889,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7515762/v1/a3dd2e60-cd51-4d0d-9a37-9008cd988d34.pdf"},{"id":104469235,"identity":"fdffa5a2-80ee-4ec9-966b-588211a9bced","added_by":"auto","created_at":"2026-03-12 07:05:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":145352466,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7515762/v1/e77819d5597527b2709a7ee9.pdf"}],"financialInterests":"Competing interest reported. E.A.J. is or has been a consultant and/or paid speaker for Bayer, FQM, Ibsa, Kanna, Menarini, Merck, Otsuka, Pfizer, Recordati, Shionogi and Viatris. The other authors declare no competing interests.","formattedTitle":"\u003cp\u003eSox2 protein stability is enhanced by BRafV600E and Pten deletion in adult neural stem/progenitor cells\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe RAS/BRAF/MEK/ERK pathway is highly mutated in cancer\u003csup\u003e1\u003c/sup\u003e, with mutations of the \u003cem\u003eBRAF\u003c/em\u003e gene occurring in about 7% of cases\u003csup\u003e2\u003c/sup\u003e. The most frequently observed BRAF mutation is the substitution of valine 600 with glutamic acid (V600E), which induces persistent, RAS-independent activation of BRAF, inhibiting its inactivation and leading to a continuous stimulation of MEK and ERK\u003csup\u003e3\u003c/sup\u003e. Such mutation is recurrent in melanomas, hairy cell leukemias, thyroid and colon carcinomas, and acts as a tumor driver in animal models for these tumors\u003csup\u003e4-7\u003c/sup\u003e. \u0026nbsp;In the nervous system,\u0026nbsp;MAPK-altered tumors are frequently found in pediatric low-grade glioneural tumors (GNTs), with \u003cem\u003eFGFR1\u003c/em\u003e or\u0026nbsp;\u003cem\u003eBRAF\u003c/em\u003e being the predominant mutated genes\u003csup\u003e8\u003c/sup\u003e. BRAF alterations\u0026nbsp;account for a restricted number of tumors and can occur as \u003cem\u003eBRAF\u003c/em\u003e fusions (\u003cem\u003eKIAA1549-BRAF\u003c/em\u003e) or substitutions (BRAFV600E).\u0026nbsp;Specifically, the \u003cem\u003eBRAFV600E\u003c/em\u003e allele has been identified in many central nervous system (CNS) tumors ranging from GNTs (such as gangliogliomas and dysembryoplastic neuroepithelial tumors)\u003csup\u003e7\u003c/sup\u003e, to astrocytic tumors (such as pleomorphic xanthoastrocytomas\u003csup\u003e9,10\u003c/sup\u003e and pilocytic astrocytomas\u003csup\u003e11\u003c/sup\u003e),\u0026nbsp;papillary craniopharyngioma\u003csup\u003e12\u003c/sup\u003e,\u0026nbsp;oligodendroglial tumors (such as oligodendroglioma-like tumors of pediatric origin\u003csup\u003e13\u003c/sup\u003e,\u0026nbsp;polymorphous low-grade neuroepithelial tumor of the young, PLNTY\u003csup\u003e14\u003c/sup\u003e), and\u0026nbsp;pediatric\u0026nbsp;or\u0026nbsp;adult glioblastoma multiforme (GBM)\u003csup\u003e15\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExploiting genetic mouse models (GEMMs) carrying cell-specific promoter/enhancer constructs to direct oncogene activation or tumor suppressor deletion in vivo, it has been shown that embryonic and adult neural stem/progenitor cells (NSPCs), heterogeneous and specialized stem cells that generate specific types of neurons and glia\u003csup\u003e16\u003c/sup\u003e, are the cells of origin of many high-grade gliomas (HGGs)\u003csup\u003e17\u003c/sup\u003e.\u0026nbsp;On the contrary, peripheral nerve sheath (PNS) tumors have been shown to arise from Schwann cell precursors (SCPs; also defined as skin cell precursors, SKPs), a cell population considered as multipotent as NSPCs.\u003csup\u003e18\u003c/sup\u003e These cells are found along outgrowing peripheral nerves\u003csup\u003e19\u003c/sup\u003e and hair bulbs\u003csup\u003e18\u003c/sup\u003e, or derive from PLP1-expressing Schwann cells\u003csup\u003e20\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMouse models of low-grade gliomas (LGGs), in particular the GNT subtypes, have primarily been obtained by in utero brain injections of oncogenes, resulting in massive transformation of fetal neural precursors and leaving open the question of whether adult NSPCs (aNSPCs) are competent to give origin to GNTs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe previously showed that Sex determining region Y-box 2 (Sox2) expression is transcriptionally regulated by BRafV600E in a melanoma mouse model\u003csup\u003e21\u003c/sup\u003e. Sox2 is a transcriptional regulator essential during early embryonic development for orchestrating stem cell pluripotency and neural commitment\u003csup\u003e22\u003c/sup\u003e. Aside from its physiological role in neural development, Sox2 plays an oncogenic role in brain tumors\u003csup\u003e23\u003c/sup\u003e, and its expression can be regulated at multiple levels through transcriptional, translational, and post-translational mechanisms\u003csup\u003e24,25\u003c/sup\u003e. Recent findings have shown that Akt-mediated phosphorylation on threonine 118 (T118) prevents the neighbor lysine (K119) ubiquitylation\u0026nbsp;in murine embryonic stem cells (ESCs)\u003csup\u003e25\u003c/sup\u003e and that Sox2 protein stability is enhanced by regulating its ubiquitylation in NSPCs\u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn the present work, we investigated the mechanism/s involved in aNSPCs transformation operated by BRafV600E alone or in combination with \u003cem\u003ePten\u003c/em\u003e deletion, both in vivo and in vitro. We found that symptomatic GNTs resembling PLNTY lesions and schwannomas develop and rapidly progress after oncogenic induction in \u003cem\u003eBRafV600E/Pte\u003c/em\u003en\u003cem\u003e\u003csup\u003edel\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eyoung adult mice in vivo, while aNSPCs shift to high proliferative activity following transduction, in vitro. We demonstrate that the \u003cem\u003eBRafV600E\u003c/em\u003e mutation, alone or in combination with \u003cem\u003ePten\u003c/em\u003e deletion, enhances Sox2 protein stability by decreasing Sox2 ubiquitylation, highlighting the essential role of concomitantly overactivated Mapk and Akt pathways in sustaining the oncogenic role of Sox2 in GNT formation.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSox2-CreERT2\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;promoter is active in telencephalic aNSPCs and in SCPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo activate the \u003cem\u003eBR\u003c/em\u003e\u003cem\u003eaf\u003c/em\u003e\u003cem\u003eV600E\u003c/em\u003e mutation and \u003cem\u003ePten\u003c/em\u003e deletion specifically in aNSPCs, we employed the \u003cem\u003eSox2-CreERT2\u003c/em\u003e deleter, proved to be active in aNSPCs from the sub-ventricular zone (SVZ) of the lateral ventricles and in the sub-granular zone (SGZ) of the hippocampus \u003csup\u003e27\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo limit the occurrence of the oncogenic mutations only to a few numbers of aNSPCs, we induced Cre-mediated recombination by a single injection of tamoxifen in one-month-old \u003cem\u003eSox2-CreERT2\u003c/em\u003e; \u003cem\u003eR26R-LacZ\u003c/em\u003e reporters (Figure 1A). Cre activation was monitored by tracing\u0026nbsp;b-\u003cem\u003egalactosidase (\u003c/em\u003eb-\u003cem\u003egal\u003c/em\u003e) activity in the brain, spinal cord, and dermis at 10 and 30 days after tamoxifen administration. On day 10, we found\u0026nbsp;b-gal-positive cells in the SVZ and SGZ neurogenic niches, as expected, in the corpus callosum and the cerebral cortex (Supplementary Figure S1A), and their numbers increased one-month post-tamoxifen injection, extending also into the caudoputamen (Figure 1B-C). We also found scattered\u0026nbsp;b-gal-positive cells in adult dorsal root ganglia (DRG) and in hair bulbs from skin sections, indicating that the \u003cem\u003eSox2\u003c/em\u003e telencephalic promoter was also active in SCP/SKPs (Figure 1D-E). \u003cem\u003eSox2-CreERT2\u003c/em\u003e activity at embryonic stage E13.5 was identified in the anterior telencephalic region (Figure 1F), as previously observed for \u003cem\u003eSox2-CreERT2; R26R-LacZ\u003c/em\u003e embryos\u003csup\u003e27\u003c/sup\u003e and within the developing DRG\u0026nbsp;\u003csup\u003e31\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBRafV600E\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and \u003cem\u003ePten\u0026nbsp;\u003c/em\u003edeletion in Sox2-expressing aNSPCs and SCPs induce the formation of brain and nerve tumors in adult mice\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo understand if Mapk overactivation might impact cell transformation and/or aberrant cell differentiation, we activated the \u003cem\u003eBRafV600E\u003c/em\u003e mutation (thereafter defined B) along with \u003cem\u003ePten\u003c/em\u003e deletion (thereafter defined P) in aNSPCs by means of the \u003cem\u003eSox2-CreERT2\u0026nbsp;\u003c/em\u003edeleter\u003cem\u003e.\u0026nbsp;\u003c/em\u003eTo trace\u003cem\u003e\u0026nbsp;Sox2-CreERT2\u0026nbsp;\u003c/em\u003eedited cells\u003cem\u003e, Sox2-CreERT2; R26R-Eyfp\u0026nbsp;\u003c/em\u003emice were crossed to \u003cem\u003eBRafV600E and\u003c/em\u003e \u003cem\u003ePten\u003csup\u003eflox/flox\u0026nbsp;\u003c/sup\u003e\u003c/em\u003emice\u003cem\u003e\u0026nbsp;\u003c/em\u003eto obtain the genotypes shown in Figure 2A\u003cem\u003e.\u0026nbsp;\u003c/em\u003eOne-month-old mice were primed with a single intraperitoneal (i.p.) injection of tamoxifen and constantly observed. Tremor was usually the first symptom appearing only in BbPP and BBPP mutants at about 30 days after tamoxifen administration and was then accompanied by weight loss, apathy, sporadic\u0026nbsp;epilepsy, kyphosis, and hindlimb retraction, indicating a neurological condition (Supplementary Video). Control (Wt) mice did not show any phenotype.\u003c/p\u003e\n\u003cp\u003eBbPP mice required euthanasia to prevent respiratory paralysis, with a median survival time of 60 days post-injection (dpi). Mice carrying the BBPP alleles exhibited a similar median survival time (60 dpi) compared to BbPP mice (Figure 2B). In contrast, Wt mice, as well as BBpp, bbPP, and most BbPp and BBPp mice, did not develop symptoms when followed up to 150 dpi (Figure 2B and Supplementary Figure S1B). Three out of eight BbPp and three out of eight BBPp mice developed a neurological phenotype at around 70 and 60 dpi, respectively,\u0026nbsp;suggesting that loss of \u003cem\u003ePten\u003c/em\u003e heterozygosity occurred (Supplementary Figure S1B).\u003c/p\u003e\n\u003cp\u003eAt autopsy, freshly isolated brains from euthanized BbPP and BBPP mice did not show gross cortical anomalies. We then evaluated the brain/body weight ratio in 6 out of 13 BbPP and 6 out of 12 BBPP mice, observing a two-fold increase in brain weight in the mutated genotypes compared to Wt (Figure 2C and Supplementary Figure S1C).\u003c/p\u003e\n\u003cp\u003eIn 100% of the affected mice from both genotypes (13/13 and 12/12, respectively),\u0026nbsp;hematoxylin and eosin (H\u0026amp;E) brain sections showed tumor masses in the telencephalic vesicles that, in some cases, induced enlargement of lateral ventricles (Figure 2D), suggesting obstructive hydrocephalus.\u003c/p\u003e\n\u003cp\u003eAll the tamoxifen-injected BbPP and BBPP mice showed a mass on the ventral surface of the brainstem associated with the trigeminal nerve (cranial nerve, CN V) (Supplementary Figure S1D). All the inspected spinal cords showed enlarged, often bilateral, DRG masses at many vertebral levels, also invading the intervertebral foramina to extend in the peri-vertebral space (Figure 2E and Supplementary Figure S1E). Such masses showed larger dimensions in BBPP compared to BbPP genotypes and signs of compression on the spinal cord, which led to animal death (Figure 2E).\u003c/p\u003e\n\u003cp\u003eWe found that the entire cohort of BbPP and BBPP mice developed DRG tumors (100%) that were accompanied by brain tumors (100%), and occasionally by skin masses (23% in BbPP and 33% in BBPP) (Figure 2A and F).\u003c/p\u003e\n\u003cp\u003eBBpp or bbPP brains and DRGs did not exhibit areas of hyperproliferation when animals were sacrificed approximately 150 days after tamoxifen injection (not shown).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further prove that aNSPCs were the tumor cells of origin, we specifically deleted \u003cem\u003eSox2\u003c/em\u003e in these cells by crossing BbPP mice with conditional \u003cem\u003eSox2\u003csup\u003eflox/flox\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003emice and generating the Sox2\u003cem\u003e\u003csup\u003eflox/flox\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eBbPP mice (Supplementary Figure S1F). Cre recombination was induced by tamoxifen in five BbPP and five Sox2\u003cem\u003e\u003csup\u003eflox/flox\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eBbPP mice. None of the injected \u003cem\u003eSox2\u003c/em\u003e-deleted (\u003cem\u003eSox2\u003csup\u003ede\u003c/sup\u003e\u003c/em\u003e\u003csup\u003el\u003c/sup\u003e) conditional mice showed symptoms 30 days after tamoxifen administration, while \u003cem\u003eSox2\u003csup\u003ewt\u003c/sup\u003e\u003c/em\u003e BbPP mice were symptomatic and were sacrificed between 60 and 75 days after tamoxifen induction (Supplementary Figure S1G). \u003cem\u003eSox2\u003csup\u003edel\u003c/sup\u003e\u0026nbsp;\u003c/em\u003emice were followed for an additional 4 months and then sacrificed. Isolated brains and spinal cords revealed that \u003cem\u003eSox2\u003csup\u003edel\u003c/sup\u003e\u003c/em\u003eBbPP mice did not develop brain tumors, nor schwannomas (data not shown). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCollectively, these findings demonstrate that BRafV600E expression combined with Pten loss can give rise to brain tumor formation in Sox2-expressing aNSPCs, and DRG and/or skin tumors in Sox2-expressing SCPs.\u0026nbsp;Moreover, our experiments confirm that Sox2 expression is a prerequisite for aNSPCs/SCPs transformation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBRafV600E\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e/\u003cem\u003ePten\u003c/em\u003e-deleted aNSPCs are prone to transform into neuroglial tumor subtypes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe telencephalic tumors from both genotypes (BbPP and BBPP) were identified as heterogeneous glioneural lesions forming neurocytic rosettes characterized by proliferating cells with rounded nuclei that were Olig2-positive and NeuN-negative with scattered neuropil islands (Figure 3A and Supplementary Figure S2A), while no gross histological alterations in BBpp, or bbPP brains were found (data not shown).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll lesions observed showed areas of uniformly round nuclei with a perinuclear halo, conferring an “oligo-like” appearance (Figure 3A\u0026nbsp;and\u0026nbsp;Supplementary Figure S2A), a feature frequently described in diffuse glioneuronal tumors with oligodendroglioma-like features\u0026nbsp;such as PLNTY\u003csup\u003e32\u003c/sup\u003e. Immunohistochemical (IHC) analysis showed Olig2-positive rounded neuroepithelial cells with scant cytoplasm and without evidence of cytoplasmic processes, intermingled with\u0026nbsp;giant\u0026nbsp;astrocytic cells showing intense Gfap and Nestin positivity, corresponding to reactive astrocytes (Figure 3A and Supplementary S2B-C). Reactive astrocytes associated with the tumor tissue had the typical stellate morphology and long, thick cytoplasmic processes\u003csup\u003e33\u003c/sup\u003e that differed morphologically from small Gfap-positive cells within the tumor, which were excluded from the oligodendroglioma-like areas (Figure 3A and Supplementary S2B-C). BbPP tumors were characterized by a low Ki67 index (5.6%\u0026nbsp;±\u0026nbsp;3.1% Ki67-positive cells) while BBPP tumors showed a higher proliferation activity with a Ki67 index reaching 15.5%\u0026nbsp;±\u0026nbsp;5.5% (Figure 3B). Tumors from both BbPP and BBPP genotypes always contained Olig2, Gfap,\u0026nbsp;as well as Sox2-positive cells (Figure 3A\u0026nbsp;and\u0026nbsp;Supplementary Figure S2D), while Nestin and Tubb3 immunoreactivity was in the non-tumoral compartment (Supplementary Figure S2D). Indeed, normal NeuN-positive neurons were engulfed by neoplastic cells (Figure 3A\u0026nbsp;and\u0026nbsp;Supplementary Figure S2D). In some cases, we observed that BBPP lesions presented with rhythmic palisades with a spongioblastic pattern (Supplementary Figure S2A), a feature described in a wide range of CNS neoplasms, including pilocytic astrocytoma and oligodendroglioma\u003csup\u003e34\u003c/sup\u003e. Staining combinations\u0026nbsp;with\u0026nbsp;Olig2-Sox2, Olig2-Gfap, and Sox2-Gfap antibodies showed that the majority of Olig2-positive tumor cells were Sox2- but not Gfap-positive, while Gfap-Sox2 positivity\u0026nbsp;mainly co-localized in\u0026nbsp;reactive astrocytes (Figure 3C).\u0026nbsp;We observed a few Gfap-Eyfp-positive cells within the tumor, suggesting that a minority of tumor cells could be of astrocytic nature (Figure 3D). Pten loss and BRafV600E expression were confirmed in serial sections of Eyfp-positive BbPP tumors by immunofluorescence (IF) and by IHC, and, accordingly, pErk1,2 immunoreactivity was also observed (Figure 3E and Supplementary Figure S2E). BBpp, but not bbPP brains, showed a few scattered BRafV600E-positive cells in the subventricular region, which were not sufficient to induce tumor formation (Supplementary Figure S2F).\u003c/p\u003e\n\u003cp\u003eAltogether, our results suggest that BRafV600E expression in a \u003cem\u003ePten\u003c/em\u003e-deleted background affects aNSPCs proliferation and differentiation, inducing the formation of LGG tumors that are more proliferative in the presence of a homozygous \u003cem\u003eBRafV600E-\u003c/em\u003emutated allele.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman PLNTY lesions express SOX2 within the oligodendroglioma-like tumor area.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether BRAFV600E-positive PLNTY lesions express SOX2, we identified two PLNTY cases diagnosed in a 16-year-old male and a 29-year-old female patient. Consistent with the literature, H\u0026amp;E staining revealed lesions with a diffuse growth pattern and oligodendroglioma-like cellular components that were strongly positive for OLIG2 (Figure 4 A-B). The Ki-67 labeling indices were approximately 10% and 5%, respectively. Notably, within the oligodendroglioma-like areas, tumor cells showed high levels of SOX2 and strong BRAFV600E immunoreactivity, and were negative for IDH1R132H, supporting the diagnosis of PLNTY. In contrast to the mouse lesions, GFAP staining was diffusely distributed throughout the tumor areas.\u0026nbsp;We also probed the lesions for PTEN and pAKT expression as readout of PI3K pathway overactivation, and pERK1,2 to assess MAPK pathway activation\u0026nbsp;(Figure 4 A-B).\u0026nbsp;These results suggest a correlation between BRAFV600E and SOX2 expression, in agreement with previous work on colon cancer (CRC) and recurrent ameloblastoma tumor cells\u003csup\u003e35,36\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBRafV600E\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eand\u003cem\u003e\u0026nbsp;Pten\u003c/em\u003e-deleted SCPs transform into paraspinal schwannomas\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistological examination of brainstem-associated masses involving CN V and enlarged DRGs revealed whorls of Schwann cells, consistent with cellular schwannomas, a tumor variant characterized by high cellularity, predominantly composed of Antoni A areas, and lacking Verocay bodies\u003csup\u003e37\u003c/sup\u003e. These tumors displayed neuronal cell bodies and axons entrapped by spindle-shaped neoplastic cells, which also invaded the spinal nerve roots. Immunohistochemical characterization showed diffuse S100b, Sox2, and Sox10 positivity in both BbPP and BBPP genotypes (Figure 5A–B and Supplementary Figure S2G). Ki67 staining indicated moderate proliferative activity, with labeling indices of 9.2% ± 2.5% and 11.2% ± 3.7% in BbPP and BBPP tumors, respectively (Figure 5A–C and Supplementary Figure S2G). Both genotypes also developed skin masses (Figure 2F and Figure 5D), spanning a spectrum from benign cutaneous neurofibromas (cNFs) to high-grade malignant peripheral nerve sheath tumors (MPNSTs), as determined by histological and immunohistochemical analyses (Figure 5D and E). cNFs consisted of spindle-shaped Schwann cells positive for S100b and Sox10, admixed with perineurial cells and fibroblasts (Figure 5D). In contrast, MPNSTs, corresponding phenotypically to sciatic nerve masses in both genotypes (one case in BbPP and one in BBPP), were composed of densely packed spindle cells with elongated, wavy nuclei and nuclear atypia. These tumors showed strong immunoreactivity for S100b, Sox2, and Sox10 (Figure 5E) while Ki67 indices showed significant differences between the two tumor genotypes (14.4%\u0026nbsp;±\u0026nbsp;1.1% vs 20.4%\u0026nbsp;±\u0026nbsp;2.9%, BbPP and BBPP, respectively; Figure 5F). Schwann cell transformation of the cranial and spinal DRG resulted in overgrowth and invasion of the posterior cranial fossa and the vertebral canal, respectively, leading to animal death.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBRafV600E-mutated aNSPCs show increased proliferation in vitro\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAttempts to establish cell cultures from brain or DRG tumors in vitro from both BbPP and BBPP genotypes\u003cem\u003e\u0026nbsp;\u003c/em\u003ewere unsuccessful; thus, we obtained in vitro deleted aNSPCs isolated from the brains of the different genotypes. To this end, SVZ cells were collected from the brains of one-month-old Wt, BbPP, and BBPP mice to obtain aNSPCs. To better dissect the independent contribution of Mapk or Akt overactivated pathways during cell transformation, we also included BBpp and bbPP aNSPCs. aNSPCs grown as neurospheres were treated with 2μM 4-hydroxytamoxifen (4HT) to induce Cre-mediated mutation. Single cell suspensions were then FACS-sorted to obtain a homogeneous Eyfp-positive cell population, genotyped to verify \u003cem\u003ePten\u003c/em\u003e deletion (Supplementary Figure S3A), and/or stained with BRafV600E-specific antibody for the inclusion of V600E mutation (Supplementary Figure S3B). To assess BRafV600E levels in BbPP and BBPP, compared to Wt neurospheres, we quantified total BRaf and BRafV600E expression in IF experiments (Supplementary Figure S3B-C). This analysis revealed increased BRafV600E fluorescence intensity in the BBPP compared with BbPP cells, consistent with successful gene editing. 5-bromo-2’-deoxyuridine (BrdU) incorporation rates in the presence of canonical Growth Factors (GFs) showed that aNSPCs from all the genotypes were more proliferative compared to Wt cells (BbPP 33.3%\u0026nbsp;±\u0026nbsp;1.7%, BBPP 37.3%\u0026nbsp;±\u0026nbsp;0.8%, BBpp 31.4%\u0026nbsp;±\u0026nbsp;3.1\u0026amp;, bbPP 31.3%\u0026nbsp;±\u0026nbsp;2.3%; Figure 6A) in vitro. BRafV600E inhibition by 0.5 or 1mM PLX-4032 (PLX) affected BRaf-mutated aNSPCs growth, and BrdU incorporation was strongly decreased in BbPP compared to Wt cells (Supplementary Figure S3D). Since GFs independence is considered a hallmark of malignancy secondary to the development of autocrine GF loops in cancer cells\u003csup\u003e38\u003c/sup\u003e, we evaluated whether mutated aNSPCs were able to proliferate in low concentrations of GFs or in their absence. Cells from all genotypes but not Wt were able to proliferate in the absence of GFs after 5 days of culture and formed large colonies in complete\u0026nbsp;GFs deprivation; however, BRafV600E-containing genotypes showed a consistently higher proliferation activity compared to \u003cem\u003ePten\u003c/em\u003e-only deleted aNSPCs (Figure 6B and Supplementary Figure S3E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBraf-mutated aNSPCs preferentially differentiate into oligodendrocytes in vitro\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt is well established that NSPCs can differentiate along the three neural lineages when exposed to serum in vitro\u003csup\u003e39\u003c/sup\u003e. To evaluate the impact of the \u003cem\u003eBRafV600E\u003c/em\u003e mutation and/or \u003cem\u003ePten\u003c/em\u003e deletion on the developmental fate of aNSPCs, we performed pseudo-differentiation experiments on cultured cells from all the genotypes in 1% FBS for 10 days. Wt aNSPCs preferentially differentiated along the astrocytic lineage with 61.37%\u0026nbsp;±\u0026nbsp;9.29% of Gfap-positive cells, compared to the neuronal lineage or oligodendrocyte lineage (30.68%\u0026nbsp;± 8.25% Tubb3-positive cells and 7.95%\u0026nbsp;± 1.05% MBP-positive cells, respectively). On the contrary, the number of Gfap-positive cells was significantly decreased in BBpp (9.54%\u0026nbsp;± 4.53%), BbPP (23.74%\u0026nbsp;± 12.9%), or BBPP (15.01%\u0026nbsp;± 8.26%) aNSPCs, in favor of MBP-positive cells, that accounted for 63.17%\u0026nbsp;± 10.47%, 31.93%\u0026nbsp;± 3.59% and 57.96%\u0026nbsp;± 5.5%, respectively. Tubb3-positive cell numbers were not significantly different in the BRafV600E-mutated genotypes (BBpp 24.39%\u0026nbsp;± 5.04%, BbPP 33.84%\u0026nbsp;± 10.31%, and BBPP 20.0%\u0026nbsp;± 0.68%) compared to the control genotype (Figure 6C).\u0026nbsp;bbPP aNSPCs differentiated preferentially toward the neuronal pathway (Tubb3 positivity: 85%\u0026nbsp;± 1.04%) as previously described\u003csup\u003e40,41\u003c/sup\u003e.\u0026nbsp;The remaining cells were positive for Gfap (13.4%\u0026nbsp;±\u0026nbsp;2.04%), but none differentiated along the oligodendrocyte lineage. Wt or bbPP aNSPCs cultures did not retain any proliferating undifferentiated cells, while in all the BRafV600E genotypes they were still present, with numbers ranging from 2.9%\u0026nbsp;±\u0026nbsp;1.3% in BBpp to 10.5%\u0026nbsp;±\u0026nbsp;2.0% in BbPP and 7.0%\u0026nbsp;±\u0026nbsp;3.0% in BBPP genotypes, respectively (Figure 6C). Treatment with PLX completely abrogated BrdU-incorporating undifferentiated cells in all the \u003cem\u003eBRafV600E\u003c/em\u003e genotypes, while Wt or bbPP were not affected (Supplementary Figure S3F).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOverall, these results suggest that, also in differentiating conditions, \u003cem\u003eBRafV600E\u003c/em\u003e-mutant aNSPCs retain their ability to proliferate, while preferentially differentiating toward the oligodendrocyte lineage at the expense of the astrocyte and neuron lineages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBRafV600E\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;mutation and \u003cem\u003ePten\u003c/em\u003e deletion both enhance Sox2 stability in aNSPCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSox2 is required to maintain aNSPCs and cancer stem cells\u003csup\u003e27,42\u003c/sup\u003e. It also acts as a glioma-reprogramming factor\u003csup\u003e23\u003c/sup\u003e,\u0026nbsp;and its deletion in PDGF-B-transformed oligodendroglioma prevents cancer cells from generating secondary tumors when orthotopically transplanted\u003csup\u003e42\u003c/sup\u003e.\u0026nbsp;By IF and western blot (WB) analysis, we found that BbPP and BBPP aNSPCs showed higher levels of Sox2 protein expression compared to Wt\u0026nbsp;cells; however, quantitative RT-PCR analysis did not show significant differences in \u003cem\u003eSox2\u003c/em\u003e mRNA levels (Figure 7A-B and Supplementary Figure S4A-B). On the contrary, \u003cem\u003eGfap\u003c/em\u003e mRNA levels were dramatically decreased in BbPP aNSPCs, as well as in all the other mutant genotypes (Supplementary Figure S4B). Treatment with PLX or PD98059 (PD) for 6 h, to inhibit Mapk activation, showed a strong reduction of Sox2 protein levels in BbPP aNSPCs\u0026nbsp;(Figure 7C and Supplementary Figure S4C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt has been shown that Akt activation decreases Sox2 ubiquitylation, enhancing its stability in ESCs and esophageal cancer cells\u003csup\u003e25,43\u003c/sup\u003e, whereas Sox2 ubiquitylation is increased upon neuronal differentiation in aNSPC\u003csup\u003e26\u003c/sup\u003e. Thus, to determine whether protein synthesis or\u0026nbsp;degradation rates could be affected by overactivated Mapk/Akt pathways,\u0026nbsp;we treated Wt, BbPP, BBpp, and bbPP aNSPCs for 5 h with the proteasome inhibitor MG132 (MG) or the protein synthesis inhibitor cycloheximide (CHX). Sox2 protein levels were upregulated by proteasome inhibition and downregulated by protein synthesis inhibition in Wt aNSPCs (Figure 7D and\u0026nbsp;Supplementary Figure S4D).\u0026nbsp;As expected, Sox2 levels were increased both in BbPP or BBpp aNSPCs compared to Wt cells, and its levels slightly changed following MG or CHX treatments, suggesting that BRafV600E promotes Sox2 protein stability by affecting the ubiquitylation process (Figure 7D and\u0026nbsp;Supplementary Figure S4D). We also found that \u003cem\u003eSox2\u003c/em\u003e mRNA levels were upregulated in Akt-deregulated bbPP aNSPCs (Supplementary Figure 4B) and, importantly, that proteasome inhibition but not CHX treatment increased Sox2 protein levels by an Akt-dependent post-translational mechanism (Figure 7D and\u0026nbsp;Supplementary Figure S4D), as previously shown in ESCs and esophageal cancer cells\u003csup\u003e25,43\u003c/sup\u003e .\u003c/p\u003e\n\u003cp\u003eWe then monitored Sox2 ubiquitylation by BRafV600E\u0026nbsp;following\u0026nbsp;transfection of HA-Ubiquitin, Sox2, and FLAG-BRafV600E- or FLAG-empty-expressing plasmids\u0026nbsp;in HEK293T cells. Notably, input extracts showed that Sox2 levels were increased following 5 h MG treatment compared to mock-treated controls in FLAG-BRafV600E/Sox2/HA-Ubiquitin cells, but not in FLAG-empty/Sox2/HA-Ubiquitin cells.\u0026nbsp;We observed that Sox2 levels were higher in FLAG-BRafV600E/Sox2 co-transfected cells compared to FLAG-Sox2-transfected cells, due to a transcriptional effect of Mek1 overactivation on CMV-promoter driven expression plasmids\u003csup\u003e44\u003c/sup\u003e. Ubiquitylated immunoprecipitated Sox2 levels, monitored by HA expression, were increased in Sox2-only transfected cells following MG treatment (2-fold), while they were decreased both in control and in MG-treated (about 0.4-fold) FLAG-BRafV600E/Sox2 transfected cells, indicating that BRafV600E expression prevented Sox2 ubiquitylation (Figure 7E and Supplementary S4E). These results corroborated the finding that inhibition of ubiquitylation by MG was a major mechanism for Sox2 stabilization either in BBpp or in BbPP aNSPCs and suggested a role for the\u0026nbsp;Mapk-activated pathway in preventing Sox2 degradation.\u003c/p\u003e\n\u003cp\u003eIt is known that phosphorylation of threonine 118 (pT118) mediated by Akt prevents Sox2 lysine 119 methylation and ubiquitylation, increasing Sox2 stability in ESCs\u003csup\u003e25,26\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe found that T118 was a target of the BRaf overactivated pathway, as mutant T118A-Sox2 was not phosphorylated in BRafV600E-transfected HEK293T\u0026nbsp;cells (Figure 7F\u0026nbsp;and\u0026nbsp;Supplementary Figure S4F). We found that FLAG-BRafV600E was present in the complex obtained from Sox2 immunoprecipitation (Figure 7E), and conversely, while Wt-Sox2 co-immunoprecipitated, T118A-Sox2 was not present in the complex obtained from FLAG-BRafV600E immunoprecipitation in HEK293T transfected cells (Figure 7G).\u0026nbsp;To assess if the T118A mutation affected Sox2 stability in FLAG-BRafV600E overexpressing cells, we transiently expressed Wt- or T118A-Sox2 in HEK293T\u0026nbsp;cells that were treated after 72 h from transfection for 1, 3, or 6 h with CHX. While Wt-Sox2 levels did not change throughout the treatment time, we found that T118A-Sox2 levels were significantly decreased after 6 h of treatment (Figure 7H\u0026nbsp;and\u0026nbsp;Supplementary Figure S4G). The role of BRafV600E in phosphorylating and stabilizing Sox2 levels was further confirmed in aNSPCs cell extracts that showed increased total- and pT118-Sox2 levels in all BRafV600E mutated genotypes (BbPP, BBPP, BBpp) (Figure 7I). As in ESCs\u003csup\u003e25\u003c/sup\u003e, T118 was a target of Akt-mediated phosphorylation, as shown in bbPP cells, confirming that the Akt-activated pathway promotes T118 phosphorylation also in aNSPCs (Figure 7I and Supplementary Figure S4H).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe next interrogated the TCGA PanCancer Atlas dataset to determine whether human high-grade tumors exhibit differential \u003cem\u003eSOX2\u003c/em\u003e mRNA expression according to BRAF mutational status. Interestingly, we found no statistically significant differences between BRAF-mutated or non-mutated samples in \u003cem\u003eSOX2\u003c/em\u003e expression levels in the LGG and GBM cohorts, although the mutated cohort in the dataset was too small compared to the non-mutated. Similar results were obtained by analyzing skin cutaneous melanomas (SKCM) (in which the two cohorts were similar in size, Supplementary Figure S4I). On the contrary, BRAF-mutated colorectal adenocarcinomas (COADs) showed significantly higher \u003cem\u003eSOX2\u003c/em\u003e levels, compared to non-mutated tumors, despite the mutated cohort representing about 1/6th of the non-mutated group (Supplementary Figure S4I). These results suggest that BRAFV600 mutations may differentially affect tumor cells depending on their cell of origin, as observed in neural precursors that give origin to GBM or to SKCM, compared with endoderm-derived epithelial cells that give origin to COAD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAltogether, these results suggest that the BRafV600E-activated pathway promotes Sox2 T118 phosphorylation in mutated aNSPCs, thereby preventing Sox2 degradation and thus enhancing its stability. As previously identified in ESCs, our results also highlight the role of the Akt-activated pathway in mediating T118-Sox2 phosphorylation in \u003cem\u003ePten\u003c/em\u003e-deleted aNSPCs.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMAPK is one of the most affected pathways in diffuse LGGs. A WHO category of diffuse LGG with MAPK pathway alterations has been recently described, in which \u003cem\u003eBRAF\u003c/em\u003e alterations represent more than 80% of cases\u003csup\u003e45\u003c/sup\u003e, the majority being of paediatric age\u003csup\u003e10,46\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWhile susceptibility of the cell-of-origin and deregulation of signaling pathway/s have emerged as the most critical determinants in GBM, it has not been clearly demonstrated whether these factors can also be responsible for LGGs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe tested the hypothesis that MAPK overactivated signaling, operated by BRafV600E mutation, plays a transforming role in mouse aNSPCs, introducing the homonymous BRafV600E mutation, through CreERT2 recombineering specifically in Sox2-expressing aNSPCs. The stemness nature of the \u003cem\u003eSox2\u003c/em\u003e promoter was validated by analysis of\u0026nbsp;b-galactosidase (b-gal?) expression in the brain, DRGs, and skin of adult mice following tamoxifen induction. While no overt pathological phenotypes were elicited in \u003cem\u003eBRafV600E\u003c/em\u003e-only or \u003cem\u003ePten\u003c/em\u003e-only deleted animals during the observation period, as also previously reported\u003csup\u003e40,47\u003c/sup\u003e,\u0026nbsp;both mutations were required for tumor transformation of aNSPCs\u0026nbsp;in the lateral ventricles and in the DRGs, leading animals to death very early\u0026nbsp;from the recombination events. Brain tumors were identified as low-grade neuroepithelial tumors with oligodendroglioma-like features and peripheral nerve tumors as\u0026nbsp;schwannomas (WHO, 2021),\u0026nbsp;highlighting the role of concomitant MAPK and PI3K/AKT overactivated pathways in directing LGG and schwannoma tumorigenesis. BRafV600E has been described as an oncogenic driver when oncosuppressors such as \u003cem\u003eTp53\u003c/em\u003e or \u003cem\u003eInk4/Arf\u0026nbsp;\u003c/em\u003ewere concomitantly\u0026nbsp;deleted\u003cem\u003e\u0026nbsp;\u003c/em\u003ein\u003cem\u003e\u0026nbsp;\u003c/em\u003efetal brains, promoting the development of malignant ganglioglioma or astrocytoma, respectively\u003csup\u003e48,49\u003c/sup\u003e. Here we demonstrate\u0026nbsp;that, in the presence of concomitant \u003cem\u003ePten\u003c/em\u003e deletion, BRafV600E expression specifically in aNSPCs and SCPs promotes cell transformation leading to low-grade oligodendroglioma-like lesions and to schwannomas.\u0026nbsp;Indeed, GNTs and PLNTY with oligodendrocyte-like differentiation are driven by aberrant members of the MAPK-activated pathway, including genetic anomalies of \u003cem\u003eBRAF\u003c/em\u003e\u003csup\u003e14,32,50\u003c/sup\u003e. Consistent with these findings, oligodendroglial-like tumor cells from PLNTY lesions harboring the BRAFV600E mutation express both OLIG2 and SOX2. Notably, an association between BRAFV600E and SOX2 expression has been reported in two other human tumors, CRC and ameloblastoma, in which inhibition of MAPK signaling led to SOX2 downregulation\u003csup\u003e35,36\u003c/sup\u003e. Consistent with this, in our brain tumor model Olig2-positive tumor cells were Sox2- but not Gfap- positive, and Gfap-Sox2-co-staining was observed mainly in reactive astrocytes. While GFAP staining was diffusely positive throughout the human tumor areas, mouse lesions exhibited only scant Gfap expression within tumor cells, with strong labeling restricted to the reactive astroglial component. This discrepancy may reflect differences in tumor kinetics and in the timing of tissue collection and analysis between mouse models and human samples. Whereas mouse tumors were\u0026nbsp;collected at an early stage following the initial transformation event induced by CreERT2 activation, human lesions were obtained after an unknown and likely prolonged period of tumor evolution. This temporal difference may have allowed the accumulation of downstream events associated with increased GFAP expression in the human samples. In agreement, another mouse model resembling PLNTY lesions reported scant GFAP expression in tumor cells\u003csup\u003e48\u003c/sup\u003e.\u0026nbsp;Biallelic\u0026nbsp;\u003cem\u003eBRafV600E\u003c/em\u003e mutation significantly increased the Ki67 index compared to the monoallelic condition in the brain tumors, indicating that reinforced MAPK pathway activation enhances tumor proliferative activity, as it has been recently reported for biallelic RasG12V-induced hepatocarcinomas\u003csup\u003e51\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite a higher Ki67 index in homozygous compared with heterozygous \u003cem\u003eBRafV600E\u0026nbsp;\u003c/em\u003etumors, we did not observe a significant difference in median survival time. Such \u0026nbsp;discrepancy was likely due to the rapid worsening of the symptomatology in both genotypes induced by the DRG tumors that compressed the spinal cord, requiring euthanasia for ethical concerns at very close intervals, in a matter of days.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is well established that \u003cem\u003ePten\u003c/em\u003e-deleted aNSPCs gain enhanced self-renewal, proliferative ability, and decreased GF dependency\u003csup\u003e52\u003c/sup\u003e. As expected, in vitro-cultured \u003cem\u003ePten\u003c/em\u003e-deleted aNSPCs were more proliferative than Wt cells\u003csup\u003e40\u003c/sup\u003e, but, most interestingly, the BRafV600E mutation alone also increased proliferative ability in aNSPCs, strongly enhancing GF independence. \u0026nbsp;While it has previously been shown that in vivo homozygous deletion of \u003cem\u003ePten\u003c/em\u003e in aNSPCs does not result in tumor formation\u003csup\u003e40\u003c/sup\u003e, BRafV600E overexpression in fetal neural cells guided by a strong viral promoter leads to circumscribed lesions composed of oligoid cells\u003csup\u003e48\u003c/sup\u003e. We did not detect any brain anomalies in BBpp mice at the time of sacrifice; however, we cannot exclude the possibility that limited, asymptomatic lesions may have developed at later time points. These results suggest that, in vivo, two BRafV600E mutant alleles are insufficient to drive tumor formation unless an additional genetic hit, such as Pten inactivation, occurs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn agreement with tumor histology, Mapk overactivation in vitro primed aNSPCs toward oligodendrocyte-like differentiation, as shown by MBP positivity at the expense of the astrocytic fate. Forced Erk1,2 activation during developmental myelination of the CNS and PNS promotes oligodendrocyte precursor cells expansion and myelin production from both oligodendrocyte and Schwann cells in animal models\u003csup\u003e53\u003c/sup\u003e. Since the BRafV600E mutation has been identified\u0026nbsp;in 10% of sporadic schwannomas and in MPNSTs not associated with \u003cem\u003eNF1/2\u003c/em\u003e mutations\u003csup\u003e54,55\u003c/sup\u003e, our results further demonstrate that schwannomas or MPNSTs can originate from postnatal SCPs following BRafV600E\u003cem\u003e\u0026nbsp;\u003c/em\u003emutation and concomitant Pten inactivation.\u003c/p\u003e\n\u003cp\u003eIn many brain tumors, Sox2 is expressed at high levels and is required for GBM stem cell propagation\u003csup\u003e56\u003c/sup\u003e. A recently described mouse model for skin tumors has clearly shown that BRafV600E expression in skin progenitor cells, along with Mapk activation, induces upregulation of Sox2 expression and tumor formation, making these cells permissive to rapid transformation\u003csup\u003e57\u003c/sup\u003e . Consistently, we found that \u003cem\u003eSox2\u003c/em\u003e deletion in BbPP mice abrogated tumor formation, indicating that Sox2 acts as a stemness factor essential for aNSPC survival and transformation.\u0026nbsp;We found that aNSPCs obtained from all the \u003cem\u003eBRafV600E\u003c/em\u003e genotypes showed enhanced Sox2 expression at the protein but not mRNA levels, while bbPP aNSPCs mediated Sox2 expression mainly at the transcriptional level. Sox2 increase mediated by BRafV600E, instead, was not linked to increased protein synthesis, but rather to the decrease of Sox2 ubiquitylation, as revealed by proteasomal inhibition experiments. In agreement, BRafV600E expression reduced Sox2 ubiquitylation in HEK293T transfected cells, corroborating the results obtained in aNSPCs. A mechanism that involves an ubiquitylation switch mediated by Cul4a/Det1/Cop1 has been shown to regulate Sox2 levels during aNSPCs development, while a phosphorylation/methylation/ubiquitylation switch in residues T118/K119 of Sox2, mediated by Akt1/Set7/WWP2, has been shown to be involved in the control of ESC stemness\u003csup\u003e25\u003c/sup\u003e. Moreover, phosphorylated levels of Sox2-T118A were not increased following BRAFV600E transfection in HEK293T cells, supporting the role of this residue as a substrate for BRaf-induced phosphorylation. Our results indicate that PI3K/AKT signaling promotes both Sox2 transcription and protein stabilization, while Mapk activation reinforces Sox2 expression by enhancing protein stability via T118 phosphorylation, highlighting a cooperative interplay between the two pathways in aNSPCs. Even if none of the tested downstream members of the MAPK pathway, such as pMek1,2, pErk1,2, Rsk1, and Rsk2, were found to interact with Sox2 in immunoprecipitation (IP) experiments (not shown), we cannot exclude that these downstream members or other unknown BRaf-interacting kinase/s could mediate Sox2 T118 phosphorylation and stabilization.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn summary, this study identifies the \u003cem\u003eBRafV600E\u003c/em\u003e and MAPK-activated pathways as essential events together with \u003cem\u003ePten\u003c/em\u003e loss for aNSPC/SCP transformation that lead to LGG and schwannoma formation, as outlined in the model presented in Figure 8. Although the direct phosphorylation of Sox2 by BRafV600E was not demonstrated, our results uncover a novel downstream event initiated by BRafV600E that leads to increased Sox2 protein stabilization through T118 phosphorylation and impaired Sox2 ubiquitylation in mutated aNSPCs. Our findings suggest that this mechanism may represent a parallel oncogenic process downstream of MAPK/PI3K signaling, contributing to cellular transformation.\u003c/p\u003e\n"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eReagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll reagents used throughout the experiments are listed in Supplementary Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMice and Tamoxifen treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice carrying the \u003cem\u003eSox2-CreERT2\u003c/em\u003e transgene\u003csup\u003e27\u003c/sup\u003e were crossed with transgenic mice carrying conditional \u003cem\u003eBRafV600E\u003c/em\u003e\u003cem\u003e, \u003c/em\u003e\u003cem\u003ePten\u003csup\u003eflox/flox\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003ealleles, and the \u003cem\u003eR2R6-Eyfp \u003c/em\u003ereporter or \u003cem\u003eR26R-LacZ \u003c/em\u003e(Jackson Laboratories, Bar Harbour, ME, USA ) and with Sox2\u003csup\u003eflox/flox\u003c/sup\u003e\u003csup\u003e27\u003c/sup\u003e. Animals were backcrossed into the C57BL/6 strain. Experimental animals carried the \u003cem\u003eSox2-CreERT2\u003c/em\u003e in heterozygosity.\u003c/p\u003e\n\u003cp\u003eTransgenic 1-month-old mice were injected i.p. with 225mg/kg tamoxifen dissolved in a 9:1 corn oil:ethanol mixture, once. For oral gavage, 11.5 days post coitum (dpc) pregnant \u003cem\u003eSox2-CreERT2;\u003c/em\u003e \u003cem\u003eR26R-LacZ \u003c/em\u003eanimals were treated with 100\u0026mu;L of 5mg/ml tamoxifen for two consecutive days and sacrificed at 13.5 dpc to obtain recombined embryos. Experiments were carried out on both male and female mice, in similar ratios.\u003c/p\u003e\n\u003cp\u003eAnimals were individually identified and monitored daily using a predefined clinical scoring system to assess disease progression. Scoring was performed by trained personnel blinded to group allocation. Phenotypic signs were recorded at each observation. Ruffled fur was assigned a score of 1, and kyphotic posture a score of 2. Body weight was measured regularly, with weight loss \u0026lt;5% assigned a score of 1 and weight loss \u0026lt;10% a score of 3. Clinical signs included respiratory distress (score 2) and paralysis (score 5). Behavioral alterations were also assessed, with reduced mobility and ataxia each assigned a score of 3. Animals exhibiting severe clinical signs or reaching a threshold score of 5 were humanely euthanized in accordance with the guidelines of the Stazione per la Tecnologia Animale \u0026ndash; University of Rome Tor Vergata and with protocol no. 700-PR/2017 from the Italian Ministry of Health.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR and genotyping \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA was extracted from tails or aNSPCs with Proteinase K. \u003c/p\u003e\n\u003cp\u003eCre alleles and Cre-mediated recombined alleles were verified by animal genotyping. PCRs were performed using the primers listed in Supplementary Table 2. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eHistology, tumor analysis, immunohistochemistry, and immunofluorescence \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman tumor specimens were obtained for routine diagnosis. Patients\u0026rsquo; data were collected anonymously, and written informed consent, as part of the diagnostic routine and treatment procedures, was obtained from patients or their guardians in accordance with the Declaration of Helsinki, and the study adhered to Good Clinical Practice guidelines. Samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and processed. PLNTY cases were independently and blindly validated by the two pathologists (M.M. and M.G.) based on histomorphology and immunohistochemistry (IHC). For molecular analyses, two serial sections (10 \u0026micro;m thick) were obtained from formalin-fixed paraffin-embedded (FFPE) tissue blocks. Tumor tissue represents greater than 50% of the sample. After deparaffinization, genomic DNA was extracted from microdissected FFPE tissue using the QIAamp DNA FFPE Tissue Kit on the QIAcube automated platform, according to the manufacturer\u0026rsquo;s instructions. DNA concentration and quality were assessed using the QIAxpert system. The mutational status of the BRAF gene was assessed using the BRAF Codon 600 Mutation Analysis Kit II, a diagnostic assay for qualitative detection of mutations at codon 600 in exon 15. The assay specifically identifies five clinically relevant mutations: V600E, V600K, V600D, V600R, and V600M. Interpretation of the mutational status was performed according to the manufacturer\u0026rsquo;s recommendations. BRAF mutation (c.1799T\u0026gt;A; p.V600E) was detected. Mouse tumor specimens were obtained following animal perfusion and post-fixed with 4% paraformaldehyde. Five mm sections from tumor samples were processed for H\u0026amp;E, IHC, and IF stainings. For IHC and IF analysis, antigen retrieval on tissue sections was performed using citrate buffer pH 6.0. For IF analysis of cultured aNSPCs, cells were fixed in 4% PFA and permeabilized with 0.1% Triton X-100. For \u0026beta;-galactosidase staining, embryos and tissues were processed as previously described\u003csup\u003e28\u003c/sup\u003e. All antibodies used, and relative dilutions are listed in Supplementary Table 3.\u003c/p\u003e\n\u003cp\u003eKi-67 assessment was obtained as the ratio of the number of tumor cells stained by Ki-67 antibodies to the total number of tumor cells counted in at least 5 non-overlapping microscopic fields at 400x magnification. The fluorescence intensity measurements were performed using LAS-X software (Leica Microsystems, Wetzlar, Germany).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eaNSPCs culture and assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eaNSPCs were isolated as previously described by Donegà et al.\u003csup\u003e29\u003c/sup\u003e aNSPCs were obtained from pools of 3 adult (1-month-old) brains cultured in NeuroCult Basal Medium, Pen/Strep, 20 ng/ml EGF, 15 ng/ml bFGF, and 0.0002% Heparin. Experiments were run utilizing at least 3 batches of aNSPCs between passage 5 and 20. All aNSPCs cultures were tested negative for Mycoplasma spp.\u003c/p\u003e\n\u003cp\u003eIn vitro, tamoxifen was administered by adding 0.2 \u0026mu;M 4HT for 10 consecutive days to cell cultures. Sox2-CreERT2 activation was reported by Eyfp expression in cells.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFACS sorting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle cell suspension of Cre-activated aNSPCs was loaded onto BD FACS Aria III cell sorter (BD Biosciences, Franklin Lakes, NJ, USA) and gated for Eyfp fluorescence to obtain Eyfp-positive cell population. To increase the sorting stringency, a purity criterion was set. Eyfp-negative cells were discarded. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eIn vitro assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the differentiation assays, 12000 cells/cm\u003csup\u003e2\u003c/sup\u003e were seeded on plates pre-coated with 1:200 Geltrex and cultivated in Neurobasal+ Medium with B27+, 0.5 mM GlutaMAX, 1% FBS, Pen/Strep for 10 days.\u003c/p\u003e\n\u003cp\u003eFor the proliferation assay, single cells were incubated with 100 \u0026mu;M BrdU for 30 minutes. Cells were fixed and immunostained with anti-BrdU antibody.\u003c/p\u003e\n\u003cp\u003eFor the clonogenic assay, 3000 cells/cm\u003csup\u003e2\u003c/sup\u003e were plated and allowed to form secondary spheres for 10 days. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eWestern blot and immunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell extraction was performed in RIPA buffer. Primary antibodies listed in Supplementary Table 3 were all used at a 1:1000 dilution. \u003c/p\u003e\n\u003cp\u003eFor IP, cells were homogenized in lysis buffer, and 1 mg of cell extract was incubated with 3 \u0026mu;g of mouse anti-Sox2 antibody, mouse anti-FLAG, or mouse immunoglobulin G (IgG) (negative control) and Dynabeads Protein G in 1 ml, overnight at 4\u0026deg;C under constant rotation. \u003c/p\u003e\n\u003cp\u003eAll samples were resuspended in 4x SDS sample buffer and processed for WB analysis. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eRNA extraction and qPCR \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was collected from aNSPCs and extracted using the RNeasy Kit. \u003c/p\u003e\n\u003cp\u003ecDNA was synthesized from 1 mg of total RNA with SuperScript III Reverse Transcriptase and random primers. \u003cem\u003eActb\u003c/em\u003e was used as a control for mRNA normalization. \u003c/p\u003e\n\u003cp\u003eThe relative amounts of each substrate were calculated by the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;CT\u003c/sup\u003e method, and expression levels were represented as fold increases relative to the control sample, which was set to 1. All primers were provided by Sigma-Aldrich and are listed in Supplementary Table 2. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003ePlasmids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003epMT2-HAubq\u003csup\u003e30\u003c/sup\u003e was a gift from Dr. A. Peschiaroli (National Research Council, CNR, Rome), p3XFLAG-BRAFV600E was purchased from Addgene, and p3XFLAG-CMV-14 from Sigma-Aldrich. \u003c/p\u003e\n\u003cp\u003eFull-length Sox2 was PCR-amplified, subcloned into the TOPO TA cloning kit, and then into pcDNA3 using XhoI/EcoRI restriction enzymes (see Supplementary Table 1-2). T118A mutation was inserted by PCR-directed mutagenesis from pcDNA3-Sox2 as starting template, subcloned in TOPO TA cloning kit, and then into pcDNA3 using XhoI/EcoRI restriction enzymes. Plasmids were Sanger-sequenced to verify the DNA sequence.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eStatistical and bioinformatic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were conducted using GraphPad Prism version 10 software. Data are presented as means and standard deviations; tests were considered significant for relative values with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All experiments were performed on at least three biological replicates. For the bioinformatic analysis, \u003cem\u003eSOX2\u003c/em\u003e gene expression and BRAF somatic mutation data of Colorectal Adenocarcinoma (TGCA, PanCancer Atlas), Brain Lower Grade Glioma (TGCA, PanCancer Atlas), Glioblastoma Multiforme (TGCA, PanCancer Atlas), and Skin Cutaneous Melanoma (TGCA, PanCancer Atlas) were retrieved from the cBioPortal database (https://www.cbioportal.org/). Boxplot visualization and statistical analyses were conducted using R software (version 4.1.1). p-values were calculated by the Wilcoxon test with Benjamini-Hochberg (BH) adjustment using the R package ggpubr 0.4.0.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data reported in this paper will be shared by the lead contact upon request.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors thank\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eDr\u0026nbsp;A. Peschiaroli (Istituto di Farmacologia Traslazionale - (IFT); CNR- Rome, Italy) for sharing pMT2-HAubq plasmid.\u003c/p\u003e\n\u003cp\u003eThis paper was supported by grants from Italian Ministry of University and Research Prin2022CE79J_004 to SD; Prin 2022AWB8T4 and P2022SE38P_004, Mnesys NRRP PE0000006 to EAJ; Prin\u0026nbsp;2022X4FWZ5\u0026nbsp;to MS.\u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: E.G. and S.D. \u0026nbsp;wrote the main manuscript text\u003c/p\u003e\n\u003cp\u003eMethodology: E.G. (Figs 1-7), A.C. (Figs 1,2,3,4,6,7) V.C. \u0026nbsp;(Phenotype analysis, Fig.1), L.M. and \u0026nbsp;M.P. (Fig.5), S.D.C (FACS analysis and sorting) M.M. and M.G. (Fig.4)\u003c/p\u003e\n\u003cp\u003eResources: S.K.N., S.D. (animal models)\u003c/p\u003e\n\u003cp\u003eFormal analysis: E.G., A.C., M.M., M.G, M.S. S.D.\u003c/p\u003e\n\u003cp\u003eFunding: E.A.J., M.S., S.D.\u003c/p\u003e\n\u003cp\u003eOriginal Draft Preparation: E.G., A.C, S.D. wrote the main \u0026nbsp;manuscript text\u003c/p\u003e\n\u003cp\u003eSupervision and Project administration: S.D., E.A.J.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript\u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.A.J. is or has been a consultant and/or paid speaker for Bayer, FQM, Ibsa, Kanna, Menarini, Merck, Otsuka, Pfizer, Recordati, Shionogi and Viatris. The other authors declare no competing interests.\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBurotto, M., Chiou, V. L., Lee, J. M. \u0026amp; Kohn, E. C. 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P.\u003cem\u003e et al.\u003c/em\u003e SOX2 confers tumour permissiveness in a specific skin progenitor population. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 304, doi:10.1038/s41467-025-66251-4 (2026). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-precision-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjprecisiononcology","sideBox":"Learn more about [npj Precision Oncology](http://www.nature.com/npjprecisiononcology/)","snPcode":"41698","submissionUrl":"https://submission.springernature.com/new-submission/41698/3","title":"npj Precision Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"BRafV600E, Mapk, Pten, Sox2, PLNTY, schwannoma","lastPublishedDoi":"10.21203/rs.3.rs-7515762/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7515762/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"BRAF mutations are oncogenic drivers present in about 7% of human cancers, with the V600E substitution being the most frequent. In the nervous system, BRAFV600E has been identified in both low- and high-grade gliomas (LGG and HGG) and in tumors of the peripheral nervous system. To investigate the mechanisms underlying BRafV600E-driven tumorigenesis, we generated a mouse model in which the BRafV600E mutation and Pten deletion can be induced through the Sox2-CreERT2 system. This inducible deleter is active in adult neural stem/progenitor cells (aNSPCs) and Schwann cell precursors (SCPs). In this model, BRafV600E-mutated/Pten-deleted telencephalic aNSPCs give rise to diffuse LGG with oligodendroglioma-like features resembling the human diffuse LGG, MAPK pathway–altered subtype. In contrast, mutated SCPs develop schwannomas, cutaneous neurofibromas, and malignant peripheral nerve sheath tumors (MPNSTs). Sox2 deletion in BRafV600E/Ptendel mice markedly reduces tumor formation, indicating that aNSPCs act as tumor cells of origin. In vitro analyses show increased proliferation of BRafV600E/Ptendel aNSPCs and preferential differentiation toward oligodendroglia-like tumor cells. BRaf-mutant aNSPCs display increased Sox2 protein, but not mRNA levels, suggesting post-transcriptional regulation. Sox2 phosphorylation at T118 is promoted by BRafV600E, potentially stabilizing the protein during aNSPC transformation, although the involvement of downstream kinases cannot be excluded.","manuscriptTitle":"Sox2 protein stability is enhanced by BRafV600E and Pten deletion in adult neural stem/progenitor cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-12 07:05:48","doi":"10.21203/rs.3.rs-7515762/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2026-04-08T17:37:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-05T21:52:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272287996386582956609063044482799807470","date":"2026-04-05T21:38:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-03T15:21:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"182285180457544119980791094091872513502","date":"2026-04-03T13:20:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-03T12:48:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-01T08:58:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Precision Oncology","date":"2026-03-01T23:50:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-precision-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjprecisiononcology","sideBox":"Learn more about [npj Precision Oncology](http://www.nature.com/npjprecisiononcology/)","snPcode":"41698","submissionUrl":"https://submission.springernature.com/new-submission/41698/3","title":"npj Precision Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4335b2b9-fddb-4a3d-bfd0-b20df6966a8b","owner":[],"postedDate":"March 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":64368969,"name":"Biological sciences/Cancer"},{"id":64368970,"name":"Biological sciences/Cell biology"},{"id":64368971,"name":"Biological sciences/Molecular biology"},{"id":64368972,"name":"Biological sciences/Neuroscience"},{"id":64368973,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-04-27T16:15:32+00:00","versionOfRecord":{"articleIdentity":"rs-7515762","link":"https://doi.org/10.1038/s41698-026-01439-5","journal":{"identity":"npj-precision-oncology","isVorOnly":false,"title":"npj Precision Oncology"},"publishedOn":"2026-04-20 15:59:56","publishedOnDateReadable":"April 20th, 2026"},"versionCreatedAt":"2026-03-12 07:05:48","video":"","vorDoi":"10.1038/s41698-026-01439-5","vorDoiUrl":"https://doi.org/10.1038/s41698-026-01439-5","workflowStages":[]},"version":"v1","identity":"rs-7515762","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7515762","identity":"rs-7515762","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}