The RASopathies are a group of congenital disorders with overlapping clinical manifestations that are caused by pathogenic germline or early somatic variants that result in the hyperactivation of the RAS/mitogen-activated protein kinase (MAPK) signaling pathway. Given the heterogeneous clinical presentations of these disorders that involve abnormalities across multiple organ systems, multidisciplinary clinical management and progress in scientific research are essential for optimal patient diagnosis and care. The 9th International RASopathies Symposium, a biennial meeting, was organized by the patient advocacy group RASopathies Network and showcased recent discoveries, case studies, and advances in preclinical research. Participants, who included scientists, clinicians, industry representatives, patients, and family advocates, explored knowledge gaps, innovative clinical approaches, and lived experiences of individuals with a RASopathy. Sessions centered around organ systems were introduced with a patient perspective to highlight the burden of disease, continued with presentations from established and early-career investigators. Overall, the RASopathies Symposia serve as a catalyst for sustained community collaboration focused on enhancing patient health and accelerating the translation of discoveries into effective treatments.

Supplementary Table 3 shows the combination index determination results of small molecules used in this study when combined with a MEK inhibitor.

Neurofibromatosis type 1 (NF1) patients are predisposed to develop plexiform neurofibromas (PNFs). By cross-comparison of RNA sequencing and RUNX1-CHIP sequencing data on mouse PNFs, we found that transcripts encoding the NF1-interacting p97/valosin-containing protein (VCP) gene are overexpressed in PNFs. Co-immunoprecipitation confirmed that VCP bounded to neurofibromin. Western blot and immunostaining confirmed VCP overexpression in both mouse and human PNFs. Treatment of primary mouse PNF Schwann cells with CB-5083, a p97/VCP inhibitor, led to accumulation of poly-ubiquitinated proteins and generation of irresolvable proteotoxic stress. Pharmacological or genetic inhibition of VCP reduced mouse PNF cell-derived sphere number, and genetic inhibition of Vcp in Schwann cell precursors decreased tumor-like lesion numbers in a cell transplantation model. In vivo treatment with CB-5083 in Nf1fl/fl;DhhCre PNF mice significantly inhibited cell proliferation, increased cell apoptosis and reduced PNF volume. The combination with a MEK inhibitor did not increase efficacy compared to the single agent, supporting the hypothesis that VCP functions in parallel to, and may be modulated by, RAS-MAPK signaling under stress or oncogenic conditions. The significant effects of VCP inhibition in this pre-clinical study suggest a potential novel therapy for patients with PNFs.

BACKGROUND:Neurofibromatosis type 1 (NF1) is a heterogeneous neurodevelopmental disorder where motor deficits and attention-deficit/hyperactivity disorder (ADHD) occur at higher rates than typical populations. To characterize network-level changes associated with these impairments, we compared functional connectivity in youth with NF1 to typically developing controls. In the NF1 cohort, we analyzed relationships between functional connectivity, motor impairment, and ADHD severity. METHODS:Thirty-two participants (16 NF1, 16 typically developing; 8-16 years) underwent resting-state functional magnetic resonance imaging. Seed-to-voxel analyses were conducted for network seeds (sensorimotor, default mode, salience, and dorsal attention). Group differences were tested across brain voxels, and we examined brain-behavior associations within the NF1 group by correlating functional connectivity with motor scores from the Physical and Neurological Examination of Subtle Signs and ADHD severity from the parent-reported ADHD rating scale. RESULTS:Youth with NF1 displayed cortico-cortical hyperconnectivity and cortico-subcortical hypoconnectivity within the sensorimotor network, and hyperconnectivity within and between default mode, dorsal attention, frontoparietal, and visuospatial networks. In youth with NF1, poorer motor performance was associated with reduced cortico-cortical intrasensorimotor and sensorimotor-visuospatial connectivity. Greater inattentive symptoms were linked to decreased default mode-sensorimotor connectivity, increased default mode-visuospatial connectivity, and increased dorsal attention-frontoparietal connectivity. Default mode-sensorimotor/visuospatial hyperconnectivity correlated with worse total ADHD symptoms. CONCLUSIONS:Ineffective integration across default mode, sensorimotor, and visuospatial networks may be linked to motor and attentional phenotypes in NF1 and may serve as a candidate biomarker, pending replication in larger, more heterogeneous samples. We also demonstrate preliminary evidence of compensatory hyperconnectivity in youth with NF1 presenting with co-occurring neurodevelopmental difficulties.

In neurofibromatosis type 1 (NF1), loss-of-function mutations in the NF1 gene increase activation of the RAS-MEK-ERK signaling cascade, driving tumorigenesis. MEK inhibitors (MEKi) inhibit tumor growth and significantly shrink nerve tumors (neurofibromas). However, MEKi treatment alone fails to eradicate tumor cells, and tumor regrowth occurs after drug withdrawal, highlighting the limitations of targeting the single MEK pathway. An alternative strategy is to promote dephosphorylation of hyperactive kinases that drive tumor growth by enhancing phosphatase activity. We identified deregulated expression of genes encoding subunits of the PP2A phosphatase in neurofibroma and neurofibroma Schwann cells. We confirmed significant reductions in both the expression and enzymatic activity of the PP2A A and C subunits. FTY720, a compound known to restore PP2A phosphatase activity, inhibited tumor sphere formation by mouse and human neurofibroma Schwann cell progenitor cells, suppressed the proliferation of both primary and immortalized neurofibroma-derived Schwann cells, and induced cell apoptosis in vitro. Furthermore, treatment with FTY720-alone or in combination with MEKi-significantly suppressed tumor number and reduced tumor burden in remaining tumors in a murine model of NF1, highlighting the promise of using FTY720 as a novel therapeutic strategy in NF1.

Abstract Rationale: NF1-/- Schwann cells are the cells of origin in plexiform neurofibroma (PNF). This benign tumor formation occurs long after Nf1 loss in mouse models of the disease, suggesting that Schwann cells undergo secondary changes during tumor formation. However, additional genetic hits are not observed in this tumor type. Single cell RNA-sequencing implicated NF-κB signaling as upregulated in established tumor formation. Consistent with the idea that NF-κB signaling is a tumor driving signal, in human and mouse neurofibromas p-65 was nuclear (active) in neurofibroma cells, some of which were Schwann cells (Kershner et al., 2022). Methods: To test if activation of NF-κB signaling is a second step in neurofibroma formation we used a combination of multiplexed antibody staining, flow cytometry and RNA sequencing in tumors over their development. In vitro assays were utilized to determine the effect of NF-κB pathway modulation in Nf1-/- Schwann cells. Finally, we tested if blocking IKK2 activity in vivo reduces tumor formation or growth. Results: We identified markers of Schwann cells, fibroblasts and immune cells in PNF by multiplex imaging and flow cytometry. Tumor Schwann cells, but not pre-tumor Schwann cells in the same mice, expressed the cell surface markers CD44 and CD49f; cultured Nf1-/- Schwann cells upregulate these markers and nuclear (active) p65 when exposed to stressors known to activate NF-κB signaling, including prolonged serum depletion, Poly I:C, IL1β, and TNFα, or when infected with activated IKK2, which activates the NF-κB pathway; these Schwann cells increased secretion of cytokines that are immune cell chemoattracts. Concurrent increases in EMT genes were observed in vivo and in vitro. Treatment of DhhCre;Nf1fl/fl mice with the NF-κB pathway inhibitor BAY 11-7082 combined with MEK inhibitor Mirdametinib reduced the CD44+ CD49f+ Schwann cell population, tumor cell proliferation, the tumor immune cell population, and tumor cytokines. Conclusion: A two-step process to PNF formation is proposed, with Nf1 loss in Schwann cells an initiating step and the formation of an inflammatory microenvironment via activation of the NF-κB pathway and Schwann cell reprogramming as a second step. (Supported by DOD-HT9425-1-0435 (to NR and JS), NIH NS115438R01 (to DAL and NR) and a Children’s Tumor Foundation Young Investigator Award to RR) Citation Format: Ramya Ravindran, Noemi Kedei, Eui-Kyung Youn, Kwangmin Choi, Avery Volz, Jay Pundavela, David A. Largaespada, Jack F. Shern, Nancy Ratner. Two-step mechanism of plexiform neurofibroma formation: Role of the NF-κB pathway in neurofibroma formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2269.

BACKGROUND:Children and youth with neurofibromatosis type 1 (NF1) commonly experience motor, behavioral, and cognitive problems. Promising findings from an NF1 mouse model suggested that the antioxidant N-acetylcysteine (NAC) might address underlying mechanistic deficits. METHODS:We conducted a 12-week (8-week treatment, 4-week washout), double-blind, placebo-controlled randomized trial of NAC in 8-16-year-olds with NF1. We screened 481 children, identified 203 eligible, and randomized 25; 23 received at least one dose (10 NAC at ∼70 mg/kg/day; 13 placebo). Outcomes were safety, tolerability, motor function (primary: Physical and Neurological Examination for Subtle Signs), cognitive function (secondary: attention deficit/hyperactivity disorder symptom scores and executive function measures), and exploratory biomarkers (transcranial magnetic stimulation and magnetic resonance spectroscopy measures). RESULTS:In the modified intention to treat analysis, no significant difference in Physical and Neurological Examination for Subtle Signs was observed between groups. In completer analyses, NAC did not improve any secondary or exploratory outcome at 8 weeks. NAC was generally well tolerated, although two participants were withdrawn by parents because of worsening behavior. Test-retest reliability of scales, measures, and biomarkers over 12 weeks was generally moderate. CONCLUSIONS:NAC was generally well tolerated but did not improve motor or behavioral outcomes in youth with NF1. Larger, longer trials with preceding dose-escalation and target-engagement studies are needed to evaluate new therapies.

Pyroptosis, a lytic and immunogenic form of cell death, holds broad therapeutic potential, yet its selective induction in specific cell populations remains a fundamental challenge. Loss of the NF1 tumor suppressor, one of the most frequent events across pediatric and adult cancers, elevates RAS-GTP and drives tumorigenesis through hyperactivated RAS signaling. Here we demonstrate that protein kinase Cδ (PKCδ) agonism selectively triggers pyroptosis in NF1-deficient cells by exploiting their dependency on KRAS. PKCδ directly phosphorylates KRAS at S39 and S181, inducing KRAS-GDP accumulation and driving endoplasmic reticulum translocation. The dually phosphorylated KRAS-GDP interacts with caspase-8 and competitively displaces inhibitory BCL2, promoting caspase-8/caspase-3/gasdermin-E-mediated pyroptosis. This vulnerability is conserved across multiple NF1-deficient tumor types, and PKC agonism suppresses NF1-deficient neurofibroma and malignant peripheral nerve sheath tumor growth in vivo . These findings establish the inactive KRAS-GDP as a functionally active signaling molecule and PKCδ agonism as a selective therapeutic strategy for NF1-deficient cancers.

Patients with neurofibromatosis type 1 develop Schwann cell tumors called neurofibromas that arise within peripheral nerves, driven by loss of neurofibromin and consequent increased RAS/RAF/MEK signaling. MEK inhibitors achieve partial responses for benign neurofibromas but are limited by toxicity and incomplete efficacy, necessitating alternative approaches. Using the Dhh-Cre;Nf1fl/fl neurofibroma mouse model, we found that genetic ablation of Kras, but not Hras, markedly reduced neurofibroma development, inhibited MAPK activation, and rescued disrupted Remak bundles that are a morphologic hallmark of neurofibromas. These findings reveal a RAS paralog-specific requirement for KRAS in NF1-deficient neurofibroma initiation. Pharmacological KRAS inhibition with BI6674, an orally bioavailable KRASmulti inhibitor, reduced tumor volume and proliferation in established neurofibromas and remodeled the tumor immune microenvironment, decreasing macrophages and dendritic cells. Combining KRAS and MEK inhibition further enhanced tumor regression. These findings demonstrate that KRAS is essential for neurofibroma formation and represents a promising therapeutic target, supporting clinical evaluation of KRAS inhibition for neurofibromas in patients with neurofibromatosis type 1.

Germline pathogenic variants that activate the Ras/mitogen-activated protein kinase (MAPK) pathway cause neurodevelopmental disorders called 'Rasopathies'. Because many affected proteins directly regulate Ras, causative mutations may alter other Ras-dependent pathways in addition to MAPK signaling. To better understand which Rasopathy sequelae result from hyperactivation of downstream MAP kinases, we engineered mice with a gain-of-function mutation in the terminal MAP kinase gene Mapk1, which encodes ERK2 and is associated with the recently described genetic syndrome MAPK1-related Rasopathy (MRR). Mapk1 mutant mice successfully modeled key aspects of the human MRR phenotype, including small stature, facial dysmorphism, and impaired cognitive function. Importantly, they recapitulated phenotypes identified in Rasopathy models with upstream Ras activation, such as neurofibromatosis type 1 (NF1): oligodendrocyte lineage defects, reactive astrogliosis, memory deficits, and hypersensitivity to sensory stimuli. These findings emphasize the importance of downstream MAPK signaling in the pathophysiology of neurocognitive symptoms observed in Rasopathy syndromes.

Supplementary Figure 1 illustrates the concept of synthetic lethality and ways to implement this for therapeutics discovery.

Neurofibromatosis type I (NF1) is a common cancer predisposition syndrome caused by heterozygous loss-of-function mutations in the tumor-suppressor gene NF1. Individuals with NF1 develop benign tumors of the peripheral nervous system (neurofibromas), originating from the Schwann cell (SC) lineage after somatic loss of the wild-type NF1 allele, some of which progress further to malignant peripheral nerve sheath tumors (MPNST). There is only one FDA-approved targeted therapy for symptomatic plexiform neurofibromas and none approved for MPNSTs. The genetic basis of NF1 syndrome makes associated tumors ideal for using synthetic drug sensitivity approaches to uncover therapeutic vulnerabilities. We developed a drug discovery pipeline to identify therapeutics for NF1-related tumors using isogeneic pairs of NF1-proficient and NF1-deficient immortalized human SCs. We utilized these in a large-scale high-throughput screen for drugs that preferentially kill NF1-deficient cells, through which we identified 23 compounds capable of killing NF1-deficient SCs with selectivity. Multiple hits from this screen clustered into classes defined by the method of action. Four clinically interesting drugs from these classes were tested in vivo using both a genetically engineered mouse model of high-grade PNSTs and human MPNST xenografts. All drugs tested showed single-agent efficacy in these models as well as significant synergy when used in combination with the MEK inhibitor selumetinib. This high-throughput screen platform yielded novel therapeutically relevant compounds for the treatment of NF1-associated tumors and can serve as a tool to rapidly evaluate new compounds and combinations in the future.

Neurofibromatosis 1 (NF1) is an inherited tumor-predisposition syndrome in which mutations in the neurofibromin gene NF1 cause various dysfunctions in the nervous system, including pain that substantially diminishes quality of life. Pain can be both tumor-dependent and tumor-independent and is challenging to manage therapeutically. Mice with homozygous loss of Nf1 in Schwann cells exhibit pain before tumors form, and this pain is associated with increased gene expression of glial cell line-derived neurotrophic factor (GDNF). Here, we investigated the cause of increased GDNF production and its downstream target cells in a mouse model of NF1. We found that Schwann cells were the dominant source of GDNF in NF1 mice. Schwann cell-derived GDNF activated the receptor GFRα1 on high-threshold mechanoreceptors and polymodal C-fibers, which mediated mechanical hypersensitivity in mice. Treating the mice with pharmacological inhibitors of mitogen-activated protein kinase (MAPK) signaling reduced pain-like behaviors and the expression of GDNF at the mRNA and protein levels in Schwann cells. The findings provide insight into the signaling pathways that underlie tumor-independent pain in NF1 and identify a targetable pathway for therapeutic intervention.

Taqman probes used for quantitative PCR.

Loss of NF2 tumor suppressor activity causes NF2-related schwannomatosis. Proximity biotinylation identified proteins proximal to Merlin isoform 1 and isoform 2 at confluence, when Merlin is active, but not in sub-confluent, growing cells. These data confirmed Merlin involvement in cell-cell and cell-substrate junctions, identified new signal transduction pathways, and highlighted a role for Merlin in intracellular transport. Direct binding assays identified the small GTPases RalA and RalB as high affinity PIP2-dependent Merlin binding proteins that co-localized with RalA/B on the plasma membrane. Merlin loss resulted in aberrant activation of RalA and RalB at high cell density. Merlin competitively inhibited RalB binding to its exocyst effectors Sec5 and Exo84 and regulated the kinetics of exocytosis in a RalB dependent manner. Thus, RalB is a novel binding partner for active Merlin, and the RalA/B pathway is a possible therapeutic target to treat NF2-related schwannoma.

Dual inhibition of ENG and MEK efficiently blocks ENG-Smad1/5 and MAPK/ERK pathway activation in ST88-14 cells.

Dual inhibition of ENG and MEK efficiently blocks ENG-Smad1/5 and MAPK/ERK pathway activation in ST88-14 cells.

ENG knockdown does not affect MPNST cell apoptosis in vivo.

Significant deregulated genes in shENG versus shScramble STS26T cells.