Emerging RAS-targeted therapies offer new opportunities to investigate therapeutic vulnerabilities in NF1-associated tumours and schwannomatosis. This talk will explore how reliance on RAS signalling may confer therapeutic sensitivity even in tumours without RAS mutations. Building on these insights, the discussion will consider rational combination strategies to improve treatment responses in these settings.

Background: Vestibular schwannomas (VS) are the hallmark tumour of NF2-related schwannomatosis (NF2-SWN), causing significant morbidity including hearing loss, balance dysfunction and brainstem compression. Tumour-associated macrophages (TAMs) are among the most abundant non-neoplastic cells in VS; alternatively activated TAMs associate with faster tumour growth, and inflammatory mediators including NLRP3 and IL-1β have been linked to sensorineural hearing loss. Despite this, the effect of NF2 mutation has not previously been studied in macrophages.
Methods: We generated CRISPR/Cas9-edited Nf2-null murine immortalised bone marrow-derived macrophages (iBMDMs) to model merlin loss. Merlin expression, cell morphology and proliferation were assessed alongside macrophage functions relevant to tumour inflammation. Phagocytosis was assayed, NLRP3 inflammasome priming was induced with LPS and activation with nigericin, imiquimod or ATP. Responses were quantified by cell death assays, ELISA for IL-6 and IL-1β, and Western blotting for NLRP3, (pro)-IL-1β and pro-caspase-1.
Results: Nf2-null iBMDMs showed loss of merlin expression but no detectable change in morphology or proliferation compared with wild-type macrophages. Phagocytic capacity was unchanged. Following LPS priming, Nf2-null macrophages expressed similar NLRP3 and pro-IL-1β and released comparable IL-6. Subsequent NLRP3 activation induced equivalent cell death and IL-1β release.
Conclusion: Direct Nf2 loss does not appear sufficient to intrinsically reprogramme macrophage inflammatory or phagocytic function in vitro, supporting a model in which VS TAM behaviour is more likely shaped by the tumour microenvironment. Future work will explore tumour-derived signals that recruit, polarise and position macrophages in NF2-SWN and sporadic VS, while investigating how NLRP3/IL-1β signalling contributes to VS pathology, its cellular targets and therapeutic tractability.

Background:
MPNST are highly aggressive soft tissue sarcomas with poor clinical outcomes largely driven by recurrence. This study investigates molecular mechanisms of recurrence to identify prognostic markers and potential therapeutic vulnerabilities.

Methods:
We gathered a cohort of 22 matched primary and recurrent MPNST from individuals with neurofibromatosis type 1, including local and distant relapses as well as 40 primary and 11 relapsed unmatched cases. Of these, we selected 13 matched primary and recurrent samples and performed global DNA methylation profiling, followed by copy number inference and clustering with a large reference cohort of peripheral nerve sheath tumors (PNSTs). Single-nucleus RNA sequencing was conducted on eight primary tumors and matched recurrent or metastatic lesions.

Results:
In our cohort, the median time to first recurrence was nine months. Methylation analysis revealed high CNV burden and similar epigenetic profiles in matched primary and recurrent tumors. Preliminary results from single-nucleus RNA sequencing showed distinct separation of malignant cells for each individual tumor. Notably, matched primary-relapse pairs formed neighboring malignant cell clusters in most cases, suggesting a conserved patient-specific malignant program during progression, while also implying additional transcriptional remodeling between primary and relapse. Differential gene expression analysis indicate enrichment of inflammatory, immune-related, and stress-response pathways in malignant cells of primary tumors compared with metastatic lesions.

Conclusion:
Preliminary results from analyses indicate that MPNST recurrence appears to preserve patient-specific epigenetic and gene expression signatures while undergoing progressive transcriptomic remodeling. Further analyses focus on tumor–microenvironment interactions and conserved malignant cell populations between primary and relapse tumors as therapeutic targets.

Background: RAS inhibition through the GTPase-Activating Protein (GAP)-Related Domain (GRD) is considered neurofibromin’s key biological activity. However, many NF1 mutations occur outside the GRD or other well-characterized domains, suggesting additional, largely unexplored functions. We recently found that somatic NF1 loss enhances cancer cell sensitivity to DM1, a microtubule-targeting agent. This prompted us to study neurofibromin in microtubule biology.
Methods: we investigated the physical and functional interaction between neurofibromin and microtubules in i) breast cancer cells (TP53-deficient), engineered to knock out NF1 and/or re-express WT or mutant NF1; ii) in vitro-reconstituted microtubules imaged by either fluorescence (TIRF) or electron microscopy, together with insect-purified WT/mutant, or mScarlet-tagged neurofibromin.
Results: Neurofibromin showed defining features of Microtubule-Associated Proteins (MAP), as it: i) bound microtubules in vitro (figure 1A); ii) co-localized with the mitotic spindle (figure 1B); iii) accelerated tubulin polymerization and microtubule dynamics, in particular intra-tubular damage repair in vitro and in vivo (figure 1C-D). Intriguingly, the RAS-GAP-deficient K1423E mutant was completely deficient in microtubular repair both in vitro and in vivo, suggesting biochemical overlap between GAP and MAP functions. However, MAP activity was RAS-independent since it i) was not replicated by RASG12V expression; ii) was observed in vitro in the absence of RAS proteins. An in silico model of neurofibromin-microtubule interaction, grounded on preliminary structural data, suggests a vast interaction surface at the large HEAT/ARM domain (figure 1E), whose function was uncharacterized to date.
Conclusion: NF1 deficiency generates microtubular dysfunction that likely contributes to neurofibromatosis pathogenesis, given the established role of microtubules in neural development and tumorigenesis.

Although RNA-based therapeutics are expanding treatment options for many rare and common diseases, they remain largely unexplored in neurofibromatosis type 1 (NF1).
We have developed engineered exosomes for gene-replacement strategies and demonstrate their ability to differentially modulate MAPK–ERK signaling and Schwann cell growth.
In parallel, we are evaluating RNA oligonucleotides designed to restore NF1 expression and/or compensate for neurofibromin deficiency in NF1-deficient Schwann cells.