Abstract 265: NF1 is a novel MAP that modulates microtubule damage repair: rediscovering a tumor suppressor

Abstract Neurofibromin 1 (NF1) is a tumor suppressor widely mutated across several cancers and primarily recognized as a GTPase-activating protein (GAP) that inhibits RAS signaling. Other potential functions have been suggested but still not fully explored, due to its large size and complex domain structure. It remains unclear whether somatic mutations in NF1 can be used to inform clinical decision-making. We previously showed that NF1 plays a novel RAS-independent role in microtubule damage repair in response to the microtubule destabilizing drug Mertansine (DM1), the payload in the commonly used Antibody-Drug Conjugate T-DM1. We showed that NF1 exhibits biochemical properties of a bona fide Microtubule-Associated Protein (MAP) and specifically enhances microtubule (MT) repair, a recently discovered phenomenon whose regulation remains poorly characterized. A key unresolved question is whether the MT-repairing function is dependent or uncoupled from the GAP activity; this requires the development of novel methods to quantify MT damage in vitro and in vivo. We developed a novel analysis method to systematically identify and quantify areas of decreased tubulin intensity in MT stabilized in vitro in conditions known to lead to intratubular damage, using TIRF microscopy. Our method allowed us to asses MT repair as the amount of free-tubulin incorporation spatially overlapping with damaged sites. DM1 increased the number of intra-lattice discontinuities and abolished repair of damaged sites. This was rescued by the addition of NF1, which led to a strong increase in the fraction of repaired MTs, with or without DM1.To further confirm the role of NF1 in modulating DM1-dependent microtubule damage, we quantified tubulin staining patterns by high-resolution immunofluorescence (MT length, mean staining intensity and other derived parameters) in HER2+ breast cancer cell lines, in which we managed to obtain, for the first time, stable expression of WT NF1 in a KO background by lentiviral transduction. NF1 ablation or rescue did not lead to visible differences in MT signal in the absence of treatment. Upon addition of DM1, MTs were significantly shorter and more damaged in NF1 KO cells; this was completely rescued by WT NF1 re-expression. This system allows to directly measure the activity of NF1 or its mutants on MT damage, allowing the quantification of structure-function relationships in a relevant cellular system. We generated a collection of NF1 mutants, selected based on clinical associations and prior literature, that impact various domains of NF1, either affecting its GAP activity, its MT binding or its structural stability. Preliminary results suggest that GAP and MT-repairing activity can be partially but not entirely uncoupled. We speculate that some effects of NF1 somatic loss in cancer may be revisited as due to MT dysfunction and not to altered RAS signaling. Funded by Next Generation EU- PNRR-MAD-2022-12376934 Citation Format: Eleonora Messuti, Giulia Tini, Alessia Castiglioni, Ambra Dondi, Giuseppe Ciossani, Silvia Monzani, Daria Khuntsariya, Bruno Achutti Duso, Marcus Braun, Zdenek Lansky, Simona Rodighiero, Luigi Scietti, Luca Mazzarella. NF1 is a novel MAP that modulates microtubule damage repair: rediscovering a tumor suppressor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 265.