NF1 AS A REGULATOR OF CYTOSKELETON DYNAMICS AND BIOMARKER FOR DECISION-MAKING IN HER2-POSITIVE BREAST CANCER

The tumour suppressor NF1 is best characterised as a canonical negative Ras regulator, but sparse evidence suggests additional Ras-independent roles. The interaction of its product neurofibromin with both microtubule (MT) and actin cytoskeleton remains poorly characterised to date but may be of particular therapeutic interest as NF1 is somatically mutated across multiple tumour types. We identified NF1 as the second most differentially mutated gene between metastatic and primary HER2+ breast cancer (BC), so we investigated the biological consequences of NF1 loss on sensitivity to antineoplastic treatment and on the metastatic process. We generated NF1 knockout (KO) HER2+ BC cells (i.e.: BT-474, SK-BR-3, HCC1954) by CRISPR/Cas9. NF1KO cells demonstrated exquisite sensitivity to the antibody-drug conjugate (ADC) trastuzumab emtansine (T-DM1). This hypersensitivity was specific to the mertansine MT-targeting component (DM1) since it was i) replicated by the naked payload but not the antibody alone; ii) absent with other ADCs (i.e.: trastuzumab deruxtecan [T-DXd]); iii) not accompanied by increased T-DM1 uptake; and iv) associated with increased drug-target engagement in NF1KO cells. The mechanism is likely Ras-independent, as oncogenic Ras overexpression did not alter T-DM1 sensitivity. By combining molecular biology with confocal and live-cell imaging, we discovered that NF1KO cells exhibited marked signs of altered mitosis with longer G2/M duration, supernumerary centrosomes, chromosome misalignment and frequent aneuploidy. In agreement, we found that BC patients bearing NF1 mutations from the AACR-GENIE cohort exhibited a higher aneuploidy score (AS). MTs in NF1KO cells were severely hypodynamic and showed imbalanced expression of plus/minus end MT-associated proteins (MAPs). This was also associated with an increased abundance of GTP-bound tubulin, a conformational state known to cause MT stability. This raises the intriguing possibility that NF1 may directly regulate tubulin intrinsic GTP-hydrolysis, similar to its GTPase activity on Ras. Finally, we show that NF1KO cells present with markedly anchorage-independent growth advantage, a feature associated with acquisition of metastatic potential. Assessment of biophysical properties of NF1KO models showed enhanced actin subcortical mesh with upregulation of cell junction markers and marked global stiffening by atomic force microscopy. This study provides extensive mechanistic evidence for a previously underappreciated role of NF1 in MT and actin dynamics, which reshapes our understanding of its tumour-suppressive activity and provides a rationale for the pharmacological targeting of NF1-mutated tumours in prospective clinical trials.