Abstract P5-13-04: NF1 mutations render HER2+ breast cancer highly sensitive to T-DM1 by altering microtubule dynamics
Authors: Duso Bruno A , Dorronzoro Elena Gavilán , Tini Giulia , de Filippo Maria R , Bonetti Emanuele , Ippolito Maria R , Soriani Chiara , D'Amico Paolo , Rodighiero Simona , Curigliano Giuseppe , Santaguida Stefano , Cristofanilli Massimo , Pelicci Pier Giuseppe , Mazzarella Luca 
Published: March 2022

Abstract
Background: Despite major technological and conceptual advancements, treatment decisions in HER2+ metastatic breast cancer (mBC) remain largely based on clinical evidence, with no established predictive biomarkers to direct treatment for individual patients. The tumour suppressor Neurofibromatosis 1 (NF1) has been implicated in endocrine resistance but its role remains incompletely characterized in mBC. NF1 is best known as a GTPase-activating protein (GAP) that attenuates RAS signalling. However, the GAP function is likely not limited to RAS, and NF1 has been involved in other GTP-dependent processes including cytoskeletal dynamics. Data mining and analysis of public mutational registries revealed NF1 mutations as particularly enriched in HER2+ mBC compared to other molecular subtypes. Methods: To investigate the biological consequences of NF1 loss, we generated NF1 KO HER2+ mBC cell lines (BT474 and SKBR3) by CRISPR-Cas9 and both 2D and 3D proliferations assays were used for drug sensitivity profiling; live-cell imaging, high-resolution confocal microscopy and an ad-hoc computational algorithm were employed to study cell fate and microtubule conformational changes. Patient data were obtained from the Northwestern University through a prospective observational study in mBC patients. Results: Screening of several compounds approved for HER2+ mBC showed that response was generally equal or reduced in NF1 KO vs WT cells. However, response to trastuzumab-emtansine (T-DM1) was significantly increased in NF1 KO cells (IC50 ~0,3 vs 1,6 μg/mL in NF1WT). This sensitization was not observed with other antibody drug conjugates (ADCs) like DS-8201 and was reproducible with maytansine alone, suggesting a pharmacologically relevant NF1 activity on microtubules. Using the FUCCI(Ca) reporter, which tracks cell cycle progression at single-cell level, we saw a more prominent G2/M phase arrest and cell death upon T-DM1 treatment in NF1 KO compared to WT cells. Notably, NF1 KO cells exhibited a higher frequency of aberrant mitotic figures (chromosome alignment defects and multipolar spindle formation) and stronger β-galactosidase activity, an established marker of senescence. Collectively, these results suggest that NF1 KO cells become particularly subject to T-DM1-triggered mitotic catastrophe. Dephosphorylation of GTP-bound tubulin is required for appropriate microtubular dynamics; so-called “GTP islands” within the inner microtubule region are prone to rapid repolymerization and are normally kept at low levels. We hypothesize that expanded GTP-tubulin islands generated by the loss of NF1 GAP activity is a major cause of microtubular instability in NF1 KO cells. Preliminary evidence in support of this model was obtained by quantification of GTP-tubulin with a specific antibody. Finally, we assessed the predictive role of NF1 as a biomarker for T-DM1 response in a cohort of 300 mBC patients with mutational data in circulating tumour DNA (Guardant 360); we identified 13 heavily pretreated patients (&gt;4 prior lines) who received T-DM1, of which 3 had loss-of-function NF1 mutations and 10 were NF1 WT. Median progression-free survival was higher in NF1-mutated than WT patients (334 vs 80 days); given the small sample size, these results cannot yet be considered significant (p=0.14). Conclusions: These results provide preliminary mechanistic and clinical evidence supporting the use of NF1 loss to guide treatment in HER2+ mBC. As novel HER2-specific agents are being rapidly added to the therapeutic arsenal, we propose biology-driven criteria to identify patients that may benefit specifically from T-DM1. In addition, NF1 dependence for correct microtubular dynamics may be exploited by other inhibitors of microtubular polymerization in use as ADC payloads, further extending the potential usefulness of NF1 determination.
Citation Format: Bruno A Duso, Elena Gavilán Dorronzoro, Giulia Tini, Maria R de Filippo, Emanuele Bonetti, Maria R Ippolito, Chiara Soriani, Paolo D'Amico, Simona Rodighiero, Giuseppe Curigliano, Stefano Santaguida, Massimo Cristofanilli, Pier Giuseppe Pelicci, Luca Mazzarella. NF1 mutations render HER2+ breast cancer highly sensitive to T-DM1 by altering microtubule dynamics [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-04.