Neural Crest-Like Cell State in MPNSTs is Dependent on SHH Pathway Activation

Toronto Western Hospital, University of Toronto

Severa Bunda, Jeff Liu, Vikas Patil Mira Li, Gelareh Zadeh

INTRODUCTION: The molecular mechanism that drives malignant transformation from a neurofibroma to a MPNST in NF1 individuals has yet to be fully understood. Our previous work has demonstrated that SHH pathway activation drives malignant transformation in a subset of malignant peripheral nerve sheath tumors (MPNSTs). Here, we demonstrate that SHH pathway activation induces a neural crest cell-like state in a subset of MPNSTs.

METHODS: We performed single nuclear RNA sequencing on 6 tumors (5 MPNSTS and 1 atypical neurofibroma). Data was generated using the 10X Chromium platform and analyzed using the Seurat 3.0 pipeline. Cellular trajectories were inferred by the generating of relative pseudotime through a linear transformation relative to cells with lowest and highest pseudotimes with Moncole2. We performed in vitro experiments to assess the correlation between SHH pathway activation and neural crest signatures. We inhibited SHH pathway with sonidegib, an SMO inhibitor, in MPNST cell lines and performed RT-PCR to assess markers of neural crest cell signatures. In addition, we assessed cellular viability with treatment.

RESULTS: A total of 43,365 cells were analyzed, with 30,518 tumoral cells and 12,847 non-tumoral cells characterized. Notably, we observed that cells that express SMO at high levels, with SHH pathway activation, express neural crest cell makers. These cell populations are negative for S100B but overexpress TWIST1. Single cell trajectory analysis further demonstrates a pseudotemporal continuum from atypical neurofibromas (Schwann cells), MPNSTs (Schwann cell precursor cells) and other MPNSTs (neural crest cells), which supports that these tumors fall along the developmental trajectory of neural crest cell lineage. SHH pathway activity was high in S462TY MPNST cell line and low in STS-26T MPNST cell line. S462TY also demonstrated prominent elevation in expression of neural crest cell markers TWIST1, SOX9, SNAI2, OTX2, PAX4 and PAX6. We found that treatment with sonidegib treatment attenuates the expression of neural crest cell signatures. Finally, inhibiting SHH pathway activation reduced cellular viability in MPNST cell lines.