Malignant Peripheral Nerve Sheath Tumors Activate Distinct Immunosuppressive Pathways Following Radiotherapy and are Associated with Immune Depletion In Vivo

Abstract

Patients with neurofibromatosis type I, caused by NF1 loss, develop benign plexiform neurofibromas (pNF) in their peripheral nervous system (PNS). Malignant transformation of pNFs into malignant peripheral nerve sheath tumors (MPNSTs) occurs following CDKN2A/B and SUZ12 loss, a process associated with radiotherapy (RT). However, the molecular mechanisms underlying RT responses by different PNS cell types remain unclear. We hypothesized normal peripheral nerve cells, pNFs, and MPNSTs harbor distinct RT responses. Patient derived NF1 WT immortalized peripheral nerve cells (iPNs), NF1 mutant pNF cells, and NF1/CDKN2AB/SUZ12 mutant MPNST cells were used to study RT responses in vitro. CRISPRi was used to assess the functional effects of candidate gene repression. In vitro viability was measured by cell counts. Transcriptomic signatures were measured by bulk RNA-sequencing and integrated with single-cell RNA sequencing (scRNA-seq) data from patient-derived pNF and MPNST resection specimens. Radiation dose response curves revealed pNF cells (IC50 0.61 Gy) were more radiosensitive than MPNST cells (4.15 Gy). WT iPNs, NF1 deficient iPNs, and pNFs cells displayed no difference in cell viability (p = 0.67; t-test) following initiation of 2 Gy x 5 fractions, while MPNST cells were significantly more viable (p = 0.02; t-test). Principal component analysis of bulk RNA-sequencing data at 5 or 14 days following 2 Gy x 5 fractions revealed cell line of origin accounted for the greatest inter-sample variation (64.9% variance), with additional components separating samples based on radiation presence and timing. Using the most variable genes in PCA space to identify markers of RT response, iPNs and pNFs upregulated pro-apoptotic pathways (BAD, DAPK3) at 5 days post-radiation while MPNST cells alone upregulated pro-survival growth factor signaling). At 14 days post radiation, MPNST cells uniquely upregulated TGFβ signaling and interferon response circuits. Incorporation of scRNA-seq data revealed enrichment of growth factor signaling and TGFβ signatures in MPNSTs compared to pNF. Moreover, MPNST harbored significantly fewer immune cells than pNFs (p = 0.008, t-test), suggesting cell-autonomous signaling and crosstalk with the microenvironment are both critical to MPNST radioresistance. Our data indicate additional genetic hits beyond NF1 loss may be required for RT-associated malignant transformation of pNFs and radioresistance in MPNSTs. Analysis of transcriptomic responses to RT suggests that upregulated growth factor signaling and TGFβ-associated immunosuppression are distinct features of MPNST. Future work will focus on CRISPRi screens to unbiasedly nominate functional modifiers of RT response in NF1/CDKN2AB deficient tumors, which may be broadly useful in cancer.