Table of mice enrolled in drug study

Kaplan Meier Survival Curve and Tolerability

Gene set enrichment analysis of RAS signatures

Reactome G1 Phase Heatmap and Top DEGs

Raw log2LFQ values for kinases profiled by MIB/MS

Plexiform neurofibromas (PNFs) are benign tumors of the peripheral nervous system that represent a major source of morbidity in neurofibromatosis type 1 (NF1). A substantial proportion of patients do not respond to current therapies or experience intolerable side effects. Transcriptomic characterization of murine and human PNF at bulk and single-cell resolution identified transforming growth factor–β (TGFβ) signaling as a key upstream regulator, driving aberrant basement membrane (BM) protein production by neoplastic Schwann cells and Fbs. Conditional TGFβ1 overexpression in Nf1 -deficient Schwann cells driven by Hoxb7- Cre promoted PNF growth and malignant transformation in vivo. Conversely, pharmacologic inhibition of the type I TGFβ receptor (TGFβRI) reduced PNF tumor burden in Nf1 mutant mice. Proteomic characterization of the extracellular matrix (ECM) showed reduced BM proteins upon TGFβRI inhibition. These findings implicate TGFβ as a potential therapeutic target in PNF and provide insights into the role of TGFβ signaling in orchestrating ECM dynamics in the PNF microenvironment.

PathView normalized signature scores for biological pathways relevant to tumor progression, immune response and tumor microenvironment for each sample.

Normalized count matrix. Probes with raw counts below threshold were excluded.

Raw count matrix and probe annotations.

Background/Objective:Neurofibromatosis type 1 (NF1) is a multisystem disorder that affects ~1/3000 newborns. Plexiform neurofibromas (PN) are present in about half of cases and can transform (lifetime risk of 8-13%) into malignant peripheral nerve sheath tumor (MPNST), a highly aggressive and metastatic sarcoma with poor survival. Unfortunately, there are currently no reliable biomarkers to identify PN at risk of undergoing malignant transformation. Our research has revealed that a subset of benign-appearing and atypical PN exhibit deregulated immune surveillance and T-cell infiltration that precede malignant transformation. In this study, we are analyzing tumor microenvironment and immune landscape in NF1-related tissue specimens to identify biomarkers of disease progression. To power these studies, this project focused on constructing a dataset of NF1-related samples to be analyzed. Methods:701 patients were identified via Cerner billing codes and pathology archives. De-identified clinical data, including presenting symptoms, relevant clinical history, pathology diagnoses, disease features, prior chemotherapeutics/radiation, prior gene profiling, and imaging features were collected. Results: We selected 86 patients with a total of 175 samples. 81% of patients had a clinical diagnosis of NF1, and 6% had a history of MPNST. Out of the 175 samples, 54 were in the head and neck, 42 in the thorax, 28 in the lower extremity, 27 in the upper extremity, 26 in the pelvis/abdomen, and 15 in the paraspinal region. The leading causes of procedures were pain (41%), growth (40%), and concern for malignancy (27%). The most common tissue diagnoses were neurofibroma (51.4%), PN (20%), and undefined-grade MPNST (11%). Out of the 46 MPNSTs, 30 were primary tumors, 4 metastases, and 12 local recurrences. Conclusion and Potential Impact:The results of this study will provide valuable insights to inform preclinical models of NF1-tumorigenesis to validate these findings and identify novel treatment approaches for individuals affected by these rare but devastating tumors.

Supplemental Figure 10. Survivin inhibition with LQZ-7I abrogates viability of human MPNST and murine primary Schwann cell precursors.

Supplemental Figure 11. Diagnostic performance of a three gene signature (CXCL8, HLA-DQB1 and IL6) to identify neurofibromas contiguous with MPNST.

Abstract Purpose: Plexiform neurofibromas (PNF) are benign peripheral nerve sheath tumors (PNST) associated with neurofibromatosis type 1 (NF1). Despite similar histologic appearance, these neoplasms exhibit diverse evolutionary trajectories, with a subset progressing to malignant peripheral nerve sheath tumor (MPNST), the leading cause of premature death in individuals with NF1. Malignant transformation of PNF often occurs through the development of atypical neurofibroma (ANF) precursor lesions characterized by distinct histopathologic features and CDKN2A copy-number loss. Although genomic studies have uncovered key driver events promoting tumor progression, the transcriptional changes preceding malignant transformation remain poorly defined. Experimental Design: Here we resolve gene-expression profiles in PNST across the neurofibroma-to-MPNST continuum in NF1 patients and mouse models, revealing early molecular features associated with neurofibroma evolution and transformation. Results: Our findings demonstrate that ANF exhibit enhanced signatures of antigen presentation and immune response, which are suppressed as malignant transformation ensues. MPNST further displayed deregulated survival and mitotic fidelity pathways, and targeting key mediators of these pathways, CENPF and BIRC5, disrupted the growth and viability of human MPNST cell lines and primary murine Nf1-Cdkn2a-mutant Schwann cell precursors. Finally, neurofibromas contiguous with MPNST manifested distinct alterations in core oncogenic and immune surveillance programs, suggesting that early molecular events driving disease progression may precede histopathologic evidence of malignancy. Conclusions: If validated prospectively in future studies, these signatures may serve as molecular diagnostic tools to augment conventional histopathologic diagnosis by identifying neurofibromas at high risk of undergoing malignant transformation, facilitating risk-adapted care.

Differentially expressed genes comparing GTEx Normal Nerve and TCGA MPNST obtained from XenaBrowser.net

Supplemental Figure 7. Gene expression analysis of harmonized RNAseq data from normal nerve and MPNST samples obtained from GTEx and TCGA.

Log2 transformed normalized count matrix. Probes with raw counts below threshold were excluded.

List of Normal Nerve and MPNST samples obtained from XenaBrowser.net

Supplemental Figure 1. Heatmap of raw counts.

Supplemental Figure 8. Core molecular features of human ANNUBP and MPNST are retained in two independent genetically engineered mouse models driven by combined inactivation of Nf1 and Ink4a/Arf.

Supplemental Figure 2. H3K27me3 immunohistochemical staining of MPNSTs.