Abstract 2269: Two-step mechanism of plexiform neurofibroma formation: Role of the NF-κB pathway in neurofibroma formation.

Abstract Rationale: NF1-/- Schwann cells are the cells of origin in plexiform neurofibroma (PNF). This benign tumor formation occurs long after Nf1 loss in mouse models of the disease, suggesting that Schwann cells undergo secondary changes during tumor formation. However, additional genetic hits are not observed in this tumor type. Single cell RNA-sequencing implicated NF-κB signaling as upregulated in established tumor formation. Consistent with the idea that NF-κB signaling is a tumor driving signal, in human and mouse neurofibromas p-65 was nuclear (active) in neurofibroma cells, some of which were Schwann cells (Kershner et al., 2022). Methods: To test if activation of NF-κB signaling is a second step in neurofibroma formation we used a combination of multiplexed antibody staining, flow cytometry and RNA sequencing in tumors over their development. In vitro assays were utilized to determine the effect of NF-κB pathway modulation in Nf1-/- Schwann cells. Finally, we tested if blocking IKK2 activity in vivo reduces tumor formation or growth. Results: We identified markers of Schwann cells, fibroblasts and immune cells in PNF by multiplex imaging and flow cytometry. Tumor Schwann cells, but not pre-tumor Schwann cells in the same mice, expressed the cell surface markers CD44 and CD49f; cultured Nf1-/- Schwann cells upregulate these markers and nuclear (active) p65 when exposed to stressors known to activate NF-κB signaling, including prolonged serum depletion, Poly I:C, IL1β, and TNFα, or when infected with activated IKK2, which activates the NF-κB pathway; these Schwann cells increased secretion of cytokines that are immune cell chemoattracts. Concurrent increases in EMT genes were observed in vivo and in vitro. Treatment of DhhCre;Nf1fl/fl mice with the NF-κB pathway inhibitor BAY 11-7082 combined with MEK inhibitor Mirdametinib reduced the CD44+ CD49f+ Schwann cell population, tumor cell proliferation, the tumor immune cell population, and tumor cytokines. Conclusion: A two-step process to PNF formation is proposed, with Nf1 loss in Schwann cells an initiating step and the formation of an inflammatory microenvironment via activation of the NF-κB pathway and Schwann cell reprogramming as a second step. (Supported by DOD-HT9425-1-0435 (to NR and JS), NIH NS115438R01 (to DAL and NR) and a Children’s Tumor Foundation Young Investigator Award to RR) Citation Format: Ramya Ravindran, Noemi Kedei, Eui-Kyung Youn, Kwangmin Choi, Avery Volz, Jay Pundavela, David A. Largaespada, Jack F. Shern, Nancy Ratner. Two-step mechanism of plexiform neurofibroma formation: Role of the NF-κB pathway in neurofibroma formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2269.