Abstract 699: NF-kB is a master regulator of resistance to therapy in high-risk neuroblastoma
Authors: Grossmann Liron D. , Uzun Yasin , Lindsay Jarrett , Chen Chia-Hui , Wingrove Catherine , Gao Peng , Thadi Anusha , Marshall Quinlen , Kendsersky Nathan M. , Surrey Lea , Martinez Daniel , Mycek Emily , Casey Colleen , Krytska Kateryna , Tsang Matthew , Wolpaw Adam , Groff David N. , Runbeck Erin , McDevitt Jayne , Diep Dinh , Patel Tasleema , Bernt Kathrin M. , Dang Chi , Zhang Kun , Mosse Yael P. , Tan Kai , Maris John M. 
Published: June 2022

Abstract
BACKGROUND High-risk neuroblastoma is a pediatric cancer arising from the developing sympathetic nervous system with a 50% relapse rate that is typically fatal. At least two subpopulations of neuroblastoma cells exist that can transdifferentiate, adrenergic and mesenchymal, the latter being more resistant to chemotherapy. Mechanisms of therapy resistance are largely unknown and the cells responsible for relapse have not been identified.
METHODS We used single nucleus RNA and ATAC sequencing to identify and characterize the cells that survive chemotherapy, termed here “persister cells”, from a cohort of 20 matched diagnostic and post induction chemotherapyhigh-risk neuroblastoma patients and two patient derived xenograft (PDX) models from diagnostic tumors. Eight representative cell lines derived from neuroblastomas at diagnosis were treated with standard-of-care chemotherapy, and flow cytometry was used to sort for live cells. ML120B and CRISPR-CAS9 were used to modulate NF-kB signaling. An RNA-seq dataset of 153 high-risk neuroblastoma patients was used to determine differentially activated pathways between adrenergic and mesenchymal tumors.
RESULTS Residual malignant cells in the post-chemotherapy tumor samples clustered into three main groups separated by the response to therapy. The most prevalent group of persister cells in responders (N=16/20) displayed low MYC(N) activity even in the presence of MYCN amplification. This group also demonstrated decreased expression of the adrenergic core regulatory circuit genes including PHOX2B, ISL1, HAND2, along with marked activation of TNF-alpha via NF-kB signaling. High NF-kB activity was found in a subpopulation of diagnostic cells in two chemo-refractory patients. We validated decreased expression of MYCN (2-fold decrease, p&lt;0.0001) and PHOX2B (3.13-fold decrease, p&lt;0.0001) in PDXs following chemotherapy. MYCN protein levels were decreased and nuclear p65 levels were increased in cell lines treated with chemotherapy. Pharmacologic inhibition of NF-kB signaling and genetic depletion of p65 resulted in increased killing (3.58-fold increase, p=0.0012) of neuroblastoma cell lines in response to chemotherapy. Finally, we classified 153 diagnostic high-risk neuroblastomas as predominantly adrenergic or mesenchymal using RNA-seq, showing that mesenchymal tumors were enriched with NF-kB pathway activation signatures. We then validated high nuclear p65 levels in 3 mesenchymal cell lines. We tested 6 adrenergic lines, 4 of which had no detectable nuclear p65. Notably, the 2 cell lines with detectable nuclear p65 were derived from diagnostic specimens that showed de novo chemotherapy resistance.
CONCLUSIONS NF-kB activation is a major mediator of de novo and acquired chemotherapy resistance in high-risk neuroblastoma. We postulate that concomitant silencing of this pathway could eliminate persister cells and prevent disease relapse.
Citation Format: Liron D. Grossmann, Yasin Uzun, Jarrett Lindsay, Chia-Hui Chen, Catherine Wingrove, Peng Gao, Anusha Thadi, Quinlen Marshall, Nathan M. Kendsersky, Lea Surrey, Daniel Martinez, Emily Mycek, Colleen Casey, Kateryna Krytska, Matthew Tsang, Adam Wolpaw, David N. Groff, Erin Runbeck, Jayne McDevitt, Dinh Diep, Tasleema Patel, Kathrin M. Bernt, Chi Dang, Kun Zhang, Yael P. Mosse, Kai Tan, John M. Maris. NF-kB is a master regulator of resistance to therapy in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 699.