Abstract LB067: NF1 deletion potentiates tumorigenesis and activates expression of cancer-related kinases in an iPSC-based model of H3.3K27M diffuse intrinsic pontine glioma
Authors: Skinner Kasey , Koga Tomoyuki , Miki Shunichiro , Gruener Robert F. , Huang R. Stephanie , Furnari Frank , Miller Ryan 
Published: June 2022

Abstract
Diffuse intrinsic pontine glioma (DIPG) is a subset of high-grade glioma that occurs predominantly in children and has no cure. Up to 80% of DIPG harbor a heterozygous point mutation that results in a lysine 27 to methionine substitution in histone variant H3.3 (H3.3K27M). However, H3.3K27M alone is insufficient for tumorigenesis in existing DIPG models, suggesting that it interacts with co-occurring oncogenic mutations. NF1 deletion co-occurs with TP53 mutation and H3.3K27M in DIPG, but the impact of these mutations individually and together are understudied. To address this gap, we designed a model of DIPG based on human induced pluripotent stem cells (iPSC) edited via CRISPR to express either wild-type or deleted NF1 in conjunction with heterozygous H3.3K27M and a loss of function mutation in TP53. Edited iPSC were chemically differentiated into neural progenitor cells (iNPC), which upon implantation into the brainstems of immunodeficient mice formed diffusely invasive tumors that were histologically consistent with high-grade glioma. Mice with TP53 mut;H3.3K27M tumors survived significantly (p&lt;0.05) longer than those harboring TP53 mut;NF1 -/-;H3.3K27M tumors. In vitro proliferation assays of neurospheres generated from these tumors (termed iDIPG) confirmed this result, suggesting that NF1 deletion synergizes with the other two mutations to accelerate tumor growth. To determine which transcriptional pathways could be involved in this faster tumorigenesis, we performed RNA-sequencing TP53 mut;H3.3K27M (TK) and TP53 mut;NF1 -/-;H3.3WT (TNK) iDIPG neurospheres as well as their iNPC precursors. iDIPG and iNPC clustered by NF1 status. Differential expression analysis comparing TNK and TK iDIPG neurospheres revealed 7226 differentially expressed genes (p&lt;0.05). Gene set enrichment analysis showed upregulation of transcriptional programs related to cell cycle and kinase signaling cascades in TNK iDIPG neurospheres; therefore, we focused our analysis on differentially expressed kinases to determine whether NF1 deletion significantly impacts expression of the kinome. We found 141 kinases upregulated in TNK iDIPG neurospheres, including several members of the MAP kinase family and EGFR, which is amplified or activated in a large number of high-grade gliomas and therefore represents an attractive therapeutic target. Taken together, these data show that NF1 deletion is associated with a significant alteration of kinase expression in H3.3K27M iDIPG, potentially opening up a new therapeutic avenue in these devastating tumors. Further work using this model will focus on screening for kinases necessary for TNK iDIPG neurospheres survival in culture and investigating synergy between targeted kinase inhibition and HDAC inhibitors, which have shown promise in H3.3K27M DIPG.
Citation Format: Kasey Skinner, Tomoyuki Koga, Shunichiro Miki, Robert F. Gruener, R. Stephanie Huang, Frank Furnari, Ryan Miller. NF1 deletion potentiates tumorigenesis and activates expression of cancer-related kinases in an iPSC-based model of H3.3K27M diffuse intrinsic pontine glioma [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 LB067.