Tumor heterogeneity underlies clinical outcome and MEK inhibitor response in somatic NF1-mutant glioblastoma
Sixuan Pan, Kanish Mirchia, Emily Payne, S. John Liu, Nadeem Al-Adli, Zain Peeran, Poojan Shukla, Jacob S. Young, Rohit Gupta, Jasper Wu, Joanna Pak, Tomoko Ozawa, Brian Na, Alyssa T. Reddy, Steve E. Braunstein, Joanna J. Phillips, Susan Chang, David A. Solomon, Arie Perry, David R. Raleigh, Mitchel S. Berger, Adam R. Abate, Harish N. Vasudevan
Sixuan Pan, Kanish Mirchia, Emily Payne, S. John Liu, Nadeem Al-Adli, Zain Peeran, Poojan Shukla, Jacob S. Young, Rohit Gupta, Jasper Wu, Joanna Pak, Tomoko Ozawa, Brian Na, Alyssa T. Reddy, Steve E. Braunstein, Joanna J. Phillips, Susan Chang, David A. Solomon, Arie Perry, David R. Raleigh, Mitchel S. Berger, Adam R. Abate, Harish N. Vasudevan
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Research Article Genetics Oncology

Tumor heterogeneity underlies clinical outcome and MEK inhibitor response in somatic NF1-mutant glioblastoma

Abstract

Tumor suppressor NF1 is recurrently mutated in glioblastoma, leading to aberrant activation of Ras/rapidly accelerated fibrosarcoma (RAF)/MEK signaling. However, how tumor heterogeneity shapes the molecular landscape and efficacy of targeted therapies remains unclear. Here, we combined bulk and single-cell genomics of human somatic NF1-mutant, isocitrate dehydrogenase (IDH) wild-type glioblastomas with functional studies in cell lines and mouse intracranial tumor models to identify mechanisms of tumor heterogeneity underlying clinical outcome and MEK inhibitor response. Targeted DNA sequencing identified CDKN2A/B homozygous deletion as a poor prognostic marker in somatic NF1-mutant, but not NF1 wild-type, glioblastoma. Single-nucleus RNA sequencing of human patient NF1-mutant glioblastomas demonstrated that mesenchymal-like (MES-like) tumor cells were enriched for MEK activation signatures. Single-cell RNA-sequencing of mouse intracranial glioblastomas treated with the MEK inhibitor selumetinib identified distinct responses among tumor subpopulations. MEK inhibition selectively depleted MES-like cells, and selumetinib-resistant MES-like cells upregulated Ras signaling while resistant non-MES cells expressed markers of glial differentiation. Finally, genome-wide CRISPR interference screens validated Ras/RAF/MEK signaling as a key mediator of selumetinib response. Repression of the RAF regulator SHOC2 sensitized glioblastomas to selumetinib in vitro and in vivo, suggesting a synergistic treatment strategy. Taken together, these results highlighted the heterogeneity of NF1-mutant glioblastomas and informed future combination therapies.

Authors

Sixuan Pan, Kanish Mirchia, Emily Payne, S. John Liu, Nadeem Al-Adli, Zain Peeran, Poojan Shukla, Jacob S. Young, Rohit Gupta, Jasper Wu, Joanna Pak, Tomoko Ozawa, Brian Na, Alyssa T. Reddy, Steve E. Braunstein, Joanna J. Phillips, Susan Chang, David A. Solomon, Arie Perry, David R. Raleigh, Mitchel S. Berger, Adam R. Abate, Harish N. Vasudevan

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Figure 2

SnRNA-Seq of human NF1-mutant, IDH wild-type glioblastoma (n = 9) reveals MES-like cells exhibit Ras pathway activation.

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SnRNA-Seq of human NF1-mutant, IDH wild-type glioblastoma (n = 9) reveal...
(A) SnRNA-Seq of 21,959 nuclei from 9 patient-derived human NF1-mutant, IDH wild-type glioblastomas identifies 5 tumor cell clusters and 9 nontumor cell clusters. (B) Tumor cells were distributed across 4 cell states (OPC-like, NPC-like, AC-like, MES-like), with relative enrichment for MES-like and OPC-like cell states. (C) Individual NF1-mutant glioblastomas samples are divided into MES-like enriched (samples S3, S4, S5, S6, and S8) and non-MES enriched (sample S1, S2, S7, S9) tumors. (D) MES-like cells express significantly increased MEK activation compared with non-MES cells (** P < 0.001, Wilcoxon rank-sum test). (E) Single-cell dot plot expression analysis of 31 genes comprising the MEK activation gene set confirms enrichment in MES-like compared with non-MES cells. (F) Integration with published glioblastoma snRNA-Seq data (45) comparing NF1-mutant and NF1 wild-type tumors reveals the MEK activation signature is significantly enriched in MES-like cells within NF1-mutant glioblastomas compared with both non-MES cells in NF1-mutant glioblastomas and MES-like cells in NF1 wild-type glioblastomas (** P < 0.001, Wilcoxon rank-sum test). UMAP, uniform manifold approximation and projection; SNV, single nucleotide variant.