A transcriptomic, proteomic, and functional genetic atlas dissects neurofibromin function in the peripheral nervous system

TL;DR

This study explores how losing the NF1 gene affects peripheral nervous system tumors and suggests that directly inhibiting KRAS could be a new treatment approach.

Contribution

The study provides a comprehensive functional and molecular analysis of NF1 loss in peripheral nerve cells and identifies KRAS inhibition as a novel therapeutic strategy.

Findings

NF1 loss increases Ras GTP levels and promotes cell proliferation and dedifferentiation.

KRAS inhibition, but not HRAS or NRAS inhibition, effectively blocks ERK and CDK1/2 activation in NF1 mutant cells.

PTPN11 repression decreases proliferation and increases sensitivity to MEK inhibitors in iPN cells.

Abstract

Personalized medicine based on pharmacologically targeting specific mutations have revolutionized cancer. In contrast to gain of function oncogenes that can be directly inhibited, such approaches are challenging for loss of function tumor suppressors. One such example is the NF1 tumor suppressor gene encoding neurofibromin, which negatively regulates Ras small GTPases and thus leads to RAF/MEK/ERK activation. Although MEK inhibitors are approved for some NF1 mutant tumors, better therapies are needed for people who do not respond to or cannot tolerate MEK inhibitors. To identify additional therapeutic strategies for NF1 mutant tumors, we systematically analyze upstream inputs and downstream outputs to Ras following NF1 loss. Our data suggest direct KRAS inhibition may be a promising approach for neurofibromatosis type 1.

The NF1 tumor suppressor gene is recurrently mutated in human cancers and is associated with the neurofibromatosis type 1 (NF-1) cancer predisposition syndrome. NF1 encodes neurofibromin, a Ras guanosine triphosphate (GTPase) activating protein that negatively regulates Ras signaling. NF1 mutation accordingly leads to Ras misactivation and downstream activation of RAF/MEK/ERK signaling, leading to the approval of the MEK inhibitor selumetinib for NF-1 associated peripheral nervous system (PNS) tumors. However, how NF1 loss modifies response to selumetinib and the utility of targeting additional upstream inputs or downstream outputs of Ras these tumors remain unclear. Here, we perform RNA-sequencing, phosphoproteomic, pharmacologic, and proximal proteomic analysis across a panel of CRISPR interference immortalized peripheral nerve (iPN) cells to systematically dissect the function of neurofibromin loss. Small guide NF1 (sgNF1) repression is sufficient to increase Ras GTP levels and alter gene expression to promote cell proliferation and dedifferentiation, with sgNF1 iPNs showing decreased sensitivity to selumetinib due to altered feedback regulation to Ras/RAF/MEK/ERK. Upstream small guide PTPN11 (sgPTPN11) repression leads to the inverse gene expression signature, decreasing cell proliferation and promoting differentiation, and sgPTPN11 iPNs are more sensitive to selumetinib. However, upstream sonof sevenless 1 inhibition shows limited efficacy in iPNs due to compensation by SOS2. Finally, proximal proteomics reveals Kirsten rat sarcoma virus (KRAS), but not Harvey rat sarcoma virus (HRAS) or neuroblastoma Ras viral oncogene homolog (NRAS), associates with neurofibromin in iPN cells, and pan-KRAS inhibition is sufficient to block ERK activation and CDK1/2 activation in NF1 mutant cells, suggesting blocking KRAS may be a therapeutic approach for NF1 mutant PNS tumors.

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