Neurofibromatosis type 1 (NF1) is a common genetic disorders in humans, occurring in 1 in 3500 live births. NF1 is caused by inherited or de novo
mutation in the NF1
tumor suppressor gene, which encodes a GTPase activating protein (GAP) for Ras signaling proteins. NF1 has a broad clinical spectrum: affected individuals can develop benign nervous system tumors called neurofibromas, low-grade astrocytomas, pheochromocytoma, and juvenile myelomonocytic leukemia (1
). Plexiform neurofibromas occurring in deep nerves can degenerate into malignant peripheral nerve sheath tumors (MPNSTs), a life-threatening consequence of NF1 (2
). The lifetime risk of MPNST in NF1 patients is estimated to be 8–15%, and the 5-year survival is approximately 20% (4
Plexiform neurofibromas are heterogeneous, consisting of fibroblasts, perineurial cells, mast cells, and Schwann cells, but only Schwann cells have biallelic inactivation of NF1
). In mouse models, targeted deletion of NF1
from the Schwann cell lineage gives rise to neurofibromas (8
). Thus, loss of NF1
from Schwann cell precursors is thought to initiate plexiform neurofibroma. Aberrant signaling occurs between NF1
-deficient Schwann cells and NF1
heterozygous mast cells, which generates a tumorigenic microenvironment (8
). Because of their role in the initiation of plexiform neurofibroma and progression to MPNST, NF1
-deficient Schwann cells represent an ideal population for targeted molecular therapies.
Chemical screens have revolutionized the discovery process for targeted molecular therapies. However, primary Schwann cells are difficult to culture and present a challenge for high throughput screening. Another challenge in drug discovery is the rapid and efficient identification of the receptor for a novel compound – either the physical ligand, or the biological process that is being modified. Approaches addressing these challenges are needed to identify new compounds and target pathways for the devastating tumors that afflict NF1 patients.
The budding yeast Saccharomyces cerevisiae
has two NF1
, which encode Ras-GTPase activating proteins (13
). Deletion of an IRA
gene increases Ras-GTP and activates of two pathways: a MAPK pathway that modifies cell morphology, and the cAMP-dependent protein kinase (PKA) pathway (14
). Schwann cells lacking NF1
have increased intracellular cAMP, and display PKA-dependent phenotypes (16
). The fact that Schwann cells lacking NF1
and budding yeast lacking IRA2
share the high-PKA phenotype suggests that the yeast model might be useful for targeting the cell-autonomous effects of NF1
loss in Schwann cells. The yeast platform enables rapid and cost-effective high-throughput chemical screening and allows for the use of powerful yeast genetics to identify new drug targets.
To identify therapeutic agents and target pathways for NF1-associated tumors, we performed a high-throughput chemical screen in mammalian MPNST cell lines and in the yeast. Here we describe a novel compound that preferentially inhibits both a NF1
-deficient MPNST cell line and IRA2
-deficient yeast. In yeast, growth inhibition is partially alleviated by high-copy expression of NAB3
, which encodes an RNA-binding protein that regulates transcript termination of non-polyadenylated Pol II transcripts (18
). This finding led us to a novel genetic interaction between IRA2
, suggesting a functional interaction between Ras signaling and the non-poly(A)-dependent termination pathway which may have relevance to mammalian cells and the treatment of MPNST.