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Target Identification Reveals Lanosterol Synthase As a Vulnerability in Glioma

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Target Identification Reveals Lanosterol Synthase As a Vulnerability in Glioma Target identification reveals lanosterol synthase as a vulnerability in glioma Richard E. Phillipsa,b, Yanhong Yangc, Ryan C. Smithc, Bonne M. Thompsond, Tomoko Yamasakie, Yadira M. Soto-Felicianob, Kosuke Funatoc, Yupu Liangf, Javier Garcia-Bermudezg, Xiaoshi Wangh, Benjamin A. Garciah, Kazuhiko Yamasakie, Jeffrey G. McDonaldd, Kivanç Birsoyg, Viviane Tabarc, and C. David Allisb,1 aDepartment of Neurology and Brain Tumor Center, Memorial Sloan Kettering Cancer Center, New York, NY 10065; bLaboratory of Chromatin Biology and Epigenetics, The Rockefeller University, New York, NY 10065; cDepartment of Neurosurgery, Center for Stem Cell Biology and Brain Tumor Center, Memorial Sloan Kettering Cancer Center, New York, NY 10065; dCenter for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, TX 75390; eBiomedical Research Institute, National Institute of Advanced Industrial Science and Technology, 305-8566 Tsukuba, Japan; fCenter for Clinical and Translational Science, The Rockefeller University, New York, NY 10065; gLaboratory of Metabolic Regulation and Genetics, The Rockefeller University, New York, NY 10065; and hEpigenetics Program, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 Contributed by C. David Allis, February 18, 2019 (sent for review December 10, 2018; reviewed by Philip A. Cole, Michael R. Green, and Jun O. Liu) Diffuse intrinsic pontine glioma (DIPG) remains an incurable childhood loss of oncogenic gene transcription, suppression of cell growth, and brain tumor for which novel therapeutic approaches are desperately cell differentiation (7). needed. Previous studies have shown that the menin inhibitor MI-2 How MI-2 exerts an antitumor effect in gliomas remains un- exhibits promising activity in preclinical DIPG and adult glioma known. Neither driver mutations in menin, nor MLL fusions occur models, although the mechanism underlying this activity is unknown. in adult glioma or DIPG (2, 3, 14), and the role of wild-type menin Here, using an integrated approach, we show that MI-2 exerts its in driving oncogenic gene transcription in different glioma subtypes antitumor activity in glioma largely independent of its ability to has not been systematically evaluated. Whether the mechanism of target menin. Instead, we demonstrate that MI-2 activity in glioma is MI-2 activity is shared in different molecularly distinct glioma mediated by disruption of cholesterol homeostasis, with suppression subtypes such as DIPG and adult glioma remains to be determined. of cholesterol synthesis and generation of the endogenous liver X Here, we set out to characterize the mechanism of action of MI-2, receptor ligand, 24,25-epoxycholesterol, resulting in cholesterol de- reasoning that this knowledge would reveal an important pathway pletion and cell death. Notably, this mechanism is responsible for MI-2 MEDICAL SCIENCES vulnerability in these tumors. activity in both DIPG and adult glioma cells. Metabolomic and biochemical analyses identify lanosterol synthase as the direct Results molecular target of MI-2, revealing this metabolic enzyme as a vul- Menin-Independent Activity of MI-2 in Glioma. Since the develop- nerability in glioma and further implicating cholesterol homeostasis ment of MI-2, novel menin inhibitors have been developed as an attractive pathway to target in this malignancy. with increased potency at disrupting the menin–MLL interface SI Appendix glioma | MI-2 | menin inhibitor | target identification | lanosterol synthase in biochemical and cellular assays (13) ( ,Fig. S1A). To further explore the importance of the menin–MLL interaction for glioma cell growth, we treated DIPG and adult iffuse intrinsic pontine glioma (DIPG) is a uniformly fatal glioma patient-derived cell lines (DIPG-VI and GBM-0401, Dbrain tumor that remains the leading cause of brain tumor death in children (1). No drugs have shown efficacy in this ma- lignancy, despite >250 clinical trials. Recent exome-sequencing Significance studies have revealed oncogenic drivers in DIPG, most notably somatic hotspot mutations in histone H3, leading to a lysine-27 Diffuse intrinsic pontine glioma (DIPG) is an incurable childhood to methionine substitution (H3K27M) in >80% of DIPG tumors cancer with a median survival of less than 1 y. Characterization (2, 3). This discovery has facilitated the development of pre- of druggable targets in this disease remains a longstanding goal, clinical animal models of DIPG (4–6); however, the H3K27M as no pharmacological agents have proven efficacy in this ma- mutation is not yet directly targetable, and thus there remains a lignancy. We recently identified the menin inhibitor, MI-2, as need to identify actionable vulnerabilities in these tumors. exhibiting potent antitumor activity in preclinical models of To this end, we recently identified a small molecule, menin DIPG. Here, we show that MI-2 exerts its activity in glioma inhibitor MI-2 (7), as a potential therapy for DIPG in preclinical largely independent of its ability to target the epigenetic regulator animal models of this malignancy (4). Subsequently, work by menin, but instead by disrupting cholesterol homeostasis through others demonstrated promising antitumor effects of a structur- direct inhibition of the cholesterol biosynthesis enzyme, lanosterol ally similar analog (MI-2-2) (8) in patient-derived adult glioma synthase, revealing this metabolic enzyme as an actionable vul- xenograft models (9, 10), pointing toward antiglioma activity, nerability in glioma and implicating cholesterol homeostasis as an which likely extends beyond DIPG tumors harboring the H3K27M attractive pathway to target in malignant gliomas. mutation and broadening the potential applicability of MI-2 and its analogs. Author contributions: R.E.P., J.G.M., K.B., V.T., and C.D.A. designed research; R.E.P., Y.Y., R.C.S., B.M.T., T.Y., K.F., Y.L., J.G.-B., X.W., B.A.G., and K.Y. performed research; Y.M.S.-F. MI-2 was developed as a menin inhibitor for use in leukemia, contributed new reagents/analytic tools; R.E.P., Y.Y., R.C.S., B.M.T., T.Y., Y.M.S.-F., K.F., Y.L., acting by disrupting the interaction between menin and its J.G.-B., X.W., B.A.G., K.Y., J.G.M., K.B., V.T., and C.D.A. analyzed data; and R.E.P., Y.Y., R.C.S., binding partner MLL1 (mixed lineage leukemia 1) (7), a histone- B.M.T., Y.M.S.-F., K.F., B.A.G., K.Y., J.G.M., K.B., V.T., and C.D.A. wrote the paper. methyltransferase that positively regulates gene expression by Reviewers: P.A.C., Harvard Medical School; M.R.G., University of Massachusetts Medical establishing methylation at histone H3 lysine-4 (H3K4) (11). In School; and J.O.L., John Hopkins School of Medicine. certain leukemias, MLL1 undergoes chromosomal rearrange- The authors declare no conflict of interest. ment, leading to generation of a fusion protein (MLL-fusion Published under the PNAS license. leukemia), which drives oncogenic transcription (11). Importantly, 1To whom correspondence should be addressed. Email: [email protected]. the N-terminal portion of MLL fusions interact with menin to re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. main chromatin-bound (12), and blocking this interface using MI-2, 1073/pnas.1820989116/-/DCSupplemental. or other menin inhibitors targeting the same interface (13), leads to Published online March 28, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1820989116 PNAS | April 16, 2019 | vol. 116 | no. 16 | 7957–7962 Downloaded by guest on September 30, 2021 respectively) with MI-2 and a more potent, structurally distinct A – ABCA1 ABCG1 SREBF1 inhibitor of the menin MLL interface called MI-503 (13) (Fig. 20 A SI Appendix A ABCA1 1 and , Fig. S1 ). We reasoned a more potent ) ABCG1 menin–MLL inhibitor might exhibit increased activity in glioma 15 p-value cells if this interaction were critical in this cellular context, and ( 10 10 could also represent a more powerful tool to probe downstream SREBF1 5 mechanisms. To our surprise, MI-2 was up to 15-fold more po- -Log tent in glioma cell-lines than MI-503, and notably there was no 0 -2 -1 0 1 2 therapeutic window between normal neural progenitors (NPCs) Log2 fold change and tumor cell lines with MI-503 in dose-inhibition viability as- A B C says (Fig. 1 ). The superior activity of MI-2 in glioma cells DIPG-VI GBM-0401 NPC compared with MI-503 led us to speculate that the mechanism of action of MI-2 may be menin-independent in this setting. To formally test this hypothesis, we generated multiple MEN1 knockout (KO) clones of glioma cells, using CRISPR/Cas9 (Fig. 1 B and C), and compared their sensitivity to MI-2 with their MEN1 wild-type counterparts (Fig. 1D). Cells were stably transduced with Cas9, followed by transduction with either sgRNAs targeting the MEN1 gene or, as a control, targeting B C D Renilla luciferase (Fig. 1 and ). Remarkably, MI-2 retained DIPG-VI GBM-0401 nanomolar potency in MEN1 KO clones, with IC50 concentra- tions comparable to those of wild-type clones (transduced with sgRNA targeting Renilla luciferase) and parental cells (Fig. 1D), supporting our hypothesis that menin engagement in this context was not responsible for the highly potent activity of MI-2 in A MI-2 MI-503 Fig. 2. (A, Left) Volcano plot of genome-wide RNA-Seq data, with choles- terol homeostasis transcripts highlighted in red. (A, Right) qPCR validation of most significant up-regulated transcripts in DIPG-VI, GBM-0401, and NPCs. (B) Ingenuity pathway analysis of significantly up-regulated genes (P < 0.05) in DIPG-VI cells treated with 0.4 μM MI-2 compared with 0.4 μM MI-nc (control). (C) Relative free cholesterol levels measured by LC-MS in cells treated with 0.4 μM MI-2 compared with 0.4 μM MI-nc (n = 3).
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