Exploiting mitochondrial and metabolic homeostasis as a vulnerability in NF1 deficient cells

Abstract

Neurofibromatosis type 1 is a disease caused by mutation of neurofibromin 1 (NF1), loss of which results in hyperactive Ras signaling and a concomitant increase in cell proliferation and survival. Patients with neurofibromatosis type 1 frequently develop tumors such as plexiform neurofibromas and malignant peripheral nerve sheath tumors. Mutation of NF1 or loss of the NF1 protein is also observed in glioblastoma, lung adenocarcinoma, and ovarian cancer among other sporadic cancers. A therapy that selectively targets NF1 deficient tumors would substantially advance our ability to treat these malignancies. To address the need for these therapeutics, we developed and conducted a synthetic lethality screen to discover molecules that target yeast lacking the homolog of NF1, IRA2. One of the lead candidates that was observed to be synthetic lethal with ira2Δ yeast is Y100. Here, we describe the mechanisms by which Y100 targets ira2Δ yeast and NF1-deficient tumor cells. Y100 treatment disrupted proteostasis, metabolic homeostasis, and induced the formation of mitochondrial superoxide in NF1-deficient cancer cells. Previous studies also indicate that NF1/Ras-dysregulated tumors may be sensitive to modulators of oxidative and ER stress. We hypothesize that the use of Y100 and molecules with related mechanisms of action represent a feasible therapeutic strategy for targeting NF1 deficient cells.

Keywords: RAS; mitochondria; neurofibromin 1; proteostasis; synthetic lethal.

Figure 1. Y100 and Y100B are synthetic lethal with a yeast model of NF1 loss

(A) A schematic of the high throughput screen for small molecules that target a yeast model of NF1 deficiency. erg6Δ and erg6Δnf1Δ yeast were screened against ~5,100 small molecules. Cell death/growth was determined by measuring OD₆₀₀. Small molecules that induced slow growth or death of the erg6Δnf1Δ yeast without affecting the erg6Δ yeast strain were considered hits. (B) Structure of Y100 and Y100B. (C–D) Treatment of yeast strains for 18 h with 0.2–100 μM Y100 or Y100B results in selective inhibition of nf1Δ strains. Control strains are unaffected. Error bars are standard deviation; graph is the average of three (Y100) or two (Y100B) independent experiments.

Figure 2. Y100 inhibits NF1 deficient mammalian cells and modulates markers of proteostasis

(A) NF1-deficient U251-MG and U87-MG glioblastoma cells were treated with 0.039–20 μM Y100 for 72 hours. Error bars represent the standard deviation of four technical replicates. IC₅₀ Y100 U87: 1.13 μM, IC₅₀ Y100 U251-MG: 2.33 μM. (B) The effect of Y100 on cell growth/viability appears to be rapid and irreversible. U87-MG cells were treated with 0.039–20 μM Y100 for 30 minutes-72 hours. At timepoints, cells were rinsed with PBS and replaced with drug-free culture media. Data for all timepoints was collected at 72 h. Error bars represent the standard deviation of four technical replicates. (C) Y100 treatment induces accumulation of the autophagy marker p62. U87-MG cells were treated for 24 hours with vehicle, Y100, or the autophagy inhibitor HCQ. Cells were also treated with 2 h of MG132 and bortezomib (BTZ) as a control for proteasome inhibition. (D) Y100 does not result in accumulation of the lipidated form of the autophagy marker LC3 (LC3-II). U87-MG cells were treated for 24 hours with vehicle, Y100, HCQ, or the autophagy inducer/mTOR inhibitor rapamycin. (E) Y100 treatment induces accumulation of p62, but not LC3B. U87-MG cells were treated with vehicle, Y100 or HCQ for 24 hours and immunolabeled for LC3B (green) and p62 (red). DNA was stained with DAPI. (F–G) Y100 treatment causes the accumulation of K48- and K63-polyubiquitin linked protein. U87-MG cells were treated with DMSO (−), 1.7/3.4 μM Y100 or HCQ for 24 hours or for 2 hours with 10 μM Y100 or 1 μM BTZ/10 μM MG-132. K48/K63 linked proteins and alpha-tubulin (loading control) were detected by western blotting.

Figure 3. Y100 treatment causes U87-MG cells to develop polarized mitochondrial “hotspots” and cells lacking Tid1

(A) U87-MG cells were treated for 24 hours with vehicle, Y100 or HCQ and immunolabeled for Tom20 (green), a total mitochondrial marker, and Mitotracker (red), a marker of polarized mitochondria, and imaged on a confocal microscope. Treatment of U87-MG cells with Y100 induced the formation of polarized mitochondrial “hotspots” along the mitochondrial network (hotspot positive cells indicated with arrows). These hotspots are not observed with autophagy inhibition. (B) Hotspot-positive cells have reduced, or lack, Tid1, a mitochondrial heat shock chaperone protein. U87-MG cells were treated with vehicle or Y100 for 30 minutes-24 hours, immunolabeled for mitochondrial heat shock chaperone protein (Tid1, green) and polarized mitochondria (Mitotracker, red), and imaged on a wide-field microscope. Mitochondrial puncta are first observable at 8 hours of Y100 treatment. Cells with polarized mitochondrial puncta contain low to no observable Tid1 signal (indicated with arrows).

Figure 4. Y100 treatment causes mitochondrial superoxide and DNA damage

ROS scavenging reagents abrogate the effect of Y100. (A) U87-MG cells were treated with vehicle, CCCP, or Y100 for a range of times from 30 minutes to 24 hours. Cells were stained with the mitochondria superoxide specific reagent MitoSOX Red, and analyzed by flow cytometry. Y100 and CCCP treatment induce mitochondrial superoxide. Y100 induced superoxide is detectable within 30 minutes of treatment. (B) Y100 induces DNA damage. U87-MG cells were treated for 24 hours with DMSO, Y100, or the nucleotide synthesis inhibitor hydroxyurea (HU). γH2AX (green) was immunolabeled as a marker of DNA damage, imaged with a wide-field microscope, and quantified in (C), where 100 cells per condition were scored as H2AX positive (3 or more nuclear foci, or pan-nuclear staining) and H2AX negative (less than three nuclear foci). Values are the average of three experiments. (*) indicates a significant difference as compared to the DMSO control, p < 0.005, error bars are standard error of the mean. (D) The GSH synthesis inhibitor buthionine sulfoximine (BSO) slightly potentiates the effect. U87-MG cells were pretreated with vehicle or BSO for 2 hours. Pretreatment media was replaced with media containing media of the same composition with 0.39–2 μM Y100 added. Cells were incubated for 72 h with drug. Viability/growth was measured with alamarBlue fluorescence.

Figure 5. Y100 disrupts mitochondrial homeostasis

(A–B) Y100 rapidly induces increased oxygen consumption, and slightly increases extracellular acidification. Metabolic function (oxygen consumption rate, OCR; extracellular acidification rate, ECAR) was measured at baseline and after injection of mitochondrially targeted agents (oligomycin, CCCP, antimycin A) as well as vehicle and Y100 (injected after 3 baseline measurements). (C) Cells lacking IRA2 have increased sensitivity to the mitochondrial uncoupling agent CCCP. Yeast were treated for 18 hours in the presence of CCCP, and growth/cell death was measured with OD₆₀₀. (D) Y100 increases oxygen consumption by mechanisms independent of electron transport chain uncoupling. We profiled the mitochondrial bioenergetic capacity of Y100 treated cells by performing a modified Mito Stress Test, which utilizes serial injections of oligomycin, FCCP, and a combination of antimycin A and rotenone to characterize cellular bioenergetic potential. In some samples, FCCP was substituted with Y100.