Ponatinib promotes a G1 cell-cycle arrest of merlin/NF2-deficient human schwann cells

Abstract

Neurofibromatosis type 2 (NF2) is a genetic syndrome that predisposes individuals to multiple benign tumors of the central and peripheral nervous systems, including vestibular schwannomas. Currently, there are no FDA approved drug therapies for NF2. Loss of function of merlin encoded by the NF2 tumor suppressor gene leads to activation of multiple mitogenic signaling cascades, including platelet-derived growth factor receptor (PDGFR) and SRC in Schwann cells. The goal of this study was to determine whether ponatinib, an FDA-approved ABL/SRC inhibitor, reduced proliferation and/or survival of merlin-deficient human Schwann cells (HSC). Merlin-deficient HSC had higher levels of phosphorylated PDGFRα/β, and SRC than merlin-expressing HSC. A similar phosphorylation pattern was observed in phospho-protein arrays of human vestibular schwannoma samples compared to normal HSC. Ponatinib reduced merlin-deficient HSC viability in a dose-dependent manner by decreasing phosphorylation of PDGFRα/β, AKT, p70S6K, MEK1/2, ERK1/2 and STAT3. These changes were associated with decreased cyclin D1 and increased p27Kip1levels, leading to a G1 cell-cycle arrest as assessed by Western blotting and flow cytometry. Ponatinib did not modulate ABL, SRC, focal adhesion kinase (FAK), or paxillin phosphorylation levels. These results suggest that ponatinib is a potential therapeutic agent for NF2-associated schwannomas and warrants further in vivo investigation.

Keywords: PDGFR; SRC; STAT3; neurofibromatosis type 2; schwannoma.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors reported no potential conflicts of interest.

Figures

Figure 1. Ponatinib decreases HSC viability

(A) Characterization of primary HSC. Confocal Images of HSC expressing human and SC linage markers: human nuclear antigen (HNA, red), S100 (green), GAP43 (red), proteolipid protein (PLP, green), negative nestin (red), DAPI stained nuclei (blue) and F-actin was visualized with phaloidin-Alexa633 (white). Scale bar: 50μm. (B) Representative Western blots of primary HSC and merlin-deficient HSC (MD-HSC) lysates, merlin silencing increased levels of phosphorylated SRC and PDGFRα/β. (C) Western blotting for merlin and β-actin in control HSC and merlin deficient cells at increasing cell passages. (D) Western blot for merlin and β-actin in three control HSC lines and four merlin-deficient (knock-down) HSC lines. (E) Ponatinib dose-response CellTiter-Fluor viability assay. Control HSC lines: SCR-HSC, GFP-HSC and WT-HSC and merlin-deficient HSC: #45, 74, 75, 77 were treated with increasing concentrations of ponatinib in constant 0.1% DMSO or vehicle alone for 48h. Viability is presented as a % of the DMSO control. Graph represents the mean ± SEM of three independent experiments. (F-G) WT-HSC and MD-HSC (#45) were maintained in serum free medium for 5-10 days and then treated with 0.25μM ponatinib for one week. Relative cell numbers was assessed using a crystal violet assay: (F) Representative 20X phase contrast images of cells. Scale bar= 100μm. (G) Cell viability calculated as a % of their respective DMSO control. Graph represent mean ± SEM of three independent experiments (** p<0.01, unpaired t-test, two tailed). (H) Viability of primary human VS cells treated for 48h with increasing ponatinib concentrations. Relative cell viability was assessed using a crystal violet assay and presented as % viability normalized to DMSO group. Plot of mean ± SEM of 6 replicates. VS1 (heterozygous deletion of 23 nucleotides in exon 8 of the NF2 gene, non-irradiated, passage 2); VS2 (heterozygous missense c.1460T>A and p. I487N in exon 14 of the NF2 gene, non-irradiated, passage 2).

Figure 2. Ponatinib decreased merlin-deficient HSC viability independent of ABL/SRC/FAK pathway inhibition

Representative ponatinib dose-response Western blots (n=3). MD- HSC#45 plated in 12-well plates were treated with increasing concentrations of ponatinib for 2h as indicated. Cells were harvested, lysed, resolved by SDS-PAGE and blotted for: (A) p-ABL-Tyr452, c-ABL and β-actin as a loading control; (B) p-SRC-Tyr416 and total SRC, FYN, YES, p-FAK-Tyr576, p-FAK-Tyr577, total FAK, p-Paxillin-Tyr118, and total paxillin. The β-actin levels were used as loading controls.

Figure 3. Ponatinib inhibited PDGFRα/β phosphorylation in merlin-deficient HSC

Representative ponatinib dose-response Western blots (n=3). MD-HSC#45 plated in 12-well plates were treated with increasing concentrations of ponatinib for 2h as indicated. Cells were harvested, lysed, resolved by SDS-PAGE and blotted for p-PDGFRα/β-Tyr849/Tyr857, p-PDGFRβ-Tyr740, p-PDGFRβ-Tyr771, p-PDGFRβ-Tyr1021 and total PDGFRα and PDGFRβ. The β-actin levels were used as loading controls.

Figure 4. Downstream signaling pathways inhibited by ponatinib in merlin-deficient HSC

Western blots of extracts prepared from merlin-deficient HSC#45 treated with increasing ponatinib concentrations. Quantitation was done by fluorescence intensity analysis, normalized to β-actin, and plotted as mean ± SEM (n=3). One-way analysis of variance and Dunnett's multiple comparison post-test were used for statistical analysis (* p<0.1; ** p<0.01 and *** p<0.001). Representative Western blots of dose response experiments at 2h for: (A) p-AKT-Thr308 and AKT; (B) p-p70S6K-Thr229; (C) MEK1/2, p-MEK1/2-Ser217/Ser221, ERK1/2, and p-ERK1/2-Thr202/Tyr204; (D) p-STAT3-Tyr705, and total STAT3. (E) Ponatinib dose-responseCellTiter-Fluor viability assay. MD- HSC#45 were treated with semi-log serial dilutions of ponatinib, NSC-74859, selumetinib, perifosine in 0.1% DMSO for 48h, or vehicle alone. Viability is presented as % of the DMSO control. Graph represents the mean ± SEM of three independent experiments.

Figure 5. Ponatinib arrests merlin-deficient HSC at the G1 phase of the cell cycle

(A) Representative Western blots for Cyclin D1of lysates prepared from merlin-deficient HSC#45 treated 24h with increasing concentrations of ponatinib. Plotted below as mean ± SEM (n=3). One-way analysis of variance and Dunnett's multiple comparison post-test were used for statistical analysis (** p<0.01 and *** p<0.001). (B,C) Merlin-deficient HSC were treated with 3 and 5μM ponatinib for 24h and during the last 3h, 10μM EdU was added. Cells were harvested, labeled with live/dead fixable dye, and analyzed by flow cytometry. (B) Representative plots of the distribution of EdU- and FxCycle-labelled cells of 0.1% DMSO vehicle control and ponatinib treated cells. (C) Graph of the distribution of the cell cycle phases (gated for the live population) of all the experiments as mean ± SEM, n=4; **p<0.01 and ***p<0.001 were determined by two-way ANOVA and Bonferroni multiple comparisons post-test. (D) Representative plots of the distribution of live and dead cell population in these experiments with increasing concentrations of ponatinib as indicated.

Figure 6. Analysis of G1 regulatory proteins during the cell-cycle in merlin-deficient HSC treated with ponatinib

MD-HSC were treated with 3μM ponatinib or vehicle control for 24h. Cells were harvested, fixed, and permeabilized. DNA was stained with FxCycle, and intracellular G1 regulatory proteins were immunostained and analyzed by flow cytometry. (A) Distribution plots of cells with positive/negative p27Kip1 immunostain vs DNA content. Data shown are representative plots of four independent experiments. (B) Distribution of cells analyzed by flow cytometry with positive/negative cyclin D1 immunostain vs DNA content. Shown are representatives of four independent experiments. (C) Diagram of signaling pathways inhibited by ponatinib in merlin-deficient HSC. Merlin deficiency leads to activation of PDGFR, SRC and PI3K. Activation of PI3K potentiates AKT and p70S6K phosphorylation and leads to cell survival, growth and G1 cell cycle progression. PDGFR activity triggers ERK and STAT3 activation, leading to cyclin D1 expression and cell proliferation. SRC activation of FAK and paxillin is not modulated by ponatinib. Ponatinib decreases cell viability through downstream inhibition of AKT, ERK, and STAT3.

Figure 7. PDGFRα/β, SRC, MEK and STAT3 are overactive in human schwannomas

(A) Phospho-RTK membrane profile of schwannomas and control cultured primary HSC. (B) Bar graph of the quantitation ofp-PDGFRα and p-PDGFRβ membrane dot intensity normalized to positive controls. **p<0.005; ****p<0.0001 determined using unpaired t-test of control HSC vs. VS populations, two-tailed. (C) Phospho-kinase membrane profile of schwannomas and control cultured primary HSC samples. (D) Bar graph of the quantitation of phospho-SRC, phospho-MEK1/2 and phospho-STAT3 membrane intensity normalized to positive controls. *p<0.05; ***p<0.001 determined using unpaired t-test of control HSC vs. VS populations, two-tailed.