Factor quinolinone inhibitors alter cell morphology and motility by destabilizing interphase microtubules

Factor quinolinone inhibitors are promising anti-cancer compounds, initially characterized as specific inhibitors of the oncogenic transcription factor LSF (TFCP2). These compounds exert anti-proliferative activity at least in part by disrupting mitotic spindles. Herein, we report additional interphase consequences of the initial lead compound, FQI1, in two telomerase immortalized cell lines. Within minutes of FQI1 addition, the microtubule network is disrupted, resulting in a substantial, although not complete, depletion of microtubules as evidenced both by microtubule sedimentation assays and microscopy. Surprisingly, this microtubule breakdown is quickly followed by an increase in tubulin acetylation in the remaining microtubules. The sudden breakdown and partial depolymerization of the microtubule network precedes FQI1-induced morphological changes. These involve rapid reduction of cell spreading of interphase fetal hepatocytes and increase in circularity of retinal pigment epithelial cells. Microtubule depolymerization gives rise to FH-B cell compaction, as pretreatment with taxol prevents this morphological change. Finally, FQI1 decreases the rate and range of locomotion of interphase cells, supporting an impact of FQI1-induced microtubule breakdown on cell motility. Taken together, our results show that FQI1 interferes with microtubule-associated functions in interphase, specifically cell morphology and motility.


Table of Contents
, 1e, S1a, S1b, S1c, S1d, S2c, S2d, and S5g.. 1 H NMR spectra were obtained at 400 MHz and referenced to the CHCl3 singlet at 7.26 ppm, or the DMSO singlet at 2.50 ppm. 13 C NMR spectra were obtained at 100 MHz, and referenced to the center peak of the CDCl3 triplet at 77.16 ppm, or the center of the DMSO-d6 septet at 39.51 ppm. Chemical shifts are reported in parts per million as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constant, and integration. High resolution mass spectrometry data were obtained on a Waters Qtof (hybrid quadrupolar/time-offlight) API US system by electrospray (ESI) in the positive mode. Mass correction was done by an external reference using a Waters Lockspray accessory. Mobile phases were water and acetonitrile with 0.1% formic acid. The MS settings were: capillary voltage = 3kV, cone voltage = 35, source temperature = 120 °C and dissolvation temperature = 350 °C. Flash column chromatography was performed on Sorbent Technologies 60 Å silica gel.
FQI-34 and FQI2-34 were generally prepared according to patented procedures (Hansen et al., 2019;Schaus et al., 2020), with a few modifications as detailed in the following step-by-step protocol (Scheme S1).
(Note: 3,4-(methylenedioxy)aniline was recrystallized from hexanes prior to use). Vacuum ovendried potassium carbonate (8.30 g, 60 mmol, 1.40 equiv) was added, and the reaction was cooled to 0 ºC. Bromoacetyl chloride (4.64 mL, 55.7 mmol, 1.30 equiv) was added via syringe and the reaction was stirred at 0 ºC for 20 min and then allowed to warm to room temperature. Saturated aqueous sodium bicarbonate was added (100 mL) and the mixture was extracted with dichloromethane (3 x 150 mL). The combined organic layers were washed with saturated aqueous sodium chloride (100 mL), and dried over anhydrous sodium sulfate (Na2SO4

(E)-N-(benzo[d][1,3]dioxol-5-yl)-3-(4-(dimethylamino)-2-ethoxyphenyl)acrylamide:
A flame dried 25-mL round-bottomed flask equipped with a Teflon-coated magnetic stirbar was 2.50 mmol) and THF (8.3 mL, 0.30 M). The flask was flushed with argon and fitted with a rubber septum and argon balloon, then cooled to 0 °C in an ice-water bath. n-Butyllithium (1.6 M in hexanes, 1.35 equiv) was added dropwise, and the mixture was allowed to warm to RT and stir for 30 min, at which time 4-(dimethylamino)-2-ethoxybenzaldehyde (387 mg, 2.0 mmol) was added as a single portion. The flask was fitted with a reflux condenser and argon balloon, and the mixture was heated to reflux for 22 h. The mixture was cooled to RT and quenched with saturated ammonium chloride (5 mL). The mixture was transferred to a 500-mL separatory funnel, and diluted with dichloromethane (150 mL) and water (50 mL). The dichloromethane layer was removed and washed with water (3 x 50 mL), then dried over anhydrous sodium sulfate (Na2SO4), filtered, and concentrated by rotary evaporation. The product was isolated as a yellow brown solid (671 mg, 95% yield, 5:1 E:Z) and used without further purification. 1 H NMR (CDCl3, 400 MHz) 1H), 5.93 (s, 2H), 4.09 (q, J = 6.9 Hz, 2H), 3.00 (s, 6H), 1.47 (t, J = 6.9 Hz, 3H). 13 C NMR (CDCl3, 100 MHz) 165.9, 159.7, 152.7, 147.6, 138.0, 135.2, 133.2, 130.3, 115.8, 112.8, 112.2, 108.0, 104.6, 102.7, 101.1, 97.2, 95.5, 63.7, 40.3, 14.9  and refluxed for 20 h. The resulting mixture was cooled to room temperature, and transferred to a 500-mL Erlenmeyer flask. The mixture was diluted with dichloromethane (150 mL) and cooled to 0 ºC in an ice bath. The reaction mixture was quenched with saturated aqueous sodium bicarbonate (~100 mL) and then transferred to a 500-mL separatory funnel. The organic layer was removed, and the aqueous layer was extracted with dichloromethane (2 x 50 mL). The combined organic layers were washed with saturated aqueous sodium chloride (50 mL   plus the respective treatment. Pelleted cells were resuspended in 500 µL of PBS buffer with 1 mM Pefabloc plus the respective treatment. Aliquots of 50 µL of cell suspension from each treatment sample were incubated separately at the indicated temperatures for 3 minutes in a thermal cycler (Bio-rad, T100 Thermocyler), then cooled at the room temperature for 3 minutes. Lysates were prepared by four rounds of snap freezing and thawing. Soluble protein was separated from aggregates by centrifugation at 20,000 x g for 10 minutes. 40 µL of each supernatant was used for immunoblot analysis. Lysates were separated by electrophoresis through 10% SDS polyacrylamide gels, in 25 mM Tris, 192 mM glycine, and 0.1% SDS. Gels were transferred to PVDF membranes and incubated in blocking buffer with 5% milk in TBST for 1 hour. Membranes were incubated overnight with anti-LSF antibody (1:1,000, BD Bioscience, 610818) at 4°C, and subsequently with goat anti-mouse HRP antibody (Thermo Fisher Scientific, 62-6520; 1:7,000)

Figure S1. FQI1-induced decrease in stable microtubule levels is reversible and not prevented by ROCK inhibitor.
(a) FH-B cells were treated with fresh media containing indicated inhibitors (1 µM Taxol, 4 µM FQI1, 1 µM nocodazole) or vehicle (0.01% DMSO) for 1 hour. Cells were either harvested immediately (pre washout) or incubated with fresh media for 1 hour or 2 hours, followed by the microtubule sedimentation assay. Quantitation of the levels of pelleted tubulin relative to total tubulin are shown underneath each immunoblot. The experiment was performed once. (b) FH-B cells were treated with 4 µM FQI1, 1 µM nocodazole, 10 µM Y-27632, or vehicle (0.01% DMSO) for 2, 5, 10, or 30 minutes. Cell lysates were blotted for pMLC2 and β-actin. (c,d) FH-B and RPE cells were pretreated with 10 µM Y-27632 or vehicle (0.01% DMSO) for 30 minutes, followed by treatment with either 4 µM FQI1 or vehicle (0.02% DMSO) for another 30 minutes. As positive and negative controls, taxol ("T", 1 µM) and nocodazole ("N", 1 µM), respectively, were also added after the pretreatment. Cells were then fractionated using the microtubule sedimentation assay, followed by an analysis of soluble and insoluble α-tubulin. Two independent biological replicates were performed for each cell line. Left panels: Representative α-tubulin immunoblots of FH-B (c) and RPE (d) lysate fractions from the DMS0-and FQI1-treated cells. Right panels: quantitation of immunoblots. Numbers above brackets represent p-values, which were calculated using an unpaired two-sample t-test. Bars represent means and circles represent individual data points.    Visible microtubule ends, marked with asterisks, were counted as in Fig. 2d,f, and partitioned into percentages in the two sectors, which were averaged and plotted as stacked bar charts (b). P-values for the centrosome distal and centrosome proximal percentages are indicated above and below the brackets, respectively and were determined using an unpaired two-sample t-test comparing the averages for each group; numbers in stacked bar plots represent the indicated biological replicates as designated in Fig. 2d.

Figure S5. Effects of FQI1 on RPE cells in wound healing and cell motility assays.
(a,b) Confluent RPE cells treated with high concentrations of thymidine to limit proliferation both before and during the wound healing assay were analyzed for cellular DNA content at the final, 12-hour endpoint of the experiment (Fig. 4a,b). (a) Representative histograms of DNA content are shown across all treatment groups at 12 hours, plus controls that were harvested at 0 hours of an asynchronous population and a population of cells treated with 2 mM thymidine block. (b) Quantitation of the proportion of cells in G1, S, and G2/M cell cycle phases derived from the histograms presented in (a). Bar graphs and error bars represent the mean ± s.e.m. Three independent biological replicates were performed. Numbers above brackets represent p-values, which were calculated using an unpaired two-sample t-test. (c,d) RPE cells were synchronized, treated, and imaged the same way as FH-B cells in Fig. 5. Images of representative nuclei of migrating RPE cells from both treatment groups, taken at 20-minute intervals, are shown in (c) and the swarmplot of distances travelled by migrating RPE cells is shown in (d). Given only 2 biological replicates and the possibility of the apparent trend occurring by chance between the DMSO and FQI1-treated samples, a t-test was viewed as inappropriate. When calculated in batch using the Mann-Whitney U test, p =4.5x10 -6 . The total number of cells analyzed in each condition is indicated by "n". Representative fluorescence images are shown in (c). The areas covered by individual cells in each treatment group were quantified using ImageJ and displayed as swarmplots in (d). P-values were calculated using an unpaired two-sample t-test on medians from two biological replicates. When calculated in bulk using the Mann-Whitney U test, the p value was < 2.2x10 -16 between DMSO and FQI1 treatments. The number of cells analyzed in each condition is indicated by "n".  DMSO (right), after incubations at the indicated temperatures. Top panels: Quantitation of thermal stability of LSF after treatment of cells with each condition, as indicated. Data points are the mean ± standard deviations from a total of 3 independent experiments. For full immunoblots of (g), see Supplementary Fig. S7. Figures 1a, 1c, 1e, S1a, S1b, S1c, S1d, S2c, S2d, and S5g.

Figure S8. Synthesis of FQI2-34.
Indicated is a summary of the series of steps for synthesizing the final product, FQI2-34. Details are included in the Supplementary Methods (above). The yields for each step are indicated underneath each arrow.

Movies 1 & 2: FQI1-treated FH-B cells undergo rapid morphological compaction.
FH-B cells were stained with CellBrite Steady Membrane and treated with either 0.01% DMSO (Movie 1) or 4 µM FQI1 (Movie 2). Cells were imaged by fluorescence time-lapse imaging every 30 seconds using a 20x objective. Asterisks denote cells that are presented in Fig. 3c. Frame rate was set to ten frames per second. Timestamps denote minutes:seconds. Scale bars are 50 µm.

Movies 3-6: FQI1 inhibits motility of FH-B and RPE cells.
FH-B and RPE cells were treated as described in Fig. 5c,d and Supplementary Fig. S5c,d, respectively. Briefly, cells were synchronized by a single thymidine block, treated with 4 µM FQI1 or vehicle (0.01% DMSO) for 1 hour, stained with NucSpot Live 650 dye and monitored by time-lapse fluorescence microscopy for 2 hours. Movies 3 and 4 show DMSO-and FQI1-treated FH-B cells, respectively; movies 5 and 6 show DMSO-and FQI1-treated RPE cells, respectively. Asterisks denote cells that are presented in Fig. 5c,d and Supplementary  Fig. S5c,d. Frame rate was set to ten frames per second. Timestamps denote hours:minutes. Scale bars are 50 µm.