The tropomyosin 3.1/3.2 inhibitor ATM-3507 alters B-cell actin dynamics and impairs the growth and motility of diffuse large B-cell lymphoma cell lines

Figures

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  Figure 1. ATM-3507 treatment alters BCR-induced actin remodeling and impairs B cell spreading. (A) A20 cells were pre-treated with either 3 µM ATM-3507, an equivalent volume of DMSO (0.03% final concentration), or 50 µM snBB for 1 h at 37°C. The cells were added to coverslips coated with 2.4 μg/cm2 anti-IgG and allowed to spread for 30 min before being stained with rhodamine-phalloidin to visualize F-actin. For each condition, more than 105 cells from 3 independent experiments were imaged by confocal microscopy. Representative confocal microscopy images are shown (top panel; scale bar is 10 µm). Cell areas (middle panel) and cell circularity (lower panel) were quantified from the confocal microscopy images using ImageJ. The graphs on the left show one of three independent experiments (biological replicates) with similar results. Each dot represents one cell, and the medians and interquartile ranges are shown for >35 cells per condition. p-values were calculated using the Mann–Whitney U test. The graphs on the right show combined data from the 3 independent experiments. Each symbol is an individual experiment, and the data are presented as the mean ± SEM for the median values from the 3…

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  Figure 2. Tpm3 co-localizes with the peripheral F-actin ring during B cell spreading. A20 cells were pre-treated for 1 h with 0.03% DMSO or 3 µM ATM-3507 and then allowed to spread for 30 min on coverslips that had been coated with 2.4 µg/cm2 anti-IgG. Cells were stained for F-actin (rhodamine-phalloidin) and Tpm3 and then imaged by confocal microscopy. (A) Representative images from one of 3 independent experiments. Scale bar: 10 µm. (B) The Manders’ coefficient represents the fraction of Tpm3 staining that co-localized with F-actin staining. The super-plot displays combined data for >25 cells per condition from two independent experiments. Each dot is one cell, and the different colors represent the two experiments. The large symbols represent the median values for each experiment. p-values for DMSO versus ATM-3507-treated cells were calculated using the Mann–Whitney U test.

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  Figure 3. Tpm3 co-localizes with actin arcs, and Tpm3.1/3.2 function is important for their formation. (A) A20 cells were allowed to spread for 30 min on coverslips that had been coated with 0.625 µg/cm2 anti-IgG plus 0.15 µg/cm2 ICAM-1. Cells were stained for F-actin (rhodamine-phalloidin) and Tpm3 and then imaged by confocal microscopy. Representative images from one of 3 independent experiments are shown, in which yellow arrows indicate an actin arc. Scale bar: 10 µm. (B-D) A20 cells were pre-treated for 1 h with 0.03% DMSO or 3 µM ATM-3507 and then allowed to spread for 30 min on coverslips coated with 0.625 µg/cm2 anti-IgG plus 0.15 µg/cm2 ICAM-1. F-actin staining was used to define the cell periphery, quantify cell areas, and visualize actin arcs. In (B), each symbol represents the median value from one of 3 independent experiments in which n >30 cells per condition were analyzed. The mean ± SEM for the median values from the 3 experiments is shown. Representative images of DMSO- and ATM-3507-treated cells are shown in (C), with an actin arc indicated by a yellow arrow. The percent of DMSO- and ATM-3507-treated cells in which actin arcs were clearly visible is shown in (D). Each…

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  Figure 4. ATM-3507 inhibits the growth of DLBCL cell lines. NU-DUL-1 (A), TMD8 (B), Toledo (C), or SU-DHL-8 DLBCL cells (D) were cultured for 48 h or 72 h with the indicated concentrations of ATM-3507. The Alamar Blue assay was used to measure metabolic activity. In each experiment, each condition was analyzed in triplicate wells (technical replicates), and the mean value was used for data analysis. The data are expressed as a percent of the metabolic activity for cells cultured in the absence of ATM-3507 (control; dotted black lines). Each symbol is the average ± range or the mean ± SEM from 2 or 3 independent experiments. Where no error bars are shown, they were smaller than the symbol. The dotted red lines indicate 50% inhibition. Approximate IC50 values are shown. DMSO concentrations equivalent to those for the highest ATM-3507 concentrations had no effect on metabolic activity.

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  Figure 5. ATM-3507 causes G2/M cell cycle arrest in DLBCL cells. (A) NU-DUL-1 or Toledo cells were cultured with 0.03% DMSO or the indicated concentrations of ATM-3507 for 24 h or 72 h before being stained with rhodamine-phalloidin and DAPI. Cells were imaged by confocal microscopy. Representative images are shown. (B) NU-DUL-1 or Toledo cells were cultured for 24 h with 3 µM ATM-3507 or 0.03% DMSO before being analyzed by flow cytometry. Single live cells were identified by FSC, side scatter, and 7-AAD staining. Geometric means of the FSC values from 3 independent experiments are shown along with the mean ± SEM. (C, D) Propidium iodide (PI) staining was used to quantify the DNA content of live NU-DUL-1 (C) or Toledo (D) cells. Representative histograms showing peaks corresponding to cells with 2N or 4N DNA content. The graphs show the percent of live cells with 4N DNA content. Each symbol is an independent experiment, and the data are presented as the mean ± SEM from 3 experiments. p-values were calculated using two-tailed paired t-tests.

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  Figure 6. ATM-3507 inhibits the migration of DLBCL cells toward CXCL12. (A, B) NU-DUL-1 or Toledo cells were pre-treated with ATM-3507 or 0.03% DMSO (equivalent to the highest ATM-3507 concentration) for 1 h before being added to the upper chamber of a Transwell. The lower chamber contained 100 nM CXCL12 plus the same concentration of ATM-3507 or DMSO as in the upper chamber. After 4 h, the number of cells that had migrated into the lower chamber was determined using flow cytometry. The data are expressed as a percent of the number of DMSO-treated cells that migrated into the bottom chamber. The 100% values (percent of DMSO-treated cells that migrated into the lower chamber) in individual experiments ranged from 15-30% for NU-DUL-1 cells and from 50-70% for Toledo cells. Each symbol is an independent experiment. Means ± SEM are shown for 3 independent experiments. (C) NU-DUL-1 or Toledo cells were treated with 3 µM ATM-3507 or 0.03% DMSO for 1 h or 5 h before quantifying cell surface levels of CXCR4 by flow cytometry. Means ± SEM are shown for 3 independent experiments. p-values were calculated using two-tailed paired t-tests.

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  Figure 7. ATM-3507 inhibits the 2D motility of DLBCL cells on FN. Toledo DLBCL cells were added to FN-coated coverslips in the presence of 100 nM CXCL12 and either 10 µM ATM-3507 or 0.1% DMSO. After a 1 h pre-treatment period, time-lapse images were acquired every 30 s for 1 h. Cell tracks were generated from the time-lapse videos. Representative videos of DMSO-treated cells ( Supplementary Video 1 ) and ATM-3507-treated cells ( Supplementary Video 2 ) are in the Supplementary Material . (A) Individual cell tracks from a representative experiment. In each experiment, 40–100 cell tracks were analyzed per condition. (B) Compiled data from 3 independent experiments. Each symbol represents the median value from an individual experiment. Means ± SEM are shown. p-values were calculated using two-tailed paired t-tests.

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  Figure 8. Tpm3.1/3.2 regulates actin organization and actin-dependent processes in B cells. Tpm3.1/3.2 dimers associate with actin filaments, form homopolymers along the filament, stabilize the actin filament, and recruit myosin II. Myosin II dimers can crosslink actin filaments and mediate actomyosin contractility. Inhibition of Tpm3.1/3.2 by ATM-3507 impairs multiple actin-dependent processes in B cells, including the assembly of a peripheral actin ring that drives BCR-induced cell spreading via the formation of protrusive lamellipodia, and the formation of actomyosin arcs (indicated by the yellow arrow) at the inner face of the peripheral actin ring. ATM-3507 also inhibits the growth of DLBCL cells as well as their CXCL12-dependent migration and motility. Created with BioRender.com.