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. 2023 Feb;161(3):593-604.
doi: 10.1007/s11060-023-04260-3. Epub 2023 Feb 15.

The PYK2 inhibitor PF-562271 enhances the effect of temozolomide on tumor growth in a C57Bl/6-Gl261 mouse glioma model

Jescelica Ortiz-Rivera  1 , Rebeca Nuñez  2 , Yuriy Kucheryavykh  2 , Lilia Kucheryavykh  2
Affiliations

The PYK2 inhibitor PF-562271 enhances the effect of temozolomide on tumor growth in a C57Bl/6-Gl261 mouse glioma model

Jescelica Ortiz-Rivera et al. J Neurooncol. 2023 Feb.

Abstract

Background: The development of resistance to temozolomide (TMZ), a standard chemotherapeutic, limits the effective treatment of glioblastoma (GBM). Focal adhesion kinase (FAK) and proline rich tyrosine kinase 2 (Pyk2) regulate proliferation and invasion of GBM cells. We found that TMZ activates FAK and Pyk2 signaling in GBM. We hypothesized that pharmacological inhibitors of Pyk2/FAK together with TMZ can enhance the inhibitory effect of TMZ on tumor growth and dispersal and improve the treatment outcome.

Methods: Primary human GBM cell cultures and a C57Bl/6-GL261 mouse glioma implantation model were used. Pyk2 (Tyr579/580) and FAK (Tyr925) phosphorylation was analyzed by western blotting. Viability, cell cycle, migration, invasion and invadopodia formation were investigated in vitro. Animal survival, tumor size and invasion, TUNEL apoptotic cell death and the Ki67 proliferation index were evaluated in vivo upon treatment with TMZ (50 mg/kg, once/day, orally) and the Pyk2/FAK inhibitor PF-562271 (once/daily, 50 mg/kg, orally) vs. TMZ monotherapy.

Results: In vitro studies revealed significantly reduced viability, cell cycle progression, invasion and invadopodia with TMZ (100 µM) + PF-562271 (16 nM) compared with TMZ alone. In vivo studies demonstrated that combinatorial treatment led to prominent reductions in tumor size and invasive margins, extensive signs of apoptosis and a reduced proliferation index, together with a 15% increase in the survival rate in animals, compared with TMZ monotherapy.

Conclusion: TMZ + PF-562271 eliminates TMZ-related Pyk2/FAK activation in GBM and improves the treatment efficacy.

Keywords: FAK; GBM; PF-562271; Pyk2; Temozolomide.

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Conflict of interest statement

The authors have no relevant financial or nonfinancial interests to disclose.

Figures

Fig. 1
Fig. 1
PF-562271 reverses the stimulatory effect of TMZ on Pyk2 and FAK phosphorylation in glioma cells. Representative western blots and quantifications for average levels of total and phosphorylated Pyk2 and FAK protein expression for each treatment group relative to control are presented for glioma cells purified from tumors generated in the C57BL/6-GL261 glioma mouse model (a) and for primary human GBM cultured CL-2 (b) and CL-3 cells (c). Images are presented for the control, PF-562271, TMZ, and PF-562271 combined with TMZ groups. Tumors were analyzed after 14 days of treatment, given beginning the 5th day after tumor implantation. Six animals per experimental group was used. In vitro data are shown for 24 h of treatment in each experimental group. Actin was used as a loading control. Mean ± S.E. and significant differences from the control (*) or TMZ groups (**) are shown (p < 0.005). N = 6
Fig. 2
Fig. 2
TMZ and PF-562271 combinatorial treatment reduced cell viability in primary human glioma cell lines. a–c The number of dead cells was calculated as a percent of dead cell relative to total number of cells using the viability assays after 72 h of treatment with vehicle (control), PF-562271, TMZ, and PF-562271 + TMZ in CL-2, CL-3 and GL261 cell lines. Live and dead cells were quantified based on calcein and ethidium homodimer-1 staining for live and dead cells. d–f Cell cycle analysis was performed for CL-2, CL-3 and GL261 cells using flow cytometric evaluation of propidium iodide as a nuclear marker. The percentage of cells in the G0/G1, S and G2/M phases was determined based on DNA content. Bar graphs represent the total distribution of cells at different phases of the cell cycle. g–l Relative expression of Bcl-2 and cyclin D1 in CL-2 (g, j), CL-3 (h, k) and GL261 (i, l) evaluated by western blots and analyzed as fold change relative to control are presented. Actin was used as a loading control. N = 4. Mean ± S.E. and significant differences from the control (*) and (**) from TMZ are shown (p < 0.05)
Fig. 3
Fig. 3
TMZ and PF-562271 combinatorial treatment reduced tumor cell proliferation and increased apoptosis in the C57BL/6-GL261 mouse glioma implantation model compared with TMZ monotherapy. Immunofluorescence confocal images of tumors (a) and quantification (b) of cells expressing the Ki67 marker are presented for animals that received vehicle, TMZ, PF-562271, and combined TMZ and PF562271 treatment for 7 days, beginning the 10th day after tumor implantation. Number of Ki67-positive cells in a field of view is presented. c, d Immunohistochemical images of tumors and quantification of the induction of apoptosis using TUNEL assays for animals that received vehicle, TMZ, PF-562271, and combined treatment with TMZ and PF562271. Arrows indicate the TUNEL stained individual cells or cells aggregations. TUNEL signal intensity was quantified as average gray measurements, normalized to the background, with use of ImageJ program. Data are presented as relative to control. Scale bar is 100 µm. Mean ± S.E. and significant differences from the control (*) or TMZ group (**) are shown (p < 0.005). N = 6
Fig. 4
Fig. 4
TMZ and PF-562271 combinatorial treatment decreases functional invadopodia formation, migration and invasion in primary human GBM cells. Invadopodia formation assays, performed for CL-2, CL-3 and GL261 cells, are presented as confocal images (confocal images for CL-2 are presented in (a), images for CL-3 and GL261 are presented in Online Recourse 5) and calculations of the number of cells forming invadopodia (b–d), together with calculations of area fraction gelatin matrix (invadopodia activity, e–g) for cells treated with vehicle, TMZ, PF-562271, and TMZ and PF-562271 in combination. Study duration was 16 h. Cells with invadopodia are presented as a ratio of cells forming invadopodia to the total number of nucleuses in each image. Invadopodia activity is presented as a ratio of area fraction to the total area and normalized to the total number of cells in each image. F-actin, stained with rhodamine–phalloidin (red), FITC-conjugated gelatin (green), and DAPI, used for nuclei staining (blue), are shown. Degraded areas of FITC-labeled gelatin are shown as black patches. Transwell migration (h–j) and invasion (k–m) assays were performed for CL-2, CL-3 and GL261 cells. The relative number of migrating and invading glioma cells, compared to control, is presented. Scale bar: 60 µm. Mean ± S.D. with significant differences from controls (*) and TMZ (**) are shown (p < 0.05). N = 5
Fig. 5
Fig. 5
TMZ combined with PF-562271 reduces tumor growth and invasion margins and increases animal survival rates in a C57BL/6-GL261 mouse glioma implantation model compared with TMZ monotherapy. Hematoxylin and eosin staining of mouse brain slices encompassing implanted tumors (a, c) and quantification of tumor size (b) and invasion distance (d) in animals that received vehicle, PF-562271, TMZ, and PF-562271 + TMZ combinatorial treatments for 14 days after tumor implantation. Tumor size evaluation was performed immediately after treatment course termination. e Kaplan‒Meier estimates of overall survival probability for animals that received vehicle, PF-562271, TMZ and PF-562271 + TMZ combinatorial treatments. Six animals per group were used in all presented studies. Curve comparison was performed using the log-rank (Mantel‒Cox) test (p < 0.05). The results are presented as the mean ± S.D. with significant differences from control (*) and TMZ (**) (p < 0.05). Scale bar: 100 µm
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