CDK4/6 Inhibition With Lerociclib is a Potential Therapeutic Strategy for the Treatment of Pediatric Sarcomas

Abstract

Background: Sarcomas are a heterogenous collection of bone and soft tissue tumors. The heterogeneity of these tumors makes it difficult to standardize treatment. CDK 4/6 inhibitors are a family of targeted agents which limit cell cycle progression and have been shown to be upregulated in sarcomas. In the current preclinical study, we evaluated the effects of lerociclib, a CDK4/6 inhibitor, on pediatric sarcomas in vitro and in 3D bioprinted tumors.

Methods: The effects of lerociclib on viability, proliferation, cell cycle, motility, and stemness were assessed in established sarcoma cell lines, U-2 OS and MG-63, as well as sarcoma patient-derived xenografts (PDXs). 3D printed biotumors of each of the U-2 OS, MG-63, and COA79 cells were utilized to study the effects of lerociclib on tumor growth ex vivo.

Results: CDK 4/6, as well as the intermediaries retinoblastoma protein (Rb) and phosphorylated Rb were identified as targets in the four sarcoma cell lines. Lerociclib treatment induced cell cycle arrest, decreased proliferation, motility, and stemness of sarcoma cells. Treatment with lerociclib decreased sarcoma cell viability in both traditional 2D culture as well as 3D bioprinted microtumors.

Conclusions: Inhibition of CDK 4/6 activity with lerociclib was efficacious in traditional 2D sarcoma cell culture as well as in 3D bioprints. Lerociclib holds promise and warrants further investigation as a novel therapeutic strategy for management of these heterogenous groups of tumors.

Keywords: 3D bioprinting; CDK 4/6 inhibitors; Lerociclib; Pediatric sarcomas.

Figure 1.. Increased expression of CDK4 correlates with worse outcomes for patients with sarcoma.

(A) Schematic demonstrating the CDK4/6 cyclin D1 complex which phosphorylates Rb, permitting the release of Rb from E2F. E2F promotes transcription of cell cycle genes and allows progression of the cell cycle from G₁ to S phase. (B) Characteristics of the cell lines and PDXs utilized in the current study. (C) Query of the TCGA-SARC database evaluating the level of CDK4 abundance and patient vital status at follow-up. Patients with lower CDK4 abundance (n = 184) were more likely to be alive at follow-up compared to those with higher amounts (n = 75) (p = 0.043). (D, E) Immunoblotting of whole cell lysates confirmed the expression of CDK4 and CDK6 in U-2 OS (D) and COA79 (E) cell lines. Rb protein expression was mostly unchanged after treatment with lerociclib, but phosphorylation of Rb at the S780 and S795 sites was decreased. Vinculin served as an internal loading control.

Figure 2:. Lerociclib treatment decreases proliferation and attenuates cell cycle progression.

(A) Proliferation was assessed using cell titer assay. U-2 OS (5 × 10³ cells per well), MG-63 (5 × 10³ cells per well), COA30 (1 × 10⁴ cells per well) or COA79 (1 × 10⁴ cells per well) were plated in 96-well plates, allowed to attach overnight, and treated with lerociclib at increasing concentrations (0–10 μM) for 72 hours. There was a statistically significant decrease in proliferation in all cell lines beginning at 5.0 μM. (B) Flow cytometry evaluated the effects of lerociclib treatment on the cell cycle. U-2 OS, MG-63, COA30 or COA79 cells (1 × 10⁶) were plated in 6 well plates and treated with 0–4 μM lerociclib for 24 hours. Cells were stained with 20 μg/mL propidium iodine and analyzed by flow cytometry. Treatment with lerociclib led to a significant increase in the percentage of cells in G₁ phase in U-2 OS, MG-63, and COA79 cells and a decrease in the percentage of cells in S phase in U-2 OS, COA30, and COA79 cells. (C) Cell cycle data presented in graphic form. Bolded numerals indicate statistically significant values. (D) Representative histogram for cell cycle presented. All experiments were repeated with at least three biologic replicates. Data are reported as mean ± SEM. *p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Figure 3.. Lerociclib treatment affects sarcoma viability in 2D and 3D cultures.

Viability was measured using alamarBlue assay. (A) Sarcoma cells were cultured in standard 2D conditions. U-2 OS (1.5 × 10⁴ cells per well), MG-63 (1.5 × 10⁴ cells per well), COA30 (3 × 10⁴ cells per well), or COA79 (3 ×10⁴ cells per well) were plated in 96-well plates, allowed to attach overnight, and treated with lerociclib at increasing concentrations (0–10 μM) for 72 hours. Treatment with increasing concentrations of lerociclib resulted in decreased viability in all four sarcoma cell lines. (B) COA79 cells (1 × 10⁶) were mixed in in 100 μL of 1% sodium alginate and 6% gelatin bioink and printed as a mixed print onto a 96-well plate. Prints were incubated overnight and treated with increasing doses of lerociclib (0–10 μM) for 72 hours. Lerociclib significantly decreased bioprint viability, but the 3D bioprints were less sensitive than the COA79 cells cultured in 2D conditions (A). (C) U-2 OS cells (1 × 10⁶) were mixed in100 μL of 1% sodium alginate and 6% gelatin bioink and printed as a mixed print onto a 96-well plate. Prints were incubated overnight and treated with increasing doses of lerociclib (0–5 μM) for 24 hours. Lerociclib significantly decreased viability. Viability of the U-2 OS 3D bioprints was affected at similar lerociclib concentrations as the U-2 OS cells cultured in 2D conditions. Experiments for A and C were repeated with at least three biologic replicates. Data for B represent two biologic replicates. Data are reported as mean ± SEM. *p ≤ 0.05, ** p ≤ 0.01

Figure 4:. Lerociclib decreases sarcoma cell migration.

Migration assays were conducted using 8 μm micropore Transwell inserts from 24-well culture plates coated with fibronectin (U-2 OS, MG-63, and COA79) or human laminin (COA30). U-2 OS (4 × 10⁴), MG-63 (5 × 10⁴), COA30 (1 × 10⁵) or COA79 (1 × 10⁵) cells were plated and treated with lerociclib (0–4 μM) at concentrations below the calculated LD₅₀ and allowed to migrate through the membrane. The inserts were fixed with 3% paraformaldehyde for 10 minutes and stained with 1% crystal violet for 30 minutes. A light microscope was used to obtain images of the inserts and the number of cells in seven random fields per insert were counted using ImageJ. (A) After treatment with 2.5 μM lerociclib, there was a significant decrease in U-2 OS cell migration (1 v. 0.45 ± 0.04, p ≤ 0.0002). (B) In COA30 cells, treatment with increasing concentrations of lerociclib resulted in a significant decrease in migration at both 2 μM (1 v. 0.18 ± 0.05, p ≤ 0.001) and 4 μM (1 v. 0.45 ± 0.04, p ≤ 0.001). (C) Treatment with 1 μM lerociclib resulted in a decrease in motility of COA79 cells (1 v. 0.81 ± 0.05, p ≤ 0.05). All experiments were repeated with at least three biologic replicates. Data are reported as mean ± SEM. Scale bars represent 100 μm. *p ≤ 0.05, *** p ≤ 0.001

Figure 5:. Lerociclib treatment decreases invasion.

For invasion assays, the bottom side of the Transwell plate was treated as described for migration and the top of the plate was coated with 50 μL of Matrigel for 4 hours at 37 °C. U-2 OS (4 × 10⁴), MG-63 (5 × 10⁴), COA30 (1 × 10⁵) or COA79 (1 × 10⁵) cells were plated and treated with increasing concentrations (0–2.5 μM) of lerociclib. Cells were allowed to invade through the Matrigel coating, fixed with 3% paraformaldehyde for 10 minutes and stained with 1% crystal violet for 30 minutes. A light microscope obtained images of the inserts and the number of cells in seven random fields per insert were counted using ImageJ. (A) In the U-2 OS cell line, there was a significant decrease in invasion after treatment with both 1 μM (1 v. 0.48 ± 0.04, p ≤ 0.0002) and 2.5 μM (1 v. 0.45 ± 0.12, p ≤ 0.01) concentrations of lerociclib. (B) There was a decrease in MG-63 invasion following 1 μM (1 v. 0.46 ± 0.04, p ≤ 0.001) and 2 μM (1 v. 0.37 ± 0.06, p ≤ 0.001) lerociclib concentrations. (C) In COA30 cells, there was a significant decrease in invasion following treatment with both 1 μM (1 v. 0.37 ± 0.10, p ≤ 0.001) and 2 μM (1 v. 0.38 ± 0.14, p ≤ 0.05) doses of lerociclib. (D) In COA79 cells, treatment with lerociclib significantly decreased invasion at 1 μM (1 v. 0.62 ± 0.14, p ≤ 0.05) and 2.5 μM (1 v. 0.37 ± 0.07, p ≤ 0.01) concentrations. All experiments were repeated with at least three biologic replicates. Data are reported as mean ± SEM. Scale bars represent 100 μm. *p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Figure 6:. Treatment with lerociclib decreases sarcoma stemness.

Extreme limiting dilution assays were conducted under non-adherent conditions using serial dilutions from 5000 to 1 cell per well to assess the ability of sarcoma cell lines to form spheres after treatment with lerociclib. (A) U-2 OS and (B) MG-63 cells were plated in serum-free DMEM with B-27 without vitamin A, L-glutamine, epidermal growth, fibroblast growth factor, and bovine serum albumin. Treatment media (100 μL) with either 0 or 2 μM lerociclib was added to each well. After 1 week, a single blinded researcher evaluated the wells for the presence or absence of spheres and extreme limiting dilution analysis software was used to analyze the results. There was a statistically significant decrease in the ability of U-2 OS (p ≤ 0.001) (A) or MG-63 (p ≤ 0.001) (B) cells to form spheres. Non-adherent (C) COA30 and (D) COA79 cells were plated in NB media in 96 well ultra-low attachment plates using serial dilutions from 5000 to 1 cell per well. Treatment media (100 μL) with either 0, 1 or 2 μM lerociclib was added to each well. After 1 week, a single blinded researcher evaluated the wells for the presence or absence of spheres and extreme limiting dilution analysis software was used to analyze the results. Lerociclib treatment reduced sphere forming ability in COA30 (p ≤ 0.001) (C) and COA79 (p ≤ 0.001) (D) cells. All experiments were repeated with at least three biologic replicates. Solid lines represent sphere forming capacity with no lerociclib treatment. Dashed lines represent sphere forming ability after lerociclib treatment.