Figure S4 from Tumor Suppressors Condition Differential Responses to the Selective CDK2 Inhibitor BLU-222

figure

posted on 2025-04-03, 07:21 authored by Adam P. Dommer, Vishnu Kumarasamy, Jianxin Wang, Thomas N. O’Connor, Michelle Roti, Sidney Mahan, Karen McLean, Erik S. Knudsen, Agnieszka K. Witkiewicz

Figure S4. Loss of RB1 shifts phase of arrest and sensitivity to BLU-222. A, Heatmap of RNA sequencing data comparing differential gene expression between BLU-222-treated Kuramochi (125 nM BLU-222) and OVCAR-8 (250 nM BLU-222) cells normalized to respective DMSO-treated controls. B, Cell cycle profiling from univariate flow cytometric analysis from OVCAR-8 cells at baseline, after 1 to 5 days of treatment with 500 nM BLU-222, and after 1 to 5 days of drug washout. C, Western blot for abrogation of G2/M checkpoint signaling in OVCAR-8 cells following doxycycline (Dox) induction of the RB1 expression vector and treatment with BLU-222 compared with RB1 deficient controls. D, Live cell proliferative monitoring of OVCAR-8 cells following Dox induction of the RB1 expression vector (RB1 Dox +) with non-induced controls (RB1 Dox -) and treatment with serial concentrations of palbociclib (n = 3–4 per group; error bars represent SEM). E, Table summarizing effect of RB1 induction on IC50 to BLU-222 in OVCAR-8 cells. F, Live cell proliferative monitoring of OVCAR-8 cells following doxycycline induction of the RB1 expression vector (RB1 Dox +) with non-induced controls (RB1 Dox -) and transfection with siNT, siCCNE1, or siCCNA2 [n = 3–4 per group; two-way ANOVA, significance (shown as #) for combined RB1 and siRNA effect; error bars represent SEM]. G, Live-cell imaging endpoint stills for the OVCAR-8 cells in D (scale bar = 100 µm). H, Western blot of RB1-E2F pathway activity in Kuramochi cells with RB1 knockdown or siNT control and treatment with BLU-222. I, Cell cycle profiling from univariate flow cytometric analysis from Kuramochi cells with RB1 knockdown or siNT control and treatment with BLU-222. J, Western blot of RB1-E2F pathway activity in MDA-MB-157 cells with and without deletion (DEL) of RB1 and treatment with BLU-222. K, Cell cycle profiling from univariate flow cytometric analysis from MDA-MB-157 cells with and without deletion (DEL) of RB1 and treatment with BLU-222. L, Table summarizing effect of RB1 knockdown/deletion on IC50 to BLU-222 in Kuramochi and MDA-MB-157 cells, respectively. ####, P < 0.0001. PI, propidium iodide.

Funding

National Cancer Institute (NCI)

United States Department of Health and Human Services

Find out more...

Roswell Park Alliance Foundation

History

ARTICLE ABSTRACT

Cyclin-dependent kinase 2 (CDK2) inhibitors have recently been developed and have entered clinical trials. Combination approaches can help broaden the use of therapeutic agents and establish more effective treatments. Here, we evaluated the selective CDK2 inhibitor BLU-222 for mechanisms of response in the context of ovarian and breast cancer models. Sensors of cellular CDK activity indicated that sensitivity to either CDK4/6 or CDK2 inhibition was related to the differential dependence on a single CDK for G1–S transition. Unlike CDK4/6 inhibitors, BLU-222 was able to robustly inhibit proliferation through cell-cycle inhibition in both G1 and G2 phases. However, it remained possible for cells to reenter the cell cycle upon drug withdrawal. The antiproliferative strength and impact on G1–S versus G2–M accumulation was mediated by the RB tumor suppressor. To broaden the sensitivity to CDK2 inhibition, combinatorial drug screens were performed that identified both synergistic (e.g., CDK4/6 inhibitors) and antagonistic (e.g., WEE1 inhibitors) relationships. Models that were exceptionally sensitive to CDK2 inhibition displayed coordinate expression of cyclin E1 and P16INK4A, an endogenous CDK4/6 inhibitor. Functional studies demonstrated that P16INK4A and CDK4/6 activity were key mediators of sensitivity to BLU-222. Clinical gene and protein expression analyses revealed a positive correlation between cyclin E1 and P16INK4A and identified that ∼25% of ovarian cancers exhibited coordinate expression of cyclin E, P16INK4A, and RB, indicative of strong sensitivity to CDK2 inhibition. Together, this work advances a precision strategy for the use of CDK2 inhibitors in the context of ovarian and breast cancers.Significance: The CDK2-specific inhibitor BLU-222 shows preclinical efficacy in breast and ovarian cancer with select determinants of response and holds promise in combinatorial strategies.See related article by House and colleagues, p. 1297