WEE1 inhibition delays resistance to CDK4/6 inhibitor and antiestrogen treatment in estrogen receptor-positive breast cancer

Abstract

Although endocrine therapies and Cdk4/6 inhibitors have produced significantly improved outcomes for patients with estrogen receptor positive (ER+) breast cancer, continuous application of these drugs often results in resistance. We hypothesized that cancer cells acquiring drug resistance might increase their dependency on negative regulators of the cell cycle. Therefore, we investigated the effect of inhibiting WEE1 on delaying the development of resistance to palbociclib and fulvestrant. We treated ER+ MCF7 breast cancer cells with palbociclib alternating with a combination of fulvestrant and a WEE1 inhibitor AZD1775 for 12 months. We found that the alternating treatment prevented the development of drug resistance to palbociclib and fulvestrant compared to monotherapies. Furthermore, we developed a mathematical model that can simulate cell proliferation under monotherapy, combination or alternating drug treatments. Finally, we showed that the mathematical model can be used to minimize the number of fulvestrant plus AZD1775 treatment periods while maintaining its efficacy.

Fig. 1. Schematic representation of four 12-month treatment strategies. MCF7 cells are treated by monotherapy or alternating treatment over 12 months, 28 days per month.

MCF7 cells are re-plated at the beginning of each month in the mono and alternating treatments. Mono 1: palbociclib monotherapy at 750nM. Mono 2: ICI monotherapy at 750nM. Alter 1: alternating treatment of palbociclib 750nM with ICI 750nM, drug used is altered at the beginning of each month. Alter 2: alternating treatment of palbociclib 750nM with ICI 750nM plus AZD1775 250nM, drug used is altered at the beginning of each month. Palbo: palbociclib, AZD: AZD1775.

Fig. 2. Cell proliferation and protein level changes after 12 months mono and alternating treatments.

a Experimental cell proliferation data (mean ± s.d., n = 3, technical replications) for palbociclib monotherapy (solid magenta line), ICI monotherapy (solid black line), alternating treatment of palbociclib (dashed magenta line) with ICI (dashed black line), and alternating treatment of palbociclib (dashed orange line) with ICI plus AZD1775 (dashed green line). Experimental settings are the same as Fig. 1. The cell number is normalized to the cell number at re-plating. b Experimental cell proliferation data (mean ± s.d., n = 3) for AZD (solid purple line) and ICI plus AZD1775 (solid green line) at 1 month (28 days). c Bar plot of western blot data for total CyclinE1 (mean ± s.d., n = 3) level changes in palbociclib monotherapy and alternating treatment of palbociclib with ICI plus AZD1775 at different time points. Statistical testing was performed by one-way ANOVA. Only the significant differences between treatment timepoints and t=0 are shown. d Bar plot of western blot data for total Cdk6 (mean ± s.d., n = 3) level changes in ICI monotherapy and alternating treatment of palbociclib with ICI plus AZD1775 at different time points. Statistical testing was performed by one-way ANOVA. Only the significant differences between treatment timepoints and the t=0 are shown. Palbo: palbociclib, AZD: AZD1775.

Fig. 3. Palbociclib and AZD1775 dose response for MCF7 cells under mono and alternating treatments.

a Palbociclib dose response normalized to vehicle after 12 months palbociclib monotherapy, alternating treatment of palbociclib with ICI, alternating treatment of palbociclib with ICI plus AZD1775 compared to parental cells. The palbociclib monotherapy and alternating treatments are the same as Fig. 1. b Palbociclib dose response normalized to t=0, otherwise same as a. c The GR value of palbociclib dose response, otherwise same as a. d AZD1775 dose response normalized to vehicle after 12 months palbociclib monotherapy compared to parental cells. The palbociclib monotherapy is the same as Fig. 1. e AZD1775 dose response normalized to t=0, otherwise same as d. f The GR value of AZD1775 dose response, otherwise same as d. Palbo: palbociclib, AZD: AZD1775.

Fig. 4. Signaling diagram of the biological mechanism and model structure.

a Interactions of the biological mechanism. Reversible binding interactions are represented by dots on the components and an arrow to the complex. Arrows pointing from one protein to another protein represent phosphorylation, dephosphorylation, enhancement (arrow) or inhibition (blunt head) of the protein. Line pointing to another line represents enhancement (arrow) or inhibition (blunt head) of the interactions. Applied drugs are colored in red. The biological mechanism consists of the following numbered processes: 1. E2 binds to ER; 2. ICI binds to ER; 3. E2:ER increases transcription of c-Myc; 4. E2:ER increases transcription of CyclinE1; 5. E2:ER increases transcription of CyclinD1; 6. c-Myc inhibits transcription of p21; 7. CyclinD1 binds to Cdk4; 8. CylinD1 binds to Cdk6; 9. p21 binds to CyclinD1:Cdk4; 10. p21 binds to CyclinD1:Cdk6; 11. CyclinE1 binds to Cdk2; 12. p21 binds to CyclinE1:Cdk2; 13. Palbociclib binds to Cdk4; 14. Palbociclib binds to Cdk6; 15. Palbociclib binds to CyclinD1:Cdk4; 16. Palbociclib binds to CyclinD1:Cdk6; 17. Palbociclib binds to CyclinD1:Cdk4:p21; 18. Palbociclib binds to CyclinD1:Cdk6:p21; 19. CyclinD1:Cdk4/6 phosphorylates RB1 to RB1-p; 20. CyclinE1:Cdk2 phosphorylates RB1-p to RB1-pp; 21. RB1 binds to E2F; 22. RB1-p binds to E2F; 23. E2F up-regulates RB1; 24. E2F up-regulates c-Myc; 25. E2F up-regulates CyclinE1. 26. E2F drives cell proliferation; 27. ICI increases Cdk6; 28. Palbociclib increases CyclinE1; 29. AZD decreases CyclinE1, the increased CyclinE1 effect caused by palbociclib and the increased Cdk6 effect caused by ICI. b Structure of the mathematical model, a simplified version of the biological mechanism in a. Palbo: palbociclib, AZD: AZD1775.

Fig. 5. Mathematical model simulation compared to proliferation data for various treatments.

a Palbociclib monotherapy. The experimental data are shown in magenta and the simulation results are shown in brown (the shaded regions encompass the entire range of simulations within the cohort). b ICI monotherapy. The experimental data are shown in black and the simulation results are shown in brown. c Palbociclib alternating with ICI treatment. The experimental data are shown in magenta (palbociclib arm) and black (ICI arm). d Palbociclib alternating with ICI plus AZD1775 treatment. The experimental data are shown in magenta (palbociclib arm) and green (ICI plus AZD1775 arm). e AZD1775 treatment for 1 month. The experimental data are shown in purple. f ICI plus AZD1775 treatment for 1 month. The experimental data are shown in green. Palbo: palbociclib, AZD: AZD1775.

Fig. 6. Mathematical model simulation of protein level changes and comparison to western blot data for various treatments.

a Model simulation of normalized Cdk6 level changes for palbociclib (magenta) and ICI (black) monotherapy over 12 months. The shaded regions encompass the entire range of simulations for the cohort. b Model simulation of normalized CyclinE1 level changes for palbociclib (magenta) and ICI (black) monotherapy over 12 months. c Model simulation of normalized Cdk6 level changes for palbociclib (magenta) alternating with ICI (black), and palbociclib (orange) alternating with ICI plus AZD1775 (green) over 12 months. d Model simulation of normalized CyclinE1 level changes for palbociclib (magenta) alternating with ICI (black), and palbociclib (orange) alternating with ICI plus AZD1775 (green) over 12 months. e Bar plot of model simulation of Cdk6 level compared to experimental data for ICI (black) monotherapy, and palbociclib alternating with ICI plus AZD1775 (green) at different timepoints. The simulation results shown in brown are the average results from all cohort simulations. f Bar plot of model simulation of CylinE1 level compared to experimental data for palbociclib (magenta) monotherapy, and palbociclib alternating with ICI plus AZD1775 (green) at different timepoints. Palbo: palbociclib, AZD: AZD1775.

Fig. 7. Minimize the number of ICI plus AZD1775 treatment intervals within an alternating regimen while ensuring that the maximum proliferation does not exceed 20-fold per month over 24 months.

a-c Proposed Palbociclib (magenta) alternating with ICI plus AZD1775 (green) protocol to reduce the frequency of ICI plus AZD1775. The shaded regions encompass the entire range of simulations within the cohort. a Normalized cell number. b Normalized total Cdk6 level. c Normalized total CyclinE1 level. d-f Proposed Palbociclib (magenta), ICI (black) and ICI plus AZD1775 (green) alternating treatment. d Normalized cell number. e Normalized total Cdk6. f Normalized total CyclinE1. Palbo: palbociclib, AZD: AZD1775.