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. Author manuscript; available in PMC: 2019 Dec 15.
Published in final edited form as: Clin Cancer Res. 2018 Sep 4;24(24):6594–6610. doi: 10.1158/1078-0432.CCR-18-1446

Figure 4. Sequential combination treatment with CDK2 inhibitor followed by AZD1775 in cyclin E–low TNBC is synergistic in vitro and in vivo.

Figure 4.

(A) Combination index (CI) of sequential (D→AZD) and concomitant (D+AZD) combination of dinaciclib and AZD1775 in MDA231 cyclin E–inducible cells with (DOX) or without (CNL) 10 ng/mL doxycycline. CI=1.0 indicates an additive effect (red line). (B) Mean CI values of sequential (D→AZD) and concomitant (D+AZD) treatment using dinaciclib and AZD1775 in TNBC cells by HTSA. CI=1.0 indicates an additive effect (red line). The concentrations of inhibitors of this assay are listed in table S5. (C) The clonogenic assay of TNBC cells with dinaciclib (D) and/or AZD1775 (AZD), as single agents or in combination sequentially (D→AZD). (D) Cells were treated with vehicle, dinaciclib (24 hours, D), AZD1775 (48 hours, AZD), or sequential dinaciclib (24 hours) followed by AZD1775 (48 hours) (D→AZD) at concentrations listed in table S6. Cells subjected annexin V assay (top), quantification of the sub-G1 population (middle), or immunoblot with PARP antibody (bottom). Differences between combination treatment and other groups were analyzed statistically for significance. (E) Mean CI values of sequential (CDK2i→AZD) and concomitant (CDK2i+AZD) treatment using CDK2 inhibitor (meriolin5, SNS032, or roscovitine) and AZD1775 in MDA231 and SUM159 by HTSA as in (B). CI=1.0 indicates an additive effect (red line). (F, G, H) Tumor volumes (top) and mouse survival (bottom) of mice harboring xenograft tumors derived from (F) SUM149 cells or patient tumors, (G) PDX1, or (H) PDX2 were treated with 25 mg/kg dinaciclib once per day for 2 days and 50 mg/kg AZD1775 twice per day for 2 days of each 7-day cycle. (Schematic in fig. S12A.) V, mouse treated by vehicle only; D, mouse treated by dinaciclib and vehicle of AZD1775; AZD, mouse treated by AZD1775 and vehicle of dinaciclib; D+AZD, mouse concomitantly treated by dinaciclib and AZD1775; D→AZD, mouse sequentially treated by dinaciclib and AZD1775; N.S., not significant. SUM149 xenografts were treated for 6 cycles; PDX1 and PDX2 were treated for 4 cycles, with the exception of the D+AZD arm and the D→AZD arm in PDX1, which were treated for 6 cycles and 10 cycles respectively. A two-tailed unpaired t-test was use to compare two groups (n=4–20). Error bars represent standard error of the mean. The log-rank (Mantel-Cox) test was used in survival experiments. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.