CDK 4/6 inhibitors sensitize PIK3CA mutant breast cancer to PI3K inhibitors

Abstract

Activation of the phosphoinositide 3-kinase (PI3K) pathway occurs frequently in breast cancer. However, clinical results of single-agent PI3K inhibitors have been modest to date. A combinatorial drug screen on multiple PIK3CA mutant cancers with decreased sensitivity to PI3K inhibitors revealed that combined CDK 4/6-PI3K inhibition synergistically reduces cell viability. Laboratory studies revealed that sensitive cancers suppress RB phosphorylation upon treatment with single-agent PI3K inhibitors but cancers with reduced sensitivity fail to do so. Similarly, patients' tumors that responded to the PI3K inhibitor BYL719 demonstrated suppression of pRB, while nonresponding tumors showed sustained or increased levels of pRB. Importantly, the combination of PI3K and CDK 4/6 inhibitors overcomes intrinsic and adaptive resistance leading to tumor regressions in PIK3CA mutant xenografts.

Trial registration: ClinicalTrials.gov NCT01219699.

Copyright © 2014 Elsevier Inc. All rights reserved.

Figures

Figure 1. Viability, cell cycle profiles, and signaling in PIK3CA mutant breast cancer cell lines with acquired resistance to PI3Ki

A) Parental and resistant MCF7 were treated with either vehicle or GDC-0941 1 μM. Parental and resistant 453 and T47D cell lines were treated with either vehicle or BYL719 1 μM. When vehicle treated cells grew to confluence, all cells were fixed and stained for nucleic acid with Syto-60 and absorbance was quantified. Representative plates are shown. Data are mean of three independent experiments performed in triplicate. *indicates p

Figure 2. Combinatorial Drug Screen to identify sensitizers to PI3K inhibition in resistant PIK3CA mutant cell lines

A) An outline of the drug screening protocol. Resistant cells were seeded in 96 well plates and treated in triplicate with escalating doses of each of the 42 compounds in the drug screen, either singly or in combination with 1 μM dose of PI3Ki. B) For each of the resistant cell lines, dose response curves to the targeted agents were generated, in the absence or presence of 1 μM of BYL719 for T47DR and 453R or GDC-0941 for MCF7R. The agents were ranked according to greatest difference in area under the curve (AUC) between these two dose response curves for each cell line. Targets of each agent, ranked by difference in AUC, are depicted for each cell line. Darkened values indicate statistically significant sensitizers (FDR

Figure 3. The correlation between post-treatment PIP3 levels and the capacity of Akt inhibition to re-sensitize resistant cells to PI3Ki

A) Each resistant line was treated with escalating doses of the agent to which it was made resistant for 24 hours (BYL719 for 453R and T47DR, and GDC-0941 for MCF7R). Lysates were prepared and were probed with the indicated antibodies. B) Phospholipids in parental and resistant cells were isolated after 24 hr treatment with vehicle or the indicated PI3Ki (for parental lines) or continuous treatment with PI3Ki (for resistant lines. PIP3 and PIP2 levels were measured and the data shown as mean of three independent experiments. *indicates p<0.05 by student's t test. C) Viability was assessed in parental and resistant 453 and T47D lines treated with escalating doses of MK2206 for 5 days. Resistant lines were also treated with escalating doses of MK2206 in the presence of 1 μM BYL719, and dose response curves were generated. Viability values for each curve were normalized to the measured inhibition value at zero dose of MK2206 (-/+ BYL719 1 μM for 453R and T47DR). Data represent mean of 3 replicates. D) MCF7 and MCF7R cells were treated with escalating doses of MK2206. MCF7R was also treated with MK2206 in the presence of 1 μM dose of GDC-0941 or BYL719. Viability values for each curve were normalized to the measured inhibition value at zero dose of MK2206 (-/+ GDC-0941 1 μM for MCF7R). Data represent mean of 3 replicates. E-F) Parental and resistant 453, T47D (E), and MCF7 (F) cells were treated for 24 hours with the specified agents. Lysates were made after 24 hours, and probed with the indicated antibodies. All error bars in this figure represent +/- SEM. See also Figure S3.

Figure 4. Combined CDK 4/6 and PI3K inhibition in the treatment of resistant PIK3CA breast cancer lines

A) A dose matrix of PI3Ki, either GDC-0941 (GDC) or BYL719 (BYL) as indicated, and CDK 4/6 inhibitor LEE011 was created in all 3 pairs of parental and resistant cell lines. Viability was assessed after 5 days. Percent inhibition at each dose of drug is presented. B) Cell lines were treated with vehicle (VEH) or GDC0941 (GDC) and LEE011 (LEE), in the case of MCF7R, or BYL719 (BYL) and LEE011, in the case of 453R and T47DR, at 1 μM doses for 24 hours. Cell cycle analysis was then performed using propidium iodide staining followed by flow cytometry and percentage of cells in subG1, G1, S, and G2 phases were quantified. Data are mean of three replicates. * indicates pPIK3CA mutant (MT) and 10 PIK3CA wild-type (WT) breast cancer cell lines were treated with BYL719 and LEE011 in dose matrices across 7 concentrations for each agent, and assessed for proliferation after 5 days. The response of each cell line to the combination was scored for synergy (Supplemental Experimental Procedures), and ranked by synergy score. E) Median synergy scores between the PIK3CA mutants and wild-type lines were compared. * indicates p<0.05 by ANOVA. All error bars in this figure represent +/- SEM. See also Figure S4.

Figure 5. Comparison of RB phosphorylation between resistant lines and sensitive lines following PI3K inhibition, PI3K/mTORC inhibition, and PI3K/CDK inhibition

A) Parental and resistant cells were seeded and treated with either AZD-8055 (8055) 500 nM or the indicated PI3Ki 1 μM either alone or in combination. Lysates were made after 24 hours of treatment and probed with the indicated antibodies. B) Cells were prepared and treated as in (A), but lysed with buffer containing 0.1% SDS. C) Cells were treated with PI3Ki 1μM or LEE011 1μM alone or in combination and cell lysates were prepared and probed as in (A). D) Cells were prepared and treated as per C, but lysed with buffer containing 0.1% SDS. See also Figure S5.

Figure 6. Correlation between post-treatment pRB levels and sensitivity to single-agent PI3Ki in vitro and in patient biopsy specimens

A) 5 sensitive PIK3CA mutant cell lines (IC50≤400nM to BYL719) and 5 de novo resistant PIK3CA cell lines (IC50≥800nM to BYL719) were treated with media or with BYL719 1 μM (BYL) for 24 hours, and lysates were probed with the indicated antibodies. The fraction of remaining pRB (relative to actin) was calculated for both the sensitive and resistant lines and a student's t test was performed. *indicates p<0.05 by student's t test. B) Eight patients enrolled in early clinical trials of BYL719 were classified as responders or nonresponders as previously described (Elkabets et al., 2013) (n=4 in each group). Biopsy specimens were collected within 2 weeks prior to start of treatment and while on treatment. Slides were prepared, stained and scored for pRB (see Supplemental Experimental Procedures). The percentage change relative to pretreatment levels of pRB for both the responders and the nonresponders is depicted. * indicates p

Figure 7. PI3K/ CDK 4/6 inhibitor combination in PIK3CA mutant breast cancer xenografts in vivo

A) MCF7 xenografts were treated with LEE011 75 mg/kg/d, GDC-0941 100 mg/kg/d or the combination. At day 28, three MCF7 xenografts that had progressed on GDC-0941 monotherapy had LEE011 75 mk/kg/d added to their regimen. *indicates p

Figure 8. Inhibition at vertical nodes within the PI3K pathway improves efficacy of PI3Ki

Combined inhibition of PI3K and Akt (indicated by the dashed bracket) is synergistic only in resistant models that maintain PIP3 and phosphorylation of Akt substrates (453R, T47DR). Combined inhibition of PI3K and mTOR or PI3K and CDK 4/6 (indicated by the solid brackets) is synergistic in all resistant models; including those that maintain PIP3 and phosphorylation of Akt substrates (453R, T47DR) and those that do not (MCF7R). * indicates that some resistant models maintain low level flux through the PI3K and Akt pathways.