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. 2023 Aug 5;9(1):64.
doi: 10.1038/s41523-023-00571-w.

Combining the AKT inhibitor capivasertib and SERD fulvestrant is effective in palbociclib-resistant ER+ breast cancer preclinical models

Lorna Hopcroft  1 , Eleanor M Wigmore  2 , Stuart C Williamson  1 , Susana Ros  1 , Cath Eberlein  1 , Jennifer I Moss  1 , Jelena Urosevic  1 , Larissa S Carnevalli  1 , Sara Talbot  1 , Lauren Bradshaw  1 , Catherine Blaker  1 , Sreeharsha Gunda  3 , Venetia Owenson  4 , Scott Hoffmann  4 , Daniel Sutton  4 , Stewart Jones  4 , Richard J A Goodwin  4 , Brandon S Willis  5 , Claire Rooney  6 , Elza C de Bruin  6 , Simon T Barry  7
Affiliations

Combining the AKT inhibitor capivasertib and SERD fulvestrant is effective in palbociclib-resistant ER+ breast cancer preclinical models

Lorna Hopcroft et al. NPJ Breast Cancer. .

Erratum in

Abstract

Combining the selective AKT inhibitor, capivasertib, and SERD, fulvestrant improved PFS in a Phase III clinical trial (CAPItello-291), treating HR+ breast cancer patients following aromatase inhibitors, with or without CDK4/6 inhibitors. However, clinical data suggests CDK4/6 treatment may reduce response to subsequent monotherapy endocrine treatment. To support understanding of trials such as CAPItello-291 and gain insight into this emerging population of patients, we explored how CDK4/6 inhibitor treatment influences ER+ breast tumour cell function and response to fulvestrant and capivasertib after CDK4/6 inhibitor treatment. In RB+, RB- T47D and MCF7 palbociclib-resistant cells ER pathway ER and Greb-1 expression were reduced versus naïve cells. PI3K-AKT pathway activation was also modified in RB+ cells, with capivasertib less effective at reducing pS6 in RB+ cells compared to parental cells. Expression profiling of parental versus palbociclib-resistant cells confirmed capivasertib, fulvestrant and the combination differentially impacted gene expression modulation in resistant cells, with different responses seen in T47D and MCF7 cells. Fulvestrant inhibition of ER-dependent genes was reduced. In resistant cells, the combination was less effective at reducing cell cycle genes, but a consistent reduction in cell fraction in S-phase was observed in naïve and resistant cells. Despite modified signalling responses, both RB+ and RB- resistant cells responded to combination treatment despite some reduction in relative efficacy and was effective in vivo in palbociclib-resistant PDX models. Collectively these findings demonstrate that simultaneous inhibition of AKT and ER signalling can be effective in models representing palbociclib resistance despite changes in pathway dependency.

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Conflict of interest statement

All authors declare a financial conflict. All authors are AstraZeneca employees and shareholders.

Figures

Fig. 1
Fig. 1. RB− and RB+ palbociclib-resistant cell lines have distinct signalling and transcriptional profiles.
A Western blot profiling of PalboR cells for cell cycle, ER-regulated and PI3K pathway markers. Cells cultured long-term with continuous palbociclib (CP) 3 μM T47D, 1 μM MCF7, palbocilib withdrawn (PW) for 1 (MCF7) or 2 (T47D) weeks. Parental cells treated for 24 h with DMSO (C) or palbociclib (T) 3 μM T47D, 1 μM MCF7. B Effect of palbociclib on cell proliferation in parental versus PalboR cells plated without palbociclib overnight and then treated for 5 days with palbociclib at concentrations indicated. Mean ± SD of three independent experiments performed with duplicates represented. C Stability of resistance phenotype measured by removing palbociclib for 1 and 7 days, then retreating palbociclib for 5 days. Mean duplicate cell counts ± SD representative of three independent experiments. D PalboR cells in continuous palbociclib compared to parental cell lines treated with DMSO. Pathway heatmaps of the top 30 pathways ordered by combined log p-value across treatment groups as indicated; upregulated pathways red (top heatmap), downregulated pathways blue (bottom heatmap). Shade represents log p-value. Numbers to the right of the heatmap represent the total number of genes in the pathway signature, yellow numbers in boxes DEG found in signature. RET respiratory electron transport, PROD production, GF growth factor. E, F Heatmap showing E T47D and F MCF7 mRNA z-scores of (i) differentially expressed genes in T47D RB− and MCF7 RB− following palbociclib withdrawal compared to continuous treatment, (ii) differentially expressed genes in T47D CDK6H and MCF7 PacqR following palbociclib withdrawal compared to continuous treatment. Each row represents a gene with significant differential expression between groups in one or both resistant cells (−1 > log2FC > 1 and p-value (FDR) < 0.01). No genes were found in common between (i) and (ii) for T47D or MCF7-resistant cell lines.
Fig. 2
Fig. 2. Palbociclib exposure impacts PI3K pathway, ER pathway and cell cycle response.
A Treatment schedule. B Analysis of PI3K/AKT and ER pathway biomarkers and C cell cycle and apoptosis biomarkers of parental and PalboR T47D & MCF7 cells plated in the absence of palbociclib for 96 h prior to treatment with capivasertib, fulvestrant and the combination for 24 h at the doses indicated. Representative example of two independent experiments. D Cell cycle analysis of the EdU stained parental and PalboR T47D & MCF7 cells following palbociclib removal for 96 h prior to a 48-h treatment with capivasertib, fulvestrant and the combination at the doses indicated. Mean cell distribution of each cell cycle phase plotted ± SD (n = 4 biological replicates).
Fig. 3
Fig. 3. Transcriptomic profile of the capivasertib fulvestrant combination in parental versus palbociclib-resistant cells.
A Significant differentially expressed genes after combination treatment in parental T47D (z-scores of the mean expression). Each row represents a single gene. Columns represent relative expression for DMSO and combination treatment in parental, T47D RB−, T47D CDK6H cells. The number of genes that were significantly differentially expressed in PalboR lines as well as parental for combination vs DMSO are shown as the common genes below each column (the total number of DEG found in T47D RB− was 411 and for T47D CDK6H 484). B Significant differentially expressed genes after combination treatment in parental MCF7 are shown as z-scores, with each row representing a single gene with significant differential expression. Columns representing the changes in the same genes are shown for DMSO and capivasertib fulvestrant combination treatment comparing parental, MCF7 RB−, MCF7 PacqR cells. The number of genes that were also found to be significantly differentially expressed in PalboR lines are shown as the common genes below the relevant columns. MCF7 RB− had a total of 1531 DEG in comparison of capivasertib fulvestrant vs DMSO and MCF7 PacqR had a total 2300 DEG. C, D Overlap of downregulated genes enriched in pathways (blue) and upregulated genes enriched in pathways (red) in the comparison of combination treatment with DMSO in C T47D parental, T47D RB−, T47D CDK6H cells and D MCF7 parental, MCF7 RB− and MCF7 PacqR. E, F Pathway heatmaps representing the top 35 pathways ordered by combined log p-value across the groups. Pathways that are enriched in the downregulated DEG are shown in blue. All comparisons are combination vs DMSO (control) in each of E the three T47D cell lines and F the three MCF7 cell lines. Shade represents log p-value, the numbers to the right of the heatmap the total number of genes in the pathway and yellow numbers in boxes the DEG found in the pathway.
Fig. 4
Fig. 4. Sensitivity of palbociclib-resistant cells to the combination of capivasertib and fulvestrant.
A T47D and B MCF7 parental and PalboR cells plated without palbociclib overnight, then treated with capivasertib and fulvestrant at concentrations indicated for 5 days. Cell count normalised to DMSO control (100%) and cell count at the time of dosing (0%). Mean of A 4 and B 3 independent experiments. C T47D and D MCF7 parental and PalboR cells treated with capivasertib and fulvestrant at concentrations indicated for 5 days. Normalised cell counts analysed by Genedata Screener Compound Synergy Extension. Representative combination clusters are shown (0 represents DMSO control, values 1–100 represent cell growth inhibition, values > 100 represent cell death) with Highest Single Agent (HSA) excess heatmaps (highlights dose range where combination benefit was detected) from representative experiment. Tables summarise the range of HSA scores for 3+ independent experiments. E Parental and PalboR T47D and MCF7 cell confluence measured by Incucyte S3. Cells treated 4 days on 3 days off 500 nM capivasertib, continuous 1 nM fulvestrant, or combination for ≥14 days as indicated. F Relative growth inhibition following monotherapy and combination treatment. Confluence calculated at day 18 or 80% control confluence (time point represented by dotted vertical lines on (E). Representative data of ≥3 independent experiments shown with ordinary 1-way ANOVA (Analysis of Variance) statistical analysis *p < 0.05; **p < 0.01; ***p < 0.005, ****p < 0.001.
Fig. 5
Fig. 5. In vivo anti-tumour activity of the combination of capivasertib and fulvestrant in palbociclib insensitive models.
A In vivo activity of capivasertib, fulvestrant and the combination in PI3K pathway mutated PDX tumour models (ST3632, ST3932, CTC174 and ST1799/HI/PBR) and PI3K pathway unaltered models ST3164B and ST941/HI/PBR. Tumours treated with vehicle (closed circles), 130 mg/kg capivasertib BID 4 days on 3 days off (open circles), 5 mg/animal fulvestrant, QW (open triangle), or combination (closed squares). Geomean tumour volumes ± SEM (*p < 0.05, **p < 0.005, ***p < 0.0005) are shown. B (i) Pharmacodynamic changes phosphorylation and total protein levels of PRAS40 (Thr246), S6 (Ser235/236) and p-4EBP1 (Thr37/46), pRb1 or ER in the CTC174 PDX PI3KCAm model after 28 days of treatment. Tumours treated with vehicle, 100 mg/kg capivasertib BID 4 days on 3 days off, 5 mg/animal fulvestrant, or combination. Data normalised to the geomean of β-actin and vinculin, percentage change from control plotted as mean ± SEM (n = 5). Statistical analysis ANOVA test vs vehicle-treated, *p < 0.05; **p < 0.005; ***p < 0.0005. (ii) Ki-67 was visualised using immunohistochemistry of tumour samples; represented as mean ± SEM (n = 5). Statistical analysis ANOVA test, *p < 0.05; **p < 0.01; ***p < 0.005, ****p < 0.001.
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