The effect of the alpha-specific PI3K inhibitor alpelisib combined with anti-HER2 therapy in HER2+/PIK3CA mutant breast cancer

Abstract

Background: HER2 is amplified or overexpressed in around 20% of breast cancers (BC). HER2-targeted therapies have significantly improved the prognosis of patients with HER2+ BC, however, de novo and acquired resistance to anti-HER2 treatment is common. Activating mutations in the PIK3CA gene are reported in ∼30% of HER2+ BC and are associated with resistance to anti-HER2 therapies and a poor prognosis. Here, we investigated the in vitro and in vivo antitumor efficacy of the alpha-specific PI3K inhibitor alpelisib alone or in combination with anti-HER2 therapy using a panel of HER2+ BC cell lines. We also generated models of acquired resistance to alpelisib to investigate the mechanisms underlying resistance to alpha-specific PI3K inhibition.

Materials and methods: PIK3CA mutant (HCC1954, KPL4 and JMT1) and wild-type (BT474 and SKBR3) HER2+ BC cell lines were used. The HCC1954 and KPL4 cells were chronically exposed to increasing concentrations of alpelisib or to alpelisib + trastuzumab in order to generate derivatives with acquired resistance to alpelisib (AR) and to alpelisib + trastuzumab (ATR). The transcriptomic profiles of HCC1954, KPL4 and their AR and ATR derivatives were determined by RNA sequencing. Cell growth was assessed by MTT assay. Changes in the protein levels of key PI3K pathway components were assessed by Western blotting. Gene expression, cellular and patients' data from the Cancer Dependency Map (DepMap) and KMPlot datasets were interrogated.

Results: HER2+ BC cell lines harboring activating mutations in PIK3CA were less sensitive to single or dual anti-HER2 blockade compared to PIK3CA wild-type cells. Alpelisib treatment resulted in dose-dependent inhibition of the growth of cells with or without PIK3CA mutations and enhanced the antitumor efficacy of anti-HER2 therapies in vitro. In addition, alpelisib greatly delayed tumor growth of HCC1954 xenografts in vivo. Functional annotation of the significantly differentially expressed genes suggested the common activation of biological processes associated with oxidation reduction, cell proliferation, immune response and RNA synthesis in alpelisib-resistant models compared with native cells. Eight commonly upregulated genes (log2 fold-change >1, False Discovery Rate [FDR] <0.05) in models with acquired resistance to alpelisib or alpelisib + trastuzumab were identified. Among these, AKR1C1 was associated with alpelisib-resistance in vitro and with a poor prognosis in patients with HER2+ BC.

Conclusions: Our findings support the use of an alpha-selective PI3K inhibitor to overcome the therapeutic limitations associated with single or dual HER2 blockade in PIK3CA-mutant HER2+ breast cancer. Future studies are warranted to confirm the potential role of candidate genes/pathways in resistance to alpelisib.

Keywords: AK1RC1; HER2; PIK3CA; alpelisib; breast cancer; resistance.

Figure 1

Cell growth inhibition in vitro of HER2+ breast cancer cell lines treated with monoclonal anti-HER2 antibodies. Cells were plated in 24-well plates and treated with vehicle (DMSO), 10 μg/ml trastuzumab, or 10 μg/ml trastuzumab + 10 μg/ml pertuzumab. Cell growth was assessed after 6 days. Data were normalized to vehicle. Data represent means ± SEM. *p-value <0.05, **p-value <0.01; ***p-value <0.001; ****p-value <0.0001; ns, not significant according to multiple t test with Bonferroni correction.

Figure 2

Responses of HER2+ breast cancer cell lines to the alpha-specific PI3K inhibitor alpelisib. (A), Cells were plated in 24-well plates and treated with increasing doses of alpelisib. Cell growth was assessed after 6 days. Data were analyzed by GraphPad Prism (version 9.0) to generate drug response curves and relative IC50 values using the log (inhibitor) versus response-variable slope model (bars, SEM) with normalization of data defining the biggest number in each dataset as 100% and the smallest number in the same dataset as 0%. (B), Estimated IC50 values of alpelisib in HER2 + breast cancer cell lines. CI, confidence of interval.

Figure 3

The alpha-specific PI3K inhibitor alpelisib increases the efficacy of anti-HER2 therapy in HER2+ breast cancer cell lines. (A), Cells were plated in 24-multiwell and treated with vehicle (DMSO), 10μg/ml trastuzumab, 10μg/ml trastuzumab + 10μg/ml pertuzumab, 1μM alpelisib, 1μM alpelisib + 10μg/ml trastuzumab, 1μM alpelisib + 10μg/ml trastuzumab + 10μg/ml pertuzumab. Cell growth was assessed after 6 days. Data were normalized to vehicle. Data represent means ± SEM. *p-value <0.05; ***p-value <0.001; ****p-value <0.0001 according to multiple t test with Bonferroni correction. (B), Western blot analysis of total and phosphorylated proteins of the HER2/PI3K pathway signaling. Cells were plated in 6-multiwell and treated with vehicle (DMSO), 10μg/ml trastuzumab (T), 10μg/ml trastuzumab + 10μg/ml pertuzumab (T+P), 1μM alpelisib (A), 1μM alpelisib + 10μg/ml trastuzumab (A+T), 1μM alpelisib + 10μg/ml trastuzumab + 10μg/ml pertuzumab (A+T+P).

Figure 4

Pharmacological synergy between alpelisib and trastuzumab in HER2+/PIK3CA mutant breast cancer cell lines. HCC1954 and KPL4 cells were seeded in 96 well plate treated with increasing concentrations of each drug alone or in combination (up to 10μM alpelisib and 100μg/ml trastuzumab) for 7 days. Combination indices were determined using the Chou-Talalay test by CompuSyn software. Numbers inside each box indicate the ratio of viable treated cells to untreated cells, from three independent experiments. Synergy is defined as combination index (CI)<1, an additive effect as CI=1, and antagonism effect as CI>1.

Figure 5

In vivo efficacy alpelisib alone or in combination with trastuzumab. (A), Mice were injected with 5 x106 HCC1954 cells and randomized to receive vehicle, trastuzumab, alpelisib, alpelisib + trastuzumab (see Materials and Methods for details). (B), Tumor growth curves obtained by measuring the volumes of the tumors in the study groups. Data points represent mean ± SEM tumor volume. Table reported the rate-based T/C computed using the data up to day 15. (C), Growth rates of HCC1954 xenografts by treatment arm. Each dot represents a different mouse. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001; ns, not significant according to multiple t test with Bonferroni correction.

Figure 6

Venn diagram of significantly up-regulated genes in HER2+/PIK3CA mutant alpelisib-resistant (AR) and alpelisib + trastuzumab-resistant (ATR) derivatives.

Figure 7

Association between gene expression and in vitro sensitivity to alpelisib in breast cancer cell lines. Bars indicate fold-change between mRNA expression of the 8 genes commonly overexpressed in our alpelisib resistant models and in vitro cell viability upon treatment with alpelisib measured as median fluorescence intensity (MFI) in 26 breast cancer cell lines. Data were downloaded from the “Cancer Dependency Map Portal” (https://depmap.org/portal/achilles/).

Figure 8

Kaplan-Meier curves showing relapse-free survival (RFS) of HER2+ breast cancer patients. Log-rank test was used for comparing two groups with high (red) and low (black) expression of ARL4A, SNRPG, ODC1, AKR1C1, TRIM16L, AKR1C2, GLB1L2, and BDH1 genes. Number of patients at risk are indicated below each panel. HR, hazard ratio.

Figure 9

Effect of AKR1C1 siRNA knockdown in parental and alpelisib-resistant HER2+/PIK3CA mutant breast cancer cell lines. (A), HCC1954 and KPL4 parental and alpelisib-resistant (AR) cell lines were transfected with siRNA targeting the AKR1C1 or siRNA control (si-Cnt). Culture medium was replaced the next day with regular medium or alpelisib-containing medium, and replaced again at 4 days. Cell growth was assessed at day 6. Data were normalized to si-Cnt transfection. Data represent means ± SEM. *p-value <0.05; ****p-value <0.0001 according to multiple t test with Bonferroni correction. (B), Protein levels of AKR1C1 in HER2+/PIK3CA mutant breast cancer cell lines transfected with siRNA targeting siRNA control or against AKR1C1 were tested by Western blot.