Targeting tumour re-wiring by triple blockade of mTORC1, epidermal growth factor, and oestrogen receptor signalling pathways in endocrine-resistant breast cancer

Background Endocrine therapies are the mainstay of treatment for oestrogen receptor (ER)-positive (ER+) breast cancer (BC). However, resistance remains problematic largely due to enhanced cross-talk between ER and growth factor pathways, circumventing the need for steroid hormones. Previously, we reported the anti-proliferative effect of everolimus (RAD001-mTORC1 inhibitor) with endocrine therapy in resistance models; however, potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB, and mTORC1) may provide enhanced long-term clinical utility. Methods A panel of ER+ BC cell lines adapted to long-term oestrogen deprivation (LTED) and expressing ESR1wt or ESR1Y537S, modelling acquired resistance to an aromatase-inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan-ERBB inhibitor) in the presence or absence of oestradiol (E2), tamoxifen (4-OHT), or fulvestrant (ICI182780). End points included proliferation, cell signalling, cell cycle, and effect on ER-mediated transactivation. An in-vivo model of AI resistance was treated with monotherapies and combinations to assess the efficacy in delaying tumour progression. RNA-seq analysis was performed to identify changes in global gene expression as a result of the indicated therapies. Results Here, we show RAD001 and neratinib (pan-ERBB inhibitor) caused a concentration-dependent decrease in proliferation, irrespective of the ESR1 mutation status. The combination of either agent with endocrine therapy further reduced proliferation but the maximum effect was observed with a triple combination of RAD001, neratinib, and endocrine therapy. In the absence of oestrogen, RAD001 caused a reduction in ER-mediated transcription in the majority of the cell lines, which associated with a decrease in recruitment of ER to an oestrogen-response element on the TFF1 promoter. Contrastingly, neratinib increased both ER-mediated transactivation and ER recruitment, an effect reduced by the addition of RAD001. In-vivo analysis of an LTED model showed the triple combination of RAD001, neratinib, and fulvestrant was most effective at reducing tumour volume. Gene set enrichment analysis revealed that the addition of neratinib negated the epidermal growth factor (EGF)/EGF receptor feedback loops associated with RAD001. Conclusions Our data support the combination of therapies targeting ERBB2/3 and mTORC1 signalling, together with fulvestrant, in patients who relapse on endocrine therapy and retain a functional ER

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Additional file 4: of Targeting tumour re-wiring by triple blockade of mTORC1, epidermal growth factor, and oestrogen receptor signalling pathways in endocrine-resistant breast cancer

Figure S4. Anti-proliferative effect combination of RAD001 and neratinib together with endocrine agents (a) 4-OHT and (b) ICI. Endocrine-resistant and -sensitive BC cell lines were treated with a combination of RAD001 and neratinib and increasing concentrations of (a) 4-OHT or (b) ICI for 6 days with media change at day 3. Cell viability was analysed using a cell titer-glo assay. Data are expressed as fold-change relative to vehicle control. Error bars represent mean ± SEM. wt-MCF7 (1.5 nM RAD001; 200 nM neratinib); MCF7-LTED (1.5 nM RAD001; 300 nM neratinib); wt-SUM44 (0.37 nM RAD001; 450 nM neratinib); SUM44-LTED (0.37 nM RAD001; 250 nM neratinib); wt-HCC1428 (1.5 nM RAD001; 500 nM neratinib); HCC1428-LTED (3 nM RAD001; 250 nM neratinib). (PDF 208 kb

Additional file 2: of Targeting tumour re-wiring by triple blockade of mTORC1, epidermal growth factor, and oestrogen receptor signalling pathways in endocrine-resistant breast cancer

Figure S2. Anti-proliferative effect of RAD001 in combination with endocrine agents (a) 4-OHT and (b) ICI. Endocrine-resistant and -sensitive BC cell lines were treated with a combination of RAD001 (3 nM) and increasing concentrations of (a) 4-OHT or (b) ICI for 6 days with media change at day 3. Cell viability was analysed using a cell titer-glo assay. Data are expressed as fold-change relative to vehicle control. Error bars represent mean ± SEM. (PDF 196 kb

Additional file 6: of Targeting tumour re-wiring by triple blockade of mTORC1, epidermal growth factor, and oestrogen receptor signalling pathways in endocrine-resistant breast cancer

Figure S6. Assessment of dynamic changes in gene expression in response to RAD001, neratinib, or the combinations with fulvestrant. (a) MCF72a cells, which were previously engineered to express aromatase (CYP19) [33] were implanted into ovariectomised mice under androstenedione support. In this setting, MCF72a cells convert androstenedione in to oestrogen to drive proliferation. Once tumours developed, androstenedione was withdrawn. After a lag phase, tumour growth occurred synonymous with ligand independence. Assessment of the MCF72a-LTED showed continued expression of ER and proliferation in the absence of exogenous oestrogen providing a model of AI relapse. (b) Changes to gene expression (log2 difference drug – vehicle) as detected by RNA-seq for five drug combinations (neratinib, RAD001, ICI, neratinib + RAD001, and neratinib + RAD001 + ICI) were mapped to KEGG pathway graphs using Pathview ( https://bioconductor.org/packages/release/bioc/html/pathview.html ). Genes with a fold-change greater than 50% compared with vehicle in any condition were selected in order to expand the list of differentially expressed genes, allowing the identification of subtle changes in gene expression; for example, kinases or transcription factors that might have significant impact on downstream gene expression. A heatmap for each gene in shown. (c) Assessment of expression of E2F target genes in response to neratinib and RAD001. (PDF 1861 kb

Additional file 7: of Targeting tumour re-wiring by triple blockade of mTORC1, epidermal growth factor, and oestrogen receptor signalling pathways in endocrine-resistant breast cancer

Figure S7. Assessment of dynamic changes in expression of cell cycle regulatory genes. (a) Log2 differences in CCNE1, CCNL1, CDK3, CDK7, and CDK9 gene expression following treatment with RAD001, RAD001 + neratinib, and RAD001 + neratinib + fulvestrant (ICI), compared with vehicle. (b) GSEA enrichment plots for 198 genes known to be induced by sustained activation of ERK in response to EGF activity. Plots show the profile of the running Enrichment Score and positions of GeneSet Members on the Rank Ordered List for rank gene lists generated from the comparison of: ICI vs. vehicle; neratinib + ICI vs. ICI; RAD001 + ICI vs. ICI; and RAD001 + neratinib + ICI vs. RAD001 + ICI. (PDF 721 kb

Abstract B169: Neratinib has clinical activity in HER2-amplified breast cancer patients with tumors that have acquired activating mutations in HER2

Abstract Overexpression/amplification of HER2/ERBB2 occurs in 20% of breast cancers. Thanks to specific anti-HER2 agents, the prognosis of HER2-positive breast cancer has improved considerably. However, acquired resistance inevitably emerges over time and tumors escape pharmacologic pressure. In this work, we propose that acquisition of activating somatic mutations in HER2 upon anti-HER2 therapy may be more frequent than commonly reported and can reduce sensitivity to these agents. Moreover, we tested whether neratinib, an irreversible pan-HER inhibitor, is effective in tumors bearing both amplification and mutations of ERBB2. By targeted exome sequencing, we found that samples from metastatic breast cancer (MBC) patients relapsing to multiple lines of anti-HER2 therapy presented the acquisition of HER2 mutations. These mutations spanned from the extracellular domain (L313I, R456C) to the kinase domain (L755S, D769Y) of the receptor. To investigate the role of these mutations in drug resistance, we conducted functional studies by stably transducing the L755S HER2 mutation (the most frequent HER2 mutation in breast cancer) in two ERBB2-amplified breast cancer cell lines intrinsically sensitive to HER2 inhibition. In both models, we found that expression of L755S mutant-HER2 was sufficient to limit sensitivity to trastuzumab, lapatinib, or the combination of both agents. Consistently, neither trastuzumab nor lapatinib was effective in inhibiting tumor growth of patient-derived xenografts established from a patient with ERBB2-amplified/mutant (D769Y) breast cancer. However, neratinib treatment demonstrated marked sensitivity in this tumor model, resulting in significant tumor growth inhibition. The antitumor activity of neratinib was also explored in breast cancer patients with coexisting ERBB2 amplification and mutation, either by compassionate use after failure of standard-of-care therapy or as part of a “basket” trial (NCT01953926) enrolling ERBB2-mutant patients. In both settings, we observed durable clinical response to neratinib. MBC case #HER2 co-mutationResponse to neratinibDurability of response (mo)1D769YSD62L313ISD93Y772_A775dupSD44L755SSD55V777LPR6 Our findings indicate that acquired HER2 mutations may reduce the effectiveness of therapeutic agents commonly used for the management of ERBB2-amplified MBC. Moreover, we propose neratinib as an effective treatment option for patients whose tumors harbor both ERBB2 amplifications and mutations. Citation Format: Emiliano Cocco, F. Javier Carmona, Helen H. Won, Michael F. Berger, David M. Hyman, Valentina Rossi, Carmen Chan, Alyssa Moriarty, Kyriakos P. Papadopoulos, Michael J. Wick, James Cownie, Ivana Sarotto, Richard E. Cutler, Francesca Avogadri-Connors, Peter Savas, Alshad S. Lalani, Valentina Boni, Sherene Loi, Jose Baselga, Filippo Montemurro, Maurizio Scaltriti. Neratinib has clinical activity in HER2-amplified breast cancer patients with tumors that have acquired activating mutations in HER2 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B169

Neratinib is effective in breast tumors bearing both amplification and mutation of ERBB2 (HER2)

Mutations in , the gene encoding epidermal growth factor receptor (EGFR) family member HER2, are common in and drive the growth of "HER2-negative" (not amplified) tumors but are rare in "HER2-positive" ( amplified) breast cancer. We analyzed DNA-sequencing data from HER2-positive patients and used cell lines and a patient-derived xenograft model to test the consequence of HER2 mutations on the efficacy of anti-HER2 agents such as trastuzumab, lapatinib, and neratinib, an irreversible pan-EGFR inhibitor. HER2 mutations were present in ~7% of HER2-positive tumors, all of which were metastatic but not all were previously treated. Compared to HER2 amplification alone, in both patients and cultured cell lines, the co-occurrence of HER2 mutation and amplification was associated with poor response to trastuzumab and lapatinib, the standard-of-care anti-HER2 agents. In mice, xenografts established from a patient whose HER2-positive tumor acquired a D769Y mutation in HER2 after progression on trastuzumab-based therapy were resistant to trastuzumab or lapatinib but were sensitive to neratinib. Clinical data revealed that six heavily pretreated patients with tumors bearing coincident HER2 amplification and mutation subsequently exhibited a statistically significant response to neratinib monotherapy. Thus, these findings indicate that coincident HER2 mutation reduces the efficacy of therapies commonly used to treat HER2-positive breast cancer, particularly in metastatic and previously HER2 inhibitor-treated patients, as well as potentially in patients scheduled for first-line treatment. Therefore, we propose that clinical studies testing the efficacy of neratinib are warranted selectively in breast cancer patients whose tumors carry both amplification and mutation of /HER2