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Combination of CDK4/6 inhibitors and endocrine therapy improves clinical outcome in advanced oestrogen receptor (ER)-positive breast cancer, however relapse is inevitable. Here, we show in model systems that other than loss ofRB1few gene-copy number (CN) alterations are associated with irreversible-resistance to endocrine therapy and subsequent secondary resistance to palbociclib. Resistance to palbociclib occurred as a result of tumour cell re-wiring leading to increased expression ofEGFR, MAPK, CDK4, CDK2, CDK7, CCNE1andCCNE2. Resistance altered the ER genome wide-binding pattern, leading to decreased expression of ‘classical’ oestrogen-regulated genes and was accompanied by reduced sensitivity to fulvestrant and tamoxifen. Persistent CDK4 blockade decreased phosphorylation of tuberous sclerosis complex 2 (TSC2) enhancing EGFR signalling, leading to the re-wiring of ER. Kinome-knockdown confirmed dependency on ERBB-signalling and G2/M–checkpoint proteins such as WEE1, together with the cell cycle master regulator, CDK7. Noteworthy, sensitivity to CDK7 inhibition was associated with loss of ER andRB1CN. Overall, we show that resistance to CDK4/6 inhibitors is dependent on kinase re-wiring and the redeployment of signalling cascades previously associated with endocrine resistance and highlights new therapeutic networks that can be exploited upon relapse after CDK4/6 inhibition.

Resistance to endocrine therapy remains a major clinical problem in breast cancer. Genetic studies highlight the potential role of estrogen receptor-α (ESR1) mutations, which show increased prevalence in the metastatic, endocrine-resistant setting. No naturally occurring ESR1 mutations have been reported in in vitro models of BC either before or after the acquisition of endocrine resistance making functional consequences difficult to study. We report the first discovery of naturally occurring ESR1 Y537C and ESR1 Y537S mutations in MCF7 and SUM44 ESR1-positive cell lines after acquisition of resistance to long-term-estrogen-deprivation (LTED) and subsequent resistance to fulvestrant (ICIR). Mutations were enriched with time, impacted on ESR1 binding to the genome and altered the ESR1 interactome. The results highlight the importance and functional consequence of these mutations and provide an important resource for studying endocrine resistance., ESR1 mutations occur in endocrine-resistant patients but have not yet been reported in in vitro models of breast cancer. Here, the authors report the discovery of naturally occurring ESR1 Y537Cand ESR1 Y537S mutations in two breast cancer cell lines after acquisition of resistance to long-term-estrogen-deprivation.

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 â vehicle) as detected by RNA-seq for five drug combinations (neratinib, RAD001, ICI, neratinib + RAD001, and neratinib + RAD001â +â 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)

Figure S5. Effect of RAD001, neratinib, or their combination with endocrine agents on ER-mediated transactivation and ER signalling. Cell lines were co-transfected with EREIItkLuc and pCH110, and treated for 24 h with the drug combinations indicated. IC50 values were used for RAD001 and neratinib together with standard concentrations of E2 (0.01 nM), 4-OHT (0.1 nM), and ICI (0.1 nM). Luciferase activity was normalized by β-galactosidase from triplicate wells and fold-changes expressed relative to the E2 control. Error bars represent mean ± SEM. *p

Figure S3. Anti-proliferative effect of neratinib in combination with endocrine agents (a) 4-OHT and (b) ICI. Endocrine-resistant and -sensitive BC cell lines were treated with a combination of neratinib (500 nM in wt-MCF7 and MCF7-LTED; 700 nM in wt-SUM44, SUM44-LTED, and wt-HCC1428; 300 nM in HCC1428-LTED) 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 195 kb)

Figure S1. IC50 values for the anti-proliferative effect of RAD001 and neratinib in relation to the ESR1, ERBB2, and PIK3CA mutational status in endocrine-resistant and -sensitive BC cell lines. (a) Mutational or wt status is depicted in grey and white, respectively, for ESR1, ERBB2, and PIK3CA. (b) Varying degrees of expression of genes encoding proteins targeted by fulvestrant, neratinib, and RAD001 showing heterogeneity in the cell lines tested. (c,d) Cells were treated in the absence or presence of exogenous oestradiol (E2) (0.01Â nM) and doubling concentrations of (c) RAD001 or (d) neratinib. Treatments were performed at day 1 and day 3 after seeding. After 6Â days of treatment, cell viability was analysed using a cell titer-glo assay and IC50 values were plotted. (PDF 156 kb)

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)

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)

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)