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AR ER protein densitometry and dose response to PI3K/mTORi

Gene changes in MCF7aro. The impact of cyp19 overexpression on SGK3 expression in an independent study. Impact of siRNA-AR/ER on SGK3 mRNA

Supplemental tables 1& 2

Impact of SiSGK3 in ZRaroLetR. MTS following siSGK3 under various hormonal stimuli.

Characterisation of ZR75aro-LetR. Validation of ER AR siRNA

Correlation between CYP19 amplification and SGK3 expression.

AR and ER binding events in the proximal promoters of SGK3, MYBL1, GREB1 and PKIB.

Supplemental methods

Divergent roles for androgen receptor (AR) in breast cancer have been reported. Following aromatase inhibitor (AI) treatment, the conversion of circulating androgens into estrogens can be diminished by >99%. We wished to establish whether the steroid environment can dictate the role of AR and the implications of this for subsequent therapy. This study utilizes models of AI resistance to explore responsiveness to PI3K/mTOR and anti-AR therapy when cells are exposed to unconverted weak androgens. Transcriptomic alterations driven by androstenedione (4AD) were assessed by RNA-sequencing. AR and estrogen receptor (ER) recruitment to target gene promoters was evaluated using ChIP, and relevance to patient profiles was performed using publicly available data sets. Although BEZ235 showed decreased viability across AI-sensitive and -resistant cell lines, anti-AR treatment elicited a decrease in cell viability only in the AI-resistant model. Serum and glucocorticoid-regulated kinase 3 (SGK3) and cAMP-dependent protein kinase inhibitor β (PKIB) were confirmed to be regulated by 4AD and shown to be mediated by AR; crucially, reexposure to estradiol suppressed expression of these genes. Meta-analysis of transcript levels showed high expression of SGK3 and PKIB to be associated with poor response to endocrine therapy (HR = 2.551, P = 0.003). Furthermore, this study found levels of SGK3 to be sustained in patients who do not respond to AI therapy. This study highlights the importance of the tumor steroid environment. SGK3 and PKIB are associated with poor response to endocrine therapy and could have utility in tailoring therapeutic approaches.

Table S1. Cell line authentication. Cell line DNA profiling was carried out using ATCC MCF7 DNA profile as reference. Table S2: List of ChIP and mRNA primers used for DNA and mRNA quantification on the Lightcycler (Roche). Table S3: Patient information for endocrine treated patients. Table S4. Over represented TF binding motifs in 666 ER ChIPseq peaks from LetR cells (using the Universe as background, q-value

Purpose: Disease recurrence is a common problem in breast cancer and yet the mechanisms enabling tumor cells to evade therapy and colonize distant organs remain unclear. We sought to characterize global expression changes occurring with metastatic disease progression in the endocrine-resistant setting.Experimental Design: Here, for the first time, RNAsequencing has been performed on matched primary, nodal, and liver metastatic tumors from tamoxifen-treated patients following disease progression. Expression of genes commonly elevated in the metastases of sequenced patients was subsequently examined in an extended matched patient cohort with metastatic disease from multiple sites. The impact of tamoxifen treatment on endocrine-resistant tumors in vivo was investigated in a xenograft model.Results: The extent of patient heterogeneity at the gene level was striking. Less than 3% of the genes differentially expressed between sequential tumors were common to all patients. Larger divergence was observed between primary and liver tumors than between primary and nodal tumors, reflecting both the latency to disease progression and the genetic impact of intervening therapy. Furthermore, an endocrine-resistant in vivo mouse model demonstrated that tamoxifen treatment has the potential to drive disease progression and establish distant metastatic disease. Common functional pathways altered during metastatic, endocrine-resistant progression included extracellular matrix receptor interactions and focal adhesions.Conclusions: This novel global analysis highlights the influence of primary tumor biology in determining the transcriptomic profile of metastatic tumors, as well as the need for adaptations in cell–cell communications to facilitate successful tumor cell colonization of distant host organs. Clin Cancer Res; 21(23); 5371–9. ©2015 AACR.

Supplementary Figure S1 from Coassociation of Estrogen Receptor and p160 Proteins Predicts Resistance to Endocrine Treatment; SRC-1 is an Independent Predictor of Breast Cancer Recurrence

Table containing all brain metastatic cell lines in the BrMPanel

Figure S1. Characterization of MM134, SUM44, CAMA-1 and OCUB-M ILC cell lines.

Detailed description of the materials and methods

supplementary data figure legends updated to include wnt11 knockdown data in FigS3 (untracked)

Spread of cancer to the brain remains an unmet clinical need in spite of the increasing number of cases among patients with lung, breast cancer, and melanoma most notably. Although research on brain metastasis was considered a minor aspect in the past due to its untreatable nature and invariable lethality, nowadays, limited but encouraging examples have questioned this statement, making it more attractive for basic and clinical researchers. Evidences of its own biological identity (i.e., specific microenvironment) and particular therapeutic requirements (i.e., presence of blood–brain barrier, blood–tumor barrier, molecular differences with the primary tumor) are thought to be critical aspects that must be functionally exploited using preclinical models. We present the coordinated effort of 19 laboratories to compile comprehensive information related to brain metastasis experimental models. Each laboratory has provided details on the cancer cell lines they have generated or characterized as being capable of forming metastatic colonies in the brain, as well as principle methodologies of brain metastasis research. The Brain Metastasis Cell Lines Panel (BrMPanel) represents the first of its class and includes information about the cell line, how tropism to the brain was established, and the behavior of each model in vivo. These and other aspects described are intended to assist investigators in choosing the most suitable cell line for research on brain metastasis. The main goal of this effort is to facilitate research on this unmet clinical need, to improve models through a collaborative environment, and to promote the exchange of information on these valuable resources.

Figure S1. SRC-1 dependent transcriptome in endocrine resistant LY2 cells. Figure S2. Pathways enriched in SRC-1 repressed gene set. Figure S3. Repression gene set is directly regulated and suppressed by SRC-1. Figure S4. SRC-1 mediates endocrine treatment resistant phenotype Figure S5. Expression validation for functional experiments. Figure S6. Target gene set expression in untreated ER positive population Figure S7. RG-108 treatment can drive re-expression of suppressed target gene set. Figure S8: Nuclear receptor binding activity to the SRC-1 target genes.

Purpose: Acquired resistance to aromatase inhibitor (AI) therapy is a major clinical problem in the treatment of breast cancer. The detailed mechanisms of how tumor cells develop this resistance remain unclear. Here, the adapted function of estrogen receptor (ER) to an estrogen-depleted environment following AI treatment is reported.Experimental Design: Global ER chromatin immuno-precipitation (ChIP)-seq analysis of AI-resistant cells identified steroid-independent ER target genes. Matched patient tumor samples, collected before and after AI treatment, were used to assess ER activity.Results: Maintained ER activity was observed in patient tumors following neoadjuvant AI therapy. Genome-wide ER–DNA-binding analysis in AI-resistant cell lines identified a subset of classic ligand-dependent ER target genes that develop steroid independence. The Kaplan–Meier analysis revealed a significant association between tumors, which fail to decrease this steroid-independent ER target gene set in response to neoadjuvant AI therapy, and poor disease-free survival and overall survival (n = 72 matched patient tumor samples, P = 0.00339 and 0.00155, respectively). The adaptive ER response to AI treatment was highlighted by the ER/AIB1 target gene, early growth response 3 (EGR3). Elevated levels of EGR3 were detected in endocrine-resistant local disease recurrent patient tumors in comparison with matched primary tissue. However, evidence from distant metastatic tumors demonstrates that the ER signaling network may undergo further adaptations with disease progression as estrogen-independent ER target gene expression is routinely lost in established metastatic tumors.Conclusions: Overall, these data provide evidence of a dynamic ER response to endocrine treatment that may provide vital clues for overcoming the clinical issue of therapy resistance. Clin Cancer Res; 22(11); 2765–77. ©2016 AACR.

PDF file, 14KB, Supplementary Table 1: Demographics of patients according to AIB1 status.

PDF file, 320KB, Supplementary Figures 1-9 Supplementary Figure 1: Expression levels of aromatase in our cell lines and aromatase activity assay Supplementary Figure 2: Proliferation of Aro and LetR in response to treatment Supplementary Figure 3: Kaplan Meier estimates of disease free survival in the entire ER positive population Supplementary Figure 4:Interactions between ERalpha and AIB1 in letrozole sensitive Aro cells Supplementary Figure 5: Analysis of protein levels in Anastrozole resistant cell line (ANAR) Supplementary Figure 6: ChiP showing recruitment of ERalpha and AIB1 to distal region promoters Supplementary Figure 7: Basal protein levels of Myc and cyclinD1 protein and basal mRNA levels of pS2 Supplementary Figure 8:Effect of Lapatinib treatment on pS2 and Myc mRNA levels Supplementary Figure 9: Pathway analysis of ERalpha-AIB1 target genes.

Supplementary Table S4: Patient information for patients A-L

Contains experimental therapies tested on brain metastatic cell lines and associated clinical trials

Purpose: The use of aromatase inhibitors (AI) in the treatment of estrogen receptor (ER)-positive, postmenopausal breast cancer has proven efficacy. However, inappropriate activation of ER target genes has been implicated in the development of resistant tumors. The ER coactivator protein AIB1 has previously been associated with initiation of breast cancer and resistance to endocrine therapy.Experimental Design: Here, we investigated the role of AIB1 in the deregulation of ER target genes occurring as a consequence of AI resistance using tissue microarrays of patients with breast cancer and cell line models of resistance to the AI letrozole.Results: Expression of AIB1 associated with disease recurrence (P = 0.025) and reduced disease-free survival time (P = 0.0471) in patients treated with an AI as first-line therapy. In a cell line model of resistance to letrozole (LetR), we found ERα/AIB1 promoter recruitment and subsequent expression of the classic ER target genes pS2 and Myc to be constitutively upregulated in the presence of both androstenedione and letrozole. In contrast, the recruitment of the ERα/AIB1 transcriptional complex to the nonclassic ER target cyclin D1 and its subsequent expression remained sensitive to steroid treatment and could be inhibited by treatment with letrozole. Molecular studies revealed that this may be due in part to direct steroid regulation of c-jun-NH2-kinase (JNK), signaling to Jun and Fos at the cyclin D1 promoter.Conclusion: This study establishes a role for AIB1 in AI-resistant breast cancer and describes a new mechanism of ERα/AIB1 gene regulation which could contribute to the development of an aggressive tumor phenotype. Clin Cancer Res; 18(12); 3305–15. ©2012 AACR.

Supplementary Figures S1-7. Supplementary Figure S1: Gene expression profiles of classic biomarkers in matched tumour samples from 3 patients. Supplementary Figure S2: Network map showing the enriched KEGG pathways from upregulated differentially expressed genes (DEGs). Supplementary Figure S3: Network map showing the enriched KEGG pathways from downregulated differentially expressed genes (DEGs). Supplementary Figure S4: Protein expression in matched primary and metastatic tissue from endocrine treated breast cancer patients. Supplementary Figure S5: Phosphorylated mTOR signaling in endocrine resistant breast cancer tumours. Supplementary Figure S6: Interaction plot showing functional roles for 21 genes commonly upregulated in metastatic liver tumours of all three patients. Supplementary Figure S7: Genes upregulated in liver metastases are also strongly expressed in breast metastases to the brain.

Figure S1. Ligand independent ER drives growth of AI resistant cells. Figure S2. Confirmation of transient knockdown using siRNA. Figure S3: pSer118 ER expression significantly increases during neoadjuvant AI therapy. Figure S4: Androgen Receptor (AR) expression in patient tumours before and after endocrine therapy. Figure S5. Reduced ligand dependency is a feature of multiple endocrine resistant cells compared to endocrine sensitive MCF7 cells. Figure S6. FoxA1 expression and interactions with ER are comparable between MCF7 and LetR cells treated with either estrogen (E2) or androstenedione (A). Figure S7. Steroid-sensitive recruitment of ER to a selection of target genes in MCF7-Aro cells. q-PCR of ER ChIP in MCF7-Aro cells treated with vehicle (V) or steroid (S) for 45 minutes. IgG was used as an internal control. Bar charts show relative recruitment to each target gene with n=3. Heat map shows relative recruitment of ER to each target gene as a fold increase above IgG control. Figure S8. ER ChIPseq binding peak profiles in 7 genes of the ER target gene signature. ER binding activity is observed at the same location in both LY2 and LetR cells. Images were captured from UCSC Genome Browser. V, vehicle; E, estrogen; A, androstenedione. Figure S9. ER binding does not affect expression of PEBP4. Figure S10. A, EGR3 knockdown decreases cell growth in LetR cells. Figure S11: ER target gene signature is more accurate than ESR1 alone for predicting outcome on AI therapy.

Purpose: Despite the clinical utility of endocrine therapies for estrogen receptor–positive (ER) breast cancer, up to 40% of patients eventually develop resistance, leading to disease progression. The molecular determinants that drive this adaptation to treatment remain poorly understood. Methylome aberrations drive cancer growth yet the functional role and mechanism of these epimutations in drug resistance are poorly elucidated.Experimental Design: Genome-wide multi-omics sequencing approach identified a differentially methylated hub of prodifferentiation genes in endocrine resistant breast cancer patients and cell models. Clinical relevance of the functionally validated methyl-targets was assessed in a cohort of endocrine-treated human breast cancers and patient-derived ex vivo metastatic tumors.Results: Enhanced global hypermethylation was observed in endocrine treatment resistant cells and patient metastasis relative to sensitive parent cells and matched primary breast tumor, respectively. Using paired methylation and transcriptional profiles, we found that SRC-1–dependent alterations in endocrine resistance lead to aberrant hypermethylation that resulted in reduced expression of a set of differentiation genes. Analysis of ER-positive endocrine-treated human breast tumors (n = 669) demonstrated that low expression of this prodifferentiation gene set significantly associated with poor clinical outcome (P = 0.00009). We demonstrate that the reactivation of these genes in vitro and ex vivo reverses the aggressive phenotype.Conclusions: Our work demonstrates that SRC-1-dependent epigenetic remodeling is a ’high level’ regulator of the poorly differentiated state in ER-positive breast cancer. Collectively these data revealed an epigenetic reprograming pathway, whereby concerted differential DNA methylation is potentiated by SRC-1 in the endocrine resistant setting. Clin Cancer Res; 24(15); 3692–703. ©2018 AACR.

Table S3: Cox regression analysis on RATHER RNA-seq data

Purpose: This study investigates the role of the p160 coactivators AIB1 and SRC-1 independently, and their interactions with the estrogen receptor, in the development of resistance to endocrine treatments.Experimental Design: The expression of the p160s and the estrogen receptor, and their interactions, was analyzed by immunohistochemistry and quantitative coassociation immunofluorescent microscopy, using cell lines, primary breast tumor cell cultures, and a tissue microarray with breast cancer samples from 560 patients.Results: Coassociation of the p160s and estrogen receptor α was increased in the LY2 endocrine-resistant cell line following treatment with tamoxifen in comparison with endocrine-sensitive MCF-7 cells. In primary cultures, there was an increase in association of the coactivators with estrogen receptor α following estrogen treatment but dissociation was evident with tamoxifen. Immunohistochemical staining of the tissue microarray revealed that SRC-1 was a strong predictor of reduced disease-free survival (DFS), both in patients receiving adjuvant tamoxifen treatment and untreated patients (P < 0.0001 and P = 0.0111, respectively). SRC-1 was assigned a hazard ratio of 2.12 using a Cox proportional hazards model. Endocrine-treated patients who coexpressed AIB1 with human epidermal growth factor receptor 2 had a significantly shorter DFS compared with all other patients (P = 0.03). Quantitative coassociation analysis in the patient tissue microarray revealed significantly stronger colocalization of AIB1 and SRC-1 with estrogen receptor α in patients who have relapsed in comparison with those patients who did not recur (P = 0.026 and P = 0.00001, respectively).Conclusions: SRC-1 is a strong independent predictor of reduced DFS, whereas the interactions of the p160 proteins with estrogen receptor α can predict the response of patients to endocrine treatment.

Table S1: 736 genes downregulated in LY2 shNT (Ensemble ID)

PDF file, 55KB, Supplementary table 2: Rate of incidence of disease recurrence according to AIB1, HER2, tumour grade, tumour size and nodal status in breast cancer patients treated with first-line AI.

PDF file, 213KB, Supplementary Table 3b ER/AIB1 overlap genelist - partial ERE.

Supplementary Materials and Methods

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Supplementary Figures 1-3, Table 1 from Interaction of Developmental Transcription Factor HOXC11 with Steroid Receptor Coactivator SRC-1 Mediates Resistance to Endocrine Therapy in Breast Cancer

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The development of breast cancer resistance to endocrine therapy results from an increase in cellular plasticity that permits the emergence of a hormone-independent tumor. The steroid coactivator protein SRC-1, through interactions with developmental proteins and other nonsteroidal transcription factors, drives this tumor adaptability. In this discovery study, we identified ADAM22, a non-protease member of the ADAM family of disintegrins, as a direct estrogen receptor (ER)-independent target of SRC-1. We confirmed SRC-1 as a regulator of ADAM22 by molecular, cellular, and in vivo studies. ADAM22 functioned in cellular migration and differentiation, and its levels were increased in endocrine resistant-tumors compared with endocrine-sensitive tumors in mouse xenograft models of human breast cancer. Clinically, ADAM22 was found to serve as an independent predictor of poor disease-free survival. Taken together, our findings suggest that SRC-1 switches steroid-responsive tumors to a steroid-resistant state in which the SRC-1 target gene ADAM22 has a critical role, suggesting this molecule as a prognostic and therapeutic drug target that could help improve the treatment of endocrine-resistant breast cancer. Cancer Res; 72(1); 220–9. ©2011 AACR.

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Fixed-tissue ChIP-seq for H3K27 acetylation (H3K27ac) profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65 °C in a higher concentration of detergent and a minimized sonication step, to produce robust genome-wide H3K27ac maps from clinical samples. FiTAc-seq generates high-quality enhancer landscapes and super-enhancer (SE) annotation in numerous archived FFPE samples from distinct tumor types. This approach will be of great interest for both basic and clinical researchers. The entire protocol from FFPE blocks to sequence-ready library can be accomplished within 4 d. This protocol describes a ChIP-seq procedure optimized for profiling H3K27 acetylation in archived formalin-fixed, paraffin-embedded (FFPE) tissues sampled through whole or macrodissected sectioning or from punched cores.

Invasive lobular carcinoma (ILC) is the second most common type of breast cancer after invasive ductal carcinoma (IDC), accounting for approximately 10-15% of all breast tumors. ILC is characterized by inactivation of E-Cadherin and neoplastic cells that invade the stroma in a "single-file" pattern. Women with ILC are usually older, have used hormone replacement therapy and are more likely to have hormone receptor-positive disease. ILCs have similar survival to IDCs at both five and 10 years, but despite this, the clinical course is distinct: ILCs are three times more likely to metastasize to the peritoneum, gastrointestinal tract, and ovaries and are more frequently bilateral. Therefore, tailored therapeutic options for this distinct, hard-to-treat subtype of breast cancer are required. As part of the RATHER FP7 HEALTH consortium (www.ratherproject.com), we carried out RNA-Seq analysis of 61 primary ILC samples and identified that high expression of the BET family protein Brd3 (uniquely among BRD family members) was associated with poor recurrence free survival (p=0.03, HR 8.63, CI 1.22-60.85). This observation was further validated in the independent METABRIC cohort (n=99), where again, high Brd3 expression (and not other BRD members) was associated with poor recurrence-free survival (p Citation Format: Darran P O'Connor, Louise Walsh, Kathryn Haley, Bruce Moran, Brian Mooney, Stephen Madden, Sudipto Das, Oscar Rueda, Catriona Dowling, Damir Vareslija, Suet-Feung Chin, Sabine Linn, Leonie Young, Karin Jirstrom, John P Crown, Rene Bernards, Carlos Caldas, William M Gallagher, Triona Ni Chonghaile. Combination of BET and FGFR inhibition as a rational therapeutic strategy for invasive lobular breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-06.

Purpose: Invasive lobular carcinoma (ILC) is a subtype of breast cancer accounting for 10% of breast tumors. The majority of patients are treated with endocrine therapy; however, endocrine resistance is common in estrogen receptor–positive breast cancer and new therapeutic strategies are needed. Bromodomain and extraterminal inhibitors (BETi) are effective in diverse types of breast cancer but they have not yet been assessed in ILC. Experimental Design: We assessed whether targeting the BET proteins with JQ1 could serve as an effective therapeutic strategy in ILC in both 2D and 3D models. We used dynamic BH3 profiling and RNA-sequencing (RNA-seq) to identify transcriptional reprograming enabling resistance to JQ1-induced apoptosis. As part of the RATHER study, we obtained copy-number alterations and RNA-seq on 61 ILC patient samples. Results: Certain ILC cell lines were sensitive to JQ1, while others were intrinsically resistant to JQ1-induced apoptosis. JQ1 treatment led to an enhanced dependence on antiapoptotic proteins and a transcriptional rewiring inducing fibroblast growth factor receptor 1 (FGFR1). This increase in FGFR1 was also evident in invasive ductal carcinoma (IDC) cell lines. The combination of JQ1 and FGFR1 inhibitors was highly effective at inhibiting growth in both 2D and 3D models of ILC and IDC. Interestingly, we found in the RATHER cohort of 61 ILC patients that 20% had FGFR1 amplification and we showed that high BRD3 mRNA expression was associated with poor survival specifically in ILC. Conclusions: We provide evidence that BETi either alone or in combination with FGFR1 inhibitors or BH3 mimetics may be a useful therapeutic strategy for recurrent ILC patients.

Introduction Brain metastasis (BrM) occurs in 10-30% of patients with advanced breast cancer (BC). BrM is increasing in incidence and confers a poor prognosis. We aimed to investigate the contribution of global epi-transcriptomic alterations in N6-methyladenosine (m6A) RNA-methylation as a therapeutic target in brain metastatic breast cancer. Method In preliminary studies we have demonstrated m6A demethylase – FTO as the main contributor to the progression of ER+ breast cancer. Furthermore an association between FTO and reduced disease-free-survival (n=870, p=0.018) was observed. Here we conducted an epigenetic inhibitor screen using two therapeutic agents, ethyl-ester-meclofenamic acid (MA2) and FB23-2 on matched 2D cell line, 3D organoid cultures and patient-derived xenografts (PDX) explant models of brain metastasis. Result Upon integration of mapped global RNA methylation landscape with matched proteomic analysis, we observed genome-wide RNA hypo-methylation of key pluripotency genes, including SOX2 and KLF4, as key players underlying tumour progression to the brain. Genetic and pharmacological inhibition of FTO in novel ex vivo models of BrM significantly reduced protein expression levels of KLF4 and SOX2. Moreover, pharmacological inhibition of FTO with MA2 and FB23-2, inhibited cell proliferation in endocrine-resistant BC and patient BrM cells. We translate our findings to the clinic by demonstrating the efficacy of anti-FTO therapies in several unique PDX and 3D organoid BrM models. Conclusion Our results reveal epi-transcriptional remodelling events as a key mechanism in BrM. This study establishes an early role for targeting RNA methylation in the management of disease progression and presents FTO as a potential therapeutic target in BrM. Take-home message This study establishes an early role for targeting RNA methylation in the management of disease progression and presents FTO as a potential therapeutic target in brain metastatic breast cancer.

Introduction The incidence of brain metastases is increasing despite longer survival rates for patients with advanced breast cancer. The identification of novel therapeutic targets for these patients is an urgent unmet clinical need. Sequencing of metastatic tumours have largely focused on mutations however gene fusions have an important, yet underappreciated role in tumorigenesis and disease progression. In this study, we investigate the role of gene fusions in brain metastatic disease and their impact on altered therapeutic responses. Method RNA sequencing was performed on the largest reported cohort of patient matched primary and resected brain metastatic tumours (45 patients n=90 samples). Expressed gene fusions were detected computationally using STAR-Fusion and Arriba. Result We identified differential gene fusion burden in brain metastatic tumours (medium of 58) vs. primary breast tumours (medium of 38) (p Conclusion These results highlight the significant role of gene fusions in breast cancer brain metastases. Abbreviations MAPK Mitogen Activated Protein Kinase, HER Human Epidermal Receptor, CDK12 Cyclin Dependent Kinase 12 Take-home message We highlight the significant role of gene fusions in breast cancer brain metastases and offer specific actionable genomic alterations to be exploited.