Mutations In Breast Cancer Research Articles

Genome engineering for estrogen receptor mutations reveals differential responses to anti-estrogens and new prognostic gene signatures for breast cancer

Mutations in the estrogen receptor (ESR1) gene are common in ER-positive breast cancer patients who progress on endocrine therapies. Most mutations localise to just three residues at, or near, the C-terminal helix 12 of the hormone binding domain, at leucine-536, tyrosine-537 and aspartate-538. To investigate these mutations, we have used CRISPR-Cas9 mediated genome engineering to generate a comprehensive set of isogenic mutant breast cancer cell lines. Our results confirm that L536R, Y537C, Y537N, Y537S and D538G mutations confer estrogen-independent growth in breast cancer cells. Growth assays show mutation-specific reductions in sensitivities to drugs representing three classes of clinical anti-estrogens. These differential mutation- and drug-selectivity profiles have implications for treatment choices following clinical emergence of ER mutations. Our results further suggest that mutant expression levels may be determinants of the degree of resistance to some anti-estrogens. Differential gene expression analysis demonstrates up-regulation of estrogen-responsive genes, as expected, but also reveals that enrichment for interferon-regulated gene expression is a common feature of all mutations. Finally, a new gene signature developed from the gene expression profiles in ER mutant cells predicts clinical response in breast cancer patients with ER mutations.

Comparison of Clinical, Pathological, and Prognostic Features in BRCA Mutant and Wild-Type Male Breast Cancer Patients.

Published studies on male breast cancer (MBC) and BRCA mutations are scarce and usually include,a small number of patients. The clinicopathological characteristics of BRCA mutant and wild-type MBC patients were compared in more than forty patients in this study. A retrospective review of MBC patients' clinical and histopathological data was conducted. To compare the patients' characteristics, chi-square test and Fisher's Exact test were utilized. Kaplan-Meier analysis was used to examine the survival analysis. In total 43 cases were reviewed. The average duration of follow-up was 35.8 months. BRCA mutations were found in 11 (25.6%) of the patients. BRCA1 mutations were found in four patients (9.3%), BRCA2 mutations in six patients (14%), and BRCA1 and BRCA2 mutations in one patient (2.3%). The median age at diagnosis was 58 years old, and there was no statistically significant difference between groups (p = 0.7). Tumor location (p = 0.3), human epidermal growth factor receptor 2 overexpression (p = 0.5), estrogen receptor status (p = 0.05), progesterone receptor status (p = 0.6), tumor stage (p = 0.9), lymph node positivity (p = 0.5), tumor histology (p = 0.06), and recurrence status (p = 0.6) were similar between BRCA-wild type and -mutated patients. Overall survival averaged 115.6 months (range: 76.0-155.3), with no statistically significant differences between groups (p = 0.6). This study investigated clinical and pathological characteristics and prognoses of BRCA wild and mutant-type MBC and these were similar in all groups studied.

Combination of ligand and structure based virtual screening approaches for the discovery of potential PARP1 inhibitors.

Poly (ADP-ribose) polymerase 1 (PARP1) has high therapeutic value as biomolecular target for research and development of small molecules with antineoplastic activity, since it is upregulated in many cancers, especially in ovarian and BRCA 1/2 mutated breast cancers. Decades of investigation of PARP inhibitors (PARPi) have led to the approval of several drug compounds, however clinical application of PARPi in cancer therapy is limited due to a number of factors, including low selectivity, weak affinity and undesired side effects. Thus, identification of novel drug-like chemical compounds with alternatives to the known PARPi chemical scaffolds, binding modes and interaction patterns with amino acid residues in the active site is of high therapeutic importance. In this study we applied a combination of ligand- and structure-based virtual screening approaches with the goal of identification of novel potential PARPi.

Impact of BRCA Mutation Status on Tumor Dissemination Pattern, Surgical Outcome and Patient Survival in Primary and Recurrent High-Grade Serous Ovarian Cancer: A Multicenter Retrospective Study by the Ovarian Cancer Therapy-Innovative Models Prolong Survival (OCTIPS) Consortium

BackgroundThis study seeks to evaluate the impact of breast cancer (BRCA) gene status on tumor dissemination pattern, surgical outcome and survival in a multicenter cohort of paired primary ovarian cancer (pOC) and recurrent ovarian cancer (rOC).Patients and MethodsMedical records and follow-up data from 190 patients were gathered retrospectively. All patients had surgery at pOC and at least one further rOC surgery at four European high-volume centers. Patients were divided into one cohort with confirmed mutation for BRCA1 and/or BRCA2 (BRCAmut) and a second cohort with BRCA wild type or unknown (BRCAwt). Patterns of tumor presentation, surgical outcome and survival data were analyzed between the two groups.ResultsPatients with BRCAmut disease were on average 4 years younger and had significantly more tumor involvement upon diagnosis. Patients with BRCAmut disease showed higher debulking rates at all stages. Multivariate analysis showed that only patient age had significant predictive value for complete tumor resection in pOC. At rOC, however, only BRCAmut status significantly correlated with optimal debulking. Patients with BRCAmut disease showed significantly prolonged overall survival (OS) by 24.3 months. Progression-free survival (PFS) was prolonged in the BRCAmut group at all stages as well, reaching statistical significance during recurrence.ConclusionsPatients with BRCAmut disease showed a more aggressive course of disease with earlier onset and more extensive tumor dissemination at pOC. However, surgical outcome and OS were significantly better in patients with BRCAmut disease compared with patients with BRCAwt disease. We therefore propose to consider BRCAmut status in regard to patient selection for cytoreductive surgery, especially in rOC.

Targeting ESR1 mutation-induced transcriptional addiction in breast cancer with BET inhibition.

Acquired mutations in the ligand-binding domain (LBD) of the gene encoding estrogen receptor α (ESR1) are common mechanisms of endocrine therapy resistance in patients with metastatic ER+ breast cancer. The ESR1 Y537S mutation, in particular, is associated with development of resistance to most endocrine therapies used to treat breast cancer. Employing a high-throughput screen of nearly 1,200 Federal Drug Administration–approved (FDA-approved) drugs, we show that OTX015, a bromodomain and extraterminal domain (BET) inhibitor, is one of the top suppressors of ESR1 mutant cell growth. OTX015 was more efficacious than fulvestrant, a selective ER degrader, in inhibiting ESR1 mutant xenograft growth. When combined with abemaciclib, a CDK4/6 inhibitor, OTX015 induced more potent tumor regression than current standard-of-care treatment of abemaciclib + fulvestrant. OTX015 has preferential activity against Y537S mutant breast cancer cells and blocks their clonal selection in competition studies with WT cells. Thus, BET inhibition has the potential to both prevent and overcome ESR1 mutant–induced endocrine therapy resistance in breast cancer.

Immunometabolism characteristics and a potential prognostic risk model associated with TP53 mutations in breast cancer.

TP53, a gene with high-frequency mutations, plays an important role in breast cancer (BC) development through metabolic regulation, but the relationship between TP53 mutation and metabolism in BC remains to be explored. Our study included 1,066 BC samples from The Cancer Genome Atlas (TCGA) database, 415 BC cases from the Gene Expression Omnibus (GEO) database, and two immunotherapy cohorts. We identified 92 metabolic genes associated with TP53 mutations by differential expression analysis between TP53 mutant and wild-type groups. Univariate Cox analysis was performed to evaluate the prognostic effects of 24 TP53 mutation-related metabolic genes. By unsupervised clustering and other bioinformatics methods, the survival differences and immunometabolism characteristics of the distinct clusters were illustrated. In a training set from TCGA cohort, we employed the least absolute shrinkage and selection operator (LASSO) regression method to construct a metabolic gene prognostic model associated with TP53 mutations, and the GEO cohort served as an external validation set. Based on bioinformatics, the connections between risk score and survival prognosis, tumor microenvironment (TME), immunotherapy response, metabolic activity, clinical characteristics, and gene characteristics were further analyzed. It is imperative to note that our model is a powerful and robust prognosis factor in comparison to other traditional clinical features and also has high accuracy and clinical usefulness validated by receiver operating characteristic (ROC) and decision curve analysis (DCA). Our findings deepen our understanding of the immune and metabolic characteristics underlying the TP53 mutant metabolic gene profile in BC, laying a foundation for the exploration of potential therapies targeting metabolic pathways. In addition, our model has promising predictive value in the prognosis of BC.

Synthesis and evaluation of a new series of spiro aryl dioxolane compounds: A new scaffold as potential poly(ADP‐ribose)polymerase‐1 (PARP‐1) inhibitors

AbstractA new series of spiro aryl dioxolane derivatives were synthesized and evaluated to find a new scaffold as potential poly(ADP‐ribose)polymerases‐1 (PARP‐1) inhibitors. This key starting compound, 2,6‐bis(methoxybenzylidene)cyclohexanone, was functionalized with different nucleophilic reagents via cyclocondensation reactions to obtain new benzylidene scaffolds containing diverse aryl groups such as spiroindazoleorquinazoline [1,3]dioxolane compounds. Furthermore, the Michael addition reaction of bis‐benzylidene with some active methylene compounds afforded spirochromene‐ or naphthalene‐[1,3]dioxolane compounds. All the synthesized compounds revealed promising inhibition with IC50 values in the nanomolar range (0.997–2.698 nM), not significantly different from that of Olaparib (IC50 = 0.861 nM). Compounds 5b and 15 (IC50 = 1.009 and 0.997 nM) showed the highest potency among all the prepared compounds and accordingly their ability to inhibit the growth of BRCA1 mutated breast cancer cell line MDA‐MB‐436 was tested, where compound 5b (IC50 = 0.67 μM) showed a potency ten folds higher than that of Olaparib but compound 15 (IC50 = 7.47 μM) exerted lower activity but still comparable to that of Olaparib (IC50 = 6.84 μM). Both resulted in the arrest of cell cycle at S phase and caused cell apoptosis. In silico studies including absorption, distribution, metabolism, excretion (ADME) properties, drug‐likeness, and molecular docking were carried out to support the above‐mentioned findings. This work presents compounds 5b and 15 as promising scaffolds as PARP‐1 inhibitors.

Poziotinib Inhibits HER2-Mutant-Driven Therapeutic Resistance and Multiorgan Metastasis in Breast Cancer.

Evaluation of the functional impact of HER2 mutations on therapy-induced resistance and metastasis identifies robust antitumor activity of poziotinib and supports the clinical evaluation of poziotinib in ER+ HER2 mutant breast cancer.

Abstract 648: A novel small molecule targeting oncogenic PELP1 demonstrates anti-tumor activity in wild-type and mutant ER-positive breast cancer

Abstract A significant proportion of estrogen receptor positive breast cancers (ER+BC) will initially respond to treatment, but many eventually develop therapy resistance (TR-BC), and progress to incurable metastases. Oncogenic ER coregulators overexpressed in BC can contribute to constitutive, ligand-independent and ligand-dependent signaling which drives growth, resistance to therapy and metastasis. Proline-, glutamic acid, and leucine-rich protein 1 (PELP1), is a known coregulator that plays a critical role in ER oncogenic functions. Its expression is deregulated in BC and is a prognostic indicator of poor BC survival. The lack of a small molecule inhibitor that directly targets PELP1 represents a major knowledge gap. Therefore, we conducted a large scale peptide library screening and identified novel Peptide Inhibitor of PELP1 (PIP1). We demonstrated that PIP1 directly interacts with PELP1, promotes its degradation and has the potential to block PELP1 oncogenic functions in vitro. Using innovative peptidomimetic technology, we modeled PIP1 and synthesized several derivatives as Small Molecule Inhibitors of PELP1 (SMIPs). Using MTT assay and multiple BC cell lines, we identified a lead compound, SMIP34 with an IC50 of 5-10µM and with minimal effect on human mammary epithelial cells. SMIP34 in vitro activity was assessed by colony formation and Matrigel invasion assays. Knockdown of PELP1 using shRNA in BC cells significantly reduced SMIP34 activity, indicating target specificity. Further, MST and biotin-SMIP34 pulldown confirmed direct binding of SMIP34 to PELP1. Using ER+WT, mtER (mutant ER), and TR-BC cell lines we demonstrated that SMIP34 exhibits antiproliferative effects and reduces invasiveness. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation via the proteasome pathway. Thus MG132 treatment attenuated SMIP34 mediated degradation. RTqPCR analyses confirmed SMIP34 treatment reduced expression of PELP1 target genes. RNAseq analyses showed SMIP34 treatment altered the expression of genes associated with Estrogen response, Cell cycle and Apoptosis pathways. Cell cycle analyses revealed SMIP34 treatment promoted S phase arrest of BC cell lines. Using ER+WT, mtER, and PDX tumor tissues ex vivo, we demonstrated that SMIP34 significantly decreased tumor proliferation as measured by Ki67 staining. Further, SMIP34 (20mg/kg/IP) treatment in vivo significantly reduced tumor progression in mouse models of ER+WT, mtER, and mtER patient-derived xenograft BC. Our results using in vitro, ex vivo, and in vivo models showcase SMIP34 as a first-in-class inhibitor of oncogenic PELP1 signaling and may serve as a potential therapeutic molecule for treating ER+, mtER, and TR-BC. Supported by NIH 1F31CA257298 (KA) and VA I01BX004545 (RV). Citation Format: Kristin Ann Altwegg, Monica Mann, Dimple Chakravarty, Zexuan Liu, Junhao Liu, Uday P. Pratap, Behnam Ebrahimi, John R. Sanchez, Ben H. Park, Hariprasad Vankayalapati, Gangadhara R. Sareddy, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi. A novel small molecule targeting oncogenic PELP1 demonstrates anti-tumor activity in wild-type and mutant ER-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 648.

Abstract 3356: Comparative analysis of approved and undertrial SERDs in estrogen receptor-α (ERα): An in-silico approach

Abstract Background: Despite recent advancements in early detection and treatment, breast cancer is the second most common cancer affecting women globally, with 11.7% of total new cases. The disease is the fifth leading cause of cancer mortality worldwide, with 685,000 deaths in 2020. Approximately 75% of all breast cancer are ERα positive and resistant to current therapies like aromatase Inhibitors (AIs) and selective receptors modulators (SERMs). The selective estrogen receptor degrader (SERDs) ability to antagonize and degrade ERα makes them promising therapeutic agents for hormone-refractory breast cancer treatment. Fulvestrant is the only SERD with steroidal core currently approved for ER-positive breast cancer treatment. However, its poor physicochemical properties limit its efficacy. Many non-steroidal SERDs such as Amcenestrant, Elacestrant, Camizestrant, Giredestrant, and LSZ102 are under various advanced development stages. However, their long-term efficacy is yet to be established. Our in- silico studies aim to evaluate and compare multiple SERDs (approved and under trial) in terms of their shape similarity, binding mechanism, and stability with ERα receptor. Methods: Present work focuses on evaluating and comparing the shape similarity and electrostatic potential of various SERDs with respect to ERα endogenous ligand; estradiol (E2). Docking studies were performed to understand the binding mode of selected SERDs within the active site of ERα. Additional parameters like root-mean square-square deviation (RMSD), root-mean square-fluctuation (RMSF), pocket volume fluctuations, and protein-ligand binding energy were calculated using Molecular Dynamics (MD) simulation techniques to understand the stability and binding mechanism of various SERDs. Result: Among various evaluated SERDs, Fulvestrant shares the highest whereas Elacestrant shares the lowest shape similarity with E2. All docked SERDs cores occupy the same binding pocket and tend to bind in a similar fashion as E2, with their tail protruding towards helix 12 (H12). As observed interactions with H12 are mainly responsible for ERα downregulation. However, in MD studies analysis, it was found that non-steroidal core molecules were less stable and showed more fluctuations (within active site) than steroidal core molecule (Fulvestrant). Moreover, protein-ligand binding energy for Fulvestrant was found to be lower which corresponds to its higher stability with ERα. Conclusion: The current study helps us to understand the binding mechanism, antagonism, and downregulation of multiple SERDs, among which Fulvestrant showed the highest binding affinity towards ERα (due to its highest shape similarity with E2) and can interact with and disrupt H12 (due to its long tail) which is a critical component of SERD. This knowledge could further be used to develop novel SERDs for ESR1 mutant and HR-positive breast cancer treatment. Citation Format: Vishal Unadkat, Shishir Rohit, Parva Purohit, Chirag Mehta, Vishal Goswami, Mahesh Barmade, Sonam Sinha, Ganesh Sangle. Comparative analysis of approved and undertrial SERDs in estrogen receptor-α (ERα): An in-silico approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3356.

Abstract 3237: Combining PARP inhibition with the polo-like kinase 1 (PLK1) inhibitor onvansertib overcomes PARP inhibitor resistance

Abstract Background: BRCA1/2 mutant tumors cells are deficient for homologous recombination (HR)-mediated DNA repair and are particularly sensitive to PARP inhibitors (PARPi). PARPi have proved efficacy in breast, ovarian, prostate, and pancreatic cancers, particularly in HR-deficient tumors, while their activity is limited in HR-proficient tumors. However, PARPi resistance is inevitable and therapeutic resistance resulting from restoration of HR repair is a pressing clinical problem. Identifying combination treatments to sensitize tumors cells to PARPi and/or overcome PARPi resistance is critical to expand the benefit of these therapies. The Polo-like kinase 1 (PLK1), a serine threonine kinase, is a master regulator of mitosis, overexpressed in many cancers. PLK1 is also involved in the DNA damage response through the promotion of HR-mediated DNA repair and the recovery from the G2/M checkpoint. PLK1 roles in HR repair suggest that PLK1 inhibition may reverse PARPi resistance. Methods: To test the effect of PLK1 and PARP inhibitors combination, we used onvansertib, a highly selective, ATP-competitor PLK1 inhibitor currently in clinical development and the FDA-approved PARPi olaparib. The antitumor effect of the single and combined drug treatments was tested in 2 BRCA1 mutated high-grade serous ovarian cancer (HGSOC) patient derived (PDX) models resistant to olaparib. Orthotopically PDX transplanted mice were treated for 4 weeks and followed for survival. Results: The combination of onvansertib and olaparib was well tolerated and showed strong anti-tumor activity in both PDX models. The combination significantly increased mice survival in comparison to vehicle, olaparib and onvansertib, and showed that onvansertib can re-sensitize PARPi-resistant tumors to olaparib. Median survival increased by 2.7-fold and 8.1-fold respectively in the 2 PDX models in the combination group versus vehicle and the Kaplan Meyer survival curves of mice treated with the combination showed a statistically survival advantage versus control and single agent treated mice. Pharmacodynamic analyses showed an increase in mitotic, apoptotic and DNA damage markers in tumors treated with the combination versus vehicle. Conclusions: The combination of the PLK1 inhibitor onvansertib and the PARPi olaparib showed potent anti-tumor activity in olaparib-resistant BRAC1 mutant HGSOC PDX models. Additional studies are ongoing to further assess the potential of the combination in BRCA wild-type and mutant ovarian, prostate, pancreatic and breast cancer preclinical models. Citation Format: Michela Chiappa, Federica Guffanti, Alessandra Decio, Alessandro Aliverti, Francesca Ricci, Eugenio Scanziani, Federica Camin, Ilaria Craparotta, Maria Chiara Barbera, Marco Bolis, Maya Ridinger, Giovanna Damia. Combining PARP inhibition with the polo-like kinase 1 (PLK1) inhibitor onvansertib overcomes PARP inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3237.

Abstract 1797: Bcl-2 up-regulation mediates taxane resistance downstream of APC loss

Abstract Patients with triple negative breast cancer (TNBC) are treated with traditional chemotherapy, such as paclitaxel (PTX). Despite the efficacy of taxanes, many tumors will recur due to drug resistance. Therefore, the need to understand the mechanism behind drug resistance is critical to improve patient outcome and survival. Our lab was the first to show that loss of the Adenomatous Polyposis Coli (APC) tumor suppressor caused resistance to PTX in the MDA-MB-157 human TNBC cell line. In the absence of APC, apoptosis induction was decreased, as measured through cleaved caspase 3 and annexin/PI staining. To understand the molecular mechanisms behind APC-mediated PTX response, we analyzed the BCL-2 family of proteins and found a robust increase of the pro-survival family member, Bcl-2. The BH3 mimetic, ABT-199 (Venetoclax), which specifically targets Bcl-2, has been used as a single or combination therapy in multiple hematologic malignancies. In addition, ABT-199 has shown promise in multiple subtypes of breast cancer. Therefore, we used ABT-199 in combination with PTX to address the hypothesis that APC-induced Bcl-2 increase is responsible for PTX resistance. Combination treatment in three CRISPR-mediated APC knockout TNBC cell lines (MDA-MB-157, MDA-MB-231, and SUM159) caused changes in cell proliferation and apoptosis. Combined, these data suggest restored sensitivity to PTX using ABT-199. Our studies are the first to show that Bcl-2 inhibition can restore PTX-sensitivity in APC mutant breast cancer cells. These studies are critical to advance better treatment regimens in patients with TNBC. Citation Format: Sara Maloney, Sarah Zabala, Grace Richmond, Jenifer R. Prosperi. Bcl-2 up-regulation mediates taxane resistance downstream of APC loss [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1797.

Abstract 3726: Functional genomic dissection of PIK3CA hotspot mutations using a multi-lineage isogenic breast cell model

Abstract PIK3CA is the most commonly mutated gene in breast cancer, and in breast cancer there are two hotspot mutations that represent an outsized proportion of PIK3CA mutations. Previous research suggests that there are distinct prognostic and cell signaling differences induced by these mutational isoforms, however there is currently only one targeted treatment approved for PIK3CA mutant breast cancer. The goal of our research is to leverage a comprehensive multi-lineage isogenic breast cell line model to further interrogate the unique effects of these PIK3CA mutations on breast cells using assays to assess gene expression and chromatin state. Insights gained from these assays shed light on disrupted pathways that may prove to be new targets for PIK3CA mutation-specific treatment of breast cancer. Citation Format: Adam X. Miranda, Emily Hodges, Ben Ho Park. Functional genomic dissection of PIK3CA hotspot mutations using a multi-lineage isogenic breast cell model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3726.

Abstract 2955: SMYD2 regulates chromatin modifier KMT2D in ER+/PIK3CA mutant breast cancer

Abstract Breast cancer leads current cancer diagnoses worldwide, accounting for 1 in 4 cancer cases and 1 in 6 cancer deaths in women. Over 70% of breast tumors are hormone receptor positive and can be treated with endocrine therapy, however tumor recurrence remains a major obstacle. Oncogenic mutations in the catalytic subunit of PI3K occur in up to 40% of ER+/HER2- negative breast tumors, making it a promising therapeutic target. Clinically, PI3K inhibitors have shown limited efficacy as monotherapy due to a number of resistance mechanisms, including an upregulation of ER-dependent signaling. Identification of the ER-PI3K signaling crosstalk led to clinical trials which resulted in the approval of the PI3K inhibitor alpelisib in combination with fulvestrant in patients with hormone-refractory ER+/PIK3CA-mutant breast cancer. Further work uncovered a key role for the chromatin modifier KMT2D in regulating ER-dependent transcription upon PI3K inhibition. PI3K effectors AKT1/SGK1 negatively regulate KMT2D through direct S1331 phosphorylation. When PI3K is inhibited, this downstream regulatory event is lost, leading to increased chromatin recruitment of KMT2D and an upregulation of ER-dependent transcription. We have identified a novel methylation site on KMT2D, at K1330 directly adjacent to S1331, catalyzed by the lysine methyltransferase SMYD2. Knockdown of SMYD2 sensitizes ER+/PIK3CA mutant breast cancer cell lines to PI3K inhibition. The combination of SMYD2 knockdown and alpelisib leads to a significant reduction in tumor growth compared to alpelisib alone in a xenograft model. Mechanistically, SMYD2 knockdown is able to attenuate alpelisib-induced KMT2D chromatin binding globally and at ER loci. Accordingly, loss of SMYD2 abrogates alpelisib-induced changes in gene expression, including ER-dependent transcription. Altogether our results demonstrate a role for epigenetic therapy using SMYD2 inhibitors in combination with alpelisib for the treatment of ER+/PIK3CA mutant breast cancer. Citation Format: Ryan Blawski, Mirna Sallaku, Xinyu Guo, Srushti Kittane, Maurizio Scaltriti, Minkui Luo, Eneda Toska. SMYD2 regulates chromatin modifier KMT2D in ER+/PIK3CA mutant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2955.

Abstract CT562: PARTNER: Randomised, phase II/III trial to evaluate the safety and efficacy of the addition of Olaparib to Platinum-based neoadjuvant chemotherapy in triple negative and/or germline BRCA mutated breast cancer patients

Abstract Background: Triple Negative Breast Cancers (TNBC) are a biologically diverse and aggressive subgroup lacking targeted therapy. TNBC and Germline BRCA (gBRCA) breast cancer share certain phenotypic and molecular similarities, with gBRCA mutations seen in 10% to 20% of TNBC patients. Homologous recombination deficient tumours, especially those caused by germline or somatic BRCA mutations, are thought to be particularly sensitive to PARP inhibitors. Aim: To establish if the addition of Olaparib to neoadjuvant Platinum-based chemotherapy in the treatment of basal TNBC and/or gBRCA breast cancer is safe and increases efficacy. Trial design: 3-stage open label randomised phase II/III trial of neoadjuvant Paclitaxel and Carboplatin +/- Olaparib, followed by clinicians' choice of Anthracycline regimen. Stages 1 and 2: Randomisation (1:1:1) to control (3-weekly carboplatin AUC5/weekly with paclitaxel 80mg/m2 for 4 cycles), or to one of two research arms. These use an identical chemotherapy regimen and also include different treatment schedules of Olaparib 150mg BD for 12 days. Stage 3: Randomisation (1:1) to either the control or research arm chosen following stage 2. End-points: Stage 1: Safety; Stage 2: Schedule selection based on pCR rate and Olaparib completion rate using a “pick-the-winner” design. Stage 3: pCR rate. This trial includes an optional pathway (PARTNERING) for patients with evidence of residual disease after six chemotherapy cycles. This aims to establish if the addition of new agents (ATR inhibitor and PD-L1 inhibitor) improves treatment response. Eligibility criteria: Aged 16-70; histologically confirmed invasive breast cancer; ER-negative, HER2-negative with TNBC basal phenotype or gBRCA positive, HER2-negative irrespective of hormone status; stage T1-4 N0-2; performance status 0-1; treatment within 6 weeks of diagnostic biopsy; biomarker scores: TILs, CK 5/6, EGFR +/- AR. Statistical methods: The recruitment of TNBC non-gBRCA and gBRCA patients is independent. Enrichment design is applied with an overall significance level 0.05(α) and 80% power. A minimum of 780 patients will be included to detect an absolute improvement of 15% (all patients and TNBC non-gBRCA cohort) and 20% (gBRCA patients) by combining Olaparib with Platinum based chemotherapy. A minimum of 478 TNBC non-gBRCA and 188 gBRCA patients will be recruited. Each PARTNERING cohort will consist of 15 patients. Current Enrollment: Since May 2016, 756 patients from 30 sites have been enrolled. Stages 1 and 2 are completed. An IDSMC review identified no safety concerns and Research Arm 2 was selected. This arm involves Olaparib administration on days 3-14. Stage 3 Phase I (recruitment of non-gBRCA and gBRCA patients) completed in December 2021. Stage 3 Phase II (recruitment of gBRCA patients only) remains open to patients to UK and internationally. 5 patients have been enrolled in PARTNERING. ClinicalTrials.gov Identifier: NCT03150576 Citation Format: Lynsey Drewett, Karen A. Pinilla, Louise Grybowicz, Jerome Wulff, Alimu Dayimu, Nikolaos Demiris, Rebecca Lucey, Anne-Laure Vallier, Wendi Qian, Andrea Machin, PARTNER Research Team, Karen McAdam, Rebecca Roylance, Ellen R. Copson, Anne Armstrong, Nicola Levitt, Elena Provenzano, Marc Tischkowitz, Emma McMurtry, Helena Earl, Jean E. Abraham. PARTNER: Randomised, phase II/III trial to evaluate the safety and efficacy of the addition of Olaparib to Platinum-based neoadjuvant chemotherapy in triple negative and/or germline BRCA mutated breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT562.

Genetic epidemiology of breast cancer (BC) risk genes from a diverse real-world oncology practice in Brazil.

10594 Background: As germline testing in BC is becoming more accessible, a large body of evidence from distinct populations is revealing different prevalence of affected genes or recurrent and founder mutations. We evaluated a series of Brazilian BC patients from diverse regions of the country that were studied in a single laboratory facility for likely pathogenic/pathogenic variants (PV) and variants of unknown significance (VUS) in BRCA1/2 and other BC susceptibility genes (ATM, BARD1, CDH1, CHEK2, PALB2, RAD51C, RAD51D and TP53). Methods: We used multi-gene NGS panels covering from 35 to 105 genes, including copy number alterations, to perform sequencing in patients with diagnosis of BC from 2016 to 2021. With patient consent, we extracted clinical and pathological data from curated Real-World Database of Oncoclínicas Group to classify tumors according to hormone receptor (HR) and HER2 status and assess family history of cancer. Primary endpoint was prevalence of PV and VUS in BRCA1/2 and other breast cancer risk genes. Results: In total, 1,520 patients with BC were included in the analysis, 99% were female, median age at genetic testing was 46 years and 84% had family history of cancer. BC subtype was available in 717 cases (43% HR+/HER2-, 26% HER2+, 31% triple negative). Median age at genetic testing in HR+/HER2- population was 49 years and 44 years in HER2+ and triple-negative cases. Overall, 11.7% (CI95% 10.1-13.4) of the population had at least one PV in any BC risk gene: 50 in BRCA1, 49 in BRCA2, 22 in TP53 (17 had Brazilian founder p.R337H haplotype), 19 in PALB2, 18 in CHEK2, 15 in ATM, 7 in other genes (BARD1, CDH1, RAD51C, RAD51D) and 4 patients had co-existing PV in 2 BC risk genes. BRCA1/2 PVs were found in 7% of BC patients and BRCA1/2 VUS in 3%, being higher in triple-negative cases (11% PVs) than HR+/HER2- (5% PVs) and HER2+ (3%). For other BC risk genes, 5% of the patients had a PV, without differences according to BC subtype, and the prevalence of VUS was 14%. The prevalence of TP53 Brazilian founder mutation in BC was 1.2%. Median age of genetic testing was younger in population with BRCA1 PV (43 years) than BRCA2 PV (47 years) and other genes PV (46 years). Conclusions: In this real-world cohort of BC patients enriched for high-risk features such as young age at diagnosis, positive family history and triple-negative disease, the prevalence of BRCA1 and BRCA2 PV was very similar and represented only 56% of all PV detected. Different from other studies, TP53 mutations are the third most common germline alteration in the Brazilian population, with close to 80% of the cases presenting the founder mutation p.R337H. BC with epidemiologic high-risk features should be offered a germline test in Brazilian patients due to the considerable chance of a PV detection.

Predominance of BRCA2 mutation and estrogen receptor-positive breast cancer among BRCA1/2 mutation carriers.

551 Background: PARP inhibitor (PARPi) agents can improve progression-free survival of patients with breast cancer (BC) who carry a germline BRCA1 or BRCA2 pathogenic or likely pathogenic variant (gBRCA1/2) in both the metastatic and adjuvant setting. Therefore, we need to redefine the criteria of women and tumor phenotype that should be tested for gBRCA1/2. Methods: We studied the relative distribution of gBRCA1 and gBRCA2 in unselected populations of women with BC and in unaffected individuals. We also analyzed the proportion of estrogen receptor (ER)-positive (ER+) tumors in unselected BC patients with gBRCA1/2.We performed a meta-analysis of studies of unselected BC that analyzed the relative contribution of gBRCA1 versus gBRCA2 and ER+ tumors among gBRCA1/2 carriers. We then performed a meta-analysis of gBRCA1/2 carriage in unaffected individuals, from genome-wide population studies, the gnomAD databank, and case–control studies. Results: The BRCA2 gene was involved in 54% of BC in unselected patients with gBRCA1/2 (n=108,699) and 59% of unaffected individuals (n=238,973) as compared with 38% of gBRCA1/2 family cohorts (n=29,700). The meta-analysis showed that 1.66% (95% CI 1.08-2.54) and 1.71% (95% CI 1.33-2.2) of unselected BC patients carried a gBRCA1 and gBRCA2, respectively. In unaffected individuals, the frequency of heterozygosity for gBRCA1 and gBRCA2 was estimated at 1/434 and 1/288, respectively. Nearly 0.5% of unaffected individuals in the studied populations carried a gBRCA1/2. Carriage of a gBRCA was 2.5% for patients with ER+ tumors (95% CI 1.5-4.1) and 5.7% (95% CI 5.1-6.2) for those with ER- tumors. Overall, 58% of breast tumors occurring in women carrying a gBRCA1/2 were ER+ (n=86,870). Conclusions: This meta-analysis showed that gBRCA2 carriage is predominant in unselected BC and in unaffected individuals. ER+ tumors among women with gBRCA1/2-related BC is predominant and has been underestimated. Because PARPi agents improve progression-free survival with ER+ gBRCA1/2 BC in both the adjuvant and metastatic setting, BC should be considered regardless of ER status for BRCA1/2 screening for therapeutic purposes.

Establishment of a novel BRCAness score that predicts response to PARP inhibitors.

549 Background: BRCAness is a generic term used to describe characteristic features of homologous recombination deficiency (HRD) mimicking mutations in BRCA genes. Although clinical genetic testing has increased the detection of mutations in BRCA1 and BRCA2, we hypothesized that a measure to quantify BRCAness will identify the responders to PARP inhibitors that cause synthetic lethality in BRCA mutation tumors. Methods: The BRCAness score was established by using gene set variation analysis (GSVA) algorithm on 34 BRCA-mutation related genes. We investigated the clinical relevance of the score by performing silico analyses of 6245 breast cancer patients using multiple independent large cohorts in this study. Results: A score to quantify BRCAness was generated using gene set variation analysis algorithm on 34 BRCA1-mutation related genes selected by high AUC levels in ROC curve between BRCA1 mutation and wildtype breast cancer. The score was significantly associated with BRCA1 mutation, high overall mutation load and intratumoral heterogeneity as well as high HRD, DNA repair and MKI67 expression regardless of mutation in BRCA gene. High score tumor enriched not only DNA repair, but also five cell proliferation-related gene sets (E2F targets, G2M checkpoint, MYC targets v1 and v2, and MITOTIC signaling) in Hallmark collection (all false discovery rate < 0.10). Breast cancers with high score were significantly associated with higher infiltration of anti-cancerous immune cells and higher cytolytic activity. Not all breast cancer cell lines with BRCA-mutation showed high score and the other cells in human breast cancer tumor microenvironment were contributing to the score. We found that the BRCAness score was the highest in triple-negative among subtypes consistently in all cohorts (all p < 0.001). Finally, BRCAness was associated with response to chemotherapy and correlated strongly with response to PARP inhibitor in both triple-negative (AUC = 0.815) and ER-positive/HER2-negative breast cancer (AUC = 0.715). Conclusions: We established a novel BRCAness score using mRNA expression of BRCA-mutation-related genes and found that it associates with DNA repair and response to PARP inhibitor regardless of BRCA mutation.

Stereospecific lasofoxifene derivatives reveal the interplay between estrogen receptor alpha stability and antagonistic activity in ESR1 mutant breast cancer cells.

Chemical manipulation of estrogen receptor alpha ligand binding domain structural mobility tunes receptor lifetime and influences breast cancer therapeutic activities. Selective estrogen receptor modulators (SERMs) extend estrogen receptor alpha (ERα) cellular lifetime/accumulation. They are antagonists in the breast but agonists in the uterine epithelium and/or in bone. Selective estrogen receptor degraders/downregulators (SERDs) reduce ERα cellular lifetime/accumulation and are pure antagonists. Activating somatic ESR1 mutations Y537S and D538G enable resistance to first-line endocrine therapies. SERDs have shown significant activities in ESR1 mutant setting while few SERMs have been studied. To understand whether chemical manipulation of ERα cellular lifetime and accumulation influences antagonistic activity, we studied a series of methylpyrollidine lasofoxifene (Laso) derivatives that maintained the drug's antagonistic activities while uniquely tuning ERα cellular accumulation. These molecules were examined alongside a panel of antiestrogens in live cell assays of ERα cellular accumulation, lifetime, SUMOylation, and transcriptional antagonism. High-resolution x-ray crystal structures of WT and Y537S ERα ligand binding domain in complex with the methylated Laso derivatives or representative SERMs and SERDs show that molecules that favor a highly buried helix 12 antagonist conformation achieve the greatest transcriptional suppression activities in breast cancer cells harboring WT/Y537S ESR1. Together these results show that chemical reduction of ERα cellular lifetime is not necessarily the most crucial parameter for transcriptional antagonism in ESR1 mutated breast cancer cells. Importantly, our studies show how small chemical differences within a scaffold series can provide compounds with similar antagonistic activities, but with greatly different effects of the cellular lifetime of the ERα, which is crucial for achieving desired SERM or SERD profiles.

Molecular Characterization of BRCA1 c.5339T>C Missense Mutation in DNA Damage Response of Triple-Negative Breast Cancer

Simple SummaryRecent studies re-classified the c.5339T->C; p.Leu1780Pro (L1780P) BRCA1 missense mutation as “likely pathogenic” in ACMG classification, which shows a high prevalence in the Korean population. This study aims to reveal the molecular mechanisms and therapeutic relevance of BRCA1 L1780P mutation in DNA damaging response of triple-negative breast cancer (TNBC).BRCA1 L1780P BRCT domain mutation has been recognized as a pathogenic mutation in patients with breast cancer. However, the molecular significance of this mutation has not yet been studied in triple-negative breast cancer (TNBC) cells in vitro. We established MDA-MB 231, HCC1937, and HCC1395 TNBC cell lines expressing BRCA1 L1780P mutant. BRCA1 L1780P mutant TNBC cells showed increased migration and invasion capacity, as well as increased sensitivity to olaparib and carboplatin compared to BRCA1 wild-type cells. BRCA1 L1780P mutant TNBC cells showed decreased RAD51 expression and reduced nuclear RAD51 foci formation following carboplatin and olaparib treatment. The molecular interaction between p-ATM and BRCA1 was abrogated following introduction of BRCA1 L1780P mutant plasmid in TNBC cells, suggesting that the BRCA1 L1780P mutation disrupts the p-ATM-BRCA1 protein–protein interaction. We established an olaparib-resistant BRCA1 L1780P mutant TNBC cell line by chronic drug treatment. Olaparib-resistant cell lines showed upregulation of RAD51 expression upon olaparib treatment, and reduction in RAD51 expression in olaparib-resistant cells restored olaparib sensitivity. Collectively, these results suggest that the BRCA1 L1780P mutation impairs RAD51 recruitment by disrupting p-ATM-BRCA1 interaction, which is a crucial molecular factor in homologous recombination and olaparib sensitivity. Further therapeutic targeting of RAD51 in BRCA1 L1780P mutant breast cancer is warranted.