Cosgrove, Nicola

Abstract Background Cyclin-dependent kinase 12 (CDK12) regulates general gene transcription elongation and plays multiple roles in RNA splicing, DNA damage-response, cell cycle, and genomic stability. However, transcriptional partners that guide CDK12-specific gene programs have not been identified. Genomic alterations in CDK12 have been observed in multiple cancers, exhibiting both pro-tumorigenic and tumor-suppressive functions, suggesting a context-dependent mechanism of action. Methods CDK12 copy number alterations and gene expression levels were analyzed in matched primary and brain metastatic patient tumors. Clinical significance was assessed by immunohistochemistry in a large cohort of primary breast cancer patient tumors. RNA sequencing, ChIP sequencing, and molecular studies were conducted to explore CDK12’s mechanism of action, and pharmacological studies were performed both in vitro and in vivo using models of advanced (endocrine-resistant and metastatic) estrogen receptor positive (ER+) disease. Results CDK12 amplifications and gene overexpression were observed in brain metastatic tumors. In ER+ primary patient tumors, high CDK12 protein expression was significantly associated with poor overall survival, particularly within the ER+/HER2-negative group. In ER+ endocrine resistant models, CDK12 regulated estrogen signaling pathways, with ER/MED1 identified as the master transcriptional complex directing CDK12-specific pro-tumorigenic gene programs. Pharmacological inhibition of CDK12 significantly reduced viability in endocrine resistant and metastatic cell and organoid models in vitro, and decreased metastatic spread in vivo. Conclusion This work describes a novel mechanism for CDK12, suggesting a potential vulnerability in ER+ breast cancer. These findings provide a basis for further investigation into the role of CDK12 inhibition as a therapeutic approach, particularly in advanced disease settings.

Abstract 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 < 0.05). Enrichment for fusions in pathways associated with tumour cell plasticity and proliferation with recurrent fusions in known cancer driver genes related to MAPK, HER signaling identified. Of note, a fusion in CDK12 is of clinical importance. Increased genomic alterations and over expression of CDK12 is associated with brain metastases free survival in an independent cohort of primary breast tumours with a recorded history of brain metastases. It has been proposed that inhibition of CDK12 may induce BRCAness in tumours making them sensitive to PARP inhibition. 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.

Abstract BACKGROUND: Brain metastatic disease occurs in 10-30% of metastatic breast cancer cases. The incidence of brain metastases is increasing, yet overall survival remains < 2 years. Treatment of brain metastases is limited with current clinical practice centered on radiation, chemotherapy and surgery. Although these treatments may prolong survival in the short term, targeting oncogenic alterations in brain metastases may deliver a more sustained clinical benefit. In this multicenter study, we comprehensively characterized DNA and RNA alterations that aberrantly drive specific oncogenic pathway activity pertinent to breast cancer brain metastases (BCBM). METHODS: RNA sequencing was performed on a cohort of patient-matched primary and resected brain metastatic tumours (45 patients; N=90 samples). Whole exome DNA sequencing (WXS) was performed for 18/45 patients (54 trios consisting of primary tumor, brain metastasis and matched normal tissue). An independent brain metastatic WXS cohort (N=21 patients) (PMID: 26410082) was also analysed resulting in a total of 39 patient samples. Recurrent somatic copy number alterations (SCNA), somatic single nucleotide variants (SNVs) and mutational signatures were identified from WXS data. Expressed gene fusions were detected computationally from RNA-Seq (N=45 cases) RESULTS: Of the 45 BCBM patients, median age at diagnosis was 51 years [25, 67], median overall survival 57 months (range 18-255) with median brain metastases free survival 34 months (range 5-216).Clinical molecular subtype of the primary tumour included 13 ER+/HER2- (29%), 16 HER2+ (35.5%) and 16 TNBC (35.5%). Regions of significant recurrent amplifications and deletions in BCBM (N=39 patients) were identified in 4q12, 10q11.21, 8p11.23, 8q23.3 and 17q12 (FDR < 0.10). Recurrent expressed gene fusions identified in known cancer driver genes were associated with chromatin modification, MAPK and HER signaling pathways. Mutational signature analysis of SNVs identified signatures associated with ageing, mismatch repair and homologous recombination deficiency (HRD) mutational processes. The relative contribution of the HRD signature was significantly increased in brain metastases compared to matched primary tumour (p < 0.05). Concordantly increased HRD in the brain metastatic transcriptome was confirmed in these patients by gene set variation analysis (GSVA) of homologous recombination pathway genes from KEGG database in RNA-Seq data. Moreover, in the extended BCBM RNA-Seq (N=45 patients) GSVA pathway scores were elevated in brain metastases relative to primary tumours, validating the functional significance of altered DNA repair defects in brain metastases CONCLUSIONS: Here, we report recurrent genetic drivers, supported by altered functional transcriptome, unique to brain metastasis that may have clinical implications for prognosis and treatment choice. Specifically, targeting defects in the homologous recombination repair mechanism may represent new therapeutic strategies and management opportunities for breast cancer brain metastases patients. Citation Format: Nicola S Cosgrove, Damir Varešlija, Stephen Keelan, Simon Furney, Jodi M Carter, Steven N Hart, Siddhartha Yadav, Arnold DK Hill, Steffi Oesterreich, Adrian V Lee, Fergus J Couch, Leonie Young. Homologous recombination deficiency represents a new therapeutic strategy for breast cancer brain metastases [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD13-01.