Cyclin E1 protein is stabilized inBRCA1mutated breast cancers leading to synergy between CDK2 and PARP inhibitors

Abstract

26Basal-like breast cancers (BLBC) are aggressive breast cancers that respond poorly 27 to targeted therapies and chemotherapies. In order to define therapeutically 28 targetable subsets of BLBC we examined two markers: cyclin E1 and BRCA1 loss. 29 In high grade serous ovarian cancer (HGSOC) these markers are mutually exclusive, 30 and define therapeutic subsets. We tested the same hypothesis for BLBC. 31Using a BLBC cohort enriched for BRCA1 loss, we identified convergence between 32 BRCA1 loss and high cyclin E1 expression, in contrast to HGSOC in which CCNE1 33 amplification drives increased cyclin E1 gene expression. Instead, BRCA1 loss 34 stabilized cyclin E1 during the cell cycle. Using siRNA we showed that BRCA1 loss 35 leads to stabilization of cyclin E1 by reducing phospho-cyclin E1-T62, and 36 conversely the overexpression of BRCA1 increased phospho-T62. Mutation of cyclin 37 E1-T62 to alanine increased cyclin E1 stability. We showed that tumors with high 38 cyclin E1/BRCA1 mutation in the BLBC cohort had decreased phospho-T62, 39 supporting this hypothesis. 40Since cyclin E1/CDK2 protects cells from DNA damage and cyclin E1 is elevated in 41 BRCA1 mutant cancers, we hypothesized that CDK2 inhibition would sensitize these 42 cancers to PARP inhibition. CDK2 inhibition induced DNA damage and synergized 43 with PARP inhibitors to reduce cell viability in BRCA1 mutated cell lines. Treatment 44 of BLBC patient-derived xenograft models with combination PARP and CDK2 45 inhibition led to tumor regression and increased survival. We conclude that BRCA1 46 status and high cyclin E1 have potential as predictive biomarkers to dictate the 47 therapeutic use of combination CDK inhibitors/PARP inhibitors in BLBC. 48 3 instability in cancer cells, and is frequently elevated in BLBC (9). Perplexingly, in 65 high grade serous ovarian cancer (HGSOC) cyclin E1 amplification and BRCA1/2 66 mutation are mutually exclusive, presumably because both aberrations drive 67 genomic instability and together they precipitate lethal genomic damage (10-12). 68We recently described two subsets of HGSOC, one where cyclin E1 gene 69 amplification and BRCA1 mutation were mutually exclusive, and another where high 70 cyclin E1 protein expression was due to post-transcriptional deregulation rather than 71 4 gene amplification, and was often concurrent with BRCA1/2 mutation (12). Cyclin E1 72 protein stability is regulated by a multi-step process of specific phosphorylation and 73 ubiquitination, leading to its cyclic expression and turnover (13). Key regulators in the 74 turnover of cyclin E1, such as the ubiquitin ligase component FBXW7 and the 75 deubiquitinase USP28, are frequently dysregulated in cancer (13-15) leading to 76 altered stability of the cyclin E1 protein. 77In this study, we examined whether BRCA1 loss and cyclin E1 gain occurred 78 concurrently or independently in breast cancer. We also explored the mechanisms 79 underpinning high cyclin E1 expression in BRCA1 mutated breast cancer including 80 gene amplification and p...

Targeting Replicative Stress and DNA Repair by Combining PARP and Wee1 Kinase Inhibitors Is Synergistic in Triple Negative Breast Cancers with Cyclin E or BRCA1 Alteration

“…The association between cyclin E gene amplification and BRCA1/2 mutations have been examined previously and in studies with high grade serous ovarian cancer, such associations were found to be mutually exclusive [ 58 , 59 ]. However, when correlating cyclin E protein levels to BRCA1/2 mutations, studies in both ovarian and breast cancer patients show a significant level of correlation [ 60 , 61 , 62 ]. While mechanisms of regulation between BRCA1/2 and cyclin E remain unclear, a recent study suggested that BRCA1 mutation may stabilize cyclin E1 protein by decreasing phosphorylation T62 on cyclin E1 [ 62 ].…”

“…However, when correlating cyclin E protein levels to BRCA1/2 mutations, studies in both ovarian and breast cancer patients show a significant level of correlation [ 60 , 61 , 62 ]. While mechanisms of regulation between BRCA1/2 and cyclin E remain unclear, a recent study suggested that BRCA1 mutation may stabilize cyclin E1 protein by decreasing phosphorylation T62 on cyclin E1 [ 62 ]. Phosphorylation T62 of full-length cyclin E1 [ 63 ], but not its LMWE forms, leads to its proteasome mediated degradation in the nucleus by FBWX7 [ 64 ].…”

High cyclin E1 protein, but not gene amplification, is prognostic for basal‐like breast cancer

“…We explored the relationship between cyclin E1 protein expression and survival in BLBC by quantifying cyclin E1 expression across the kConFab familial breast cancer cohort [7]. Cyclin E1 expression was significantly higher in BLBC compared to non‐BLBC cases ( p < 0.0001) (Figure 1A).…”

“…Molecular and clinical data were obtained for breast cancer patients from the Kathleen Cunningham Foundation Consortium for research into Familial Breast cancer (kConFab), as described in Ref. [7], and for HGSOC from the Australian Ovarian Cancer Study (AOCS), as described in Ref. [8].…”

“…Recently, we described separate subsets of HGSOC, one where CCNE1 amplification and BRCA1/BRCA2 mutation were mutually exclusive (the 19q12 amplified subset), and another where BRCA1/BRCA2 mutation co‐occurred with high cyclin E1 protein overexpression (the high cyclin E1 protein subset) [8]. We subsequently investigated a patient cohort that is highly enriched for BLBC and identified the co‐occurrence of BRCA1 mutation and high cyclin E1 protein [7]. However, an unresolved question is whether CCNE1 amplification and cyclin E1 protein expression in BLBC demarcate patients into subgroups that are similar to those that have been detected in HGSOC.…”