Single-Cell Dynamics Determines Response to CDK4/6 Inhibition in Triple-Negative Breast Cancer

Abstract

Purpose: Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of breast cancer that is associated with a poor prognosis. We evaluated the activity of CDK4/6 inhibitors across the TNBC subtypes and investigated mechanisms of sensitivity.Experimental Design: A panel of cell lines representative of TNBC was tested for in vitro and in vivo sensitivity to CDK4/6 inhibition. A fluorescent CDK2 activity reporter was used for single-cell analysis in conjunction with time-lapse imaging.Results: The luminal androgen receptor (LAR) subtype of TNBC was highly sensitive to CDK4/6 inhibition both in vitro (P < 0.001 LAR vs. basal-like) and in vivo in MDA-MB-453 LAR cell line xenografts. Single-cell analysis of CDK2 activity demonstrated differences in cell-cycle dynamics between LAR and basal-like cells. Palbociclib-sensitive LAR cells exit mitosis with low levels of CDK2 activity, into a quiescent state that requires CDK4/6 activity for cell-cycle reentry. Palbociclib-resistant basal-like cells exit mitosis directly into a proliferative state, with high levels of CDK2 activity, bypassing the restriction point and the requirement for CDK4/6 activity. High CDK2 activity after mitosis is driven by temporal deregulation of cyclin E1 expression. CDK4/6 inhibitors were synergistic with PI3 kinase inhibitors in PIK3CA-mutant TNBC cell lines, extending CDK4/6 inhibitor sensitivity to additional TNBC subtypes.Conclusions: Cell-cycle dynamics determine the response to CDK4/6 inhibition in TNBC. CDK4/6 inhibitors, alone and in combination, are a novel therapeutic strategy for specific subgroups of TNBC.

Insights into Molecular Classifications of Triple-Negative Breast Cancer: Improving Patient Selection for Treatment

“…Combination of PI3K/mTOR inhibition and AR antagonism has demonstrated synergistic activity in AR-positive TNBC preclinical models, and a phase I trial is planned to explore enzalutamide plus alpelisib, an α-specific PI3K inhibitor, in patients with AR-positive, PTEN lo (IHC 0%) TNBC (NCT03207529). Additional studies have revealed that, in contrast to basal-like and mesenchymal subtypes, LAR TNBC cell lines are highly sensitive to CDK4/6 inhibitors, with comparable sensitivity to that observed in the ER-positive MCF7 cell line (25). LAR cell lines exhibit lower transcriptomic levels of CCNE1 and CDK2 compared with basal-like TNBC and, thus, are dependent on CDK4/6 to phosphorylate RB1 and reenter the cell cycle.…”

“…In core-basal tumors, the prevalence of AR positivity defined by ≥1% of tumor cell nuclei IHC staining has been reported to be 32% (24). Interestingly, other studies have suggested that LAR tumors are characterized by a quiescent cell state (25), as opposed to rapidly proliferative basal tumors, raising the question of the optimal method of testing for AR positivity and possibly lack of a robust approach due to limited sample size. Altogether, this has prompted interest in exploring the role of antiandrogens in this subgroup.…”

Apocrine lesions of the breast: part 2 of a two-part review. Invasive apocrine carcinoma, the molecular apocrine signature and utility of immunohistochemistry in the diagnosis of apocrine lesions of the breast

“…Using a fluorescent CDK2 activity reporter for single cell analysis together with time-lapse imaging to test a panel of cell lines representative of TNBC (including an MDA-MB-453 LAR cell line xenograft), Asghar et al 40 have recently demonstrated that the LAR subset of TNBC proved highly sensitive to CDK4/6 inhibition both in vivo and in vitro. Clinical trials are required to test the efficacy of PI3K and CGK4/6 inhibition in these tumours.…”

Identifying Biomarkers to Pair with Targeting Treatments within Triple Negative Breast Cancer for Improved Patient Stratification

“…Previously, triple negative breast cancers were not thought to be a good candidate for treatment with CDK4/6 inhibitors due to approximately 20% of these tumours lacking functional Retinoblastoma-like protein (Rb) [41]. Pre-clinical data however has indicated the potential for sensitive subtypes of TNBC; in particular, a study by Asghar et al showed that the LAR subtype of TNBC was CDK4/6 inhibitor sensitive in vitro and in vivo [42]. Other TNBC tumours with high RB expression, androgen receptor positivity, or associated clinical characteristics are also considered potential candidates [43] and some pre-clinical research suggests a benefit of combination treatment including CDK4/6 inhibition [41].…”