Researchers have identified a subtype of TNBC that may be responsive to cyclin-dependent kinases 4 and 6 (CDK4/6) inhibition, according to findings presented at the IMPAKT Breast Cancer Conference, held in Brussels, Belgium, 4 to 6 May, 2017. This study also sheds light on the mechanisms of resistance to CDK4/6 inhibition seen in other subtypes of TNBC.
Inhibitors of CDK4/6, such as palbociclib and ribociclib, are indicated in combination with an aromatase inhibitor for the treatment of hormone receptor positive breast cancer. However, TNBC has demonstrated resistance to these agents, prompting investigators to use several preclinical models to identify subtypes of TNBC that may be sensitive to CDK4/6 inhibition and to define the mechanisms of resistance in TNBC.
Dr Uzma Asghar of Breast Cancer Now, Institute of Cancer Research, London, UK headed a research team that used in vitro assays on 18 TNBC cell lines to quantify sensitivity to palbociclib and ribociclib. These agents were evaluated for in vivo drug toxicity, efficacy, and pharmacodynamics in mouse MDAMB453 xenograft models. The investigators performed single cell phenotypic analysis using a CDK2 live cell reporter to understand resistance mechanisms to palbociclib using time-lapse imaging technology. Additional experimental techniques employed included immunofluorescence, western blotting, and immunohistochemistry (IHC) to determine molecular determinants of sensitivity.
LAR subtype of TNBC is highly sensitive to CDK4/6 inhibition
Luminal Androgen Receptor (LAR) Subtype of Triple Negative Breast Cancer (TNBC) Demonstrates in vitro and in vivo Sensitivity to CDK4/6 Inhibition.
Figure legends A. Sensitivity to 500nmol Palbociclib across 13 TNBC cell lines in clonogenic assays with LAR subtype highly sensitive to CDK4/6 inhibition [p<0.0001 Student’s T test LAR vs. basal-like]. ER+ve MCF7cells are shown as positive control of sensitivity.
B. Mouse xenografts from LAR MDAMB453 cells, treated daily with vehicle (n=10) and Palbociclib (n=10) demonstrating in vivo efficacy [p<0.0001 Student’s T test vehicle vs. palbociclib]. Error bars = mean tumour volume (mm3) and SD.
C. Post-mitotic (2 hours) CDK2 activity in individual cells from five TNBC cell lines. LAR MFM223 vs. Basal-like SUM149 cell lines (Student’s T-test p<0.0001). LAR MDAMB453 vs. Basal-like SUM149 (p<0.0002). Error bars = mean CDK2 ratio and SD. LAR=Luminal androgen receptor subgroup (light blue); MES=mesenchymal (grey) and Basal-like (dark blue).
D. Immunofluorescent staining of nuclear Cyclin E1 (red) in CDK2L positive (GFP) MDAMB453 cells and SUM149 cells 1 hour after mitosis; DAPI (blue). Cyclin E1 nuclear intensity was assessed by immunofluorescence in individual cells transfected with the CDK2-L sensor, 1-2 hours post-mitosis in LAR MDAMB453 and Basal-like SUM149 cell lines (p value =0.094 Student’s T-test). Error bars = mean nuclear intensity and SD.
E. Mouse xenografts from MDAMB453 cells, treated daily with Vehicle (n=10), Palbociclib (n=10), Taselisib (n=10) or combination (n=10) (p=0.02 Palbociclib vs. combination p=0.02; Taselisib vs. combination p=0.01). Error bars are mean tumour volume and SD.
F: Left: Schematic of cell cycle dynamics for Palbociclib-sensitive model. CDK4/6 inhibition sensitive cell (CDK2low) exits mitosis into a quiescent state (G0), requiring CDK4/6 activity (blue) to pass through the restriction point (yellow circle) after which CDK2 activity (red) promotes S phase entry. Right: Schematic of cell cycle dynamics for Palbociclib-resistant model. CDK4/6 inhibitor resistant cells (CDK2high) exit mitosis in an active proliferating state with high CDK2 activity, bypassing the restriction point, making CDK4/6 activity redundant to enter S phase. Credit: Uzma Asghar
The in vitro assays demonstrated that the luminal androgen receptor (LAR) subtype of TNBC was highly sensitive to CDK4/6 inhibition compared to basal-like subtypes, which were resistant to CDK4/6 inhibition (p < 0.001). This sensitivity to palbociclib at 50 mg/kg was confirmed in vivo using MDAMB453 xenografts wherein 7 partial responses (total 10 xenographs) were observed.
Furthermore, IHC analysis revealed a reduction in tumour phosphorylated retinoblastoma (pRB) levels, a key factor in cell cycle progression, as early as 4 hours after treatment.
Dysregulation of cyclin E1 expression in TNBC cell-lines is implicated in resistance to CDK4/6 inhibition
The mechanism of resistance was studied by phenotypic single cell analysis using 3 palbociclib sensitive and 3 palbociclib resistant TNBC cell lines, which revealed distinct cell cycle dynamics. Cells sensitive to palbociclib exited mitoses in a CDK2low quiescent state that required CDK4/6 activity for cell cycle re-entry. However, cells resistant to palbociclib exited mitoses directly into a CDK2high proliferative state with rapid transition to S phase with shorter cell cycles. Higher cyclin E1 levels were observed during the early G1 phase in resistant CDK2high cells than measured in palbociclib sensitive CDK2low cells. Interestingly the investigators demonstrated that these CDK2high resistant cells could become sensitive to palbociclib by silencing cyclin E1.
This study also demonstrated that CDK4/6 inhibitors used in concert with PI3kinase inhibitors in PIK3CA mutant TNBC cell lines acted synergistically and could make other TNBC subtype cell lines sensitive to palbociclib.
Conclusions
Based on these pre-clinical findings, the authors concluded that the LAR subgroup of TNBC demonstrated high sensitivity to CDK4/6 inhibition, and noted that an ongoing clinical trial is currently assessing CDK4/6 inhibition in TNBC (phase Ib PIPA: NCT02389842).
They further determined distinct differences between cell cycle exit from mitosis between palbociclib sensitive and resistant TNBC cell lines, with resistant cells having higher levels of cyclin E1. Blocking cyclin E1 activity made resistant cells, sensitive to palbociclib, leading to the conclusion that dysregulation of cyclin E1 expression is a key determinant of sensitivity to CDK4/6 inhibition.
Disclosure
Funding from Breast Cancer Now, the Avon Breast Cancer Crusade, the Institute of Cancer Research, and Genentech was disclosed