Abstract 1788: The CDK inhibitor dinaciclib sensitizes triple-negative breast cancer cells to PARP inhibition.
Authors: Johnson Shawn F. , Johnson Neil , Chi David , Primack Benjamin , D'Andrea Alan D. , Lim Elgene , Shapiro Geoffrey I. 
Published: August 2014

Background: Triple Negative Breast Cancer (TNBC) comprises 15% of all breast cancers, and has a poor prognosis relative to other breast cancer subtypes. Inhibition of poly (ADP-ribose) polymerase (PARP) causes the degeneration of single-strand DNA breaks to more lethal double-strand breaks (DSBs), and also traps PARP-DNA complexes, which must be repaired or bypassed by homologous recombination (HR). BRCA1 plays a critical role in HR and its activity is in part regulated by cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation. PARP inhibition results in synthetic lethality in cells that have impaired HR, such as BRCA-deficient cells. Other CDK family members activate additional components of the HR pathway. Here, we show that HR-proficient TNBC cells can be sensitized to PARP inhibition through use of CDK inhibition to impair BRCA1 function and disrupt HR.

Methods: We examined the effects of dinaciclib (CDK1, 2, 5 and 9 inhibitor), γ-irradiation and veliparib (PARP inhibitor) on a panel of BRCA1 wild type TNBC cell lines (MDA-MB-231, MDA-MB-468, and BT549). Levels of phospho-S1497 BRCA1 (pBRCA1; CDK phosphorylation site), γ-H2AX, and RAD51 were measured by western blot to assess effects on HR proteins. A direct assessment of DSB repair was measured using the U2OS-DR-GFP reporter system. Cell viability was measured using Cell-Titer Glo assays. Patient-derived TNBC xenograft models were established in immunocompromised NOD-SCID-IL2γ-/- mice, and implanted orthotopically into cohorts of mice for in vivo efficacy studies.

Results: There was a significant reduction in total and pBRCA1 and RAD51 protein levels with increasing concentrations of dinaciclib in all TNBC cell lines. In response to 10 Gy γ-irradiation treatment, pretreatment with dinaciclib (20 nM) reduced the percentage of cells with greater than five BRCA1 foci from 54% to 5% (P &lt; 0.001); and the percentage of cells with greater than 5 RAD51 foci from 26% to 4% (P = 0.001). In U2OS-DR-GFP assays, expression of GFP was detected in 3.4% of the vehicle group and in 1.9% of dinaciclib treated cells. Cell viability assays following five days of dinaciclib, veliparib or the combination revealed in vitro cytotoxic synergy in all three TNBC cell lines. In an orthotopic TNBC model derived from a primary patient sample, the relative tumor volumes over a 30-day treatment period for vehicle, dinaciclib, veliparib and combination- treated mice were 5.6-fold, 3.3-fold, 3.2-fold and 0.66-fold (P &lt; 0.001) that of the initial tumor volume at the start of treatment. Tumor volume decreased only in the combination arm.

Conclusion: CDK inhibition effectively inhibits HR, and renders BRCA-proficient TNBC cells sensitive to PARP inhibition. This combination represents an effective strategy for the treatment of TNBC.

Citation Format: Shawn F. Johnson, Neil Johnson, David Chi, Benjamin Primack, Alan D. D'Andrea, Elgene Lim, Geoffrey I. Shapiro. The CDK inhibitor dinaciclib sensitizes triple-negative breast cancer cells to PARP inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1788. doi:10.1158/1538-7445.AM2013-1788