PD 0332991, a selective CDK 4/6 inhibitor, preferentially inhibits proliferation of luminal ER+ breast cancer cells and acts synergistically with tamoxifen and trastuzumab in vitro.

Abstract

Abstract Abstract #5064 Background: Cell cycle dysregulation is a common molecular finding in malignancy and the cyclin-D kinases (CDK) represent an attractive target in this pathway. PD 0332991 (Pfizer Inc.) is an orally active potent and highly selective inhibitor of CDK 4 and CDK6 kinases with the ability to block pRb phosphorylation at serine 780 and 795 in the low nanomolar range. To identify predictors of response to PD 0332991, we determined the in vitro sensitivity to PD 0332991 of a large panel of molecularly characterized breast cancer cell lines, and then used baseline expression profiles to determine genes associated with response. Methods: 44 human breast cancer cell lines representing the known molecular subgroups of breast cancer (i.e. luminal, HER2, basal, etc) and 3 immortalized breast lines were treated in duplicate with PD 0332991 using two-fold dilutions over 12 concentrations. Dose response curves were generated using a cell count assay to calculate the IC50 across the panel. These data were then analyzed against baseline gene expression data (Agilent microarray) to identify genes associated with sensitivity and resistance to PD 0332991. ANOVA analysis identified genes associated with response and/or resistance to PD 0332991. Western blot analysis was performed analyzing the effects of PD 0332991 on pRb phosphorylation. Cell cycle analysis was performed using NIM-DAPI staining and flow-cytometry. In addition, combination studies were performed to analyze the interaction between PD 0332991 and tamoxifen and trastuzumab. These data were analyzed using Calcusyn software to generate a combination index (CI) to define the interaction as additive (CI=1), synergisitc (CI<1), or antagonistic (CI>1). Results: Cell lines representing the luminal ER+ subtype (including HER2 amplified) were most sensitive to inhibition by PD 0332991 and non-luminal/basal (triple-negative) were most resistant. ANOVA analysis identified 563 differentially expressed genes between the sensitive and resistant groups. While, several of these genes are associated with breast cancer subtype, pRb was elevated and CDKN2A (p16) was decreased in the most sensitive lines. Cell lines treated with PD 0332991 showed clear G0/G1 arrest and a decrease in S-phase fraction in sensitive but not in resistant cell lines. In addition, Western blot showed that pRb phosphorylation is blocked in a time dependent manner. Finally, the combination of tamoxifen and PD 0332991 was strongly synergistic in three ER+ cell lines evaluated and appeared synergistic with trastuzumab as well. Conclusion: These studies suggest that a subgroup of breast cancers may be more likely to benefit from treatment with the PD 0332991 CDK4/6 inhibitor than others. This group represents ER+ luminal breast cancer and is associated with elevated pRb and low p16. The combination of PD 0332991 and tamoxifen and trastuzumab shows promising biologic activity in ER+ and HER2 amplified breast cancer cell lines, respectively. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5064.