Abstract B39: Identification of kinases that compensate for PI3K inhibition in breast cancer cells

The phosphatidylinositol 3-kinase (PI3K) pathway is one of the most commonly altered pathways in breast cancer. A number of PI3K inhibitors have recently entered clinical trials for the treatment of breast and other cancers. However, studies with similar targeted therapies, such as mTORC1 inhibitors, suggest that inhibition of key signaling nodes may relieve negative feedback pathways that limit the clinical efficacy of these inhibitors. Likewise, we anticipate that the response to PI3K inhibitors may be attenuated by relief of negative feedback pathways and upregulation of compensatory signaling nodes. The purpose of this study was to identify kinases and kinase- regulated signaling pathways that compensate for PI3K inhibition in human breast cancer cells. To this end, we employed a siRNA screen targeting 779 human kinases in order to determine if downregulation of specific kinases increases the sensitivity of HCC1937 human breast cancer cells to the PI3K inhibitor XL147. XL147 is an ATP-mimetic that inhibits the p110α catalytic subunit of PI3K with an in vitro IC50 of 39 nM. HCC1937 cells lack PTEN, the negative regulator of the PI3K pathway, and are growth inhibited by XL147 with an IC50 of 10 µM. Following reverse transfection with the siRNA library, we treated cells with either DMSO or 10 µM XL147 for 72 hours in medium containing 2.5% fetal bovine serum; the alamar blue assay was used to measure cell viability. In parallel, we performed gene expression microarrays, phospho-receptor tyrosine kinase (RTK) arrays, and phospho- intracellular kinase arrays using RNA or lysates from HCC1937 cells ± XL147 in order to determine which kinases and pathways are upregulated upon inhibition of PI3K. XL147 treatment resulted in increased expression and/or phosphorylation of many RTKs and intracellular kinases within 8-48 hours. Eight and 24 hours treatment with XL417 suppressed phospho-Akt at both S473 and T308, but at 48 hours, phosphorylation at S473 was partially restored. From these approaches, we identified several kinases and pathways that are upregulated by XL147 and that, when downregulated by siRNA, increased sensitivity to XL147. These include several RTKs, such as HER3, MSPR, and Axl, and members of the MAP kinase signaling pathway, such as several MAP3Ks/MEKKs. Western blotting confirmed that XL147 treatment decreased phospho-Akt at S473 and T308 and increased phospho- and total HER3. XL147 also increased phosphorylation of the MAP kinases ERK, JNK, and p38 MAPK. Combined treatment with XL147 and HER3 siRNA inhibited HCC1937 cell growth to a larger degree than each intervention alone, suggesting that the upregulation of phospho-HER3 compensated for growth inhibition as a result of pharmacological inhibition of PI3K. These preliminary results suggest that compensatory upregulation of RTK and MAP kinase pathways may limit the antitumor efficacy of PI3K inhibitors. Therefore, therapies combining compounds that inhibit these kinases or pathways along with PI3K inhibitors should improve the anti-tumor activity of PI3K inhibitors in breast cancer. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B39.