Abstract P6-08-03: Functional detection and inhibition of the targetable oncogenic kinome of chemotherapy-treated triple-negative breast cancer cells
Authors: Pan B , Olow A , Sun Q , Mori M , Lee PRE , Hartog M , Wang C , Wolf D , Yau C , van 't Veer L , Coppé J-P 
Published: May 2016

Background: Triple-negative breast cancer (TNBC) accounts for 10-15% of all breast cancer cases. A major area of innovation in TNBC is identifying potential treatment targets, especially in TNBC cells which survive chemotherapy. Our previous study showed that TNBC cells displayed deregulated kinase-dependent signaling cascades, and uniquely divergent phospho-circuits could be distinguished between TNBC vs non-TNBC cell lines [2014 SABCS abstract 1672, poster P2-05-09]. We further hypothesized that specific dysfunctional phospho-signaling network played a key role in the early adaptive changes in DNA damage response of TNBC cells exposed to DNA damaging chemotherapeutic agents.

Methods: TNBC cell lines MDA-MB-231 and MDA-MB-436 were treated with 5-fluorouracil (5-Fu), carboplatin and doxorubicin at their respective half maximal inhibitory concentrations (ic50s). MiSeq gene sequencing of the untreated vs treated TNBC cells was performed to investigate whether exposure to chemotherapy agents for 3-day's duration would induce additional adaptive genetic mutation. Apoptosis and cell-cycle distribution of the untreated and treated TNBC cells were analyzed with flow cytometry. The functional phospho-signature of each TNBC cell sample was analyzed using a high throughput experimental platform that monitors the level of activity of myriad kinases at once. This technique used over 450 phospho-sensing probes, including over 150 controls in an aqueous-based assay to simultaneously and directly measure the phospho-catalytic activity of phosphorylating enzymes in cell lysates. The kinome activities of the untreated vs treated TNBC cell lines were compared respectively, and the most significantly deranged and functionally altered phospho-signaling cascades and their related kinases were identified as the early adaptive changes of the survived TNBC cells after the 3-day exposure to DNA damage chemotherapies.

Results: Using the two TNBC cell lines treated with the three chemotherapies, we made 8 cell line samples, including 6 treated and 2 untreated as the control. MiSeq gene sequencing showed no significant additional adaptive genetic mutations in the treated TNBC cells after the 3-day short-term exposure to 5-Fu, carboplatin and doxorubicin. 36 phospho-signatures were generated and validated for repeatability and robustness. The kinase activity signature of each TNBC sample was analyzed and compared to each other using unsupervised hierarchical clustering. The phospho-sensing assay revealed that phospho-signaling cascades related to CHK1/2 and IKK kinases were differentially altered in the untreated vs treated TNBC cell lines, which, when respectively inhibited by AZD7762 and IKK16, successfully increased growth inhibition and cell death of TNBCs.

Conclusions: We identified specific phospho-fingerprints of the early adaption of TNBC cell lines and combinatorial targeted therapies that improve treatment outcome. Our next goal is to identify specific phosphorylation cascades in a broader range of cell lines and tumor tissues, to explore the actionable, kinase-dependent mechanisms critical to the DNA damage-induced adaptive reprogramming of TNBCs and early changes driving drug-resistance.

Citation Format: Pan B, Olow A, Sun Q, Mori M, Lee PRE, Hartog M, Wang C, Wolf D, Yau C, van 't Veer L, Coppé J-P. Functional detection and inhibition of the targetable oncogenic kinome of chemotherapy-treated triple-negative breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-08-03.