(A-B) Lung cancer cell lines were treated with 2.5 μM AZD1775 alone for 72 h and linear correlation (R2) between percentage cell viability and WEE1 and pY15-CDK1 expression was determined. (C) Immunoprecipitation of BRCA1 using a monoclonal antibody. Note that BRCA1 is shown as two light bands (hypo- and hyper phosphorylated forms) and that the antibody preferentially immunoprecipitates the hypo-phosphorylated form (faster migrating band). (D) Control western blots for PAXIP1 antibodies (#370 and #369). (E) Immunoprecipitation of PAXIP1 using #369 antibody and blotted against the #370 antibody. Ectopically expressed TAP-tagged PAXIP1 is also recognized by the #370 antibody. (F) AZD1775 does not disrupt the interaction between PAXIP1 tBRCT C2 and WEE1. (biological replicate of Figure 2E). TAP-tBRCT C2 or a TAP-GFP were ectopically expressed in 293FT cells. Equal amounts of lysates expressing TAP-tBRCT C2 were used to incubate in the presence (1 µM) or absence of AZD1775 for 1 h. Lysates were pulled down with streptavidin beads and blotted against CBP to demonstrate equivalent pull down of the ectopic proteins (bottom panel). Incubation with AZD1775 slightly increased binding of WEE1 to PAXIP1 tBRCT C2 (right panel).
ARTICLE ABSTRACT
The DNA damage response (DDR) involves a complex network of signaling events mediated by modular protein domains such as the BRCA1 C-terminal (BRCT) domain. Thus, proteins that interact with BRCT domains and are a part of the DDR constitute potential targets for sensitization to DNA-damaging chemotherapy agents. We performed a pharmacologic screen to evaluate 17 kinases, identified in a BRCT-mediated interaction network as targets to enhance platinum-based chemotherapy in lung cancer. Inhibition of mitotic kinase WEE1 was found to have the most effective response in combination with platinum compounds in lung cancer cell lines. In the BRCT-mediated interaction network, WEE1 was found in complex with PAXIP1, a protein containing six BRCT domains involved in transcription and in the cellular response to DNA damage. We show that PAXIP1 BRCT domains regulate WEE1-mediated phosphorylation of CDK1. Furthermore, ectopic expression of PAXIP1 promotes enhanced caspase-3–mediated apoptosis in cells treated with WEE1 inhibitor AZD1775 (formerly, MK-1775) and cisplatin compared with cells treated with AZD1775 alone. Cell lines and patient-derived xenograft models expressing both PAXIP1 and WEE1 exhibited synergistic effects of AZD1775 and cisplatin. In summary, PAXIP1 is involved in sensitizing lung cancer cells to the WEE1 inhibitor AZD1775 in combination with platinum-based treatment. We propose that WEE1 and PAXIP1 levels may be used as mechanism-based biomarkers of response when WEE1 inhibitor AZD1775 is combined with DNA-damaging agents. Mol Cancer Ther; 15(7); 1669–81. ©2016 AACR.
