Abstract 3859: Peptidomimetic Src kinase inhibitor KX-01 sensitizes estrogen receptor α-negative breast tumor xenografts to tamoxifen by inducing ERα re-expression
Authors: Anbalagan Murali , Sheng Mei , Fleischer Brian , Hangauer David , Rowan Brian G. 
Published: July 2016

Unlike breast cancer that is positive for estrogen receptor α (ERα) or amplified/overexpressed HER2/neu, there are no targeted therapies for triple negative breast cancer (TNBC). ERα is silenced in TNBC through epigenetic changes including DNA methylation and histone acetylation. Restoring ERα expression in TNBC tumors may sensitize patients to endocrine therapy. Expression of c-Src kinase and ERα are inversely correlated in breast cancer suggesting that c-Src inhibition may lead to re-expression of ERα in TNBC. KX-01 is a peptide substrate targeted Src kinase/pretubulin inhibitor in clinical trials for solid tumors. At low dose, KX-01 is an effective Src kinase inhibitor in breast tumor xenografts in mice, but does not affect microtubules. MDA-MB-231 and MDA-MB-468 TNBC xenografts in NUDE mice were treated with low dose (1 mg/kg b.wt. BID) KX-01 for 40 days. KX-01 treatment decreased Src kinase activity in tumors and increased ERα mRNA and protein. Immunohistochemistry and Western blot demonstrated that the epithelial markers progesterone receptor and E-cadherin were increased, whereas the mesenchymal markers vimentin and nuclear β-catenin were decreased, suggesting a mesenchymal to epithelial transition (MET) in the tumors. At the ERα promoter in MDA-MB-231 tumors, KX-01 treatment led to enrichment of transcriptionally active (acetyl-H3, acetyl-H3Lys9) chromatin marks. There was no change in ERα promoter methylation as assessed by methylation PCR analysis and bisulfite sequencing. KX-01 treatment did not alter histone deacetylase 1 (HDAC1) levels or activity in tumors, but did result in HDAC1 dissociation from the ERα promoter, and a concomitant recruitment of RNA Polymerase II as assessed by chromatin immunoprecipitation (ChIP). Tamoxifen-resistant MDA-MB-231 xenografts in NUDE mice were treated with vehicle, tamoxifen alone (5 mg pellet; 60 day release), KX-01 alone (1 mg/kg b.wt. BID), or KX-01 + tamoxifen for 40 days. KX-01 alone and KX-01 + tamoxifen reduced tumor volume by 59% and 70%, respectively, compared to vehicle. Tumor volume for the KX-01 + tamoxifen group was significantly reduced compared to the KX-01 alone group (P = .023), and tumor weight of the KX-01 + tamoxifen group was 32% lower compared to the KX-01 alone group (P&lt; 0.01). Only the KX-01 + tamoxifen group exhibited reduced levels of ERα targets pS2, c-Myc and cyclin D1 indicating that estradiol signaling was attenuated by tamoxifen only in tumors treated with KX-01. In MDA-MB-468 tumors, tamoxifen alone (10 mg pellet; 60 day release) and KX-01 alone (1 mg/kg b.wt. BID) had no effect on tumor volume compared to vehicle, but KX-01 + tamoxifen reduced tumor volume 67% compared to vehicle (P = 0.0025). Collectively, these data demonstrate that the peptidomimetic Src inhibitor KX-01 can restore ERα expression in TNBC through changes in histone acetylation that sensitize TNBC tumors to endocrine therapy (tamoxifen).

Citation Format: Murali Anbalagan, Mei Sheng, Brian Fleischer, David Hangauer, Brian G. Rowan. Peptidomimetic Src kinase inhibitor KX-01 sensitizes estrogen receptor α-negative breast tumor xenografts to tamoxifen by inducing ERα re-expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3859.