Background: We have recently shown that acquired resistance to aromatase inhibitors (AI) can, in some instances, be reversed low-dose estradiol treatment until progression followed by repeat treatment with an aromatase inhibitor (Ellis, M. JAMA 302:774, 2009). We have also demonstrated that simultaneous inhibition of phosphoinositol-3-kinase (PI3K) and ER (though estrogen deprivation) activates cell death through apoptosis, which is reversed by estradiol, revealing a link between ER activation and the efficacy of PI3 kinase inhibition (Crowder, R. Cancer Res 69:3955, 2009). Consistent with this concept we have also shown that long term estrogen deprived (LTED) cells are co-resistant to both classes of agent (SABCS 2009 Abs#3131). We therefore studied ways to overcome resistance to PI3 kinase inhibitors in LTED cell lines using second line endocrine therapy strategies that could be offered to patients with AI resistant advanced disease. Methods: ER+ MCF7 and T47D (PIK3CA mutant) cells were cultured in phenol-red free media containing charcoal-stripped serum for 12 months to create long term estrogen deprivation (LTED) variants that mimicked clinical endocrine therapy resistance. A variant of the MCF7 LTED line that regained estrogen-dependent proliferation was created by re-exposure to estradiol for 4 months (MCF7 LTED-R). The effects of the PI3K isoform inhibitor BKM120, the mTOR inhibitor RAD001 and the dual PI3K/mTOR inhibitor BGT226 in MCF7 LTED and T47D LTED were assessed in combination with estrogen deprivation and fulvestrant. MCF7 LTED-R cells were treated with PI3 kinase pathway inhibitors after acute estradiol withdrawal. Apoptosis was measured using TUNEL flow cytometry. Results: MCF7 LTED were resistant to all three PI3 kinase inhibitors, but combination treatment with fulvestrant restored apoptosis to levels seen with parental MCF7 cells after acute estrogen-deprivation. Acute estrogen deprivation in MCF7 LTED-R cells was also effective in restoring an apoptotic response to BGT226. T47D LTED cells, which, unlike MCF7 cells, have markedly down-regulated ER expression, become more sensitive to PI3 kinase monotherapy than the parental T47D cell line, particularly in response to BKM120, and fulvestrant did not increase the apoptotic response. Conclusions: The MCF7 cell results suggest that PI3 kinase inhibitors will be effective in endocrine therapy resistant advanced breast cancer if these agents are administered with appropriate second line endocrine strategies. Potentially efficacious approaches include fulvestrant combinations and second-line aromatase inhibition, but only after restoration of AI sensitivity with estradiol. The T47D experiments suggest that in the setting of ER+ tumors that lose ER upon disease progression PI3 kinase inhibitors may be very effective. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-19-01.