Abstract GATA3 is an essential mediator of mammary gland development and is expressed at high levels in Estrogen Receptor positive tumors. This gene has recently emerged as one of the most frequently mutated genes in Estrogen Receptor positive breast cancer. GATA3 mutant tumors tend to retain and express the wild-type GATA3 allele which, together with the established role for GATA3 in maintaining luminal epithelial differentiation, has led to the suggestion that GATA3 is a haploinsufficient tumor suppressor gene in the mammary gland. We describe a striking contrast between the GATA3 mutation spectrum in breast cancer with that found in a true GATA3 haploinsufficiency syndrome and propose that this is indicative of a strong selective pressure to mutate and retain expression of a substantial portion of the GATA3 coding sequence. We demonstrate that the proteins encoded by GATA3 mutant alleles lack the capacity to bind to canonical GATA sequence motifs and acquire a new recognition specificity governed by the N-terminal zinc finger domain. Transgenic expression of the mutant protein results in a pronounced precocious lobuloalveolar development in 9 week old female mice, suggesting that the mutant gene is not a null allele and can regulate cell proliferation. In addition, we demonstrate that Estrogen Receptor positive GATA3 mutant human breast tumors arise at earlier ages than Estrogen Receptor GATA3 wild-type tumors, which further emphasizes the clinical relevance of these mutations. Based on the estimated mutation prevalence from the TCGA study, approximately 22,000 new cases of GATA3-mutant invasive breast cancer are diagnosed in the US each year. Collectively, our data indicate that GATA3 mutant tumors may comprise a breast cancer subset with distinct biological and clinical behavior and that a detailed understanding of the novel repertoire of target genes may yield useful clinical targets for this population. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-02.