Abstract Background: TNBC comprises about 15% of breast cancer cases and, with no approved targeted therapy, is commonly treated with adjuvant chemotherapy. We have described a novel targeted therapy for basal-like TNBC combining EGFR and sphingosine kinase (SphK) inhibitors, which induce a strongly synergistic cytostatic effect in six TNBC cell lines (Martin et al, Breast Cancer Res 19:90, 2017). IGFBP-3, which is highly expressed in basal-like TNBC and prognostic for poor recurrence-free survival, initiates an oncogenic pathway involving SphK1 and EGFR activation. Since IGFBP-3 downregulation in vitro prevents the synergistic cytostasis caused by EGFR and SphK inhibitors, we aimed in this study to evaluate nuclear IGFBP-3 as a response biomarker in TNBC tumors after treatment with the inhibitor combination, with or without doxorubicin (dox), a common component of TNBC chemotherapy. Methods: Human basal-like TNBC cell lines HCC1806 and MDA-MB-468 were grown as orthotopic xenograft tumors in female BALB/c nude mice, and treated when tumors reached 100 mm3 with the SphK inhibitor and S1P receptor modulator, fingolimod, plus the EGFR kinase inhibitor, gefitinib (F+G) ± dox at the maximum tolerated dose of 2 mg/Kg weekly. Tumors were analyzed by immunohistochemistry (IHC). Cell proliferation was studied in vitro by IncuCyte live-cell imaging. Results: In both cell lines in vitro, F+G at doses that caused minimal cytostasis, acted synergistically with a sub-cytostatic dox dose (10 nM), to cause almost complete inhibition of proliferation. In contrast, in xenograft mouse models, in which F+G administered 3x weekly significantly inhibited tumor growth and enhanced mouse survival, dox administration for 6 weeks showed minimal growth inhibition alone, and no significant incremental effect with F+G. Examined by IHC, TNBC tumor IGFBP-3 staining showed predominantly nuclear localization, was positively correlated with Ki67, and was significantly downregulated by F+G treatment, with no added dox effect. By Kaplan-Meier analysis, high (> median) nuclear IGFBP-3 IHC scores were strongly associated with worse mouse survival, similar to high Ki67 scores, while high apoptosis (cleaved caspase-3) scores were associated with better survival. Conclusion: The synergism between dox and combination F+G, observed in vitro, was not seen in vivo, possibly because of dose-limiting dox toxicity. This might be improved by alternative dox delivery methods. However, the F+G combination alone is highly inhibitory to basal-like TNBC tumor growth in both xenograft models. IGFBP-3 is known to interact with nuclear hormone receptors and DNA damage response intermediates in the nucleus. Nuclear IGFBP-3 staining may have utility as a biomarker of treatment response in TNBC, alone or together with Ki67 and cleaved caspase-3. Supported by Cancer Council NSW. Citation Format: Sohel M. Julovi, Janet L. Martin, Robert C. Baxter. Nuclear IGFBP-3 is a potential biomarker for response to EGFR-sphingosine kinase targeted therapy in basal-like triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 789.