Abstract Introduction: Histone deacetylases (HDACs) are dysregulated in human cancers, making them a valuable therapeutic target. The pan-HDAC inhibitor (HDACi) panobinostat is an approved anticancer drug for patients with multiple myeloma. It has been shown that triple negative breast cancer (TNBC), as compared to other breast cancer (BC) subtypes, is more sensitive to panobinostat. Despite the positive data observed in experimental models, to date this HDACi (panobinostat) has not yielded promising results in clinical trials for most solid tumors, including BC. Thus, a better understanding of the molecular mechanisms underlying the action of panobinostat is required to design effective therapeutic strategies against TNBC. Methods: Cell growth MTS assays and LIVE/DEAD cell viability assays were used to determine cell viability. Co-immunoprecipitation (Co-IP) and Western blot were performed to assess the expression, interaction and activation of proteins. Quantitative real-time PCR (qRT-PCR) assays were carried out to measure the expression levels of mRNAs. Lentiviral vectors containing cDNA or specific shRNAs were used to overexpress or knockdown gene expression. Tumor xenograft models via subcutaneous inoculation of MDA-MB-231 cells into nude mice were established to test the in vivo antitumor activity of panobinostat alone or its combination with the EGFR inhibitor gefitinib. Results: Elevated expression of HDAC1/2/3 was observed in BC and significantly associated with poor prognosis in BC patients. In general, all TNBC cells were sensitive to panobinostat-induced growth inhibition and apoptosis. However, in the TNBC cells with HER3-low/undetectable expression, treatment with panobinostat induced upregulation of HER3, and increased the levels of phosphorylated HER3 (p-HER3) and Akt (p-Akt). Specific knockdown of HER3 via shRNAs or inhibition of HER3 by our monoclonal antibody (4A7) sensitized TNBC cells to panobinostat-induced growth inhibition and apoptosis. Further studies revealed an enhanced interaction between HER3 and EGFR due to panobinostat-induced upregulation of HER3. Combinations of panobinostat and the EGFR inhibitor gefitinib exhibited a synergistic effect inhibiting cell growth and promoting apoptosis in TNBC cells. Moreover, in the tumor xenograft models established with MDA-MB-231 cells, panobinostat in combination with gefitinib significantly inhibited tumor growth and induced apoptosis in vivo. Conclusion: While panobinostat exhibits potent anti-proliferative/anti-survival activity in TNBC cells, upregulating HER3 may be an adaptive response compromising the therapeutic potential of panobinostat. Simultaneous targeting of HER3 and its oncogenic dimerization partner, such as EGFR holds promise to combat the compensatory signaling-initiated by HER3. This new therapeutic strategy potentially improves the outcomes of TNBC patients. Citation Format: Hui Lyu, Sanbao Ruan, Congcong Tan, Bolin Liu. Attenuation of HER3-EGFR signaling augments antitumor activity of panobinostat in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3272.