Trastuzumab emtansine (T-DM1) was the first and one of the most successful antibody–drug conjugates (ADC) approved for treating refractory HER2-positive breast cancer. Despite its initial clinical efficacy, resistance is unfortunately common, necessitating approaches to improve response. Here, we found that in sensitive cells, T-DM1 induced spindle assembly checkpoint (SAC)-dependent immunogenic cell death (ICD), an immune-priming form of cell death. The payload of T-DM1 mediated ICD by inducing eIF2α phosphorylation, surface exposure of calreticulin, ATP and HMGB1 release, and secretion of ICD-related cytokines, all of which were lost in resistance. Accordingly, ICD-related gene signatures in pretreatment samples correlated with clinical response to T-DM1–containing therapy, and increased infiltration of antitumor CD8+ T cells in posttreatment samples was correlated with better T-DM1 response. Transforming acidic coiled-coil containing 3 (TACC3) was overexpressed in T-DM1–resistant cells, and T-DM1 responsive patients had reduced TACC3 protein expression whereas nonresponders exhibited increased TACC3 expression during T-DM1 treatment. Notably, genetic or pharmacologic inhibition of TACC3 restored T-DM1–induced SAC activation and induction of ICD markers in vitro. Finally, TACC3 inhibition in vivo elicited ICD in a vaccination assay and potentiated the antitumor efficacy of T-DM1 by inducing dendritic cell maturation and enhancing intratumoral infiltration of cytotoxic T cells. Together, these results illustrate that ICD is a key mechanism of action of T-DM1 that is lost in resistance and that targeting TACC3 can restore T-DM1–mediated ICD and overcome resistance. Significance: Loss of induction of immunogenic cell death in response to T-DM1 leads to resistance that can be overcome by targeting TACC3, providing an attractive strategy to improve the efficacy of T-DM1.

Objective: AMP-activated-protein-kinase (AMPK) regulates cellular energy-homeostasis. Eukaryotic-elongation-factor-2-kinase (eEF2K) plays important roles in regulating protein-synthesis, functions similarly to AMPK to protect cancer-cells from metabolic stress. Tumor-hypoxia induces angiogenesis with activation of hypoxia-inducible-factor-1-alpha (HIF-1α). AMPK might be implicated both in regulation of protein-translation and response-to-hypoxia. We aimed to investigate the effects of AMPK modulation on regulation of protein translation and response-to-hypoxia by evaluating eEF2K and HIF-1α proteins in breast and hepatocellular cancers. Materials and methods: Hepatocellular (Huh-7, HepG2) and breast (SKBR-3, MDA-MB-453) cancer-cells were incubated with AMPK-activator (A769662) or inhibitor (dorsomorphin) for 8/24 h. Alterations in eEF2K/HIF-1α protein expressions were examined. Results: Cancer-cells slightly increased eEF2K expression after 24-h of AMPK-activation. Significant decreases in eEF2K expressions were observed with AMPK inhibition in all cancer-cells except Huh-7. Slight transient decrease in HIF-1α expression was observed after 8-h of AMPK-activation in all cancer-cells except MDA-MB-453. AMPK-inhibition decreased HIF-1α expression, especially in HepG2-cells. Conclusion: The effects of AMPK modulation on eEF2K/HIF-1α protein expressions were investigated. Cells with varying molecular-expression-profiles demonstrate different metabolic activities. AMPK-activation may provide adaptive advantage to cancers and such an advantage may be reverted with an AMPK-inhibitor. The current study contributes to the literature in determining the effects of therapeutic strategies targeting AMPK on cancer-cell metabolic-pathway regulation.