Mitochondria are hubs of metabolism and signaling. We previously demonstrated the importance of mitochondrial structure and function in chemotherapy-refractory triple-negative breast cancer (TNBC). Herein, we present the first 3D analysis of mitochondrial networks in human tumor tissues. Using serial block face scanning electron microscopy, we reconstructed 3,750 mitochondria and 800 lipid droplets (LDs) in naive and residual tumors persisting after conventional chemotherapies in two orthotopic patient-derived xenografts (PDX). Chemotherapies administered as monotherapy or in combination produced residual tumors that harbored mitochondria with significantly increased areas, volumes, and perimeters. We observed substantial reduction of mitochondrial intratumor heterogeneity following all treatments. Further, mitochondrial complexity was significantly elevated after single-agent treatments in one model, but was reduced in the other PDX model. Mitochondria-LD significantly increased contacts in residual tumors, congruent with our previous studies providing evidence for rewiring of lipid metabolism in residual TNBC. These results highlight the potential for structure-based monitoring of chemotherapy-induced metabolic rewiring in TNBC.

Abstract Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype in which neoadjuvant chemotherapy (NACT) is the backbone of standard of care. Unfortunately, ∼45% of patients have residual tumor burden following NACT, which is strongly associated with poor prognoses. Our group previously demonstrated that mitochondrial oxidative phosphorylation is upregulated and is a therapeutic vulnerability of chemo-refractory TNBC. We used metabolomic flux tracing to show a heightened contribution of glucose oxidation to the tricarboxylic acid (TCA) cycle in residual human TNBC cells surviving several conventional chemotherapies. We found significantly elevated abundance of citrate and acetyl-coA (AcCoA) in residual cells. Further, glucose, but not palmitate, glutamine, or acetate, derived heavy carbon was more strongly incorporated into citrate and AcCoA in residual relative to naïve cells. Concomitantly, we observed drastic lipidomic remodeling, largely characterized by elevation of triglycerides, long chain fatty acids, and poly unsaturated fatty acids (PUFAs) in cultured TNBC cells and orthotopic patient-derived xenograft (PDX) tumors following chemotherapy relative to their treatment naïve counterparts. This was accompanied by a significant increase in the number of lipid droplets (LDs) in residual cells. Together, these data suggest glucose oxidation supports fatty acid synthesis (FAS) and storage in chemoresistant TNBC. Our analyses of human TNBC proteomic and transcriptomic data affirmed the significant association of fatty acid metabolism with TNBC chemoresistance, as well as its upregulation in chemotherapy-treated tumors relative to their pre-treated counterparts. Mining those data led us to ATP citrate lyase (ACLY), the rate limiting enzyme for cytosolic AcCoA production from citrate. We hypothesized that ACLY converts excess citrate, generated by heightened TCA cycling, to AcCoA to promote FAS and storage in chemoresistant TNBC. We found elevated protein levels of ACLY and an activating phosphorylation mark in TNBC cells surviving treatment with conventional chemotherapies doxorubicin and carboplatin. ACLY knockdown or inhibition potently reduced chemotherapy-induced accumulation of AcCoA and LDs and elicited lipidomic rewiring largely characterized by increased PUFAs. Notably, combining ACLY inhibition or KD with conventional chemotherapy treatments provided significant improvement of tumor cell growth inhibition. These data indicate that NACT can cause accumulation of citrate, AcCoA, LDs, and overall lipidomic rewiring through ACLY. ACLY is a novel functional dependency of chemo-refractory TNBC and should be further explored as a potential therapeutic target. We posit that TNBC cells adapt to the stress of NACT by upregulating lipid synthesis and storage in conjunction with glucose catabolism, enhancing metabolic flexibility and cell survival. Citation Format: Katherine E. Pendelton, Mokryun L. Baek, Mariah J. Berner-Wu, Steven W. Wall, Audra Lane, Jonathan T. Lei, Iqbal Mahmud, Lin Tan, Lacey E. Dobrolecki, Philip L. Lorenzi, Michael T. Lewis, Blake R. Rushing, Gloria V. Echeverria. ATP citrate lyase fuels lipid storage to support triple negative breast cancer chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2027.

BCL-xL inhibition does not change mitochondrial fusion and fission related proteins.

Abstract Nearly 45% of patients with triple negative breast cancer (TNBC) treated with chemo-immunotherapy have residual cancer burden, which is associated with relapse and mortality. Previously, our group reported that TNBCs rely heavily on mitochondrial structural and functional adaptations to survive treatment (PMIDs 30996079, 36813854). Additionally, mitochondrial translation-related proteomic profiles of TNBC patients (n=55, PMID 36001024) and orthotopic PDX tumors (n=42, PMID 39713418) reveal a significant association with resistance to carboplatin (CRB), docetaxel (DTX), or their combination. Furthermore, on-treatment biopsies were collected from a subset of patients, and mitochondrial translation proteomic signatures were significantly elevated relative to their matched pre-treatment counterparts. Leading-edge proteins in those pathways included 30 mitoribosome proteins and the accessory protein Oxidase (Cytochrome C) Assembly 1-Like (OXA1L). OXA1L plays two crucial roles in the mitochondria: 1) it promotes translation termination for the 13 mtDNA-encoded mitochondrial respiratory chain (MRC) proteins, and 2) it aids in inner mitochondrial membrane insertion of mtDNA- and nDNA-encoded MRC proteins. Therefore, we hypothesized that mitochondrial translation, supported by OXA1L, is critical for maintaining mitochondrial function and chemoresistance in TNBC. Knock-down (KD) of OXA1L in TNBC cells significantly reduced MRC protein levels, mitochondrial ‘respirasome’ supercomplex formation, and oxidative phosphorylation (oxphos). Notably, the characteristic elevation of oxphos in ‘residual’ cells surviving CRB treatment was abolished by the KD. Concomitantly, OXA1L KD cells exhibited significantly improved CRB sensitivity relative to control cells. These results underscore the significance of OXA1L in MRC assembly and function in TNBC, as well as its role in supporting chemoresistance. Though there are no existing inhibitors of OXA1L, we tested the translational potential of our findings by leveraging the mitochondria’s bacterial ancestry through repurposing FDA-approved antibiotics to inhibit mitochondrial translation (PMID 25625193). We demonstrated inhibition of mitochondrial translation, MRC protein production, respirasome formation, and oxphos with low-dose tigecycline (TIG) treatment in TNBC cells. While we observed minimal toxicity to TNBC cells from TIG as a single agent, we found a significant enhancement of sensitivity to conventional chemotherapies, both in vitro and in vivo, in a PDX. In summary, our data reveal the vital role of mitochondrial translation for the metabolic adaptation and survival of chemo-refractory TNBC. Moreover, these data provide evidence that disrupting MRC assembly by inhibiting mitochondrial translation may be a promising approach to overcome mitochondrial vulnerabilities in chemo-refractory TNBC. Citation Format: Mariah Joy Berner, Steven W. Wall, Mokryun L. Baek, Audra Lane, Allison S. Greer, Karen Wang, Lacey E. Dobrolecki, Bing Zhang, Jonathan T. Lei, Michael T. Lewis, Gloria Vittone Echeverria. Mitochondrial protein translation supports metabolic rewiring of chemo-refractory triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2013.

Triple negative breast cancer (TNBC) patients harboring residual cancer burden following completion of conventional neoadjuvant chemo-immunotherapy regimens have poor relapse-free and overall survival rates and limited therapeutic options. We and others have demonstrated that mitochondrial function is required for the survival of chemo-refractory TNBC. Here, we define the mitochondrial translation machinery as a critical and targetable dependency underlying chemo-refractory TNBC. Analyses of human and orthotopic patient-derived xenograft (PDX) mass spectrometry proteomics datasets revealed that mitochondrial protein translation-related signatures were among the top associated with chemoresistance. These signatures included core mitoribosome components and the mitoribosome-associated factor Oxidase (Cytochrome C) Assembly 1-Like (OXA1L), which was consistently enriched in chemoresistant versus chemosensitive TNBC across datasets. OXA1L, a key mediator of mitochondrial translation and electron transport chain (ETC) assembly, has not been functionally characterized in cancer. We therefore tested whether OXA1L-dependent mitochondrial translation sustains mitochondrial function and chemoresistance in TNBC. Knockdown (KD) of OXA1L in human TNBC cells reduced ETC protein levels, mitochondrial respirasome supercomplex levels, ATP production, and oxidative phosphorylation (oxphos), establishing a requirement for OXA1L in maintaining mitochondrial bioenergetics in TNBC. OXA1L was required for the characteristic oxphos elevation induced by carboplatin (CRB), and KD significantly enhanced CRB sensitivity, demonstrating that mitochondrial translation supports adaptive metabolic responses to chemotherapy. To explore the translational potential of targeting the mitoribosome in TNBC, we leveraged the bacterial ancestry of mitochondria to repurpose the FDA-approved antibiotic tigecycline (TIG) as a mitochondrial translation inhibitor. Direct measurement of mitochondrial nascent peptide levels revealed that, while CRB elevated mitochondrial translation, TIG potently suppressed mitochondrial translation as monotherapy and in combination with CRB or docetaxel (DTX). TIG abolished CRB-induced oxphos, decreased oxphos in combination with DTX, and significantly improved chemotherapy sensitivity in human TNBC cell lines, PDX-derived spheroids, and in vivo. TIG sensitivity associated with mitochondrial translation-related proteomic signatures, concordant with PDX and patient-derived signatures associated with chemoresistance. Together, these data identify OXA1L-dependent mitochondrial translation as a targetable dependency that sustains mitochondrial function and chemoresistance in TNBC, demonstrate that its inhibition enhances chemotherapeutic response, and nominate a mitochondrial translation-related protein signature as a candidate predictive biomarker of TIG sensitivity and chemoresistance. These findings support mitochondrial translation inhibition as a potential therapeutic strategy in chemo-refractory disease.

Synergistic cell death induction by MCL-1 inhibitor, AZD5991 and BCL-xL inhibitor, A1155463, in DM cell lines and PDC.

Advancements in transmission electron microscopy (TEM) have enabled in-depth studies of biological specimens, offering new avenues to large-scale imaging experiments with subcellular resolution. Mitochondrial morphology is of growing interest in cancer biology due to its crucial role in regulating the multi-faceted functions of mitochondria. We and others have established the crucial role of mitochondria in triple-negative breast cancer (TNBC), an aggressive subtype of breast cancer with limited therapeutic options. Building upon our previous work demonstrating the functional role of mitochondrial morphology dynamics in the metabolic adaptations and survival of chemotherapy-refractory TNBC cells, we sought to extend those findings to analysis of transmission electron micrographs. Here, we present a novel U-Net artificial intelligence (AI) model for automatic annotation and assessment of mitochondrial morphology and feature quantification. Our model was trained on 11,039 manually annotated mitochondria across 125 micrographs derived from a variety of orthotopic patient-derived xenograft (PDX) mouse model tumors and adherent cell cultures. The model achieves an F1 score of 0.85 on test micrographs at the pixel level. To validate the ability of our model to detect expected mitochondrial morphology changes, we utilized micrographs from mouse primary skeletal muscle cells genetically modified to lack Dynamin-related protein 1 (Drp1), a key mitochondrial fission protein. We subjected in vitro and in vivo TNBC models to conventional chemotherapy treatments commonly used for clinical management of TNBC, including doxorubicin, carboplatin, paclitaxel, and docetaxel. We found substantial within-sample heterogeneity of mitochondrial morphology in both in vitro and in vivo. In four of five PDX models, in vivo treatment with DTX elicited significant alteration in mitochondrial elongation and/or area. We went on to compare mammary tumors and matched lung metastases in a highly metastatic PDX model of TNBC, revealing altered mitochondrial elongation in metastatic lesions compared to their matched primary mammary tumor. The successful application of our AI model provides a framework for high-throughput quantitative analysis of mitochondrial morphology and enables future studies investigating how mitochondrial structural changes relate to chemotherapy response and mechanism of action. Our publicly available manually curated electron micrograph dataset serves as a unique resource for developing, benchmarking, and applying computational models, while further advancing investigations into mitochondrial morphology in breast cancer. This study provides proof of concept that mitochondrial structural remodeling is an additional layer of cellular reprogramming accompanying therapeutic resistance in TNBC that merits further investigation.

Characterization of DM patient derived cell lines (PDC) and in vitro growth features.

Co-targeting MCL-1 and BCL-xL synergistically increase cell death through the intrinsic mitochondrial apoptotic pathway in PDC.