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Mitochondria are morphologically plastic organelles that cycle between fission and fusion to maintain mitochondrial integrity and metabolic homeostasis. We found that DNA-damaging agents increased mitochondrial fusion, mitochondrial content, flux of glucose through the TCA cycle, and OXPHOS, whereas taxanes instead decreased mitochondrial fusion and OXPHOS. These events are mediated by the mitochondrial inner membrane fusion protein optic atrophy 1 (OPA1). Using TNBC cell lines and an in vivo PDX model of residual TNBC, we found that sequential treatment with DNA damaging chemotherapy, thus inducing mitochondrial fusion and OXPHOS, followed by MYLS22, a specific inhibitor of OPA1, was able to suppress mitochondrial fusion and OXPHOS and significantly inhibited residual tumor regrowth. Our findings suggest that TNBC mitochondria can optimize OXPHOS through modulation of mitochondrial structure. This may provide an opportunity to therapeutically target mitochondrial adaptations of chemoresistant TNBC.

We are using mass-spectrometry metabolomics and proteomics do elucidate the molecular evolution of TNBC cells that are not killed by some of the most common clinical chemotherapeutic regimens. We are using several PDX models for these experiments. Computational analyses of metabolites, proteins, and pathways will reveal novel vulnerabilities that arise in chemoresistant cell populations, allowing us new opportunities for mechanistic studies and therapeutic testing.

Cells can metabolize fatty acids via fatty acid oxidation in the mitochondria, generating acetyl-CoA which is necessary to drive the TCA and OXPHOS. We are studying the effects of chemotherapy on inducing heightened fatty acid metabolism in chemoresistant TNBC. We have observed that DNA-damaging agents chemotherapies induce greater accumulation of lipid droplets in TNBC cells (top panel) and PDXs (bottom panel) compared to vehicle. We are investigating this question by genetically perturbing fatty acid genes, generating high resolution microscopy images, utilizing pharmacologic agents, and assessing mitochondrial function in cells treated with chemotherapy. Our goal is to identify a therapeutic vulnerability within fatty acid synthesis or oxidation that can be leveraged to treat residual TNBC.

Our lab’s prior studies demonstrate that chemoresistant tumors have increased mitochondria metabolism. Key oxphos components are encoded in the mitochondrial genome (mtDNA) and thus require the mitochondrial ribosome for their production. We have identified mitochondrial translation and electron transport chain (ETC) assembly as potential mediators of metabolic adaptations in chemoresistant TNBCs. We are investigating the potential to therapeutically target mito-ribosome activity using conventional antibiotics to overcome chemoresistance in TNBC. We aim to elucidate the roles of these pathways in the metabolic adaptations in TNBC cell lines and PDX models resistant to conventional chemotherapy.

Many enzymes, namely metabolic enzymes, carry out oxidation-reduction (redox) reactions using high-energy electron carriers such as flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), and nicotinamide adenine dinucleotide phosphate (NADP). One of the hallmarks of cancer and a major driver of chemotherapy resistance is metabolic rewiring. Yet the therapeutic potential of modulating high-energy electron carriers has not been thoroughly investigated. Our previous work established TNBC cells that survive DNA-damaging chemotherapy (e.g., doxorubicin and carboplatin) are dependent on oxidative phosphorylation (oxphos). Given NADH is the primary electron donor for the electron transport chain (ETC) we are investigating the utility of perturbing the oxidation state of NAD to enhance chemotherapy sensitivity. Moreover, NAD-dependent enzymes such as PARP and the Sirtuin proteins may contribute to chemotherapy resistance by a variety of mechanisms. Thus, this approach is also potentially viable for re-sensitizing TNBC cells to non-DNA-damaging chemotherapy (e.g., taxanes).

Extensive fibrotic desmoplasia is observed in residual TNBC tumors following chemotherapy treatment. As shown on the left, PDX residual tumors have infiltration of fibroblasts and deposition of various ECM components that is reverted as tumors relapse. Our goal is to elucidate the functional contributions of tumor cell-ECM interactions to survival and relapse of post-chemotherapy residual disease. Furthermore, we are investigating mechanisms of metabolic crosstalk between tumor and stroma cell populations. We are answering these questions using multi-spectral quantitative pathology imaging, tumor-stroma co-culture organoid models, genetic, and pharmacologic tools in PDX models and TNBC cell lines. Based on expression of cell-surface antigens, we are investigating the anti-tumor activity of chimeric antigen receptor (CAR)- T cells against treatment-naive and chemoresistant TNBC cells.

Using metabolomics, we found that increased OXPHOS in residual TNBCs surviving conventional chemotherapy treatment is accompanied by elevated levels of mitochondrial TCA cycle intermediates. To deduce the mechanism driving increased OXPHOS in residual TNBCs, we are investigating specific proteins involved in regulating TCA cycle intermediates with a particular focus on enzymes crucial for pyruvate production- the pyruvate dehydrogenase complex and malic enzyme 2. We aim to determine whether these proteins 1) play a role in the increased levels of TCA cycle intermediates and OXPHOS, as well as 2) play a functional role in TNBC chemoresistance.

We have observed that residual TNBC cells after chemotherapy treatment have different metabolic and signaling features compared to treatment naïve cells. Our goal is to complement these findings by generating TNBC cell lines, by long-term exposure to increasing concentrations of conventional chemotherapeutics, that are stably resistant to chemotherapies. We hypothesize these resistant lines will display distinguishing characteristics from each other and treatment naïve cells. Further, we are generating these for the four most commonly used chemotherapeutics in TNBC, providing us with the opportunity to discern their unique molecular impacts. These characteristics will help us understand the mechanisms behind chemotherapeutic resistance in TNBC and elucidate molecular targets for these cells.

We are conducting functional genomics screens using pooled CRISPR/Cas9 screening libraries targeting candidate metabolic regulators. Screening libraries will be used in vitro using TNBC cell lines and PDX-derived organoid cultures and ultimate in vivo to conduct screens in mice bearing orthotopic PDX tumors. We are complementing this approach with barcode-mediated lineage tracking of primary and metastatic tumors throughout development of therapeutic resistance to delineate the clonal architecture of TNBC.