Douglas B. Fox

Summary: The survival of residual tumor cells following therapy and their eventual recurrence constitutes one of the biggest obstacles to obtaining cures in breast cancer, but it remains unclear how the clonal composition of tumors changes during tumor relapse. We used cellular barcoding to directly monitor clonal dynamics during tumor recurrence in a genetically engineered mouse model. We found that the clonal diversity of tumors progressively decreased during tumor regression, residual disease, and recurrence. Only a fraction of subclones survived oncogene withdrawal and persisted in residual tumors. The minimal residual disease phase itself was accompanied by a continued attrition of clones, suggesting an ongoing process of selection during dormancy. The reactivation of dormant residual cells into recurrent tumors followed several distinct evolutionary routes. Approximately half of the recurrent tumors exhibited a striking clonal dominance in which one or two subclones comprised the vast majority of the tumor. The majority of these clonal recurrent tumors exhibited evidence of de novo acquisition of Met amplification, and were sensitive to small-molecule Met inhibitors. A second group of recurrent tumors exhibited marked polyclonality, with thousands of subclones and a clonal architecture very similar to primary tumors. These polyclonal recurrent tumors were not sensitive to Met inhibitors, but were instead dependent upon an autocrine IL-6 – Stat3 pathway. These results suggest that the survival and reactivation of dormant tumors can proceed via multiple independent routes, producing recurrent tumors with distinct clonal composition, genetic alterations, and drug sensitivities.

Summary: Oncogenic signaling pathways both directly and indirectly regulate anabolic metabolism, and this is required for tumor growth. Targeted therapies that inhibit oncogenic signaling have dramatic impacts on cellular metabolism. However, it is not known whether the acquisition of resistance to these therapies is associated with – or driven by – alterations in cellular metabolism. To address this, we used a conditional mouse model of Her2-driven breast cancer to study metabolic adaptations following Her2 inhibition, during residual disease, and after tumor recurrence. We found that Her2 downregulation caused widespread changes in cellular metabolism, culminating in oxidative stress. Tumor cells adapted to this metabolic stress by upregulation of the antioxidant transcription factor, NRF2. Constitutive NRF2 expression persisted during residual disease and tumor recurrence, and NRF2 was both sufficient to promote tumor recurrence, and necessary for recurrent tumor growth. These results are supported by clinical data showing that the NRF2 transcriptional program is activated in recurrent breast tumors, and that NRF2 is associated with poor prognosis in patients with breast cancer. Mechanistically, NRF2 signaling in recurrent tumors induced metabolic reprogramming to re-establish redox homeostasis and upregulate de novo nucleotide synthesis. Finally, this NRF2-driven metabolic state rendered recurrent tumor cells sensitive to glutaminase inhibition, suggesting that NRF2-high recurrent tumors can be therapeutically targeted. Together, these data provide evidence that NRF2-driven metabolic reprogramming is required for breast cancer recurrence following oncogene inhibition. Significance: Although tumor recurrence is the leading cause of mortality in breast cancer, the cellular properties that allow tumor cells to evade therapy and form recurrent tumors remain largely uncharacterized. Similarly, very little is known about how tumor metabolism changes following therapy, or whether alterations in cellular metabolism drive tumor recurrence. In this study, we identify the antioxidant transcription factor NRF2 as a critical positive regulator of breast cancer recurrence. We find that NRF2-dependent metabolic reprogramming is both sufficient and required to promote tumor recurrence. Additionally, we demonstrate that the NRF2-driven metabolic state renders recurrent tumors sensitive to glutaminase inhibitors, suggesting a novel therapeutic approach for the treatment of recurrent breast cancer.