Faculty Bibliography

The KEAP1-NRF2 system is a sulfur-employing defense mechanism against oxidative and electrophilic stress. NRF2 is a potent transcription activator for genes mediating sulfur-involving redox reactions, and KEAP1 controls the NRF2 activity in response to the stimuli by utilizing reactivity of sulfur atoms. In many human cancer cells, the KEAP1-mediated regulation of NRF2 activity is abrogated, resulting in the persistent activation of NRF2. Persistently activated NRF2 drives malignant progression of cancers by increasing therapeutic resistance and promoting aggressive tumorigenesis, a state termed as NRF2 addiction. In NRF2-addicted cancer cell, NRF2 contributes to metabolic reprogramming in cooperation with other oncogenic pathways. In particular, NRF2 strongly activates cystine uptake coupled with glutamate excretion and glutathione synthesis, which increases consumption of intracellular glutamate. Decreased availability of glutamate limits anaplerosis of the TCA cycle, resulting in low mitochondrial respiration, and nitrogen source, resulting in the high dependency on exogenous non-essential amino acids. The highly enhanced glutathione synthesis is also likely to alter sulfur metabolism, which can contribute to the maintenance of the mitochondrial membrane potential in normal cells. The potent antioxidant and detoxification capacity supported by abundant production of glutathione is achieved at the expense of central carbon metabolism and requires skewed metabolic flow of sulfur. These metabolic features of NRF2 addiction status provide clues for novel therapeutic strategies to target NRF2-addicted cancer cells.

The unregulated proliferative capacity of many tumors is dependent on dysfunctional nutrient utilization and ROS (reactive oxygen species) signaling to sustain a deranged metabolic state. Although it is clear that cancers broadly rely on these survival and signaling pathways, how they achieve these aims varies dramatically. Mutations in the KEAP1/NRF2 pathway represent a potent cancer adaptation to exploit native cytoprotective pathways that involve both nutrient metabolism and ROS regulation. Despite activating these advantageous processes, mutations within KEAP1/NRF2 are not universally selected for across cancers and instead appear to interact with particular tumor driver mutations and tissues of origin. Here, we highlight the relationship between the KEAP1/NRF2 signaling axis and tumor biology with a focus on genetic mutation, metabolism, immune regulation, and treatment implications and opportunities. Understanding the dysregulation of KEAP1 and NRF2 provides not only insight into a commonly mutated tumor suppressor pathway but also a window into the factors dictating the development and evolution of many cancers.
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Rewiring of metabolic pathways is a hallmark of tumorigenesis as cancer cells acquire novel nutrient dependencies to support oncogenic growth. A major genetic subtype of lung adenocarcinoma with KEAP1/NRF2 mutations, which activates the endogenous oxidative stress response, undergoes significant metabolic rewiring to support enhanced antioxidant production. We demonstrate that cancers with high antioxidant capacity exhibit a general dependency on exogenous non-essential amino acids (NEAAs) that is driven by the Nrf2-dependent secretion of glutamate through system x_c^- (XCT), which limits intracellular glutamate pools that are required for NEAA synthesis. This dependency can be therapeutically targeted by dietary restriction or enzymatic depletion of individual NEAAs. Importantly, limiting endogenous glutamate levels by glutaminase inhibition can sensitize tumors without alterations in the Keap1/Nrf2 pathway to dietary restriction of NEAAs. Our findings identify a metabolic strategy to therapeutically target cancers with genetic or pharmacologic activation of the Nrf2 antioxidant response pathway by restricting exogenous sources of NEAAs.

Despite substantial progress in lung cancer immunotherapy, the overall response rate in KRAS-mutant lung adenocarcinoma (ADC) patients remains low. Combining standard immunotherapy with adjuvant approaches that enhance adaptive immune responses-such as epigenetic modulation of anti-tumor immunity-is therefore an attractive strategy. To identify epigenetic regulators of tumor immunity, we constructed an epigenetic-focused sgRNA library, and performed an in vivo CRISPR screen in a KrasG12D/P53-/- (KP) lung ADC model. Our data showed that loss of the histone chaperone Asf1a in tumor cells sensitizes tumors to anti-PD-1 treatment. Mechanistic studies revealed that tumor cell-intrinsic Asf1a deficiency induced immunogenic macrophage differentiation in the tumor microenvironment by upregulating GM-CSF expression and potentiated T cell activation in combination with anti-PD-1. Our results provide rationale for a novel combination therapy consisting of ASF1A inhibition and anti-PD-1 immunotherapy.

Lung cancer remains one of the most genetically complex, aggressive, and lethal solid malignancies. Understanding how distinct lung cancer mutations give rise to altered nutrient requirements and promote immune evasion in the context of a heterogeneous lung tumor microenvironment is vital for the development of novel personalized therapeutic strategies.

Approximately 30% of human lung cancers acquire mutations in either Keap1 or Nfe2l2, resulting in the stabilization of Nrf2, the Nfe2l2 gene product, which controls oxidative homeostasis. Here, we show that heme triggers the degradation of Bach1, a pro-metastatic transcription factor, by promoting its interaction with the ubiquitin ligase Fbxo22. Nrf2 accumulation in lung cancers causes the stabilization of Bach1 by inducing Ho1, the enzyme catabolizing heme. In mouse models of lung cancers, loss of Keap1 or Fbxo22 induces metastasis in a Bach1-dependent manner. Pharmacological inhibition of Ho1 suppresses metastasis in a Fbxo22-dependent manner. Human metastatic lung cancer display high levels of Ho1 and Bach1. Bach1 transcriptional signature is associated with poor survival and metastasis in lung cancer patients. We propose that Nrf2 activates a metastatic program by inhibiting the heme- and Fbxo22-mediated degradation of Bach1, and that Ho1 inhibitors represent an effective therapeutic strategy to prevent lung cancer metastasis.

Thioredoxin reductase-1 (TrxR1)-, glutathione reductase (Gsr)-, and Nrf2 transcription factor-driven antioxidant systems form an integrated network that combats potentially carcinogenic oxidative damage yet also protects cancer cells from oxidative death. Here we show that although unchallenged wild-type (WT), TrxR1-null, or Gsr-null mouse livers exhibited similarly low DNA damage indices, these were 100-fold higher in unchallenged TrxR1/Gsr-double-null livers. Notwithstanding, spontaneous cancer rates remained surprisingly low in TrxR1/Gsr-null livers. All genotypes, including TrxR1/Gsr-null, were susceptible to N-diethylnitrosamine (DEN)-induced liver cancer, indicating that loss of these antioxidant systems did not prevent cancer cell survival. Interestingly, however, following DEN treatment, TrxR1-null livers developed threefold fewer tumors compared with WT livers. Disruption of TrxR1 in a marked subset of DEN-initiated cancer cells had no effect on their subsequent contributions to tumors, suggesting that TrxR1-disruption does not affect cancer progression under normal care, but does decrease the frequency of DEN-induced cancer initiation. Consistent with this idea, TrxR1-null livers showed altered basal and DEN-exposed metabolomic profiles compared with WT livers. To examine how oxidative stress influenced cancer progression, we compared DEN-induced cancer malignancy under chronically low oxidative stress (TrxR1-null, standard care) vs. elevated oxidative stress (TrxR1/Gsr-null livers, standard care or phenobarbital-exposed TrxR1-null livers). In both cases, elevated oxidative stress was correlated with significantly increased malignancy. Finally, although TrxR1-null and TrxR1/Gsr-null livers showed strong Nrf2 activity in noncancerous hepatocytes, there was no correlation between malignancy and Nrf2 expression within tumors across genotypes. We conclude that TrxR1, Gsr, Nrf2, and oxidative stress are major determinants of liver cancer but in a complex, context-dependent manner.

Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice. The CTC isolation and sorting technology presented here provides a research tool to help reveal details of how CTCs evolve over time, allowing studies to credential changes in CTCs as biomarkers of drug response and facilitating future studies to understand the role of CTCs in metastasis.

Pineoblastoma is a rare and highly aggressive brain cancer of childhood, histologically belonging to the spectrum of primitive neuroectodermal tumors. Patients with germline mutations in DICER1, a ribonuclease involved in microRNA processing, have increased risk of pineoblastoma, but genetic drivers of sporadic pineoblastoma remain unknown. Here, we analyzed pediatric and adult pineoblastoma samples (n = 23) using a combination of genome-wide DNA methylation profiling and whole-exome sequencing or whole-genome sequencing. Pediatric and adult pineoblastomas showed distinct methylation profiles, the latter clustering with lower-grade pineal tumors and normal pineal gland. Recurrent variants were found in genes involved in PKA- and NF-κB signaling, as well as in chromatin remodeling genes. We identified recurrent homozygous deletions of DROSHA, acting upstream of DICER1 in microRNA processing, and a novel microduplication involving chromosomal region 1q21 containing PDE4DIP (myomegalin), comprising the ancient DUF1220 protein domain. Expresion of PDE4DIP and DUF1220 proteins was present exclusively in pineoblastoma with PDE4DIP gain.