Faculty Bibliography

Simultaneous measurement of surface proteins and gene expression within single cells using oligo-conjugated antibodies offers high-resolution snapshots of complex cell populations. Signal from oligo-conjugated antibodies is quantified by high-throughput sequencing and is highly scalable and sensitive. We investigated the response of oligo-conjugated antibodies towards four variables: concentration, staining volume, cell number at staining, and tissue. We find that staining with recommended antibody concentrations causes unnecessarily high background and amount of antibody used can be drastically reduced without loss of biological information. Reducing staining volume only affects antibodies targeting abundant epitopes used at low concentrations and is counteracted by reducing cell numbers. Adjusting concentrations increases signal, lowers background, and reduces costs. Background signal can account for a major fraction of total sequencing and is primarily derived from antibodies used at high concentrations. This study provides new insight into titration response and background of oligo-conjugated antibodies and offers concrete guidelines to improve such panels.

Pancreatic ductal adenocarcinoma is a lethal disease characterized by late diagnosis, propensity for early metastasis and resistance to chemotherapy. Little is known about the mechanisms that drive innate therapeutic resistance in pancreatic cancer. The ataxia-telangiectasia group D-associated gene (ATDC) is overexpressed in pancreatic cancer and promotes tumor growth and metastasis. Our study reveals that increased ATDC levels protect cancer cells from reactive oxygen species (ROS) via stabilization of nuclear factor erythroid 2-related factor 2 (NRF2). Mechanistically, ATDC binds to Kelch-like ECH-associated protein 1 (KEAP1), the principal regulator of NRF2 degradation, and thereby prevents degradation of NRF2 resulting in activation of a NRF2-dependent transcriptional program, reduced intracellular ROS and enhanced chemoresistance. Our findings define a novel role of ATDC in regulating redox balance and chemotherapeutic resistance by modulating NRF2 activity.

In lung cancer, enrichment of the lower airway microbiota with oral commensals commonly occurs and ex vivo models support that some of these bacteria can trigger host transcriptomic signatures associated with carcinogenesis. Here, we show that this lower airway dysbiotic signature was more prevalent in group IIIB-IV TNM stage lung cancer and is associated with poor prognosis, as shown by decreased survival among subjects with early stage disease (I-IIIA) and worse tumor progression as measured by RECIST scores among subjects with IIIB-IV stage disease. In addition, this lower airway microbiota signature was associated with upregulation of IL-17, PI3K, MAPK and ERK pathways in airway transcriptome, and we identified Veillonella parvula as the most abundant taxon driving this association. In a KP lung cancer model, lower airway dysbiosis with V. parvula led to decreased survival, increased tumor burden, IL-17 inflammatory phenotype and activation of checkpoint inhibitor markers.

Takahashi et al. (2020) conduct a focused CRISPR/Cas9 screen against NRF2 target and other redox regulatory genes in both 2D- and 3D-culture systems, uncovering a vulnerability of spheroid cancer cells deprived of extracellular matrix to undergo ferroptosis.

Targeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.

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.