Glutamine antagonist DRP-104 suppresses tumor growth and enhances response to checkpoint blockade in KEAP1 mutant lung cancer.

Loss-of-function mutations in KEAP1 frequently occur in lung cancer and are associated with poor prognosis and resistance to standard of care treatment, highlighting the need for the development of targeted therapies. We previously showed that KEAP1 mutant tumors consume glutamine to support the metabolic rewiring associated with NRF2-dependent antioxidant production. Here, using preclinical patient-derived xenograft models and antigenic orthotopic lung cancer models, we show that the glutamine antagonist prodrug DRP-104 impairs the growth of KEAP1 mutant tumors. We find that DRP-104 suppresses KEAP1 mutant tumors by inhibiting glutamine-dependent nucleotide synthesis and promoting antitumor T cell responses. Using multimodal single-cell sequencing and ex vivo functional assays, we demonstrate that DRP-104 reverses T cell exhaustion, decreases Tregs, and enhances the function of CD4 and CD8 T cells, culminating in an improved response to anti-PD1 therapy. Our preclinical findings provide compelling evidence that DRP-104, currently in clinical trials, offers a promising therapeutic approach for treating patients with KEAP1 mutant lung cancer.

KEAP1 mutation in lung adenocarcinoma promotes immune evasion and immunotherapy resistance.

Lung cancer treatment has benefited greatly through advancements in immunotherapies. However, immunotherapy often fails in patients with specific mutations like KEAP1, which are frequently found in lung adenocarcinoma. We established an antigenic lung cancer model and used it to explore how Keap1 mutations remodel the tumor immune microenvironment. Using single-cell technology and depletion studies, we demonstrate that Keap1-mutant tumors diminish dendritic cell and T cell responses driving immunotherapy resistance. This observation was corroborated in patient samples. CRISPR-Cas9-mediated gene targeting revealed that hyperactivation of the NRF2 antioxidant pathway is responsible for diminished immune responses in Keap1-mutant tumors. Importantly, we demonstrate that combining glutaminase inhibition with immune checkpoint blockade can reverse immunosuppression, making Keap1-mutant tumors susceptible to immunotherapy. Our study provides new insight into the role of KEAP1 mutations in immune evasion, paving the way for novel immune-based therapeutic strategies for KEAP1-mutant cancers.

Tumor-intrinsic LKB1-LIF signaling axis establishes a myeloid niche to promote immune evasion and tumor growth.

Tumor mutations can influence the surrounding microenvironment leading to suppression of anti-tumor immune responses and thereby contributing to tumor progression and failure of cancer therapies. Here we use genetically engineered lung cancer mouse models and patient samples to dissect how LKB1 mutations accelerate tumor growth by reshaping the immune microenvironment. Comprehensive immune profiling of LKB1 -mutant vs wildtype tumors revealed dramatic changes in myeloid cells, specifically enrichment of Arg1 + interstitial macrophages and SiglecF Hi neutrophils. We discovered a novel mechanism whereby autocrine LIF signaling in Lkb1 -mutant tumors drives tumorigenesis by reprogramming myeloid cells in the immune microenvironment. Inhibiting LIF signaling in Lkb1 -mutant tumors, via gene targeting or with a neutralizing antibody, resulted in a striking reduction in Arg1 + interstitial macrophages and SiglecF Hi neutrophils, expansion of antigen specific T cells, and inhibition of tumor progression. Thus, targeting LIF signaling provides a new therapeutic approach to reverse the immunosuppressive microenvironment of LKB1 -mutant tumors.

Glutamine antagonist DRP-104 suppresses tumor growth and enhances response to checkpoint blockade in KEAP1 mutant lung cancer.

Loss-of-function mutations in KEAP1 frequently occur in lung cancer and are associated with resistance to standard of care treatment, highlighting the need for the development of targeted therapies. We have previously shown that KEAP1 mutant tumors have increased glutamine consumption to support the metabolic rewiring associated with NRF2 activation. Here, using patient-derived xenograft models and antigenic orthotopic lung cancer models, we show that the novel glutamine antagonist DRP-104 impairs the growth of KEAP1 mutant tumors. We find that DRP-104 suppresses KEAP1 mutant tumor growth by inhibiting glutamine-dependent nucleotide synthesis and promoting anti-tumor CD4 and CD8 T cell responses. Using multimodal single-cell sequencing and ex vivo functional assays, we discover that DRP-104 reverses T cell exhaustion and enhances the function of CD4 and CD8 T cells culminating in an improved response to anti-PD1 therapy. Our pre-clinical findings provide compelling evidence that DRP-104, currently in phase 1 clinical trials, offers a promising therapeutic approach for treating patients with KEAP1 mutant lung cancer. Furthermore, we demonstrate that by combining DRP-104 with checkpoint inhibition, we can achieve suppression of tumor intrinsic metabolism and augmentation of anti-tumor T cell responses.

EMSY inhibits homologous recombination repair and the interferon response, promoting lung cancer immune evasion.

Non-small cell lung cancers (NSCLCs) harboring KEAP1 mutations are often resistant to immunotherapy. Here, we show that KEAP1 targets EMSY for ubiquitin-mediated degradation to regulate homologous recombination repair (HRR) and anti-tumor immunity. Loss of KEAP1 in NSCLC induces stabilization of EMSY, producing a BRCAness phenotype, i.e., HRR defects and sensitivity to PARP inhibitors. Defective HRR contributes to a high tumor mutational burden that, in turn, is expected to prompt an innate immune response. Notably, EMSY accumulation suppresses the type I interferon response and impairs innate immune signaling, fostering cancer immune evasion. Activation of the type I interferon response in the tumor microenvironment using a STING agonist results in the engagement of innate and adaptive immune signaling and impairs the growth of KEAP1-mutant tumors. Our results suggest that targeting PARP and STING pathways, individually or in combination, represents a therapeutic strategy in NSCLC patients harboring alterations in KEAP1.

Activation of the NRF2 antioxidant program sensitizes tumors to G6PD inhibition.

The KEAP1/NRF2 pathway promotes metabolic rewiring to support redox homeostasis. Activation of NRF2 occurs in many cancers, often due to KEAP1 mutations, and is associated with more aggressive disease and treatment resistance. To identify metabolic dependencies in cancers with NRF2 activation, we performed a metabolism-focused CRISPR screen. Glucose-6-phosphate dehydrogenase (G6PD), which was recently shown to be dispensable in Ras-driven tumors, was a top dependency. G6PD catalyzes the committed step of the oxidative pentose phosphate pathway that produces NADPH and nucleotide precursors, but neither antioxidants nor nucleosides rescued. Instead, G6PD loss triggered tricarboxylic acid (TCA) intermediate depletion because of up-regulation of the alternative NADPH-producing enzymes malic enzyme and isocitrate dehydrogenase. In vivo, G6PD impairment markedly suppressed KEAP1 mutant tumor growth, and this suppression was further augmented by TCA depletion by glutaminase inhibition. Thus, G6PD inhibition­induced TCA depletion is a therapeutic vulnerability of NRF2-activated cancer.

The Center Cannot Hold: NRF2 Battles Ferroptosis in the 3rd Dimension.

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.

Activation of Oxidative Stress Response in Cancer Generates a Druggable Dependency on Exogenous Non-essential Amino Acids.

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 xc- (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.

Activation of the NRF2 antioxidant program generates an imbalance in central carbon metabolism in cancer.

During tumorigenesis, the high metabolic demand of cancer cells results in increased production of reactive oxygen species. To maintain oxidative homeostasis, tumor cells increase their antioxidant production through hyperactivation of the NRF2 pathway, which promotes tumor cell growth. Despite the extensive characterization of NRF2-driven metabolic rewiring, little is known about the metabolic liabilities generated by this reprogramming. Here, we show that activation of NRF2, in either mouse or human cancer cells, leads to increased dependency on exogenous glutamine through increased consumption of glutamate for glutathione synthesis and glutamate secretion by xc- antiporter system. Together, this limits glutamate availability for the tricarboxylic acid cycle and other biosynthetic reactions creating a metabolic bottleneck. Cancers with genetic or pharmacological activation of the NRF2 antioxidant pathway have a metabolic imbalance between supporting increased antioxidant capacity over central carbon metabolism, which can be therapeutically exploited.

Imprinted DNA methylation reconstituted at a non-imprinted locus.

BACKGROUND: In mammals, tight regulation of cytosine methylation is required for embryonic development and cellular differentiation. The trans-acting DNA methyltransferases that catalyze this modification have been identified and characterized; however, these proteins lack sequence specificity, leaving the mechanism of targeting unknown. A cis-acting regulator within the Rasgrf1 imprinting control region (ICR) is necessary for establishment and maintenance of local imprinted methylation. Here, we investigate whether 3-kb of sequence from the Rasgrf1 ICR is sufficient to direct appropriate imprinted methylation and target gene expression patterns when ectopically inserted at the Wnt1 locus. RESULTS: The Rasgrf1 ICR at Wnt1 lacked somatic methylation when maternally transmitted and was fully methylated upon paternal transmission, consistent with its behavior at the Rasgrf1 locus. It was unmethylated in the female germline and was enriched for methylation in the male germline, though not to the levels seen at the endogenous Rasgrf1 allele. Wnt1 expression was not imprinted by the ectopic ICR, likely due to additional sequences being required for this function. CONCLUSIONS: We have identified sequences that are sufficient for partial establishment and full maintenance of the imprinted DNA methylation patterns. Because full somatic methylation can occur without full gametic methylation, we infer that somatic methylation of the Rasgrf1 ICR is not simply a consequence of maintained gametic methylation.

Slug contributes to cancer progression by direct regulation of ERα signaling pathway.

Hormone therapy targeting estrogen receptor α (ERα) is the most effective treatment for breast cancer. However, this treatment eventually fails as the tumor develops resistance. Although reduced expression of ER-α is a known contributing factor to endocrine resistance, the mechanism of ER-α downregulation in endocrine resistance is still not fully understood. The present study shows that Slug has an inverse relationship with ERα in breast and prostate cancer patient samples. Also the inhibition of Slug blocks mammary stem cell activity in primary mammary epithelial cells. We hypothesize that Slug may be a key transcription factor in the regulation of ERα expression. To understand the Slug-ERα signaling pathway, we employed resistant cell line MCF-TAMR (ERα relatively negative) derived from its parental MCF-7 (ERα positive) cell line and assessed changes in cell phenotype, activity and response to therapy. Conversely, we performed knockdown of Slug in the high-Slug expressing cell line MDA-MB-231 and assessed reversal of the mesenchymal phenotype. Microarray analysis showed that Slug is overexpressed in high grade breast and prostate cancer tissues. Additionally, Slug overexpression leads to drug resistance. Furthermore, we demonstrated that Slug binds directly to ERα promoter E-boxes and represses ERα expression. This resulted in decrease in epithelial-to-mesenchymal transition in cancer cells. These findings demonstrate that Slug, by regulation of ERα expression, contributes to tumor progression and could serve as an important target for cancer therapy.