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Sarah Ann Best



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    MA24 - Genomic Evolution, KEAP 3 and More Non-Coding RNA (ID 928)

    • Event: WCLC 2018
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 2
    • Now Available
    • Moderators:
    • Coordinates: 9/26/2018, 10:30 - 12:00, Room 205 BD
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      MA24.09 - Synergy Between the KEAP1/NRF2 and PI3K Pathways Drives Non-Small Cell Lung Cancer with an Altered Metabolism (Now Available) (ID 13734)

      11:30 - 11:35  |  Presenting Author(s): Sarah Ann Best

      • Abstract
      • Presentation
      • Slides

      Background

      The lung is a highly oxidative environment, tolerated through the engagement of tightly controlled stress response pathways. A critical stress response mediator is the transcription factor Nuclear Factor Erythroid-2-Related Factor 2 (NFE2L2/NRF2), which is negatively regulated by Kelch-like ECH-Associated Protein 1 (KEAP1). Alterations in the KEAP1/NRF2 pathway have been identified in 23% of lung adenocarcinomas, suggesting that deregulation of the pathway is a major driver in lung cancer.

      Method

      We generated a novel genetically engineered mouse model (GEMM) whereby Keap1 (Keap1f/f) and Pten (Ptenf/f) were conditionally deleted in the lung, utilising intranasal inhalation of Adenovirus-Cre. The effects on lung pathology were investigated using histopathology, metabolomics and flow cytometry.

      Result

      We found that, while loss of Keap1 alone displayed no abnormalities in the lung, loss of Keap1 combined with loss of the tumour suppressor Pten, promoted malignant transformation. We further monitored tumour progression and immune infiltration in the lung, and metabolite profile changes in the serum of the Keap1f/f/Ptenf/f mouse model. Notably, a tumour-specific metabolite signature was identified in the plasma of Keap1f/f/Ptenf/f tumour-bearing mice, which indicated that tumourigenesis is associated with metabolic reprogramming. Furthermore, the immune milieu was dramatically changed by Keap1 and Pten deletion, and tumour regression was achieved utilising immune checkpoint inhibition.

      Conclusion

      Our study highlights the ability to exploit both metabolic and immune characteristics in the detection and treatment of lung adenocarcinomas harbouring KEAP1/NRF2 pathway alterations.

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      MA24.10 - Interrogating the Metabolic Effects of Keap1 Inactivation in Adenocarcinoma (Now Available) (ID 13764)

      11:35 - 11:40  |  Presenting Author(s): Sarah Ann Best

      • Abstract
      • Presentation
      • Slides

      Background

      The most frequently altered gene in lung adenocarcinoma (ADC) is the KRAS oncogene. There are currently no effective treatments to target KRAS-mutant ADC. Loss of function in Kelch-like ECH-associated protein 1 (KEAP1) is co-mutated in 18% of KRAS-mutant ADC and is mutually exclusive with inactivating mutations in Tumour protein 53 (TP53). KEAP1 is a negative regulator of the transcription factor NRF2, which regulates cellular antioxidant and metabolic pathways. Metabolic dysregulation is considered a hallmark of cancer cells, which utilize anaerobic glycolysis and anabolic glucose metabolism in preference to aerobic oxidative phosphorylation in a phenomenon termed the Warburg effect.

      Method

      We interrogated the consequences of Keap1 loss in KrasG12D-induced ADC using conditional genetically engineered mouse models (GEMMs). To examine metabolic features unique to Keap1-mutant ADC, GEMMs with KrasLSL-G12D/+ alone or with either the p53flox/flox allele (Kras/p53) or Keap1flox/flox allele (Kras/Keap1) were investigated. Gene and protein expression of key enzymes involved in glucose metabolism were measured in spontaneous lung tumors. Glycolytic functional assays were performed in live FACS-isolated tumor cells using a novel protocol.

      Result

      Major alterations in glycolytic function were identified as unique features of Keap1 inactivation in lung adenocarcinomas carrying oncogenic activation of Kras. Loss of Keap1 function in Kras-mutant ADC therefore created a pro-oncogenic metabolic environment to drive lung tumourigenesis.

      Conclusion

      Targeting metabolic dependency in KRAS-mutant tumors may provide a unique method of treating this aggressive subset of lung ADC.

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    MTE01 - Preclinical Models of Lung Cancer (Ticketed Session) (ID 811)

    • Event: WCLC 2018
    • Type: Meet the Expert Session
    • Track: Biology
    • Presentations: 1
    • Now Available
    • Moderators:
    • Coordinates: 9/24/2018, 07:00 - 08:00, Room 206 F
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      MTE01.01 - GEMM of Lung Cancer (Now Available) (ID 11546)

      07:00 - 07:30  |  Presenting Author(s): Sarah Ann Best

      • Abstract
      • Presentation
      • Slides

      Abstract

      The primary induction of lung cancer is difficult to study in humans because patients often present very late in the course of their disease. Genetically engineered mouse models (GEMMs) have therefore emerged as crucial bridging strategies between understanding pathogenic mechanisms and clinical translation. Importantly, they reveal insights on the events and processes underlying tumor initiation and progression, studies which are not possible when employing transplantation or chemically-induced model systems.

      The recent advent of next-generation sequencing technologies has provided us with an in-depth characterization of the cancer genome of lung adenocarcinoma (LUAD) (1), squamous cell carcinoma (LUSC) (2) and small cell lung cancer (SCLC) (3). While these studies have highlighted the genetic complexities of lung cancers, attention is now focused on elucidating “driver” mutations that confer a growth advantage, from “passenger” mutations that have little impact on malignant transformation. Investigating the loss or gain-of-function of individual genes, alone or in combination, can be directly addressed using GEMM systems.

      The “gold-standard” lung cancer models are based on Cre-LoxP recombination technology that enable the formation of autochthonous tumors from a limited number of somatic cells in a spatial and temporal fashion. Critically, tumors arise sporadically within the lung, in the setting of an intact immune microenvironment. GEMMs are designed to harbor genetic mutations frequently identified in human lung cancer. Cre-inducible alleles are engineered to disrupt tumor suppressor genes (LoxP sites flanking key exons (floxed), that are removed upon recombination) and/or activate oncogenes (LoxP-flanked stop codons (lox-stop-lox) that result in gene expression upon recombination). Cre-recombinase is delivered to the lung via inhalation or intra-tracheal injection of a recombinant adenovirus (Ad5) expressing Cre-recombinase under the control of a ubiquitous cytomegalovirus (CMV) promoter. Expression of Cre-recombinase directs the recombination of floxed alleles in a variety of epithelial cell types in the adult mouse lung (4,5). Utilizing this approach enabled investigators to interrogate the functional consequences of genetic alterations found in human lung cancer through the generation of models of LUAD, SCLC and more recently lung LUSC (6). Moreover, the recent advent of CRISPR-Cas9 gene-editing technology now enables us to interrogate the functional interaction between multiple genetic alterations in a high-throughput setting (7). Furthermore, the generation of cell type specific Ad5-Cre viruses, that restrict Cre expression, and thus recombination, to alveolar type II (ATII) (Ad5-SPC-Cre), club (Ad5-CC10-Cre), neuroendocrine (Ad5-CGRP-Cre) and basal (Ad5-K5-Cre, Ad5-K14-Cre) (8) cells, have provided insights into the cellular origins of different subtypes of lung cancer (9,10). Critically, unlike patient-derived xenograft (PDX) models, one additional advantage of GEMMs is the ability to interrogate the interplay between tumor cells and immune cells present in the tumor microenvironment. Such studies are crucial given the success of immune checkpoint inhibitors in lung cancer patients.

      This presentation will outline lung cancer GEMMs commonly used in the field and how these models can be utilized to identify cancer initiating cells, understand the molecular pathways underlying tumorigenesis, the immune microenvironment of lung cancer, and importantly to identify vulnerabilities that can be exploited for the design of improved treatment modalities for patients.

      References

      1. The Cancer Genome Atlas Research Network, Comprehensive molecular profiling of lung adenocarcinoma (2014) Nature, 511 (7511) 543-550.

      2. The Cancer Genome Atlas Research Network, Comprehensive genomic characterization of squamous cell lung cancers (2012) Nature, 489 (7417) 519-525.

      3. George et al., Comprehensive genomic profiles of small cell lung cancer (2015) Nature, 524 (7563) 47-53.

      4. Best et al., Combining cell type-restricted adenoviral targeting with immunostaining and flow cytometry to identify cells-of-origin of lung cancer (2018) Methods in Molecular Biology, 1725 15-29.

      5. DuPage et al., Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase (2009) Nature Protocols, 4 (7) 1064-1072.

      6. Farago et al., SnapShot: Lung cancer models (2012) Cell, 149 (1) 246-246e1.

      7.Rogers et al., A quantitative and multiplexed approach to uncover the fitness landscape of tumor suppression in vivo (2017) Nature Methods, 14 (7) 737-742.

      8. Ferone et al., SOX2 is the determining oncogenic switch in promoting lung squamous cell carcinoma from different cells of origin (2016) Cancer Cell, 30 (4) 519-532.

      9. Sutherland et al., Cell of origin of small cell lung cancer: inactivation of Trp53 and Rb1 in distinct cell types of adult mouse lung (2011) Cancer Cell, 19 (6) 754-764.

      10. Sutherland et al., Multiple cells-of-origin of mutant K-Ras-induced mouse lung adenocarcinoma (2014) Proc. Natl. Acad. Sci. USA, 111 (13) 4952-2957.

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