Edward Gabrielson

BACKGROUND: Nuclear factor erythroid-2 related factor 2 (NRF2) is a redox-sensitive transcription factor that positively regulates the expression of genes encoding antioxidants, xenobiotic detoxification enzymes, and drug efflux pumps, and confers cytoprotection against oxidative stress and xenobiotics in normal cells. Kelch-like ECH-associated protein 1 (KEAP1) negatively regulates NRF2 activity by targeting it to proteasomal degradation. Increased expression of cellular antioxidants and xenobiotic detoxification enzymes has been implicated in resistance of tumor cells against chemotherapeutic drugs. METHODS AND FINDINGS: Here we report a systematic analysis of the KEAP1 genomic locus in lung cancer patients and cell lines that revealed deletion, insertion, and missense mutations in functionally important domains of KEAP1 and a very high percentage of loss of heterozygosity at 19p13.2, suggesting that biallelic inactivation of KEAP1 in lung cancer is a common event. Sequencing of KEAP1 in 12 cell lines and 54 non-small-cell lung cancer (NSCLC) samples revealed somatic mutations in KEAP1 in a total of six cell lines and ten tumors at a frequency of 50% and 19%, respectively. All the mutations were within highly conserved amino acid residues located in the Kelch or intervening region domain of the KEAP1 protein, suggesting that these mutations would likely abolish KEAP1 repressor activity. Evaluation of loss of heterozygosity at 19p13.2 revealed allelic losses in 61% of the NSCLC cell lines and 41% of the tumor samples. Decreased KEAP1 activity in cancer cells induced greater nuclear accumulation of NRF2, causing enhanced transcriptional induction of antioxidants, xenobiotic metabolism enzymes, and drug efflux pumps. CONCLUSIONS: This is the first study to our knowledge to demonstrate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC. Loss of KEAP1 function leading to constitutive activation of NRF2-mediated gene expression in cancer suggests that tumor cells manipulate the NRF2 pathway for their survival against chemotherapeutic agents.

The p16(INK4a) (p16) tumor suppressor gene can be inactivated by promoter region hypermethylation in many tumor types including lung cancer, the leading cause of cancer-related deaths in the U.S. We have determined the timing of this event in an animal model of lung carcinogenesis and in human squamous cell carcinomas (SCCs). In the rat, 94% of adenocarcinomas induced by the tobacco specific carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone were hypermethylated at the p16 gene promoter; most important, this methylation change was frequently detected in precursor lesions to the tumors: adenomas, and hyperplastic lesions. The timing for p16 methylation was recapitulated in human SCCs where the p16 gene was coordinately methylated in 75% of carcinoma in situ lesions adjacent to SCCs harboring this change. Moreover, the frequency of this event increased during disease progression from basal cell hyperplasia (17%) to squamous metaplasia (24%) to carcinoma in situ (50%) lesions. Methylation of p16 was associated with loss of expression in both tumors and precursor lesions indicating that both alleles were functionally inactivated. The potential of using assays for aberrant p16 methylation to identify disease and/or risk was validated by detection of this change in sputum from three of seven patients with cancer and 5 of 26 cancer-free individuals at high risk. These studies show for the first time that an epigenetic alteration, aberrant methylation of the p16 gene, can be an early event in lung cancer and may constitute a new biomarker for early detection and monitoring of prevention trials.

Abstract Immune checkpoint inhibitors have shown significant therapeutic responses against tumors containing increased mutation-associated neoantigen load. We have examined the evolving landscape of tumor neoantigens during the emergence of acquired resistance in patients with non–small cell lung cancer after initial response to immune checkpoint blockade with anti–PD-1 or anti–PD-1/anti–CTLA-4 antibodies. Analyses of matched pretreatment and resistant tumors identified genomic changes resulting in loss of 7 to 18 putative mutation-associated neoantigens in resistant clones. Peptides generated from the eliminated neoantigens elicited clonal T-cell expansion in autologous T-cell cultures, suggesting that they generated functional immune responses. Neoantigen loss occurred through elimination of tumor subclones or through deletion of chromosomal regions containing truncal alterations, and was associated with changes in T-cell receptor clonality. These analyses provide insight into the dynamics of mutational landscapes during immune checkpoint blockade and have implications for the development of immune therapies that target tumor neoantigens. Significance: Acquired resistance to immune checkpoint therapy is being recognized more commonly. This work demonstrates for the first time that acquired resistance to immune checkpoint blockade can arise in association with the evolving landscape of mutations, some of which encode tumor neoantigens recognizable by T cells. These observations imply that widening the breadth of neoantigen reactivity may mitigate the development of acquired resistance. Cancer Discov; 7(3); 264–76. ©2017 AACR. See related commentary by Yang, p. 250. This article is highlighted in the In This Issue feature, p. 235