Abstract 2324: FRA1 contributes to ERK-mediated increased PD-L1 expression in KRAS mutated premalignant human bronchial epithelial cells
Authors: Lee Mi-Heon , Yanagawa Jane , Walser Tonya C , Goldman Jonathan W. , Garon Edward B. , Zeng Gang , Sharma Sherven , Gao Boning , Minna John , Dubinett Steven M. , Lee Jay M 
Published: July 2016

BACKGROUND: Activating KRAS mutations are common driver mutations in non-small cell lung carcinoma. Patients with mutated KRAS demonstrate less benefit from adjuvant chemotherapy and resistance to tyrosine kinase inhibitors. KRAS mutations are known to activate the RAF-MEK-ERK pathway. Fos-related antigen-1 (FRA1) is a MEK/ERK-dependent transcription factor and member of the AP-1 transcription factor superfamily. Immune checkpoint pathways including the PD-1/PD-L1 pathway are involved in immune-mediated tumor evasion. We hypothesize that KRAS mutation directly regulates the PD-1/PD-L1 pathway through ERK activation and FRA1 may be an important transcriptional mediator.

METHODS: In order to assess the role of KRAS mutation independent of other somatic mutations and to begin to understand potential immune suppressive pathways operative in pulmonary premalignancy, premalignant human bronchial epithelial cell lines (HBEC) were used instead of human lung cancer cell lines. Four immortalized HBEC (2, 3, 7, and 11 cell lines) with KRAS v12-mutation (HBEC-KRAS) compared to control (HBEC-vector) were used to assess mRNA and protein expression levels of PD-L1 by RT-qPCR, flow cytometry, and western blot. Cell-lines were treated with MEK (ERK kinase) inhibitor (PD0325901) for 24 hours (h) up to 1μM. FRA1 was silenced by transfection with siRNA at 100nM.

RESULTS: In comparing HBEC-KRAS to HBEC-vector (wild-type) cells, PD-L1 mRNA (1.3∼3.4 fold) and surface protein expressions (1.2∼2.3 fold by flow cytometry) were increased in all 4 cell lines and confirmed by western blot analyses. PD-L1 mRNA and protein levels were highest in HBEC3-KRAS. MEK inhibition resulted in decreased PD-L1 expression by 10 fold (HBEC3-vector) and 11 fold (HBEC3-KRAS) in mRNA levels and 3 fold (HBEC3-vector and HBEC3-KRAS) in surface protein levels. In comparing HBEC3-KRAS to HBEC3-vector, FRA1 increased mRNA (2.7 fold) and protein level expression (2.8 fold). In comparing HBEC3-KRAS to HBEC3-vector cells, FRA1 silencing (FRA1 siRNA) resulted in reduced PD-L1 (53% at 48 h; 73% at 72 h) and FRA1 (94% at 48 h; 99% at 72 h) by western blot. In comparing HBEC3-KRAS to HBEC3-vector cells, ERK phosphorylation increased 7.9 fold and FRA1 silencing led to inhibition of ERK phosphorylation by 2.8 fold at 72 h by western blot.

CONCLUSIONS: Here, we demonstrate that PD-L1 expression is elevated in premalignant KRAS mutated human bronchial epithelial cells, and ERK activation mediates KRAS mutation driven up regulation of PD-L1 and at least in part through FRA1 dependence. Our data suggest that KRAS mutation may directly regulate the PD-1/PD-L1 immune checkpoint pathway through FRA1 and MEK/ERK-dependent transcriptional regulation. Further understanding of KRAS driven molecular pathways that modulate immune checkpoints may elucidate therapeutic targets for potential new combination immune therapies.

Citation Format: Mi-Heon Lee, Jane Yanagawa, Tonya C Walser, Jonathan W. Goldman, Edward B. Garon, Gang Zeng, Sherven Sharma, Boning Gao, John Minna, Steven M. Dubinett, Jay M Lee. FRA1 contributes to ERK-mediated increased PD-L1 expression in KRAS mutated premalignant human bronchial epithelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2324.