Abstract B072: MEK inhibition modulates cytokine response to mediate therapeutic efficacy in lung cancer
Authors: Xie Mengyu , Zheng Hong , Madan-Lala Ranjna , Dai Wenjie , Gimbrone Nicholas T. , Chen Zhihua , Kinose Fumi , Blackstone Sarah A. , Smalley Keiran S. M. , Cress Douglas , Haura Eric B. , Rix Uwe , Beg Amer A. 
Published: March 2020

Abstract
Background: Activating mutations in BRAF, a key mediator of RAS signaling, are present in ~50% of melanoma patients. Pharmacological inhibition of BRAF or the downstream MAP kinase MEK are highly effective in treating BRAF-mutant melanoma. In contrast, RAS pathway inhibitors have been less effective in treating epithelial malignancies, such as lung cancer. Immunotherapeutics, especially those targeting checkpoint receptors on T cells, have revolutionized treatment of many cancer types. It has been proposed that MEK inhibitors(MEKi) may help generate a tumor microenvironment that enhances response to immunotherapy. The aim of this study was to investigate a novel cross-talk mechanism between MEKi and cytokine signaling response which could be used to enhance therapeutic efficacy against cancer types that are minimally responsive to MEKi. Methods: The association between MEK inhibition, NF-κB signaling and clinical response was analyzed using publicly available RNA-sequencing data from pre-treatment and on-treatment biopsies of patients (EGA S00001000992). To identify clinical and pre-clinical compounds that enhance cytokine response, we performed drug library screening for CCL5 and CXCL10 expression. 289 different agents (0.1 mM and 1 mM) were incubated with A549 cells in 96 well plates, with or without the presence of 0.2 ng/ml TNFα and 1 ng/ml IFNγ. Chemokine levels in supernatant were detected using Bead-Based Multiplex Assays. To investigate cytokine response modulatory mechanisms of MEKi, qPCR, Western Blot and flow cytometry were used to determine NF-κB activity and targeting gene expressions in lung cancer cells lines. The anti-tumor effect of MEKi in combination with TNFα and IFNγ was assessed using viable cell counting, cell cycle assay and apoptosis marker (cleaved caspase 3) expression in vitro. For in vivo studies, we performed combinatory therapies using MEKi plus intratumoral cytokine injection, MEKi plus PD-1 blockade in LKR subcutaneous tumor model. Results: We show that treatment of melanoma patients with BRAF and MEK inhibitors activated tumor NF-κB activity. MEKi potentiated the response to TNFα, a potent activator of NF-κB. MEKi increased cell surface expression of TNFα receptor 1 (TNFR1), which enhanced NF-κB activation and augmented expression of genes regulated by TNFα and IFNγ. Drug screening results demonstrated that this was a general activity of inhibitors of MEK and ERK kinases. Treatment with MEKi led to acquisition of a novel vulnerability to TNFα and IFNγ-induced apoptosis in lung cancer cells that were refractory to MEKi killing and augmented cell cycle arrest. Abolishing the expression of TNFR1 on lung cancer cells impaired the anti-tumor efficacy of MEKi while the administration of TNFα and IFNγ in MEKi-treated mice enhanced the anti-tumor response. Furthermore, immunotherapeutics known to induce expression of these cytokines synergized with MEKi in eradicating tumors. Conclusions:These findings define a novel cytokine response modulatory function of MEKi which can be therapeutically exploited. We show that lung cancer cells are rendered sensitive to MEKi by TNFα and IFNγ, providing strong mechanistic rationale for combining MEKi with immunotherapeutics, such as checkpoint blockers, in lung cancer.
Citation Format: Mengyu Xie, Hong Zheng, Ranjna Madan-Lala, Wenjie Dai, Nicholas T. Gimbrone, Zhihua Chen, Fumi Kinose, Sarah A. Blackstone, Keiran S. M. Smalley, Douglas Cress, Eric B. Haura, Uwe Rix, Amer A. Beg. MEK inhibition modulates cytokine response to mediate therapeutic efficacy in lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B072. doi:10.1158/1535-7163.TARG-19-B072