EZH1/2 inhibition improves immunotherapy response through MHC Class II de-repression and neutrophil reprogramming

Squamous cell carcinoma of the lung is a difficult-to-treat cancer with high prevalence in the US, and particularly in Kentucky. The goals of this work were to test if the EZH1/2 inhibitor valemetostat improves anti-PD1 responses in squamous cell lung cancer models, and to develop ex vivo models to test immunotherapy drug combinations. We found that valemetostat produced augmented anti-tumor responses to anti-PD1 therapy through up-regulation of tumor cell specific Major Histocompatibility Complex Class II (MHC Class II), and a shift towards activated CD8+ T cells. Neutrophils predominated in these tumors regardless of therapy, but examination of bone marrow revealed that valemetostat treated mice and mice that rejected tumors both had more mature neutrophils. Likewise, Ezh2 knock-out mice produced neutrophils that were more apoptotic, less migratory, and less able to produce extracellular nets, but had similar ability to kill bacteria as Ezh2-WT neutrophils. To test tumor responses to differing neutrophil populations, we engineered three-dimensional air-liquid interface cultures with tumoroids, lung mesenchymal cells, and T cells, with and without bone marrow containing neutrophils and myeloid progenitors from distinct donors. Bone marrow from tumor-naive or mice with actively growing untreated tumors boosted tumoroid growth, while bone marrow from tumor-rejected or mice with tumors treated with valemetostat was anti-tumor. MHC Class II blockade lowered the ability of bone marrow to boost tumor growth, and reduced the ability of valemetostat with anti-PD1 to reduce tumoroid growth. Patient samples revealed a strong negative correlation between EZH2 and MHC Class II, suggesting that targeting EZH2 activity could lead to marked increase in MHC Class II and improve treatment responses in lung squamous cell carcinomas.