Dabrafenib alters MDSC differentiation and function by activation of GCN2

The BRAF inhibitor dabrafenib has been reported to activate the integrated stress response (ISR) kinase GCN2, and the therapeutic effect has been partially attributed to GCN2 activation. Since ISR signaling is a key component of myeloid-derived suppressor cell (MDSC) development and function, we measured the effect of dabrafenib on MDSC differentiation and suppressive activity. Our data showed that dabrafenib attenuated MDSC ability to suppress T cell activity, which was associated with a GCN2-dependent block of the transition from monocytic progenitor to polymorphonuclear (PMN)-MDSCs and proliferative arrest resulting in PMN-MDSC loss. Transcriptional profiling revealed that dabrafenib-driven GCN2 activation altered metabolic features in MDSCs enhancing oxidative respiration, and attenuated transcriptional programs required for PMN development. Thus, ourdata reveals transcriptional networks that govern MDSC developmental programs, and the impact of GCN2 stress signaling on the innate immune landscape in tumors, providing novel insight into potentially beneficial off target effects of dabrafenib. Overall design: Bone marrow was flushed out of the tibia and femur of C57BL/6 mice. Red cells were depleted with ACK lysing buffer. Cells were seeded at concentration 6x10^5 cells/mL in RPMI 1640 medium supplemented with 10% FBS, 100 units/mL of penicillin, 100 µg/mL of streptomycin and 55 µM 2-Mercaptoethanol. GM-CSF (50 ng/mL) and IL-6 (50 ng/mL) were used to drive MDSC differentiation in vitro in presence of 1.5 µM Dabrafenib or matching volume of Dimethyl sulfoxide (DMSO). On day 4, cells were harvested and processed for scRNA-seq using Chromium Next GEM Single Cell 5' Kit v2 workflow (10x Genomics).