Transcriptomic studies on ruxolitinib with or without checkpoint inhibitors as a cancer therapy

Unleashing anti-tumor T cell activity by checkpoint inhibition is effective in many cancer patients but clinical response rates remain limited. Myeloid derived suppressor cells erode antitumor lymphocyte numbers and function, and correlate with resistance to checkpoint inhibitors. By screening small molecule libraries, we identified JAK inhibitors’ ability to rescue T cell function. Despite its documented immune suppressive properties, the prototypical JAK inhibitor ruxolitinib enhanced the efficacy of immune checkpoint blockade in cancer. This effect correlated with loss of suppressive gene expression, and acquisition of immunostimulatory molecular markers and T cell stimulatory activity in myeloid cells. In preclinical models, ruxolitinib significantly improved the function and increased the total numbers of activated tumor-infiltrating NK and CD4 T cells compared to checkpoint blockade alone and the efficacy was conditional on granulocytic cells. In addition to myeloid reprogramming in the tumor, ruxolitinib blunts G-CSF signaling in the bone marrow to prevent expression of suppressive and chemotaxis genes in neutrophils. Immunocompetent mice bearing wt MC38, JAK1ko MC38, LLC1 or EL4 tumors were treated with ruxolitinib or vehicle (daily gavage) and anti-PD1 plus anti-CTLA4 (ICB), or isotype control (intraperitoneal injection every 7 d). Treatment was initiated once tumors became palpable. At a pre-selected timepoint tumors were harvested and processed into single-cell suspension. Live single CD45+ cells were sorted and subjected to CITE-seq using a standard protocol for 10X Genomics 3' single-cell transcriptome with antibody-derived protein barcoding. Bone marrow and blood CD45+ were harvested as single-cell suspensions and processed using the same CITE-seq protocol. To test the effect of ruxolitinib on splenocytes during persistent viral infection, mice infected with LCMV Clone 13 (2x10^6 pfu) were treated with ruxolitinib or vehicle by daily gavage from the day of infection and at 10 d post infection single-cell splenocyte suspension sorted by flow cytometry. Live single CD45+ splenocytes were subjected to the same CITE-seq protocol.