NK cell-mediated cytotoxicity contributes to tumor control by a cytostatic drug combination

Accumulating evidence suggests that molecularly targeted therapies, which were originally developed to target the underlying cell autonomous genetic drivers of tumorigenesis, can provoke tumor specific immune responses. Using immunocompetent mouse models of KRAS mutant lung adenocarcinoma, we show that a combination of MEK and CDK4/6 inhibitors can induce natural killer (NK) cell immune surveillance that is necessary for its full anti-tumor effect. Mechanistically, we show that the drug combination – but neither agent alone – drives RB-mediated cellular senescence induction leading to potent cell cycle arrest and activation of the immunomodulatory senescence-associated secretory phenotype (SASP). This, in turn, leads to an increase in tumor-specific NK cell ligand expression and secretion of TNF-α that provokes NK cell-mediated targeting of senescent tumor cells, culminating in tumor regressions and long-term survival in genetically engineered mouse models of lung cancer. These studies identify a means and method of invoking a unique form of NK cell mediated immune surveillance in lung tumors through molecularly targeted therapies that induce senescence. For RNA-seq analysis of transcriptional changes after drug treatment of human KRAS mutant lung and pancreas cancer cell lines, total RNA was extracted using the RNeasy Mini Kit (Qiagen) from cell lines following 8 day treatment with vehicle (DMSO), trametinib (25 nM), palbociclib (500nM), or both in combination. For RNA-seq analysis of transcriptional changes in Natural Killer (NK) cells in vivo, total RNA was extracted as described above from CD45+CD3-NK1.1+ NK cells sorted from the lungs of tumor bearing KP transplant mice following 1 week treatment with vehicle or combined trametinib (1 mg/kg body weight) or palbociclib (100 mg/kg body weight). PolyA mRNA was selected using beads coated with polyT oligonucleotides. Purified polyA mRNA was subsequently fragmented, and first and second strand cDNA synthesis performed using standard Illumina mRNA TruSeq library preparation protocols. Double stranded cDNA was subsequently processed for TruSeq dual-index Illumina library generation. For sequencing, pooled multiplexed libraries were run on a HiSeq 2500 machine on RAPID mode. Approximately 10 million 76bp single-end reads were retrieved per replicate condition. Resulting RNA-Seq data was analyzed by removing adaptor sequences using Trimmomatic (29), aligning sequencing data to GRCh37.75(hg19) with STAR (30), and genome wide transcript counting using HTSeq (31) to generate a RPKM matrix of transcript counts.