Tumorigenicity of KRAS-ablated pancreatic cancer cells requires STAT3 function

Oncogenic KRAS mutations underlie some of the deadliest human cancers. Genetic or pharmacological KRAS inactivation produces mixed outcomes, ranging from complete regression to frequent relapse. Mechanisms underpinning the resistance of cancer cells to KRAS inactivation remain to be understood. Here we investigate a conceptual framework of pancreatic ductal adenocarcinoma showing that CRISPR-mediated KRAS ablation impedes tumor growth contingent on the concomitant inactivation of the STAT3 transcription factor. Mechanistically, the incurred losses of KRAS and STAT3 disrupt a temporal balance between tumor cell differentiation, proliferation, and self-renewal. This in turn impairs tumor growth in mice and enhances their immune rejection, resulting in tumor clearance. Our findings identify a specific role for STAT3 in supporting cancer cell fitness with a particular focus on KRAS-inhibited tumors and provide a rationale for developing therapies targeting mutant KRAS and STAT3. Overall design: Orthotopic injections into the pancreas of FVB male mice were performed with 104 iKRAS parental control (A9993) or iKRAS STAT3 KO cells in Matrigel diluted 1:10 with Opti-MEM using standard procedures (Kim et al 2009). For tumor formation, the drinking water was provided ad libitum and supplemented with 2% sucrose, doxycycline hyclate (0.5 mg/ml), neomycin (0.5 mg/mL) and ampicillin (1 mg/mL). The animals were observed for tumor development by palpation and orthotopic tumors were pooled from 2-3 individual mice at day 0 (~150-350mg) or following doxycycline removal for 4 days (day 4) (100-200mg).Single cell suspensions were prepared by treating tumor tissues with 1x collagenase/hyaluronidase (StemCell Technology), 0.5 mg/ml Liberase-DL (Roche), and 0.1 mg/ml DNase I (Sigma) for 45 minutes at 37?C with continuous mixing. Single-cell RNA sequencing was performed by the Cold Spring Harbor Laboratory Single-Cell Biology Shared Resource, Genomics Technology Development.