STAT3 sustains tumorigenicity following mutant KRAS ablation

Mechanisms underpinning the residual tumorigenicity of mutant KRAS cancers following KRAS inactivation remain to be completely understood. Our studies show that KRAS ablation in pancreatic ductal adenocarcinoma (PDAC) prevents tumor growth contingent on the concomitant inactivation of the transcription factor STAT3. Mechanistically, the incurred losses of KRAS and STAT3 disrupt a temporal balance between tumor cell differentiation, proliferation, and self-renewal. 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 both mutant KRAS and STAT3. RNA sequencing data are presented for PDAC cells following CRISPR-mediated STAT3 and KRAS double knockouts compared to single knockouts, and with double knockouts following enhanced expression of the FOXA1 and FOXA2 genes. Chromatin immunoprecipitation analyses are presented with antibodies to phosphorylated STAT3 in parental and KRAS knockout cells and control antibodies in parental cells. Excel files of sequencing results prepared by Novogene Corp. are provided. In addition, Excel files are included for single-cell RNA-seq analyses of tumors formed by doxycycline-induced mutant KRAS (iKRAS) pancreatic cells and derived STAT3 knockout cells in mice treated with doxycycline and following removal of doxycycline for 4 days. The data was acquired by Cold Spring Harbor Laboratory Genome Center from the Loupe browser software.

Methods
Total RNA was isolated from parental and genetically modified murine pancreatic ductal adenocarcinoma cell lines (KPC) using the PureLink RNA kit (ThermoFisher) and phenol-extracted. Whole exome RNA sequencing with bioinformatics was performed by Novogene Corp, and Excel files are provided. 

Data also include chromatin immunoprecipitation results of control KPC cells and cells deleted for mutant KRAS by CRISPR-mediated gene ablation (KRAS KO). These experiments were performed with antibodies to phosphorylated STAT3 and control antibodies. Cell Signaling Technology SimpleChIP Plus enzymatic chromatin IP kit with magnetic beads was used. Briefly, formaldehyde treatment of tissue culture cells was used to crosslink proteins to DNA, chromatin was digested with micrococcal nuclease and sonicated, and crosslinked chromatin was immunoprecipitated overnight with antibodies and reagents from Cell Signaling Technology (control rabbit antibody or phospho-STAT3 antibody (Tyr705). Immunocomplexes were collected with ChIP-grade protein G magnetic beads, stringently washed, reverse cross-linked, and DNA libraries were prepared with NEBNext Ultra II DNA library prep kit for Illumina for next generation sequencing by Novogene Corp. Bioinformatics of genome mapping, peak calling, and peak annotation was performed by Novogene Corp.