Axon guidance receptor ROBO3 modulates subtype identity and prognosis via AXL-associated inflammatory network in pancreatic cancer

Metastatic pancreatic cancer (PDAC) has a poor clinical outcome with a 5-year survival rate below 3%. Recent transcriptome profiling of PDAC biopsies has identified 2 clinically distinct subtypes — the “basal-like” (BL) subtype with poor prognosis and therapy resistance compared with the less aggressive and drug-susceptible “classical” (CLA) subtype. However, the mechanistic events and environmental factors that promote the BL subtype identity are not very clear. Using preclinical models, patient-derived xenografts, and FACS-sorted PDAC patient biopsies, we report here that the axon guidance receptor, roundabout guidance receptor 3 (ROBO3), promotes the BL metastatic program via a potentially unique AXL/IL-6/phosphorylated STAT3 (p-STAT3) regulatory axis. RNA-Seq identified a ROBO3-mediated BL-specific gene program, while tyrosine kinase profiling revealed AXL as the key mediator of the p-STAT3 activation. CRISPR/dCas9-based ROBO3 silencing disrupted the AXL/p-STAT3 signaling axis, thereby halting metastasis and enhancing therapy sensitivity. Transcriptome analysis of resected patient tumors revealed that AXLhi neoplastic cells associated with the inflammatory stromal program. Combining AXL inhibitor and chemotherapy substantially restored a CLA phenotypic state and reduced disease aggressiveness. Thus, we conclude that a ROBO3-driven hierarchical network determines the inflammatory and prometastatic programs in a specific PDAC subtype.This work was supported by Max-Eder Research grants of German Cancer Aid to SKS (70112999) and to AN (70113213) and the Wilhelm-Sander-Stiftung (2021.159.1) to SKS. The study was also supported by the DFG grant (KFO 5002) to AN, EH, VE, and SKS and the Volkswagen-Stiftung/Ministry for Culture and Science in Lower Saxony (MWK) to VE (11-25 76251-12-3/16). This work was also supported by the German Ministry of Science and Education (BMBF) project CancerScout (FKZ: 13GW0451A) to HB, SK, and PS. The MD research fellowship of NK was supported by DGVS. The BMBF e:Med program for systems biology (PANC-STRAT consortium, grant no. 01ZX1305C and 01ZX1605C); the Dietmar-Hopp Foundation; the BioRNSpitzen cluster “Molecular- and Cell-based Medicine” and Heidelberger Stiftung Chirurgie supported our work for collecting and processing of patients’ specimens. We thank Stephan A. Hahn (Ruhr University Bochum) for supporting this study with PDX materials. We sincerely thank Waltraut Kopp, Kristina Reutlinger, Eva Schmitt, Dana Wörz, and Dimitra Spyropoulou for their excellent technical support. We thank N.A. Giese, T. Hackert, O. Strobel, and M. Büchler for organizing resected primary PDAC specimens from the EPZ-Biobank and Department of General, Visceral and Transplantation Surgery of the University Hospital Heidelberg