The autocrine loop of ALK receptor and ALKAL2 ligand is an actionable target in Consensus Molecular Subtype 1 colon cancer

The Consensus Molecular Subtypes (CMSs) classification stratifies colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, thus providing an important support to personalized medicine1. In this work, we performed a bioinformatic meta-analysis of a cohort of 1700 CMS-stratified CRC patients. We identified a negative correlation between high level of anaplastic lymphoma kinase (ALK) expression and relapse-free survival, exclusively in CMS1 subtype. The unexpected association of ALK with the CMS1 led to the hypothesis that ALK pharmacological inhibition may elicit therapeutic potential in this subgroup. Stemming from these observations we tested two ALK inhibitors, crizotinib and alectinib, on several CRC in vitro models, stratified according to the CMS classification, and in CRC patient-derived organoids2. Proliferation assays performed both in monolayer and in 3D growing conditions on CMS1 cells and patient-derived organoids firmly supported the results of the preliminary bioinformatic data. Indeed, ALK interception by crizotinib and alectinib strongly inhibited cell proliferation already at nanomolar doses, specifically in CMS1 cells, while no effect was found in CMS2/3/4. Consistently, ALK inhibitors mediated a pro-apoptotic effect in CMS1 cells and were responsible for the dampening of ALK activation along with the downstream AKT cascade. In line, the treatment with ALK ligand ALKAL2 induced ALK phosphorylation in CMS1 cells, effect that was completely nullified by the combination with ALK inhibitors. Also, ALKAL2 prompted CMS1 cell invasion in gelatin degradation assays, supporting the hypothesis of a dependency of CMS1 invasion from ALK activation. Mechanistically, we found that CMS1 cells display several copies of ALKAL2 ligand and ALK-mRNAs, suggesting an autocrine loop mediated by ALKAL2 in the activation of ALK pathway, responsible for the invasive phenotype. Finally, crizotinib effect was confirmed by in vivo experiments, where it strongly dampened tumorigenic growth of CMS1 cells and doubled the mice survival. In agreement, an ALK signature encompassing 65 genes statistically associated to worse relapse-free survival in CMS1 subtype. Collectively, these findings support the hypothesis that ALK may represent an attractive target for colorectal cancer therapy, and CMSs classification may provide a useful tool to identify patients who could benefit from the treatment with ALK inhibitors.