Pharmacological inhibition of the ALK axis elicits therapeutic potential in Consensus Molecular Subtype 1 colon cancer patients

Abstract

In the last years, several efforts have been made to classify colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, known as Consensus Molecular Subtypes (CMSs). In this work, we performed a meta-analysis of 1700 CRC patients stratified into four CMSs. We identified a negative correlation between a high level of anaplastic lymphoma kinase (ALK) expression and relapse-free survival, exclusively in CMS1 subtype. Stemming from this observation, we tested several CMSs in vitro models with crizotinib (CZB) or alectinib (ALC), potent ALK inhibitors, already approved for clinical use. ALK interception strongly inhibits cell proliferation already at nanomolar doses, specifically in CMS1 cell lines, while no effect was found in CMS2/3/4 groups. Furthermore, in vivo imaging identified a role for ALK in the dynamic formation of 3D spheroids, which was impaired by the pharmacological inhibition of ALK. Consistently, CZB was responsible for the dampened activation of ALK along with the downstream AKT cascade. Mechanistically, we found a specific pro-apoptotic effect of ALK inhibition in CMS1 cell lines, both in 2D and 3D. Confocal analysis suggests that inhibition in CMS1 cells enhances cell-cell adhesion when growing in 3D. In agreement with our findings, an ALK signature encompassing 65 genes statistically associated with worse relapse-free survival in CMS1 subtype. Finally, the efficacy of ALK inhibition treatment was demonstrated in patient-derived organoids. Collectively, our findings suggest that ALK inhibition may represent an attractive therapy for CRC, and CMS classification may provide a useful tool to identify patients who could benefit from this treatment. These findings offer rationale and pharmacological strategies for the treatment of CMS1 CRC.

A new method for the study of biophysical and morphological parameters in 3D cell cultures: Evaluation in LoVo spheroids treated with crizotinib

“…Accordingly, we were able to document that CZB treatment has an impact on the mass density, which represents an important marker characterizing cancer cell treatment. We speculate that the possible mechanism responsible for the increased spheroid mass density may be related to the enhanced cell-cell adhesion, as previously reported [27]. Indeed, we found that CZB treated cells displayed increased e-cadherin production, with consequent improved tight-junction organization.…”

“…We found that in live samples CZB treatment significantly decreases weight and size ( Fig 2B and 2C ) at concentrations of 250 and 500 nM, compared to CTRL. This is in line with an antiproliferative activity of the inhibitor [ 27 ]. On the other hand, under the same treatments, mass density displays an opposing result, with a significant increase in the density of the spheroids’ bulk ( Fig 2A ).…”

“…Cells were seeded at the concentration of 1x10 6 /wells and cultured for 10 days in DMEM (Sigma-Aldrich, St. Louis, MO) with 10% FCS in presence of 250 and 500 nM of CZB (MedChem Express), at 37°C and 5% CO 2 . The inhibitor was added to the medium simultaneously with the seeding as previously described [ 27 ]. LoVo cells grown in DMEM (Sigma-Aldrich) with 10% FCS were used as control (CTRL).…”

“…In this study, the LoVo colon cancer cell line forming spheroids was treated with crizotinib (CZB) an ATP-competitive small-molecule inhibitor of the receptor tyrosine kinases. This inhibitor, already been proved to impact LoVo spheroids morphology [ 26 , 27 ], was employed to assess the correlation between biophysical and morphological parameters in both live and fixed cells ( Fig 1 ).…”