Abstract 4715: Characterization of potential co-drivers of pathogenesis in ALK positive ALCL
Authors: Mologni Luca , Sharma Geeta G. , Villa Matteo , Mauri Mario , Arosio Giulia , Lobello Cosimo , Larose Hugo , Pirola Alessandra , Bombelli Silvia , Cordani Nicoletta , Massimino Luca , Pospíšilová Šárka , Turner Suzanne D. , Inghirami Giorgio , Pagni Fabio , Piazza Rocco , Perego Roberto , Chiarle Roberto , Gambacorti-Passerini Carlo 
Published: September 2020

Abstract
ALK positive Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin lymphoma characterized by expression of the NPM/ALK fusion tyrosine kinase essential for neoplastic cells growth. When resistant or relapsed to front-line chemotherapy, ALK+ ALCL prognosis is very poor. Although ALK+ ALCL cells are mainly driven by the constitutively active NPM/ALK kinase, presence of additional mutations might affect the disease course and response to treatment, leading to different outcomes in different patients. We employed whole exome sequencing to characterize the co-mutational burden in ALK+ ALCL samples at diagnosis. Diagnosis biopsy and matched healthy control DNA samples from 24 ALK+ ALCL patients were sequenced. Somatic variants present at high frequency within the tumor (at least 25% variant allele frequency), which can be viewed as potential co-drivers, were annotated; the identified mutations were weighed using in silico tools to predict their oncogenic potential and their effects on protein function. All identified variants were confirmed by Sanger sequencing. Gene ontology and pathway analyses identified cell adhesion and actin cytoskeleton among the most enriched biological functions. Using the above-mentioned criteria, we found recurrent somatic variants in 7 genes including PPP1R9A, RUNX1T1, FAT4 and RBBP8. Here we report the initial characterization of the role of FAT4 in ALCL cells. FAT4 is a member of the cadherin superfamily with tumor suppressor functions. In our cohort, 3/24 patients (13%) were found to carry FAT4 mutations, including nonsense mutations and missense changes predicted to be deleterious, suggesting a loss of function effect. To characterize the biological consequences of these mutations, we generated FAT4 knockdown ALCL cell lines. FAT4-silenced cells grew faster and showed increased migratory properties compared to parental cells. They also differed from parental cells in terms of morphology, having bigger size and modulated F-actin rearrangement. Tumor suppressing activity of FAT4 has been linked to Wnt/β-catenin pathway; we found that FAT4-silenced cells had decreased levels of serine/threonine phosphorylated β-catenin and higher nuclear localization, which insinuates activation of Wnt/β-catenin signalling and henceforth, increased cellular proliferation. To explore therapeutic implications of FAT4 mutations, we analyzed the sensitivity of FAT4 silenced cells to chemotherapeutic agents. FAT4 knockdown cells were less sensitive to a CHOP combination treatment as compared to parental cells. No difference was seen in terms of sensitivity to crizotinib. Finally, we investigated the effects of RUNX1T1 somatic variants found in our study. Preliminary luciferase assay results showed that the variants significantly increase the transcriptional repression ability of RUNX1T1. Altogether, our data provides information about genes other than NPM-ALK that might play a role in the pathogenesis of ALCL.
Citation Format: Luca Mologni, Geeta G. Sharma, Matteo Villa, Mario Mauri, Giulia Arosio, Cosimo Lobello, Hugo Larose, Alessandra Pirola, Silvia Bombelli, Nicoletta Cordani, Luca Massimino, Šárka Pospíšilová, Suzanne D. Turner, Giorgio Inghirami, Fabio Pagni, Rocco Piazza, Roberto Perego, Roberto Chiarle, Carlo Gambacorti-Passerini. Characterization of potential co-drivers of pathogenesis in ALK positive ALCL [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4715.