Introduction: Anaplastic large-cell lymphomas (ALCL) frequently carry oncogenic fusions involving the anaplastic lymphoma kinase (ALK) gene. The ALK fusions activate several signaling pathways, promoting cell growth, migration, and survival. Chemotherapy is the standard treatment for ALCL patients, but about 30% of patients relapse. Targeting ALK using tyrosine kinase inhibitors (TKIs) has shown promising results, and the FDA recently granted breakthrough therapy designation to crizotinib for use in patients with relapsed/refractory ALK+ ALCL. However, resistance to crizotinib develops in ALK+ ALCL patients secondary to ALK mutations or unknown mechanisms. In this study, we aimed at elucidating unknown bypass mechanisms of crizotinib resistance in ALK+ ALCL.
Methods: We used Genome-wide CRISPR-Cas9 Knockout Screening (GeCKO.v2) to identify candidate genes that contribute to resistance to crizotinib. Four different ALCL cell lines were infected with Lenti-GeCKO libraries. After treatment with crizotinib for 14 days to select for resistant cells, next-generation sequencing was performed on crizotinib-resistant cells to identify candidate resistance genes. Top candidates were selected for validation assays and further analyses.
Results: Genomic loss-of-function screens identified two phosphatases, PTPN1 and PTPN2, in all ALCL cell lines as consistent top hits driving resistance to crizotinib. Functional validation of these candidate genes showed that single loss of either PTPN1 or PTPN2 generates immediate resistance to crizotinib and other ALK TKIs such as alectinib and lorlatinib. Consistently, RNA-seq in patients who developed resistance to crizotinib showed downregulation of PTPN1 or PTPN2 expression. By multiple assays, we demonstrate that PTPN1 and PTPN2 are phosphatases that de-phosphorylate ALK, thereby regulating its overall phosphorylation levels and activity. In addition, we found that PTPN1, but not PTPN2, is also a phosphatase of SHP2, a key mediator of oncogenic ALK signaling. Downstream signaling analysis showed that deletion of PTPN1 or PTPN2 induces resistance to crizotinib by hyperactivating the MAPK and JAK/STAT pathways. A treatment that combined crizotinib and a recently developed SHP2 inhibitor (SHP099) completely blocked the sustained MAPK activation and reverted crizotinib resistance in vitro and in vivo.
Conclusions: We discovered that PTPN1 and PTPN2 are ALK phosphatases that control sensitivity to ALK TKIs in ALCL. Combined inhibition of SHP2 is a potential therapeutic approach to overcome resistance to ALK TKIs in ALCL.
Citation Format: Elif Atabay, Qi Wang, Taek-Chin Cheong, Nina Prokoph, Chiara Ambrogio, Ines Mota, Achille Pich, Enrico Patrucco, Claudia Voena, Roberto Chiarle. Phophatases modulate resistance to ALK inhibitors in anaplastic large-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-50.