Identifying Novel Mechanisms of Resistance to Tyrosine Kinase Inhibitors in Anaplastic Large Cell Lymphoma

INTRODUCTION: Anaplastic large cell lymphomas (ALCL) frequently carry oncogenic fusion proteins as a consequence of chromosomal translocations of the anaplastic lymphoma kinase (ALK) gene. The fusion protein resulting from the translocation between dimerization domain of nucleophosmin (NPM) and intracellular tyrosine kinase domain of ALK activates several signaling pathways, promoting cell growth, transformation, migration, and survival of the cells. Chemotherapy has been used as a standard treatment approach for ALCL patients, yet about 30% of patients relapse. A more specific treatment method is based on targeting ALK tyrosine kinase using tyrosine kinase inhibitors (TKIs). Crizotinib is an ALK TKI that is approved for the treatment of ALK-rearranged lung cancer and has received Breakthrough Therapy designation for lymphoma because of its high activity in chemo refractory ALCL. However, as for lung cancer, also ALCL patient develop crizotinib resistance due to ALK mutations or unknown mechanisms. In this study, we aimed at elucidating unknown by-pass mechanisms of crizotinib resistance in ALCL.

METHODS: We used Genome-wide CRISPR-Cas9 Knockout Screening (GeCKO) 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, DNA isolation and next generation sequencing was performed on crizotinib resistant cells to identify candidate genes depleted by the GeCKO screening. Top candidates were selected for validation assays and further analyses.

RESULTS: We identified two phosphatases, PTPN1 and PTPN2, in different ALCL cell lines as consistent top hits. Functional validation of these candidate genes showed that single loss of either PTPN1 or PTPN2 generate immediate resistance to crizotinib in ALCL cell lines. Analysis of downstream pathways showed that while loss of PTPN1 activates primarily the MAPK pathway, loss of PTPN2 promotes persistent STAT3 and MAPK activation in ALK inhibited cells. Remarkably, in PTPN1 knockout cells we observed hyperactivation of SHP2, an oncogenic phosphatase that positively regulates the RAS-MAPK pathway. On the other hand, over-expression of PTPN1 and PTPN2 partially inhibited SHP2 phosphorylation. A treatment that combined crizotinib and the recently developed SHP2 inhibitor completely blocked the sustained ERK phosphorylation and reverted the crizotinib resistance observed in PTPN1 and PTPN2 deficient lymphoma cells.

CONCLUSIONS: GeCKO library screen identified PTPN1 and PTPN2 as specific genes that mediate crizotinib resistance in ALCL cell lines. Loss of PTPN1 and PTPN2 drives resistance by activating MAPK and/or JAK-STAT pathway. Combined inhibition of SHP2 is a potent therapeutic approach to overcome resistance to crizotinib in ALCL cells.