Novel Resistance Mutations to ALK Tyrosine Kinase Inhibition Arising During Cell‐Culture Drug Selections in T‐Cell Lymphoma

Anaplastic Large Cell Lymphoma (ALCL) is a rare T‐cell lymphoma. The Anaplastic Lymphoma Kinase (ALK)‐positive molecular subtype of ALCL is driven by constitutive activation of the ALK tyrosine‐kinase domain, typically through chromosomal translocation. The most common rearrangement, t(2;5)(p23;q35), results in the NPM‐ALK fusion kinase. Recently, two ALK‐specific tyrosine kinase inhibitors (TKIs), crizotinib and ceritinib, have been approved for clinical use in ALK‐positive lung cancer while another inhibitor, alectinib, is in early‐stage clinical trials. We developed TKI‐resistant ALK + ALCL cell line subclones from Karpas‐299, SUP‐M2 and SU‐DHL‐1. Each line was cultured in increasing concentrations of crizotinib or ceritinib until viable at 500nM and 150nM, respectively. We identified three novel mutations in our resistant lines affecting the ALK kinase domain: R1192P, I1171S, and F1174L. We cloned wild‐type and mutant NPM‐ALK into a murine retroviral vector and infected an IL3‐dependent murine pro‐B cell line FL5‐12. The infected lines were withdrawn from IL‐3, leading to their IL3‐independent, oncogene‐dependent transformation. We evaluated the half‐maximal inhibitory concentration (IC 50 ) of crizotinib, ceritinib and alectinib for wild‐type ALK‐dependent and mutant ALK dependent FL5‐12 cells, through luminescence based viability assays (Table 1). We found that all three mutations confer resistance to both crizotinib and ceritinib to varying degrees. I1171S was the only mutation to confer resistance to alectinib, while R1192P and F1174L remain sensitive. Our findings have significant clinical implications in matching specific TKIs to patient disease based on ALK mutational status. NPM‐ALK Genotypes Average Crizotinib IC 50 (nM) Crizotinib Standard Error of the Mean Average Ceretinib IC 50 (nM) Ceretinib Standard Error of the Mean Average Alectinib IC 50 (nM) Alectinib Standard Error of the Mean Wild Type 171.85 7.19 34.03 2.00 2.84 .33 R1192P 366.8 17.56 97.19 5.40 5.47 .29 I1171S 479.825 28.9 57.7 1.24 43.185 4.34 F1174L 245.05 15.9 57.015 3.62 3.87 .68