Abstract 902: In vitro andin vivocharacterization of resistance to lorlatinib treatment in ALK mutated cancers

The development of targeted therapy has significantly improved the treatment of Anaplastic Lymphoma Kinase (ALK) dependent neoplasias such as Anaplastic Large Cell Lymphoma (ALCL) and Non-Small Cell Lung Cancer (NSCLC). Despite the efficacy of ALK inhibitor crizotinib, acquired resistance is a major challenge. Lorlatinib is a second generation inhibitor with improved activity and selectivity against ALK. To investigate resistance mechanisms that may arise on lorlatinib therapy, we selected in vitro and in vivo lorlatinib resistant tumor cells. In vivo, 10 mice carrying subcutaneous Karpas299 xenografts received vehicle or lorlatinib at increasing doses, starting at 0.1 mg/kg BID. As expected, tumor size was significantly reduced in lorlatinib group compared to controls. After initial partial responses, tumors relapsed and mice were shifted to an increased dose (0.25 mg/kg). Tumors relapsed again and mice were randomized into three groups, to achieve a maximum dose of 0.5, 1 or 2 mg/kg. In all cases tumors regrew after an initial response, indicating acquired resistance to lorlatinib. Upon mice sacrifice, tumors were collected and characterized. All ex vivo cell lines were resistant to lorlatinib as assessed by proliferation assay and 2/10 showed moderate drug addiction. However, in several cases, ALK phosphorylation did not fully correlate with the observed high drug resistance, suggesting the involvement of ALK-independent mechanisms. ALK kinase domain sequencing revealed N1178H mutation in 5/10 mice, alone or in combination with other mutations and L1196M substitution in 2/10 cases. Since both mutants have been described as sensitive or moderately resistant to lorlatinib in murine BaF3 cells, further characterization of these cell lines was performed. ALK N1178H mutant was found to be predominantly localized in the cytoplasm, possibly mimicking overexpression, in human cells. Whole-exome and mRNA sequencing of cells carrying the L1196M substitution showed significant alterations in the PI3K/AKT and RAS/MAPK pathways, as well as perturbations of RAC1-related small GTPases. Functional validation by small molecule inhibitors confirmed the involvement of these pathways in resistance to lorlatinib. In vitro selected lorlatinib-resistant cell lines, representing ALCL, NSCLC and neuroblastoma, showed both ALK mutations, including G1202R and C1156F/L1198F, and ALK-independent mechanisms of resistance. In one case, NSCLC cells acquired hyper-activation of EGFR; its blockage by erlotinib restored sensitivity to lorlatinib. Whole-exome sequencing and proteomic profiling of sensitive and resistant neuroblastoma cell lines revealed NF1 and EP300 mutations, as well as hyper-activation of ErbB4, in lorlatinib-resistant cells. Altogether, our data provide an extensive characterization of resistance mechanisms that may arise in different ALK positive cancers upon lorlatinib exposure