Abstract 4320: Interrogation of autophagy inhibition in anaplastic large cell lymphoma (ALCL)
Authors: Katie L. Buelow, Stephanie Celano, Gregory Poole, Katie Martin, Jeffrey MacKeigan, David J. Hoogstra
Journal: Cancer Research (2024)

Abstract The objectives of this study are to evaluate if anaplastic lymphoma kinase (ALK) inhibition activates autophagy in ALK+ ALCL cells and to determine if that activation can be mitigated using autophagy inhibition. ALCL is an aggressive T-cell non-Hodgkin lymphoma that often presents as advanced stage disease. With the addition of crizotinib or brentuximab vedotin to the ALCL99 chemotherapy backbone, the recently reported Children’s Oncology Group ANHL12P1 study demonstrated a 2-year event-free survival for ALCL patients of 79%. Thus, even with multi-agent chemotherapy combined with targeted therapy, improved therapeutic efficacy is needed. Autophagy is activated when mTORC1 is inhibited, and as ALK signaling is known to promote mTORC1 activity, ALK inhibition is hypothesized to activate autophagy. If present, ALK inhibitor-induced autophagy activation would represent a novel therapeutic opportunity targetable with emerging autophagy inhibitors. An ALK+ ALCL cell line, Karpas 299, was treated with drug combinations for 3 hours, lysates prepared, and proteins separated by molecular weight using standard procedures. ALK inhibitor target engagement was assessed by western blot using phospho-ALKY1604 intensity in 9-point dose response curves. Target engagement of autophagy inhibition (ULK-101 directed against ULK1/2 and hydroxychloroquine (HCQ) targeting the lysosome) was assessed by western blot using phospho-ATG14S29 and LC3B-II intensity in 6-point dose response curves. Cell viability was evaluated using a luminescent CellTiter-Glo assay. Karpas299 cells resistant to crizotinib at 50nM (KCrizR50) were generated by gradually increasing crizotinib concentration in the media until cells grew at a rate similar to parental cells. Crizotinib, ceritinib, and lorlatinib all effectively inhibit ALK phosphorylation in Karpas 299 cells with low nanomolar potency. ULK-101 effectively inhibited pATG14S29 at 5 µM and HCQ effectively inhibited autophagic flux, as evidenced by increased LC3B-II intensity, at 20 µM. When treated with the lowest ALK inhibitor dose needed to achieve complete pALK inhibition (IC99), pATG14 intensity increased representing increased autophagic activity. When combined with ALK inhibition, ULK-101 effectively decreased pATG14S29, blocking the ALK inhibitor-induced autophagy activation. Decreased viability in Karpas 299 cells was observed after crizotinib, ceritinib and lorlatinib treatments with nanomolar potency. KCrizR50 cells had a potency higher than parental Karpas 299 cells for all three ALK inhibitors. ALK inhibition activates autophagy in ALCL cells and autophagy activation is effectively mitigated through concurrent autophagy inhibition. ALK inhibitor resistance in Karpas 299 cells will be further evaluated to determine whether autophagy contributes to the resistant phenotype. Citation Format: Katie L. Buelow, Stephanie Celano, Gregory Poole, Katie Martin, Jeffrey MacKeigan, David J. Hoogstra. Interrogation of autophagy inhibition in anaplastic large cell lymphoma (ALCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4320.