Abstract 2851: ALK-inhibitors as a novel potential therapy for proteasome inhibitor-resistant multiple myeloma

Abstract

Abstract Background: Proteasome inhibitors (PI) disrupt the equilibrium between production and disposal of excess and/or misfolded proteins in multiple myeloma (MM) cells, leading to apoptosis. Resistance to PI bortezomib (BTZ) and carfilzomib (CFZ) is a major obstacle to the successful treatment of MM. The drug repurposing approach offers a promising strategy to classical drug development in particular in MM. Previously, ALK-inhibitors were shown to have anti-MM activity ex vivo. Therefore, we aimed to identify the mechanism of action of ALK-inhibitors against MM cells and to identify promising drug therapy combinations of ALK-inhibitors for refractory MM. Methods: A set of PI-naïve and PI-resistant cells was used in the study. Viability of the cells was determined by CCK8 viability assay. Genome-wide CRISPR/Cas9-based loss-of-function screening with Brunello library was used in MM AMO-1 cell line to identify mechanism of action of ceritinib. Western blot was used to assess levels of total and phosphorylated proteins. RNA-seq was used to determine the amount of transcripts in PI-sensitive and PI-resistant cells. Results: Initially, 7 different ALK-inhibitors were tested across PI-naïve and PI-adapted MM cell lines. Based on the IC50 values, the most effective ALK-inhibitors to induce cytotoxicity in MM were ceritinib > brigatinib > crizotinib > entrectinib. The combination of ceritinib, brigatinib and entrectinib showed synergistic cytotoxicity with the PI BTZ and CFZ and overcame PI-resistance in 4 different sets of PI-adapted cells. The strongest synergistic cytotoxicity was observed between ceritinib and CFZ in CFZ-adapted cells. CRISPR/Cas9-screening identified genes involved in negative regulation of mTORC signalling (NPRL2, NPRL3, TSC1, TSC2) and transcription factor FOXO1 as the major resistance candidates to ceritinib. Subsequently, ceritinib treatment significantly inhibited mTORC signaling, suggesting a shut-down of cellular proliferation due to targeting of the upstream receptor-tyrosine kinases (RTK), essential for cell viability and thus not identified by the screening. Further search identified InsR and IGF1R as the overlapping RTK inhibited by ceritinib and brigatinib. Co-treatment of PI-resistant cells with the combination of InsR/IGF1R inhibitors and CFZ induced strong synergistic cytotoxicity, resembling the synergistic cytotoxicity observed for ceritinib and CFZ, whereas InsR/IGF1R inhibitors did not show any synergistic cytotoxicity with ceritinib, suggesting targeting the same pathway. Our data further show that PI-naïve and resistant cells are negative for ALK, but positive for InsR and IGF1R expression. Conclusion: Crizotinib inhibits InsR/IGF1R signaling in MM, which is essential for MM survival and together with CFZ it overcomes PI-resistance. Therefore, it represents a promising and already available therapy for PI-resistant MM patients. Citation Format: Lenka Besse, Andrej Besse, Marianne Kraus, Matej Jasik, Ondrej Slaby, Christoph Driessen. ALK-inhibitors as a novel potential therapy for proteasome inhibitor-resistant multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2851.