Abstract 3338: Concurrent treatment with Pim kinase inhibitor enhances efficacy of FLT3 inhibitors in AML with FLT3-ITD through GSK-3βactivation and GSK-3β-mediated enhanced proteasomal degradation of c-Myc and Mcl-1
Authors: Lee Jonelle , Scarpa Mario , Chatterjee Aditi , Ali Mooath Mustafa , Singh Prerna , Kapoor Shivani , Trotta Rossana , Baer Maria 
Published: June 2022

BACKGROUND: fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) cells in 30% of patients, with poor patient outcomes. FLT3 inhibitors are in clinical use, but with incomplete responses and onset of resistance. New targeting strategies are needed. The oncogenic serine/threonine kinase proviral integration site for Moloney murine leukemia virus (Pim-1) is upregulated downstream of FLT3-ITD, directly stimulates cell growth and inhibits apoptosis, and also phosphorylates and stabilizes FLT3 in a positive feedback loop. We previously showed that concurrent treatment of FLT3-ITD cells with Pim kinase and FLT3 inhibitors enhanced apoptosis induction through post-translational downregulation of the master transcription factor c-Myc and the anti-apoptotic protein Mcl-1. The serine/threonine kinase GSK-3β regulates c-Myc and Mcl-1 protein via phosphorylation at T58 and S159, respectively; we hypothesized downregulation of both proteins by combination treatment through phosphorylation by GSK-3β.

METHODS: FLT3-ITD cells lines (Ba/F3-ITD, MV4-11, and MOLM-14) and primary FLT3-ITD AML patient blasts were cultured with the pan-Pim kinase inhibitor AZD1208, the FLT3 inhibitors gilteritinib or quizartinib and the GSK-3β inhibitor TCG-24. Protein expression was measured by immunoblotting. To measure protein half-lives, cells were pretreated with cycloheximide with and without the proteasome inhibitor MG-132. Ba/F3-ITD cells were infected with Myc-T58A plasmid, preventing c-Myc T58 phosphorylation, or Mcl-1 S159A plasmid, preventing Mcl-1 S159 phosphorylation, or empty vector controls. Apoptosis was measured by Annexin V labeling, measured by flow cytometry.

RESULTS: Combined treatment of FLT3-ITD-expressing cell lines and AML patient blasts with Pim and FLT3 inhibitors caused rapid downregulation of c-Myc, and then Mcl-1, protein expression, relative to single drugs. Combined Pim and FLT3 inhibitor treatment caused marked decrease in half-lives of both proteins, which was rescued by proteasome inhibition, as the mechanism of protein downregulation. Combined Pim and FLT3 inhibitor treatment rapidly activated (dephosphorylated) GSK-3β and culture with GSK-3β inhibitor prevented induction of c-Myc and Mcl-1 downregulation, increased c-Myc and Mcl-1 protein turnover by concurrent Pim and FLT3 inhibitor treatment. Finally, combination treatment of Ba/F3-ITD cells infected with T58A c-Myc or S159A Mcl-1 plasmids did not downregulate these proteins or increase their turnover, with reduced apoptosis induction.

CONCLUSION: Pim inhibitors enhance efficacy of FLT3 inhibitors via GSK-3β activation and GSK-3β-mediated enhanced proteasomal degradation of c-Myc and Mcl-1, highlighting GSK-3β as a key target in cells with FLT3-ITD.

Citation Format: Jonelle Lee, Mario Scarpa, Aditi Chatterjee, Mooath Mustafa Ali, Prerna Singh, Shivani Kapoor, Rossana Trotta, Maria Baer. Concurrent treatment with Pim kinase inhibitor enhances efficacy of FLT3 inhibitors in AML with FLT3-ITD through GSK-3βactivation and GSK-3β-mediated enhanced proteasomal degradation of c-Myc and Mcl-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3338.