Concurrent Inhibition of Pim-1 and FLT3 Kinases in FLT3-ITD Acute Myeloid Leukemia Post-Translationally Downregulates the Anti-Apoptotic Protein Mcl-1 through Downregulation of the Mcl-1 Deubiquitinase USP9X
Authors: Baldwin Patrick R. , Kapoor Shivani , Natarajan Karthika , Trotta Rossana , Tron Adriana , Huszar Dennis , Davila Eduardo , Perrotti Danilo , Baer Maria R. 
Published: October 2019

Abstract
Internal tandem duplication (ITD) mutations of the receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) are present in acute myeloid leukemia (AML) cells in 30% of cases and are associated with high relapse rate and short disease-free survival following both chemotherapy and allogeneic hematopoietic stem cell transplantation. Inhibitors of FLT3 signaling have shown activity in clinical trials in FLT3-ITD AML, but efficacy has generally been limited and transient. Concurrent inhibition of other targets in FLT3-ITD signaling pathways is being explored as an approach to increasing the depth and duration of responses to FLT3 inhibitors.
The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD and phosphorylates and stabilizes FLT3, thereby promoting FLT3 signaling in a positive feedback loop in cells with FLT3-ITD. Pim kinase inhibitors are in clinical trials. We previously showed that combinations of clinically active Pim kinase and FLT3 inhibitors at pharmacologically relevant concentrations enhance apoptosis and decrease clonogenic growth of FLT3-ITD AML cell lines and primary patient cells in vitro and suppress growth of FLT3-ITD AML cells in vivo, in relation to treatment with FLT3 or Pim inhibitors alone. Here we studied the mechanistic effects of concurrent Pim kinase and FLT3 inhibition, demonstrating a novel mechanism of Mcl-1 downregulation in FLT3-ITD AML cells.
Ba/F3-ITD cells, transfected with FLT3-ITD, were cultured with the pan-Pim kinase inhibitor AZD1208 at 1 μM, a concentration chosen based on in vitro and phase I clinical trial data, and/or the FLT3 inhibitor quizartinib at 1 nM, its IC50 concentration, and expression of the anti-apoptotic proteins Mcl-1, Bcl2 and Bcl-xL and the pro-apoptotic proteins BAD/S112 p-BAD, BAK, BAX and Bim was measured by western blot analysis. Mcl-1 expression decreased in a time-dependent manner with AZD1208 and quizartinib co-treatment, but not with treatment with either inhibitor alone, while levels of the other proteins did not change. Mcl-1 downregulation with Pim kinase and FLT3 inhibitor combination treatment was then confirmed in the human FLT3-ITD AML cell lines MV4-11 and MOLM-14.
Mcl-1 expression is regulated at multiple levels, and we next sought to determine the mechanism(s) by which it is downregulated by concurrent Pim and FLT3 inhibition. While Mcl-1 protein levels decreased, Mcl-1 mRNA levels did not change, indicating post-transcriptional regulation. Additionally, levels of miR-29b, a negative regulator of Mcl-1 translation,decreased similarly in Ba/F3-ITD cells treated with AZD1208 and quizartinib, compared to quizartinib alone. Polysome profiling showed decreased total mRNA translation, but no selective reduction in Mcl-1 translation. In contrast, the progressive decrease in Mcl-1 protein expression with AZD1208 and quizartinib co-treatment was abrogated by addition of the proteasome inhibitor MG-132, demonstrating that Mcl-1 protein is downregulated by enhanced Mcl-1 proteasomal degradation. This mechanism was further confirmed by demonstration of an increase in ubiquitinated Mcl-1 prior to Mcl-1 downregulation in cells co-treated with AZD1208 and quizartinib, but not with each inhibitor alone or with DMSO control.
The deubiquitinase USP9X decreases Mcl-1 ubiquitination and consequent proteasomal degradation, and we found that USP9X expression is downregulated prior to the increase in ubiquitinated Mcl-1 and the subsequent decrease in Mcl-1 protein levels during AZD1208 and quizartinib co-treatment, but was not altered by treatment with either inhibitor alone. In contrast, expression of the ubiquitin E3 ligases Mule/ARF-BP1, SCFβ-TrCP and Trim17, which mediate Mcl ubiquitination, did not change prior to Mcl-1 downregulation.
Preclinical studies in our laboratory and others have shown in vitro and in vivo efficacy of combination treatment with Pim kinase and FLT3 inhibitors in FLT3-ITD AML, suggesting clinical promise of this approach. Here we show that, mechanistically, concurrent Pim kinase and FLT3 inhibition causes a post-translational decrease in expression of the anti-apoptotic protein Mcl-1 via enhanced proteasomal degradation, preceded by downregulation of the Mcl-1 deubiquitinase USP9X and an increase in ubiquitinated Mcl-1, a novel mechanism of Mcl-1 downregulation in FLT3-ITD AML cells.

Disclosures
Tron: AstraZeneca: Employment; AstraZeneca: Employment. Huszar:AstraZeneca: Employment.