Internal Tandem Duplications of FLT3 Induces Increased ROS Production, DNA Damage and Misrepair: Implications for Genomic Instability and Disease Resistance in Myeloid Malignancies.
Authors: Fan Jinshui , Sallmyr Annahita , Kim Kyu-Te , Datta Kamal , Shapiro Paul , Small Donald , Rassool Feyruz V. 
Published: April 2020

Abstract
The FMS-like tyrosine kinase (FLT3), which belongs to the class III receptor tyrosine kinase family, is primarily expressed by hematopoietic cells and plays an important role in hematopoiesis. Activating mutations of FLT3 occur in approximately 30% of myeloid malignancies and, at least for the internal tandem duplications (ITDs) of the juxtamembrane region, are an independent predictor of poor clinical outcome. FLT3 signaling in leukemia includes activation of RAS, PI3 kinase/AKT pathways, and signal transducer and activator of transcription-5 (STAT5), leading to increased cellular proliferation and survival. We have previously shown that N-RAS activation can initiate a cycle of genomic instability whereby increased reactive oxygen species (ROS) leads to double strand breaks (DSB) and error-prone repair in myeloid malignancies. Virtually nothing is known about the role of FLT3 mutations in genomic instability. Here we report that FLT3/ITD signaling can lead to genomic instability that may be driven by increased ROS production. We find that mouse myeloid progenitor cells (32D) stably expressing FLT3/ITD (32D/ITD), demonstrate significantly increased ROS production compared with isogenic controls, as measured by flow cytometric analysis of the fuorescent probe, 2’,7’-dichlorofluoresecein (H2DCHF-DA) which is oxidized in the presence of ROS. FLT3/ITD cells treated with small molecule tyrosine kinase inhibitors (TKI) of FLT3 significantly reduced ROS levels. Intriguingly, we find that phosphorylated STAT5 (pSTAT5), which is greatly stimulated by mutant FLT3 signaling, binds RAC1-GTP, an essential component of ROS-producing NADPH oxidase. This novel finding suggests that STAT5 may have unique non-genomic functions involved in regulating RAC1 activation and ROS generation. FLT3 mutant cells treated with siRNA to down-regulate STAT5, show significantly reduced ROS. Inhibition of RAC1 and NADPH oxidase in FLT3 mutant cells also leads to reduced ROS levels, providing further evidence for the involvement of RAC1 in ROS production. While RAC1 activity is unchanged by FLT3 inhibition, significantly less RAC1 binds NADPH oxidase protein GP91phox in the cell membrane. Therefore pSTAT5 binding to RAC1-GTP may be necessary for NADPH oxidase activity and ROS production. Importantly, human leukemia cell lines (MOLM-14 & MV-4-11) and primary samples (N=3) with naturally occurring FLT3/ITD also show significantly decreased ROS in a dose- and time-dependent manner, following treatment with FLT3 TKI, confirming the mouse cell line data. Blocking ROS production by inhibition of FLT3 in AML cells significantly decreased γH2AX foci formation, suggesting reduced DSB. An in vitro DSB repair assay based on plasmid rejoining in nuclear extracts was used to assess repair efficiency and fidelity. Higher repair efficiency and fidelity was observed following inhibition of the FLT3-mediated endogenous ROS production in FLT3/ITD-positive human AML cells. Our data serve as a model system to further study the role of FLT3-mediated increased ROS in propagating genomic instability and for therapeutic targeting in an effort to stabilize the genome and reduce the accumulation of additional mutations in these cases of AML.