Cap Analysis of Gene Expression (CAGE) Sequencing Reveals Alterations of the Transcriptional Signatures of FLT3-ITD with Secondary D835 TKD Mutations in Acute Myeloid Leukemia
Authors: Tabe Yoko , Sekihara Kazumasa , Saitoh Kaori , Monma Norikazu , Ikeo Kazuho , Kaczkowski Bogumil , Zhang Weiguo , Yamanaka Yasunari , Miida Takashi , Shah Neil P. , Harada Hironori , Hayashizaki Yoshihide , Konopleva Marina , Andreeff Michael 
Published: October 2019

Abstract
Mutations in the gene encoding Fms-like tyrosine kinase-3 (FLT3) are found in approximately 30% of acute myeloid leukemia (AML) patients, with two distinct groups of mutations; the internal tandem duplication mutations in juxtamembrane domain (FLT3-ITD) and the point mutations within the tyrosine kinase domains (FLT3-TKD).
The second-generation tyrosine kinase inhibitor (TKI) quizartinib which primarily targets an inactive (type II) kinase conformation, has demonstrated responses in relapsed/refractory FLT3-ITD AML patients. However, duration of response to quizartinib is limited largely by secondary mutations in FLT3-TKD, most commonly at D835 activation loop, emerging in at least 20% of patients.
To elucidate the alterations of transcript signatures of quizartinib-resistant FLT3-ITD/D835 mutants in AML, we performed cap analysis of gene expression (CAGE) sequencing utilizing 11 FLT3-ITD and 6 FLT3-ITD/D835 primary AML cells with wild type NPM1. CAGE identifies and quantifies the 5' ends of capped mRNA transcripts, which enables the identification of transcription start sites (TSS) and allows investigating promoter structures necessary for gene expression. The TSS of genes altered in AML cells with FLT3-ITD/D835 compared with FLT3-ITD were mapped, and CAGE detected upregulation of 124 genes and downregulation of 25 genes in FLT3-ITD/D835 cells (FDR < 0.05). Specifically, several genes closely linked with activation of MAPK signaling (MAPK6, TCF4), AKT/mTOR signaling (NBN) and both (TNFAIP6) were upregulated in the D835 acquired cells. Expression of inflammatory response cytokine genes IL1B and IL8 were also increased in FLT3-ITD/D835 cells.
The FLT related functional pathway analysis by KEGG ontology indicated that Ras pathway with its downstream Raf/MAPK and PI3K/AKT/mTOR signaling including transcription factor NFKB are the most significantly activated pathways in FLT3-ITD/D835 cells compared to FLT3-ITD cells. No additional activation of STAT5 pathway, a direct downstream signaling of mutated FLT3, was found in FLT3-ITD/D835 cells.
To assess the underlying molecular basis of transcription signature, we performed an unbiased search for enriched sequence motifs using HOMER software. HOMER revealed upregulation of 80 motifs and downregulation of 51 motifs in AML cells with FLT3-ITD/D835 compared with FLT3-ITD (q < 0.05). Motifs of AP-1, JUN, CEBP, ATF and NFKB were substantially enriched in FLT3-ITD/D835 compared to FLT3-ITD cells. ETS, SPI-1 and RUNX1 were the most common binding motifs altered (both increased and decreased) by D835 mutation acquisition. Of note, AP-1 modulates inflammatory response and is tightly regulated by MAPK.
To validate the transcriptional changes and the highlighted gene ontology results of AML primary samples, we utilized paired isogenic FLT3-ITD or FLT3-ITD/D835Y transfected Ba/F3 cell lines. As predicted, type II kinase inhibitor quizartinib was active against FLT3-ITD Ba/F3 cells (IC50; 0.6nM) but not FLT3-ITD/D835Y Ba/F3 cells (IC50; > 5000nM). Type I kinase inhibitor crenolanib was highly active against both FLT3-ITD (IC50; 2.1nM) and FLT3-ITD/D835Y cells (IC50; 16.4nM). Focusing on FLT related Ras pathway, we first investigated MAPK activation in these cells. Immunoblot analysis demonstrated substantial reduction of phospho- (p-) ERK after quizartinib (5nM) or crenolanib (50nM) treatment both in FLT3-ITD and FLT3-ITD/D835Y cells, irrespective of their susceptibility. We next examined p-S6, the target of PI3K/AKT/mTOR and MAPK signaling, and observed that crenolanib but not quizartinib successfully diminished p-S6 expression in FLT3-ITD/D835Y cells. FLT3-ITD/D835 cells showed higher baseline expression of NFkB regulator p-IkB compared to FLT3-ITD cells, which was not repressed by crenolanib.
In conclusion, our study showed that the acquired D835 mutation in FLT3-ITD AML cells facilitates transcriptional network activation to induce proinflammatory genes through Ras-MAPK-NF-κB/AP-1 pathway and Ras-PI3K-AKT-mTOR signaling. This transcriptional modulation promotes oncogenic pro-survival and proliferative signaling in AML cells and defines differential sensitivity against Type I and Type II kinase inhibitors.

Disclosures
Shah: ARIAD: Research Funding; BMS: Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Research Funding; Plexxikon: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.