Browsing by Author "Hirade, Tomohiro"

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    Internal Tandem Duplication in FLT3 Attenuates Proliferation and Regulates Resistance to the FLT3 Inhibitor AC220 by Modulating p21Cdkn1a and Pbx1 in Hematopoietic Cells
    (Public Library of Science (PLoS), 2016) Abe, Mariko; Pelus, Louis M.; Singh, Pratibha; Hirade, Tomohiro; Onishi, Chie; Purevsuren, Jamiyan; Taketani, Takeshi; Yamaguchi, Seiji; Fukuda, Seiji; Department of Microbiology and Immunology, IU School of Medicine
    Internal tandem duplication (ITD) mutations in the Fms-related tyrosine kinase 3 (FLT3) gene (FLT3-ITD) are associated with poor prognosis in patients with acute myeloid leukemia (AML). Due to the development of drug resistance, few FLT3-ITD inhibitors are effective against FLT3-ITD+ AML. In this study, we show that FLT3-ITD activates a novel pathway involving p21Cdkn1a (p21) and pre-B cell leukemia transcription factor 1 (Pbx1) that attenuates FLT3-ITD cell proliferation and is involved in the development of drug resistance. FLT3-ITD up-regulated p21 expression in both mouse bone marrow c-kit+-Sca-1+-Lin- (KSL) cells and Ba/F3 cells. The loss of p21 expression enhanced growth factor-independent proliferation and sensitivity to cytarabine as a consequence of concomitantly enriching the S+G2/M phase population and significantly increasing the expression of Pbx1, but not Evi-1, in FLT3-ITD+ cells. This enhanced cell proliferation following the loss of p21 was partially abrogated when Pbx1 expression was silenced in FLT3-ITD+ primary bone marrow colony-forming cells and Ba/F3 cells. When FLT3-ITD was antagonized with AC220, a selective inhibitor of FLT3-ITD, p21 expression was decreased coincident with Pbx1 mRNA up-regulation and a rapid decline in the number of viable FLT3-ITD+ Ba/F3 cells; however, the cells eventually became refractory to AC220. Overexpressing p21 in FLT3-ITD+ Ba/F3 cells delayed the emergence of cells that were refractory to AC220, whereas p21 silencing accelerated their development. These data indicate that FLT3-ITD is capable of inhibiting FLT3-ITD+ cell proliferation through the p21/Pbx1 axis and that treatments that antagonize FLT3-ITD contribute to the subsequent development of cells that are refractory to a FLT3-ITD inhibitor by disrupting p21 expression.
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    Internal tandem duplication mutations in FLT3 gene augment chemotaxis to Cxcl12 protein by blocking the down-regulation of the Rho-associated kinase via the Cxcl12/Cxcr4 signaling axis
    (American Society for Biochemistry and Molecular Biology, 2014-11-07) Onish, Chie; Mori-Kimachi, Satomi; Hirade, Tomohiro; Abe, Mariko; Taketani, Takeshi; Suzumiya, Junji; Sugimoto, Toshitsugu; Yamaguchi, Seiji; Kapur, Reuben; Fukuda, Seiji; Department of Pediatrics, IU School of Medicine
    Internal tandem duplication mutations in the Flt3 gene (ITD-FLT3) enhance cell migration toward the chemokine Cxcl12, which is highly expressed in the therapy-protective bone marrow niche, providing a potential mechanism underlying the poor prognosis of ITD-FLT3(+) acute myeloid leukemia. We aimed to investigate the mechanisms linking ITD-FLT3 to increased cell migration toward Cxcl12. Classification of the expression of Cxcl12-regulated genes in ITD-FLT3(+) cells demonstrated that the enhanced migration of ITD-FLT3(+) cells toward Cxcl12 was associated with the differential expression of genes downstream of Cxcl12/Cxcr4, which are functionally distinct from those expressed in ITD-FLT3(-) cells but are independent of the Cxcr4 expression levels. Among these differentially regulated genes, the expression of Rock1 in the ITD-FLT3(+) cells that migrated toward Cxcl12 was significantly higher than in ITD-FLT3(-) cells that migrated toward Cxcl12. In ITD-FLT3(-) cells, Rock1 expression and Mypt1 phosphorylation were transiently up-regulated but were subsequently down-regulated by Cxcl12. In contrast, the presence of ITD-FLT3 blocked the Cxcl12-induced down-regulation of Rock1 and early Mypt1 dephosphorylation. Likewise, the FLT3 ligand counteracted the Cxcl12-induced down-regulation of Rock1 in ITD-FLT3(-) cells, which coincided with enhanced cell migration toward Cxcl12. Rock1 antagonists or Rock1 shRNA abolished the enhanced migration of ITD-FLT3(+) cells toward Cxcl12. Our findings demonstrate that ITD-FLT3 increases cell migration toward Cxcl12 by antagonizing the down-regulation of Rock1 expression. These findings suggest that the aberrant modulation of Rock1 expression and activity induced by ITD-FLT3 may enhance acute myeloid leukemia cell chemotaxis to the therapy-protective bone marrow niche, where Cxcl12 is abundantly expressed.
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    The magnitude of CXCR4 signaling regulates resistance to quizartinib in FLT3/ITD+ cells via RUNX1
    (Elsevier, 2023) Fukuda, Seiji; Matsuda, Nozomi; Shoji, Tsukimi; Onishi, Chie; Hirade, Tomohiro; Taketani, Takeshi; Pelus, Louis M.; Microbiology and Immunology, School of Medicine
    CXCR4 antagonists sensitize FLT3/ITD+ AML cells to FLT3 inhibitors; however, CXCR4 signaling can induce apoptosis in AML cells, raising the question of whether CXCR4 signaling exerts divergent effects on FLT3/ITD+ cells. The present study investigated the paradoxical function of CXCR4 in resistance to FLT3 inhibitors. The FLT3 inhibitor quizartinib significantly decreased the number of FLT3/ITD+ Ba/F3 cells, whereas 1 ng/ml CXCL12 showed a significant protective effect against quizartinib. In contrast, CXCL12 over 100 ng/ml significantly decreased FLT3/ITD+ cell viability with concomitant downregulation of Runx1. Moreover, the survival of FLT3/ITD+ Ba/F3 or MOLM13 cells with low surface CXCR4 expression incubated with quizartinib was significantly enhanced by 100 ng/ml CXCL12; however, this protective effect of CXCL12 against quizartinib was barely detected in cells with high surface CXCR4 expression. Although silencing Runx1 downregulated CXCR4 expression, RUNX1 expression levels were significantly higher in CXCR4LOW FLT3/ITD+ Ba/F3 cells incubated with 100 ng/ml CXCL12 than in CXCR4HIGH cells, coincident with an increase in FLT3 phosphorylation. Silencing RUNX1 partially abrogated resistance to quizartinib in CXCR4LOW cells incubated with CXCL12, whereas ectopic RUNX1 significantly restored resistance in CXCR4HIGH cells. These results indicate that CXCR4 signaling of different magnitudes paradoxically regulates resistance to quizartinib in FLT3/ITD+ cells via RUNX1.