Follistatin is a novel therapeutic target and biomarker in FLT 3/ ITD acute myeloid leukemia
2020; Springer Nature; Volume: 12; Issue: 4 Linguagem: Inglês
10.15252/emmm.201910895
ISSN1757-4684
AutoresBai‐Liang He, Ning Yang, Cheuk Him Man, Nelson Ka‐Lam Ng, Chae‐Yin Cher, Ho‐Ching Leung, Leo Lai‐Hok Kan, Bowie Yik‐Ling Cheng, Stephen Lam, Michelle Lu‐Lu Wang, Chunxiao Zhang, Hin Kwok, Grace Cheng, Rakesh Sharma, Alvin Chun‐Hang, Chi Wai Eric So, Yok‐Lam Kwong, Anskar Y.H. Leung,
Tópico(s)Retinoids in leukemia and cellular processes
ResumoArticle5 March 2020Open Access Follistatin is a novel therapeutic target and biomarker in FLT3/ITD acute myeloid leukemia Bai-Liang He Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Guangdong Provincial Key Laboratory of Biomedical Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, Guangdong Province, China Search for more papers by this author Ning Yang Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Cheuk Him Man Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Nelson Ka-Lam Ng Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Chae-Yin Cher Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Ho-Ching Leung Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Leo Lai-Hok Kan Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Bowie Yik-Ling Cheng Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Stephen Sze-Yuen Lam Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Michelle Lu-Lu Wang Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Chun-Xiao Zhang Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Hin Kwok Centre for Genomic Sciences, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Grace Cheng Centre for Genomic Sciences, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Rakesh Sharma Centre for Genomic Sciences, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Alvin Chun-Hang Ma Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong SAR, China Search for more papers by this author Chi-Wai Eric So orcid.org/0000-0002-4117-0036 Leukemia and Stem Cell Biology Group, Division of Cancer Studies, Department of Hematological Medicine, King's College London, London, UK Search for more papers by this author Yok-Lam Kwong Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Anskar Yu-Hung Leung Corresponding Author [email protected] orcid.org/0000-0001-9975-8687 Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Bai-Liang He Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Guangdong Provincial Key Laboratory of Biomedical Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, Guangdong Province, China Search for more papers by this author Ning Yang Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Cheuk Him Man Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Nelson Ka-Lam Ng Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Chae-Yin Cher Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Ho-Ching Leung Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Leo Lai-Hok Kan Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Bowie Yik-Ling Cheng Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Stephen Sze-Yuen Lam Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Michelle Lu-Lu Wang Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Chun-Xiao Zhang Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Hin Kwok Centre for Genomic Sciences, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Grace Cheng Centre for Genomic Sciences, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Rakesh Sharma Centre for Genomic Sciences, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Alvin Chun-Hang Ma Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong SAR, China Search for more papers by this author Chi-Wai Eric So orcid.org/0000-0002-4117-0036 Leukemia and Stem Cell Biology Group, Division of Cancer Studies, Department of Hematological Medicine, King's College London, London, UK Search for more papers by this author Yok-Lam Kwong Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Anskar Yu-Hung Leung Corresponding Author [email protected] orcid.org/0000-0001-9975-8687 Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Search for more papers by this author Author Information Bai-Liang He1,2,‡, Ning Yang1,‡, Cheuk Him Man1, Nelson Ka-Lam Ng1, Chae-Yin Cher1, Ho-Ching Leung1, Leo Lai-Hok Kan1, Bowie Yik-Ling Cheng1, Stephen Sze-Yuen Lam1, Michelle Lu-Lu Wang1, Chun-Xiao Zhang1, Hin Kwok3, Grace Cheng3, Rakesh Sharma3, Alvin Chun-Hang Ma4, Chi-Wai Eric So5, Yok-Lam Kwong1 and Anskar Yu-Hung Leung *,1 1Division of Hematology, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China 2Guangdong Provincial Key Laboratory of Biomedical Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, Guangdong Province, China 3Centre for Genomic Sciences, The University of Hong Kong, Hong Kong SAR, China 4Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong SAR, China 5Leukemia and Stem Cell Biology Group, Division of Cancer Studies, Department of Hematological Medicine, King's College London, London, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +86 (852)22553347; Fax: +86 (852)29741165; E-mail: [email protected] EMBO Mol Med (2020)12:e10895https://doi.org/10.15252/emmm.201910895 Correction(s) for this article Follistatin is a novel therapeutic target and biomarker in FLT3/ITD acute myeloid leukemia07 August 2020 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Internal tandem duplication of Fms-like tyrosine kinase 3 (FLT3/ITD) occurs in about 30% of acute myeloid leukemia (AML) and is associated with poor response to conventional treatment and adverse outcome. Here, we reported that human FLT3/ITD expression led to axis duplication and dorsalization in about 50% of zebrafish embryos. The morphologic phenotype was accompanied by ectopic expression of a morphogen follistatin (fst) during early embryonic development. Increase in fst expression also occurred in adult FLT3/ITD-transgenic zebrafish, Flt3/ITD knock-in mice, and human FLT3/ITD AML cells. Overexpression of human FST317 and FST344 isoforms enhanced clonogenicity and leukemia engraftment in xenotransplantation model via RET, IL2RA, and CCL5 upregulation. Specific targeting of FST by shRNA, CRISPR/Cas9, or antisense oligo inhibited leukemic growth in vitro and in vivo. Importantly, serum FST positively correlated with leukemia engraftment in FLT3/ITD AML patient-derived xenograft mice and leukemia blast percentage in primary AML patients. In FLT3/ITD AML patients treated with FLT3 inhibitor quizartinib, serum FST levels correlated with clinical response. These observations supported FST as a novel therapeutic target and biomarker in FLT3/ITD AML. Synopsis FLT3/ITD is one of the most commonly mutated genes in acute myeloid leukemia, an aggressive hematological malignancy with poor prognosis. Here, embryonic morphogen FST is identified as a novel biomarker and therapeutic target for human FLT3/ITD-mutated acute myeloid leukemia. The embryonic morphogen FST was overexpressed in FLT3/ITD-expressing zebrafish, Flt3/ITD knock-in mice and human FLT3/ITD AML. A novel FLT3/ITD-p90RSK-CREB-FST signaling cascade was uncovered in human AML. FST is a novel therapeutic target and biomarker in FLT3/ITD AML. The paper explained Problem Acute myeloid leukemia (AML) is an aggressive hematological malignancy with distinct cytogenetic, genetic, and clinicopathogenic features. Intensive chemotherapy and hematopoietic stem cell transplantation are the mainstays of treatment; however, treatment outcome is dismal. FLT3/ITD mutation occurs in about 30% of AML and is associated with poor prognosis. FLT3 inhibitors have been shown to be effective in FLT3/ITD AML; however, additional mutations of FLT3 confer drug resistance and treatment failure. It would be critical to identify new pathogenetic signals in FLT3/ITD AML. Results The present study demonstrates the following: (i) Ectopic expression of FLT3/ITD induces axis duplication in zebrafish embryos; (ii) the axis-duplicated embryos are associated with upregulation of a secreted morphogen FST; (iii) FST is a CREB target gene, which is consistently overexpressed in FLT3/ITD-expressed zebrafish, Flt3/ITD knock-in mice, and human FLT3/ITD AML; (iv) overexpression of FST potentiates MAPK/ERK signaling to promote leukemia cell growth; and (v) targeting FST reduces FLT3/ITD+ AML cell growth in vitro and in vivo. Impact This study suggests that FST is a novel therapeutic target and biomarker in human FLT3/ITD-mutated AML. Introduction Acute myeloid leukemia (AML) is characterized by an abnormal increase in myeloblasts in peripheral blood (PB) and bone marrow (BM). It is a heterogeneous disease with distinct clinicopathologic, cytogenetic, and genetic features in individual patients (Dohner et al, 2015). In young patients, intensive chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT) are the mainstays of treatment and 30–40% patients can achieve long-term remission (Papaemmanuil et al, 2016). In elderly patients not eligible for standard therapy, the outcome is poor. Specific driver genetic mutations occur in different subtypes of AML, and therapies targeting mutations in AML have emerged: midostaurin (Stone et al, 2017) in combination with induction chemotherapy for upfront treatment and gilteritinib monotherapy (Perl et al, 2017) for relapsed or refractory disease in AML with FMS-like tyrosine kinase 3 (FLT3) mutations; and ivosidenib (DiNardo et al, 2018) and enasidenib (Stein et al, 2017) for relapsed or refractory AML with isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) mutations. Strategies targeting other genetic mutations and their activated cellular pathways are urgently needed (Lam et al, 2017). Internal tandem duplication of FLT3 (FLT3/ITD) occurs in about 30% of AML and is associated with poor treatment outcome (Patel et al, 2012; Smith et al, 2012). Constitutive activation of FLT3 arising from ITD activates downstream signals including PI3K/AKT, STAT5, and Ras/MEK/ERK (Mizuki et al, 2000; Martelli et al, 2006; Zhang et al, 2016). FLT3 inhibitors have been shown to be effective in FLT3/ITD AML (Assi & Ravandi, 2018). However, tyrosine kinase domain (TKD) mutations in FLT3 confer drug resistance and are an important cause of treatment failure (Man et al, 2012; Smith et al, 2017). Identifying new pathogenetic signals in FLT3/ITD AML may result in novel therapeutic strategies. Follistatin (FST) was first identified from follicular fluid in the ovary and shown to suppress secretion of follicle-stimulating hormone from the anterior pituitary gland (Ueno et al, 1987). FST has been shown to express in different tissues (Phillips & de Kretser, 1998) and antagonizes activin A, a member of the TGF-β (transforming growth factor-β) superfamily (Cash et al, 2012). Alternative splicing of FST generates FST317 and FST344 that encode FST288 and FST315 proteins (Shimasaki et al, 1988), the latter being the predominant circulatory form. Fst had been reported to be regulated by CREB, FoxL2, and Smad3 in mouse gonadotrophic cell lines (Winters et al, 2007; Blount et al, 2009), by FoxO1 in mouse hepatocytes (Tao et al, 2018), and by Nrf2 in pulmonary epithelial cells (Lin et al, 2016). In mice, Fst knockout resulted in early postnatal mortality with multiple defects in muscles, skin, and bones (Matzuk et al, 1995). In zebrafish and Xenopus embryos, fst overexpression led to a dose-dependent dorsalization phenotype and, when ventrally expressed, induced a secondary body axis (Fainsod et al, 1997). In humans, FST has been implicated in the pathogeneses of prostate (Sepporta et al, 2013), ovarian (Di Simone et al, 1996), and liver (Rossmanith et al, 2002) cancers, and hyperglycemia (Tao et al, 2018). Mechanistically, both antagonism of TGF-β signaling and inhibition of cellular rRNA synthesis during glucose deprivation have been reported (Gao et al, 2010). In this study, we demonstrated a hitherto undescribed pathogenetic role of FST in human AML with particular reference to FLT3/ITD. It originated from an unexpected finding that human FLT3/ITD induced ectopic fst expression during early embryonic development in zebrafish and caused axis duplication and dorsalization. Induction of FST expression by FLT3/ITD could be recapitulated in adult FLT3/ITD-transgenic zebrafish, Flt3/ITD knock-in mice, and human FLT3/ITD primary AML-derived murine xenografts, which was mediated by CREB phosphorylation. FST upregulated expression of RET, IL2RA, and CCL5 that collectively potentiated MAPK signaling and promoted leukemia growth in vitro and in vivo. FST targeting by shRNA, CRISPR/Cas9, and antisense oligo suppressed leukemia growth in vitro and in vivo. Serum FST correlated with clinical response to specific FLT3 inhibitor and subsequent leukemic progression in FLT3/ITD AML patients. These findings underscored the potential role of FST in AML as a new therapeutic target and biomarker during treatment with FLT3 inhibitors. Results Ectopic expression of FLT3/ITD induced axis duplication in zebrafish embryo Previously, we demonstrated that expression of human FLT3/ITD by plasmid DNA injection in zebrafish embryos induced expansion of myelopoiesis reminiscent of the hematopoietic phenotype of Flt3/ITD knock-in mouse (He et al, 2014). In this study, FLT3/ITD was ectopically expressed in zebrafish embryos by mRNA injection. Intriguingly, axis duplication and dorsalization were observed in 15.2 ± 1.3 and 34.7 ± 3.2% of FLT3/ITD, but not FLT3/WT mRNA-injected embryos on 1 dpf (day post-fertilization) (Fig 1A–D; Appendix Fig S1A–J). This phenotype was not observed in FLT3/ITD plasmid DNA-injected embryos in our previous study (He et al, 2014). Axis duplication was confirmed by whole-mount in situ hybridization (WISH) of notochord-specific marker col9a2 (Fig 1E–H). Constitutive phosphorylation and activation of FLT3 downstream signals STAT5, AKT, and ERK were confirmed in 293FT transfectant (Fig 1I) and zebrafish embryos (Fig 1J). Importantly, a specific FLT3 inhibitor quizartinib ameliorated the dorsalization and axis duplication anomalies in a dose-dependent fashion (Fig 1K), confirming the link between activation of flt3 signaling and the morphologic anomalies. Figure 1. Overexpression of FLT3/ITD induced axis duplication and ectopic expression of FST in zebrafish embryos A–D. The morphology of uninjected, FLT3/WT mRNA, and FLT3/ITD mRNA-injected (150 ng per embryo) embryos on day 2 post-fertilization (dpf). E–H. Whole-mount in situ hybridization (WISH) of notochord-specific marker col9a2 in uninjected, FLT3/WT mRNA, and FLT3/ITD mRNA-injected embryos on 2 dpf. I, J. FLT3 signaling was detected by Western blotting in 293FT cells transfected with FLT3/ITD mRNA (I) or in zebrafish embryos injected with FLT3/ITD mRNA (J). K. The effect of FLT3 inhibitor quizartinib (Qui) on the dorsalization and axis duplication phenotype induced by FLT3/ITD mRNA injection in zebrafish. L–N. Quantification of fst expression by RT–qPCR (L), Western blotting (M), and WISH (N) after FLT3/ITD overexpression in zebrafish embryos at 6 hpf. Data information: ov: otic vesicles; cm: cephalic mesoderm; nc: notochord. Scale bar = 500 μm. In (K and L), the experiments were performed in triplicates and the data are presented as mean ± SEM. *P < 0.05 and **P < 0.01 (Student's t-test). NS, not significant. Source data are available online for this figure. Source Data for Figure 1 [emmm201910895-sup-0007-SDataFig1.tif] Download figure Download PowerPoint FLT3/ITD upregulated FST expression in zebrafish and human AML These developmental defects suggested disruption of normal morphogen gradients critical for dorsoventral (D-V) patterning and axis formation in vertebrates (De Robertis et al, 2000), including follistatin (fst) (Fainsod et al, 1997), goosecoid (gsc) (Steinbeisser et al, 1995), and chordin (chd) (Sasai et al, 1994). Therefore, their expression in FLT3/ITD-injected embryos was examined. The mRNA and protein expression of fst was significantly increased at shield stage (6 hpf) by 1.7- and 1.9-fold (Fig 1L and M). Ectopic expression of fst (Fig 1N) and goosecoid (gsc) was also shown by WISH (Appendix Fig S1K) and GFP reporter assay (Appendix Fig S1L–N). Consistently, upregulation of fst was also observed in FLT3/ITD plasmid DNA-injected embryos at 36 hpf, which could be effectively blocked by quizartinib treatment (Fig 2A–C). The relevance of fst to adult hematopoiesis was examined in transgenic zebrafish where human FLT3/ITD was expressed in hematopoietic stem cells (HSCs) (Fig 2D–K). FLT3/ITD expanded the myeloid and hematopoietic progenitor cell populations (Fig 2L–N) from whole kidney marrow (KM) (equivalence of mammalian BM) of adult transgenic zebrafish, where fst expression was significantly increased (Fig 2O). Figure 2. FST was increased in FLT3/ITD-transgenic zebrafish and FLT3/ITD-mutated AML A–C. WISH of fst in FLT3/WT (A), and FLT3/ITD plasmid DNA-injected zebrafish embryos without (B) or with (C) quizartinib treatment (2.5 μM) from 6 to 36 hpf. fst expression was expanded by FLT3/ITD DNA in 86% of embryos (B, arrow, 32/37) which could be effectively blocked by treating with FLT3 inhibitor quizartinib in 83% of embryos (C, 29/35). D–K. Generation and characterization of FLT3/ITD-transgenic zebrafish. Diagrammatic representation (D and E) of the generation of Runx1-FLT3/ITD-transgenic zebrafish (see Materials and Methods section). GFP expression was detected by fluorescent microscopy (F–H) and in blood circulation and thymus by WISH (I and J, blue arrow) in WT sibling and Runx1-FLT3/ITD-transgenic zebrafish (F1) embryos at 4 dpf. FLT3/ITD-positive zebrafish (F1) were confirmed by PCR genotyping of GFP and FLT3/ITD using genomic DNA from fin clip of WT siblings and Runx1-FLT3/ITD-transgenic zebrafish (F1) at 2 months old. Fish 4, 5, and 6 showed germline transmission of FLT3/ITD transgene (K). L–N. Kidney marrow (KM) was collected from Runx1-FLT3/ITD-transgenic zebrafish (F1) at 18 months old. The morphology and hematopoietic composition of KM from WT siblings (n = 6) and Runx1-FLT3/ITD-transgenic (n = 6) zebrafish were examined by Giemsa staining (L) and flow cytometry (M, N) (abbreviation for panel M: M, myeloid cells; P, progenitor cells; L, lymphoid cells; E, erythroid cells). Data are presented in box plot. The whiskers, boxes, and central lines in panel N represented the minimum-to-maximum values, 25th-to-75th percentile, and the 50th percentile (median), respectively. **P < 0.01 (Student's t-test). O. Expression of fst was detected by RT–qPCR in KM from WT sibling and Runx1-FLT3/ITD-transgenic zebrafish at 18 months old. The RT–qPCR experiments were performed in triplicates, and data were presented as mean ± SEM. **P < 0.01. P. Detection of FST expression, p-ERK1/2, and p-CREB in mononuclear cells from normal peripheral blood stem cell (PBSC) and FLT3/ITD AML patients (diagnostic samples with leukemia blasts > 80%) by Western blotting. ^: non-specific staining of p-ATF1 protein due to the conserved motif. Data information: Scale bar = 500 μm. Source data are available online for this figure. Source Data for Figure 2 [emmm201910895-sup-0008-SDataFig2.tif] Download figure Download PowerPoint In silico gene expression analysis based on BloodSpot database showed that total FST expression was upregulated in different cytogenetically defined AML subtypes relative to normal HSC (Appendix Fig S2A). Consistent with previous studies, isoform-specific RT–PCR showed that FST344 was the predominant FST transcript in FLT3/ITD AML cell lines MOLM-13 and MV4-11 (Appendix Fig S2B and C). Endogenous FST was expressed preferentially in the cytoplasm of FLT3/ITD-positive MOLM-13 cells at the perinuclear zone (Appendix Fig S2D). Total FST was significantly upregulated in primary myeloblasts obtained from FLT3/ITD AML patients relative to those in mobilized peripheral blood stem cells (PBSCs) from healthy donors (Fig 2P) and FLT3/WT AML samples (Appendix Fig S2E–G). Moreover, transfection of FLT3/ITD led to a significant upregulation of total FST in HeLa cells (Appendix Fig S2H). FST is a CREB target gene in FLT3/ITD AML In silico analysis of transcription factor (TF) binding sites in FST promoter was performed. Binding sites for cAMP-response element binding protein (CREB) are over-represented (Fig 3A). Direct binding of p-CREB to FST promoter in FLT3/ITD-positive MOLM-13 cells was confirmed by ChIP-qPCR. Compared with normal IgG control, there was a sixfold increase in DNA binding to p-CREB in FST promoter (Fig 3B and C). Dual-luciferase reporter assay demonstrated that deletion of CREB-binding site on FST promoter abolished the effect of CREB-mediated FST upregulation (Fig 3D). Isogenic Ba/F3 cells transduced with FLT3/ITD was used to test the regulation between FLT3/ITD and FST. Consistently, FLT3/ITD, its downstream signals including p-STAT5, p-ERK1/2, p-AKT, and p-4E-BP1, and FST expression were increased in Ba/F3-FLT3/ITD cells compared to the parental cells (Fig 3E). CREB phosphorylation was also significantly increased (Fig 3F). Quizartinib that suppressed FLT3 signaling (Appendix Fig S2I) reduced CREB phosphorylation (Fig 3G), Fst transcription, and expression (Fig 3H) in Ba/F3-FLT3/ITD cells. CREB had been reported to be activated by p90RSK (a.k.a. RSK1) (Sakamoto & Frank, 2009), which is a known downstream effector of FLT3/ITD-ERK signaling cascade (Elf et al, 2011). Indeed, inhibition of p90RSK by BRD7389 effectively reduced FST expression and phosphorylation of CREB in MOLM-13 (Fig 3I and J) and Ba/F3-FLT3/ITD cells (Fig 3J and K). Consistently, a doxycycline-inducible system showed that knockout of p90RSK by CRISPR/Cas9 resulted in significant decrease in FST expression in MOLM-13 cell line (Fig 3L). Specifically, CREB inhibitor 666-15 (Kang et al, 2015) or knockout by CRISPR/Cas9 also decreased FST expression (Fig 3M and N) and CREB inhibitor 666-15 reduced cell growth of Ba/F3-FLT3/ITD without supplemental IL-3, but not Ba/F3 parental cells or Ba/F3-FLT3/ITD cells supplemented by IL-3 (Fig 3O). Furthermore, 666-15 treatment partially rescued the morphologic anomalies induced by FLT3/ITD in zebrafish embryos (Fig 3P) without observable toxicity, underscoring the preferential role of CREB in FLT3/ITD signaling. These observations demonstrated the presence of a FLT3-p90RSK-CREB-FST signaling axis that might be relevant in the pathogenesis of FLT3/ITD. Figure 3. FLT3/ITD upregulated FST through phosphorylation of CREB A. In silico analysis (DECipherment of DNA Elements, SABiosciences) and schematic model of transcription factor binding sites on human FST promoter. CBP: CREB-binding protein; CRE: cAMP-response element; TSS: transcription start site. B, C. The direct binding of p-CREB to human FST promoter was detected by ChIP-PCR (B) and ChIP-qPCR (C). c-Fos was used as positive control of p-CREB target gene. Normal IgG was used as negative control of ChIP. D. Dual-luciferase assay demonstrating the direct binding of p-CREB on human FST promoter. pRL-CMV, Renilla luciferase vector; pGL-CRE− and pGL-CRE+, firefly luciferase expression driven by human FST promoter with deleted CRE site (CRE−) or wild type (CRE+); p-GFPSpark, GFP-expressing vector; p-CREBY134F, CREBY134F-GFP-expressing vector. E. FST expression and FLT3/ITD signaling were detected by Western blotting in Ba/F3-parental (P in short) and Ba/F3-FLT3/ITD (ITD in short) cells. F–H. Phospho-flow analysis of p-CREB in Ba/F3-parental, Ba/F3-FLT3/ITD, and Ba/F3-FLT3/ITD cells treated with FLT3 inhibitor quizartinib (Qui in short). Isotype antibody was used as control to calculate the mean fluorescence intensity (MFI) ratio (F, G). The transcription and expression of Fst were detected by RT–qPCR after quizartinib treatment (10 nM) in Ba/F3-FLT3/ITD cells for 1 day (H). I–K. The expression of FST and phosphorylation of CREB were detected by Western blotting (I and K) and phospho-flow analysis (J) in MOLM-13 (I) and Ba/F3-FLT3/ITD (K) cells treated with quizartinib and BRD7389 for 1 day, respectively. L. RSK expression and FST expression were detected by Western blotting after p90RSK knockout by CRISPR/Cas9 in MOLM-13 cells. M. The phosphorylation of CREB and FST expression was detected by Western blotting in Ba/F3-FLT3/ITD cells treated with CREB inhibitor 666-15 for 1 day. ^: non-specific staining of p-ATF1 protein due to the conserved motif. N. CREB expression and FST expression were detected by Western blotting after CREB knockout by CRISPR/Cas9 in MOLM-13 cells. O. The growth of Ba/F3-parental (with IL-3), Ba/F3-FLT3/ITD (without IL-3), and Ba/F3-FLT3/ITD (with IL-3) cells was measured after 3 days treatment of CREB inhibitor 666-15 in vitro. P. The rescue effect of CREB inhibitor 666-15 on FLT3/ITD-induced dorsalization and axis duplication in zebrafish embryos at 1 dpf. Data information: In (C, D, G, H, J, and O), the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P < 0.01 and ***P < 0.001 (Student's t-test). Source data are available online for this figure. Source Data for Figure 3 [emmm201910895-sup-0009-SDataFig3.tif] Download figure Download PowerPoint FST potentiates MAPK/ERK signaling to promote leukemia cell growth To delineate the pathogenetic roles of FST in AML, FST was overexpressed in the AML line ML-2, which showed the lowest endogenous FST expression (Fig 4A). Overexpression of the two spliced forms of FST (FST317 and FST344) was confirmed at mRNA and protein levels (Fig 4B) and was shown to enhance cell growth (Fig 4C) and clonogenicity (Fig 4D and E) in vitro, respectively. Transplantation of ML-2 cells overexpressing either of these spliced variants into NSG mice demonstrated increased leukemia engraftment (Fig 4F and G) and shortened animal survival (Fig 4H). Figure 4. FST promoted leukemia growth by activating ERK A. FST expression in different AML cell lines was detected by Western blotting. B, C. FST317 and FST344 overexpression resulted in significant increases in FST transcription by RT–qPCR and protein by Western blot (B) and promoted ML-2 cell growth in vitro (C). Green, ML-2-GFP; blue, ML-2-FST317; red, ML-2-FST344. The RT–qPCR experiments were performed in triplicates (B). D, E. The clonogenicity of ML-2 overexpressing GFP, FST317, and FST344 in vitro for 14 days. The CFU experiments were performed in triplicates
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