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The Actin Binding Protein Plastin-3 Is Involved in the Pathogenesis of Acute Myeloid Leukemia

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The Actin Binding Protein Plastin-3 Is Involved in the Pathogenesis of Acute Myeloid Leukemia cancers Article The Actin Binding Protein Plastin-3 Is Involved in the Pathogenesis of Acute Myeloid Leukemia Arne Velthaus 1, Kerstin Cornils 2,3, Jan K. Hennigs 1, Saskia Grüb 4, Hauke Stamm 1, Daniel Wicklein 5, Carsten Bokemeyer 1, Michael Heuser 6, Sabine Windhorst 4, Walter Fiedler 1 and Jasmin Wellbrock 1,* 1 Department of Oncology, Hematology and Bone Marrow Transplantation with Division of Pneumology, Hubertus Wald University Cancer Center, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; [email protected] (A.V.); [email protected] (J.K.H.); [email protected] (H.S.); [email protected] (C.B.); fi[email protected] (W.F.) 2 Department of Pediatric Hematology and Oncology, Division of Pediatric Stem Cell Transplantation and Immunology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; [email protected] 3 Research Institute Children’s Cancer Center Hamburg, 20246 Hamburg, Germany 4 Center for Experimental Medicine, Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; [email protected] (S.G.); [email protected] (S.W.) 5 Department of Anatomy and Experimental Morphology, University Cancer Center, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; [email protected] 6 Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, 20246 Hannover, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-40-7410-55606 Received: 29 September 2019; Accepted: 25 October 2019; Published: 26 October 2019 Abstract: Leukemia-initiating cells reside within the bone marrow in specialized niches where they undergo complex interactions with their surrounding stromal cells. We have identified the actin-binding protein Plastin-3 (PLS3) as potential player within the leukemic bone marrow niche and investigated its functional role in acute myeloid leukemia. High expression of PLS3 was associated with a poor overall and event-free survival for AML patients. These findings were supported by functional in vitro and in vivo experiments. AML cells with a PLS3 knockdown showed significantly reduced colony numbers in vitro while the PLS3 overexpression variants resulted in significantly enhanced colony numbers compared to their respective controls. Furthermore, the survival of NSG mice transplanted with the PLS3 knockdown cells showed a significantly prolonged survival in comparison to mice transplanted with the control AML cells. Further studies should focus on the underlying leukemia-promoting mechanisms and investigate PLS3 as therapeutic target. Keywords: Acute myeloid leukemia; Plastin-3; prognostic marker 1. Introduction Acute myeloid leukemia (AML) has still a dismal prognosis due to persistence of minimal residual disease (MRD) which results in high relapse rates. Chemotherapy-resistant and therefore non-targetable leukemia-initiating cells are the cause for MRD [1,2]. The leukemia-initiating cells reside within the bone marrow (BM) in specialized niches where they undergo complex interactions with their surrounding stromal cells [3]. This microenvironment offers protection against external influences [4]. One of the major goals of modern therapeutic approaches is thus to elucidate the complex signaling within the leukemic bone marrow niche as it might represent a rich source of novel therapeutic targets. Cancers 2019, 11, 1663; doi:10.3390/cancers11111663 www.mdpi.com/journal/cancers Cancers 2019, 11, 1663 2 of 12 Cancers 2019, 11, x 2 of 12 In the current study, we investigated the impact of Plastin-3 (PLS3) in acute myeloid leukemia as we hadIn the identified current study, PLS3 aswe potential investigated player thewithin impact the of Plastin-3 leukemic (PLS3) bone marrowin acute nichemyeloid through leukemia gene expressionas we had analysis identified of PLS3 AML as and potential endothelial player cell within co-cultures. the leukemic bone marrow niche through gene expressionPlastins, analysis which areof AML also calledand endothelial fimbrins, belongcell co-cultures. to a subclass of actin-binding proteins known as actin bundlingPlastins, proteinswhich are that also can called cross-link fimbrins, actin belong filaments to a subclass into higher-order of actin-bi assembliesnding proteins like known bundles. as In mammals,actin bundling three isoformsproteins that have can been cross-link characterized: actin filaments Plastin-1 into (PLS1 higher-order or I-Plastin) assemblies is specifically like bundles. expressed inIn the mammals, small intestine, three isoforms colon and have kidney. been characterized: Plastin-2 (PLS2 Plastin-1 or L-Plastin) (PLS1 isor predominantly I-Plastin) is specifically expressed inexpressed hematopoietic in the cells,small whileintestine, Plastin colon 3 (PLS3and kidney or T-plastin). Plastin-2 is (PLS2 described or L-Plastin) to be expressed is predominantly in cells of non-hematopoieticexpressed in hematopoietic origin [5,6 cells,]. In addition,while Plastin to its 3 actin-bundling(PLS3 or T-plastin) capacity, is descri PLS3bed may to be also expressed control actinin turnover,cells of stabilizationnon-hematopoietic and assembly origin [5,6]. [7]. In addition, to its actin-bundling capacity, PLS3 may also control actin turnover, stabilization and assembly [7]. 2. Results 2. Results 2.1. The Actin Binding Protein PLS3 Is Expressed in Primary AML Cells 2.1. The Actin Binding Protein PLS3 Is Expressed in Primary AML Cells In order to identify genes being implicated in the interaction of acute myeloid leukemia (AML) cells andIn order stromal to identify cells, we genes performed being implicated co-cultures in the of primaryinteraction AML of acute cells myeloid with primary leukemia endothelial (AML) cellscells and and screened stromal cells, for geneswe perfor thatmed were co-cultures up- or down-regulated of primary AML undercells with the primary co-culture endothelial conditions cells by microarrayand screened analysis for (Humangenes that Gene were 2.0 up- ST Array,or down-r Affymetrix,egulated Thermo under Fisherthe co-culture Scientific, conditions Waltham, by MA, USA).microarray Co-cultures analysis of CD34(Human positive Gene hematopoietic2.0 ST Array, Affymetrix, progenitor Thermo cells from Fisher healthy Scientific, donors Waltham, were analyzed MA, USA). Co-cultures of CD34 positive hematopoietic progenitor cells from healthy donors were in parallel. Our aim was the identification of genes that were exclusively expressed in the leukemic analyzed in parallel. Our aim was the identification of genes that were exclusively expressed in the cells. The actin binding protein PLS3 (Plastin-3) was identified to be up-regulated in the AML cells leukemic cells. The actin binding protein PLS3 (Plastin-3) was identified to be up-regulated in the co-cultured with endothelial cells (9.8 +/ 5.0 fold up-regulation) while no significant alteration was AML cells co-cultured with endothelial− cells (9.8 +/− 5.0 fold up-regulation) while no significant observed in the co-culture of CD34 positive hematopoietic progenitor cells and endothelial cells alteration was observed in the co-culture of CD34 positive hematopoietic progenitor cells and (1.6 +/ 1.3 fold up-regulation; Supplementary Table S1). Further RT-qPCR analysis revealed an endothelial− cells (1.6 +/− 1.3 fold up-regulation; Supplementary Table S1). Further RT-qPCR analysis n endogenousrevealed an PLS3endogenous expression PLS3 in expression about 50% inof about BM samples50% of BM from samples AML from patients AML ( patients= 25; Figure (n = 25;1a). TheFigure PLS3 1a). expression The PLS3 in primaryexpression AML in cellsprimary was verifiedAML cells at the was protein verified level at by the immunofluorescence protein level by (nimmunofluorescence= 9, Figure1b). In contrast, (n = 9, Figure mRNA 1b). expression In contrast, of PLS3mRNA was expression only detected of PLS3 in 2was of 12only AML detected cell lines in analyzed2 of 12 AML with cell Kasumi-1 lines analyzed showing with strong Kasumi-1 and THP-1 showing showing strong only and veryTHP-1 weak showing expression. only very Therefore, weak weexpression. concentrated Therefore, our functional we concentrated analysis our of PLS3function onal the analysis AML cellof PLS3 line on Kasumi-1. the AML cell line Kasumi-1. FigureFigure 1. 1.Expression Expression of of PLS3 PLS3 inin primaryprimary AMLAML samples. ( (aa)) The The mRNA mRNA expression expression of of primary primary AML AML samplessamples was was analyzed analyzed by by RT-qPCR RT-qPCR andand normalizednormalized to GAPDH. The The relative relative expression expression is isshown shown in in comparisoncomparison to to Kasumi-1 Kasumi-1 cells cells (expression(expression valuevalue of 1). ( (bb)) PLS3 PLS3 protein protein expression expression in in primary primary AML AML samplessamples was was analyzed analyzed by immunofluorescenceby immunofluorescence (green (green signals). signals). The panelThe panel upper upper left shows left shows the negative the controlnegative without control primary without antibody primary while antibody the lower while left the and lower right left panels and show right thepanels PLS3 show staining the ofPLS3 three primarystaining AML of three samples primary of varying AML samples PLS3 expression of varying intensity,PLS3 expression respectively. intensity, respectively. Cancers 2019, 11, 1663 3 of 12 2.2. Functional Analysis of PSL3 by Knockdown or Overexpression in Kasumi-1 Cells To study the functional effect of PLS3 on the cell biology,
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