DISSECTING THE ROLE OF CXCL4 IN PRIMARY MYELOFIBROSIS
Author(s): ,
Hélène Gleitz
Affiliations:
Hematology,Erasmus MC,Rotterdam,Netherlands
,
Aurelien Dugourd
Affiliations:
Joint Research Centre for Computational Biomedicine,RWTH Aachen,Aachen,Germany
,
Nils Leimkühler
Affiliations:
Hematology,Erasmus MC,Rotterdam,Netherlands
,
Inge Snoeren
Affiliations:
Hematology,Erasmus MC,Rotterdam,Netherlands
,
Stijn Fuchs
Affiliations:
Hematology,Erasmus MC,Rotterdam,Netherlands
,
Bella Banjanin
Affiliations:
Hematology,Erasmus MC,Rotterdam,Netherlands
,
Jessica Pritchard
Affiliations:
Hematology,Erasmus MC,Rotterdam,Netherlands
,
Remco Hoogenboezem
Affiliations:
Hematology,Erasmus MC,Rotterdam,Netherlands
,
Eric Bindels
Affiliations:
Hematology,Erasmus MC,Rotterdam,Netherlands
,
Julio Saez-Rodriguez
Affiliations:
Joint Research Centre for Computational Biomedicine,RWTH Aachen,Aachen,Germany
,
Rafael Kramann
Affiliations:
Division of Nephrology and Clinical Immunology,RWTH Aachen,Aachen,Germany
Rebekka Schneider
Affiliations:
Hematology,Erasmus MC,Rotterdam,Netherlands;Hematology, Oncology, Hemostaseology and Stem Cell Transplantation,RWTH Aachen,Aachen,Germany
EHA Library. Gleitz H. Jun 15, 2019; 267472; S887
Hélène Gleitz
Hélène Gleitz
Contributions
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Abstract

Abstract: S887

Type: Oral Presentation

Presentation during EHA24: On Saturday, June 15, 2019 from 16:15 - 16:30

Location: Elicium 1

Background

Primary myelofibrosis (PMF) is the prototypic example for bone marrow fibrosis and characterized by the progressive replacement of bone marrow (BM) cells by reticulin fibers. Most patients carry mutations in hematopoietic stem cells (HSCs) that activate the JAK-STAT pathway but the specific mechanisms that govern BM fibrosis remain unknown. We recently identified Gli1+ stromal cells as fibrosis-driving cells. The initial events that lead to Gli1+ cell expansion and activation remain to be investigated. Using genetic fate tracing experiments combined with RNA sequencing of Gli1+ cells in different stages of fibrosis we demonstrate that the platelet/megakaryocyte-specific chemokine CXCL4 is significantly up-regulated both in mutant HSCs but also Gli1+ cells in early stages, even before fibrosis is present in the bone marrow.

Aims

We now sought to investigate the role of CXCL4 in mutant hematopoietic cells in PMF in the fibrotic transformation of Gli1+ cells.

Methods

To determine whether Cxcl4 induction is one of early events leading to Gli1+ cell activation and migration, we performed genetic-fate tracing experiments using Cxcl4 genetic knockdown in hematopoietic cells in a murine model of thrombopoietin (ThPO)-induced myelofibrosis. Wild-type (WT) or Cxcl4-/- HSCs were transduced with ThPO-overexpressing or control cDNA and transplanted into bigenic Gli1CreERt2+/-;tdTomato+/- recipient mice, where Gli1-specific expression of tdTomato was induced with tamoxifen.

Results

CXCL4 knockdown completely ameliorated the MPN phenotype in ThPO-induced PMF and normalized thrombocyte and leukocyte counts. CXCL4-/- TPO mice retained normal levels of hemoglobin after transplant, together with a diminished loss of erythroid cells compared to WT TPO mice, indicating preserved hematopoiesis and less severe fibrosis. In line with this finding, grading of reticulin staining revealed less severe bone marrow fibrosis in the absence of CXCL4 in hematopoietic cells. Flow cytometric analysis of genetically-labeled Gli1+ cells  demonstrated a significant decrease in the frequency of tdTomato+ Gli1+ stromal cells in the absence of CXCL4 in HSCs, indicating a decrease in Gli1+ cell recruitment. As the pathognomonic dysplastic megakaryocytes in PMF were also restored in the absence of CXCL4, we hypothesized that megakaryocytes play a crucial role in the activation of Gli1+ cells and performed RNA sequencing of sort-purified megakaryocytes and Gli1+ cells. Knockdown of CXCL4 in megakaryocytes decreased VEGF and TGFb signaling in line with the amelioration of the fibrosis grade in the bone marrow. Gli1+ cells in ThPO induced fibrosis demonstrated a significant up-regulation of  inflammatory pathways such as NFκB, TNFα and Trail which was reduced in the absence of CXCL4, highlighting the inflammatory mediator role of Cxcl4. More detailed analysis and qt-RT-PCR validation demonstrated that Interleukin-6 (IL6) was mainly affected by CXCL4 knockdown.

Conclusion

We here propose a role for CXCL4 in the fibrotic transformation in PMF through stimulating pro-fibrotic programs in megakaryocytes and inflammation in the bone marrow microenvironment.

Session topic: 15. Myeloproliferative neoplasms - Biology & Translational Research

Keyword(s): Inflammation, Myelofibrosis

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