EHA Library - The official digital education library of European Hematology Association (EHA)

JAK1-STAT3 SIGNALING AXIS SUPPORTS LEUKEMIC STEM CELL PERSISTENCE IN CML
Author(s): ,
Maja Kim Kuepper
Affiliations:
Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Faculty of Medicine,RWTH Aachen University,Aachen,Germany
,
Oliver Herrmann
Affiliations:
Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Faculty of Medicine,RWTH Aachen University,Aachen,Germany
,
Ivan G. Costa
Affiliations:
Institute for Computational Genomics,RWTH Aachen University,Aachen,Germany
,
Steffen Koschmieder
Affiliations:
Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Faculty of Medicine,RWTH Aachen University,Aachen,Germany
,
Tim H. Bruemmendorf
Affiliations:
Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Faculty of Medicine,RWTH Aachen University,Aachen,Germany
,
Gerhard Mueller-Newen
Affiliations:
Institute of Biochemistry and Molecular Biology,RWTH Aachen University,Aachen,Germany
Mirle Schemionek
Affiliations:
Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Faculty of Medicine,RWTH Aachen University,Aachen,Germany
(Abstract release date: 05/17/18) EHA Library. Schemionek-Reinders M. 06/17/18; 214571; S1551
Mirle Schemionek-Reinders
Mirle Schemionek-Reinders
Contributions
Abstract

Abstract: S1551

Type: Oral Presentation

Presentation during EHA23: On Sunday, June 17, 2018 from 08:15 - 08:30

Location: Room A2

Background
Chronic myeloid leukemia (CML) is driven by the chromosomal translocation t(9;22) which transforms hematopoietic stem cells into leukemic stem cells (LCS). Blocking the oncogenic Bcr-Abl kinase by tyrosine kinase inhibitors (TKI) has greatly improved CML therapy, but is rarely curative as the disease-initiating LCS survive within the bone marrow (BM) niche independently of Bcr-Abl kinase activity. Recently, the signal transducer and activator of transcription 3 (STAT3) was shown to be highly activated in TKI-resistant CML cells that were supported by the BM microenvironment. However, while targeting STAT3 in combination with Bcr-Abl was potent to kill TKI-resistant LCS, the activating mechanism of persisting STAT3 activation remains unclear.

Aims
In this study, we first analyzed STAT3 activation in TKI treated leukemic cells, and how it is influenced by the BM microenvironment. Subsequently, we aimed to identify the mechanism allowing for TKI-persisting STAT3 activation and evaluated the therapeutic potential of combined Bcr-Abl and JAK1 inhibition using human cell lines, primary murine cells, as well as primary CML CD34+ cells.

Methods
Gene Set Enrichment Analysis (GSEA) was performed using transgenic murine CML and control LSK cells. We generated conditioned medium (CM) by harvesting the supernatant of primary human mesenchymal stroma cells (MSC). qRT-PCR was performed to analyze target gene expression. Phosphorylation of STAT3 (pSTAT3Y705) was evaluated by western blot upon Bcr-Abl (imatinib) and JAK1 (filgotinib/itacitinib) inhibition. Furthermore, an IL-6-blocking receptor fusion protein (RFP) was applied. Finally, colony forming unit (CFU) assay, CSFE staining and apoptosis analysis via flow cytometry of primitive CML CD34+ cells were performed.

Results
GSEA pathway analysis revealed increased JAK/STAT signaling in transgenic CML LSK cells. We observed elevated STAT3 mRNA expression in KCL-22 (1.9-fold, p≤0.001) and CML MNC (1.6-fold, p≤0.05), as well as increased pSTAT3Y705 levels in murine Bcr-Abl BM, KCL-22 and CML-MNC upon imatinib treatment, exclusively in presence of MSC-derived CM. In the presence of JAK1- but not JAK2-specific inhibitors, persisting pSTAT3Y705 was decreased to basal levels in murine Bcr-Abl BM, KCL-22 and primary CML MNC. A similar effect was observed when applying an IL-6-blocking RFP. Combined inhibition of Bcr-Abl and JAK1 strongly reduced the CFU capacity of murine (2.2-fold, p≤0.001) and human CML cells (6.9-fold, p≤0.001) compared to IM treatment alone. Tracking the proliferation of CML CD34+ cells by CSFE staining revealed that Bcr-Abl- and JAK1-inhibited cells showed increased quiescence (3.2-fold, p≤0.001) and decreased proliferation (3.6-fold, p≤0.001) compared to single treatments. Interestingly, combined Bcr-Abl and JAK1 inhibition strongly induced apoptosis even in quiescent LSC (2.4-fold, p≤0.001).

Conclusion
In the presence of BM microenvironment-derived CM, STAT3 is upregulated in Bcr-Abl leukemic cells upon oncogene inhibition. Here, we identified JAK1 as the STAT3-activating kinase in CML cells. JAK1 inhibition by TKIs decreases pSTAT3Y705 levels, blocks proliferation and reduces the CFU capacity. Upon Bcr-Abl and JAK1 inhibition, apoptosis is strongly induced compared to IM treatment alone in quiescent LCS. Our data demonstrate that persistent STAT3 activation is observed under IM treatment and supported by the microenvironment via JAK1 thus promoting LSC survival. As a consequence, JAK1 emerges as a potential therapeutic target for curative CML therapies.

Session topic: 7. Chronic myeloid leukemia – Biology & Translational Research

Keyword(s): STAT3, Janus Kinase inhibitor, Leukemic Stem Cell

Abstract: S1551

Type: Oral Presentation

Presentation during EHA23: On Sunday, June 17, 2018 from 08:15 - 08:30

Location: Room A2

Background
Chronic myeloid leukemia (CML) is driven by the chromosomal translocation t(9;22) which transforms hematopoietic stem cells into leukemic stem cells (LCS). Blocking the oncogenic Bcr-Abl kinase by tyrosine kinase inhibitors (TKI) has greatly improved CML therapy, but is rarely curative as the disease-initiating LCS survive within the bone marrow (BM) niche independently of Bcr-Abl kinase activity. Recently, the signal transducer and activator of transcription 3 (STAT3) was shown to be highly activated in TKI-resistant CML cells that were supported by the BM microenvironment. However, while targeting STAT3 in combination with Bcr-Abl was potent to kill TKI-resistant LCS, the activating mechanism of persisting STAT3 activation remains unclear.

Aims
In this study, we first analyzed STAT3 activation in TKI treated leukemic cells, and how it is influenced by the BM microenvironment. Subsequently, we aimed to identify the mechanism allowing for TKI-persisting STAT3 activation and evaluated the therapeutic potential of combined Bcr-Abl and JAK1 inhibition using human cell lines, primary murine cells, as well as primary CML CD34+ cells.

Methods
Gene Set Enrichment Analysis (GSEA) was performed using transgenic murine CML and control LSK cells. We generated conditioned medium (CM) by harvesting the supernatant of primary human mesenchymal stroma cells (MSC). qRT-PCR was performed to analyze target gene expression. Phosphorylation of STAT3 (pSTAT3Y705) was evaluated by western blot upon Bcr-Abl (imatinib) and JAK1 (filgotinib/itacitinib) inhibition. Furthermore, an IL-6-blocking receptor fusion protein (RFP) was applied. Finally, colony forming unit (CFU) assay, CSFE staining and apoptosis analysis via flow cytometry of primitive CML CD34+ cells were performed.

Results
GSEA pathway analysis revealed increased JAK/STAT signaling in transgenic CML LSK cells. We observed elevated STAT3 mRNA expression in KCL-22 (1.9-fold, p≤0.001) and CML MNC (1.6-fold, p≤0.05), as well as increased pSTAT3Y705 levels in murine Bcr-Abl BM, KCL-22 and CML-MNC upon imatinib treatment, exclusively in presence of MSC-derived CM. In the presence of JAK1- but not JAK2-specific inhibitors, persisting pSTAT3Y705 was decreased to basal levels in murine Bcr-Abl BM, KCL-22 and primary CML MNC. A similar effect was observed when applying an IL-6-blocking RFP. Combined inhibition of Bcr-Abl and JAK1 strongly reduced the CFU capacity of murine (2.2-fold, p≤0.001) and human CML cells (6.9-fold, p≤0.001) compared to IM treatment alone. Tracking the proliferation of CML CD34+ cells by CSFE staining revealed that Bcr-Abl- and JAK1-inhibited cells showed increased quiescence (3.2-fold, p≤0.001) and decreased proliferation (3.6-fold, p≤0.001) compared to single treatments. Interestingly, combined Bcr-Abl and JAK1 inhibition strongly induced apoptosis even in quiescent LSC (2.4-fold, p≤0.001).

Conclusion
In the presence of BM microenvironment-derived CM, STAT3 is upregulated in Bcr-Abl leukemic cells upon oncogene inhibition. Here, we identified JAK1 as the STAT3-activating kinase in CML cells. JAK1 inhibition by TKIs decreases pSTAT3Y705 levels, blocks proliferation and reduces the CFU capacity. Upon Bcr-Abl and JAK1 inhibition, apoptosis is strongly induced compared to IM treatment alone in quiescent LCS. Our data demonstrate that persistent STAT3 activation is observed under IM treatment and supported by the microenvironment via JAK1 thus promoting LSC survival. As a consequence, JAK1 emerges as a potential therapeutic target for curative CML therapies.

Session topic: 7. Chronic myeloid leukemia – Biology & Translational Research

Keyword(s): STAT3, Janus Kinase inhibitor, Leukemic Stem Cell

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