POTENTIAL COMBINATIONAL THERAPY OF LEUKEMIC STEM CELLS USING BONE MORPHOGENETIC PROTEIN AND TYROSINE KINASE INHIBITORS.
Author(s): ,
Caroline Busch
Affiliations:
The Centre for the Cellular Microenvironment,University of Glasgow,Glasgow,United Kingdom
,
Helen Wheadon
Affiliations:
Paul O'Gorman Leukaemia Research Centre,University of Glasgow,Glasgow,United Kingdom
,
Mhairi Copland
Affiliations:
Paul O'Gorman Leukaemia Research Centre,University of Glasgow,Glasgow,United Kingdom
Catherine Berry
Affiliations:
The Centre for the Cellular Microenvironment,University of Glasgow,Glasgow,United Kingdom
EHA Library. Busch C. Jun 14, 2019; 266266; PF466
Caroline Busch
Caroline Busch
Contributions
×
Abstract

Abstract: PF466

Type: Poster Presentation

Presentation during EHA24: On Friday, June 14, 2019 from 17:30 - 19:00

Location: Poster area

Background

Chronic myeloid leukemia (CML) disease outcomes have improved since the introduction of tyrosine kinase inhibitors (TKIs), however a proportion of patients will have TKI resistance and TKIs are not curative in the majority of patients. Low-levels of Bcr-Abl transcripts persist and a major clinical aim is to understand mechanism underlying disease persistence and the role of leukemic stem cells (LSCs) in this. To date it has proved difficult to both understand how LSCs dominate and alter the niche, and to target LSCs with current therapies. Previous studies have highlighted the importance of the bone morphogenetic protein (BMP) pathway in CML LSCs with altered stem cell fate, persistence and the response to BMP antagonists affecting cell behavior with altered cell cycle, apoptosis and expansion (Laperrousaz, B. et al, Blood, 2013; Grockowiak, E. et al, Blood, 2017).

Aims
To investigate the effect of BMP pathway inhibition in combination with TKIs in primary CML samples to improve insights and identify new potential therapeutic approaches.

Methods
Primary CML CD34+cells were treated with Imatinib (IM) or Saracatinib (SC), a Src/Abl dual inhibitor, alone or in combination with BMP inhibitor Dorsomorphin (DOR). Additionally, cell cultures were supplemented with soluble BMP4 or co-cultured with the human stromal cell line HS-5. Apoptosis, cell cycle and proliferation were investigated by flow cytometry. RNA was harvested, cDNA generated and pre-amplified prior to the performance of multiplex Real-Time PCR (Fluidigm) on 89 lineage-specific, cell cycle, proliferation, and survival genes. Furthermore, we investigated tyrosine kinase activity in cell lysates of primary CML patient samples post single or dual treatment of SC and DOR using a chip-based microarray assay from PamGene containing 196 peptides. Kinase phosphorylation activities measured on peptides were further evaluated by relating them to kinases that are able to phosphorylate a particular site on the peptide sequence.

Results
Combinatorial treatments targeting the BMP pathway together with TKIs display a synergistic mode of action. Increases in late apoptosis (p<0.0002), altered cell cycle (G2-M, p=0.0058), fewer cell divisions, and reduced numbers of CD34+cells were observed in dual treatments. BMP4 supplementation revealed similar differences whereas stroma co-cultures displayed a protective effect. Furthermore, multiplex quantitative PCR has revealed significant deregulation of genes involved in cell cycle,apoptosis and differentiation, such as CDKN1a, CDKN2b, BCL6, GATA1 and SMAD5 showing differential expression in relation to response. Upstream kinase analysis using the PamGene platform, revealed kinases involved in cell growth, development, differentiation, apoptosis and cell-cell adhesion. Amongst the top 20 potential differentially expressed kinases after SC treatment, were the expected members of the Src family kinases. DOR treatment revealed an up regulation of the largest known subfamily of receptor tyrosine kinases, the Ephrin receptors and Met, a single pass tyrosine kinase receptor that had already been identified as a potential therapy target in CML for IM resistant cells.

Conclusion
Our results highlight the potential of a combined approach of targeting the BMP pathway with BMP antagonists or small inhibitors together with TKIs, which could open up new therapeutic possibilities. Future experiments will assess the potential of combinatorial treatment in a 3D bone marrow model, comprising magnetically levitated Mesenchymal Stem Cell spheroids embedded in medical-grade collagen type I.

Session topic: 23. Hematopoiesis, stem cells and microenvironment

Keyword(s): Chronic myeloid leukemia, Leukemic stem cell, Tyrosine kinase inhibitor

By continuing to browse or by clicking “Accept Terms & all Cookies”, you agree to the storing of third-party cookies on your device to enhance your user experience and agree to the user terms and conditions of this learning management system (LMS).

Cookie Settings
Accept Terms & all Cookies