Using induced pluripotent stem cells (IPSCS) as a replacement for in vivo models to screen novel therapies in chronic myeloid leukaemia (CML)

Toofan, Parto (2016) Using induced pluripotent stem cells (IPSCS) as a replacement for in vivo models to screen novel therapies in chronic myeloid leukaemia (CML). PhD thesis, University of Glasgow.

Abstract

Tyrpsine kinase inhibitors (TKIs) effectively target progenitors and mature leukaemic cells but prove less effective at eliminating leukaemic stem cells (LSCs) in patients with chronic myeloid leukaemia (CML). Several reports indicate that the TGFβ superfamily pathway is important for LSC survival and quiescence. We conducted extensive microarray analyses to compare expression patterns in normal haemopoietic stem cells (HSC) and progenitors with CML LSC and progenitor populations in chronic phase (CP), accelerated phase (AP) and blast crisis (BC) CML. The BMP/SMAD pathway and downstream signalling molecules were identified as significantly deregulated in all three phases of CML. The changes observed could potentiate altered autocrine signalling, as BMP2, BMP4 (p<0.05), and ACTIVIN A (p<0.001) were all down regulated, whereas BMP7, BMP10 and TGFβ (p<0.05) were up regulated in CP. This was accompanied by up regulation of BMPRI (p<0.05) and downstream SMADs (p<0.005). Interestingly, as CML progressed, the profile altered, with BC patients showing significant over-expression of ACTIVIN A and its receptor ACVR1C. To further characterise the BMP pathway and identify potential candidate biomarkers within a larger cohort, expression analysis of 42 genes in 60 newly diagnosed CP CML patient samples, enrolled on a phase III clinical trial (www.spirit-cml.org) with greater than 12 months follow-up data on their response to TKI was performed. Analysis revealed that the pathway was highly deregulated, with no clear distinction when patients were stratified into good, intermediate and poor response to treatment.
One of the major issues in developing new treatments to target LSCs is the ability to test small molecule inhibitors effectively as it is difficult to obtain sufficient LSCs from primary patient material. Using reprogramming technologies, we generated induced pluripotent stem cells (iPSCs) from CP CML patients and normal donors. CML- and normal-derived iPSCs were differentiated along the mesodermal axis to generate haemopoietic and endothelial precursors (haemangioblasts). IPSC-derived haemangioblasts exhibited sensitivity to TKI treatment with increased apoptosis and reduction in the phosphorylation of downstream target proteins.
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Dual inhibition studies were performed using BMP pathway inhibitors in combination with TKI on CML cell lines, primary cells and patient derived iPSCs. Results indicate that they act synergistically to target CML cells both in the presence and absence of BMP4 ligand. Inhibition resulted in decreased proliferation, irreversible cell cycle arrest, increased apoptosis, reduced haemopoietic colony formation, altered gene expression pattern, reduction in self-renewal and a significant reduction in the phosphorylation of downstream target proteins. These changes offer a therapeutic window in CML, with intervention using BMP inhibitors in combination with TKI having the potential to prevent LSC self-renewal and improve outcome for patients. By successfully developing and validating iPSCs for CML drug screening we hope to substantially reduce the reliance on animal models for early preclinical drug screening in leukaemia.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: CML, leukaemia, BMP pathway, iPSCs, stem cell.
• Subjects: Q Science > Q Science (General); R Medicine > R Medicine (General)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences > Paul O'Gorman Leukemia Research Centre
• Supervisor's Name: Wheadon, Dr. Helen and Copland, Prof. Mhairi
• Date of Award: 2016
• Embargo Date: 28 October 2020
• Depositing User: Miss Parto Toofan
• Unique ID: glathesis:2016-7729
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 04 Nov 2016 14:47
• Last Modified: 16 Feb 2020 18:41
• URI: https://theses.gla.ac.uk/id/eprint/7729