Development of novel Acute Myeloid Leukaemia therapy: targeting the leukaemic bone marrow protective niche secretome to enhance chemotherapy sensitivity

Elmarghany, Ahmed Bahaaeldin Nabih (2025) Development of novel Acute Myeloid Leukaemia therapy: targeting the leukaemic bone marrow protective niche secretome to enhance chemotherapy sensitivity. PhD thesis, University of Glasgow.

Abstract

Acute myeloid leukaemia (AML) is a heterogeneous and complex disease that is rapidly proliferative with a high rate of relapse. The bone marrow microenvironment (BMME) is known to be important in chemoresistance in AML, but the exact mechanism is not fully explored. Studies have shown that interleukin-6 (IL-6) contributes to AML survival. Our previously published data showed that AML blasts are protected from chemotherapy by interaction with the BMME. Investigating cytokines produced by bone marrow stromal cell (BMSC) using a co-culture (CC) system showed that IL-6 is present at a significantly higher concentration in CC compared to both liquid culture (LC) and BMSC cultured alone, showing that the interaction of the AML blasts and BMSC produce higher levels of IL-6 than they would in monocultures.

IL-6 is recognised as one of the key cytokines released by BMSC. Numerous studies are investigating its role in enhancing the viability of AML cells. IL-6 triggers the JAK-STAT signalling pathway, leading to the phosphorylation of STAT3, a transcription factor that regulates various genes associated with proliferation and survival. Moreover, research has identified a STAT-independent pathway involving IL-6. When IL-6 binds to its receptor and the co-receptor gp130, it not only activates the JAK-STAT pathway but also stimulates SRC family kinases.

It was hypothesised that such BMME-mediated chemoresistance is partly owing to cytokines and chemokines secreted by BMSC, the main cellular component of the BMME, such as IL-6, which can promote AML chemoresistance through stimulation of the JAK-STAT and SRC signalling. We aimed to study the prosurvival role of BMSC-secreted cytokines and signalling pathways involved in chemoprotection and how to abolish their effect to improve therapeutic outcomes. Inhibiting both pathways using ruxolitinib, a licensed JAK inhibitor, and dasatinib, a licensed SRCinhibitor, could increase AML sensitivity to standard chemotherapy.

Three core-binding factor AML cell lines (Kasumi-1 and SKNO-1 expressing t(8;21), and ME-1 expressing inv(16)), one non-core binding factor cell line (THP-1), and one human stromal cell line (HS5) were used to investigate AML cell lines and BMSC secretome. ProcartaPlex™ immunoassay with different cytokines was performed on the supernatant of Kasumi-1, ME-1, and THP-1 cell suspension alone and CC (direct contact and transwell) with HS5 cells. Moreover, we assessed the prosurvival role of IL-6, VEGF, HGF, and SDF-1a when combined with cytosine arabinoside (Ara-C) at its EC50 on the Kasumi-1 cell line using flow cytometry for apoptosis by Annexin / DAPI staining. We found a protective effect of IL-6 treatment when cells were pre-treated with IL-6 before Ara-C treatment in the Kasumi-1 cell line regarding the percentage of late apoptotic cells (Annexin +/ DAPI +), not the percentage of surviving cells (Annexin - / DAPI -). Using Luminex® technology, from 11 cytokines analysed, there were five cytokines (GM-CSF, TNF-a, IL-2, IL-6, IL-18) present at a higher level in CC compared to Kasumi-1, ME-1, THP-1 or HS5 cells cultured alone. IL-2 and IL-6 were significantly increased in CC compared to Kasumi-1 monoculture; GM-CSF, TNF-a and IL-2 were significantly higher in ME-1 CC compared to LC. With THP-1 cells, IL-2 and IL-18 were found to be higher in CC compared to LC.

Using cell lines that showed HS5 chemoprotection, we assessed SRC signalling pathway gene expression such as SRC, YAP, and CYR61 by qPCR in our in vitro CC system. Next, the concentration of dasatinib was determined by western blot.Apoptosis and cell cycle analyses elucidated the effect of CC, with and without dasatinib, on leukaemia cell viability. A significant increase in SRC gene expression (p=0.015, n=4) and total and phospho-SRC protein were observed when ME-1 cells were co-cultured on the HS5 cell line compared to suspension culture. A decrease in AML cell viability by apoptosis assay and an increase in subG1 in cell cycle analysis were noticed when AML cells within the CC system were treated with dasatinib (1nM) and Ara-C at its EC50, in comparison to treatment with Ara-C alone. This indicated that CC conferred protection to AML cells against Ara-C, which dasatinib could partly reverse.

Next, using the Kasumi-1 cell line, JAK-STAT signalling by flow cytometry using phospho-STAT3 (Y705) in our in vitro co-culture (CC) system was assessed either in direct contact or via transwell with Kasumi-1 to investigate the role of cytokines secreted by HS5. Then, the concentration of ruxolitinib required to inhibit pY705was determined. Apoptosis (Annexin / DAPI) and cell cycle analyses (Ki-67 / DAPI)elucidated the effect of CC, with and without ruxolitinib, on leukaemia cell viability.

A significant increase was observed in STAT3 pY705 when Kasumi-1 cells were co-cultured on the HS5 cell line compared to suspension culture (p=0.001, n=5). Treatment in CC with ruxolitinib (0.5µM) significantly reduced Kasumi-1 pY705 (46% inhibition of phosphorylation; p= 0.0023, n=5), proving that ruxolitinib inhibited the CC-induced JAK-STAT signalling. A significant decrease was noticed in AML cell viability by apoptosis assay (p= 0.0026), with a significant increase in subG1 in cell cycle analysis (p=0.0001) when AML cells, within the CC system, were treated with ruxolitinib (0.5µM) and Ara-C in comparison to treatment with Ara-C alone. This indicated that CC conferred protection to AML cells against Ara-C and could be partly reversed by ruxolitinib.

Overall, the BMME has an important role in providing AML chemoresistance through various cytokines secreted; we can confirm that IL-6 may provide chemoprotection for the Kasumi-1 cell line when treated with the standard chemotherapy agent,Ara-C. Targeting signalling pathways could abolish BMSC-mediated chemoresistance; ruxolitinib and dasatinib could serve as adjuvant chemotherapy to Ara-C in AML.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QH Natural history > QH301 Biology; Q Science > QR Microbiology; Q Science > QR Microbiology > QR180 Immunology; R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Supervisor's Name: Copland, Professor Mhairi and Jørgensen, Dr. Heather
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-85497
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 03 Oct 2025 11:26
• Last Modified: 03 Oct 2025 11:28
• Thesis DOI: 10.5525/gla.thesis.85497
• URI: https://theses.gla.ac.uk/id/eprint/85497

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