Investigating the potential importance of Hedgehog signalling in conferring Venetoclax resistance in AML

Abouelnaga, Hager Salah Ali (2026) Investigating the potential importance of Hedgehog signalling in conferring Venetoclax resistance in AML. PhD thesis, University of Glasgow.

Abstract

Hedgehog (Hh) signalling pathway is a highly conserved pathway, which plays a crucial role in the development, maintenance and regeneration of human tissues. Its role in the primitive and definitive haematopoiesis has been extensively studied. Its activity has been proven to be a critical mechanism in the pathogenesis of tumours, either solid or haematological. It has a vital role in promoting the proliferation and survival of acute myeloid leukaemia (AML) cells, including leukaemia stem cells (LSCs). Moreover, it contributes to therapy resistance. Several Hh signalling pathway inhibitors have been generated and are involved in AML clinical trials. The outcomes of these trials are promising. Glasdegib is a Smoothened (SMO) inhibitor, which has been approved in combination with low-dose Cytarabine (LDAC) for newly diagnosed cases of AML in patients who are not fit for extensive chemotherapy. This follows the promising results of the BRIGHT AML 1003 trial.

The introduction of Venetoclax, a BCL2 inhibitor, into clinical practice marked a revolution in the treatment of leukaemia, particularly since the anti-apoptotic B-cell lymphoma 2 (BCL2) proteins were observed to be overexpressed in different types of leukaemia and are associated with chemoresistance. Testing Venetoclax for the newly diagnosed AML patients in the VIALE-A and VIALE-C trials demonstrated promising outcomes and superiority over low-dose chemotherapy alone regimens. Thus, in AML, Venetoclax has been approved for use in combination with Hypomethylating agents (HMA) (such as Azacitidine or Decitabine) or LDAC in newly diagnosed AML patients who are unfit for intensive chemotherapy.

Before that time, Glasdegib regimens were more involved in clinical trials and clinical settings; however, the promising efficacy achieved by Venetoclax resulted in the widespread adoption of the Venetoclax-based regimens. Venetoclax resistance has been a critical challenge hindering the efficacy of several leukaemia-treatment protocols.

This thesis aimed for the repurposing of the clinically approved drugs, Glasdegib and Venetoclax, to combat therapy resistance. This work investigated the potential involvement of the Hh signalling pathway in Venetoclax resistance in AML and whether its role in Venetoclax resistance is dependent on anti-apoptotic BCL2 proteins or not.

Venetoclax-resistant (VE-RE) cell line models have been established to investigate the potential mechanisms of resistance with particular focus on the role of Hh pathway. Inducing resistance was accomplished by exposing the Venetoclax-sensitive cells of MOLM-13 cells to increasing concentrations of Venetoclax in an intermittent (group 1) or continuous (group 2) manner. Venetoclax resistance was validated in VE-RE MOLM-13 groups 1 and 2 by comparing their half-maximal inhibitory concentration (IC50) to Venetoclax with that of the wild-type MOLM-13. There was an approximately 209-fold and 418-fold increase in the Venetoclax IC50s in VE-RE MOLM-13 group 1 and 2, respectively.

VE-RE MOLM-13 groups 1 and 2 cells were profiled at the transcriptomic and genomic levels to identify potential mechanisms of Venetoclax resistance. Additionally, Venetoclax-treated (VE-TR) MOLM-13 cells at 2nM, 4nM, and 6nM were profiled, as well, to determine the trend of change and the onset of the mutations, as it is believed that Venetoclax resistance develops gradually over time, rather than immediately upon exposure to Venetoclax. The expression of BCL2 family members and Hh signalling pathway was found to be deregulated. BCL2, Myeloid cell leukaemia-1 (MCL1), BCL2 Like 1 (BCLxL), Suppressor of Fused (SUFU) and Patched 1 (PTCH1) were significantly changed from the sensitive cells to the resistant cells.

Screening VE-RE cells for the potential causes underlying Venetoclax resistance revealed interesting, promising contributors. Based on the observations reported in clinical studies, these contributors provide valuable insights into Venetoclax resistance. Thereby, further investigations are warranted to explore their therapeutic implication in VE-RE AML. Among these potential contributors are Mitogen-Activated Protein Kinase Kinase Kinase Kinase 1 (MAP4K1), KIT Proto-Oncogene (KIT), Mesothelin (MSLN), S100 Calcium Binding Protein A4 (S1004A), N-Myristoyltransferase 2 (NMT2), Mouse Double Minute 4 (MDM4) and Transmembrane BAX Inhibitor Motif Containing 4 (TMBIM4). Several deregulated genes were found to have a significant association with adverse patient survival based on multiple AML datasets.

VE-RE MOLM-13 groups 1 and 2 cells were tested against Glasdegib alone and in combination with Venetoclax. Based on our preclinical results, the combination experiments between Glasdegib and Venetoclax have improved the sensitivity of the wild-type (Bliss synergy score of 23.40) and VE-RE MOLM-13 cells of both groups to Venetoclax (Bliss synergy score of 8.59 and -5.33, respectively). Studying the cell cycle kinetics changes upon adding Glasdegib to Venetoclax revealed a significant increase in the percentage of cells in the sub G0 phase in wild-type MOLM-13 and VE-RE MOLM-13 groups 1 and 2.

This combined effect can be explained by either inducing the quiescent leukaemic cells from G0 phase by Glasdegib to be targets for the action of Venetoclax, Glasdegib-mediated apoptosis, or Glasdegib-induced downregulation of anti-apoptotic BCL2 family proteins.

The Hh signalling pathway was found to depend, to some extent, on anti-apoptotic BCL2 family proteins in inducing Venetoclax resistance. This was shown by the role of Glasdegib in the downregulation of BCL2, MCL1, and BCLxL. This was detected following the treatment of wild-type MOLM-13, VE-RE MOLM-13 groups 1 and 2 by two concentrations of Glasdegib.

Venetoclax resistance is a collaboration of multiple mechanisms that carry variable intensities upon continuous or intermittent exposure to Venetoclax. Continuous exposure was concluded to cause a higher degree of Venetoclax resistance.

Finally, this work established a valuable fact, in which Hh signalling pathway is involved in the emerging Venetoclax resistance. Furthermore, activation of Hh signalling in VE-RE cells can occur in a canonical or non-canonical manner. Glasdegib-mediated blocking of the canonical activation of the Hh signalling improves the apoptotic effect of Venetoclax on the VE-RE MOLM-13 cells.

SUFU, which encodes for a GLI Family Zinc Finger (GLI) sequestering protein, is a valuable biomarker that warrants further investigation, particularly as it is significantly downregulated in VE-RE MOLM13 of both groups. Moreover, it has been observed from studying its effect on patient survival that its low expression has a significant association with adverse patient survival in the early follow-up period in cohorts of either AML or other malignancies.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: IP rights statement: Faculty Of Medicine Menoufia University, And Ministry Of Higher Education, Egypt. The funder of my Project.
• Subjects: R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer); R Medicine > RM Therapeutics. Pharmacology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Supervisor's Name: Copland, Professor Mhairi and Wheadon, Professor Helen
• Date of Award: 2026
• Depositing User: Theses Team
• Unique ID: glathesis:2026-85723
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 28 Jan 2026 14:04
• Last Modified: 28 Jan 2026 14:05
• Thesis DOI: 10.5525/gla.thesis.85723
• URI: https://theses.gla.ac.uk/id/eprint/85723