- Title
- Molecular and phosphoproteomic characterisation of FLT3 inhibitor resistance in acute myeloid leukaemia
- Creator
- Staudt Barreto, Dilana Elisabeth
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2022
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Acute myeloid leukaemia (AML) is the most common form of acute leukaemia in adults and the second most common leukaemia in children, with current 5-year survival rates of only 29%. Driver mutations in the FMS-like tyrosine kinase 3 (FLT3) gene are the most common mutations in AML, occurring in 25-30% of patients. The two most common mutations affecting FLT3 consist of an internal tandem duplication (FLT3-ITD; 25%), followed by a missense point mutation (FLT3-TKD; 5-10%). Currently, two-thirds of AML patients achieve complete remission (CR) following high dose chemotherapies. However, patients harbouring FLT3 mutations, particularly FLT3-ITD+, often relapse with treatment resistant disease following standard of care chemotherapies, mainly due to the clonal evolution of malignant resistant clones which is, in part, influenced by chemotherapy itself. Tyrosine kinase inhibitors (TKIs) targeting FLT3 are now used clinically, and have shown clinical benefit in FLT3-mutant AML patients in combination with standard of care chemotherapy. However, sustained FLT3 inhibition is often affected due to the development of secondary resistance, primarily through the acquisition of additional FLT3 point mutations that generate a double mutant receptor (FLT3-ITD/TKD). Currently, gilteritinib is the only FLT3 inhibitor approved as single agent in the resistance/relapse (R/R) setting. However, secondary mutations promoting gilteritinib resistance have already been reported, highlighting that improved therapies are still desperately needed, particularly for R/R AML. Therefore, the studies presented herein, aimed to provide a detailed analysis of the signalling pathways that lie downstream of this double mutant receptor, to help uncover the mechanisms underpinning FLT3-mediated targeted-therapy resistance, and to identify novel therapeutic targets to be used as future treatment targets in the clinic. In addition, we hoped to demonstrate the applicability of phosphoproteomics to complement genomics in clinical treatment design by identifying potential targets that are sometimes hidden to conventional genomic approaches. In the first results chapter of this thesis (Chapter 3), isogenic models at diagnosis (FLT3-ITD), and resistance (FLT3-ITD/D835V and FLT3-ITD/D835Y) were established, and differences in cell growth and treatment sensitivity across FLT3- mutations was assessed. We then performed global, quantitative, label-based analysis of the proteome, to elucidate protein expression changes in FLT3-ITD/D835 compared to FLT3-ITD mutant cells. This comparison revealed the first insights into the signalling changes associated with FLT3-ITD/TKD-mediated resistance to type II TKIs, including loss of redox control, and the subsequent activation of stress-associated survival mechanisms, particularly the upregulation of ERK/MAPK signalling in FLT3-ITD/D835 mutants. However, these findings provided only pathway predictions, accounting solely for changes in protein expression levels, whereas protein activity is further regulated by post-translational modifications (PTMs) that warranted further investigation. Furthermore, Chapter 3 revealed limitations in the applicability of our standard label-based methodology for the analysis of AML samples in real-time, mainly due to long and complex sample preparation procedures, observations that justified further exploration. Chapter 4 addresses these limitations, by providing an optimised, rapid, flexible, and reproducible protocol that helped us to employ phosphoproteomic profiling of AML diagnosis samples compared to their parental controls, and to cells that have developed resistance to TKIs. This technique called ‘Phospho Heavy-Labeled-Spiketide FAIMS Stepped-CV DDA’ (pHASED) reduced sample preparation time by half compared to our traditional approach. Phosphoproteomic analysis of isogenic models using our pHASED protocol confirmed the predictions identified by proteome analysis, including the reactivation of ERK/MAPK signalling in resistance but, most importantly, downstream activation of DNA repair mechanisms driven by ATM signalling. Notably, we also revealed that combinatorial inhibition of FLT3 and ATM signalling were synergistically cytotoxic in both models of FLT3-ITD/D835-mediated resistance, with treatment sensitivity correlating with signal pathway activation, particularly in FLT3-ITD/D835Y mutants. In Chapter 5 we described the establishment of an in vitro model of adaptive resistance to sorafenib using human paediatric AML cell lines. We hoped that phosphoproteomic profiling of adaptive resistance would help us to identify the consequences of resistance that resembles those seen in FLT3-ITD+ patients that undergo sorafenib treatment. Next generation sequencing analysis identified that sorafenib resistance in this setting resulted in the acquisition of an additional FLT3- Y842C mutation, thus generating a double mutant FLT3-ITD/Y842C receptor. This adaptive model of resistance validated the differences in cellular growth and treatment responses observed in isogenic cellular models of FLT3-ITD/D835 mutants (Chapter 3). Furthermore, it replicated our unbiased quantitative phosphoproteomic findings, including ERK/MAPK reactivation, and ATM signalling upregulation. Notably, analysis of this model uncovered increased phosphorylation of several key MEK/ERK/MAPK proteins, as well as increased protein expression and phosphorylation of important activating sites in ATM and PRKDC (DNA-PK) kinases, key players in DNA repair signalling. To investigate the proteome predictions of alterations in redox metabolism, reactive oxygen species (ROS) detection assays were performed, and high levels of ROS-mediated oxidative stress were confirmed in sorafenib resistant cells, potentially driving the ERK/MAPK-ATM survival signalling in this setting. Finally, we revealed activation of p53-mediated cell cycle checkpoints in resistance, likely explaining the decreased cellular growth presented by these cells, and further confirming DNA repair mechanisms promoting survival signalling in FLT3-ITD/TKD mutants. Combinatorial targeting of FLT3 and ATM signalling pathways has once more proven to be effective in FLT3-ITD/Y842C cell lines, and therefore provided rationale for preclinical evaluation of this novel drug combination in the R/R setting. Together, these findings highlighted a redox-driven feedback loop of ERK/MAPK-ATM signalling activation that maintains cell survival in stressed conditions, and therefore promotes resistance to type II TKIs. Importantly, we demonstrated that combinatorial inhibition of ATM signalling was synergistic in resensitising all three models of resistance to sorafenib, providing a rationale for preclinical assessment of this new therapeutic approach in FLT3-ITD/TKD+ AML patients who relapse to TKI therapy. Overall, this body of work provides extensive evidence for the cooperation of ERK/MAPK-ATM signalling in maintaining FLT3-ITD/TKD survival in redox-driven stress conditions, highlighting a novel combinatorial therapeutic strategy that warrants further investigation.
- Subject
- acute myeloid leukemia; tyrosine kinase inhibitor (TKI); thesis by publication; FLT3; resistance; relapse; DNA damage; DNA repair; ATM; phosphoproteomics; oncogenic signaling
- Identifier
- http://hdl.handle.net/1959.13/1504548
- Identifier
- uon:55549
- Rights
- Copyright 2022 Dilana Elisabeth Staudt Barreto
- Language
- eng
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