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Exploring alternative cellular targets amenable to combination therapy in myelofibrosis
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Claudiani-S-2021-PhD-Thesis.pdf | Thesis | 22.3 MB | Adobe PDF | View/Open |
Title: | Exploring alternative cellular targets amenable to combination therapy in myelofibrosis |
Authors: | Claudiani, Simone |
Item Type: | Thesis or dissertation |
Abstract: | Myelofibrosis (MF) is the most aggressive subtype of the classical Philadelphia (Ph)-negative chronic myeloproliferative neoplasms (MPN), characterised by bone marrow fibrosis, splenomegaly, anaemia, debilitating symptoms and a high risk of evolution into acute leukaemia. It originates in clonal haematopoietic stem cells which are transformed by hyperactivation of JAK/STAT pathway resulting from JAK2, CALR or MPL mutations. The disease progresses through a complex network of contributors, which include the bone marrow and spleen microenvironment, non-clonal haematopoietic cells and an abnormal inflammatory background. The modern therapy of MF is ruxolitinib, a JAK2 inhibitor. However, this drug is only able to reduce the symptom burden and the splenomegaly, without significantly modifying the disease course and overall survival. In view of this, there is an urgent need for new therapeutic strategies. In order to identify the genes essential for the survival of MPN cells and those that protect them against ruxolitinib, we applied pooled-shRNA library screening on HEL cells, an MPN cell model, and primary CD34+ cells from MF patients. While the latter proved to be technically very challenging, mainly due to the limited transduction efficiency of the lentiviral system and did not lead to statistically significant candidates, the screen of HEL cells revealed several potential targets. Among the top hits, the most represented were the genes encoding for the ubiquitin-proteasome family, which were essential for HEL cell survival in the presence and absence of ruxolitinib and microenvironment-mimicking conditioned medium derived from HS-5 cells. This finding was validated through the exposure of both HEL cells and primary MF cells to the proteasome inhibitors (PI) carfilzomib (CFZ) and bortezomib (BTZ), which were both effective in inhibiting cell proliferation. Considering the pharmacokinetic profile of BTZ and its lack of clinical efficacy as monotherapy in MF, CFZ was chosen for investigation of primary MF cells. Pulsed-exposure experiments confirmed that adding CFZ to ruxolitinib augmented the growth inhibition, the extent of apoptosis and the reduction of clonogenicity of MF CD34+ cells, compared to ruxolitinib alone. Gene set enrichment analysis showed that ruxolitinib downregulated the expression of proteasome family genes in MF CD34+ cells, suggesting that this mechanism could make these cells more sensitive to PI. We also provided preliminary evidence that CFZ alone and its combination with ruxolitinib decrease the activation of NF-κB canonical pathway in MF CD34+ cells, which, if confirmed by a larger study, may constitute another important disease-promoting pathway targeted by CFZ. In summary, starting from the pooled-shRNA screening on HEL cells identifying proteasome genes among the top hits, we found that CFZ enhances the efficacy of ruxolitinib against primary MF CD34+ cells. Further research should investigate the exact mechanistic basis and consider further exploration of this combination as a potential new therapeutic strategy for MF. |
Content Version: | Open Access |
Issue Date: | Jun-2021 |
Date Awarded: | Dec-2021 |
URI: | http://hdl.handle.net/10044/1/97992 |
DOI: | https://doi.org/10.25560/97992 |
Copyright Statement: | Creative Commons Attribution NonCommercial NoDerivatives Licence |
Supervisor: | Apperley, Jane Khorashad, Jamshid |
Department: | Department of Immunology and Inflammation |
Publisher: | Imperial College London |
Qualification Level: | Doctoral |
Qualification Name: | Doctor of Philosophy (PhD) |
Appears in Collections: | Department of Immunology and Inflammation PhD Theses |
This item is licensed under a Creative Commons License