A study of the pathogenic role of CAMK1D and its potential as therapeutic target in Hodgkin lymphoma and diffuse large B cell lymphoma
Creators: name: Lles, Eva
Publisher: name: University of Limerick
Year: 2024
Subjects: classificationCode: 42
subject: Health sciences
subject: FOS: Health sciences

description: Lymphoma is a heterogeneous group of malignancies divided in two groups: Hodgkin lymphoma and non-Hodgkin lymphoma, with the commonest subform being Classical Hodgkin lymphoma and diffuse large B cell lymphoma within each group, respectively. Although research efforts over the past decade have improved overall survival for these tumours (~90% for HL and ~55% for DLBCL), up to 35% of patients relapse within the first two years following therapy, and there is a need to improve them, as secondary malignancies and long-term toxicities among survivors are common and have a significant impact on their quality of life. Therefore, it is essential to improve our understanding of these diseases and to identify potential novel targets for the development of more effective and specific treatments.This project thus focusses on one such potential target, CAMK1D, a protein kinase that belongs to the Calcium/Calmodulin-dependent protein kinases and is regulated by calcium signalling. As calcium is crucial for normal B cell function and is tightly regulated, it is not surprising that CAMK1D is dysregulated in lymphoma pathogenesis. Our group has shown that it is overexpressed in HL and DLBCL tumour samples. Moreover, CAMK1D expression correlates with prognosis in DLBCL; Activated-B cell (ABC) tumours present higher CAMK1D expression and have a worse prognosis. To study the role of CAMK1D and explore its potential as therapeutic target, I investigated its signalling network through activation of CAMK1D signalling combined with/without chemical inhibition of CAMK1D (via PROTAC or phospho-inhibitor treatment) and examined the expression of potential downstream targets by Western blotting. Among the targets tested, only pCREB was found as downstream target of CAMK1D and only in one cell line, which exposes the common heterogeneity found in DLBCL pathogenesis. The examination of the upstream regulators presented the BCR as an activator of CAMK1D and the subsequent activators presented heterogeneity within the cell lines, which could depend on the DLBCL subtype. Interestingly, a role for Bcl2 was identified in the activation of CAMK1D within the BCR signalling pathway and the inhibition of Bcl2 through venetoclax treatment presented a complete inhibition of CAMK1D activation in multiple DLCBL cell lines.Following this, I investigated CAMK1D contribution in the cell cycle progression. Its contribution was equally observed in both DLBCL and HL pathogenesis. Firstly, with an increase of the number of cells in G1 phase, observed by flow cytometry when cells were treated with CAMK1D phospho-inhibitor, but not when the total protein was removed. This suggested that pCAMK1D (T180) participates in G1/S transition as an accumulation in G1 was seen upon inhibition. Secondly with the examination of pCAMK1D (T180) during G1/S transition (i.e cells synchronised in G1 and re-entering the cell cycle) which was observed increased through Western blotting analysis.Lastly, I examined the sensitivity to the conventional chemotherapy vincristine and CAMK1D inhibitor of HL and DLBCL cell lines through luminescent cell viability assay. I observed that all cell lines presented sensitivity to vincristine while very low sensitivity to CAMK1D inhibitors was detected. Although no cell death was observed after treatment with 1µM of pCAMK1D inhibitor (CS640) or 1nM of vincristine as single agent, their combination presented synergetic effect and demonstrated a substantial decrease of cell viability. Interestingly, this finding seems to be specific to HL pathogenesis as the synergetic effect was not observed in DLBCL cells.Overall, CAMK1D appears to participate in the driving of carcinogenesis through its implication in the cell cycle progression and more work is required to fully understand its specific function and partners. The signalling network of CAMK1D especially within the BCR signalling pathway has been examined and needs further investigation as it is a crucial pathway involved in lymphomagenesis. Regarding the translational side of this research, the synergetic effect observed with vincristine+CS640 requires optimisation and testing (i.e toxicity on healthy cells, specificity, optimisation of drug concentrations, etc) but provides a foundation for a potential new therapy in HL patients.<br>