Identifying the role(s) of GPR56 and GPR97 in difficult-to-treat acute myeloid leukaemia cells to find new treatments for the disease

Precision Medicine Project - Identifying the role(s) of GPR56 and GPR97 in difficult-to-treat acute myeloid leukaemia cells to find new treatments for the disease

Supervisor(s): Dr Samanta Mariani, Dr Andrew Wood, Prof Vignir Helgason (University of Glasgow) & Prof Simon Arthur (University of Dundee)
Centre/Institute: Centre for Inflammation Research

Background

Acute myeloid leukaemia (AML) arises from abnormal proliferation and differentiation of myeloid cells. Current therapies are not curative to all patients and around 40% of patients relapse one year post transplant. There is a strong unmet need to identify novel therapeutic targets for AML.

High-risk treatment-resistant AML patients show a molecular signature with upregulated expression of the G-protein coupled receptor 56 (GPR56). High GPR56 protein expression has been linked to poor survival (<3 years) and higher risk of relapse post transplantation.GPR56 is a 7-transmembrane receptor in the adhesion G-coupled protein receptor family. 

At steady-state, the absence of Gpr56 in mouse cells is compensated by the upregulation of Gpr97 (genetically-linked and highly-homologous to Gpr56).

GPR56 is a druggable receptor that might lead to the production of new antibodies for the treatment of GPR56-expressing high-risk leukaemia patients, but their effectiveness might be hindered by a compensatory up-regulation of GPR97, as seen in mouse haematopoiesis. Similar to GPR56, GPR97 has been associated with poor prognosis in AML.

Assessing the roles of and the possible redundancy between GPR56 and GPR97 in high-risk AML cells is needed to further stratify AML patients and to design new drug therapies, such as GPR56 mono- or GPR56/97 bi-specific antibodies.

HypothesisGPR56 and GPR97 (aberrantly expressed in subgroups of high-risk AML patients) have redundant/complementary functions in AML cells.

AimDefine the role(s) of GPR56 and GPR97 in AML, with the final goal of further stratifying high-risk AML patients, and assessing GPR56/GPR97 suitability as druggable receptors for the disease.

Objectives1. Knock-out line generation: To understand if the redundancy between GPR56 and GPR97 is present in leukaemia cells, four leukaemia cell lines, MUTZ-3, MOLM13, MV4-11 and NOMO1 (characterised by different oncogenic mutations) will be transfected (CRISPR/Cas9 constructs) to generate single and double knock-out lines.

2. Test the effect of GPR56 and GPR97 loss-of-function: Proliferation curves of wild type (WT) and CRISPR-engineered MUTZ-3, MOLM13, MV4-11 and NOMO1 cells will be generated to understand whether the absence of either or both receptors affects leukaemic cell proliferation, and if the effects are mutation specific (i.e. different outputs in different AML cell lines). Progenitor potential/lineage bias, differences in cell cycle and/or apoptosis, and the effect of different anti- cancer treatments (5-Fluoruracil, Cytarabine, Radiation, Daunorubicin) will also be tested.

3. Define the intracellular signalling pathways of GPR56 and GPR97: The downstream pathways of GPR56 and GPR97 in the haematopoietic compartment are yet unknown. To assess GPR56 and GPR97 signalling directly at protein level, high-resolution quantitative proteomics will be performed on WT and GPR56-/-, GPR97-/-, GPR56-/-/97-/- MUTZ-3, MOLM13, MV4-11 and NOMO1 cells. Results will be validated by flow cytometry and western blotting.

4. Translation to human primary BM-derived CD34+ leukaemia cells: Thanks to an active collaboration with the Erasmus MC, we have access to microarray, RNAseq and prognosis data of >600 AML patients, among which there are AML patients showing overexpression of GPR56 and/or GPR97. The available data will be integrated with the proteomic data and will be bioinformatically analysed to find specific GPR56/GPR97-related signature(s) to further stratify high-risk AML patients. Moreover, key experiments to validate the data obtained on cell lines will be performed on primary AML samples expressing or not GPR56 and/or GPR97 (4-5 samples per group, Erasmus MC cohort - ethical approval obtained by Prof Delwel at Erasmus MC. Approval number: MEC-2015-155)

Training outcomesThe student will learn different wet and dry laboratory techniques, including but not limited to: Cell culture (both cell lines and primary cells) 

CRISPR/Cas9 targetingHigh-resolution proteomicsBioinformatic analysis of proteomic and RNAseq data Multi-colour flow cytometryWestern blottingProliferation assayHaematopoietic progenitor assay

References

1- Daga S, Rosenberger A, Quehenberger F, Krisper N, Prietl B, Reinisch A, et al. High GPR56 surface expression correlates with a leukemic stem cell gene signature in CD34-positive AML. Cancer Med. 2019 04;8(4):1771-8.

2- Yang J, Wu S, Alachkar H. Characterization of upregulated adhesion GPCRs in acute myeloid leukemia. Transl Res. 2019 Oct;212:26-35.

3- Maglitto A, Mariani SA, de Pater E, Rodriguez-Seoane C, Vink CS, Piao X, et al. Unexpected redundancy of Gpr56 and Gpr97 during hematopoietic cell development and differentiation. Blood Adv. 2021 Feb 09;5(3):829-42.

Apply Now

Click here to Apply Now

  • The deadline for 25/26 applications is Monday 13th January 2025
  • Applicants must apply to a specific project. Please ensure you include details of the project on the Recruitment Form below, which you must submit to the research proposal section of your EUCLID application. 
  • Please ensure you upload as many of the requested documents as possible, including a CV, at the time of submitting your EUCLID application.  

 

Q&A Sessions

Supervisor(s) of each project will be holding a 30 minute Q&A session in the first two weeks of December. 

If you have any questions regarding this project, you are invited to attend the session on Tuesday 10th December at 10am via Microsoft Teams. Click here to join the session.

This article was published on