Succinate-induced leukemic stem cell formation via epigenetic remodeling of hematopoietic stem cell

Project Information

SuccHSC

Grant agreement ID: 101031817

DOI

10.3030/101031817

Project terminated on 6 February 2024

EC signature date 12 March 2021

Start date 11 March 2022

End date 10 March 2024

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 202 158,72

EU contribution

€ 202 158,72

Coordinated by

UNIVERSITETET I TROMSOE - NORGES ARKTISKE UNIVERSITET
Norway

Periodic Reporting for period 1 - SuccHSC (Succinate-induced leukemic stem cell formation via epigenetic remodeling of hematopoietic stem cell)

Reporting period: 2022-03-11 to 2024-03-10

Summary of the context and overall objectives of the project

1) What is the problem/issue being addressed?
Hematopoietic stem cells (HSC) are blood cells responsible for generating all types of blood cells. In acute myeloid leukemia (AML), HSC are transformed into malignant or leukemia stem cells (LSC), responsible for the onset of the disease. HSC are found in a tightly regulated microenvironment inside the bone marrow cavity, which has shown to be essential in AML progression. Growing amount of data, including work from the lab of the Supervisor, links chronic inflammation, metabolism and intercellular communication to the onset of myeloid malignancies. In this context, the main question that we wanted to address in SuccHSC was the mechanisms of transformation of HSC into LSC, focussing on the potential role of succinylation, as a major effector event involved in the epigenetic remodeling of HSC into LSC.

2) Why is it important for society?
Finding new treatment options for an aggressive blood cancer like AML requires acquisition of new knowledge on the pathogenic mechanisms. The current best treatment option is chemotherapy and stem cell transplant, which offer 5-year survival to 20% of patients above the age of 60 and 40% to the younger patients. Stem cell transplantation is not performed in older patients due to their frailty. However, the incidence of AML increases with age with a median age at diagnosis of 67 years of age, with 55% of cases occurring in people over the age of 65.

3) What are the overall objectives?
The goal was to identify and compare the DNA sequences which have succinate bound to histones, between HSC obtained from healthy donors and LSC obtained from AML patients. Using an experimental mouse model, we would demonstrate the causal association between succinylation and leukemic transformation.

The questions we wanted to address were:
1. What is the amount of succinate metabolite inside human bone marrow cells?
2. How much succinate is bound to histones at specific genes?
3. Does gene succinylation influence expression and cell function?
4. How does that correlate with inflammatory parameters?
5. How does succinylation impact AML origin and progression?
6. Can we capitalize this knowledge to design a treatment strategy?

Conclusions

These objectives could only be partially accomplished due to delays, technical challenges and early termination of the grant.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The Researcher received extensive lab and animal training, and obtained the FELASA C certificate as stated in the project. We obtained the ethical approval for the planned mouse experiments proposed in SuccHSC.

Experiments for measuring TCA intermediates in human samples were planned by the Researcher and the Supervisor, following the original plan stated in the project. Since the human samples from the Biobank are precious, it was agreed that no tests would be performed with those samples before all the necessary optimizations were setup. The Supervisor and the Researcher decided to use WT C57BL/6J mouse samples for biochemistry analysis tests. Given the technical issues encountered, no experiments were executed with human cells.

ChIP-seq optimization was initiated as well with mouse C57BL/6J cells, as stated in the project. The protocol included validation steps with immunofluorescence and ChIP-RT-PCR on reference gene, using H3K4me3 and isotype control antibody, as well as sequencing. John Arne Dahl shared all relevant protocols with the Researcher and the Supervisor, and the Researcher visited Dr. Dahl lab to discuss the technique and any doubt with members of his team. No successful optimization was done beyond DNA sonification fragmentation, therefore no success was made towards reaching 10 000 cell sample size for control antibody ChIP-Seq nor for antibody against succinylated lysine’s on histones, due to technical issues.

Experiments were planned in the project with frozen patient samples from Francesco Di Raimondo, at Catania University Hospital (Italy), and Sdhb fl/fl mouse model from other collaborators. Administrative delays prevented access to those samples.

There will be no research articles derived from the work of the Researcher, but the MSCA-IF program will be acknowledged in future publications of the lab of the Supervisor related to this project.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The work performed only reached early stage and no progress beyond the state of the art has been done.

Project Description