HETEROchromatin and RETROelements: devils of aging and leukemic development – vadeRETRO

Mutated HSCs amplify in the context of age-related inflammation and in response to ionizing radiation, used in radiotherapy. To test the impact of these stresses on HSC epigenetics and TE expression, we worked on mouse models presenting or not a loss of function of the TET2 gene, and subjected them to radiation or chronic inflammation by regularly injecting them with a bacterial product for 1 month. We then sorted the HSCs from these mice and studied the epigenetics and expression of genes and TEs using various genome sequencing techniques. We studied the impact of stresses and TEs on gene expression alterations by sequencing, and the induction of DNA breaks by microscopy. Finally, we tested the impact of stresses on HSC function using competitive engraftments experiments. These experiments involved grafting HSC from tested mice (mutated or not/stressed or not), into recipient mice that had lost the ability to produce normal hematopoiesis. Supportive HSCs are also grafted to ensure the good health of the recipient mice. The function of the transplanted cells is measured by their ability to produce all blood cells.

We show that stresses induce loss of epigenetic control and expression of TEs in non-mutated HSCs. These TEs induce alterations in gene expression and DNA breaks, leading to loss of HSC function. Conversely, mutated HSCs resist stress by epigenetically controlling TEs. We also show that if we prevent TEs from being expressed in unmutated HSCs, then mutated HSCs are no longer able to expand in response to inflammation.

Our current aim is to confirm these data in human HSCs, and to identify the epigenetic factors involved in TE control in mutated cells and their resistance to stress.

We have published a study in a high-impact scientific journal showing that radiations induce a loss of the epigenetic control of TEs, and alterations in gene expression, inducing loss of function of HSCs. (Pelinski et al. JEM 2022. DOI: 10.1084/jem.20211356 )

We are currently preparing a second paper showing the impact of inflammation on the epigenetics of mutated and non-mutated HSCs, and the impact of TEs in the expansion of mutated HSCs.

Finally, we are developing projects aimed at confirming these data in humans, and identifying epigenetic factors that can be targeted to prevent mutated HSC resistance and expansion.

These data pave the way for possible epigenetic therapies to prevent TE expression in unmutated HSC, amplification of mutated HSC and development of leukemia.

We are developing projects aimed at confirming these data in humans, and identifying epigenetic factors that can be targeted to prevent mutated HSC resistance and expansion.

We also show in this study that Tet2 KO HSC present a loss of heterochromatin at TEs. Interestingly, TE losing H3K9me3 are enriched for binding sites motifs for transcription factors (TFs) involved in HSC self-renewal and myeloid differentiation (data not shown). Loss of heterochromatin at these motifs may make them accessible for the corresponding TFs, and be involved in the myeloid bias observed in Tet2 KO HSCs. Increased production of myeloid cells and cytokine they produce was proposed to be involved in cardiovascular and aged-related diseases upon clonal hematopoiesis7. Understanding the mechanisms involved in this myeloid bias and cytokine production is crucial to prevent these diseases. We aim at submitting a grant at AAPG-2025 in order to decipher the role of TEs in myeloid differentiation upon TET2 loss of function.

Pelinski, Y. et al. NF-?B signaling controls H3K9me3 levels at intronic LINE-1 and hematopoietic stem cell genes in cis. Journal of Experimental Medicine 219, e20211356 (2022).

Understanding how exposure to ionizing radiations (IR) induces premature aging and installs a pre-leukemic state is a major public health concern.
Hematopoietic stem cells (HSC) give rise to all blood cell lineages. Maintenance of their integrity throughout life is thus crucial. HSC are particularly sensitive to Ionizing Radiations (IR). The persistence of panhematopoietic defects as late as 50 years after initial IR exposure in Japanese atomic bomb survivors suggests that HSC are the primary targets of IR. IR and aging induce similar defects in HSCs: DNA damage accumulation, loss of self-renewal, and a biased differentiation towards the myeloid lineage leading to increased myeloid cell counts and decline of the adaptive immune response. These changes are likely contributing to many IR-induced premature aging disorders and to the high risk of developing therapy-related myeloid neoplasms (t-MN). T-MN are associated with very poor survival and are one of the most devastating consequences of treatments applied to otherwise curable cancers. This has become a major public concern in the last decades as the number of long-term survivors of cancer treatments is continuously growing, consequently increasing the incidence of t-MN. However, the mechanisms governing HSC loss of function upon IR are unknown.
IR induces DNA double strand breaks. DNA damage is thought to be one of the main driving forces of aging. However a direct link between DSBs and long-lasting defects in HSCs function induced by IR is still lacking. On another hand, IR has been shown to induce epigenetic alterations in different tissues. Epigenetic factors controlling DNA and histone methylation are key regulators of HSC function and are often mutated in leukemia. These mutations also increase with age in otherwise healthy individuals where they confer clonal hematopoiesis. Thus epigenetic defects may be the first molecular events leading to HSC loss of function and leukemia.
Alteration of heterochromatin, that represses lineage inappropriate genes and retrotransposable elements (RTEs) expression, was reported in aging. RTE are repetitive sequences elements that can spread in the genome through a copy/paste mechanism. They constitute a great source of genomic instability. RTE uncontrolled expression is also involved in major transcriptomic changes. Recently, our team uncovered a new molecular mechanism involved in IR-induced HSC defects involving RTE. We showed that RTE are overexpressed upon IR. This is linked to persistent DNA-damage. The team also showed increased RTE expression with age in mouse and human HSCs. However, the impact of RTE derepression on HSC transcriptome and function is unknown.
We propose to explore a novel concept: that IR directly affects HSC heterochromatin. Heterochromatin alterations, by unleashing RTE, will rewire the transcriptome and act as the driving force behind HSC loss of function and the establishment of a preleukemic state.
Loss of function mutations in TET2 are among the most frequent mutations observed in clonal hematopoiesis, upon aging or in hematopoietic malignancies, and are associated with an increased risk of t-MN. WE also propose to investigate the consequences of loss of function mutations in TET2 on HSC
We will finally investigate the hypothesis that IR-induced heterochromatin alterations and TET2 loss of function may cooperate to synergistically deregulate common and/or different sets of genes/RTE finally increasing and accelerating the risk of developing t-MN.
Together, this research proposal should provide novel insights in the molecular mechanisms involved in HSC loss of function upon irradiation or aging, and the development of t-MN. It should also provide some clues on the first events involved in leukemogenesis.
The final aim of this project is to identify pathways that could be targeted therapeutically in order to alleviate the effects of genotoxic therapies and aging.