P202259EM5 - Unravelling the role of DNMT3A loss-of-function in driving haematopoietic stem cell expansion upon microbial-driven inflammation in clonal haematopoiesis and in cooperation with JAK2V617F gain-of-function mutation in myeloproliferative neoplasms

Clonal haematopoiesis of indeterminate potential (CHIP) is a common aging-related phenomenon caused by the presence of a clonally-expanded mutated hematopoietic stem cell (HSC) in individuals without any haematological abnormalities. However, CHIP is associated with an increased risk of haematologic malignancies. The most frequently mutated gene in CHIP is the epigenetic regulator DNMT3A and its mutations are also commonly found as early-occurring mutations in patients with myeloproliferative neoplasm (MPNs). Investigating the contribution of extrinsic and intrinsic factors in the expansion of CHIP and MPN HSCs is an expanding field of research. Extrinsically, inflammation seem to have a crucial role in the expansion of CHIP clones. Among intrinsic factors, secondary lesions seem paramount: the gain-of-function mutation JAK2V617F (JAK2VF, the most frequent lesion found in MPN) is also one of the most common “second hits” in DNMT3A-mutant CHIP. The main aim of this project is to investigate how microbial-driven intestinal inflammation can drive expansion of DNMT3A-deficient haematopoiesis, which we observed in preliminary experiments, and if this finding is recapitulated in “two-hit” models where JAK2VF cooperates with Dnmt3a loss. Specifically, our grant proposal is composed of two different but integrated MILESTONES (M1 and M2). M1 aims to characterize the HSC behaviour in response to microbial-driven inflammation in a mouse model of Dnmt3a-loss haematopoiesis as well as in a “two-hit” model with JAK2VF and Dnmt3a loss. We first plan to extensively characterize the cellular response to such inflammation in mouse. Furthermore, we aim to address the functional, transcriptional and epigenetic change seen in mutant HSCs upon inflammation and to identify specific promoting factors. In M2, we intend to investigate the contribution of DNMT3A deficiency in human JAK2VF-driven MPN and the role of inflammation in disease onset and progression. We plan to characterize the phylogenetic hierarchy and correlation of the order-of-mutation acquisition with biological properties of mutant HSCs as well as with disease phenotype through the use of in vitro functional assays on primary patient samples. Similarly to M1, we also propose to evaluate what pro-inflammatory cytokines enhance the pathological behaviour of single- and double-hit HSCs, whether the added complexity of order-of-mutation acquisition plays a role in influencing mutant HSC response to inflammation, and how transcriptomics and epigenetics are affected. Overall, this project proposes to untangle previously uncharted aeras at the interface between microbial-driven inflammation, mutations in HSCs and order-of-mutation acquisition, aiming for a unifying model of clonal expansion for the first time.