Investigating Germline Predisposition to Clonal Hematopoiesis through Perturbation of a Variant-Harboring Enhancer of TET2

Clonal hematopoiesis (CH) is the age-related expansion of hematopoietic stem and progenitor cells (HSPCs) due to acquired genetic changes and is associated with increased blood cancer risk. Despite considerable progress in understanding how specific acquired somatic mutations result in clonal expansion, we have a limited understanding of the role of germline mutations that also predispose to clonal expansion.

Recent work has revealed a low frequency germline variant found exclusively in individuals of African diasporic descent that confers a 2.4-fold increased risk for CH (Bick et al., Nature, 2020). Remarkably, this variant is found within a putative enhancer of the TET2 gene, which encodes a key epigenetic modifier that catalyzes conversion of methylated cytosines to 5-hydroxymethylcytosine, thereby facilitating DNA demethylation. However, the precise role of this enhancer variant in altering TET2 activity and human hematopoiesis is poorly understood. We specifically hypothesize that this germline variant may alter TET2 activity subtly in hematopoietic stem cells (HSCs) to modify DNA methylation at a number of HSC regulatory elements and subsequently gene expression, which are likely mediated through selective changes in transcription factor (TF) activity.

To explore this hypothesis, we performed deletions of the putative TET2 enhancer in adult CD34 ⁺HSPCs using CRISPR/Cas9 editing through the introduction of Cas9 ribonucleoproteins. Following deletion of this enhancer, we observed a moderate increase in the total number of phenotypic long-term reconstituting hematopoietic stem cells (LT-HSCs; as marked by CD34 ⁺CD45RA ^-CD90 ⁺CD201 ⁺CD133 ⁺CD49c ⁺) and in primary colony formation unit (CFU) assays compared to control editing (AAVS1 edited). Interestingly, the enhancer deletion did not cause an increase in the number of phenotypic short-term HSCs (CD34 ⁺CD45RA ^-CD90 ⁺CD201 ⁺CD133 ⁺CD49c ^-), HSPC proliferation, or secondary CFU plating. Assessment of the deletion stability showed selection against enhancer deleted cells during myeloid differentiation, however some cells could still be identified after more than 30 days following editing. This result suggests that the enhancer likely functions in a selective manner within HSCs. The overall phenotypes we observe suggest some overlap, but distinct presentations in comparison to concomitant TET2 coding disruption that we have also performed.

Ongoing studies seek to use these promising phenotypic results to define changes in accessible chromatin and DNA methylation states in this isogenic perturbed model. This will enable insights into the specific enhancers altered through this perturbation and we plan to examine TF motif alterations at these regulatory elements. While the perturbation performed may represent a larger perturbation than is seen through the naturally-occurring variant, this provides a platform for defining how this CH predisposition arises with a larger impact perturbation. Use of base editors in a similar manner should enable more selective perturbations, as well. Together, these results will further reveal potential germline genetic and molecular origins of CH and further explain broader mechanisms of TET2 function and the importance of proper DNA methylation during human hematopoiesis that may provide clinical relevance for the potential prevention of blood cancers.