Investigating the Role of ZBTB33 Loss in Clonal Hematopoiesis and Hematologic Malignancies

Abstract

Clonal hematopoiesis describes a state in which an individual’s blood cells are disproportionately derived from one hematopoietic stem cell (HSC). Clonal somatic mutations in specific genes mutated in blood cancers can be found in the blood of otherwise healthy individuals and are associated with age and an increased risk of hematologic malignancy, cardiovascular disease, and overall mortality. However, clonal hematopoiesis also occurs in individuals where no mutation can be identified. To discover novel candidate driver genes, we analyzed >54,000 normal blood exomes, which revealed recurrent mutations in ZBTB33. We validated that CRISPR/Cas9 editing of Zbtb33 in mouse hematopoietic stem and progenitor cells (HSPCs) leads to clonal expansion and a competitive reconstitution advantage. Furthermore, we discovered ZBTB33 mutations in myelodysplastic syndromes (MDS) and noted a significant co-occurrence with mutations in SF3B1. Together, these findings led us to nominate ZBTB33 as a candidate driver of clonal hematopoiesis and MDS and motivated us to study its role in hematopoietic cells. DNA methylation can modulate mRNA splicing, and mutations in genes encoding proteins involved in these pathways occur frequently in clonal hematopoiesis and MDS. Since ZBTB33 is a proposed reader of 5-methylcytosine (5mC) and ZBTB33 mutations co-occur with mutations in genes encoding splicing factors, we investigated the intersection of these pathways. To assess the impact of combinatorial genetic lesions on clonal expansion and transformation, we generated mouse models of Zbtb33 loss in combination with genetic alterations in Sf3b1 and other genes. Next, we performed RNA-sequencing to evaluate the effects of Zbtb33 loss on gene expression and alternative splicing. In mouse HSPCs, we observed an increase in intron retention in Zbtb33 edited cells, which also occurs upon knockout of another 5mC reader, Mecp2. Consistent with a role in modulating splicing, we noted a physical interaction between ZBTB33 and splicing factors as well as RNA polymerase II in cell lines. Zbtb33/ZBTB33 loss also led to widespread gene expression changes in mouse HSPCs and K562 cells. Overall, this work establishes a role for ZBTB33 in regulating HSC clonal expansion and identifies potential processes governing this phenotype, providing foundational insight for future biological investigation and clinical studies.