Modeling Clonal Hematopoiesis Due to Chromosome 13q Deletion in Human Hematopoietic Stem Cells

Before diagnosis, a pre-leukemic state precedes leukemia, where a single mutated hematopoietic stem cell (HSC) with a competitive growth advantage expands clonally (termed clonal hematopoiesis (CH)). Autosomal mosaic chromosome alterations (mCAs) are common CH drivers but are unexplored mechanistically. Chromosome 13q deletion (13q-) is one of the most common mCAs, increasing leukemia risk 29.5-fold and chronic lymphocytic leukemia (CLL) 185-fold.

We informatically determined that a 260kb region containing two lincRNAs, DLEU1 and DLEU2 is the 13q minimum deleted region (MDR). With CRISPR, the 13q MDR was deleted in human cord blood hematopoietic stem and progenitor cells (HSPCs). Then, detailed in vitro single-cell erythro-myeloid (EM) and B cell stromal differentiation assays and in vivo xenograft assays determined the effects of 13q- on HSC function. In vitro studies with 1,385 colonies showed a significantly increased CD14+ monocyte and decreased B cell output in MDR knock-out (KO) long-term (LT-) HSC-derived colonies. However, detailed analysis of the B cell lineage showed MDR KO increased proportion of primitive CD10+ B cells followed by decreased proportion of more mature CD19+ B cells, pointing to a downstream differentiation block; this was seen in LT-, short-term (ST-) HSC, and multi-lymphoid progenitors (MLPs) derived colonies. MDR KO also increased the number of LT-HSC-derived colonies expressing CD34+. Together, MDR KO in HSPCs increases monocyte output, retains the primitiveness of HSCs, and delays B cell maturation in vitro.

13q- CH humanized models were established by xenotransplanting MDR KO LT-HSCs or CD34+CD38- HSPCs into 174 NSG, NSG-SGM3, and NSG-Quad mice. Comparing the proportion of cells with MDR KO before and after 10-20 weeks of transplantation revealed a doubling in the percentage of KO cells in the human graft at endpoint, indicating a competitive growth or output advantage conferred by 13q- in HSCs. Supporting this, MDR KO cells generated larger CD45+ human grafts than control in injected femurs and peripheral lymphoid organs such as spleens and livers. There were 10% greater MDR KO cell prevalence in male recipients, correlating to higher male prevalence of autosomal mCA in CH and CLL, suggesting the male environment enhanced advantages in MDR KO HSCs. Aligning with in vitro studies, both monocytes and more primitive CD10+CD20- pro-B cells were increased and more mature CD10+/-CD20+ B cells were decreased in MDR KO xenografts. Moreover, a decrease in neutrophils and splenomegaly were observed, but with no evidence of CLL. To examine mature myeloid subsets in more detail, we adopted NSG-Quad mice. These express transgenic huCSF1 that promotes monocyte mobilization and macrophage differentiation. Liver and lung homogenates from NSG-Quad showed a significant, male-specific reduction in tissue resident macrophages upon MDR KO. Together, 13q- endows a competitive growth advantage in HSPCs, perturbs monocyte, neutrophil, and tissue resident macrophages output, and promotes primitiveness of B cells.

Although extending outside the MDR, the lincRNA DLEU2 contains miR-15a and miR-16-1, two intronic miRNAs proposed to drive CLL in mouse models. To identify potential genes responsible for the MDR phenotypes, we repeated all the in vitro and xenotransplantation assays using miR-15a/16-1 KO human HSPCs. Most findings phenocopied the MDR results, suggesting that miR-15a/16-1 contributes to 13q- CH. However, the magnitude of the effect was less than MDR, suggesting that not all the impact of MDR can be explained by miR-15a/16-1. This data also points to a potential regulatory element within the MDR that impacts DLEU2/miR-15a/16-1, which is our active objective to uncover.

Here we establish that a large chromosomal alteration 13q- provides a competitive growth advantage to human HSC and alters their differentiation output. Mechanistic studies into how 13q hijacks normal stemness programs to give a growth advantage are ongoing, and this could inform potential therapeutic targets for 13q- CH and CLL. Broadly, our 13q example offers a roadmap for modeling other mCAs and evaluating their effect on human HSC. These will be valuable for studying mCA-driven CH and the malignant and non-malignant diseases that derive from them.