Abstract

Hematopoietic stem cell (HSC) regulation is controlled by extrinsic and intrinsic factors adapting blood cell production to the need of the organism. To search for novel HSC regulatory genes, our group has established a unique screening approach. By systematically analyzing the entire integration site (IS) repertoire of ten Wiskott-Aldrich syndrome (WAS) patients enrolled in clinical gene therapy trials, we hypothesize to identify novel key regulatory genes in hematopoiesis. By applying our screening pipeline based on the number and distance of IS to the transcription start sites (TSS) of genes, we observed a statistically significant number of therapeutic vector insertions close to 32 single genes in nine out of ten WAS patients including the Evl/miR-342 gene locus which has not been linked to hematopoiesis so far. Common insertion sites close to Evl/miR-342 accounted for up to 1.2% of relative sequencing reads within the peripheral blood (PB) of patients and clones harboring such integrations contributed to hematopoiesis for up to six years. We therefore hypothesized that the protein-coding gene Evl and/or its intronic miR-342 - which share a common genomic locus - may regulate hematopoiesis. Evl has been shown to play a pivotal role in actin cytoskeleton remodeling, and to interact with RAD51 complexes within homologous recombination. MiR-342 is a direct target of the transcription factor PU.1, which drives myeloid differentiation, and accelerates all-trans retinoic acid (ATRA)-induced differentiation of APL blasts.

First of all, we investigated the candidate gene RNA expression in purified murine hematopoietic cell populations. Interestingly, we observed that Evl and miR-342 are not highly expressed in murine Lineage- Sca1+ ckit+ (LSK) cells but their expression increases profoundly with blood cell differentiation. While Evl expression was highest in lymphocytes (20-30 fold higher as compared to LSK cells), miR-342 was expressed at the highest level in macrophages (300 fold higher compared to LSK cells). To study the role of the candidates in hematopoiesis, we overexpressed Evl and miR-342 by using lentiviral vectors in murine primary LSK cells. Gene expression profiling of LSK cells overexpressing Evl revealed that 32% (62 out of 190) of the deregulated transcripts were involved in hematopoietic system development and function. Moreover, the top deregulated canonical pathways detected are essential for the development of B-cells (p=2.59*10-11). However, pathways important for myeloid cells such as immune cell trafficking and, more specifically, granulocytic adhesion and diapedesis (p=2.59*10-3) were significantly upregulated within miR-342- positive LSK cells. Functional analysis showed that Evl overexpression leads to a three- to fourfold increase of preB-cell colonies compared to control vector-transduced LSK cells. By contrast, miR-342 overexpressing cells formed a twofold higher number of myeloid colonies in semisolid medium. Next, the influence of Evl and its intronic miRNA on self-renewal and multilineage differentiation in vivo was investigated in serial bone marrow (BM) transplantation experiments. Within the PB of primary recipient mice, we detected a decrease of Evl-positive cells over time (week 4: 20.6 ± 9.6%; week 20: 4.7 ± 2.4%). Within the spleens a significantly higher donor-derived B-cell frequency was detectable (Evl: 63.6 ± 17.1%; Mock: 39.9 ± 18.3%). In line with our in vitro results, we detected a 4.3 fold higher frequency of Evl positive B-cells four weeks after secondary transplantation (Evl: 68.1 ± 9.8 %; Mock: 15.9 ± 7.5%).

In summary, our data show that different hematopoietic differentiation programs are driven by one common gene locus depending on the expressed gene product. While the protein-coding gene Evl drives B-cell lymphopoiesis, its intronic miR-342 promotes myeloid differentiation.

Disclosures

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.