Recurrent Germline Variant in RAD21 Predisposes Children to Lymphoblastic Leukemia or Lymphoma.

Somatic loss of function mutations in cohesin genes are frequently associated with various cancer types, while cohesin disruption in the germline causes cohesinopathies such as Cornelia-de-Lange syndrome (CdLS). Here, we present the discovery of a recurrent heterozygous RAD21 germline aberration at amino acid position 298 (p.P298S/A) identified in three children with lymphoblastic leukemia or lymphoma in a total dataset of 482 pediatric cancer patients. While RAD21 p.P298S/A did not disrupt the formation of the cohesin complex, it altered RAD21 gene expression, DNA damage response and primary patient fibroblasts showed increased G2/M arrest after irradiation and Mitomycin-C treatment. Subsequent single-cell RNA-sequencing analysis of healthy human bone marrow confirmed the upregulation of distinct cohesin gene patterns during hematopoiesis, highlighting the importance of RAD21 expression within proliferating B- and T-cells. Our clinical and functional data therefore suggest that RAD21 germline variants can predispose to childhood lymphoblastic leukemia or lymphoma without displaying a CdLS phenotype.

The bulk of the hematopoietic stem cell population is dispensable for murine steady-state and stress hematopoiesis.

Long-term repopulating (LT) hematopoietic stem cells (HSCs) are the most undifferentiated cells at the top of the hematopoietic hierarchy. The regulation of HSC pool size and its contribution to hematopoiesis are incompletely understood. We depleted hematopoietic stem and progenitor cells (HSPCs) in adult mice in situ and found that LT-HSCs recovered from initially very low levels (<1%) to below 10% of normal numbers but not more, whereas progenitor cells substantially recovered shortly after depletion. In spite of the persistent and massive reduction of LT-HSCs, steady-state hematopoiesis was unaffected and residual HSCs remained quiescent. Hematopoietic stress, although reported to recruit quiescent HSCs into cycle, was well tolerated by HSPC-depleted mice and did not induce expansion of the small LT-HSC compartment. Only upon 5-fluorouracil treatment was HSPC-depleted bone marrow compromised in reconstituting hematopoiesis, demonstrating that HSCs and early progenitors are crucial to compensate myeloablation. Hence, a contracted HSC compartment cannot recover in situ to its original size, and normal steady-state blood cell generation is sustained with <10% of normal LT-HSC numbers without increased contribution of the few residual cells.

Activation of the cGAS/STING Axis in Genome-Damaged Hematopoietic Cells Does Not Impact Blood Cell Formation or Leukemogenesis.

Genome damage is a main driver of malignant transformation, but it also induces aberrant inflammation via the cGAS/STING DNA-sensing pathway. Activation of cGAS/STING can trigger cell death and senescence, thereby potentially eliminating genome-damaged cells and preventing against malignant transformation. Here, we report that defective ribonucleotide excision repair (RER) in the hematopoietic system caused genome instability with concomitant activation of the cGAS/STING axis and compromised hematopoietic stem cell function, ultimately resulting in leukemogenesis. Additional inactivation of cGAS, STING, or type I IFN signaling, however, had no detectable effect on blood cell generation and leukemia development in RER-deficient hematopoietic cells. In wild-type mice, hematopoiesis under steady-state conditions and in response to genome damage was not affected by loss of cGAS. Together, these data challenge a role of the cGAS/STING pathway in protecting the hematopoietic system against DNA damage and leukemic transformation. SIGNIFICANCE: Loss of cGAS/STING signaling does not impact DNA damage-driven leukemogenesis or alter steady-state, perturbed or malignant hematopoiesis, indicating that the cGAS/STING axis is not a crucial antioncogenic mechanism in the hematopoietic system. See related commentary by Zierhut, p. 2807.

Fate mapping of hematopoietic stem cells reveals two pathways of native thrombopoiesis.

Hematopoietic stem cells (HSCs) produce highly diverse cell lineages. Here, we chart native lineage pathways emanating from HSCs and define their physiological regulation by computationally integrating experimental approaches for fate mapping, mitotic tracking, and single-cell RNA sequencing. We find that lineages begin to split when cells leave the tip HSC population, marked by high Sca-1 and CD201 expression. Downstream, HSCs either retain high Sca-1 expression and the ability to generate lymphocytes, or irreversibly reduce Sca-1 level and enter into erythro-myelopoiesis or thrombopoiesis. Thrombopoiesis is the sum of two pathways that make comparable contributions in steady state, a long route via multipotent progenitors and CD48hi megakaryocyte progenitors (MkPs), and a short route from HSCs to developmentally distinct CD48-/lo MkPs. Enhanced thrombopoietin signaling differentially accelerates the short pathway, enabling a rapid response to increasing demand. In sum, we provide a blueprint for mapping physiological differentiation fluxes from HSCs and decipher two functionally distinct pathways of native thrombopoiesis.

Continuous mitotic activity of primitive hematopoietic stem cells in adult mice.

The proliferative activity of aging hematopoietic stem cells (HSCs) is controversially discussed. Inducible fluorescent histone 2B fusion protein (H2B-FP) transgenic mice are important tools for tracking the mitotic history of murine HSCs in label dilution experiments. A recent study proposed that primitive HSCs symmetrically divide only four times to then enter permanent quiescence. We observed that background fluorescence due to leaky H2B-FP expression, occurring in all H2B-FP transgenes independent of label induction, accumulated with age in HSCs with high repopulation potential. We argue that this background had been misinterpreted as stable retention of induced label. We found cell division-independent half-lives of H2B-FPs to be short, which had led to overestimation of HSC divisional activity. Our data do not support abrupt entry of HSCs into permanent quiescence or sudden loss of regeneration potential after four divisions, but show that primitive HSCs of adult mice continue to cycle rarely.

SCA-1 Expression Level Identifies Quiescent Hematopoietic Stem and Progenitor Cells.

Blood cell generation depends on continuous cellular output by the sequential hierarchy of hematopoietic stem cell (HSC) and progenitor populations that all contain quiescent and actively cycling cells. Hematopoietic stem and progenitor cells (HSPCs) express the surface molecule Stem cell antigen 1 (SCA-1/LY6A). Using histone 2B-red fluorescent fusion protein label retention and cell-cycle reporter mice, we demonstrate that high SCA-1 expression (SCA-1hi) identifies not only quiescent HSCs but quiescent cells on all hierarchical levels within the lineage-SCA-1+KIT+ (LSK) population. Each transplanted SCA-1hi HSPC population also displayed self-renewal potential superior to that of the respective SCA-1lo population. SCA-1 expression is inducible by type I interferon (IFN). We show, however, that quiescence and high self-renewal capacity of cells with brighter SCA-1 expression at steady state were independent of type I IFN signaling. We conclude that SCA-1 expression levels can be used to prospectively isolate functionally heterogeneous HSPC subpopulations.