Multiple mechanisms disrupt the let-7 microRNA family in neuroblastoma.

Poor prognosis in neuroblastoma is associated with genetic amplification of MYCN. MYCN is itself a target of let-7, a tumour suppressor family of microRNAs implicated in numerous cancers. LIN28B, an inhibitor of let-7 biogenesis, is overexpressed in neuroblastoma and has been reported to regulate MYCN. Here we show, however, that LIN28B is dispensable in MYCN-amplified neuroblastoma cell lines, despite de-repression of let-7. We further demonstrate that MYCN messenger RNA levels in amplified disease are exceptionally high and sufficient to sponge let-7, which reconciles the dispensability of LIN28B. We found that genetic loss of let-7 is common in neuroblastoma, inversely associated with MYCN amplification, and independently associated with poor outcomes, providing a rationale for chromosomal loss patterns in neuroblastoma. We propose that let-7 disruption by LIN28B, MYCN sponging, or genetic loss is a unifying mechanism of neuroblastoma development with broad implications for cancer pathogenesis.

Interferon-α signaling promotes embryonic HSC maturation.

In the developing mouse embryo, the first hematopoietic stem cells (HSCs) arise in the aorta-gonad-mesonephros (AGM) and mature as they transit through the fetal liver (FL). Compared with FL and adult HSCs, AGM HSCs have reduced repopulation potential in irradiated adult transplant recipients but mechanisms underlying this deficiency in AGM HSCs are poorly understood. By co-expression gene network analysis, we deduced that AGM HSCs show lower levels of interferon-α (IFN-α)/Jak-Stat1-associated gene expression than FL HSCs. Treatment of AGM HSCs with IFN-α enhanced long-term hematopoietic engraftment and donor chimerism. Conversely, IFN-α receptor-deficient AGMs (Ifnαr1(-/-)), had significantly reduced donor chimerism. We identify adenine-thymine-rich interactive domain-3a (Arid3a), a factor essential for FL and B lymphopoiesis, as a key transcriptional co-regulator of IFN-α/Stat1 signaling. Arid3a occupies the genomic loci of Stat1 as well as several IFN-α effector genes, acting to regulate their expression. Accordingly, Arid3a(-/-) AGM HSCs had significantly reduced transplant potential, which was rescued by IFN-α treatment. Our results implicate the inflammatory IFN-α/Jak-Stat pathway in the developmental maturation of embryonic HSCs, whose manipulation may lead to increased potency of reprogrammed HSCs for transplantation.

Flow-induced protein kinase A-CREB pathway acts via BMP signaling to promote HSC emergence.

Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta-gonad-mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)-cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA-CREB and BMP pathways in isolated AGM VE-cadherin(+) cells from mid-gestation embryos, we demonstrate that PKA-CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA-CREB-BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition.

Effect of developmental stage of HSC and recipient on transplant outcomes.

The first hematopoietic stem cells (HSCs) that engraft irradiated adult mice arise in the aorta-gonad-mesonephros (AGM) on embryonic day 11.5 (E11.5). However, at this stage, there is a discrepancy between the apparent frequency of HSCs depicted with imaging and their rarity when measured with limiting dilution transplant. We have attempted to reconcile this difference using neonatal recipients, which are more permissive for embryonic HSC engraftment. We found that embryonic HSCs from E9.5 and E10.5 preferentially engrafted neonates, whereas developmentally mature, definitive HSCs from E14.5 fetal liver or adult bone marrow (BM) more robustly engrafted adults. Neonatal engraftment was enhanced after treating adult BM-derived HSCs with interferon. Adult BM-derived HSCs preferentially homed to the liver in neonatal mice yet showed balanced homing to the liver and spleen in adults. These findings emphasize the functional differences between nascent and mature definitive HSCs.