Purpose of review The hematopoietic compartment is tasked with the establishment and maintenance of the entire blood program in steady-state and in response to stress. Key to this process are hematopoietic stem cells (HSCs), which possess the unique ability to self-renew and differentiate to replenish blood cells throughout an organism's lifetime. Though tightly regulated, the hematopoietic system is vulnerable to both intrinsic and extrinsic factors that influence hematopoietic stem and progenitor cell (HSPC) fate. Here, we review recent advances in our understanding of hematopoietic regulation under stress conditions such as inflammation, aging, mitochondrial defects, and damage to DNA or endoplasmic reticulum. Recent findings Recent studies have illustrated the vast mechanisms involved in regulating stress-induced hematopoiesis, including cytokine-mediated lineage bias, gene signature changes in aged HSCs associated with chronic inflammation, the impact of clonal hematopoiesis and stress tolerance, characterization of the HSPC response to endoplasmic reticulum stress and of several epigenetic regulators that influence HSPC response to cell cycle stress. Summary Several key recent findings have deepened our understanding of stress hematopoiesis. These studies will advance our abilities to reduce the impact of stress in disease and aging through clinical interventions to treat stress-related outcomes.

Canonical and non-canonical Wnt signaling, as well as the Pax/Six gene network, are involved in patterning the freshwater sponge aquiferous system. Using computational approaches to identify transcription factor binding motifs in a freshwater sponge genome, we located putative PaxB binding sites near a Secreted Frizzled Related Protein (SFRP) gene in Ephydatia muelleri . EmSFRP is expressed throughout development, but with highest levels in juvenile sponges. In situ hybridization and antibody staining show EmSFRP expression throughout the pinacoderm and choanoderm in a subpopulation of amoeboid cells that may be differentiating archeocytes. Knockdown of EmSFRP leads to ectopic oscula formation during development, suggesting that EmSFRP acts as an antagonist of Wnt signaling in E . muelleri . Our findings support a hypothesis that regulation of the Wnt pathway by the Pax/Six network as well as the role of Wnt signaling in body plan morphogenesis was established before sponges diverged from the rest of the metazoans.

Hematopoiesis is tightly regulated by a network of transcription factors and complexes that are required for the maintenance and development of HSCs. In a screen for epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement of the tumor suppressor protein, Ing4, in hematopoietic stem and progenitor cell (HSPC) specification. Though the Ing4 mechanism of action remains poorly characterized, it has been shown to promote stem-like cell characteristics in malignant cells and is a frequent target of inactivation in various cancer types. The tumor suppressive activity is, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. In zebrafish, loss of Ing4 results in loss of HSC specification and a significant increase in NF-kB target gene expression. Knockdown of NF-kB expression in Ing4 deficient zebrafish recovered HSC marker expression in the aorta suggesting that NF-kB inhibition could remediate the loss of Ing4 expression. Small molecule NF-kB pathway inhibitors with varying mechanisms were also observed to rescue of HSC marker staining in the zebrafish aorta. Ing4 deficient embryos incubated with a lower dose of inhibitor had a 31% recovery of marker staining and 82% of embryos incubated in the highest dose recovered HSC marker staining emphasizing a dose dependent rescue of HSC specification through NF-kB suppression. As in the zebrafish, we have identified a requirement for Ing4 in murine hematopoiesis. Ing4-/- bone marrow has aberrant hematopoiesis resulting in an increase in the number of short term-HSCs (ST-HSCs) (11.4% vs 31.7%) and a dramatic decrease in multipotent progenitor cells (MPPs) (47.9% vs 19.3%) along with a concurrent modest increase in the population of long-term HSCs (LT-HSCs) (2.4% vs 5.5%). Analysis of differentiation in Ing4 null bone marrow also reveals skewed hematopoiesis. We see a 14% increase in granulocytes in the null mouse marrow and observe similar skewing in CFU assays. Additionally, there were alterations in stress hematopoiesis following hematopoietic stem cell transplant. Sorted LT-HSCs fail to engraft, suggesting an evolutionarily conserved requirement for Ing4 in HSCs. Surprisingly, competitive transplantation assay with Ing4-defecient MPPs versus wild-type showed dramatic increase in peripheral blood multilineage chimerism up to 9 months post-transplantation (19% vs. 59%). This lends to the hypothesis that Ing4 deficient MPPs gain self-renewal capabilities. In further characterization of these cells, we found an increase in MPPs that express lower levels of CD34 (55.5% vs 67.7%). CD34 expression is a marker of HSCs. This CD34+/mid population also express CD229 (85% positive), which is barely detectable in wildtype marrow (less that 0.01%). CD229 is also an HSC marker. Based on these exciting findings, we hypothesize that we have identified a subset of CD34+/midCD229+ MPPs in Ing4 deficient mice that retain self-renewal characteristics. Our data suggest that Ing4 normally functions as a critical suppressor for genes required for self-renewal and developmental potency in MPPs. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Given the role of Ing4 in both normal hematopoiesis and cancer, this gene likely has a critical role in regulation of stem cell self-renewal and maintenance. Disclosures Zon: CAMP4: Equity Ownership; Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership.

Hematopoiesis is tightly regulated by a network of transcription factors and complexes that are required for the development and maintenance of hematopoietic stem cells (HSCs). We recently identified the tumor suppressor, Ing4, as a critical regulator of HSC homeostasis. Though the Ing4 mechanism of action remains poorly characterized, it has been shown to promote stem-like cell characteristics in malignant cells. This activity is, in part, due to Ing4 mediated regulation of several major signaling pathways, including NF-kB and c-Myc. In murine hematopoiesis, Ing4 deficiency induces G0 arrest in HSCs, while simultaneously promoting gene expression signatures associated with differentiation. This results in a poised state for Ing4-deficient HSCs. Long term HSCs are unable to overcome this block, but short-term HSCs convert the poised state into regenerative capacity during hematopoietic challenges, including irradiation and transplantation. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis. Our model provides key tools for further identification and characterization of pathways that control quiescence and differentiation in HSCs.

A network of transcription factors and associated complexes regulate the process of hematopoiesis and are required for maintenance and development of the hematopoietic program. Ing4, a tumor suppressor protein, was identified in a screen for epigenetic regulators of hematopoiesis in zebrafish as required for specification of hematopoietic stem and progenitor cells (HSPCs). Recent work has shown that Ing4 is inactivated in various cancer cells. This inactivation promotes stem cell-like qualities in malignant cells. Ing4 plays an inhibitory role in the NF-κB pathway, conferring, in part, Ing4's tumor-suppressor capability. Loss of Ing4 is correlated to diminished hematopoietic stem cell (HSC) specification in zebrafish and increased NF-κB target gene expression. NF-κB knockdown assays in zebrafish embryos suggest inhibition of NF-κB remediates loss of Ing4 expression, with HSC rescue efficacy varying directly with concentration of inhibitor. Similarly, the necessity of Ing4 in murine hematopoiesis has been observed. Here, Ing4 deficiency impairs HSC function, while simultaneously enhancing the regenerative capacity of multipotent progenitor cells (MPPs). Characterization of bone marrow from Ing4-deficient mice shows abnormal hematopoiesis, with a striking decrease in MPPs as compared to wildtype mice (47.9% vs 19.3%). Hematopoiesis under stress conditions is also altered in Ing4-deficient mice, as observed following competitive HSC transplantation. In a surprising finding, MPPs from Ing4-deficient mice showed a dramatic increase in peripheral blood multilineage chimerism compared to wildtype mice up to 9 months post-transplantation in a competitive transplant assay (19% vs. 59%). This supports the hypothesis that MPPs from Ing4-deficient mice have enhanced self-renewal capacity. Additionally, we have observed a subpopulation of Ing4-deficient MPPs that express lower levels of CD34, CD34+/mid. This population of CD34+/mid cells was also shown to express CD229 (85% positive), while very few WT MPPs express both CD34+/mid and CD229 (5.0%). Reduced levels of CD34 expression combined with CD299 are known to be markers of HSCs, and so we hypothesize that a subset of CD34+/midCD229+ MPPs in Ing4-deficient mice retain their self-renewal capacity. Taken together, our data suggest Ing4 typically functions as a suppressor of genes necessary for self-renewal and developmental potency of MPPs. Additionally, cell cycle analysis combined with Ki-67 expression showed Ing4-deficient MPPs have enhanced ability to maintain quiescence, with 15.2% of cells found to be in G0 phase as compared to 6.5% of wildtype MPPs in G0. Finally, after 5-FU treatment, levels of MPPs in WT mice were similar pre- and post-treatment. Future experiments will seek to elucidate this observation in consideration of the pro-inflammatory environment. These findings suggest Ing4 is a critical regulator of hematopoiesis, and these data provide important clues for further characterization of the pathways and functions of Ing4. Our data show that Ing4 deficiency promotes stem cell-like properties in MPPs, suggesting it has crucial regulatory functions in both stem cell self-renewal and maintenance. Disclosures No relevant conflicts of interest to declare.