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.
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.
Hematopoietic stem and progenitor cell (HSPC) development and maintenance is regulated through a complex regulatory network. In a screen of epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement for the tumor suppressor protein, Inhibitor of growth 4 (Ing4) in HSPC specification. Ing4 acts to regulate transcription through interactions with transcription factors, including HIF, NF-kB, and p53. It is often mis-expressed in many human cancers and has been shown to promote stem cell-like characteristics in malignant cells, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. The transcription factor NF-kB is a regulator of inflammatory response and serves an important role in embryonic HSPC emergence, survival, differentiation and proliferation. The Ing4 protein binds to the p65/RelA subunit of NF-kB, inhibiting DNA binding and suppressing NF-kB cytokines and inflammatory pathways. In the absence of Ing4 there is an overexpression of NF-kB target genes that have inhibitory effects on hematopoietic programming. Given the regulatory role of Ing4 in both hematopoiesis and cancer, it is likely critical to the regulation of stem cell self-renewal, maintenance and specialization. To better define the role of Ing4 on hematopoiesis we use two Ing4 loss-of-function models: zebrafish and mouse. For the zebrafish model of Ing4 deficiency, Ing4-deficient zebrafish embryos lose >90% of runx1+/c-myb+ cells in the aorta, gonad, mesonephros (AGM) region of the developing zebrafish embryo, demonstrating a lack of HSPC specification. 36 hours post fertilization (hpf) Ing4 morphants display increased expression of NF-kB target genes when Ing4 is absent. Genetic epistasis experiments performed to block translation of RelA, IL-1b, and additional NF-kB target gene mRNA revealed recovered HSC marker expression in the aorta. To discover small molecule inhibitors that would mimic these effects, we conducted an in vivo chemical screen of NF-kB pathway inhibitors assessing their ability to rescue HSC specification in Ing4 morphant zebrafish. Ing4 morphants treated with NF-kB inhibitors had reduced NF-kB cytokine expression, as well as a dose-dependent rescue of HSC marker expression in the aorta. These results suggest that NF-kB inhibition could remediate the effects of Ing4 loss on hematopoiesis. To more thoroughly profile the effects of Ing4 loss on HSC specification and the bone marrow niche, an Ing4-/-mouse model was used. These mice are developmentally normal but are hypersensitive to stimulation with LPS due to increased inflammatory signaling. Peripheral blood analysis reveals an increase in Mac-1 cells in the Ing4-/- mouse. Ing4-/- bone marrow progenitors are skewed toward granulocyte-myocyte progenitor cells (GMPs) lending to the shift in cell populations present in the peripheral blood. Ing4 loss further disrupts the mouse hematopoietic program resulting in a dramatic increase in the number of short term-HSCs (ST-HSC) (WT: 11.4%, Null: 31.7%), a modest increase in long term-HSCs (LT-HSC) (WT: 2.4%, Null: 5.52%), and a dramatic decrease in multipotent progenitors (MPPs) (WT: 47.9%, Null: 19.3%). We also found significant alterations in stress hematopoiesis following competitive HSC transplant where sorted Ing4-/- LT-HSCs failed to engraft. Following myeloablative insult, we found no significant change in Ing4-/- LT-HSC (-1.18%) when compared with ST-HSC (-14.43%) indicating reduced sensitivity to 5-FU ablation in the Ing4-/- LT-HSC group. Cell cycle analysis identified 92.9% of Ing4-/- LT-HSCs are in G0 compared to 76.2% of wildtype LT-HSCs. ST-HSCs were also more quiescent with 27% of Ing4-/- ST-HSCs in G0 compared to 11.1% of wildtype ST-HSCs. Previously published work reports hyper proliferative HSCs that exhibit loss of quiescence as a result of proinflammatory NF-kB signaling. We believe that the interaction between Ing4 and the HIF-1a pathway may play a role in the observed phenotype of Ing4-/- LT-HSCs resulting in increased quiescence and disruption of the balance between self-renewal and differentiation critical to reconstitution of the hematopoietic compartment. Overall, our findings suggest that the regulatory effects of Ing4 play a crucial role in hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Disclosures Zon: CAMP4 Therapeutics: Current equity holder in private company, Other: Founder; Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Amagma Therapeutics: Current equity holder in private company, Other: Founder; Cellarity: Consultancy; Celularity: Consultancy.
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, loss of Ing4 has been shown to promote stem cell-like characteristics in malignant cells and it is a frequent target of inactivation in various types of cancer. Mutations in Ing4 cause deregulation of both NF-kB and c-Myc target gene expression. We have also identified a requirement for Ing4 in murine hematopoiesis. Ing4-/- mice have aberrant hematopoiesis and elevated cytokine expression in bone marrow cells. Using RNA-sequencing, we found that Ing4-deficient HSPCs express high levels of c-Myc target genes and genes associated with oxidative phosphorylation and ribosomal biogenesis. Yet, Ing4 deficiency induces G 0 arrest in HSPCs and they have low levels of reactive oxygen species. This places Ing4-deficient HSPCs in a poised state, where they are quiescent, but express elevated levels of genes associated with differentiation. Under stress hematopoiesis following low-dose irradiation, Ing4-deficient long-term hematopoietic stem cells (LT-HSCs) do not expand, but short-term hematopoietic stem cells (ST-HSCs) function comparably to wild-type. Similarly, under transplantation stress, LT-HSCs fail to contribute to multilineage chimerism, while ST-HSCs contribute at levels equal to wild-type cells. These results are striking, particularly when compared to other models of enhanced NF-kB activity, where HSPCs cannot contribute to multilineage chimerism in transplantation. We sought to target the misregulated pathways in Ing4-deficient HSCs to rescue to effects of Ing4 deficiency. To this end, we chose to target the c-Myc pathway for several reasons: c-Myc target genes are over-represented in our RNA-seq data, c-Myc lies upstream of several of the misregulated pathways observed in Ing4-/- HSCs, and Ing4 has previously been reported to negatively regulate c-Myc activity directly. When treated with the c-Myc inhibitor, 10058-F4, both LT-HSCs and ST-HSCs are pushed into cycling, but this treatment also resulted in fewer cells overall. These results suggest that dampening of the c-Myc pathway can partially rescue Ing4 loss of function. 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 No relevant conflicts of interest to declare.
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