Ing4-Deficiency Confers Resistance to Inflammatory Stress through Enhanced Hematopoietic Stem Cell Quiescence

Abstract

In a screen of epigenetic regulators of hematopoiesis, we identified a requirement for the tumor suppressor protein, Inhibitor of growth 4 (Ing4) in hematopoietic stem and progenitor cell specification in embryonic zebrafish. To further our understanding of how Ing4 regulates adult hematopoiesis, we have extended these studies to examine murine hematopoiesis in the absence of Ing4. Though the Ing4 mechanism of action remains poorly characterized, loss of Ing4 promotes stem cell-like characteristics in malignant cells and is a frequent target of inactivation in various types of cancer. Studies in cancer cell lines have shown that mutations in Ing4 cause deregulation of NF-kB target gene expression and upregulation of c-Myc target gene expression, making it a fascinating candidate for regulation of hematopoiesis. In our mouse model of Ing4 deficiency, we have examined murine hematopoiesis. Ing4-/- mice have aberrant hematopoiesis, with a 2% increase in long-term hematopoietic stem cells (LT-HSCs) and a 12% increase in short-term HSCs (ST-HSCs). Highly elevated cytokine expression is also observed in bone marrow cells. Using bulk RNA-sequencing of HSCs, we found that Ing4-deficient HSCs express high levels of c-Myc target genes and genes associated with oxidative phosphorylation and ribosomal biogenesis. Despite these characteristics of activation, Ing4 deficiency induces a G0 arrest in HSCs. LT-HSCs show a 15% increase in cells arrested in G0, while ST-HSCs show an increase of 11%. Both populations of HSCs also contain significantly lower levels of reactive oxygen species. Taken together, these data suggest that Ing4-deficient HSCs are in a unique primed state, where they are quiescent, but express elevated levels of genes associated with activation. To test the function of Ing4-deficient HSCs, we examined hematopoiesis following low-dose irradiation (LDI). 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 multi-lineage chimerism, while ST-HSCs contribute at levels equal to wild-type cells. Exposure to inflammatory cytokines typically impairs HSC function in transplantation and renders them incapable of contributing to multi-lineage chimerism, making our results quite striking. We sought to target the dysregulated pathways in Ing4-deficient HSPCs to rescue the 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 dysregulated pathways observed in Ing4-/- HSCs, and Ing4 has been reported to negatively regulate c- Myc activity directly in cancer cell line models. Wild-type and Ing4-deficient mice were subjected to LDI, then mock or drug-treated with the c-Myc inhibitor, 10058-F4, for 7 days. Ing4-deficient HSCs are triggered into cycling upon treatment, with a 15% decrease in cells in G0. ST-HSCs show a 5% reduction of cells in G0. These data 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 hematopoietic stem cells. Our work also provides key tools for understanding how HSCs may overcome inflammatory stress by providing a model for robust HSC function under stress.