ERK1/2-c-FOS/EGR1/2 pathway in GFI1- and c/EBP -mediated granulopoiesis

The National Heart, Lung, and Blood Institute awarded the University of Toledo $561,300 under the Cardiovascular Diseases Research program (CFDA 93.837) on September 1, 2026.

The award funds research into the ERK1/2-C-FOS/EGR1/2 pathway in GFI1- and C/EBP-mediated granulopoiesis, specifically investigating how transcription factors GFI1 and C/EBPA promote neutrophil development. The research will test hypotheses that these factors instruct neutrophil fate in part through negatively regulating the ERK1/2-C-FOS/EGR1 signaling pathway and that inhibition of ERK1/2 signaling may improve granulopoiesis in GFI1-deficient mice. Understanding these mechanisms has clinical relevance to severe congenital neutropenia, a human condition associated with loss-of-function GFI1 mutations.

The period of performance runs from September 1, 2026 through August 31, 2029, with place of performance in Ottawa Hills, Ohio. This is a Project Grant, the standard assistance type for investigator-initiated research under NHLBI's portfolio.

Abstract

Cell fate choice in the hematopoietic system depends on the expression of lineage specific transcription factors. In the myeloid lineage, transcription factors GFI1 and c/ebpα promote neutrophil development whereas high levels of PU.1 favor monocyte over neutrophil development. Targeted deletion of GFI1 in mice blocks neutrophil formation accompanied by an expansion of atypical monocytes. Loss-of-function mutations in GFI1 have been associated with severe congenital neutropenia (SCN) in humans. Bone marrow (BM) cells from GFI1-/- mice or transduced with the SCN-associated GFI1N382S mutant gave rise to monocytes rather than neutrophils in response to g-CSF. C/ebpα null mice lack mature granulocytes with a specific block in common myeloid progenitor (CMP) to granulocyte-monocyte progenitor (GMP) transition. The mechanisms by which GFI1 and c/ebpα promote granulopoiesis are incompletely understood. Our preliminary studies show that GFI1 inhibited the activation of ERK1/2 and repressed the ERK1/2 downstream target genes c-FOS and EGR1. Interestingly, inhibition of ERK1/2 using the specific MEK1/2 inhibitors reduced the expression of c-FOS and EGR1, and partially rescued neutrophil development of GFI1-/- BM cells. Our preliminary results further indicate that c/ebpα downregulated the expression of c-FOS and EGR1 by the 3UTRS of their mrnas whereas PU.1 upregulated c-FOS and EGR1. Thus, IT appears that c-FOS and EGR1 are the common target genes that are regulated by lineage specific transcription factors. In this proposal, we will test the hypotheses that GFI1 and c/ebpα instruct neutrophil fate in part through negatively regulating the ERK1/2-c-FOS/EGR1 pathway and that inhibition of ERK1/2 signaling may improve granulopoiesis in GFI1-/- mice. Three specific aims are proposed to test the hypotheses. In aim 1, we will determine whether suppression of c-FOS and/or EGR1 has an effect on neutrophil development of GFI1-/- BM cells in vitro and in vivo. In aim 2, we will determine the effect of MEK1/2 inhibition on neutrophil development in mouse GFI1-/- cmps and gmps, and in human CD34+ BM cells expressing the SCN-associated GFI1N382S mutant. We will further examine whether the specific MEK1/2 inhibitor trametinib alleviates neutropenia in GFI1-/- mice. In aim 3, we will evaluate whether downregulation of c-FOS and EGR1 is required for c/ebpα-driven granulopoiesis and whether inhibition of the ERK1/2-c-FOS/EGR1/2 pathway rescues neutrophil development of BM cells from ENH(f/f); MX1-CRE mice in which the expression of c/ebpα is markedly decreased. We will also investigate the potential roles of micrornas (mirnas) and RNA binding proteins (rbps) in c/ebpα-mediated downregulation of c-FOS and EGR1 expression. The proposed research may reveal novel mechanisms by which GFI1 and c/ebpα promote granulopoiesis, and may lead to the development of new therapeutic strategies for the treatment of SCN patients with GFI1 mutations.