Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis type 1 is a genetic disease that results from either heritable or spontaneous autosomal dominant mutations in the NF1 gene, which encodes a protein serving, at least in part, to accelerate the intrinsic hydrolysis of active Ras-GTP to inactive Ras-GDP. A second-hit NF1 mutation precedes predominant NF1 neoplasms, including juvenile myelomoncytic leukemia (JMML) and plexiform neurofibroma formation, potentially fatal conditions with no medical therapy. While NF1 loss of heterozygosity (LOH) in myeloid progenitor cells sufficiently engenders leukemogenesis, plexiform neurofibroma formation depends on LOH in Schwann cells and Nf1 heterozygosity in the hematopoietic system. Specifically, recruited Nf1+/- mast cells accelerate tumorigenesis through secreted cytokines and growth factors. Nf1+/- mast cells depend upon deregulated signaling in c-kit pathways, a receptor system conserved in hematopoietic stem cells (HSCs). Accordingly, Nf1-/- myeloid progenitor cells, which can induce a JMML-like disease in mice, also demonstrate deregulated c-kit receptor signaling. C-kit-activated Nf1+/- mast cells and Nf1-/- myeloid progenitors both show increased latency and potency of active Erk1 and Erk2, the principal cytosolic-to-nuclear effectors of canonical Ras-Raf-Mek signaling. Thus, Erk represents a potential regulator of leukemogenesis and tumor-associated inflammation. However, single and combined Erk1 and Erk2 roles in HSC function, myelopoiesis, and mature mast cell physiology remain unknown, and recent hematopoietic studies relying on chemical Mek-Erk inhibitors have produced conflicting results. Here, we show that hematopoietic stability, myelopoiesis, and mast cell generation require Erk1 or Erk2, but individual isoforms are largely dispensable. Principally, Erk-disrupted hematopoietic stem cells incorporate BrdU but are incapable of dividing, a novel and cell type-specific Erk function. Similarly, mast cell proliferation requires Erk but cytokine production proceeds through other pathways, elucidating molecule-specific functions within the c-kit cascade. Based on these findings, we have reduced tumor mast cell infiltration by treating genetically-engineered tumor model mice with PD0325901, a preclinical Mek-Erk inhibitor. Moreover, we have devised a quadruple transgenic HSC transplantation model to examine dual Erk disruption in the context of Nf1 nullizygosity, testing whether diseased hematopoiesis requires Erk. These insights illuminate cell-specific Erk functions in normal and Nf1-deficient hematopoiesis, informing the feasibility of targeting Mek-Erk in NF1-associated disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Cytokines are extracellular proteins that mediate communication between cells. T helper cell subsets secrete specific cytokines that promote the development of inflammation. Naïve CD4+ T cells activated and primed in the presence of TGF-β and IL-4 predominantly secrete IL-9, a cytokine that acts as a growth factor for T cells and mast cells, and promotes allergic inflammation. The transcription factors downstream of TGF-β- and IL-4-induced signaling, and that are required for expression of IL-9, have not been previously examined. IL-4 signaling induces the expression of IRF4, a transcription factor required for the development of Th9 cells. IL-4 and the downstream-activated factor STAT6 also interfere with the expression of the transcription factors T-bet and Foxp3 that inhibit IL-9 production from Th9 cells. The TGF-β pathway induces the expression of PU.1, another transcription factor required for Th9 development. In the absence of PU.1 there is increased association of a subset of histone deacetylases to the Il9 promoter. In developing Th9 cells, PU.1 can bind to the Il9 promoter and recruit specific histone acetyltransferases, including Gcn5 to the Il9 gene. Gcn5 functionally contributes to Il9 expression as IL-9 production is diminished when Gcn5 expression is reduced, although other cytokines expressed by Th9 cells are not affected. While Gcn5 is not required for PU.1 or IRF4 binding to Il9, it is important for controlling histone acetylation at the Il9 gene promoter. Together these data define the STAT6-dependent transcription factor network in Th9 cells and the mechanism of PU.1-dependent IL-9 induction in Th9 cells and might indicate that targeting IL-9 regulation is a viable approach for treating inflammatory disease.

Indiana University-Purdue University Indianapolis (IUPUI) / CD4 T helper cells control immunity to pathogens and the development of inflammatory disease by acquiring the ability to secrete effector cytokines. Cytokine responsiveness is a critical component of the ability of cells to respond to the extracellular milieu by activating Signal Transducer and Activator of Transcription factors that induce the expression of other transcription factors important for cytokine production. STAT4 is a critical regulator of Th1 differentiation and inflammatory disease that attenuates the gene-repressing activity of Dnmt3a. In the absence of STAT4, genetic loss of Dnmt3a results in de-repression of a subset of Th1 genes, and a partial increase in expression that is sufficient to observe a modest recovery of STAT4-dependent inflammatory disease. STAT4 also induces expression of the transcription factors Twist1 and Etv5. We demonstrate that Twist1 negatively regulates Th1 cell differentiation through several mechanisms including physical interaction with Runx3 and impairing STAT4 activation. Following induction by STAT3-activating cytokines including IL-6, Twist1 represses Th17 and Tfh differentiation by directly binding to, and suppressing expression of, the Il6ra locus, subsequently reducing STAT3 activation. In contrast, Etv5 contributes only modestly to Th1 development but promotes Th differentiation by directly activating cytokine production in Th9 and Th17 cells, and Bcl6 expression in Tfh cells. Thus, the transcription factors Twist1 and Etv5 provide unique regulation of T helper cell identity, ultimately impacting the development of cell-mediated and humoral immunity.